21;:L EFFECTS OF BIOGENIC MONOAMINES. ERGOT DRUGS, ESTROGEN AND SYNTHETIC THYROTROPIN - RELEASING, HORMONE ON PITUITARY PROLACTIN RELEASE Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY KUEW-HSIUNG LU 1972 ‘IIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII I 3 1293 01104 4470 LIB RA R Y M SCIIIgan Sta to U n 1V chity "L __ This is to certify that the thesis entitled Effects of Biogenic Monoamines, Ergot Drugs, Estrogen and Synthetic Thyrotropin-Releasing Hormone on Pituitary Prolactin Release presented by Kuew-Hsiung Lu has been accepted towards fulfillment of the requirements for PhoDo degree in Physiology_ 0-7639 T‘: BINEING av “‘1' HOME 8 SUNS' II BOOK BINDERY INC. , LIBRARY emoms II smuGPont. MICHIGAN ill ___.__—-——-— MAGIC 2 MA? 118139392 .‘5‘ ‘5) q 1 O J i 43 1...; 01 I503 ABSTRACT EFFECTS OF BIOGENIC MONOAMINES, ERGOT DRUGS, ESTROGEN AND SYNTHETIC THYROTROPIN-RBLEASING HORMONE ON PITUITARY PROLACTIN RELEASE By Kuew-Hsiung Lu 1. A single intracarotid or intraperitoneal injection of dopamine, norepinephrine, or epinephrine had no effect on serum prolactin levels in proestrous female rats. The fail— ure of catecholamines given systemically to alter serum pro- lactin is due to the "blood-brain barrier" which prevents them from reaching the hypothalamus. 2. A single intraperitoneal injection of L-DOPA, the immediate precursor of dopamine, produced a rapid decrease in serum prolactin and an increase in pituitary prolactin concentrations. Monoamine oxidase inhibitors, pargyline, iproniazid and Lilly compound-15641, also significantly re- duced serum prolactin. These reductions in serum prolactin by the 4 drugs were associated with increased PIF activity in the hypothalamus. L-DOPA also produced rapid decrease in serum prolactin in hypophysectomized, pituitary-grafted rats. Kuew-Hsiung Lu This was associated with an increase in hypothalamic PIF ac- tivity and appearance of PIF in the serum. Methyl-DOPA, d- amphetamine, reserpine, chlorpromazine, and alpha-methyl- para—tyrosine, drugs known to depress hypothalamic catechol- amines, all elevated serum prolactin and reduced pituitary prolactin concentrations by increasing pituitary release of prolactin. These results indicate that hypothalamic cate- cholamines inhibit pituitary prolactin release by increasing PIF release from the hypothalamus. Drugs that decrease cate- cholamines stimulate prolactin release by decreasing PIF ac- tivity. 3. A single intravenous injection of 5—hydroxytrypto- phan, the immediate precursor of serotonin, or melatonin, a product of serotonin, produced a rapid elevation in serum prolactin in proestrous female rats. 5-Hydroxytryptophan a1- so increased serum prolactin in hypophysectomized, pituitary- grafted rats. Tryptophan produced only a small rise in serum prolactin. Serotonin itself did not significantly alter se- rum prolactin, presumably because it failed to cross the "blood-brain barrier." These results suggest that serotonin and melatonin in the brain stimulate pituitary release of prolactin. The mechanism of 5-hydroxytryptophan stimulation of prolactin release is unknown. It had no effect on hypo- thalamic PIF activity. 4. Ergocornine methanesulfonate (ERG) significantly inhibited the stimulatory effects of estradiol benzoate (EB) Kuew-Hsiung Lu on pituitary and serum prolactin concentrations, mammary growth, and increase in anterior pituitary (AP) weight in ovariectomized rats. In hypophysectomized, pituitary- grafted rats EB increased both AP and serum prolactin and stimulated mammary growth, whereas ERG reduced prolactin re- lease from the AP graft and inhibited mammary growth. When given together, ERG partially counteracted EB stimulation of pituitary prolactin secretion and mammary growth. When normal rat AP halves were incubated in_vi££2, ERG completely inhibited estrogen stimulation of prolactin release and in- creased pituitary accumulation of prolactin. These results indicate that ERG can inhibit prolactin release by a direct action on the AP, and can counteract the stimulatory effect of estrogen on prolactin secretion. 5. In female rats bearing pituitary "mammotropic" (MtT. W 15) tumor transplants, the size and number of pitu- itary tumors developed in each rat were closely related to the serum prolactin concentration. A single injection of Lilly compound-55327, an ergot derivative, into these rats produced a highly significant reduction in serum prolactin. Daily injections of ergocornine, and to a lesser extent, er- gonovine and Lilly compound-55327, also significantly inhib- ited pituitary tumor growth. Ergocornine produced a loss of nuclei and disappearance of cells in the tumor tissue. These results suggest that the major action of the ergot drugs was exerted directly on the tumor to inhibit its growth and suppress prolactin secretion. Kuew-Hsiung Lu 6. Synthetic thyrotropinvreleasing hormone (TRH) in doses of 3 and 30 ng produced no effect on prolactin re- lease by the end of 4, 8 or 12 hours of incubation when in- corporated into a medium containing an AP half from normal male rat or rat pituitary tumor tissue. TRH increased pro- lactin release by about 30% by AP halves from thyro—para— thyroidectomized rats after 4 hours of incubation, but had no effect after 8 and 12 hours of incubation. An intrave- nous injection of 5 or 7.5 ug of TRH into normal male rats had no effect on serum prolactin at 15, 30 and 60 minutes after the injection. Daily injections of 50 ug TRH for 6 days significantly increased AP prolactin concentration and produced a small elevation in serum prolactin. This stimu- lation of pituitary prolactin secretion presumably was due to activation of the TSH-thyroid system, since no such in« creases were observed when TRH was given to thyro-parathy- roidectomized rats. It is concluded from these experiments that synthetic TRH is not a specific releaser of prolactin in the rat. EFFECTS OF BIOGENIC MONOAMINES, ERGOT DRUGS, ESTROGEN AND SYNTHETIC THYROTROPIN-RELEASING HORMONE ON PITUITARY PROLACTIN RELEASE By Kuew-Hsiung Lu A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1972 Dedicated to my parents, En-Gie and Jan-Mei Lu, and to my wife, Mann ii ACKNOWLEDGMENTS Only those who have sailed on the boundless ocean of learning appreciate the importance of a great teacher, and only those who have been away from home alone in a for- eign country treasure the helping hands of a professor, who in addition to guidance, loves and cares. During his course of graduate work at this university, the author was able to reach where he is only under the capable guidance of Dr. Joseph Meites. As a foreigner from the other side of the world, fumbling in the darkness in search for advanced edu- cation in a country very much strange to him when he first came, the author is forever grateful to Dr. Meites who has rendered not only academic advice but also loving care and compassion so much needed by a foreign student. The author has been exposed to numerous science lit- erature so appropriately assigned by Dr. Meites whose en- lightening direction and profound insight have given the au- thor unique training which is second to none in this field of science. Whatever honors and credits the author has re- ceived during his graduate work at this university are due solely to Dr. Meites, to whom the author feels it extremely difficult to find proper words to express his heartfelt grat- itude and appreciation. iii The author wishes to express his sincere gratitude to the members of the guidance committee: Drs. W. D. Collings, E. P. Reineke, J. B. Scott, T. W. Jenkins, and A. J. Morris, who willingly counseled the author in prepar- ing this thesis and other academic matters toward the com- pletion of this graduate education. Sincere gratitude is expressed to Drs. E. M. Convey and G. D. Riegle who extem- porarily requested by the author upon short notice willingly served at the guidance committee. The opportunity for con- sultation with Dr. C. W. Welsch is also gratefully acknow- ledged. Appreciation is also expressed to Drs. Y. Koch, Y. Amenomori, C. L. Chen, and H. Nagasawa, Miss M. C. Gelato and Mr. C. J. Shaar, from whom the author received encour- agement and assistance during collaboration in some of his studies. The author is indebted to Mrs. Claire Twohy, Mr. Gary Kledzik, and Mr. Eldon Cassell for their invaluable technical assistance, especially in carrying out many radio- immunoassays and preparing histology sections. Special thanks are given to Mrs. Amylou Davis and Mrs. Pam Rashid for their willingness and tolerance in typing many forms, letters, and manuscripts. The author also wishes to thank Dr. Meites and the members of the Graduate Affairs Committee of Physiology De- partment, Michigan State University, for the assistantship granted him from September, 1968 to the completion of this graduate work. iv Last, but not the least, the author wishes to ex- press his many thanks to his wife, Mann, who helped and collaborated a great deal to make these graduate years at Michigan State meaningful in his career. TABLE OF CONTENTS Page LIST OF TABLES LIST OF FIGURES INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . S I. Functional Neuroanatomy of the Hypothalamic-Pituitary Axis . . . . . . . . . . S A. General Anatomy of the Hypothalamus 5 B. Hypothalamo-Pituitary Portal System . 7 C. Sites of Origin of Hypothalamic Hypophysiotropic Hormones . . . . . . . . . . 9 II. Control of Pituitary Prolactin Secretion by the Hypothalamus and by Other Systems . . . . 11 A. Hypothalamic Control of Anterior . Pituitary Function . . . . . . . . . 11 B. Hypothalamic Prolactin Release- Inhibiting Factor (PIF) . . . . . . . . . 13 C. Hypothalamic Prolactin- -Releasing. Factors (PRF)? . . . . . . . . . . . . . . 16 D. Role of Biogenic Amines . . . . . . . . . 22 E. Short- -Loop Feedback Control of Pituitary Prolactin Secretion . . . . . . . . 42 F. Direct Effects of Drugs and Hormones on Pituitary Prolactin Release . . . . . . . . . . 45 G. Multiple Control of Pituitary Prolactin Secretion . . . . . . . . . . . 52 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 54 1. Animals . . . . . . . . 54 II. Pituitary TransplantatiOn Technique . . . . . . 55 III. Blood Vessel Cannulation Technique . . . . . . . 55 vi IV. V. VI. VII. VIII. IX. Pituitary Tumor Transplantation Technique . . . Preparation of HypOthalamic Extract, Pituitary Homogenate, and Serum . . . . In Vitro Incubation Technique R§d1o1mmunoassay of Rat Prolactin . Mammary Gland Growth Rating System Methods of Statistical Analysis EXPERIMENTAL I. II. III. IV. In Vivo and In_Vitro Effects of Catecholamines on Pituitary Prolactin Release . . . . . . . . . . . . . A. Objectives . . B. Materials and Methods C. Results . . . D. Conclusions Effects of Central Acting Drugs on Serum and Pituitary Prolactin Levels A. Objectives . B. Materials and Methods C. Results . . . . . . D. Conclusions Stimulation of Pituitary Prolactin Secretion by Drugs that Depress Hypothalamic Catecholamines in Ovariectomized and Ovariectomized, Estrogen-Primed Rats A. Objectives . . B. Materials and Methods C. Results . . . D. Conclusions Effects of L-DOPA on Serum Prolactin and Prolactin Release- Inhibiting Activity in Intact and Hypophysectomized, Pituitary- -Grafted Rats . . A. Objectives . B. Materials and Methods C. Results . . . D. Conclusions vii Page 100 100 101 104 108 110 110 111 115 122 V. Effects of Serotonin, Melatonin, VI. VII. VIII. 5-Hydroxytrytophan and Tryptophan on Serum Prolactin and Hypothalamic and Serum Prolactin Releasing Activity . A. Objectives . . B. Materials and MethOds C. Results . . . . D. Conclusions Direct Inhibition by Ergocornine of Pituitary Prolactin Release A. Objectives . B. Materials and Methods C. Results . . . . D. Conclusions Secretion of Prolactin in Rats Bearing A Pituitary Mammotropic Tumor; Inhibition by Ergot Drugs of Tumor Growth and Prolactin Release A. Objectives . B. Materials and Methods C. Results . . . . D. Conclusions In Vivo and In Vitro Effects of Synthetic Pyro- Glutamyl— Histidyl- Proline Amide (TRH) on Pituitary Prolactin Release in Rats A. Objectives . . B. Materials and Methods C. Results . . . . D. Conclusions GENERAL DISCUSSION . LIST OF REFERENCES APPENDIX . viii Page 123 123 124 127 133 135 135 136 139 152 154 154 154 157 171 172 172 173 176 185 189 200 223 Table LIST OF TABLES Effects of intraperitoneal injec- tions of catecholamines on serum prolactin levels Effects of intracarotid injec- tions of catecholamines on serum and pituitary prolactin concentra- tions . . . . Effects of different doses of dopa- mine on pituitary prolactin release in vitro . . . . . . Effects of different doses of nore- pinephrine and epinephrine on pi- tuitary prolactin release in_vitro Effects of a single intraperitoneal injection of drugs on serum and pi— tuitary prolactin concentrations Effects of drugs on hypothalamic PIF activity . . . . Effects of a single intraperitoneal injection of drugs on serum and pituitary prolactin concentrations Effects of a single intraperitoneal injection of reserpine, chlorpromazine and/or L-DOPA on serum prolactin levels in proestrous rats Effects of alpha-methyl-para-tyrosine and/or L-DOPA on serum prolactin levels in diestrous rats . ix Page 73 74 75 77 86 89 91 94 96 Table Page 10. Effects of multi-injections of drugs on pituitary prolactin secretion, anterior pituitary weight, and mammary growth in ovariectomized rats . . . . . . . . . . 105 11. Effects of multi-injections of drugs on pituitary prolactin secretion, anterior pituitary weight, and mammary growth in ovariectomized, estrogen— primed rats . . . . . . . . . . . . . . . . . . . 106 12. Effects of a single iv injection of L-DOPA on serum prolactin values . . . . . . . . 114 13. ln_vitro assay of PIF activity in serum and hypothalamus from rats given L-DOPA . . . . . . . . . . . . . . . . . . . . . 118 14. Effects of a single intravenous in- jection of tryptophan, 5-hydroxytrypto- phan, serotonin and melatonin on serum prolactin values . . . . . . . . . . . . . . . . 129 15. Effects of a single intravenous injec- tion of S-hydroxytryptophan on serum prolactin values, and prolactin- releasing activity in the serum and hypothalamus of hypophysectomized and hypophysectomized, pituitary-grafted rats . . . . 130 16. Effects of estradiol benzoate (EB) and ergocornine methanesulfonate (ERG) on pituitary and serum prolactin concen- trations, pituitary weight and mammary growth in ovariectomized rats . . . . . . . . . . 141 17. Effects of estradiol benzoate (EB) and ergocornine methanesulfonate (ERG) on pituitary and serum prolactin concen- trations, pituitary weight and mammary growth in hypophysectomized-ovariectomized rats with a pituitary transplant . . . . . . . . 144 18. Effects of ergocornine methanesulfonate (ERG) and prolactin during 7 estrous cycles on ovaries . . . . . . . . . . . . . . . . 148 Table 19. 20. 21. 22. 23. 24. 25. 26. Page Body weights, tumor size and serum prolactin concentrations in rats bearing pituitary mammotropic tumor transplants at 75 days after trans- plantation . . . . . . . . . . . . . . . . . . . 158 Effects of a single injection of ergot drugs on serum prolactin- levels in rats bearing trans- planted pituitary tumors of different size . . . . . . . . . . . . . . . . . 161 Effects of subcutaneous injections of Lilly compound-55327 on body weights, tumor growth and serum prolactin levels in rats bearing transplanted pituitary tumors . . . . . . . . . 163 Effects of synthetic TRH on prolactin release by normal rat pituitary and by rat pituitary tumor tissue in_ vitro . . . . . . . . . . . . . . . . . . . . . 177 Effects of synthetic TRH on prolactin release by hypothyroid rat pituitary in vitro . . . . . . . . . 179 Effects of a single iv injection of synthetic TRH on serum prolactin levels . . . . . . . . . . . . . . . . . . . . . 180 Effects of iv injections of synthetic TRH on pituitary and serum prolactin concentration . . . . . . . . . . . . . . . . . 183 Effects of TSH injections and/or thyro- parathyroidectomy on anterior pituitary weight, and pituitary and serum pro- lactin concentrations . . . . . . . . . . . . . 186 xi Figure 10. LIST OF FIGURES Biosynthesis Pathway of Catecholamines . Catabolism Pathway of Catecholamines . View of A Rat With A Jugular Cannula Attached by an Extension Segment of Polyethylene Tubing . . . . View of a Rack Holding Tubes with Blood Samples in an Oblique Position to Increase the Contact Surface of Blood to Glass . Rat Prolactin Reference Standard Curves Showing Lack of Effect of Drugs on Antigen (Prolactin)-Antibody Reactions . Effects of Drugs That Increase Hypo- thalamic Catecholamines on Serum and Pituitary Prolactin Concentrations . Effects of Drugs That Decrease Hypo- thalamic Catecholamines on Serum and Pituitary Prolactin Concentrations . Effects of L-DOPA on Serum Prolactin Levels in Intact, Hypophysectomized, or Hypophysectomized, Pituitary- Grafted Female Rats . . . . Effects of L-DOPA on Serum Prolactin Releasing Activity in Intact, Hypo- physectomized, or Hypophysectomized, Pituitary-Grafted Female Rats Effects of Estradiol Benzoate (EB) and Ergocornine Methanesulfonate (ERG) on Serum and Pituitary Prolactin Con- centrations in Ovariectomized Rats xii Page 25 27 57 60 78 87 92 117 120 142 Figure 11. 12. 13. 14. 15. 16. 17. Effects of Estradiol Benzoate (EST) and Ergocornine Methanesulfonate (ERG) on Serum and Pituitary Pro- lactin Concentrations in Hypophy- sectomized-Ovariectomized, Pituitary- Grafted Rats . . . . . . . . . Effects of Ergocornine Methanesulfonate (ERG) Estradiol (EST) or both on Pitu- itary Prolactin Concentration and Re- lease In_Vitro . . . Effects of Lilly Compound-55327 (EGL) on the Growth of MtT W 15 Pituitary Tumors and Serum Prolactin in Rats Effects of Three Ergot Drugs on the Growth of MtT W 15 Pituitary Tumors in Rats . . . . . . . . . . (A) Section from a Transplanted Pituitary Tumor from a Control Rat Showing Cells of Diverse Size with Prominent Nuclei and Several Mitotic Figures. (B) Section from a Trans- planted Pituitary Tumor from a Rat Treated with Ergocornine, Showing Large, Separated Cells with Few Pycnotic Nuclei . . . . Effects of Multi-Injections of TRH on Pituitary and Serum Prolactin Concentrations in Intact and Thyro- Parathyroidectomized Male Rats The Relation of Biogenic Monoamines, Ergot Drugs, and Estrogen to Pituitary Release of Prolactin . xiii Page 146 151 165 168 170 184 199 INTRODUCTION It long has been recognized that the functions of the anterior pituitary are influenced by the central nervous sys- tem, particularly by the hypothalamus. Early in the 1950's when neuroendocrinology evolved as a new field of science, it was proposed that secretion of anterior pituitary hormones was controlled by specific neurohormones produced by the hy- pothalamus. Among the six anterior pituitary hormones, it is now known that synthesis and release of prolactin is uniquely regulated by a prolactin release-inhibiting factor (PIF) produced by the hypothalamus. The secretion of the other five anterior pituitary hormones requires stimulation by releasing factors produced in the hypothalamus. During recent years several significant "breakthroughs" have occurred in neuroendocrinology. The most exciting dis- covery has been the determination of the chemical structure and synthesis of two releasing factors, thyrotropin-releasing hormone (TRH) and luteinizing hormone-releasing factor (LRF). The identification of these two hypophysiotropic hormones of the hypothalamus has essentially proved the "chemotransmitter hypothesis" of hypothalamic control of anterior pituitary function as first stated by G. W. Harris (1955). These two releasing hormones are now being synthesized, are available commercially and are now employed in both basic and clinical investigations. Currently attempts also are being made to chemically characterize the remaining hypothalamic hypophy- siotropic hormones, including the prolactin-inhibiting fac- tor (PIF). Another significant advancement in neuroendocrinology has been the recent clarification of the role of biogenic amines in secretion of anterior pituitary hormones, partic- ularly in relation to prolactin and gonadotropins. Nore- pinephrine and serotonin are highly concentrated in the nerve terminals located in the basal hypothalamus. The me- dian eminence is rich in nerve endings containing dopamine and serotonin. Catecholamines have been shown to release PIF, LRF, and FRF into the pituitary stalk portal blood, with resultant inhibition of prolactin release and stimula- tion of LH and FSH release from the pituitary (Kamberi 32 al., 1969, 1970b). Catecholamines have no effects on pitu- itary release of these hormones when they are infused directly into stalk portal vessels. Thus the concept has evolved that biogenic amines, particularly catecholamines, act as neuro- transmitters to control the release of hypophysiotropic hor- mones from the hypothalamus. One aspect of this thesis is an investigation of the role of biogenic monoamines on release of PIF (and possibly prolactin-releasing factor, PRF) by the hypothalamus and on release of prolactin. One particularly significant compound employed in these studies was L-dihydroxyphenylalanine (L-DOPA), the immediate precursor of dopamine. L-DOPA has been known for its ability to ameliorate Parkinson's disease. More recently this catechol amino acid was first shown by us to inhibit prolactin release and to suppress mammary tumor growth in rats. It is now being used successfully to treat human breast cancer. In patients with Forbes-Albright syn- drome, L-DOPA administration also has been reported to in- duce cessation of persistent lactation and cause resumption of menstrual cycles. This thesis has attempted to elucidate the possible mechanism by which L-DOPA inhibits pituitary prolactin release and induces mammary tumor regression. The recent development of specific radioimmunoassays for anterior pituitary hormones permits investigators to measure hormone levels in the blood and other tissues with high accuracy. By use of the standard radioimmunoassay for rat prolactin, it is possible to detect changes in hormone levels simultaneously in three different organ-systems: PIF in the hypothalamus and its release into the systemic circulation; the amount of prolactin present in the pitui- tary; and prolactin levels in the circulation and peripheral tissues. Most of the studies in this thesis include results on changes in hormone activity in these three systems, in an effort to provide a more complete profile of changes in the hypothalamic-pituitary system under different experimen- tal conditions. There is little doubt that pituitary prolactin secre- tion is mainly under the control of hypothalamic PIF. But several drugs and hormones also can influence prolactin re- lease by a direct action on the pituitary. Part of this the- sis deals with studies on the direct action of ergot drugs and estrogen on pituitary release of prolactin, and on pro- lactin release by pituitary "mammotropic" tumor. An attempt was made to delineate the mechanisms by which ergot drugs in- duce regression of pituitary and mammary tumors. There has been much speculation recently that syn- thetic TRH, pyro-g1utamy1-histidyl-proline amide, may be iden- tical or similar to the postulated PRF in the hypothalamus. This is based on observations that synthetic TRH increases blood levels of prolactin when given to humans, monkeys and cattle. In an attempt to clarify the possible action of TRH on pituitary release of prolactin, the author has spent much of his last period of graduate work on TRH. This thesis de- scribes the findings with TRH in the rat under several dif- ferent endocrine states, and compares these with results re- ported in other mammalian species. REVIEW OF LITERATURE I. Functional Neuroanatomy of the Hypotfialamic-Pituitary_Axis A. General Anatomy of the Hypothalamus Several books and reviews have been written on the neuroanatomy of the hypothalamus (Jenkins, 1972; Truex and Carpenter, 1969; Netter, 1967; Daniel, 1966; deGroot, 1959; Gurdjian, 1927). The recent volume by Jenkins (1972) has an excellent description of the functional neuroanatomy of the mammalian hypothalamus. The colored illustrations by Netter (1967) provide a brief but clear view of the role of the hypothalamus in pituitary function. The hypothalamus is the most ventral portion of the diencephalon and is visible from the ventral surface of the brain. It comprises the lateral walls of the third ventri- cle below the hypothalamic sulcus and the structures of the ventricular floor, including the optic chiasm, the tuber cinereum and infundibulum, the neurohypophysis, and the mam- millary bodies. The region immediately in front of the optic chiasm, extending to the lamina terminalis and the anterior commissure, is known as the preoptic area. Dorsal to the hypothalamus is the thalamus, and lateral is the subthalamic areas. Caudally, the mammillary bodies are considered as the limiting border of the hypothalamus, but there is a melting into the posterior perforated substance and tegmentum of the midbrain. Medially, the vertically oriented third ventricle divides the hypothalamus into right and left halves. The pituitary is attached to the brain by the infun- dibulum (stalk of the pituitary). The latter has a hallow center, which is the ventral extension of the third ventricle. The neurohypophysis or posterior lobe of the pituitary is com- posed Of nervous tissue continuous with the basal portion of the hypothalamus. In a rostro-caudal sequence, three distinct regions can be identified in the hypothalamus: supraoptic, tuberal, and mammillary areas with their specific groups of nuclei. The supraoptic area contains two functionally well-known nu- clei, the supraoptic and paraventricular. In the tuberal re- gion, the infundibulum attaches to the median eminence of the tuber cinereum. The ventral portion of the periventricular nucleus and the arcuate nucleus collectively constitute the gray matter enveloping the base of the third ventricle. Al- so located in the tuberal region are the dorsomedial, ventro- medial, and lateral hypothalamic nuclei. The mammillary, or caudal hypothalamic, area is composed of the prominent mam- millary bodies. The hypothalamus receives projections from many biber tracts coming in from broad areas of the brain. These dif- ferent pathways include the fornix, medial forebrain bundle, thalamo-hypothalamic fibers, mammillary peduncle and stria terminalis. In addition, the hypothalamo-hypophyseal tract, periventricular fibers and mammillary body efferents are the three major efferent fiber tracts originated from specific nuclei of the hypothalamus. It long has been known that oxy- tocin and anti-diuretic hormone (ADH) are synthesized in the supraoptic and paraventricular nuclei (Bargmann and Scharrer, 1951), and subsequently traverse the hypothalamo-hypophyseal tract to the posterior pituitary. B. Hypothalamo-Pituitary Portal System The secretion of hormones by the anterior pituitary is under the direct control of the hypothalamus. This hypo- thalamic regulation is by way of the pituitary portal vessels rather than through direct innervation. No direct neural connection between the hypothalamus and the anterior pitu- itary is evident. Popa and Fielding (1930) first described the pitu- itary portal circulation and indicated that the portal blood flowed from the pituitary to the hypothalamus. However, sub- sequent studies suggested that the flow was from the hypothal- amus to the anterior pituitary (Wislocki and King, 1936;. Houssay EE.El-: 1935; Green, 1947). Green and Harris (1949) directly observed the pituitary portal circulation in living rats under the microscope. They indicated that the portal vessels originated in the median eminence of the tuberal cinereum and in the infundibular stem, and observed that the blood flowed caudally toward the anterior pituitary. Similar observations also were reported in the dog by Torok (1954) and in the mouse by Worthington (1955). Since then, the ”chemotransmitter hypothesis" of Harris (1955) has received strong support from morphological evidence (see Section II, A). The median eminence receives blood from branches of the anterior pituitary artery. These arterioles break up in- to tortuous capillary loops in the median eminence. The ven- ous blood goes from the median eminence to the stalk portal vessels which run down to the anterior pituitary tissue. The free part of the pituitary stalk (infundibular stem) is supplied by the peduncular artery which springs from the pos- terior communicating and internal carotid arteries. Stalk portal vessels also originate from the capillary loops in this region. These stalk portal vessels constitute the only venous drainage from the capillary beds in either the median eminence or the infundibular stem. The anterior pituitary derives its entire blood supply from the portal circulation. There is no arterial blood sup- ply directly perfusing the anterior pituitary tissue, except in humans and in rabbits. The dorso-caudal portion of the anterior pituitary also receives blood from the posterior pituitary through a group of small, short portal vessels. A more thorough discussion of the anatomy of the pituitary portal system is given by Daniel (1966), Green (1966), Adams gt_al. (1965), Landsmeer (1963), and Daniel and Prichard (1956). C. Sites of Origin of Hypothalamic Hypophysiotropic Hormones The anterior pituitary loses most of its histological characteristics and secretory capacity when its vascular con- nection with the median eminence of the hypothalamus is in- terrupted (Harris and Jacobsohn, 1952; Nikitovitch-Winer and Everett, 1959). Halasz 33 31. (1962) introduced the term "hypophysiotropic” to refer to the need for connections with the hypothalamus for the maintenance of normal anterior pitu- itary histology and function. The ”chemotransmitter hypothe- sis" of Harris (1955) proposed that neurohormones released by the hypothalamus are conveyed to the anterior pituitary via the stalk portal vessels. These neurohormones of the hypo- thalamus are responsible for regulating anterior pituitary function and hence have the name ”hypothalamic hypophysiotrop- ic hormones." By transplanting fragments of anterior pituitary tis- sue into different parts of the hypothalamus, it was observed that pituitary grafts showed some histological maintenance and functional activity, particularly when they were placed 10 in certain areas of the ventral hypothalamus (Halasz 31 31., 1962; Knigge, 1962; Desclin and Flament-Durand, 1963; Flament- Durand, 1964). But greater functional activity was found when the pituitary grafts were in direct contact with the me- dian eminence tissue (Halasz 31 31., 1965; Flament-Durand, 1965). Halasz 31 31. (1962) have proposed the name "hypophy- siotropic area” for the hypothalamic zone in which pituitary grafts maintained a nearly normal histological picture. This area includes the arcuate nucleus, the ventral part of the anterior periventricular nucleus, and the medial parvi- cellular region of the ”retrochiasmatic area.” Here are lo- cated the cell bodies of the tubero-infundibulular fibers to the median eminence (Halasz and Szentagothai, 1962; Szenta- gothai, 1962, 1964). It is hypothesized that the hypophysio- tropic hormones are synthesized or present in the cell bodies within the hypophysiotropic area and are normally released at the nerve endings in the median eminence (Halasz 31 31., 1962; Szentagothai and Halasz, 1964). It will be seen subse- quently that biogenic amines serve as neurotransmitters and participate in the release of hypophysiotropic hormones by the hypothalamus (see Section II, D). In recent years the evidence has essentially proved the existence of the presumed hypophysiotropic hormones in the hypothalamus. Two such substances, thyrotropin-releasing hormone (TRH) and luteinizing hormone-releasing factor (LRF), have been purified and synthesized by several laboratories. 11 The chemical structure of TRH was determined to be a tripep- tide, pyro-glutamyl-histidyl-proline amide (Burgus 31 31., 1969; Bowers 31 31., 1970). A decapeptide, pyro-glutamyl- histidyl-tryptophan-serine-tyrosine-glycine-1eucine-arginine- proline-glycine amide, was shown to be the structure of LRF (Baba 31.31,, 1971; Burgus 31_31,, 1971). These 2 hormones are now available commercially and are now employed in both basic and clinical investigations. Efforts also are being made to purify and to synthesize the remaining hypophysio- trOpic hormones, including the prolactin-inhibiting factor (PIF). II. Control of Pituitary Prolactin Secretion by the Hypothalamus and by Other systems A. Hypothalamic Control of Anterior Pituitary Function The hypothalamus, by regulating synthesis and release of specific hypophysiotropic hormones, controls the secretion of anterior pituitary hormones. Early work by Taubenhaus and Soskin (1941) suggested that the rat hypothalamus secretes an acetylcholine-like substance into the portal vessels to elic- it pituitary LH release. Later, Markee 31 31. (1948) proposed that an adrenergic mechanism also might be involved in the control of LH secretion in rats. Harris and Jacobsohn (1952) were the first to demonstrate the direct influence of anter- ior pituitary function by the hypothalamus. They observed 12 return of normal reproductive function and milk secretion in hypophysectomized rats when an anterior pituitary was grafted under the median eminence. By contrast, normal estrous cy- cles were not resumed in hypophysectomized rats when the pi- tuitary was transplanted under the temporal lobe of the brain. In 1955 Harris proposed the "chemotransmitter hypothesis," according to which neurohormones released from the hypothal- amus were responsible for regulating pituitary function. This hypothesis has been substantiated in recent years by the demonstration that specific hypophysiotropic hormones are present in the hypothalamus (see review by Guillemin and Schally, 1963; Harris, 1970); several of these have been syn- thesized by several laboratories (Burgus 31 31., 1969; Bowers 31 31., 1970; Burgus 31 31., 1971). Hypothalamic control of prolactin secretion is unique, since it is the only anterior pituitary hormone that appears to be chronically inhibited by the mammalian hypothalamus un- der most conditions. Thus, it has been demonstrated that transplantation of pituitary away from the median eminence results in sustained secretion of prolactin at a high level, whereas secretion of all other anterior pituitary hormones is sharply reduced (Everett, 1954; 1956; Nikitovitch—Winer and Everett, 1958; Chen 31_31,, 1970). Similar results also were observed in rats after sectioning the pituitary stalk (Dempsey and Uotila, 1940), after lesioning the median emi- nence or "hypophysiotropic area" of the hypothalamus (Chen 13 31 31., 1970; Welsch 31 31., 1971), by culturing or incubat- ing anterior pituitary tissue 13_11113 (Meites 31 31., 1961) and by administering appropriate drugs (Meites, 1962; Meites 31 31., 1963). These observations indicate that predominant influence of the mammalian hypothalamus on pituitary prolac- tin secretion is inhibitory in nature. B. Hypothalamic Prolactin Release- Inhibiting Factor (PIF) 1. In Vivo and in vitro demonstrations of a hypothalamic PIF Inhibition of pituitary prolactin release by the mam- malian hypothalamus is exerted via the action of a prolactin release-inhibiting factor. Addition of crude extract of rat hypothalamus to incubations or cultures of pituitary decrease prolactin release (Meites 31 31., 1961; Pasteels, 1961; Talwalker 31 31., 1963). Extracts from the hypothalami of sheep, swine, cattle, humans, but not birds also have been demonstrated to inhibit prolactin release 13_11113 (Schally 31 31., 1965; Kragt and Meites, 1965). Talwalker 31 31, (1963) were the first to propose the name "prolactin-inhibit- ing factor" (PIF) for the presumed hypothalamic hypophysio- tropic hormone which inhibits pituitary secretion of prolac- tin. They Observed a decrease of both synthesis and release of prolactin by the rat pituitary by adding a crude extract of rat hypothalamus to an incubation medium. Subsequently, Kragt and Meites (1967) demonstrated a negative dose-response 14 relationship between the quantity of hypothalamic extract added and the amount of prolactin released 13_111133 This has been confirmed by Chen (1969) by use of a specific radio- immunoassay for rat prolactin (Niswender 31 31., 1969). PIF activity also has been demonstrated 13_1113_by showing that hypothalamic extracts prevented pituitary prolactin depletion by suckling during lactation (Grosvenor 31_31., 1964) or in response to cervical stimulation during estrus in rats (Kuroshima 31_31., 1966; Arimura 31 31., 1967). Amenomori and Meites (1970) reported that an injection of crude extract of 8 rat hypothalamic equivalent markedly reduced serum pro- lactin in cycling and lactating rats. Watson 31 31. (1971) also observed that a single injection of crude hypothalamic extract decreased serum prolactin in normal and orchidecto- mized male rats. The chemistry of hypothalamic PIF has not been characterized. It has been shown that PIF activity is stable in acid medium and not destroyed by boiling, suggest- ing it may be a small peptide molecule like the other hypoe physiotropic hormones (Burgus 31 31., 1969; Bowers 31 31., 1970; Baba 31 31., 1971; Burgus 31 31., 1971). Little is known about the mechanism of action by which PIF inhibits the pituitary release of prolactin. It is believed that the membrane of the prolactin-secreting cell spontaneously de- polarizes when the pituitary is freed from hypothalamic in- fluence. Thus, Nicoll (1971) indicated that PIF may act by depressing spontaneous depolarization of prolactin cells, 15 thereby reducing the entry of Ca ions into the cells and in- hibiting the release of secretory granules. 2. Control of gypothalamic PIF secretion PIF activity in the hypothalamus is readily altered by a variety of drugs, hormones and stimuli. Perphenazine (Danon 31_31,, 1963), reserpine (Ratner 31_31., 1965), halo- peridol (Dickerman 31 31., 1972), epinephrine and acetylcho- line (Mittler and Meites, 1967), estrogen (Ratner and Meites, 1964), progesterone, testosterone and cortisol (Sar and Meites, 1968), a norethynodrel-menstranol combination (enovid) (Minaguchi and Meites, 1967a) and the suckling stimulus (Ratner and Meites, 1964; Minaguchi and Meites, 1967b) were found to decrease hypothalamic PIF activity in rats, whereas ergocornine (Wuttke 31 31., 1971), L-DOPA and monoamine oxi- dase inhibitors (pargyline, iproniazid, and Lilly compound- 15641) (Lu and Meites, 1971) and prolactin itself (Chen 31 31., 1967; Clemens and Meites, 1968; Voogt and Meites, 1971) were shown to increase PIF activity in the hypothalamus. Work by Kamberi 31 31. (1970) demonstrated that a single in- jection of dopamine into the third ventricle of rats elevated PIF activity in the pituitary stalk blood. We have extended this observation in a recent study and demonstrated that L- DOPA, the immediate precursor of dopamine, increases PIF in the hypothalamus and elicits the presence of PIF activity in 16 the systemic blood of intact and hypophysectomized rats (Lu and Meites, 1972). These results indicate that drugs that increase hypothalamic catecholamines also stimulate synthe- sis and release of PIF, whereas drugs that reduce catechol- amines in the brain also-depress hypothalamic PIF activity (Meites 31 31., 1972). Evidence has been presented that pro- lactin increases dopamine activity in the hypothalamus (Fuxe and Hokfelt, 1970). C. Hypothalamic Prolactin—Releasing Factors (PRF) ? 1. Observations suggesting the possible presence ofTa PRF in the mammalian hypothaIamus The inhibitory influence on prolactin release by the mammalian hypothalamus suggests a unique control mechanism. Demonstrations of the presence of a prolactin-releasing fac- tor (PRF) in the avian hypothalamus (Kragt and Meites, 1965; Nicoll, 1965; Gourdji and Tixier-Vidal, 1966; Meites, 1967; Chen 31 31., 1968) has raised the question that the mammalian hypothalamus also may contain a hypophysiotroPic hormone(s) that stimulates pituitary release of prolactin. Meites 31 31. (1960) observed that injections of a crude extract of rat hypothalamus initiated lactation in es- trogen-primed rats, indicating release of prolactin and prob- bably ACTH by the pituitary. However, these responses may not be specific since lactation also was induced in similar 17 rats by injecting cerebral cortical extract or other pharma- cological agents. Later, Mishkinsky 31_31. (1968) confirmed these observations and concluded that the rat hypothalamus contains a PRF. Oxytocin was considered to be responsible for pitu- itary release of prolactin (Benson and Folley, 1956) after it was found to be secreted by hypothalamic nuclei (Bargmenn and Scharrer, 1951). Peterson (1942) also postulated that the posterior pituitary may be responsible for the rapid de- crease in pituitary content observed after suckling. How- ever, no direct evidence was demonstrated that oxytocin can stimulate prolactin release. Oxytocin is ineffective in al- tering pituitary prolactin content (see review by Meites 31 31., 1963) and has little effect on serum prolactin levels in sheep (Greenwood, 1972). Inhibition of mammary gland in- volution by oxytocin appears to be via mechanisms other than release of prolactin (see review by Meites 31_31., 1963). More recently, Valverde and Chieffo (1971) reported that a single injection of a procine hypothalamic extract increased serum prolactin in estrogen, progesterone-treated male rats. This apparently conflicts with the report by Schally 31 31. (1965) that extracts from porcine hypothalami inhibited pro- lactin release under 13_1113_and 13_!1113_conditions. Several 13_g1113_studies suggested the possible pres- ence of a PRF in the rat hypothalamus. Nicoll 31 31, (1970) indicated that both prolactin-inhibiting and prolactin- 18 stimulating activities are present in the rat hypothalamus. They demonstrated that hypothalamic extract inhibited pitue itary prolactin release during the first 4 hours of incuba- tion,but stimulated prolactin release during the subsequent 4 hours. However, using a somewhat different method of incuba- tion, a consistent inhibition of pituitary prolactin release throughout 8 hours was Observed when extract from rat hypo- thalamus was added to an incubation medium (Chen, 1969; Meites, 1970). Krulich 31 31. (1971) reported that, in rat hypothalamus, the median eminence and a narrow basal portion of the preoptic area mainly contains PRF activity, while the dorsolateral region of the preoptic area is rich in PIF. These findings based on 13_11113_studies apparently conflict with results from numerous 13_1113_studies in which it was demonstrated that pituitary release of prolactin is increased and sustained by lesioning the median eminence (Chen 31_313, 1970; Welsch 31 31., 1971), indicating that this area of the hypothalamus is concerned with the release of LH rather than prolactin (Everett and Quinn, 1966). 13_11113 studies have been extended by Chen 31 31. (1972) who assayed PIF and PRF activities in fresh slices of rat hypothalamus, but their re- sults did not sustain the observation of Krulich 31_31. (1971). These studies suggest that the mammalian hypothalamus may con- tain a hypophysiotropic hormone that releases prolactin al- though the results do not permit any definite conclusion to be made at this time (Meites 31 31., 1972). 19 2. Is synthetic pyro- lutamyl-histigyl- proline amide (TRH a PRF in mammals? More recently several laboratories have reported that synthetic pyro-glutamyl-histidy1-proline amide (TRH) can induce prolactin release in humans, monkeys, bovine, and rats. Tashjian 31 31. (1971) observed that TRH increased prolactin and decreased GH release when added to cultures or short-term incubations of clonal cells from rat pituitary tumors. However, these workers also reported that extracts from rat hypothalamus, cerebral cortex, kidney or liver sig- nificantly increased prolactin and decreased GH release when added to cultures of clonal strains of rat pituitary tumor cells (Tashjian 31 31., 1970). This raises the question about the specificity of the reported effects of TRH on pro- lactin and GH release by the pituitary tumor cells 13_11113. Recent work from our laboratory (Lu 31_31., 1972) indicated that TRH had no effect on prolactin release when added to an incubation medium containing normal male rat pituitary halves or "mammotropic" pituitary tumor (MtT. W15) tissue. Single injections of TRH also failed to alter serum prolactin levels, but when TRH injected daily for 6 days there was a signifi- cant increase in pituitary prolactin concentration and a small elevation in serum prolactin. These increases of pro- lactin were not observed when TRH was injected into thyro- parathyroidectomized rats, suggesting that its effects were mediated through the TSH-thyroid system. Previous studies 20 indicated that thyroid hormones stimulated prolactin secre- tion in the rat (Chen and Meites, 1969) and could act direct- ly on the rat pituitary to increase prolactin release 13_ 11113_(Nicoll and Meites, 1963). Bowers (1971) and McCann (1971) also failed to observe any effect on prolactin release by normal rat pituitary tissue when it was added to an incu- bation system. These observations do not support the report of Tashjian 31 31. (1971) that TRH increases prolactin re- lease in the rat. The relation of these observations in the rat to re- ports that TRH can produce a rapid rise in blood prolactin in humans, monkeys, and bovine is not clear. A single intra- venous injection of TRH was reported to evoke a prompt in- crease in blood prolactin levels in humans (Hwang 31_31,, 1971; Bowers 31_31., 1971; Jacobs 31_31., 1971; Bowers 31 31., 1972) and monkeys (Knobil, personal communication). In view of the rapidity of TRH stimulation on prolactin release in the primates, and the observation that TRH is even more effective in rising blood prolactin in hypothyroid than in euthyroid humans (Bowers 31_31,, 1971; Jacobs 31_31,, 1971), it is unlikely that the stimulation of prolactin release by TRH is effected via the TSH-thyroid system in humans. A single intravenous injection of TRH also was observed to elicite a rapid increase in serum prolactin in bovine (Convey et a1., 1972). However, no increase in prolactin release was observed when TRH was added to an incubation medium containing 21 bovine pituitary slices (LaBella and Vivian, 1971; Convey 31 31., 1972). There is as yet no evidence that TRH acts directly on the human pituitary to induce prolactin release. Thus, the possibility that the synthetic pyro-glutamyl- histidyl-proline amide is identical or similar to the postu- lated PRF in the mammalian hypothalamus (Jacobs 31 31,, 1971; Tashjian 31_31,, 1971) remains to be determined. In contrast to the well established alterations of hypothalamic PIF activity by a variety of agents and stimuli (see Section II, B), changes in the activity of the presumed PRF have not been reported. A prompt and big increase in pituitary prolactin release is seen in rats after suckling (Sar and Meites, 1969; Amenomori 31_31., 1970) and after in- jecting 5-hydroxytrytophan, the immediate precursor of sero- tonin (Lu and Meites, unpublished). However, the serum or hypothalamus from rats injected with S-hydroxytryptophan or the serum of rats after suckling has no effect on pituitary prolactin release when added to an incubation medium (Lu and Meites, unpublished). Evidence has been presented that suck- ling reduces hypothalamic PIF activity (Rather and Meites, 1964; Minaguchi and Meites, 1967). Recent work from our lab- oratory (Dibbet 31 31., unpublished) has indicated that TRH does not inhibit the ability of PIF in a rat hypothalamic ex- tract to reduce pituitary release of prolactin 13_!1133, We also have observed that TRH has no ability to alter hypotha- lamic PIF activity when it is injected into normal male rats 22 (Lu and Meites, unpublished). These results from recent and earlier studies indicate that synthetic TRH is not a specif- ic releaser of pituitary prolactin in rats. It remains to be determined how synthetic TRH evokes a rapid rise in blood prolactin in primates and the bovine. There is as yet no definite evidence that synthetic pyro-glutamyl-histidyl- proline amide is the presumed hypophysiotropic hormone that stimulates pituitary release of prolactin in mammals (Lu 31 31., 1972). D. Role of Biogenic Amines l. Biogenic amines in the brain The neural signal emitted by each mammalian neuron is presumably a chemical substance, a neurotransmitter. This substance is released at the synapse and diffuses across the synaptic cleft to reach the receptor surface of the post-synaptic neuron. Upon interaction with the neurotrans- mitter, the post-synaptic neuron generates an action poten- tial and transmits a nerve impulse. The substances most gen- erally accepted as serving as neurotransmitters in the brain include acetylcholine and three monoamines, i.e., dopamine, norepinephrine, and serotonin. Serotonin is an indoleamine; dopamine and norepinephrine are catecholamines. With the de- velopment of histochemical fluorescence techniques for iden- tifying biogenic amines in the brain (see review by Hillarp 23 31 31., 1966), it has been demonstrated that norepinephrine and serotonin are highly concentrated in the hypothalamus and midbrain (Vogt, 1954; Brodie 31 31., 1959). It also has been shown that the nigrostriatal system and the median emi- nence are rich in dopaminergic nerve terminals (Dahlstrom and Fuxe, 1965; Anden 31 31., 1964). Some noradrenergic terminals are also found in the median eminence and infun- dibulum (Bjorklund 31 31., 1970). Chemical assays also have shown that relatively large amounts of norepinephrine and dopamine are present in the median eminence (Laverty and Sharman, 1965; Rinne and Sonninen, 1968). More recently, Pizzi 31_31, (1970) have observed that the bovine median em- inence also contains high concentrations of serotonin. In addition to serving as neurotransmitters as mentioned above, dopamine and norepinephrine may also function as neurohor- mones. These catecholamines may be released into the hypo- thalamo-pituitary portal system from neurons whose cell bod- ies lie in the medial hypothalamus and whose nerve endings are terminated in the median eminence and the infundibulum near the primary capillary loops of the portal system (Fuxe and Nilsson, 1967; Anton-Tay 31_31., 1969; Knigge 31_31., 1968). This has led some workers to speculate that the bio- genic amines may be the "hypophysiotropic hormones” which act directly on the anterior pituitary for hormone secretion. Based on the observations that catecholamines, including dopa- mine, norepinephrine and epinephrine inhibited pituitary 24 release of prolactin 13_X1113, MacLeod (1969) and Birge 31 31. (1970) concluded that the catecholamines in the hypo- thalamus were the not as yet defined prolactin release- inhibiting factor. There is no clear evidence that dopamine or norepinephrine is present in the portal blood which per- fuses the anterior pituitary (Wurtman, 1970). However, there is evidence that little if any of the catecholamine released from nerve endings in the brain can gain entry into the blood stream without first undergoing oxidative deamination (Glowinski 31_31., 1965). 2. Biosynthesis of brain monoamines The first biochemical transformation in the synthesis of brain catecholamines involves the hydroxylation of the amino acid, tyrosine, Tryosine is taken up from the circula- tion by neurons. The hydroxylation of tyrosine is catalyzed by the enzyme, L-tryosine hydroxylase, and results in the formation of a catechol amino acid, L-dihydroxyphenylalanine (L-DOPA). L-DOPA is rapidly converted to dopamine by the en- zyme aromatic L-amino acid decarboxylase (DOPA decarboxylase), after it is formed (Anton and Sayre, 1964). The transforma- tion of dopamine to norepinephrine is through the action of the enzyme dopamine-B-oxidase. Both tyrosine hydroxylase and dopamine-B-oxidase are only found in catecholamine-producing cells. Dopamine-B-oxidase is localized within norepinephrine storage vesicles at the nerve terminals (Wurtman, 1966). It 25 EPINEPHRINE PHENYLETHANOL- AMINE-N -METHYI. TRANSFERASE NOREPINEPHRINE DOPAMINE —B-OX|DASE DOPAMINE DOPA DECARBOXYLASE D O P A TYROSINEI HYDROXYLASE ‘I'YRO SIN E PHENYLALANINEI HYDROXYLASE PH ENYLALAN IN E Figure l. Biosynthesis Pathway of Catecholamines. 26 is believed that the action of the enzyme tyrosine hydrox- ylase is the rate-limiting step for the overall biosynthesis of catecholamines (Costa and Neff, 1970). There is as yet no clear evidence that epinephrine is synthesized by any part of the mammalian brain and serves as a neurotransmitter. However, phenylthanolamine-N-methyl transferase, an enzyme which catalyzes the conversion of norepinephrine to epine- phrine, has been identified within the olfactory bulb and the hypothalamus (Pohorecky 31_31,, 1969). The biosynthesis of brain serotonin (5—hydroxytryp- tamine) involves the transformation of tryptOphan to 5- hydroxytryptophan. This process is catalyzed by the enzyme, tryptOphan hydroxylase. S-hydroxytryptophan, like L-DOPA, is catalyzed by the enzyme, aromatic L-amino acid decarbox- ylase, to form serotonin almost immediately after it is formed (Wurtman, 1970). 3. Physiolog1cal disposition of brain monoamines Using histochemical fluorescence techniques, it has been demonstrated that catecholamines are more concentrated within synaptic vesicles at the nerve terminals than they are in other portions of the neuron (Dahlstrom and Fuxe, 1965; Potter and Axelrod, 1963; Wurtman, 1966). By contrast, serotonin is homogeneously distributed within serotonin- containing neurons (Anden 31_31., 1965). Nerve stimulation causes the release of catecholamine molecules from the 27 .monHEmHocuouwu mo kmznpmm EmHHonmumu .N oudmwm A) Al .. n u u u u u I go... 38232 no... osmozfiz . I>xo¢o>z.v§ozmz.m 1,531,154..” 1 OO O 00 k x M 00 “9.13:. 332.32 All mzaxnmzfimz All mzaxamzzm A. n I u I .. I>xo¢o>¥r>onm¥m 0< .2 I52 .5209 Imoz d O N A W $1 4 ”22:..sz mZEImmZSmZ hEco 28 synaptic vesicles. The catecholamines transverse the synap- tic cleft and interact with the post-synaptic membrane re— ceptors. The physiological actions of norepinephrine with- in the synaptic cleft are mainly terminated by the process of reuptake into the nerve terminals. Most of the catechol- amine present in a neuron is degraded within that neuron by the action of the enzyme, monoamine oxidase (MAO), and re- leased as oxidative deaminated metabolites. Norepinephrine also can be inactivated by the enzyme, catechol-O-methyl transferase (COMT) within the synaptic cleft (Anton-Tay and Wurtman, 1971). The main pathway for serotonin metabolism appears to be oxidative deamination by monoamine oxidase, then followed by oxidation or reduction. In the mammalian pineal gland, the transformation of serotonin to melatonin is of a greater significance as a pathway for its metabolism (Wurtman, 1970). Serotonin is first N-acetylated to form the compound, N- acetylserotonin. By the enzyme action of hydroxyindole-O- methyl transferase, a methyl group from S-adenosylmethionine is transferred to N-acetylserotonin resulting in the synthe- sis of 5-methoxy-N—acetyltryptamine (melatonin). 4. Alterations of brain monoamine activity by pharmacoiggical agents It is generally accepted that the enzymatic action Of tyrosine hydroxylase is the rate-limiting step for the overall biosynthesis of catecholamines. This enzyme is 29 readily attacked by methylated tyrosine analogs such as alpha-methyl-para-tyrosine and alpha-methyl-meta-tyrosine and results in the inhibition of catecholamine synthesis in the brain. Methyl-DOPA competes for DOPA-decarboxylase to synthesize methyl-dopamine and methyl-norepinephrine (false neurotransmitters) instead of dopamine and norepinephrine (normal neurotransmitters). The methylated catecholamines have much weaker function as neurotransmitters than the nor- mal catecholamines (Coppola, 1968; Innes and Nickerson, 1970). The brain catecholamine activity also can be decreased by several tranquilizers: reserpine interfers with storage and/or induces depletion of catecholamines; chlorpromazine inhibits catecholamine action by blocking the receptor sites on the post-synaptic membrane (Coppola, 1968). On the other hand, increased brain catecholamine activity can be achieved by administering precursor catecholamine substrates, such as L-DOPA. This catechol amino acid is taken up by catecholamine- containing neurons and is rapidly transformed to dopamine and norepinephrine in the brain (Wurtman, 1971), even though the amount of L-DOPA gaining entry to the brain represents only a small fraction of the total (Wurtman 31 31., 1970). Since oxidative deamination is the major pathway for monoamine me- tabolism, brain monoamine activity is readily enhanced by ad- ministering monoamine oxidase inhibitors such as pargyline, iproniazid and amphetamine (Koelle, 1971). 30 p-Chloroamphetamine and p-chlorophenylalanine induce depletion of brain serotonin by blocking the enzymatic ac- tion of tryptOphan hydroxylase (Fuller 31 31., 1965; Koe and Weissman, 1966; Sanders-Bush and Sulser, 1970). p«Chloro- phenylalanine also reduces norepinephrine levels in the brain (Koe and Weissman, 1966). 5. Chan es in brain monoamine activity durin different reproductive states and d1urnal rhythm Sandler (1968) observed no changes in hypothalamic catecholamines during the estrous cycle of rats, whereas Donoso and Stefano (1967) reported that norepinephrine lev- els are high in proestrus, low after ovulation and lowest at estrus. By contrast, Lichtensteiger (1969) and Coppola (1971) observed increased hypothalamic catecholamine content at es- trus and reduced levels at diestrus. These inconsistencies are probably due to the different method used for measuring brain catecholamine (Coppola, 1971). Nevertheless, more con- sistent results on the effects of gonadal hormones on brain norepinephrine metabolism have been published. Castration of male or female rats enhanced the synthesis of norepine- phrine in the hypothalamus (Wurtman 31 31., 1969; Anton-Tay and Wurtman, 1968; Anton-Tay 31_31,, 1970); this led to a slight increase in the steady-state concentration of nore- pinephrine (Coppola, 1968) and a marked acceleration in the turn-over rate of norepinephrine (Anton-Tay and Wurtman, 31 1968). Estrogen prevented these effects of castration (Donoso and Stefano, 1967; Coppola, 1968, 1969; Anton-Tay and wurtman, 1968). The mechanism by which castration ac- celerates norepinephrine synthesis appears to be due to a direct action of FSH, since injections of FSH to intact or hypophysectomized, ovariectomized rats mimiced the effect of castration on norepinephrine turn-over (Anton-Tay 31 31., 1969). On the day of estrus, a diurnal rhythm of brain nore- pinephrine levels was reported in rats and cats (Donoso and Stefano, 1967; Reis and Wurtman, 1968; Manshardt and Wurtman, 1968), with higher levels in the morning than in the after- noon. In cycling female rats, the prolactin and gonadotropin surges all appear approximately at the same time on the af- ternoon of proestrus (Wuttke and Meites, 1970; Gay 31 31., 1970). It remains to be determined, however, whether hypo- thalamic catecholamines participate in the control of onset of these hormonal peaks, since the former is stimulatory to pituitary release of gonadotropins but inhibitory to prolac- tin release. Preliminary results from a recent study in our laboratory have indicated that L-DOPA is ineffective in pre- venting the proestrous surge of prolactin (Lu and Meites, I unpublished). Paralleling those changes in hypothalamic catecholamines, alterations in the hypothalamic enzyme activ- ity of monoamine oxidase also were reported under different reproductive states in rats (Kobayashi 31 31., 1964; 1966). Monoamine oxidase in the rat hypothalamus was increased 32 during proestrus and estrus, but decreased at diestrus. Inv creased activity of hypothalamic monoamine oxidase was ob- served in rats with the onset of puberty. A similar increase in the enzyme activity also was seen in ovariectomized rats, but estrogen blocked this rise in monoamine oxidase. Plasma and brain levels of tryptophan, the precursor serotonin substrate, exhibit significant daily fluctuations (Wurtman 31_31,, 1968; Fernstrom and Wurtman, 1971). The serotonin content in rat brain also exhibits a diurnal rhy- thm, with a maximum at about the 8th hour after the onset of light and a minimum at the 4th hour after the onset of dark- ness (Synder 31_31., 1965; 1967); this is in opposition to the circadian rhythm of norepinephrine which shows a peak in the dark phase (Asano, 1971). A diurnal variation in serum prolactin in rats was reported by Koch 31_31, (1971), with higher serum prolactin values on the late afternoon of each day than in the morning. Similar variation was also demon- strated in pituitary prolactin content (Clark and Baker, 1964). Dunn 31_31, (1972) found a circadian periodicity in serum prolactin concentration with a peak at 11 PM. These observations suggest that there is a dual control in the hy- pothalamus over prolactin secretion, with a predominant ad- renergic tonus to inhibit prolactin release under most con- ditions, and a serotonergic system that may be responsible for the diurnal rise on the late afternoon and perhaps under other conditions in which serotonin is increased (Meites 31 31., 1972). 33 6. In vivo and in vitro fates of exggenous monoamines Any monoamines administered systematically can reach the hypothalamo-pituitary portal system only to a minor de- gree (Fuxe and Hokfelt, 1969; Steinman 31 31., 1969). Mono- amines are rapidly inactivated by enzymes in the liver and kidneys. Norepinephrine is cleared from the circulation by uptake into adrenergic nerve endings (Wurtman, 1970). Any monoamine that gained entry into brain arteries still would face great difficulty in reaching the neurons which release into the pituitary protal system. The operation of a "blood- brain barrier" hinders the entry of circulating monoamines into brain tissue. The "barrier" is relatively weak at the level of the hypothalamus and the area of postrema than in most other regions of the brain, but still is highly resis- tant to monoamines (Wurtman, 1966; 1970). Catecholamines are degraded rapidly 13_X1113_to form adrenochromes which exert powerful pharmacological effects on cellular metabolism (Wurtman, 1970). 7. Role of biogenic amines in prolactin and gonadotropin secretion Reports over many years have indicated that biogenic amines in the brain, particularly those present in the hypo- thalamus, participate in the control of secretion of several anterior pituitary hormones, including prolactin and gono- adotropins. Taubenhaus and Soskin (1941) believed that an 34 acetylcholine-like substance from the hypothalamus acted via the pituitary protal vessels to elicit LH release in rats. Markee 31_31. (1948) and Sawyer 31 31. (1949) demonstrated that adrenergic drugs produced ovulation and therefore pre- sumably elicited LH release in rabbits. These investigators proposed that an adrenergic mechanism was involved in the control of LH secretion. Anti-adrenergic drugs blocked ovu- lation in rabbits and rats (Sawyer 31_31., 1949; Everett 31 31., 1949, Markee 31_31., 1952). However, the adrenergic blockers were only effective when administered before coitus in rabbits or prior to the "critical period" on the day of proestrus in rats. Reserpine and cholropromazine were re- ported to inhibit ovulation induced by pregnant mare serum in immature rats when administered prior to the expected time of LH release (Hopkins and Pincus, 1963; Zarrow and Brown-Grant, 1964; Coppola 31 31., 1966; Coppola, 1968). It also was reported that reserpine blocked ovulation in ma- ture rats when given prior to the "critical period" on the day of proestrus (Barraclough and Sawyer, 1957; Meyerson and Sawyer, 1967). Alpha-methyl-para-tyrosine, a potent inhib- itor of catecholamine synthesis, also was shown to inhibit pregnant Aare serum-induced ovulation in immature rats (Lippmann 31 31., 1967; Coppola, 1968; Kordon and Glowinski, 1969). Subsequently, it was observed that inhibition of LH secretion by sympatholytic agents such as reserpine, alpha- methyl-DOPA, tetrabenazine and syrosingopine was associated 35 with a marked reduction in hypothalamic catecholamines; whereas drugs that specifically deplete catecholamines in peripheral tissues but not in the brain (bretylin, tyramin and guanethidine) were ineffective (Coppola 31 31., 1966; Lippmann 31 31., 1967; Coppola, 1968). It also was demon- strated that inhibition by reserpine of LH secretion could be prevented by concurrent treatment with a monoamine oxi- dase inhibitor (iproniazid) or a precursor catecholamine sub— strate (L-DOPA). These observations provide convincing evi— dence that sympatholytic agents act on a central mechanism possibly through the hypothalamic catecholamine, which con- trol the pituitary secretion of LH. In more recent studies, Kamberi 31 31. (1970) and Schneider and McCann (1970) demon- strated that catecholamines, particularly dopamine, induced release of LH and FSH by increasing hypothalamic LRF and FRF activities. Reserpine and chlorpromazine were reported to pro- duce lactation in humans (Sulman and Winnik, 1956; Rabinowitz and Friedman, 1961) and rabbits (Meites, 1957; Kanematsu 31 31., 1963), and to induce pseudopregnancy in rats (Barraclough and Sawyer, 1959). Early work from our laboratory indicated that injections of epinephrine and norepinephrine induced lac- tation in estrogen-primed rats and rabbits (Meites, 1959; 1962; Meites 31_31,, 1963). These latter responses may be non- specific, since anti-adrenergic and nonspecific agents were also effective in initiating lactation in these animals. The 36 effective drugs included acetylcholine, atropine, pilocar- pine, serotonin, morphine, amphetamine, reserpine, chlorprom- azine, and meprobamate. Since lactation is not a specific indicator of prolactin release, these early experiments did not prove that biogenic amines could induce prolactin re- lease (Meites 31 31., 1972). Nevertheless, the observation that reserpine and chlorpromazine stimulated mammary growth and lactation in rats (Meites, 1957, 1962; Meites 31_31,, 1963) suggested these two drugs stimulated prolactin release. Ratner 31 31. (1965) further demonstrated that reserpine de- creased hypothalamic production of PIF, providing an explana- tion of how reserpine evoked increase in prolactin secretion. Similar result was also reported on perphenazine, a compound related to chlorpromazine (Danon 31 31., 1963). The work by Mizuno 31 31. (1964) demonstrated that injections of ipron- iazid, a monoamine oxidase inhibitor and therefore a depres- sor of catecholamine metabolism, inhibited post-partum lac- tation in rats, suggesting decreased secretion of prolactin. This provided the first evidence that brain catecholamines are inhibitory to anterior pituitary secretion of prolactin. We have extended this in a more recent study and observed that a single injection of iproniazid decreases pituitary re- lease of prolactin by increasing hypothalamic PIF activity in cycling female rats (Lu and Meites, 1971). Alpha-methyl- para-tyrosine, a catecholamine synthesis inhibitor, and sym- patholytic agents including reserpine, alpha-methyl-DOPA, 37 tetrabenazine and syrosingopine were reported to stimulate prolactin secretion in rats (Coppola 31 31., 1965; Lippmann 31 31., 1967; Coppola, 1968). Drugs which reduce hypothala- mic catecholamines stimulated prolactin secretion, whereas agents which deplete catecholamines in tissues other than the brain were ineffective (Coppola, 1968). The stimulation of prolactin secretion by sympatholytic agents, or by cate- cholamine synthesis inhibitors could be blocked by concurr- ent treatment with monoamine oxidase inhibitors, or precur- sor catecholamine substrates respectively (Coppola 31 31., 1965; Lippmann.31_31., 1967; Coppola, 1968; van Maanen and Smelik, 1968). These observations provide evidence that catecholamines in the hypothalamus participate in the inhib- itory control of pituitary prolactin secretion. Drugs which stimulate pituitary release of prolactin exert their effects by reducing hypothalamic catecholamine levels. 13_11113_studies by Talwalker 31_31. (1963) indicated that norepinephrine and epinephrine had no direct effect on pituitary release of prolactin, whereas Gala and Reece (1965) Observed that some doses of epinephrine increased prolactin release by rat pituitary. Jacobs 31 31. (1968), MacLeod (1969), and Birge 31 31. (1970) reported that catecholamines including dopamine, norepinephrine and epinephrine profoundly inhibited prolactin release by rat pituitary tissue 12.Xl££2’ and concluded that the catecholamines may represent the not as yet defined hypothalamic PIF. In our laboratory we 38 confirmed the Observation (Koch 31_31,, 1970) that relatively large doses of catecholamines inhibited prolactin release 13 vitro, but in addition we found that the actions of catechol- amines on pituitary release of prolactin 13_vitro is dose- dependent, with high doses producing inhibition, intermed- iate doses no effect, and low doses stimulation of prolactin release. The low stimulating doses of catecholamines are ap- proximately equal to the amounts reported to be present in the rat hypothalamus (Lippmann, 1968; Donoso 31_313, 1967). Catecholamines are rapidly degraded 13_11113_to form adreno- chromes which exert powerful pharmacological effects on cell- ular metabolism (Wurtman, 1970). In our laboratory we also observed that drugs unrelated chemically to catecholamines could decrease prolactin release by rat pituitary 12.21332 (Lu and Meites, unpublished). Thus, reserpine and chlor- promazine are well known to stimulate prolactin release 13 1113 (Lu 31 31., 1970), but can inhibit prolactin release by rat pituitary tissue 13_!1113, This provides evidence that the effects of drugs 13_!1113_do not necessarily reflect their actions 13_vivo. Since there is as yet no evidence that dopamine and norepinephrine released in the median emi- nence of hypothalamus can enter the pituitary portal vessels (Wurtman, 1970), it is doubtful that hypothalamic catechol- amines exert by direct effect on pituitary release of prolac- tin. 39 By use of a specific radioimmunoassay for rat pro- lactin (Niswender 31 31,, 1969), we observed that systemic injections of a single dose of various catecholamines pro- duced no change in serum prolactin levels in the rat, al- though dopamine and epinephrine evoked a small but statisti- cally significant decrease in pituitary prolactin (Lu 31_31., 1970). Inasmuch as the "blood-brain barrier” in the hypo- thalamus effectively hinders the entry of circulating cate- cholamines into the brain tissue, no definite conclusions could be made at that time as to the role of catecholamines on prolactin release. In a more recent study, drugs that readily pass through the "blood-brain barrier" and can either increase or decrease hypothalamic catecholamines were used to assess their effects on prolactin release (Lu and Meites, 1971; Lu 31_31., 1970). Reserpine, chlorpromazine, alpha- methyl-para-tyrosine, alpha-methyl-meta-tyrosine, methyl- DOPA and d-amphetamine, each is known to reduce brain cate- cholamines, greatly stimulated pituitary release of prolac- tin in cycling female rats, thus elevating serum prolactin levels and reducing pituitary prolactin concentrations. A single injection of L—DOPA (the immediate precursor of dopa- mine) or of monoamine oxidase inhibitors (pargyline, ipron- iazid, or Lilly compound-15641), each known to enhance hypo- thalamic catecholamine activity, significantly decreased serum prolactin values. It was further demonstrated that the latter group of drugs (L-DOPA and the three monoamine 40 oxidase inhibitors) increased PIF activity in the hypothal- amus (Lu and Meites, 1971). We have extended these observa- tions in a more recent study and demonstrated that L-DOPA reduced serum prolactin values in intact and pituitary- grafted, hypophysectomized rats by increasing PIF activity in the hypothalamus and eliciting prolactin release-inhibit- ing activity in the systemic circulation (Lu and Meites, 1972). In a related study, Kamberi 31 31, (1970) reported that a single injection of dopamine into the third ventricle of rats decreased serum prolactin by increasing PIF activity in the pituitary portal blood. These results suggest that L-DOPA and dopamine increase both synthesis and release of PIF by the rat hypothalamus. 0n the other hand, reserpine and chlorpromazine reduce hypothalamic PIF and stimulate pro- lactin release. Haloperidol, another neuroleptic drug that reduces brain catecholamines also markedly elevated serum prolactin by decreasing PIF in the hypothalamus (Dickerman 31 31., 1972). From these and other related observations the concept evolved that hypothalamic catecholamines, includ- ing dopamine and norepinephrine, act as neurotransmitters to increase the release of hypophysiotropic PIF, which in turn enters the pituitary portal vessels to inhibit pituitary pro- lactin release (Meites 31 31., 1972; Wurtman, 1970; Fuxe and Hokfelt, 1969; Coppola, 1968). Reports on the role of other biogenic amines on pro- lactin secretion also have appeared recently. Work by Gala 41 31 31, (1970) indicated that hypothalamic cholinergic fibers had an inhibitory influence on pituitary prolactin secretion; they observed deciduomata formation by implanting atropine, an anti-cholinergic drug, into the rat hypothalamus. Kamberi 31 31. (1971) reported that injection of serotonin or mela- tonin into the third ventricle of rats stimulated prolactin release, suggesting involvement of indoleaminergic nerves in the hypothalamic control of prolactin secretion. In our lab- oratory we reported that a single intraperitoneal injection of serotonin had no definite effect on serum prolactin val- ues (Lu 31 31., 1970). We have extended this in a more re- cent study and demonstrated that a single intravenous injec- tion of 5-hydroxytryptophan, the immediate precursor of sero- tonin, melatonin, or to a lesser extent, tryptophan, signifi- cantly elevated serum prolactin levels in cycling female rats (Lu and Meites, unpublished). Indoleamines and catechol- amines in the rat hypothalamus appear to work in opposition on the control of pituitary prolactin secretion (Meites 31 31., 1972; Clemens and Meites, 1972). It is noteworthy that indoleamines exert an inhibitory influence on pituitary se- cretion of gonadotropins (Kamberi 31 31., 1970; 1971), a mechanism which also is in opposition to the effects of the catecholamines (Kamberi 31 31., 1970; Schneider and McCann, 1970). The mechanisms of action by which hypothalamic in- doleamines stimulate prolactin release remain to be deter- mined, since no significant change in PIF activity is observed 42 in the hypothalamus or serum from rats given a single injec- tion of tryptophan, S-hydroxytryptophan, or serotonin (Lu and Meites, unpublished). E. Short-Loop Feedback Control of Pituitary Prolactin Secretion Most anterior pituitary hormones have well defined target glands or tissues that produce hormones that inhibit secretion of their tropic hormones by the pituitary. How- ever, this classic endocrine feedback system does not appear to operate in the cases of GH and prolactin. Many years ago Sgouris and Meites (1953) postulated that prolactin may act to control its own secretion by the pituitary. This hypoth- esis has been sustained by many recent studies, although its physiological significance is not yet clear. Motta 31 31. (1969) proposed the term "short feedback loop" referring to the mechanism by which the anterior pituitary hormones them- selves act to control their own secretion. Transplantation of pituitary "mammosomatotropic" tu- mors into intact rats was reported to decrease pituitary pro- lactin content (MacLeod, 1966; 1968). Chen 31_31. (1967) made similar observations in rats with a "mammotropic" tumor and further demonstrated that these pituitary tumor trans- plants increased hypothalamic PIF content. Injections of prolactin were shown to reduce pituitary weight and prolac- tin concentration in intact female rats (Sinha and Tucker, 1968). Multiple pituitary grafts underneath the kidney 43 capsule were reported to lower pituitary weight and prolac- tin concentration of the 13.3113_pituitary in intact and ovariectomized rats (Welsch 31 31., 1968). Enormous amount of prolactin are released by the transplanted pituitary, but secretion of other anterior pituitary hormones are greatly reduced (Meites, 1966). Implants of prolactin into the me- dian eminence of rats also reduced serum prolactin values (Voogt and Meites, 1971). These results indicate that pro- lactin can act to depress its own secretion by the pituitary. Clemens and Meites (1968) showed that implantation of a minute amount of prolactin into the median eminence of intact or ovariectomized rats decreased pituitary prolactin content, inhibited mammary growth, reduced the number of corpora lutea, and increased PIF content in the hypothalamus. Inhibition of lactation was observed when prolactin was im- planted into the medium eminence of lactating rats (Clemens ‘31 31., 1969). These results suggested that prolactin im- plants into the median eminence acted by increasing PIF to inhibit both synthesis and release of prolactin. Chen 31_31, (1968) observed that an implant of pro- lactin in the median eminence shortened the length of pseudo- pregnancy and prevented the formation of deciduomata. An implant of LH or FSH had no effects. Pregnancy was termir nated when an implant of prolactin was placed into the median eminence of early pregnant rats (Clemens 31 31., 1969). How- ever, the pregnancy could be maintained by daily injections 44 of progesterone. These observations indicated that prolac- tin implants into the median eminence inhibited pituitary prolactin secretion, thereby resulting in luteal regression and reduced progesterone secretion necessary for maintenance of pregnancy and pseudopregnancy. A prolactin implant into the median eminence not only can shut off its own secretion, but stimulates release of gonadotropins by the pituitary, thus resulting in follic- ular growth, ovulation, and resumption of cycling in pseudo- pregnant, early pregnant or post-partum lactating rats (Clemens and Meites, 1968; Clemens 31 31., 1969; Voogt and Meites, 1971). Systemic injection or implantation of prolac- tin into the median eminence of immature female rats hastened the onset of puberty (Clemens 31 31., 1969) and resulted in elevated pituitary FSH levels and a probable increase in LH release (Voogt 31 31., 1969). The action of proactin implant in the median eminence is unique, since implants of other hormones such as LH, FSH, ACTH or GH each appear to inhibit selectively only their own secretion by the anterior pitu- itary, and have no effect on the secretion of other pituitary hormones (Motta 31_31,, 1969). Averill (1969) grafted a pituitary into the hypothal- amus of rats and observed no luteotropic effects by the pi- tuitary grafts. Although many of the grafts were distant from the median eminence and portal vessels, prolactin secre- tion by the grafts was inhibited. This strongly indicated 45 that there was a local feedback between the graft and hypo- thalamic tissue that secretes PIF. Thus, it becomes obvious that the site of the inhibitory action of prolactin in its own secretion is in the hypothalamus. Work by Nicoll (1971) has indicated that prolactin does not act directly on the pituitary to inhibit its own secretion 12.11232: It is sig- nificant that prolactin injections to rats markedly activated the tubero-infundibular dopaminergic neurons (Fuxe and Hokfelt, 1970). This activation of dopaminergic fibers in the hypothalamus provides an explanation of how prolactin implants in the median eminence increase the release of PIF and inhibit prolactin secretion. Evidence has been presented that L-DOPA, the immediate precursor for dopamine synthesis, increased PIF activity in the hypothalamus (Lu and Meites, 1971; 1972). This increase in dopamine activity in the hy- pothalamus also explains why prolactin implants increase pi- tuitary release of FSH and LH, since dopamine has been shown to increase the release of FSH-RF and LH-RF into the portal vessels (Kamberi 31 31., 1970). F. Direct Effects of Drugs and Hormones on Pituitary Prolactin Release The foregoing considered the control of pituitary prolactin secretion through the hypothalamus. The biogenic amines, short-loop feedback, prolactin-inhibiting activity and prolactin-stimulating activity of the hypothalamus con- stitute the principal regulatory mechanisms of prolactin 46 secretion. However, several hormones and drugs can influence prolactin secretion by a direct action on the pituitary. In addition, some agents can act both on the pituitary and hy- pothalamus to regulate prolactin secretion. Injections of estrogen were reported to stimulate prolactin secretion in hypophysectomized rats with a pitu- itary graft underneath the kidney capsule (Desclin, 1956; Chen 31 31., 1970; Lu 31 31., 1971). Although these obser- vations suggested a direct action of estrogen on the grafted pituitary, the possible intervention of the hypothalamus could not be completely excluded. Nicoll and Meites (1962) reported that estradiol could directly stimulate rate pitu- itary to release prolactin in a 3-day organ culture. Subse- quent studies confirmed this finding (Nicoll and Meites, 1963; 1964). More recently, by use of a specific radioim- munoassay for rat prolactin, a direct stimulation by estra- diol of rat pituitary prolactin release in a lZ-hour incuba— tion was demonstrated by Lu 31 31. (1971). The direct action of estrogen on the pituitary can be completely blocked when an ergot derivative, ergocornine, is administered simultaneously (Lu 31 31., 1971). Since estrogen injections 13_1113_can depress hypothalamic PIF activity in the rat (Ratner and Meites, 1964), this indicates that estrogen acts to stimulate prolactin release both via the hypothalamus and by a direct action on the anterior pituitary. The direct action of es- trogen on the pituitary may have a role in the control of 47 prolactin surge on the afternoon of proestrus. Ovariectomy (Clark and Meites, unpublished) or administration of an estradiol-antiserum on the day before proestrus prevented any rise in serum prolactin on the following day (Neill 31_ 31., 1971). It has been well demonstrated that thyroid function can influence pituitary secretion of prolactin (see Meites, 1960, 1966). Reports from our laboratory elucidated the mechanism of action by which thyroid hormones influence pi- tuitary prolactin secretion. Incorporation of small amounts of thyroxine and triiodothyronine into a culture system was found to directly increase prolactin release by the rat pi- tuitary (Nicoll and Meites, 1963). Subsequently, Chen and Meites (1969) reported that 13_1133_injections of thyroxine had no effect on hypothalamic PIF activity in rats. This suggests that thyroxine stimulates prolactin release only by a direct action on the anterior pituitary. Mention has already been made of the ability of syn- thetic TRH to increase blood prolactin levels in primates and bovine. However, there is as yet no clear evidence that TRH acts directly on the primate or bovine pituitary to re- lease prolactin. Tashjian 31 31. (1971) reported an increase in prolactin and decrease in CH release by TRH when the lat- ter was added to cultures or incubations of clonal cells from rat pituitary tumors. These workers also reported that clonal strains of rat pituitary tumor cells showed significant 48 increases in prolactin and decreases in GH release when ex- tracts of rat hypothalamus, cerebral cortex, kidney or liver were added to the culture medium (Tashjian 31 31., 1970). It was reported that TRH had no effect on prolactin release when incubated with bovine pituitary tissue 13_11113_(LaBe11a and Vivian, 1971; Convey 31 31,, 1972), despite the increase by TRH of blood prolactin levels in bovine (Convey 31 31., 1972). Bowers (1971) and McCann (1971) also failed to ob- serve any effect of TRH on prolactin release by normal rat pituitary 13.11113, A recent study from our laboratory (Lu et a1., 1972) indicated that TRH has no effect on prolactin release when added to incubation of normal rat pituitary halves or "mammotropic" pituitary tumor tissue. A single injection of large dose of TRH to rats has no effect on ser- um prolactin either. However, incorporation of TRH into an incubation medium was found to evoke a limited increase (about 30%) in prolactin release by pituitary halves from rats 5 weeks after thyro-parathysoidectomy. Thus the pitu- itary of hypothyroid rats may respond somewhat differently to TRH than the pituitary of normal rats. These observations suggest that synthetic TRH is not a specific releaser of pro- lactin in the rat. The possibility that synthetic TRH is identical with or similar to the presumed PRF of mammals re- mains to be determined (see Section II, C). Catecholamines have been shown to alter pituitary prolactin release 13_vitro, although its physiological 49 significance is not clear. Jacobs 31 31. (1968), MacLeod (1969) and Birge 31 31. (1970) reported that dopamine, nore- pinephrine and epinephrine inhibited prolactin release by the incubated rat pituitary, and suggested that catechol- amines themselves might be identical with hypothalamic PIF. Subsequently, Koch 31_31, (1970) demonstrated that the ef- fects of catecholamines on pituitary prolactin release 13_ [1119 is dose-dependent, with high doses producing inhibi- tion and low doses stimulation of prolactin release. The low stimulating doses are approximately equal to the amounts of catecholamines reported to be present in the rat hypothal- amus (Lippmann, 1968; Donoso 31 31., 1967). Catecholamines were not detected in the pituitary portal blood (Wurtman, 1970; Porter 31 31., 1970), and had no effect on prolactin release when infused into a single pituitary portal vessel (Kamberi 31 31., 1970). There is as yet no definite evidence that epinephrine is synthesized in the mammalian brain (Anton- Tay and Wurtman, 1971). In view of these observations and the reported pharmacological effects by catecholamine metab- olites on cellular metabolism 13_11113_(Wurtman, 1970), it is probable that the direct effects of catecholamines on pi- tuitary prolactin release observed 13_X1113_are non-specific in nature (Koch 31 31., 1970). Early studies in the rat indicated that injections of ergot drugs induced termination of pseudopregnancy and early pregnancy (Shelesnyak, 1955; Carlsen 31_31., 1961), 50 suppression of implantation (Shelesnyak, 1964), and inhibi- tion of lactation (Zeilmaker and Carlsen, 1962), suggesting inhibition of pituitary prolactin secretion. Later, it was found that ergot drugs inhibited mammary tumor growth in rats (Nagasawa and Meites, 1970; Yanai and Nagasawa, 1970). Definite evidence that ergocornine, an ergot derivative, re- duces pituitary and serum prolactin levels was reported by Nagasawa and Meites (1970) and by Wuttke 31 31. (1971). Subsequently, it was found that ergocornine acted directly on rat pituitary to diminish prolactin release 13.11113, resulting in an increase in pituitary prolactin stores (Lu 31_31., 1971). Ergocornine also can prevent estradiol from increasing prolactin release 13_11113, When ergocornine was injected into hypophysectomized rats with a pituitary graft underneath the kidney capsule, it significantly inhibited prolactin secretion by the graft. When ergocornine was in- jected together with estrogen into rats, it prevented estro- gen from producing enlargement of the pituitary,counteracted estrogen stimulation of pituitary prolactin secretion. Re- sults from other studies also suggest that ergot drugs act directly on the pituitary. Our laboratory recently reported that ergot drugs induced significant regression or inhibi- tion of pituitary tumor growth in rats (Quadri 31_31., 1972). Ergocornine caused a decrease in cell number and a disappear- ance or pycnosis of nuclei in the pituitary tumor tissue. It will be seen in a subsequent experiment that ergot drugs 51 inhibit prolactin release in rats bearing pituitary tumor transplants (see Experiment VII). It was reported that er- gocornine increased hypothalamic PIF content (Wuttke 31 31,, 1971), suggesting that it acted at least in part via the hy- pothalamus. Inasmuch as ergocornine inhibits prolactin re- lease from the incubated pituitary and by the pituitary transplant 13_3133, it can be assumed that inhibition of prolactin release is due mainly to its direct action on the pituitary (Lu 31 31,, 1971). Implants of sodium pentobarital into the median emi- nence of rats reduced hypothalamic PIF activity and elevated serum prolactin values (Wuttke 31_31., 1971). On the other hand, sodium pentobarbital directly inhibited prolactin re- lease by the rat pituitary 13 31113, apparently accounting for its ability to depress serum prolactin when given 13 3133 (Wuttke and Meites, 1970). It appears that sodium pentobarbital exerts a biphasic effect on pituitary prolac- tin release, initially stimulating and subsequently inhibit- ing prolactin release (Wuttke 31 31,, 1971). In addition to the above drugs, several other drugs can exert direct pharmacological effects on pituitary prolac- tin release 13 31113, Thus, reserpine, chlorpromazine, alpha-methyl-para-tyrosine, and alpha-methyl-meta-tyrosine each can inhibit prolactin release when incubated with rat pituitary 13 vitro (Lu et al., 1970b), despite their marked 52 ability to increase prolactin release 13 vivo (Lu 31 31,, 1970a). It is apparent, therefore, the effects of drugs 13_ vitro do not necessarily reflect their actions 13 vivo. G. Multiple Control of Pituitary Prolactin Secretion It is evident from the preceding discussions that pituitary secretion of prolactin is controlled mainly by the hypothalamus but also by other systems. Prolactin secretion normally is inhibited by hypothalamic PIF under most condi- tions. But it may also be influenced by the presumed PRF under some conditions. An increase in prolactin release could result from a decrease in PIF or by stimulation from PRF in the hypothalamus. Biogenic amines apparently parti- cipate in the control of pituitary prolactin secretion by regulating the release of PIF (and probably PRF) by the hy- pothalamus. There appears to be a dual control in the hypo- thalamus over pituitary secretion of prolactin, with a pre- dominant adrenergic tonus acting to depress release of pro- lactin under most conditions, and a serotonergic system that may be responsible for the diurnal rise in pituitary and serum prolactin (Clark and Baker, 1964; Koch 31'31,, 1971) on the late afternoon of each day and perhaps under other conditions (Meites 31 31., 1972). No feedback mechanism is evident for the control of prolactin secretion by its "tar- get" gland hormones. The "auto regulation" of prolactin se- cretion by a "short-loop” feedback may represent a 53 physiological means for its own control. Extrahypothalamic structures also may influence pituitary prolactin secretion (Mena and Beyer, 1968; Rubenstein and Sawyer, 1969; Tindal and Knaggs, 1969, 1970), providing an explanation of how emotional and stressful stimuli influence prolactin release (Clemens and Meites, 1972). In addition to the hypothalamic (central nervous) mechanism mentioned above, several hor- mones and drugs also can alter prolactin secretion by a di- rect action on the pituitary. Some agents can influence pi- tuitary prolactin secretion by acting both through the hypo- thalamus and directly on the pituitary. MATERIALS AND METHODS 1. Animals All intact mature male and female rats of Sprague- Dawley strain used for experiments, and as pituitary and hypothalamus donors, were purchased from Spratan Research Animals, Inc. (Haslett, Michigan). Mature female, hypophy- sectomized rats of Sprague-Dawley strain were obtained from Hormone Assay Labs., Inc. (Chicago, Illinois). Inbred fe- male rats of Wistar-Furth strain were purchased from Micro- biological Associates, Inc., Walkersville, Maryland. All rats were housed in metal wire cages in temperature-controlled (75 i 1°F) and artificially illuminated (lights on from 5:00 AM until 7:00 PM daily) rooms, and were maintained on a stan- dard diet of Wayne Lab Blox pellets (Allied Mills, Chicago, Illinois) and tap water 33_libitum. Thyro-parathyroidecto- mized rats were given 1% calcium lactate solution for one week post-operation, and hypophysectomized rats were fed a supplement of fresh orange slices and sugar cubes throughout the experimental period. All surgical procedures except blood vessel cannulation were performed with ether anesthe- sia, and surgically treated rats were given 0.2 m1 of Bicillin 54 55 (Wyeth Labs, Inc., Philadelphia, Pa.), a wide spectrum anti- biotic, post-operatively by intramuscular injection to pre— vent infection. 11. Pituitary Transplantation TTechnique Mature male rats (250-300 g bw) and female rats (200-220 g bw) of the Sprague-Dawley strain were used as pi- tuitary donors. The donor rats were decapitated with a guil- lotine. The pituitary gland was immediately removed and placed on a filter paper moistened with physiological saline in a petri dish. The anterior pituitary was carefully sep- arated from the posterior lobe using a fine forceps. Seven days after hypophysectomy, the rats were anesthetized and an abdominal skin incision (2-3 cm long) was cut just beneath the ribs to expose the left kidney. A small slit was made through the transparent renal capsule membrane, and a single anterior pituitary was placed underneath the kidney capsule close to the renal hilus. III. Blood Vessel Cannulation TTechnique Intact and thyro-parathyroidectomized mature male rats were anesthetized by a single intraperitoneal injection of sodium-pentobarbital (4.5 mg/100 g bw). The rat was placed on an operation board in a supine position. A lateral ventral neck skin incision (2—3 cm long) was cut with a razor 56 blade. The skin and fascia were retracted to expose the left carotid artery or jugular vein. With the aid of a pair Of closed forceps placed underneath, the blood vessel was pulled out and freed from surrounding tissues and nerves. Polyethylene tubing (# PE-20) filled with heparin solution was inserted into and tied on the carotid artery or jugular vein. The arterial cannula was inserted anteriorly with its opening facing to the head, whereas the tip of the jugular vein cannula was inserted down to the junction of the vena cavae. The external portion of the tubing was passed sub- cutaneously around the neck region and emerged through post- cervical skin on the neck. The cannulated rats were main- tained in separate cages to avoid intermingling and chewing of cannulas. After cannulation, at least 3 days of recovery were permitted before the rats were used for experiments, unless otherwise stated. During the experiment, each rat was kept in a cage (Figure 3). A segment of 40 cm-long poly- ethylene tubing of the same size was connected to the can- nula. Collection of blood and infusion of agents through the cannula were performed outside the cage usually without appreciable notice or disturbance to the rat. IV. Pituitary Tumor Transplantation Techn1que Furth pituitary mammotropic tumors (MtT.W 15) were removed by sterile technique from inbred Wistar-Furth strain female rats. The pituitary tumors were cut into fine pieces 57 Figure 3. View of a Rat with a Jugular Cannula Attached by an Extension Segment of Polyethylene Tubing. 58 in sterile saline solution using an iris scissors. A vol- ume of 0.2 m1 tumor mince was injected subcutaneously to 50- day-old female rats of the same strain in the postcervical region on the neck in order to develop transplantable tumors. This transplantable pituitary tumor, originally obtained through the courtesy of Dr. Jacob Furth, Department of Path- ology, Columbia University, is known to secrete large amounts of prolactin and GH (Furth, 1961). V. Pr3paration of Hypothalamic Extract, Pituitary Homggenatei_and SErum Hypothalamus-donor rats and rats at the end of the experiments were decapiated by guillotine. The hypothalamus including the stalk-median eminence was quickly removed with a small, curved forceps coated with epoxylite. The hypotha- lamic fragment was immediately placed in a plastic centri- fuge tube containing chilled 0.1 N HCl (0.15 ml/hypothalamic fragment) kept in an ice water bath. All hypothalami from each experiment were pooled in one tube and kept frozen at -20°C until assayed by 13_31113_incubation. Just prior to in- cubation, the pooled hypothalami were quickly thawed in luke- warm water and homogenized with a Sonifier cell disruptor (Heat Systems-Ultrasonics, Inc., Plainview, New York). The acid homogenates were centrifuged at 12,000 x G for 40 min- utes at 4°C in a Sorvall RCZB automatic refrigerated centri- fuge (Ivan Sorval, Inc., Norwalk, Conn.). The supernatant was then incorporated into pH 7.4 medium 199 (Difco Labs., 59 Detroit, Michigan). The acid mixture was again neutralized to pH 7.4 by adding 1.0 N NaOH a drop at a time. The volume and concentration were so adjusted that each hypothalamic equivalent was contained in 2.0 1 0.1 m. of incubation medium. Rats were killed by decapitation at the end of the experiments. The pituitary gland was quickly removed, placed in a petri dish over a filter paper moistened with physiolog- ical saline. The anterior pituitary was separated from the posterior lobe, blotted on filter paper, weighed on a Mettler electrical balance (Mettler Instrument Corp., Highstown, New Jersey) and homogenized with neutral phosphate buffer saline (PBS) in a S-ml disposable culture tube using a Sonifier cell disruptor. The volume and concentration were so prepared that each m1 of homogenate contained 0.1 mg of female, or 0.2 mg of male rat anterior pituitary tissue. The homogenates of anterior pituitary tissue after 13_31113 incubations were sim- ilarily prepared with a concentration of 0.25 mg per ml. The pituitary homogenates were kept frozen at «20°C until assayed. Collection of blood samples was performed by cardiac puncture under light ether anesthesia using a 1-ml Tuberculin syringe with a # 26 1/2 hypodermic needle. The blood was col- lected into a 5—ml disposable culture tube (Kimble Products, Owens, Illinois and Scientific Products, Mcgaw, Illinois). The blood sample tube was first placed obliquely (Figure 4) in a refrigerator (4°C) for 4 hours before centrifuging at 5,000 rpm for 10 minutes in a Sorvall refrigerated centrifuge. 60 Figure 4. View of a Rack Holding Tubes with Blood Samples in an Oblique Position to In- crease the Contact Surface of Blood to Glass. 61 Serum was pipetted into another S-ml culture tube using a l-ml Tuberculin syringe with a # 26 1/2 hypodermic needle. The serum tubes were capped with parafilm and kept frozen at -20°C until assayed. VI. In Vitro Incqbation Technique Mature male rats (250-300 g bw) used as pituitary- donor were decapitated with a guillotine. The pituitary gland was immediately removed, placed in a petri dish over a filter paper moistened with neutralized medium 199. The anterior pituitary was hemisected using a razor blade after separation from the posterior lobe. For comparison, one half of the anterior pituitary tissue was placed in a control in- cubation tube and the other half in an experimental tube. Each anterior pituitary half was blotted on filter paper, weighed on a Mettler electrical balance, and placed in a 5- ml culture tube containing 2 ml Of medium 199 at a pH of 7.4. Incubations were carried out in a Dubnoff metabolic shaking Incubator (Labline, Inc., Chicago, Illinois), 60 cycles per minute, under constant gassing with 95% 02 - 5% C02 at 37 i 0.5°C. After 30 minutes pre-incubation, the medium was re- moved and replaced with 2 ml of fresh medium 199 containing hypothalamic extract, serum, drug or hormone for incubation. During the incubation, 100 pl of medium were collected and replaced with 100 pl of fresh medium at different time inter- vals. The anterior pituitary tissue was removed from the 62 medium at the end of incubation, blotted on filter paper, weighed and homogenized according to the methods described under Section V. Both the media and pituitary homogenates were kept frozen at -20°C until assayed. VII. Radioimmunoassay of Rat Prolactin In all experiments, prolactin in individual serum samples, pituitary homogenates and incubation media was mea- sured by a double-antibody radioimmunoassay. This radioim- munoassay for rat prolactin was developed by a collaborative effort between this laboratory and Dr. Midgley's laboratory in the Department of Physiology, the University of Michigan at Ann Arbor, Michigan (Niswender 31_31,, 1969). The Ph.D. dissertation by Dr. C. L. Chen (Michigan State University, 1969), who devoted much of the initial efforts to the devel- opment of this method, describes the procedures and valida- tion for this radioimmunoassay. Purified rat prolactin (HIV-8-C and H-lO-lO-B), ob- tained from Dr. S. Ellis (NASA, Ames Research Center, Moffett Field, California), was used for radioiodination with 1125 of 1131 (Cambridge Nuclear Radiopharmaceutical Corp., Billerica, Massachusetts). The radioiodinated rat prolactin was collected by elution through a l x 15 cm Bio-Gel P-60 column, and was diluted with 0.1% egg white-neutral phosphate buffer saline (EW-PBS) to a working concentration of 30,000 cpm per 100 pl as counted under optimal conditions inside an 63 automatic gamma well counter (Nuclear-Chicago Corp., Des Plaines, Illinois). This amount of 1125_ or 1131-rat prolac- tin was added to each incubation tube for competitive bind- ing with un-labelled rat prolactin to the anti-rat prolactin antibody. The antibody against rat prolactin was developed in a rabbit, and was diluted to a working concentration of 1:4,000 or 1:5,000 which consistently bound 35-45% of the radioiodinated rat prolactin added to the incubation tube. The antiserum against rabbit gamma globulin (Anti- RGG) was developed in sheep. Optimal dilutions (1:3 to 1:9) of the antisera from different bleedings were titrated to precipitate the rabbit gamma globulin. The incubation of rat prolactin with the antibodies was carried out in 12 x 75 mm disposable culture tubes (Kimble Products, Owens, Illinois and Scientific Products, McGaw Park, Illinois) placed in a refrigerator at 4°C. On day one, 200 pl of the first antibody was added to each in- cubation tube containing known (standard) or unknown (assay sample) amounts of rat prolactin plus the diluent 1% EW-PBS in a total volume of 500 pl. Twenty four hours later, 100 pl of radioiodinated rat prolactin was added to each tube, and the incubation was carried out for another 24 hours to achieve maximal binding of the first antibody to both labelled and unrlabelled rat prolactin. On day three, 200 pl of 64 anti-RGG was pipetted into each tube, and the incubation was continued for the next 3 days to precipitate maximal amounts of both labelled and un-labelled antigen-antibody complex. At the end of the 5 day-incubation, 3 ml of PBS were added to each tube before centrifuging in a Model-K Interna- tional Centrifuge (International Equipment CO., Needham Heights, Massachusetts) at 2,000 rpm for 35 minutes. The supernatant was decanted. The tube with precipitate in the bottom was air-dried before being placed into a plastic cap- sule and counting in an automatic gamma well counter. The counting time for all tubes was set for 10,000 specific counts from the ”zero hormone"-first antibody binding tube, after subtraction of non-specific counts from the normal rab- bit serum (NRS) -antigen binding tube. Non-specific counts by NRS in each tube were both corrected on the standard curve and automatically subtracted by background setting on the gamma counter. Purified rat prolactin preparations (HIV-8-C and H-lO-lO-B obtained from Dr. 8. Ellis and NIAMD-rat prolactin RP-l received from NIH Rat Pituitary Hormone Program, Bethesda, Maryland) were used as reference standards in dif- ferent experiments of this study. 'A standard curve was drawn on semi-logarithmic paper on the results of duplicate sets of 16 different doses of standard rat prolactin ranging from 0.4 to 40 ng. The NIAMD-rat prolactin-RP-l in the radioimmunoassay, 65 using Ilzs-labelled rat prolactin as tracer, consistently gave a value of 4.0 1 0.3 ng at the 50% binding point on the standard curve. For the assay, 50-200 ul of serum sample or 25-100 ul of pituitary homogenate were pipetted into each incuba- tion tube, depending on the experiment. For 13_31113_experi- ments, 50-150 ul of diluted incubation medium were used for prolactin assay. The dilution of the incubation medium de- pends on the nature and the time of incubation. In all ex- periments, samples from comparable experiment groups were usually assayed on the same date, and each sample was assayed at 2 or 3 different dose levels to insure accurate measure- ment of prolactin values. Prolactin values were expressed in terms of purified rat prolactin reference standard as in- dicated above. VIII. Mammary Gland Growth Rati3gSystem Inguinal mammary pads were removed from rats after decapitation, spread flat on cork, and fixed in Bouin's fluid for whole mount evaluation. Each mammary pad was stained with hematoxylin by a standard procedure (Nandi, 1959) as follows, for gross examination: 1. The mammary pad on cork is fixed in Bouin's fluid overnight 2. Wash in running water for at least 24 hours until no picric acid is retained 66 . Remove extraneous tissues from the pad Stain the pad directly in Mayer's hematoxylin . Wash in running water overnight GUI-hm . Destain in acid alcohol until the right degree of stains has been obtained 7. Pass the mammary tissue through a series of alco- hol for dehydration (30% - 50% - 70% - 85% - 95% - 100% - 100% - 100%) 8. Immerse (store) the whole mammary tissue in meth- yl salicylate for clearance. Each mammary pad is rated under a dissecting micro- scope for development, according to the following scale (Talwalker and Meites, 1961): 1 Few ducts; few or no end buds; 2 Moderate duct growth; moderate number of end buds; 3 = Numerous ducts and branches; many end buds; 4 = Numerous ducts and branches; moderate lobulo- alveolar growth; U1 II Numerous ducts and branches with dense lobulo- alveolar growth, as in mid or late pregnancy. IX. Methods of Statistical Ana1ysis The prOlactin values obtained from radioimmunoassay of 2 or 3 dose levels of the same sample were averaged, and the mean value was used as the prolactin value for that 67 particular sample. Mean and standard error of the mean were calculated from the averaged prolactin value of each sample within an experimental group. Mammary gland growth ratings, organ weights, and body weights were similarly analyzed. Student's "t" test was used to determine the significance of difference between the control and the experimental groups. In experiments with 2 or more "experimental" groups, a test of significant differences was carried out by an "F" test for approximation. If the result of the "F" test showed significant differences, Duncan's new multiple range test (Duncan, 1955) was used to evaluate the significance of dif- ferences between groups. EXPERIMENTAL I. 13 Vivo and In Vitro Effects of CateEholamines on Pituitagy Prolactin‘Release A. Objectives Both 13_3133_and 13_31113_studies have indicated that catecholamines may influence prolactin release by the rat pituitary. It was considered of interest to determine the effects of catecholamines on pituitary prolactin release. Dopamine, norepinephrine and epinephrine were tested for their effects on serum and pituitary prolactin levels of fe- male rats, as measured by radioimmunoassay. The direct ef- fects of catecholamines on release of prolactin by the incu- bated pituitary also were studied. B. Materials and Methods 1. Animals Mature, 3- to 4-month-old,'virgin female Sprague- Dawley rats, weighing 200-250 g each, were used in all 13 3133_experiments. Two estrous cycles (4-5 days/cycle) were followed by taking daily vaginal smears on all female rats before they were given a drug on the day of proestrus, when 68 69 serum prolactin levels are significantly higher than during diestrus (Amenomori 31 31., 1970; Kwa and Verhofstad, 1967). Since the purpose of these experiments was to determine whether catecholamines could depress serum prolactin levels, rats with higher serum prolactin than the low basal levels present during diestrus were used. Mature male rats of Sprague-Dawley strain, weighing 250-300 g each, were used as pituitary-donors. The method for removal of anterior pituitary tissue from the rat is de- scribed under Materials and Methods. 2. Catecholamines The catecholamines used were dopamine hydrochloride (Mann Research Labs., Inc., New York, N.Y.), L-norepinephrine bitartrate hydrate (K 8 K Labs., Plainview, N.Y.), and epine- phrine chloride (Parke, Davis and CO., Detroit, Michigan). Each catecholamine was directly dissolved in 0.85% NaCl sa- line for injection, or first dissolved in saline, and then incorporated into medium 199 in volumes of 10-20 ul for in- cubation. 3. In vivo experiments The catecholamine solutions in a volume of 0.6 ml were injected intraperitoneally into cycling rats at 10:00 AM on the day of proestrus. The catecholamines also were ad— ministered intravenously via the left carotid artery, and the 70 central end of the artery was immediately ligated after the injection to prevent bleeding. The doses of catecholamines and the routes Of injection used are shown in Tables 1 and 2. Individual blood samples (0.4-0.5 ml) were taken from the rats at 0, 0.5, l and 2 hours following catecholamine injection. A pre-treatment blood sample was taken from each rat prior to catecholamine injection for comparison with sub- sequent blood samples, and the rats were killed after the last blood sample was collected. The methods for prepara- tion of pituitary homogenate and serum sample for prolactin radioimmunoassay are described under Materials and Methods. 4. In vitro experiments Each anterior pituitary half was pre-incubated with 1 ml of medium 199 at a pH of 7.4 for 30 minutes. The method for 13_31113_incubation is described under Materials and Methods. After pre-incubation, the medium was removed and replaced with 1 ml of fresh medium 199 containing a given amount of catecholamine as shown in Tables 3 and 4. Six tubes were used for each dose of catecholamine. Catechol- amines were not incorporated into the control tubes. The in- cubations were carried out for 4 hours. At the end of incu- bation, the anterior pituitary halves were removed, and the media were kept frozen at -20°C until assayed. 71 5. Prolactin ass3y Prolactin in individual serum samples, pituitary ho- mogenates and incubation media were measured by radioimmuno- assay. Each sample was assayed at 3 different dose levels, and the prolactin values were averaged and expressed in terms of the purified rat prolactin reference standard, HIV-8-C. Student's ”t" test, or analysis of variance followed by Duncan's new multiple range test was used to determine the significance of differences between groups. 6. Test for possible influence of dings on radioimmunoassay For the purpose of testing possible interactions be- tween drugs and prolactin during radioimmunoassay, 8 series of assay tubes were prepared with the same concentrations of purified rat prolactin (HIV-8-C, 0.25-64 ng) as used for plotting the standard binding curve. To each assay tube one of the following amounts of drugs was added 12 hours before initiation of the antigen-antibody reaction: 10, 20, 30 or 40 pg of norepinephrine; 10 or 20 ug of epinephrine; 235 or 470 pg of alpha-methyl-para-tyrosine. Two additional series of assay tubes with prolactin but no drugs served as controls. At the end of the 5 day-incubation, the per cent binding of labelled rat prolactin was plotted on semi-logarithmic paper, and the curves obtained from the experimental tubes were com- pared with the standard curve from the control tubes. 72 C. Results 1. Effects of a single intraperitoneal injection of cateEholamines on pituitary prolactin release The data in Table 1 show that none of the 3 catechol- amines produced any significant change in serum prolactin values as compared to pre-treatment values or saline controls by 2 hours following treatments. 2. Effects of a single intracarotid injection of’catecholamines on pituitary_prolactin reléase A single intravenous injection of catecholamines pro- duced no significant changes in serum prolactin levels after 30 minutes, 1 hour or 2 hours, when compared with pre-treat- ment or saline-control values (Table 2). The variations in the serum prolactin levels can be attributed at least in part to the surgical stress and ligation of the left carotid artery. Small but statistically significant decreases were observed in pituitary prolactin concentration after treat- ment with the larger doses of dopamine or epinephrine. 3. Effects of different doses of catechOlamines on pituitary prolactin release in vitro The data in Table 3 show that dopamine had no effect on pituitary prolactin release at doses of 2-40 ng, but pro- duced marked inhibition at doses of 80-640 ng. In two .momonunosmm cw mums mo Honesz A V .amoE may mo posse wumwcmum cam :mozm 73 3.4“”.me 4.0HHN.mm mm L o.oaww.om we m.o acaunamefimm o.mwo.Hm A.m NN.Am mm V A.OHHN.wm we mN.o onusemoefimmsoz m.wNN.Ae m.mHNH.HG mm V Q.HHHo.om we m.o enasaaoo A.mwo.os «.5 “s.ws flHHVam.s no.5m He 0.0 mmHOLOEOUV mefifimm HA N HA H udeHMOHHpIQHm 3o. HGOEHNOHH m oo~\omom He\m: .mHo>OH :fiuumaopm enhom .mHo>oH :MpUmHonm ashom co mocfiEmHoaooumo mo mcofluUOHGM Hmo:0pwuommsucH mo muoommm .H oanwfi 74 .momocucosmm :0 0000 mo ponesz .Ho.o v m .mHOHucoo may Eopm unosommfiv zfipnmoflmfinwfim u A V a .flmmfi 0&9 m0 HOHHG thfidmum @Gm fimwzm cauomaohm .900 0000 0000 0000 0000 0000 00.0000.0 0.0 00.00 0.0 00.00 0.0 00.00 0.0 00.00 00 00 00000000000 000 00 0 00 0 00 0 00 0 00.0000.0 0.0000.00 0.0000.00 0.0000.00 0.0000.00 00 0 00000000000 000 0000 0000 0000 0000 00.0000.0 0.0000.00 0.0000.00 0.0 00.00 0.0 00.00 00 00 00000000000002 000 00 0 00 0 00 0 00 0 00.0000.0 0.0000.00 0.0000.00 0.0 00.00 0.0 00.00 00 0 00000000000002 0000 0000 0000 0000 0000 00.0000.0 0.0 00.00 0.0 00.00 0.0 00.00 0.0 00.00 00 00 00000000 000 00 0 00 0 m0 0 00 0 00.0000.0 0.0000.00 0.0000.00 0.0 00.00 0.0 00.00 00 0 00000000 000 00 0 00 0 00 0 00 0 0000000000 00.0000.0 0.0000.00 0.0 00.00 0.0 00.00 00.0 00.00 00 0.0 000000 ma\w: p: N 0: H HA m.o uncapmonu 3n unoEumonH . . -000 0 000 Coaumhufiounou . m Hs\w: .mHo>OH cfluomflohm,550om \o on .mnowuwhucoocoo cwpomaohg xumpwsufia 0:0 Esuum.zo moawemaoaooumo mo 0:00000nnw vapohmomhucfi mo muoommm .m OHLMH .mo.o v 9 .00000000 on» 500m acouowmww zfiucmoflmflcmfimn .mwoe opp mo 00000 00000000 000 amozm 75 o.w0 an0mN0 O00 0.00 nmm0mm0 omm 0.00 n000o00 o00 N.om n0m0000 ow o.mw 000000 00 0.00 nmwmvw on 0.00 000000 00 0.00 0m0m00 m 0.000 0000000 0000000000 0 0.0900903 00000030000 mo we 30:95 0000006 pom commoaos unoEumoss mo acousom cwuomfioum m: .ohpw> mm ommOHOH afipumfiong 000005000 :0 00050000 mo momov u:OHOMM0w mo muoommm .m OHQmH 76 separate experiments a dose of 20 ng of norepinephrine signifi- cantly increased prolactin release by an average of 39% (Table 4). A dose of 10 ng also stimulated prolactin release by an average of 31%, but this increase was not significant because of the large standard errors. Doses of 200 or 500 ng of nore- pinephrine significantly inhibited prolactin release. A dose of 10 ng of epinephrine increased prolactin release by an aver- age of 47%, whereas 400 or 1,000 ng markedly inhibited pituitary prolactin release. 4. Effects of_presence of drugs in sample on prOlactin radioimmunoassay Dopamine, norepinephrine and epinephrine had no effect on the prolactin radioimmunoassay system when incorporated dir- ectly into the incubation tubes as shown in Figure 5. They did not alter the antigen-antibody reaction or change the binding capacity of the purified, un-labelled rat prolactin. Thus, the presence of these drugs in the radioimmunoassay system did not influence the results reported here. Alpha-methyl-para-tyrosine, a potent stimulator, and ergocornine, a potent inhibitor of pi- tuitary prolactin release (Lu 3; al., 1970; Lu 3: a1., 1971), had no effect on prolactin radioimmunoassay. D. Conclusions The present study demonstrates that an intracarotid or intraperitoneal injection of large doses of dopamine, norepine- phrine or epinephrine had no effect on serum prolactin levels. The larger of the two doses of dopamine and epinephrine given .mo.o v Q .mHOhHfiOU OS“ EOHW Hfimhmwafl kHwGwUMMMGwfimD .fimoe 03“ MO HOHHQ @Hmfifimflm Ufim Gmmzm 77 0.00 000 0000 --- 000.0 0.00 000 0000 000 0000 000 0.000 0000000 0000000 00 0.000 0000000 0000000.0 00 0.000 0000000.0 00000000.0 00 0.000 0000000.0 0000000 0 0.000 0000000 00 0000 0000000000 0 00000000000 0.00 --- 000 0000 000 0.00 000 0000 000 0000 000 0.000 00 0000 00 0000.0 00 0.000 000 0000 00000000.0 00 0.000 00 0000 0000000.0 00 0.000 00 0000 00 0000 0 0.000 00 0000 000 0000 0000000000 0 00000000000002 0.0>0U 0000000 0 0006000000 0 0000000000 000\00V mo HEQUHmm HEOEHNOMH 000000000 00000000000 000000000 00 .0000> mm 0000000 000000000 000000000 00 00000000000 000 00000000000000 00 00000 000000000 00 0000000 .0 00000 78 .00000000 0000000<-0000000000U 00M000< 00 0w000 mo 000000 00 0000 0003000 00>00u 00000000 000000000 000000000 000 .m 000w00 0:00.000 03: 0.0 00 0. 0 0 0 _ 0.0 00.0 . . . 0 . . .00.00 0. 0 0 0 _ 0.0 00.0 -0. 100% 400mH m" 0000.. my: .00d W 1000 o IIO. .00uI m 400 05200500002 oRON . ..... .. m 0550000 mlON o ..... .. < 0c_000>H0-E-u 00000.0 0550000 0000. 0230 0000.020 000000000. 5000.000 79 intracarotidly produced small reductions in pituitary pro- lactin concentrations, but the significance of this is not clear since the serum prolactin values were not altered. Catecholamines have been reported not to cross the ”blood- brain barrier" (Wurtman, 1970; Steinman 33 a1., 1969), un- doubtedly accounting for their lack of action on prolactin release. It will be shown in a subsequent eXperiment that precursors of catecholamines or inhibitors of catecholamine metabolism do cross the "blood-brain barrier" and do alter serum prolactin levels. The in_vitrg_experiments indicate that the effects of catecholamines on pituitary prolactin release in_vit£g are dose-dependent, with high doses producing inhibition, intermediate doses no effect, and low doses stimulation of prolactin release. The higher doses are within the range used by MacLeod (1969) and Birge et_al, (1970) to demonstrate inhibition of prolactin release in_vitrg, The low stimulat- ing doses (10-20 ng/ml) are approximately equal to the amounts of catecholamines reported to be present in the rat hypothalamus (Lippman, 1968; Donoso g£_al., 1967). The phy- siologic significance of the in_vi££g actions of catechol- amines is not evident, since they inhibit prolactin release only at pharmacologic dose levels (Lu 33 al., 1970a). Cate- cholamines are not present in detectable amounts in pituitary protal blood (Wurtman, 1970), and failed to alter prolactin release when presented directly to the pituitary via portal 80 vessel infusion (Kamberi g£_al., 1970b). Drugs which sig- nificantly stimulate pituitary prolactin release i§_vizg_ also profoundly inhibit prolactin release when incubated with rat pituitary in_vi££g_(Lu g£_al., 1970a). Thus, the effects of catecholamines on pituitary prolactin release in vitro do not necessarily reflect their actions in vivo. II. Effects of Central Acting Drugs on Serum andPituitary ProIacf1n*Eevels A. Objectives Inasmuch as catecholamines do not readily pass through the "blood-brain barrier" (Wurtman, 1970; Koelle, 1970; Innes and Nickerson, 1970), the present study was un- dertaken to determine whether other drugs known to enter the brain and to increase or decrease hypothalamic catecholamine levels could change serum and pituitary prolactin concentra— tions. The drugs used were L-DOPA, the immediate precursor of dopamine; three monoamine oxidase (MAO) inhibitors (pargy- line, iproniazid, and Lilly compound-15641) that depress nor- mal metabolism of catecholamines and therefore increase brain catecholamine activity; Methyl-DOPA, which competes for DOPA- decarboxylase to synthesize methyl-dopamine but reduces syn- thesis of dopamine and norepinephrine; Reserpine, which in- terfers with storage and/or induces depletion of catechol- amines; chlorpromazine, a tranquilizer which blocks catechol- amine activity at the post-synaptic receptor sites; Tyrosine 81 analogs (alpha-methyl-para-tyrosine and alpha-methyl-meta- tyrosine), which inhibit synthesis of catecholamines by blocking the enzyme tyrosine hydroxylase; And d-amphetamine, an amine releaser which inhibits amine uptake by nerve ter- mines (McGeer, 1971). B. Materials and Methods 1. Animals Mature, 4- to S-month-old, virgin female Sprague- Dawley rats, weighing 220-250 g each, were used in all ex- periments. Two complete estrous cycles of 4 to 5 days dura- tion were followed on all female rats before they were in- jected with one of the drugs on the day of proestrus or dies- trus as indicated in the experiments. 2. Drugs The drugs used were L-DOPA (Hoffmann-La Roche, Inc., Nutley, New Jersey), pargyline hydrochloride (Abbott Labs., North Chicago, Illinois), iproniazid phosphate (Hoffmann-La Roche, Inc., Nutley, New Jersey), Lilly compound-15641 (N-Z-o-Chlorophenoxy-ethy1-cyclopropy1amine) (Lilly Research Labs., Eli Lilly and Co., Indianapolis, Indiana), methyl- DOPA (Merck, Sharp and Dohme Research Labs., Merck and Co., Rahway, New Jersey), reserpine (Nutritional Biochemicals Corp., Cleveland, Ohio and Ciba Pharmaceutical Co., Summit, 82 New Jersey), chlorpromazine hydrochloride (Research Labs., Smith, Kline and French Labs., Philadelphia, Pa.), alpha- methyl-para-tyrosine (Lederle Labs., American Cyanamid Co., Pearl River, New York), alpha-methyl-meta-tyrosine (Mann Re- search Labs., Inc., New York, N.Y.), d-amphetamine sulphate (Research Labs., Smith, Kline and French Labs., Philadelphia, Pa.). 3. Treatments All drugs except L—DOPA, methyl—DOPA and tyrosine analogs were dissolved directly in 0.85% NaCl solution. The reserpine obtained from Ciba Pharmaceutical Co. was in a so~ lution form (Serpasil, reserpine USP; 2.5 mg/ml). L-DOPA and methyl-DOPA were each dissolved in a warm solution of 0.5 N HCl to which 0.5 N NaOH was added a drop at a time to bring the pH to 2.8. To avoid oxidation and precipitation, L—DOPA and methyl-DOPA solutions were administered into rats immediately after dissolving of the drugs. Tyrosine analogs were first dissolved in 0.1 N NaOH and then adjusted to pH 10.0 by adding 1.0 N HCl a drop at a time. All the drug so- lutions were injected intraperitoneally into cycling female rats at 10 AM on the day of proestrus, unless otherwise stated in the experiments. The controls received injections of physiological saline or medium of pH 2.8 or 10.0. Individual blood samples (0.5-0.7 ml) were collected by cardiac puncture under light ether anesthesia at O, 0.5, 83 1, 2 and 4 hours after each treatment. A pre-treatment blood sample was taken from each rat prior to drug injection for comparison with subsequent blood samples. The rats were killed by decapitation after the last blood samples were col- lected. Serum was separated and anterior pituitary was ho- mogenized according to the methods described under Materials and Methods. The sera and pituitary homogenates were kept frozen at -20°C until assayed. The hypothalami of the rats treated with saline (con- trols), iproniazid, pargyline, L-DOPA, or pargyline and L- DOPA together, were quickly removed after the rats were killed. The hypothalami were preserved and later homogenized according to the methods described under Materials and Meth- ods. Mature male rats, weighing 250-300 g each, were used as pituitary donors for the in vitrg_assay of hypothalamic extracts. Each anterior pituitary half was incubated with one hypothalamic equivalent in 2 ml medium 199 at a pH of 7.4. The media were assayed for prolactin at the end of 4 hours incubation. Prolactin in the medium was calculated in terms of ng released per mg of anterior pituitary tissue. Since reserpine, chlorpromazine, and alpha-methyl- para-tyrosine inhibit catecholamine activity by acting on the brain via different mechanisms (Coppola, 1968), in a separ- ate experiment it was considered of interest to compare the effects of L-DOPA, the immediate precursor of dopamine, in combination with reserpine, chlorpromazine, or alpha-methyl- 84 para-tyrosine on pituitary release of prolactin. In experi- ment I, a single intraperitoneal injection of reserpine, chlorpromazine, and/or L-DOPA was given to cycling female rats on the day of proestrus according to the time schedule shown in Table 8. A pre-treatment blood sample and subse- quent blood samples before and after drug injection were col- lected for prolactin assay. The experiment was terminated by 1:00 PM. In experiment II, cycling female rats were treated with a first dose of 50 mg alpha-methyl—para-tyrosine at 10 AM and a second dose of 30 mg alpha-methyl-para- tyrosine at 6 PM on the day of diestrus to deplete hypotha- lamic catecholamines (Creveling 33 al., 1968). First blood samples were taken at 8 PM before a single dose of 25 mg L- DOPA was given to the rats. Subsequent blood samples were collected at 9 and 11 PM. Another group of female rats re- ceived a single dose of 25 mg L-DOPA at 8 PM on the day of diestrus was used as the controls. 4. Prolactin assay Prolactin in individual serum samples and pituitary homogenates were assayed by radioimmunoassay. Each sample was assayed at 2 or 3 different dose levels, and the prolac- tin values were averaged and expressed in terms of a purified rat prolactin reference standard, NIAMD-rat prolactin-RP—l. Sample mean and standard error of the mean were cal- culated for each drug treatment group. Student's "t" test 85 was used to determine the significance of differences in se- rum prolactin values between pre-treatment and post-treatment samples, and also for the differences in pituitary prolactin concentrations between different treatment groups. C. Results 1. Effects of a single injection of drugs which increase hypothalamic catecholamines on serum and pituitary prolactin levels The data in Table 5 show that a single intraperito- neal injection of L-DOPA reduced serum prolactin about 50% by 30 minutes or 1 hour after injection as compared to pre- treatment values. By 2 hours after injection serum prolac- tin values were reduced 60% and pituitary prolactin concen- tration was significantly increased. Pargyline produced smaller reductions in serum prolactin and no significant change in pituitary prolactin concentration. When both L- DOPA and pargyline were given together, there were greater decreases in serum prolactin values than when either drug was given alone. The combination produced no significant change in pituitary prolactin concentration. Lilly compound- 15641 or iproniazid produced no effect on serum prolactin by 30 minutes or 1 hour after injection, but evoked about a 40- 50% reduction by 2 hours after injection. Lilly compound- 15641 but not iproniazid increased pituitary concentration 86 .Ho.o v a . .mo.o v a . mHogucou Eopm “cohommfiw kfluqmufimH:MHmu mHopunou thm ucohommfiw zHuchfiMchHmn .cmoE may mo youho wumwnmum can :moZm 00H.cwam.m om.Howo.mmN um.NmHo.ao~ o wa.onv¢.e om.m HQ.NN m.m “N.~e UNN.OHHw.m pa.m Hm.Hm m.o Hm.ma mo.ome.m ua.fi Hm.AH um.H Hm.mfi u m~.onno.a om.N Ho.mN UA.N Hm.oN o «H.OHoo.e m.~ Hm.ma N.o “N.me oaa.oaoa.q uo.aeao.oam um.~¢ww.mNm o noe.owao.o UN.H HA.©H UN.~ Hm.- u oa.oaaa.m a.~ Hm.Nm o.~ no.0m m.HMHH.NaH A.Hwa.mm we N.H ashampmgge<-w o.o~wa.ma a.onm.ma we ca cfiNmfleoumH N.m H~.~m N.Awo.Nm we m Loamfl wGSOQEou kHHfiq m.o “n.0H m.mwa.am we NH+ o.__pzcou n nun-nu -u >R£=Dtm 88 of prolactin. Figure 6 shows the percentage changes in se+ rum and pituitary prolactin values after administration of these drugs. 2. Effects of a single injection of drugs whichincreasehypothalamic catecholamines on PIF activity in thelhypothalamus The data in Table 6 show that the pituitary halves incubated with the hypothalami of rats treated with ipron- iazid, pargyline, L-DOPA, or pargyline and L-DOPA together, released less prolactin than that with the hypothalami of saline controls, indicating that more PIF activity was pres- ent in the hypothalamic extract from the drug-injected rats than in the controls. The combination of pargyline and L- DOPA was more effective than either alone for increasing hy- pothalamic PIF activity. 3. Effects of a single injection of drugs which decrease hypothalamic cateCholamines on serum and pituitary prolactin levels Table 5 shows that an injection of methyl-DOPA in- creased serum prolactin over pre-treatment levels about 5- fold by 30 minutes, about 6-fold by 1 hour, and about S-fold by 2 hours after injection. Methyl-DOPA significantly re- duced pituitary concentration of prolactin. A single injec- tion of d-amphetamine increased serum prolactin over 89 .Ho.o v a .maOhuaou onp anm psohommfic kapchfimficwfimu .mo.o v a .mathnou may scum uaohowmfiw zfiucmofim«cwflm n .CNGE 0:“ HO HOHHQ thfiflmum UCN Gmmzm ua.w«om Ummwmoa n«mfimma wwwwmm mmqflm.o <«on-q + on..smpmm ua>fiuom m.a u.5m.mguom»: anachm «on .Em.mgpogsn o. uaoaumonh .Apfl>fipum mHm oasmamsuomxn so manhw mo mpuommm .o oHnMH 90 pre-treatment values about 4-fold by 30 minutes, about 7- fold by 1 hour, and about 6-fold by 2 hours after injection. This drug reduced pituitary prolactin concentration by about 44%. The data in Table 7 show that a single injection of reserpine or chlorpromazine produced marked increases in se- rum prolactin concentration when compared with pre-treatment values or with saline controls. Reserpine produced no ef- fect on serum prolactin levels after one hour, a small in- crease after 2 hours, about S-fold increase by 4 hours after injection, and reduced pituitary prolactin concentration by about half of that in the saline controls. Chlorpromazine raised serum prolactin about 4-fold by one hour after injec- tion, about 7-fold by four hours after injection, and re- duced pituitary prolactin to about 1/4 of that in the saline controls. A single injection of alpha-methyl-para—tyrosine more than doubled serum prolactin levels 30 minutes later, and increased serum prolactin values about 5-fold by l and 2 hours after injection. This drug reduced pituitary con- centration of prolactin to about 1/3 of that in pH 10.0 me- dium controls. Alpha-methyl-meta-tyrosine increased serum prolactin about 8-fold over the pre-treatment values by 30 minutes after injection. Prolactin levels declined by l and 2 hours after injection but were still about four times greater than the pre-treatment values. The pituitary prolac- tin concentration was significantly increased by two hours 91 .anamopxu-mon-H>:uoE-manm .ocfimopzu-mhmm-HznuoE-mzmHm .momoguaoumm a. mum. .Ho.o v m .mHouucou Eoum «cohommfiw xflunmuflmficm«m onwmouxp-e-s-m anamouxp-m-e-m ”opoz mo honesz u A .0 n .fimmE ®£H m0 HOkhm @hmfiufimum flaw Cmmzm mHHV ocflmopxu amo.owoo.m no.mmno.mma no.«HH«.ooH am.wVHm.mmN o.H «m.om we ow -E-E-m mm V ocfimo«>u noo.oumo.H n<.omfio.fiom n«.mHHo.me LN.HHHO.OHH 0.0 Hm.m< we ow -m-s-m .m . .m.opb:ouv mH.oamH.m m.vmflo.ow w.m Hm.wm v.w Hm.wm m.o H~.Hv HE o.H Esfiwoe 0.0. mm .o.. mcHNme a...owmo.. pm.aowq.eam am.maww.o«« n¢.mmw«..m. ..m H«.ma we o.m Longho.;u .m . nmm.oamw.m n«.wvwo.omm m.mHHH.ow m.m “H.mv m.HHHo.Hm we o.~ ocflahomom 0.... .m.oppnou. em.owv~.v H.m Hm.Hm «.m Ho.o< v.« H«.wv mm.« Ho.«m HE o.o onwamm m< me\w: a: m a: N A; H :«E om unoEumohu 32 pamEumohe .mcowumuucoucou -ohm m oom :«uumaonm .ufim Hewwd .meflumhucooaou :fiuumHOHQXESHQm \omom .mnowpmuucounoo :fiuomaopm kaHMSpflm paw Enhom co mmshw mo cowuoomcw Hmocoufiuomwhpcfl oamzfim m mo mpuommm .« ofinmh 92 .cowumuunou:ou dfiuumaowm kumuHSuHm dam Edpom no modHEwHonuouwu uHemenuomzm ommouuom pagw mwdun mo muuommm .« oudmfim (cox—:— xF.:.:.u 5F._:=...30§sz:u .anlfl . N 1 u .n. a n. u — m. V N — V N I: U 4 u . _o>o._ .otcou 29502.0... —n «. 233m a 1 uuuuuuuuuuunuuuuuu-u uuuunuunuuunuulluuuuuunnnuuuoop 5.3.5:... 93 after a single injection of alpha-methyl-meta-tyrosine. The percentage changes in serum and pituitary prolactin concen- trations are shown in Figure 7. 4. Effects of L-DOPA on serum prolactin Iévels in rats_pre-treated with reserpine, cthrpromazine or alpha- methyl-para-tyrosine Table 8 shows that a single injection of L-DOPA markedly decreased serum prolactin levels by 1, 2 and 3 hours after injection as seen in previous experiments. On the other hand, reserpine raised serum prolactin about 6-fold by one hour, about 7-fold by 2 hours and about 8-fold by 3 hours after injection. Since a solution form of reserpine (Serpasil) was used, hence a quick action of drug on the pi- tuitary release of prolactin was observed. Chlorpromazine elicited 6 to 9 folds increase in serum prolactin by l, 2 and 3 hours after injection as seen in provious experiments. A single injection of L-DOPA within one hour after reserpine produced a transitory but highly significant reduction in serum prolactin levels. By 2 hours after L-DOPA, no decrease in serum prolactin was observed in reserpine-pretreated rats, indicating that the inhibition of pituitary prolactin release by L-DOPA after reserpine was short-lived. An injection of L-DOPA after chlorpromazine produced no significant reduction on serum prolactin as compared to pre-L-DOPA levels or the control values in rats treated with chlorpromazine alone. 94 .nphom ow paw .:o>.m «mom-q mo 05.. u a mmoo Nmu mocwmhomoh u mmm wagom-q u «mom mozwamm u oH cfiuumaoum Enhom paw .aofiuumna. mshw mo 05.9 .mum. machumooum :. mHo>oH cwuumHOHQ 53.0m :0 maom-q .0\w:m .oc.NmEo.m.o.go .o:.m.omo. mo :ofluuomafi Hmo:Op..ommhu:. mamcflm m mo muoommm .w oanwb 95 The data in Table 9 indicate that a single injection of L-DOPA also produced marked reduction in serum prolactin in rats on the day of diestrus. Depletion of hypothalamic catecholamines by two successive doses of alpha-methyl-para- tyrosine resulted in a lO-fold increase in serum prolactin levels by 10 or 11 hours, and a 12-fold increase by 13 hours after the first injection. A single injection of 25 mg of L-DOPA within 2 hours after the second dose of alpha-methyl- para-tyrosine reduced serum prolactin about 75% by 1 hour after injection as compared to L-DOPA pre-treatment values. By 3 hours after L-DOPA injection the serum prolactin levels were reduced by 85%. D. Conclusions These results demonstrate that a single injection of L-DOPA, the immediate precursor of dopamine, produced a rapid decrease in serum prolactin and an increase in pitu- itary prolactin concentration. The ability of L-DOPA to raise brain catecholamine levels (Fuxe and Hokfelt, 1969; Innes and Nickerson, 1970; Koelle, 1970) is believed to be responsible for the decrease in pituitary prolactin release. The MAO inhibitors, pargyline, iproniazid, and Lilly compound- 15641 (Fuller, 1968a; 1968b), all reduced serum prolactin lev- els significantly, presumably by interfering with the metab- olism of catecholamines (Koelle, 1970) and thereby increas- ing their concentration in the hypothalamus. Pargyline was 96 .ss.eme N.N mg n mm: .gunow 0m paw “:o>.m Hmz< mo 65.» n a Hmz< .oGHmOHKp-m.mm-.xcpoe-mnmaw u Hmz< ”muoz .Ho.o v a .2m oo.w um mos.m> :.uum.o.m Empom on. Scum unopoww.w kacmo.m.:mHmw .pm. pom omom u A .0 .mo.o v a .2m oo.w um mosfim> cfiuumHONQ Enhom oz. Eopm ucohommflw zapcmo.mflzwflmn .cmoe may mo .o..o whmwcmum cam cmoZm ..Ne mN. a.me on. 9 .NE om. wo.m Hm.om wo.v.Hm.mo mmanm.m Hm.mmN hmz< Hmz< maom-q + Hmz< a..e m.o. a.we on. au.ms om. o.<~wm.oom o.wv.m..om omZLw.m.Ho.oom Hm2< hmz< Hmz< ..Ne mN. n.mN ..... a... ...o. «goamN.m .N.NN «do.-. 2. co... 2. co". 2. CONN 2. co". 2< oo o. .mno.m\m.m. o. psoEumouh A.E\mcv mHo>oH :«pumaoum Enuom cam .cofiuoomc. mshw mo omov can mafia .mum. msoupmowp :. mHo>oH cfiuumHOHQ 53.6m :0 amon-q .0\pcm o:.wo.>u-mnmm-ax:uoe-wggam mo mpoommm .m oHan 97 the most effective of the three MAO inhibitors used for de- creasing serum prolactin, and when given together with La DOPA, the reduction in serum prolactin was greater than when either drug was given alone, presumably because synthesis of catecholamines was enhanced and metabolism of catecholamines was reduced by the combination of these 2 drugs. The large increase in serum prolactin and signifi- cant fall in pituitary prolactin produced by injection of methyl-DOPA is probably due to a reduction in brain catechol- amines, since false neurotransmitters (methyl-dopamine and methyl-norepinephrine) instead of normal neurotransmitters (dopamine and norepinephrine) are synthesized by nerve cells in the presence of methyl-DOPA (Coppola, 1968; Innes and Nickerson, 1970). The significant increase in serum prolac- tin and depletion of pituitary prolactin by d-amphetamine presumably is due to its actions on release of norepinephrine and inhibition of dopamine and norepinephrine reuptake by nerve terminals (McGeer, 1971), although d-amphetamine may also inhibit MAO activity. Release of an amine from nerve endings and inhibition of reuptake could make catecholamines unavailable as neurotransmitters. The present study also demonstrates that a single injection of reserpine, chlorpromazine, alpha-methyl-para- tyrosine or alpha-methyl-meta-tyrosine rapidly elevated se- rum prolactin levels. The suspension form of reserpine ap- peared to be less effective than chlorpromazine because of 98 slow absorption. When a solution form of reserpine was used in a later experiment, reserpine was shown to be as ef- fective as chlorpromazine in stimulating pituitary release of prolactin. The increases in serum prolactin values evoked by reserpine, chlorpromazine and alpha-methyl-para- tyrosine were associated with a significant decline in pi- tuitary prolactin concentration, whereas alpha-methyl-meta- tyrosine produced the largest rise in serum prolactin but also increased pituitary concentration of prolactin. This probably reflects stimulation of synthesis as well as re- lease of prolactin from the pituitary by this tyrosine an- alog. Reserpine, chlorpromazine and alpha-methyl-para- tyrosine decrease catecholamine activity by acting on the brain via different mechanisms (Coppola, 1968): reserpine interfers with storage and/or induces depletion of catechol- amines; chlorpromazine inhibits catecholamine activity by blocking the post-synaptic receptor sites; alpha-methyl- para-tyrosine inhibits the synthesis of catecholamines by blocking the enzyme tyrosine hydroxylase at the rate-limiting step. The mechanisms of action of these three central acting drugs appears to be confirmed by the findings from the pres- ent study. Injection of L-DOPA produced a rapid and sus- tained inhibition of pituitary prolactin release after de- pletion of catecholamines by two doses of alpha-methyl-para- tyrosine. The rapid syntheses of dopamine and norepinephrine 99 by administered L-DOPA apparently by-passed the inhibitory step of tyrosine hydroxylase in converting tyrosine to L- DOPA. In contrast, L-DOPA produced only a transitory reduc- tion in serum prolactin after reserpine, presumably because the strong and prolonged action of reserpine on catecholamine depletion. Reserpine quickly released the newly synthesized catecholamines from L-DOPA, thereby reducing the available catecholamines as functional neurotransmitters within the nerve terminals. Finally, injection of L-DOPA had no sig- nificant effect on serum prolactin levels in rats pre-treated with chlorpromazine. The apparent failure of L-DOPA to af- fect serum prolactin is possibly due to the blockade of re— ceptor sites by chlorpromazine, thereby rendering neurotrans- mission impossible at the synaptic junctions even though catecholamines were available in the nerve endings. In conclusion, the results of the present study are consistent with the hypothesis that hypothalamic catechol- amines inhibit pituitary release of prolactin. Drugs known to enhance brain catecholamine activity produced a decrease in pituitary prolactin release, whereas drugs that reduce hypothalamic catecholamines increased pituitary prolactin release. The work by Kamberi gt 31. (1970) demonstrated in- creased PIF activity in the hypothalamo-pituitary portal blood after intraventricular injection of dopamine in rats. The present study further demonstrates that injection of iproniazid, pargyline, L-DOPA, or L-DOPA and pargyline 100 together, increased hypothalamic PIF activity and the com- bination of pargyline and L-DOPA was more effective than either alone. These observations suggest that the increase in hypothalamic catecholamine levels by these central acting drugs results in increased hypothalamic PIF activity, and this in turn inhibits prolactin release by the anterior pi- tuitary. Thus, the hypothalamic catecholamines influence pituitary release of prolactin presumably by functioning as neurotransmitters between hypothalamic PIF and pituitary pro- lactin. III. Stimulation of Pituitary Prolactin Secretion by Drugs that Depress flypothalamic Catecholamines in Ovariectomized and Ovariecto- mized, Estrogen-Primed Rats A. Objectives Previous experiment demonstrated that central acting drugs including reserpine, chlorpromazine and tyrosine ana- logs evoked rapid release of pituitary prolactin in cycling rats (Lu _£‘_l., 1970b). Early work from our laboratory (Meites, 1962) also suggested that several pharmacological agents including reserpine and chlorpromazine stimulated pi- tuitary prolactin secretion as evident on mammary secretion and/or lactation in estrogen-primed rats. The observed stim- ulation of mammary secretion by drugs may not be due solely to increased prolactin since release of ACTH by these drugs may also be involved in the mammary response (Meites, 1966). 101 Inasmuch as none of the early studies measured blood prolac- tin, the present study was undertaken to determine whether hormones and central acting drugs, some of which are known to depress hypothalamic catecholamines, could stimulate pi- tuitary release of prolactin in ovariectomized and ovariec- tomized, estrogen-primed rats. B. Materials and Methods 1. Animals Mature, 3- to 4-month old, virgin female Sprague- Dawley rats, weighing 200-220 g each, were used in all ex- periments. The rats were bilaterally ovariectomized and later were treated with estrogen and/or drugs. Vaginal smears were followed on all rats for one week post-operation to check the completeness of ovariectomy, and no cornified vaginal epithelial cells were found. 2. Hormones and drugs The hormones and drugs used were estradiol benzoate (Nutritional Biochemicals Corp., Cleveland, Ohio), hydrocor- tisone acetate (Merck, Sharp 6 Dohme, West Point, Pa.), acetylcholine bromide (K G K Labs., Inc., Plainview, N.Y.), chlorpromazine hydrochloride (Research Labs., Smith, Kline 6 French Labs., Philadelphia, Pa.), epinephrine chloride (Parke, Davis 8 Co., Detroit, Michigan), formalin (Fisher Scientific Co., Fair Lawn, N.J.), reserpine (Nutritional 102 Biochemicals Corp., Cleveland, Ohio), serotonin creatinine sulfate (Aldrich Chemical Co., Milwaukee, Wisconsin), alpha- methyl-para-tyrosine (Regis Chemical CO., Chicago, Illinois), and alpha-methyl-meta-tyrosine (Mann Research Labs., Inc., New York, N.Y.). Estradiol benzoate was suspended in corn oil and injected subcutaneously at dorsal skin of the post- cervical region. Hydrocortisone and all drugs except tyro- sine analogs were dissolved directly in neutral PBS. Tyro- sine analogs were dissolved in pH 10.0 medium as described in previous experiment. Hydrocortisone and all drug solu- tions were given by intraperitoneal injection. 3. Treatments Seven days after ovariectomy, the rats were randomly divided into two groups for different treatments. One group of rats was treated with daily injections of one of the nine drugs for 5 days as shown in Table 10. Pre-treatment blood samples were collected from the rats for comparison with post-treatment samples. Two subsequent blood samples were taken from the rats at one and 24 hours after the last injection of drug. The rats were killed by decapi- tation after the last blood samples were collected. Pitu- itaries were removed, weighed, and homogenized in neutral PBS. Sera were separated after centrifugation of blood sam- ples. Both sera and pituitary homogenates were kept frozen at -20° C until assayed. 103 Another group of rats was first treated with estra- diol benzoate (5 pg/day) for 5 days, and then given drug in- jections for 5 days (Table 11). Pre-treatment blood samples were collected from the rats 24 hours after the last injec- tion of estradiol benzoate prior to the beginning of drug treatments. Blood samples were also collected at one and 24 hours after the last injection of drug before the rats were decapitated. Sera and pituitary homogenates were similarly prepared as shown above. Inguinal mammary pads were removed, fixed in Bouin's, stained with hematoxylin, and rated for growth according to a standard method described under Materials and Methods. 4. Prolactin assay Prolactin in individual serum samples and pituitary homogenates was measured by radioimmunoassay. Each sample was assayed at three different dose levels. The prolactin values were averaged and expressed in terms of a purified rat prolactin reference standard (HIV-8-C). 5. Statistics Mean and standard error of the mean for pituitary and serum prolactin concentrations, anterior pituitary weights, and mammary gland growth ratings were calculated for each experimental group. Student's "t” test was used to determine the significance of differences between the con- trol and experimental groups. 104 C. Results 1. Effects of ovariectomy and estrogen on pituitaryypro- lactin release Seven days after ovariectomy the serum prolactin levels were 27 i 3 ng/ml, comparable to diestrous levels in cycling female rats (Amenomori §£_al., 1970). Injection for five days of estradiol benzoate produced a 4-fold (102 i 5 ng/ml) increase in serum prolactin values. By 5 days after termination of estrogen injection, serum prolactin were sig- nificantly reduced (74 i 16 or 57 i 15 ng/ml; Table 11), but these levels were significantly higher than in the ovariec- tomized controls (Table 10).' The pituitary prolactin con- centrations were about 3-fold greater (2.44 i 0.13 or 2.56 i 0.23 ug/mg; Table 11) than that of the ovariectomized con- trols (0.74 i 0.14 or 0.80 i 0.16 ug/mg; Table 10). 2. Effects of multi-injections of drugs on pituitary prolactin release and mammary growth in ovariectomizedfrats The data in Table 10 show that injections of saline or pH 10.0 medium for 5 days had no effect on serum prolac- tin in ovariectomized rats. Serum prolactin was increased about 3-fold over saline controls after 1 hour, and was slightly elevated by 24 hours after the last injection of chlorpromazine. Reserpine produced only small increases in 105 .05.00.0p-0905-H0£u05-0gmH0 .mcho.%p-0H0Q-H03005-0AQH0 05.00.00-5-5-0 05.00.».-m-5-0 .mpoz .COHpumnaH um0H mAu Houm0 0.50: «N 050 H p0 50x0» 0.03 00H550m wOOHm « .Ho.o v m .Ho.u:ou ecu 500m uconmmme 0Hu50uHmH5meu .mo.o v a .Hopucoo 0:. 500m ucouomme >HHG0UHHH:MHm .5005 0:» mo 00.00 0.005000 050 5002M 00.0...0 00.0.0.0. 00.0.0.... 0 .N. 00 .00 0s 0. 00.00...-s-e-0 0.0.0.N 0.0.N.0. U...0.N0.. 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N...0... 00..0.0..0 0 ..0 ---- 00 . 0.00.0.00 m.o.~.v m.o.m.0H NH.owvm.~ m~.ooH ---- m5 oH 00HHoguH0p0o< u~.o.0.0 m.o.H.NH OH.o.Nm.N n0NHmvH umHHHOH m5 N.o 05HQ.000m 00.0.0.0 ..0.0.N. 0...0.00.N .N.00. 0.0.000 00 0 00.000000.o.00 0.0.0.0 m.o.m.mH mH.o.00.N mHHOo 00H.00 H5 0.0 mmHo..:ouV 05HH0w mcHu0. guzo.m m5 ...: H5\m: .: «N .g H 00w\u0. mmso.w\mu0. ow 0:0Hm 0.05505 0.00.3.Hm .50H00..50o:ou H5\m: \0mom .505p00.0 0m0.0>< .oH.0.:< 03003..H ..Hm .0o.m0.p:0o:ou :Hau0Ho.m 5:.0m .mp0. 005H.m-50mo..00 .00NH5ouu0H.0>o 5. n.30.m 0.05505 050 ..an03 0.0.HspHm .oH.0.50 .50..0.u0m 5Huu0Ho.m 0.00.5.Hm :o mms.0 mo 050Hpu0an-H.H:5 mo 0.00mmm .HH 0Hn05 107 serum prolactin levels by l or 24 hours after the last in- jection of the drug. Serotonin or formalin evoked small in- creases in serum prolactin by 24 hours after the last injec- tion. Acetycholine, epinephrine or hydrocortisone had no ef- fect on serum prolactin values by 24 hours after the last in- jection. Alpha-methyl-para-tyrosine and alpha-methyl-meta- tyrosine produced a 2- and 3-fold increase respectively in serum prolactin over medium (pH 10.0) injected controls by 1 hour after the last injection. The serum prolactin values were only slightly elevated by 24 hours after the last injec- tion of the tyrosine analogs. No significant difference in pituitary prolactin concentrations, anterior pituitary weights, or mammary growth was observed at the end of 5 days treatment with any of these drugs except the tyrosine analogs. Both alpha-methyl-para-tyrosine and alpha-methyl-meta-tyrosine significantly increased pituitary prolactin concentrations in the ovariectomized rats as compared to medium injected con- trols. Alpha-methyl-meta-tyrosine but not alpha-methyl-para- tyrosine increased anterior pituitary weights and stimulated mammary growth. 3. Effects of multi-injections of drugs onypituitary prolactin secretion and mammary growth in ovariectomized, estrogen- primed rats Serum prolactin was increased about 7-fold over sa- line controls after 1 hour, and was slightly'elevated 24 108 hours after the last injection of chlorpromazine. Reserpine produced a 2-fold increase in serum prolactin over the con- trols l or 24 hours after the last injection. Acetylcholine, serotonin, epinephrine, hydrocortisone or formalin had no ef- fect on serum prolactin 24 hours after the last injection. Both tyrosine analogs evoked S-fold increases in serum pro- lactin over the controls 1 hour after the last injection. There were small but significant increases in serum prolac- tin by 24 hours after the last injections of both tyrosine analogs. Chlorpromazine significantly reduced, whereas sero tonin and hydrocortisone increased pituitary prolactin con- centrations. All the other 6 drugs had no effect on pitu- itary prolactin concentration. Alpha-methyl-para-tyrosine was the only drug which significantly increased anterior pi- tuitary weight. Chlorpromazine, reserpine, epinephrine, formalin, and the 2 tyrosine analogs significantly stimulated mammary growth (Table 11). D. Conclusions The present study demonstrates that chlorpromazine, reserpine and tyrosine analogs, drugs known to depress hypo- thalamic catecholamine, stimulated pituitary release of pro- 1actin in estrogen-primed ovariectomized rats more effective- ly than in non-estrogen-primed controls. The difference in responsiveness to the drugs may be due to much higher initial 109 pituitary concentrations of prolactin after estrogen treat- ment and to increased sensitivity of the pituitary-hypotha- lamic system. Ovariectomy enhances norepinephrine synthesis by the hypothalamus (Wurtman §t_al,, 1969; Anton-Tay and Wurtman, 1968; Anton-Tay §£_al,, 1970), and leads to a slight increase in the steady-state concentration of the catecholamines (Coppola, 1968) and a marked acceleration in catecholamine turn-over rate (Anton-Tay and Wurtman, 1968). This would also lead to increased PIF release and reduced serum prolac- tin levels, as found here. It is possible that drugs known to depress catecholamines may not be so effective in suppres- sing hypothalamic catecholamine activity when the turn-over rate of the latter is initially accelerated. This also may account for the reduced effectiveness of chlorpromazine, re- serpine and tyrosine analogs in stimulating pituitary pro- lactin release in ovariectomized rats. A reducation in hy- pothalamic catecholamine activity would be expected to de- press hypothalamic PIF activity. Alpha-methyl-meta-tyrosine also is known to increase pituitary prolactin concentrations in cycling female rats (Lu g£_al., 1970b). The significant reduction in pituitary prolactin concentration by chlorpromazine may reflect sustained stim- ulation of prolactin release by this drug. A rapid decrease in pituitary prolactin concentration by chlorpromazine also is seen in cycling female rats (Lu 3: 51., 1970b). The 110 mechanism by which serotonin and hydrocortisone increase pi- tuitary concentration of prolactin remains to be determined, although this was previously reported for hydrocortisone (Meites, 1966) and there is evidence that serotonin stimu- lates prolactin release (Kamberi £3 11., 1971a). In most cases serum prolactin levels after drug ad- ministration were higher by one hour than after 24 hours, indicating rapid stimulation by the drugs of pituitary re- lease of prolactin. Hence, the time of blood sampling after drug administration influences the results. In general, in- creased pituitary release of prolactin results in enhanced mammary growth in estrogen-primed rats. However, drugs which have no significant effect on prolactin release such as epinephrine and formalin also stimulate mammary growth, pre- sumably due to enhanced release of ACTH by these pharmaco- logical agents. IV. Effects of L-DOPA on Serum Prolactin and Prolactin Release-Inhibiting Activity in ‘Intact and Hypophysectomized, Pituitary-Grafted Rats A. Objectives Transplantation of anterior pituitary grafts under- neath the kidney capsule of hypophysectomized rats resulted in sustained prolactin release (Chen 33 al., 1970), presum- ably due to removal of direct inhibition by the hypothalamus of prolactin release (Meites 33 11., 1963; Meites, 1970). 111 Previous experiment demonstrated that a single intraperito- neal injection of L-DOPA decreased serum prolactin and in- creased hypothalamic content of PIF in cycling female rats (Lu and Meites, 1971). It was considered of interest there- fore, to determine the effects of L-DOPA on serum prolactin levels, hypothalamic PIF content, and serum prolactin release- inhibiting activity in hypophysectomized rats with or with- out an anterior pituitary graft and in intact female rats. B. Materials and Methods 1. Animals A group of mature hypophysectomized female Sprague- Dawley rats (average body weight = 193 g each) was grafted with a single anterior pituitary underneath the left kidney capsule 7 days after hypophysectomy. Mature cycling female rats of the same strain were used as pituitary donors. An- other group of mature hypophysectomized female rats was grafted without a pituitary transplant. Mature male Sprague- Dawley rats (250-300 g each) were used as pituitary donors for in_yitrg_assay of hypothalamic PIF and serum prolactin releasing activity. Mature cycling female rats of the same strain also were injected with saline or L-DOPA at 10 AM on the day of proestrus. 112 2. Treatments Five days following pituitary-transplantation and 12 days after hypophysectomy, rats with or without an anter- ior pituitary graft were divided into 2 subgroups. After collection of a pre-treatment blood sample at 10 AM, each rat was given 0.5 ml physiological saline or 12 mg of L-DOPA by a single intraperitoneal injection. Subsequent blood samples were collected at 30 minutes, 1 hour and 2 hours af- ter saline or L-DOPA administration, and the rats were killed after the last blood samples were collected. Blood samples from the intact rats were collected just before and 2 hours after L-DOPA injection. The pituitary fossa of each hypo- physectomized rat was examined under magnification and found to be free of pituitary tissue. The hypothalami were re- moved, pooled in groups and kept frozen in 0.1 N HCl. The blood samples were centrifuged, and the sera were kept fro- zen until assayed as described under Materials and Methods. 3. In vitro assay of hypothalamic PIF—activity The pooled hypothalami from each treatment group were homogenized and extracted in 0.1 N HCl as described un- der Materials and Methods. Male pituitary-donor rats were killed by decapitation. Anterior pituitaries were removed and hemisected. After 30 minutes pre-incubation, each an- terior pituitary half was placed into a culture tube 113 containing medium 199 (pH 7.4) and extract of one hypotha- lamic fragment in a total volume of 2.1 m1. Incubations were carried out in a Dubnoff metabolic shaker as described under Materials and Methods. At the end of 4-hour incuba- tion, pituitary halves were removed and incubation media were kept frozen at -20°C until assayed. 4. In vitro assay of serum prolactin release-inhibiting activity Individual serum samples were assayed for prolactin releasing activity by incubating with single anterior pitu- itary halves, as indicated above. A total of 0.5 ml serum was incorporated into 1.5 ml medium 199 (pH 7.4) in a cul— ture tube containing one anterior pituitary half. 5. Prolactin assay Prolactin in individual serum samples and incubation media were measured by radioimmunoassay. Each sample was assayed at three different dose levels, and the prolactin values were averaged and expressed in terms of the purified rat prolactin reference standard, NIAMD-rat prolactin-RP-l. The prolactin values reported in the serum incubation exper- iments (Table 12) were corrected for the amounts of serum prolactin added to the incubation medium. Sample mean and standard error of the mean were cal- culated for each experiment group. Student's "t" test was 114 .mo.o v a .mHo..:oo 5o.m .50.0mme 0Huc0unH5mHm ..505u00.u .00 mp0. mo .02 u m V a .0005 mo .o..0 0.0000pm . 00020 .00 0 ..0 .0 0 0..00 00 N. .0000-. n 0H0..:ouv .0 .00 0 .00 .0 0 0...0 .0 0.0 .00..00 0.0. 0:0..m0o.m .uu0ucH .N.0 0 .0. .N.0 N .0. 00.N. .000m-. Ho.ucou .m .N.. N .0. .N.0 . .0. .0 0.0 .00..00 .m0.m m< o: 0.03 0.0. 00NH5opo0m0gmom0m .N.0 ...00 .N.0 0..0.. 00 N. .0000-. n fiHo.p:ouV .N .N.0 00.N0. .N.0 ...0N. .0 0.0 .00..00 .0000 .00 .0000.0. .0 0 0 .0N. 00 N. .0000-. mHo.ucouV .H .00 0N.00. .00 0.0.. .0 0.0..N0. .0 0.0 .00..00 .m0.m m< H 00H: mp0. 00~H5ouu0m0auom0m .0 N .0 . 0.0 00 .00 0.0 .00.\00000 u:05.00.u-0.m u:05u00.0 .mxm H5\m: ..uaou :Huu0Ho.a 5:.0m .003H0> :Huu0Ho.m 55.00 :0 0mo0-H mo :oHuo0nnH >0 0Hm5Hm 0 mo 0.00mmm .NH 0Hn05 115 used to determine the significance of differences between the control and.L-DOPA treated groups. C. Results 1. Effects of hypaphysectomy on body growth and serum prolactin levels Seven days after hypophysectomy, average body weight of the hypophysectomized rats was 193 t 2 g each. The final average body weight was 199 i 5 g. No significant increase in body weight was observed over the 5-day period in the hy- pophysectomized rats with no pituitary transplant. Twelve days after hypophysectomy, the serum prolac- tin values were about 14 ng/ml (Table 12) in terms of the NIAMD-rat prolactin-RP-l reference standard. This is in good agreement with our previous report of serum prolactin values of up to 10 ng/ml in hypophysectomized rats when ex- pressed in terms of HIV-8-C rat prolactin standard (Chen 23 a1., 1970). The "prolactin" in hypophysectomized rats is believed to reflect non-specific binding by rat serum pro- teins to anti-rat prolactin antibody. 2. Effects of transplantation of'a single anterior pituitary on body_g:owth and serum prolactin levels in hypophysec- tomized rats By the end of the 5-day period after a single anter- ior pituitary transplantation, body weights of the 116 hypophysectomized rats were significantly increased from an initial average of 193 t 2 g to 209 i 4 g each. The serum prolactin values were 132 i 10 ng/ml which are about 2-fold higher than that of cycling female rats (51 i 13 ng/ml) on the morning of proestrus day (Table 12). 3. Effects of L-DOPA on serum prolactin levels The data in Table 12 and in Figure 8 show that a single intraperitoneal injection of saline (controls) had no effect on subsequent serum prolactin levels in hypophysecto- mized or hypophysectomized, pituitary-grafted rats (Experi- ments 1-3). L-DOPA decreased serum prolactin concentrations by 50-60% 30 minutes, 1 or 2 hours after injection when com- pared with pre-treatment values or saline controls (Experi- ments 1-2). L-DOPA produced no effect on serum prolactin levels in hypophysectomized rats with no pituitary transplant (Experiment 3). In intact cycling female rats, L-DOPA re- duced serum prolactin values to about half of pre-treatment levels (Experiment 4). 4. Effects of L-DOPA on serum prolactin release-inhibiting activity The data in Table 13 and in Figure 9 show that the serum from the hypophysectomized and intact female rats prior to treatment with L-DOPA did not differ in their effects on pituitary prolactin release ia_vitro. An injection of saline 117 = Hypox + 'IAP craft Saline L-DOPA 120' 90‘ Scrunimr PRL "fiéhlo Hypox 110 knuct,pnooflnous 60 Pk () (L5 1 2 Figure 8. Effects of L-DOPA on Serum Prolactin Levels in Intact, Hypophysectomized, or Hypophysectomized, Pituitary-Grafted Female Rats. 118 Table 13. In vitro assay of PIF activity in serum and hypothalamus f?bm.rats given L-dopa. Exp. Treatment Pre-treatment Prolactin released (pg prolactin 30 min Hypophysectomized rats with 1 AP graft 1. Saline, 0.5 mg (controls) 1.07:0. L-Dopa, 12 mg 1.13:0. 2. Saline, 0.5 ml (controls) 1.41:0. L-Dopa, 12 mg 1.28:0. Hypophysectomized rats with no AP graft 3. Saline, 0.5 m1 (controls) 1.23:0. L-Dopa, 12 mg 1.16:0. Intact, proestrous rats 4. Saline, 0.5 m1 (controls) 1.24:0. L-Dopa, 12 mg 1.20:0. aMean : standard error of mean. b ( ) = No. of rats per treatment. 10a 03 10 OS 07 05 14 15 ( 3) C 3) (12) (12) (12) (12) ( 6) ( 6) 1.07i0.07(3) 0.87i0.17(3) Significantly different from the controls, p < 0.05. in vitro after serum incubation r€leased/mg AP) ¥ Time after treatment F“ 1 hr 2 hr 119 Prolactin released in vitro after Incubation with hypothalamic extract (ug prolactin released/mg AP) Time after treatment 1 hr 2 hr 1.23:0.11 (12) 0.47:0.03b(12) 1.3400.1s (12) 0.44:0.05b(12) 1.30:0.13~(6) 0.77:0.03b(6) 1.27:0.05 (6) 0.64:0.03b(6) 0.42:0.04 (12) 0.28:0.03b(12) 0.48:0.05 (12) 0.32:0.04b(12) 0.55:0.04 (6) 0.44:0.02b(6) 120 -f151-C-:-f'¥53:-2-:-Z-:-.-€-2-:' x L-DOPA "7P0 II- IAP gran .. + 0 d' A. 0 I .— *Wmux ua mu. '3‘ 4'. Ihhcufl /mo AP A. 0 LJ hflufldubdflflwn 1.2. SERUM ms '" w H" .6. 0 fl 0 Hr O 0.5 I 2 2 Figure 9. Effects of L-DOPA on Serum Prolactin Releasing Activity in Intact, Hypophysectomized, or Hypo- physectomized, Pituitary-Grafted Female Rats. 121 (controls) had no effect on serum prolactin releasing activ- ity in hypophysectomized or hypophysectomized, pituitary- grafted rats (Experiments 1-3). Experiment 1 shows that the incubated serum from the L-DOPA treated rats produced a small decrease in prolactin release by 30 minutes and a significant decrease of 40% by 2 hours after L-DOPA injection as compared to pre-treatment or saline control values. Experiment 2 shows that the serum from the L-DOPA treated rats produced a 65% decrease in prolactin release when incubated with pitu- itary as compared to saline controls or pre-treatment values. The serum of hypophysectomized rats with no pituitary trans- plant (Experiment 3) given L-DOPA also elicited a 65% de- crease in prolactin release by incubated pituitary. Experi- ment 4 shows that the serum from L-DOPA treated intact pro- estrous rats reduced prolactin release by almost 50%. All experiments show that injection of L-DOPA produced prolactin release-inhibiting activity in the serum. 5. Effects of L-DOPA on Hypothalamic PIF activity The data in Table 13 and in Figure 9 also show that L-DOPA increased hypothalamic PIF content by 35% in hypophy- sectomized_or hypophysectomized, pituitary-transplanted fe- male rats by 1 hour after L-DOPA injection. L-DOPA produced a 20% increase in hypothalamic PIF activity in intact female rats by 2 hours after the injection. All experiments show that L-DOPA administration increased hypothalamic PIF content. 122 D. Conclusions The present study demonstrates that transplantation of a single anterior pituitary underneath the kidney capsule results in increased body weight and sustained prolactin re- lease in hypophysectomized rats. Early work from our lab- oratory (Meites and Kragt, 1964) demonstrated that trans- plantation of a single pituitary graft induced body weight gains in immature hypophysectomized female rats. As a re- sult of removal of direct hypothalamic inhibition by pitu- itary transplantation, stimulation of prolactin turn-over rate produces an increase in serum prolactin and a decrease in prolactin concentration (or content) in the pituitary graft (Lu gt_al,, 1971). L-DOPA administration reduces serum prolactin lev- els in hypophysectomized rats with an anterior pituitary transplant and in intact rats, and this is associated with an increase in hypothalamic PIF content and the appearance of prolactin release-inhibiting activity in the serum. It is probable, therefore, that in the present study L-DOPA in- hibited pituitary prolactin release both by increasing PIF activity in the hypothalamus, and by producing prolactin release-inhibiting activity in the serum. The prolactin release-inhibiting activity in the serum after L-DOPA may represent increased synthesis and release of hypothalamic PIF into the systemic blood. The 123 possibility that some L-DOPA remained in the serum and di- rectly inhibited pituitary prolactin release remains to be studied, although L-DOPA is rapidly metabolized. V. Effects of Serotonin, Melatonin, 5* Hydroxytryptophan and Tryptophan on Serum Prolactin and Hypotha- lamic and Serum Prolactin ReléasiagyActivity A. Objectives Kamberi EE.E$° (1970a, 1971a) reported that an in- jection of serotonin or melatonin into the third ventricle of rats increased prolactin and decreased LH and FSH levels in the blood. However, Lu at_al. (1970b) observed that a single intraperitoneal injection of serotonin did not alter serum and pituitary prolactin concentrations in cycling fe- male rats. Inasmuch as serotonin, likes catecholamines, does not readily pass through the "blood-brain barrier" (Douglas, 1971), the present study was undertaken to deter- mine whether precursors of serotonin known to enter the brain and to increase brain serotonin concentrations (Fernstrom and Wurtman, 1971) could change serum prolactin levels in the rat. The precursor substrates used were L- tryptophan and the immediate precursor S-hydroxytryptophan. The effects of serotonin and melatonin on serum prolactin values also were assessed in cycling female rats. In an at- tempt to clarify the mechanism of action by which 5-hydroxy- tryptophan stimulates prolactin release, its effects on 124 serum prolactin and hypothalamic and serum prolactin releas- ing activity were assessed in hypophysectomized and hypophy- sectomized, pituitary-grafted rats. B. Materials and Methods 1. Animals Mature, 4- to S-month-old, virgin female Sprague- Dawley rats, weighing 220-250 g each, were used in the ex- periments. Two complete estrous cycles of 4 to 5 days dura- tion were followed on all female rats before they were in- jected with one of the drugs on the day of proestrus. A group of mature hypophysectomized female Sprague- Dawley rats (average body weight = 196 g each) were grafted with a single anterior pituitary (AP) underneath the left kidney capsule 7 days after hypophysectomy. Mature cycling female rats of the same strain were used as pituitary donors. Another group of mature hypophysectomized female rats were grafted without an AP transplant. Mature male Sprague- Dawley rats (250-300 g each) were used as pituitary donors for ia yitrg assay of hypothalamic and serum prolactin re- leasing activity. 2. Drugs The drugs used were L-tryptophan, DL-5-hydroxytrypto- phan, serotonin (S-hydroxytryptamine) creatinine sulfate, 125 and melatonin (N-acetyl-S-methoxytryptamine). All compounds were purchased from Nutritional Biochemicals Corp., Cleve- land, Ohio. L—tryptophan and S-hydroxytryptophan were each dissolved in slightly alkalized saline to which 0.5 N HCl was added a drop at a time to bring the pH to 6.7. Mela- tonin was first dissolved in a small amount of absolute al- cohol and then diluted with physiological saline to contain 0.3% ethanol. Serotonin was directly dissolved in 0.85% NaCl for intravenous administrations. 3. Treatments All drug solutions were injected intraperitoneally into cycling female rats via the caudal veins at 11 AM on the day of proestrus. The control rats received injections of saline-ethanol solution or pH 6.7 medium. Individual blood samples (0.5-0.7 ml) were collected by cardiac punc- ture under light ether anesthesia at 30 minutes, 1 hour and 2 hours after each treatment. A pre-treatment blood sample was taken from each rat prior to drug injection for compari- son with subsequent blood samples. Since injections of drugs were made while the rats were anesthetized lightly un- der ether, the pre-treatment blood sample was collected 30 minutes prior to drug injection to avoid prolonged ether treatment . Seven days following pituitary-transplantation and 14 days after hypophysectomy, rats with or without an AP 126 graft were given 25 mg of 5-hydroxytryptophan or 0.6 ml pH 6.7 medium by a single injection via the caudal veins at 10:30 AM. A pre-treatment blood sample was taken from each rat at 10 AM. Subsequent blood sample was taken 60 minutes after drug injection. The rats were killed by decapitation after the post-treatment blood samples were collected. The pituitary fossa of each hypophysectomized rat was examined under magnification and found to be free of pituitary tissue. The hypothalami were removed, polled in groups and kept fro- zen in 0.1 N HCl at -20° C until assayed ia.yi££g. The blood samples were kept in a refrigerator at 4° C for 4 hours before they were centrifuged. The serum was separated and preserved as described under Materials and Methods. 4. In vitro assay_of hypothalamic pr61acfin releasing activity The pooled hypothalami from each treatment group were homogenized and extracted in 0.1 N HCl as described un- der Materials and Methods. Male pituitary-donor rats were killed by decapitation. Anterior pituitaries were removed and hemisected. After 30 minutes pre-incubation, each AP half was placed into a S-ml culture tube containing medium 199 and extract of one hypothalamic fragment in a total vol— ume of 2.15 ml. Incubations were carried out in a Dubnoff metabolic shaking incubator as described under Materials and 127 Methods. At the end of 4-hour incubation, AP halves were removed and incubation media were kept frozen at -20°C until assayed for prolactin activity. 5. In vitro assay_of serum_prolactin releasifig_activity Individual serum samples were assayed for prolactin releasing activity by incubating with single AP halves. A total of 0.5 ml serum was incorporated into 1.5 ml medium 199 (pH 7.4) in a culture tube containing one AP half. 6. Prolactin assay Prolactin in individual serum samples and incubation media were measured by radioimmunoassay. Each sample was assayed at two different dose levels, and the prolactin val- ues were averaged and expressed in terms of the purified rat prolactin reference standard, NIAMD-rat prolactin-RP-l. The prolactin values reported in the serum incubation experiments (Table 15) were corrected for the amounts of serum prolactin added to the incubation medium. C. Results 1. Effects of a single intravenous injections of L-tryptophan, S-hydroxytryptophan, serotonin, and melatonin on serumapro- lactin levels in cyEling_fema1e rats The data in Table 14 show that a single intravenous injection of saline-ethanol solution or medium of pH 6.7 128 had no effect on subsequent serum prolactin values. The pro- lactin levels in the serum were slightly elevated by 1 PM on the day of proestrus. Administration of L-tryptophan pro- duced no effect on serum prolactin by 30 minutes and 1 hour, and evoked a small increase by 2 hours after injection. This increase in serum prolactin was not statistically sig- nificant due to the large standard error. By contrast, a single injection of 5-hydroxytryptophan, the immediate pre- cursor of serotonin, raised serum prolactin about 9-fold by 30 minutes, about 6-fold by 1 hour, and about 2-fold by 2 hours after injection as compared to control rats received medium of pH 6.7. Intravenous injection of serotonin pro- duced no significant changes in serum prolactin levels after 30 minutes, 1 and 2 hours, when compared with pre-treatment or saline-ethanol—control values. An injection of melatonin produced no changes in serum prolactin by 30 minutes, and elicited a 2-fold increase by 1 hour and a 3-fold increase by 2 hours after injection. 2. Effects of S-hydroxytryptophan administratiOn on serum prfilactin Iévels in hypophysectomized and hypophysectomized,apituitary- gaaftEd rats Table 15 shows that the serum prolactin values were about 14 ng/ml in hypophysectomized rats (Experiment 1). This was in good agreement with previous reports on serum prolactin values of 10-14 ng/ml in hypophysectomized rats 129 .Ho.o v a .Ho..5ou 0:. 5o.m 050.0mme 0Hp50onH5mHm0 .mo.o v a .Ho..5oo 0:. 50.m 050.0MMH0 0Hu500HmH5mHm : .5005 0:. mo .o..0 0.005000 050 50020 n.0.00.0.N.N 00.0..0.00. ..0..0.N. N.0 .0.0. 00 0 0.00.0.0: 0.0..0..0 0....0... 0.0 .0.0. 0.N..0.0. 00 0.0 0.0000000 0H0..5ouv N.0..0.00 0.0 .0..0 0.0 .0.00 0.. .0.N0 .0 0.0 .000000-00..00 0..0N.0..NN 0N....0..00 00.00.0.00. ..0 .0... 00 00 00000.00.0.xo.000-0 0.0....0.N ..0N.0..0 ..00.0..0. 0.0 .0.0. 00 00 0000000..... mHo.u5ooU 0.00.N.0.. 0.0..0.00 N.0..0..0 00.0 ...N. .0 0.0 00.000 ..0 :0 .20 00 .0 .z< 00 N.0 .20 00 ..0 .20 00 0.0 00. .000.0\000. 00 .: N .: H 5.5 cm p505.00..-0.m \00om .505u00.0 H5\m5 .05oHu0..5005ou 5Huu0Ho.m 55.0w . .005H0>.5H.00Ho.m 55.00 50 5H5ou0H05 050 5.50.0.00 .50:mopm0.u0xo.w0:-m .50:mo.50.p mo 50Hpuon5H 05050>0.p5H 0Hm5H0 0 mo 0.00mmm ..H 0H:0H 130 Table 15. Effects of a single intravenous injection of S-hydroxytryptophan on serum prolactin values, and prolactin-releasing activity in the serum and hypothalamus of hypOphysectomized and hypothysectomized, pituitary-grafted rats. Serum prolactin conc., ng/ml Time after treatment Pre- Treatment Dose/ treatment 60 min (5 rats/group) rat (10 AM) (11:30 AM) Experiment 1: Hypothysectomized rats with no AP graft pH 6.7 medium 0.6 ml 14: 1a 12: 1 (control) S-Hydroxytrayptophan 25 mg 13: l 15: 2 Experiment 2: Hypophysectomized rats with 1 AP graft pH 6.7 medium 0.6 m1 135:12 156:54 (control) S-Hydroxytryptophan 25 mg 153017 298:55b aMean and standard error of the mean. bSignificantly different from the control, p < 0.05. 131 Prolactin released in vitro after incubation—With hypothalamic extract (ug prolactin released/mg AP) Prolactin released in vitro after serum incubatiEh; pg prolactin released/mg AP Time after treatment Time after treatment Pre- treatment 60 min 60 min 1.20:0.09 1.33:0.14 0.51:0.07 1.40:0.18 1.38:0.16 0.51:0.03 1.51:0.15 1.35:0.17 0.43:0.03 1.40:0.21 1.65:0.16 0.47:0.03 132 (Chen a; a1., 1970; Lu and Meites, 1972). The "prolactin" in hypophysectomized rats is believed to reflect non-specific binding by rat serum proteins to anti-rat prolactin anti- body. An injection of pH 6.7 medium had no effect on subse- quent serum prolactin levels in hypophysectomized or hypo- physectomized, pituitary-grafted rats (Experiments l-2).‘ An injection of S-hydroxytryptophan increased serum prolactin concentrations by about 95% 1 hour after injection into hy- pophysectomized rats with a pituitary graft (Experiment 2) when compared with pre-treatment values or controls 1 hour after injection of pH 6.7 medium. It can be seen that S- hydroxytryptophan had no effect on serum prolactin levels in hypophysectomized rats with no AP graft (Experiment 1). 3. Effects of S-hydroxytryptophan administration on serum and hypothalamic‘prolactin releasing_activity The data in Table 15 show that the sera from hypo- physectomized and hypophysectomized, pituitary—grafted rats prior to treatment with 5-hydroxytryptophan or pH 6.7 medium did not differ in their effects on pituitary prolaCtin re- lease ia_yippp_(Experiments 1-2). An injection of 5- hydroxytryptophan had no effect on serum prolactin releasing activity in hypophysectomized or hypophysectomized, pituitary- grafted rats (EXperiments 1-2). Extracts of hypothalami from hypophysectomized rats treated with S-hydroxytryptOphan did not differ in their 133 effects on prolactin release when compared with medium- control values (Experiment 1). In hypophysectomized, pituitary-grafted rats, administration of S-hydroxytryptophan did not significantly alter hypothalamic prolactin releasing activity. The hypothalami from hypophysectomized and hypo- physectomized, pituitary-grafted rats did not differ in their effects on pituitary prolactin release ia_yipgp_(fixperiments 1-2). These experiments show that administration of 5- hydroxytryptophan did not change hypothalamic and serum pro- lactin releasing activity in hypophysectomized rats with or without an AP graft. D. Conclusions These results demonstrate that a single injection of S-hydroxytryptophan, the immediate precursor of serotonin, produced a rapid increase in serum prolactin in cycling fe- male rats and in hypophysectomized rats with one AP trans- plant. The ability of 5-hydroxytryptophan to raise brain serotonin concentration (Fernstrom and Wurtman, 1971) is be- lieved to be responsible for the stimulation of pituitary prolactin release. Another precursor, tryptophan (one step prior to S-hydroxytryptophan) of serotonin produced only a small but insignificant rise in serum prolactin values. The two major biochemical pathways for tryptophan oxidation to S-hydroxytryptophan and to kynurenine (White 33 a1., 1968) apparently account for the lesser ability of L-tryptophan 134 to increase serum prolactin, although intraperitoneal injec- tion of L-tryptophan was reported to increase barin serotonin concentration rapidly in rats (Fernstrom and Wurtman, 1971). Injection of melatonin also produced significant increases in serum prolactin values in cycling female rats, confirming the observations of Kamberi _£‘_l. (1971a) that an injection of melatonin into the third ventricle of rats elevated serum prolactin values. However, the results of the present and previous report (Lu ap a1., 1970b) do not substantiate the finding of Kamberi a; a1 (1971) that serotonin itself can stimulate pituitary release of prolactin. This could be due to the different routes of serotonin administration. Sys- temically administered serotonin apparently does not pass through the "blood-brain barrier" in significant amounts (Douglas, 1971). Administration of S-hydroxytryptophan also signifi- cantly stimulated prolactin release by the pituitary graft in hypophysectomized rats 1 hour after injection. The in- crease in pituitary release of prolactin by 5-hydroxytrypto- phan could be due to suppression of PIF or stimulation of PRF activity in the hypothalamus. However, there was no de- tectable change in hypothalamic and serum prolactin releas- ing activity observed after 5-hydroxytryptophan administra- tion under these experimental conditions. The 1a 11519 as- say method used in the present study may not be adequate to detect PRF activity in the hypothalamus and its release into the systemic circulation. 135 VI. Direct Inhibition by Ergocornine pf Pituitary Prolactin Release A. Objectives Recent work from our laboratory (Nagasawa and Meites, 1970; Wuttke ap a1., 1971) demonstrated that ergocornine re- duced pituitary and serum prolactin concentrations. Ergo- cornine also was shown to increase hypothalamic PIF content (Wuttke ap‘a1., 1971), suggesting that it acted at least in part via the hypothalamus. It was of interest therefore to determine whether ergocornine could also act directly on the pituitary to inhibit prolactin release. Estrogen stimulates pituitary prolactin release la yiyp (Meites a; 21': 1963; Nagasawa 33 a1., 1969; Amenomori ap‘al., 1970; Chen and Meites, 1970) and la 113:3 (Nicoll and Meites, 1962; Ratner a; a1., 1963 Nicoll and Meites, 1964) both by a direct action on the pituitary and via the hypothalamus by reducing PIF content (Ratner and Meites, 1964). It was of interest, therefore, to see whether ergo- cornine could counteract the stimulatory effect of estrogen on pituitary prolactin release. The effects of ergocornine and/or estrogen were assessed in ovariectomized rats, in hy- pophysectomized-ovariectomized rats with a single anterior pituitary transplant, in intact cycling female rats, and on pituitary tissue incubated 1a 11339. Wuttke and Meites (1971) reported that injections of ergocornine for 1 or 3 estrous cycles inhibited luteolysis 136 of corpora lutea in cycling rats, presumably due to inhibi- tion of the normal rise in blood prolactin on the days of proestrus and estrus. In the present study, it was con- sidered of interest to see whether injections of ergocor- nine into cycling rats for 4-5 weeks could interfere with the estrous cycle by inducing accumulation of corpora lutea in the ovaries. B. Materials and Methods 1. Animals Mature, 3- to 4-month-old, virgin female Sprague- Dawley rats, weighing 200-220 g, or 4- to 5-month-old fe- male rats weighing 225-240 g each, were bilaterally ovari- ectomized and later injected with estrogen and/or ergocor- nine. Intact, 3- to 4-month-old, cycling female rats of the same strain were given daily injections of ergocornine for 4-5 weeks. Mature, hypophysectomized, female rats of the same strain, weighing 190-210 g each, were ovariectom- ized and transplanted with a single anterior pituitary un- derneath the left kidney capsule 7 days after hypophysectomy. Mature male Sprague-Dawley rats, weighing 250-300 g each, served as pituitary donors for the transplantation studies and for the la vitro incubation experiments. 137 2. Drugs and Hormones Ergocornine methanesulfonate (Sandoz Ltd., Basel, Switzerland) was first dissolved in a small amount of ab- solute alcohol and then diluted with physiological saline to make up a suspension containing 0.3% alcohol. Estradiol benzoate (Nutritional Biochemicals Corp., Cleveland, Ohio) was dissolved in corn oil and injected subcutaneously in a volume of 0.3 m1 under the cervicodorsal skin, and ergocor- nine was injected intraperitoneally in a volume of 0.5 ml. Ovine prolactin (NIH-P-S 8) was dissolved in slightly alka- lized 0.85% NaCl at a concentration of l mg/0.4 m1. 3. Treatments Estrogen and/or ergocornine were administered to the experimental rats as shown in Tables l6, l7, and 18. Blood samples were collected from each rat at the end of each experiment before the rats were killed by guillotine. Inguinal mammary pads were excised from each animal and pre- pared for whole mount evaluation as described under Mater- ials and Methods. The anterior pituitaries were removed, weighed, and homogenized. Blood samples were centrifuged, and the sera were separated. Both the sera and pituitary homogenates were kept frozen at -20° C until assayed as de- scribed under Materials and Methods. 138 4. Ovarian histology Intact, cycling female rats were killed at the end of the 4- or S-week treatment with saline, ergocornine, or ergocornine plus prolactin. The ovaries were removed, cleaned, weighed, fixed in Bouin's fluid, sectioned at 10 p each, and stained with hematoxylin and eosin for microscop- ic examination. The number of cepora lutea were counted on three sections taken from the longest axis of individual ovaries from all rats of each group, and averaged. S. Pituitary_incubations Each anterior pituitary half was placed in a culture tube containing 2 m1 of medium 199 at a pH of 7.4. Incuba- tions were carried out in a Dubnoff metabolic shaker as de- scribed under Materials and Methods. After 30 minutes pre- incubation, the medium was removed and replaced with 2 ml of fresh medium 199 containing one of the following amounts of reagents: 0.5, 1.0 or 2.0 pg of estradiol (Nutritional Biochemicals Corp., Cleveland, Ohio); 0.2 or 2.0 pg of ergo- cornine; 0.5 pg estradiol and 0.2 pg ergocornine; 1.0 pg estradiol and 0.2 pg ergocornine; 2.0 pg estradiol and 0.2 pg ergocornine. Each reagent was first dissolved in a small amount of absolute alcohol and incorporated into medium 199 to a concentration of 0.3% alcohol. Six incubation tubes were used for each treatment. The control tubes contained only 2 ml of medium 199 with 0.3% alcohol. After 12-hour 139 incubation, the anterior pituitary halves were removed from the medium, weighed and homogenized. Both the incubation media and pituitary homogenates were kept frozen at -20°C until assayed as described under Materials and Methods. 6. Prolactin assay Prolactin in individual serum samples, pituitary homogenates and incubation media was measured by radioimmuno- assay. Each sample was assayed at 3 different dose levels. The prolactin values were averaged, and expressed in terms of the purified rat prolactin reference standard, H-lO-lO-B. Mean and standard error of the mean for prolactin values in anterior pituitary, serum and incubation media were calculated for each experimental group, and subjected to analysis of variance. Ovarian weights, anterior pituitary weights, number of corpora lutes, and mammary gland growth ratings were similarly analyzed. Duncan's new multiple range test (Duncan, 1955) was used to determine the signifi- cance of differences between experimental groups. C. Results 1. Effects of estradiol benzoate and ergocornine in ovariectomizedirats It can be seen (Table 16, Experiment 1; and Figure 10) that daily injections of 5 (Group 2) or 10 pg estradiol benzoate (Group 4) for five days, beginning two days after 140 ovariectomy of three— to four-month-old rats, produced a 3— to 4-fold increase in pituitary prolactin concentration and a 6- to 8-fold increase in serum prolactin levels as com- pared with the ovariectomized controls (Group 1). A dose of 0.1 mg ergocornine partially counteracted the stimulatory action of estrogen on pituitary and serum prolactin levels (Group 3 and 5), although the latter counteraction was not statistically significant. Each dose of estradiol benzoate increased anterior pituitary weight by about 50%, but injec- tions of ergocornine completely blocked this increase by the estrogen. Estradiol benzoate also signigicantly stimulated mammary growth in the ovariectomized rats, and ergocornine partially blocked this action of estrogen. Beginning on the ninth day after ovariectomy, four- to five-month-old rats were treated as shown in Table 16, Experiment 2. Daily injections for five days of 5 (Group 2) or 10 pg estradiol benzoate (Group 4) significantly increased pituitary and serum prolactin concentrations. Ergocornine partially but significantly counteracted these stimulatory effects of estrogen (Groups 3 and 5). Ergocornine also par- tially inhibited the ability of estrogen to increase anter- ior pituitary weight. The values reported in these rats are not strictly comparable to those in Experiment 1 since these rats were older initially and injections were begun nine in- stead of two days after ovariectomy. 141 .50.0mm.0 0H.500Hw05mH0 ..0:.0 :000 50.m 000.0 v 50 0.0 0.50.00.0550 .50.0mmH0 :.H3 005H0>.0 .0030“ .5005 m0 .o..0 0.0050.0 050 50020 0... .0.00 0..0.0.. 0..0.0... ..0 00 0.0 .000 + 00 0. .00 .0 00.. ...00 0..0...N 00.0.0.0. ..0 00 0. .00 .. 00.0 ...00 0..0.0.. 0..0.0... ..0 00 0.0 .000 + 00 0 .00 .0 00.0 .0.0. 0..0.0.N 0..0.0.0. ..0 00 0 .00 .N 0..0 .0..N 0..0...0 0.0.0...0 ..0 0.0.0000 .. N .505..0mxm 0..0...0 00....0.00. 0N.0...0 0..0.0.. .00 00 ..0 .000 + 00 0. .00 .0 0..0.... 00..N.0..N. 00.0.0.. 00.0.0... .00 00 0. .00 .. 0N.0.. 0 0..0 ...00 0N.0.N.0 00.0.0.. .00 00 ..0 .000 + 00 0 .00 .0 0..0.0.. 00.N..0.00 0N.0.0.0 0..0.0... .00 00 0 .00 .N 0N.0...N 0N.. .0.0. 0..0.0.. 0.0.0.... .00 0.0.0000 .. H .505H.0me mo 0. H0 H5\m5 0< m5\m: 05 ..:mH03 .505.00.H 050.0. :.30.m .5H.u0H0.m ..0500 50.00H 0< .>< .505 .>< 55.00 .>< -0.5 0< .>< .0.0. 000.50.00..0>0 5H :.30.m 0.05505 050 .:mH03 0.0.05.05 .050..0..500500 50.00H0.m 55.00 050 0.0..5.H0 50 mummy 0.050mH50050:.05 05.5.0uom.0 050 nmmv 0.00050: H0H00..00 m0 0.00mmm .0H 0H:00 142 ’ Pituitary Serum C0!" £350. £8509 £81009 £31009 OII um 0.3!»: a. no 0.30. ” 0. Flt/ml Scrum . Figure 10. Effects of Estradiol Benzoate (EB) and Ergocornine Methanesulfonate (ERG) on Serum and Pituitary Prolactin Concentrations in Ovariectomized Rats. 143 2. Effects of estradiol benzoate and ergocornine in hypophysectomized- ovariectomized rats with a single anterior pituitary transplant Beginning on the second day after ovariectomy and pituitary transplantation, the hypophysectomized rats were divided into four groups and treated for five days as shown in Table 17 and Figure 11. The animals were killed 24 hours after the last injection (between 12:00 noon and 1:00 PM) and each pituitary graft was removed from the kidney capsule, weighed, and homogenized in neutral PBS. The pituitary fos- sa of each rat was examined under magnification, and found to be free of pituitary tissue. The data in Table 17 show that control rats (Group 1) bearing one anterior pituitary graft had serum prolactin levels as high as those of rats on the morning of proestrus or estrus (Amenomori g£_al., 1970; Chen 35 al., 1970). Br- gocornine at a dose of 0.1 mg/day (Group 2) markedly de- pressed serum prolactin concentration, decreasing it only to 1/4 of the control values (Group 1). Daily injections of 5 ug estradiol benzoate for five days (Group 3) signifi- cantly increased pituitary and serum prolactin and stimu- lated mammary growth, whereas ergocornine partially counter- acted these effects of estrogen (Group 4). Ergocornine alone partially but significantly decreased the weight of the anterior pituitary graft, but had no effect on pituitary weight in the estrogen-treated hypophysectomized-ovariecto- mized rats. 144 050.00000 5H05000005000 0.0 0050.00.0550 050.00000 0003 005H0> ..0000 0000 50.0 000.0 v 50 U.U.D.N .5005 00 .o..0 0.005000 050 5002. ..0 0.. ..0 0.00 ..0 0.0 0.0 0.. we ..0 0 0 0 0 .000 + 00 0 .00 .0 0..0 0.. U..0 0.00 0..0 0.0 00.0 0.. 00 0 .00 .0 0 o o 0 o o 0 0 we 0 a 0 0H 0 H H 00 0 0 0H 00 0 N 0 00 0 0 m H 0 000 N 0..0 0.. 00.0 ..0. 00.0 ..0 0.0.0 0.. ...0000000 H00 5.00 .H no 00 H0 H5\05 5< 05\05 05 H550.0\000. 00 05000. 0030.0 .50000H0.5 .50000.0500500 .000003 5< 0505000.5 5.0550: .55.00 50000H0.5 5< .050H5050.0 5.0005005 0 50.3 000. 0000500000.0>0-00005000005050550 50 0030.0 5.05505 050 000.03 5.0005005 .050.00.0500500 50000H0.5 55.00 050 5.0005005 50 50000 0005omH500505005 05.5.0000.0 050 Hmmv 00000500 H0000.000 00 0000mmm .0H 0H005 145 Figure 11. Effects of Estradiol Benzoate (EST) and Ergocornine Methanesulfonate (ERG) on Serum and Pituitary Prolactin Concentrations in Hypophysectomized-Ovariectomized, Pituitary- Grafted Rats. 00 Flt/mg AP Gun 3 NO Flt/:l 50"”: 146 CO?" ESTsuo ERIGoJmo E5150: 0" £0" ‘ERGoJmo 147 Microscopic examination of trichrome-stained sec- tions of anterior pituitary tissue from ergocornine-treated rats indicated that this drug reduced the number and granu- lation of acidophils. 3. Effects of daily injections of ergocornine with or without pro actin during_7estrous cycles on the ovaries A total of 10 cycling female rats were each inject- ed intraperitoneally with 0.15 mg of ergocornine per rat at 12:00 noon daily, beginning on the last day of diestrus prior to the expected day of proestrus and continuing for 7 estrous cycles thereafter. Estrous cycles were followed on all female rats by examining vaginal smears daily. At 2:00 PM on the day of proestrus and again at 10:00 AM on the day of estrus during the last cycle, 5 of the 10 rats were each given a single dose of 1 mg prolactin by subcu- taneous injections. A group of 5 control cycling rats were each given daily injections of saline-ethanol solution dur- ing the same period as the above rats. The rats were killed during diestrus of the last cycle. The data in Table 18 show that daily injections of ergocornine significantly increased ovarian weight as com- pared with the controls. On the other hand, 2 injections of prolactin on the days of proestrus and estrus during the last cycle completely prevented any increase in ovarian weight. The number of corpora lutea per ovarian section 148 ..0000 5000 50.0 0m0.0 v 50 050.00000 0H050o.0.50.0 0.0 0050.00.0550 050.00000 £003 005H0> 0.0 .00de 0:“ W0 HORHO @Hmfiuflmum Ufim fidfl—Zt 0E 0.H 5.0o0H0.5 + 0.0.0000.0 0N.000.Nm pmflmom we 00.o 000 000.000..m 00.000.00. 00000N 00 00.0 000 0000.05000 0H0.0Hmn.m 00.000.00 00m000~ H5 0.0 H050£00-050H0m 50.0000 50..0>0\.05 ..05\0E E0 000\0005 0550.0\000. m0 .0005H 0.05.00 .03 50..0>o .03 0000 050E000.0 .000.0>o 50 00Ho00 050.000 0 050.50 5.0o0H0.5 050 00000 000500H50050z005 05.5.0000.0 00 0000000 .mH 0H000 .149 in the ergocornine-treated rats was significantly greater than that in the controls or rats given both ergocornine and prolactin. This appears to be due to retention of the previous as well as the most recent crop of corpora lutea. During the 4-5-week period, rats of all 3 experiment groups exhibited normal estrous cycling of 4-5 days duration. Rats given ergocornine alone or ergocornine and prolactin weighed significantly less than the controls at the end of 7—cycle treatment. 4. In vitro effects of estradiol and er ocornine on_pituitary release 0 prolactin The data in Figure 12 show that estradiol, in doses ranging from 0.5 to 2.0 ug/anterior pituitary half, in- creased pituitary release of prolactin by 30 to 40% without producing changes in pituitary prolactin concentration at the end of 12 hours of incubation. Ergocornine alone (0.2 or 2.0 ug/anterior pituitary half) inhibited pituitary pro- lactin release by about 80%, and, when incubated together with estradiol, it completely blocked estrogen stimulation of prolactin release. It can be seen in every case that in- hibition of prolactin release by ergocornine was accompan- ied by a significant increase in pituitary concentration of prolactin. When compared with the controls, it is apparent that more total prolactin was recovered from the incubation 150 .0.0.>.mw 0000H0m 050 50.00.0500500 50000.0.5 0.0005005 50 5005 .0 00000 H0.00.000 ..ommv 000500H50050500z 05.5.0000.m 00 0000000 .00 0.00.0 151 9305.93.05. 3.005.. 2......000 .... ...00 .3100 .... ...00 .... ...00 9.0.00 .3 mum—0 30.05 :30 .02: I 05:23 a 5...... n” «:1 av Bun/unsound on I ” 152 system when the pituitaries were incubated with estradiol, whereas less total prolactin was recovered when the pitu- itaries were incubated with ergocornine. D. Conclusions The present work demonstrates that ergocornine can inhibit pituitary release of prolactin, and can counteract the stimulatory effect of estrogen on prolactin release. ‘When the pituitary is removed from direct hypothalamic con- 'trol, as by grafting it underneath the kidney capsule (Chen SEE g;., 1970) or by incubating it in vi££g_(Meites g£_§l,, 1961), prolactin release is increased. The results of the jpresent study suggest that ergocornine directly inhibits jprolactin release by the transplanted pituitary and by the pituitary incubated _i_r_1_ \_r_i_1_;_r_o. In ovariectomized rats, injections of estradiol ben- zmzate increase both pituitary and serum prolactin concentra- txions. Ergocornine can partially counteract the stimula- tx>ry action of estrogen on pituitary prolactin release, and can partially or completely prevent the ability of estrogen 'to increase pituitary size. In hypophysectomized, ovariectomized rats with a Single anterior pituitary transplant, ergocornine suppresses and.estradiol benzoate stimulates prolactin release. Ergo- cornine partially counteracts the estrogen stimulation of 'Prolactin release by the pituitary graft. Inhibition by 153 ergocornine of prolactin release by the pituitary transplant is believed to be due mainly to its direct action on the pituitary. In intact, cycling female rats, injections of ergo- cornine for 7 cycles do not interfere with the normal es- trous cycle, indicating that pituitary secretion of FSH and LH was not critically impaired. It is apparent that the in- hibition by ergocornine of luteolysis of corpora lutea is due solely to suppression of prolactin release by this er- got drug, since injections of prolactin on the days of pro- estrus and estrus completely prevented any increase in ovar- ian weight in the ergocornine-treated rats. During a 12-hour incubation period, the presence of estradiol in the medium can stimulate pituitary release of prolactin. Inasmuch as incorporation of ergocornine alone into the incubation medium inhibits prolactin release from the anterior pituitary halves, it can be assumed that its action on pituitary tissue is responsible for its suppres- sion of estrogen stimulation. The data also suggest that ergocornine inhibits prolactin synthesis in_vi££g, since less total prolactin is recovered when ergocornine is pres- ent in the incubation system. 154 VII. Secretion of Prolactin in Rats Bearing A Pituitary Mammotropic *Tumor; Inhibition by Ergot Dru s of Tumor Growth an Prolactin Re1ease A. Objectives We recently reported that ergocornine, an ergot de- rivative, significantly inhibited prolactin secretion by a direct action on the pituitary (Lu 93 al., 1971). We also found that ergocornine could prevent estrogen from increas- ing the size of pituitary and secretion of prolactin in ovariectomized rats and hypophysectomized, ovariectomized rats with a pituitary graft. The Furth pituitary mammo- tropic tumor (MtT.W 15) of rats is known to secrete large amounts of prolactin and GH (Furth, 1961). The present study was undertaken to measure the change in serum prolac- tin levels in relation to growth of transplanted pituitary tumors in rats, and to see whether ergot drugs can inhibit the growth of pituitary tumors and reduce secretion of pro- lactin. B. Materials and Methods 1. Drugs 0 The ergot drugs used were ergocornine methanesul- fonate (Sandoz Ltd., Basle, Switzerland), 2-Br-(a)-ergocryp- tine mesylate (Sandoz Ltd., Basle, Switzerland), ergonovine 155 maleate (Eli Lilly and Co., Indianapolis, Indiana), and Lilly compound-55327 (dl-N-Q, 10-Didehydro-6-methyl-8a- ergolinyl-formamide) (Eli Lilly and Co., Indianapolis, Indi- ana). All drugs were first dissolved in a small amount of absolute alcohol and then incorporated into 0.85% NaCl solu- tion to contain 1% ethanol. 2. Treatment Fifty—day-old inbred female rats of the Wistar-Furth strain were given transplants of Furth pituitary mammotropic tumor (MtT.W 15) subcutaneously in order to induce growth of the tumors as described under Materials and Methods. When the tumors attained a size of 2-3 cm in diameter (7-9 weeks after transplantation), the rats were divided uniformly in- to 5 groups and given local subcutaneous injections, daily, as follows: (a) 0.05 mg of Lilly compound-55327; (b) 0.2 mg of ergocornine for the first week and 0.1 mg of ergocor- nine for the subsequent weeks; (c) 0.2 mg of ergonovine; (d) 0.3 mg of ergocryptine; (e) 0.2 m1 of 1% saline-ethanol so- lution per 100 g of body weight. Individual body weights and tumor diameter were re- corded weekly. Mean tumor diameter (average of the two larg- est diameters) in each rat was measured with calipers (Fisher Scientific Co., Fair Lawn, New Jersey) and blood samples were collected while the animal was under light ether anesthesia. At the end of 3 weeks, rats given daily 156 injections of ergocornine were killed. The pituitary tumors were removed, fixed in Bouin's fluid, sectioned and stained with Masson's trichrome stain for microscopic examination. Seventy five days after transplantation, another group of rats with transplanted MtT.W 15 pituitary tumors were tested to determine the effects of ergot drugs on serum prolactin. The rats were divided into subgroups according to the size of tumors (Tables 19 and 20). After a pre- treatment blood sample was taken and the tumor diameter was measured from each rat at 11:00 AM, a single intraperitoneal injection of Lilly compound-55327 or saline-ethanol solution was given to the rat. Subsequent blood samples were collect- ed at 1, 2 and 3 hours after injection. 3. Prolactin assay Prolactin in individual serum samples was measured by radioimmunoassay. Serum was diluted with neutral phos- phate buffer saline to a l: 10 working concentration for the assay. Each diluted serum sample was assayed at three different dose levels (20-100 pl). Prolactin values were averaged, and expressed in terms of the purified rat prolac- tin reference standard, NIAMD-rat prolactin-RP-l. Mean and standard error of the mean for serum pro- lactin values, mean tumor diameter, and body weight were calculated for each experiment group. Student's "t" test 1157 was used to determine the significance of difference between the control and experimental groups, and between the pre- treatment and post-treatment values as well. C. Results 1. The size of tumor and serum prolactin levels in rats bearing transplanted pituitary tumors Sixty days after transplantation, a group of rats bearing small, palpable, but not measurable pituitary tumors had an average body weight of 180 i 18 each and a mean serum prolactin concentration of 99 i 26 ng/ml. Table 19 shows the correlation between the body weights, the size of tumors, and serum prolactin levels in rats bearing transplanted pituitary tumors 75 days after transplantation. One, 2 or 3 MtT.W 15 tumors were found in each rat. The body weights ranged from 210 to 373 g each, mean tumor diameter from 8 to 98 mm, and prolactin concentra- tions in the serum from 280 to 22,500 ng/ml. In general, rats with larger body size and/or tumor size had higher con- centrations of prolactin in the blood. A similar trend of correlation also was shown in Table 20 (the pre-treatment serum prolactin values) and in Table 21 (the saline-ethanol controls). The number of tumors per rat and the two largest tumor diameters rather than the mean tumor diameter was closely correlated with the prolactin concentrations in the blood. 158 owm w o x OH QHN omm HH OH x NH mHN mwm w A x OH mNN mHm 0H HH x Hm omm mwA NH HH x HH HNN mmo.H AH HH x HN omN mmo.N HH HH x AH NHN mmA.N AN AH x Am ooN mAN.N mm mH x AH Nmm omN.OH mN HN x ON ASN OAN.HH mm NN x HH Hon omN.NH NH mm x Hm Nmm OHo.mH mm HN x NH HHN omN.ON Nm NN x mm OHm pmh\HoE:u 0:0 He\m: ..uqoo SE .pouoEme as x as Em :HuomHopm Eduom 008:0 cmoz «.mhopoEme woese ..0: zwom .cofipmucmHmmnmhu Houmm mxmu mA um mpcmflmwnmhu 005:0.0HmopuoEEmE humuwsuflm wcflhmon mpmp :H mcofiumpucoucou capomaonm 5:000 paw oNHm hoes“ .mpsmHoz zwom .mH oHan 159 . mhmumfimwfi HOESH uw0mhmH oz“ 0:90 omo.HN oom.NN oom.N ooH.N omm.m 000.0 oNH.HH oom.mH ooo.mH omN.oH ON0.0H omA.NH mm Hm mN om ow ow o0 me mm Hm Nm Hm 5N x mm 0 mN mH x NN + HN HH NH mH HH mH mH ON NN mH mH XKXXXXXXKX ow mN mH HN ON ON mN MN HN Hv ON 5N + .+ + -+ +- + + + + + NN wH HH mH mH mN Hm oN «N mN 5N mH XXXXKXXKKX we Hm mvm mmN wmh\mHoE:u 009:5 NN HN Nm mm mv mv NH om mm um ANN OON mwN mom Nmm NHm Nmm mum «on mom umu\mHoE:u ozh 160 2. Effects of a sin 1e injection of Lilly compound—S 327 on serum prolactin levels in ratsbearing transplanted pituitary tumors Table 20 shows that rats bearing MtT.W 15 pituitary tumors of different sizes had wide ranges of prolactin val- ues in the blood (pro-treatment values). Injection of 1% saline—ethanol solution into these rats had no effect on subsequent serum prolactin levels as compared with pre- treatment values. In 4 groups of rats bearing different sizes of tumors, 3 single intraperitoneal injection of Lilly compound-55327 significantly reduced serum prolactin levels by 30 to 60% by l, 2 and 3 hours after injection. In all 4 groups of rats, the per cent inhibition of serum prolactin by 2 or 3 hours was higher than by one hour after injection, indicating sustained inhibition by this ergot drug on pro- lactin release from the pituitary tumors. 3. Effects of daily injections of Lilly compound-55327’onggrpwth of pituitary tumors and secretion of prolactin in rats during 7 weeks Since previous experiments demonstrated that this ergot drug significantly reduced serum prolactin levels in rats bearing transplanted pituitary tumors, another experi- ment was undertaken to determine whether this ergot drug could inhibit the growth of pituitary tumors and suppress the secretion of prolactin by daily injections. Rats bearing 161 .Ho.o v m .000H0> 000E00000-00a 000 0000 000000000 0H00000000M0m0 .mo.o v a .000H0> 000E00000-00m 000 Eo0m 000000000 0H00000000w0m0 .0000 000 0o 00000 00000000 000 00020 0H0 HmAH 00H HoNH nOH “mmH 00 HmHm N N “OH 00H 0000 UOOH 000H.H ommN 000N.H omH HOH0.N N H 000 00H0.H0A0o.0 0000.HHNH0.0 nAoo HOHA.0 OHH.HHoNN.NH 0 0 “om omoo HO0N.NH 0000.HHOHA.0H uwHH.HfiomN.HH 000 HA0N.HN 0 mHHAo 0; om .ANmmm -000omEoo AHH00 00o.H00Hm.H mom Hoom.H 0H0 00H0.H O00 0000.H N N “0H O00.Nfimmo.0 O00.Nfloom.0 mNo.N0oo0.m OON.N00HO.0 N 0 000 HmHo0gcooV Ha N.0 000 000A.HH 000 000N.0H com “coo.HH 000 HOOH.HH N mH 00H .Hoamapo-mc0H00 00cc: m 000cc N 0000 H 000E00000-00m 0000 00 03m m OOH\00o0V mo .oz .00000000 000000009 H0\m0 .00o000000000ou 00000Ho00 0000m 0oes0 000: .0N00 000000000 0o 00o000 000000000 00000H000000 m00000p 0000 00 0H0>0H 000o0Ho00 00000 0o 00000 0ow00 0o 0o0000000 0Hm000 0 0o 000000m .om 0H000 162 transplanted pituitary tumors of 2-3 cm in diameter were given Lilly compound-55327 or saline-ethanol solution con- tinuously for 7 weeks. Table 21 and Figure 13 show that the initial body weights, mean tumor diameters and serum prolactin concentra— tions between the control and experimental groups were not statistically different. It is apparent that the control rats continued to gain body weights, and showed increases in size of tumors and serum levels of prolactin. During a period of 7 weeks, the body weights, mean tumor diameters and serum prolactin concentrations were increased about 2-, 3-, and 6-fold respectively in the controls. Daily injec- tions of Lilly compound-55327 produced inhibition of tumor growth and prolactin release. The mean tumor diameters of the ergot-treated rats were significantly less than in the controls by 6 and 7 weeks after injections, and by one week post-treatment. The serum prolactin concentrations in rats given the ergot drug were lower than in the controls during most of the 7 weeks, although the values were not statisti- cally different by 5 and 7 weeks after injections because of the large standard errors. At the dose given, this ergot drug had no effect on body weight gains as compared with the saline-ethanol controls. 163 Table 21. Effects of subcutaneous injections of Lilly compound-55327 on body weights, tumor growth and serum prolactin levels in rats bearing transplanted pituitary tumors. Weeks of treatmentr Treatment (7 rats/group) Pre-treatment 1 2 3 BodyweightL g Saline-ethanol 228:10a 263: 6 294:12 321:9 (controls) Lilly compound 234i 9 262:11 282: 7 30016 -55327 Mean tumor diameter, mm Saline-ethanol 23.9:3.S 31.0:4.2 37.5:5.S 46.1:7.S (controls) Lilly compound 21.5:4.6 27.5:4.3 33.1:4.7 40.0:S.2 ~55327 Serum prolactin concentrations, ng/ml Saline-ethanol 1,831i533 ---- 3,176:688 4,2251802 (controls) I b b Lilly compound 1,038i476 ---- 1,462i463 2,143i564 -55327 3Mean and standard error of the mean. bSignificantly different from the controls, p < 0.05. Note: Dose of drug used: 1% saline-ethanol 0.2 ml, or Lilly compound-55327 50 pg per 100 g body weight. 164 Weeks of treatment One week 4 5 6 7 post-treatment 349:12 369:22 394:9 416:13 435:11 322: 3 34s: 9 361:7 3840-7 414: 9 55.5:4.1 63.4:5.l 72.5:8.3 81.5:4.6 91.0:7.6 44.8:8.5 49.2:s.9 52.0.3.1b 56.5:3.2b 62.5:6.4b 6,764i1,241 7,544¢1,138 8,910:1,017 11,231:1,917 13,541:1,987 5,371: 770 6,327: 861b 7,853:1,378 9,749:1,782 3,595: 931 165 8 MEAN Control rum mmnen l2 VVooks Figure 13. Effects of Lilly Compound-55327 (EGL) on the Growth of MtT. W 15 Pituitary Tumors and Serum Prolactin in Rats. 166 4. Effects of daily_injections of er ot drugs on the growth of pituitary tumors during_§ weéks Figure 14 shows that injections of ergocornine sig- nificantly reduced the mean tumor diameter, whereas in the control group the pituitary tumors continued to increase in size. The mean tumor diameter was reduced by 15% in rats given ergocornine; in control rats there was a 58% gain in the size of tumors. Ergocornine treatment was terminated at the end of 3 weeks. Microscopic examination revealed that the transplanted pituitary tumor from control rats con- sisted of numerous cells of different size with prominent nuclei and many mitotic figures (Figure 15, A). Tumors from rats treated with ergocornine consisted of relatively few, separated, large cells with absent or pycnotic nuclei (Fig- ure 15, B). At the dose given, ergocryptine produced a small but insignificant inhibition of pituitary tumor growth by the end of 5 weeks treatment. Ergonovine significantly but not completely inhibited tumor growth. There was a 78% increase in mean tumor diameter in the controls, but only a 28% gain in tumor size in rats treated with ergonovine. At the doses given, none of the 3 ergot drugs had any significant effect on body weight, although necrosis of the tail was observed in every rat injected with ergocornine or ergonovine. 167 .00000o0om00 n omm ”000>o0ow00 u >zm M000090003900 u mum .0000 00 00o000 000000000 mH 3 B02 mo 003000 000 00 0ws0m 0om0m 00000 0o 000000m .HH 000000 16'8 mmhmifio 102:... Z00 000 00H000 000000000 0003 0000 0000000 00 0HH00 000 -3000 000 H000000 0 0000 00000 000000000 00000H000000 0 0000 000000m m mm 000000 00000 000000000 000 00 000 000000000 000 000000 00 0000000 000000000 00 :00 000000000 00 0000000 .NN 00000 ‘178 2. In vitro effects of TRH on prolactin release by hypothyroid rat pituitary Anterior pituitaries were removed from male rats 5 weeks after thyroprarthyroidectomy. Each AP half was incu- bated in medium 199 as above. The data in Table 23 show that AP halves from hypothyroid rats released only about 50% as much prolactin as released by AP halves from normal rats (see Table 22, Experiment 1). Addition of 3 or 30 ng of TRH to the incubation medium increased prolactin release by about 30% by the AP halves from thryo-parathyroidectom- ized rats after 4 hours of incubation, but had no effect on prolactin release by the end of 8 or 12 hours of incubation. It was also found that the weights of AP halves from thyro- parathyroidectomized rats were significantly higher than the AP halves from intact rats (4.92 i 0.15 vs. 3.55 t 0.19 mg/AP half). It will be seen in subsequent experiments that thyro- parathyroidectomy results in significant reduction in pitu- itary concentration of prolactin (see Tables 25 and 26). 3. Effects of a single intravenous iniEction of TRH on serum pro- lactin levels in intact male rats After a pre-treatment blood sample was collected at 11 AM, a single injection of TRH was given to normal male rats by the tail vein. Subsequent blood samples were re- moved 15 and 30 minutes after TRH administration. The data in Table 24 (Experiment 1) show that TRH did not alter serum prolactin levels. 179 .mo.o v m .Houpnou ma“ Eopm pamhmmmflw zapnmuflquMan ...:me 06:. W0 .HOHHO Uhmwfimum USN GNOZN hfifimvm mm: omv mme mmfimmm VMHmom n hvflomm mmfimnq nwfififlvm awn my axe «mamvm mvflomv mNNumnm Houpnou nm< ms\vommoaoh nwuumHOHm may mammflp thpHSHfim “my wfionxgpomxn guwz mmh mo :ofipmnsunH NH w v anachw\mon=u 0V unoEpmmne away vofihmg nowumnsunH .ouufi> mm zhmpfiSpwm pan vacuzauomxg kn ommofioh :Huumaopm :o mmh ufipmgpazm mo muuomwm .mm magma 180 .zmm H qaoZm wfimm came BHHQ am: my :me cofiumasczmu youmm h: «N um cofimsmcH “NV o.oan.o 5.0Hm.w u.oam.w am: my age :oflpmanccmu Hmumm :fie om pm :oflmzqu flaw wasnnmu vapoumu mfl> mme mo nowmzmnH “N .mxm BHNN ¢Hm~ QHmN mm: m.hv may OHNN mHNN «mflmm has m.oV onflflmm awo> Hausmu mw> axe mo cowpumnnH "H .mxm nae om :fiE ma acoEummhu-opm AmnOHM\mumh 0V unmaumohe fiHE\w:V mHo>oH :fluumaopn Enhmm .mHo>0H :wuumHOHm Enhmm no mme ufiumspcxm mo :oHuumncfi >H ofimcfim m mo muoommm .vN oflnme 181 In Experiment 2, rats with carotid cannulae were in- fused with a single dose of TRH. A pre-infusion blood sam- ple was collected 30 minutes after cannulation under sodium pentobarbital anesthesia, and 5 ug of TRH in 0.3 m1 of sa- line followed by 0.2 ml of saline rinse were infused into each rat via the cannula. Subsequent blood samples were taken at 15 and 30 minutes after TRH infusion. The data in Experiment 2 show that sodium pentobarbital depressed serum prolactin levels as reported previously by our laboratory (Nuttke and Meites, 1970), and that infusion of 5 pg of TRH dfireCtly into a carotid artery did not change serum prolac- éin values. Twenty-four hours after cannulation, serum pro- lactin was elevated to levels significantly higher than in- intact controls. This increase presumably is due to the ef- fect of sodium pentobarbital (Wuttke g£_al., 1971) and the stress from chronic cannulation (Neill, 1972). Infusion of TRH did not alter prolactin values. 4. Effects of multi-injections of TRH on pituitary prolactin release in intact and thyro-parathyroidectomized male rats Since the foregoing experiments provided no evidence for a direct stimulatory action by TRH on pituitary prolac- tin release, another eXperiment was undertaken to determine whether TRH could increase pituitary prolactin secretion by stimulation of the TRH-thyroid system. Intact and thyro- parathyroidectomized male rats were injected via the caudal 182 vein with saline or 50 ug TRH daily for 6 days. On the 7th day blood samples were collected and the rats were killed. The pituitaries, thyroids and adrenals were removed and weighed. The data in Table 25 and Figure 16 show that thyro- parathyroidectomy resulted in a significant reduction in pi- tuitary prolactin concentration and in a small but insignif- icant fall in serum prolactin. Injections of TRH into in- tact rats produced a significant increase in pituitary and serum prolactin concentration as compared to thyro-parathy- roidectomized rats treated with or without TRH. Injections of TRH into intact rats also resulted in a significant in- crease in pituitary prolactin and produced a small but in- significant elevation in serum prolactin values as compared to intact control rats. TRH injections or thyro-parathy- roidectomy had no significant effect on the weights of the pituitary, adrenals or thyroid under these experimental con- ditions. 5. Effects of multi-injections of TSH in thyrogparathyroidectomized male rats on_pituitary prolactin rélease Since injections of TSH were reported to deplete hypothalamic TRH content in thyro-parathyroidectomized rats (Motta, 1970), the effects of multi—injections of TSH on pi- tuitary and serum prolactin concentrations were measured in thyro-parathyroidectomized rats. Beginning on the 15th day after thyro-parathyroidectomy, half of the rats were given 183 .Ho.o v a .mw: om may .pumncflv 0 Echo peoumcmfie sHocmuflmfiemHmu .mo.o v m .mmHOMpcou mafiamm .pumu:HV < Eopm pqouommfiw prchMmficmHm a 2mm H cwozm umHoH UNH.onma.o n.ono.OH mm: omv may .pomflEouuowflohchmummouxce .Q eHHm ano.onfiv.fi m.on~.m mm: cmv map .uumueH .u «HBH nafl.owfla.o EH.OHN.OH “He m.ov mafifimm .won“Eeyoopfiohxcpmummouxgh .m mnem oo.oan.H mm.cnfi.m nmfiohpeouv has m.oU ocwflmm .uomuqH .< fine\weo na< ms\mnc Ame ..pzc flamv\.mouc .axm awuomaohm .cwuomaohm .uflm .p:< AQSOHM\mumh ow anhom thuflSHflm ucoEamoph Eupom pom .cofipmhucoocoo :fluumaohm xpmHMSpflm :o Ema vaponpnxm mo mnofiuuomcw >w mo muuommm .mN manme (15 30 IO 184 P“Mkwmekxfin Serum Prolactin Intact flwroidx Intact Thyroid X + TRH + TRH Figure 16. Effects of Multi-Injections of TRH on Pituitary and Serum Prolactin Concentrations in Intact and Thyro-Parathyroidectomized Male Rats. 185 a daily subcutaneous injection of 0.5 IU TSH/100 g body weight for 14 days, and the other half were given physiolog- ical saline. A group of intact male rats was given saline injections and served as controls. The rats were killed by guillotine at the end of 2 weeks treatment, and blood sam- ples were collected from the trunk. Anterior pituitaries were removed, weighed, homogenized, and later assayed for prolactin. The data in Table 26 show that thyro-parathy- roidectomy resulted in a significant increase in anterior pituitary weight and in a marked reduction in pituitary pro- lactin concentration when compared with the intact controls. Serum prolactin concentrations were not significantly re- duced in the thyro-parathyroidectomized as compared to the control rats. TSH injections in thyro-parathyroidectomized rats had no effect on pituitary or serum prolactin values. D. Conclusions The in_vit£g_results in the present study provide no evidence that synthetic TRH directly stimulates prolac- tin release from the normal male rat pituitary or from rat pituitary tumor tissue. However, it was observed that ad- dition of TRH to an incubation medium produced a 30% in- crease in prolactin release by pituitary halves from thyro- parathyroidectomized rats. An increase in prolactin and a decrease in GH release was observed by Tashjian gt il- (1971) when TRH was added to a culture or incubation system 186 .Hoo.o v a .mHOHucoo on“ Eopm pcohommwp xfiucmoflmwawflmu .Ho.o v a .maoapcoo map Eoum ucowoMMHu xfiucmofimwcmflm a 2mm H cams“ mHVN omo.onflm.o am.onm.OH 3n m OOH\:H m.o amp .eoNSgonuoefiossgpmnmmousge mHVN uNo.onw~.o pm.onm.OH He m.o maflfiwm .emufisoouoefios»;umumaopsge wnmm so.onem.o mm.OHN.w He m.o nmfiopueouv oeflfimm .numch mHE\mcv mm< mE\m:V awe .pzv xmw\omoa masoHM\mumH 0V .qmm .gmm .pflm .pfid .pa< oaosumoue Eshom .mqowwmhucoucoo :fluumaoum ezhom can zhmpwspfim paw unwwoz thuflSuwm Hoflhoucm co kaouoopwouxnumhmm-09%:p H0\vcm :ofluuonnw mmh mo mpuommm .om manna .187 containing clonal cells from rat pituitary tumor. It is possible therefore, that individual pituitary cells as well as the pituitary of hypothyroid rats may react somewhat dif- ferently than normal rat pituitary tissue to synthetic TRH. The physiological significance of these observations in the rat is difficult to assess. The in_vivg_results show that multi-injections of TRH into intact rats increased pituitary prolactin concen- tration significantly and produced a small but insignificant rise in serum prolactin values. TRH had no effect on pitu- itary or serum prolactin levels in thyro-parathyroidectomized rats. This suggests that TRH acted via the TSH-thyroid sys- tem to increase pituitary and serum prolactin values in the rat. The observation that multi-injections of TSH had no ef- fect on prolactin release in thyro-parathyroidectomized rats, suggests that its reported ability to deplete hypothalamic TRH content in thyro-parathyroidectomized rats (Motta, 1970) did not alter prolactin release. It appears therefore, that neither an increase from exogenously administered TRH nor a decrease in endogenous TRH in thyro-parathyroidectomized rats can influence prolactin release. A single injection or infusion of TRH into intact rats also failed to alter serum prolactin value by 15 or 30 minutes after injection. Multi- injections of TRH were necessary to produce even the limited increase in pituitary prolactin concentration observed in 188 this study. These results indicate that synthetic TRH is not a specific releaser of prolactin in the rat (Lu gt_al,, 1972). Thyro-parathyroidectomy resulted in significant in- crease in pituitary weight and a marked reduction in pitue itary prolactin concentration. Inasmuch as serum prolactin levels were not significantly lowered by thyro-parathyroid- ectomy, it appears that pituitary synthesis was decreased to a greater extent than release of prolactin, or that the me- tabolism of circulating prolactin was so reduced by thyro- parathyroidectomy that it remained longer in the circulation. GENERAL DISCUSSION The data presented in this thesis indicate that pituitary release of prolactin is under dual control by the hypothalamus, and also can be influenced by systems outside the hypothalamus. There is little doubt that pituitary pro- lactin release is mainly under the control of PIF, but probably is also influenced by the PRF in the hypothalamus. Hypothalamic catecholamines depress whereas serotonin stimu- lates prolactin release. The catecholamines promote PIF release and serotonin may induce PRF release. Several drugs and hormones also can influence prolactin release by a direct action on the pituitary. Thus, it was found that estrogen stimulated, and ergocornine (ERG) inhibited prolactin release by a direct action on the incubated rat pituitary 12.Xi££2' As a result of ERG inhibition of prolactin release, accumu- lation of prolactin within the pituitary tissue was observed. Estrogen increased both pituitary and serum prolac- tin levels in_vivg, indicating stimulation of both synthesis and release of pituitary prolactin. Estrogen was shown to increase the number and granulation of pituitary acidOphils (the putative prolactin cells) (Gersten and Baker, 1970). Ovariectomy resulted in a significant decrease in both pitui- tary and serum prolactin values, presumably due to a marked 189 190 acceleration of catecholamine turnover rate and increased PIF release as a result of lack of estrogen. Ovariectomy leads to a decreased sensitivity of the hypothalamo-pituitary system in response to changes in hypothalamic catecholamine activity. Thus, pituitary release of prolactin in ovariec- tomized rats is stimulated less by central acting drugs which decrease catecholamine levels in the hypothalamus than in intact female rats. Estrogen decreases PIF activity in the hypothalamus (Ratner and Meites, 1965). These results suggest that estrogen stimulates prolactin secretion by acting both on the pituitary and via the hypothalamus. When ERG was administered in viva, it counteracted the stimulatory action of estrogen on prolactin secretion, and inhibited prolactin release by a direct action on the pituitary graft underneath the kidney capsule in hypOphy- sectomized rats. When ergot drugs, particularly ERG, were given to rats bearing pituitary "mammotropic" tumor trans- plants, they produced a rapid decrease in serum prolactin and induced tumor regression. There was loss of nuclei and disappearance of cells in the pituitary tumor tissue after ERG treatment, suggesting that ERG acted directly on the pituitary tumor tissue to reduce its capacity to secrete prolactin. ERG also was shown to reduce the number and granulation of acidOphils within the pituitary graft in hypothysectomized rats with a single pituitary transplant. It was reported that ERG increased hypothalamic PIF activity 191 (Wuttke £3 31., 1971), suggesting that ERG inhibits prolactin release by acting both on the pituitary directly and via the hypothalamus. Inasmuch as ERG inhibits prolactin release from incubated pituitary tissue in vitro, from transplanted pituitary and from pituitary tumor iE.XiXE’ it can be assumed that inhibition of prolactin release by ERG is mainly due to its direct action on the pituitary. The inhibition by ERG of estrogen stimulation of pituitary prolactin release does not necessarily indicate that both agents act on the same pituitary sites. Since ERG produced a 80% inhibition of prolactin release by incubated pituitary in 11313 as compared to control pitui- tary, and resulted in accumulation of prolactin within the pituitary tissue, this strongly suggests that the major action of ERG is on the cell membrane to block the normal release of prolactin. Since ergot drugs are well known for their ability to induce vasoconstriction of peripheral blood vessels, they may similarly inhibit the blood supply to the pituitary prolactin cells. It was first shown by Nagasawa and Meites (1970) that ERG inhibited mammary tumor growth in the rat. Microscopic examinations revealed that ERG prevented the extensive proliferation of epithelial tissue usually seen in mammary tumor tissue (Lu and Meites, unpublished). ERG has been shown to be a useful agent for specific inhibition of prolactin release, since at the doese used they inhibited 192 only prolactin but not gonadotrOpins. Thus, daily injections of ERG into cycling female rats increased ovarian weight and number of corpora lutea by partially or completely preventing luteolysis of old corpora lutea (Wuttke and Meites, 1971). However, ERG did not interfer with normal estrous cycles during 5 weeks of treatment. This thesis presents additional evidence that bio- genic amines, particularly catecholamines and indoleamines, in the hypothalamus and possibly the pineal play important roles in the control of pituitary prolactin release. It long has been debated whether catecholamines have any effect on pituitary secretion of prolactin and other hormones. A survey of earlier literature indicates that inconsistent results and contradictory conclusions were reported. Insofar as prolactin is concerned, these discrepancies could be due to the different pharmacological effect of catechola- mines and their rapid metabolism in X112 and in vitro, insensitivity of pigeon crop bioassay for prolactin, inabil- ity to measure biogenic amines in the hypothalamus, etc. As shown in this thesis, pituitary prolactin concentration and serum prolactin values often go in Opposite directions shortly after stimulation of prolactin release. Measures of pituitary prolactin levels only frequently led to the wrong conclusions. Prolactin concentration in the systemic blood seems to represent the most reliable index of pituitary prolactin release. Previously, however, it was impossible 193 to detect changes in prolactin in the blood until a Specific radioimmunoassay for rat prolactin was developed. Evidence also was presented here that the duration of prolactin release or inhibition of release depends on the nature of the drug and the route of drug administration. Altered serum prolactin levels may return to normal within a few hours following drug injection. Unless sequential blood samples are measured by radioimmunoassay, investigators can easily miss the optimal inhibition of prolactin release by drugs. Erroneous conclusions also were drawn from testing the effects of catecholamines in vitro. The in vitro effects of catecholamines on pituitary prolactin release appear to be of doubtful physiologic significance, since high doses inhibited and small doses stimulated prolactin release. These amines also are rapidly degraded to form compounds which can exert powerful pharmacological action on cell membrane metabolism. Conclusions from in XiXE experiments also may be invalid, since systemically administered cate- cholamines do not pass through the "blood-brain barrier" in amounts sufficient to reach the hypothalamus. Only pre- cursors of catecholamines (tyrosine or L-DOPA) or monoamine oxidase inhibitors (i.e. pargyline, iproniazid, etc.) can reach the brain. A single injection of L-DOPA, the immediate precur- sor of dopamine, evoked a rapid decrease in serum prolactin and an increase in pituitary prolactin concentration, 194 indicating inhibition of pituitary release of prolactin. Injections of three monoamine oxidase (MAO) inhibitors, pargyline, iproniazid, and Lilly compound-15641, also sig- nificantly reduced serum prolactin vlaues. It is signifi- cant that inhibition of prolactin release by L-DOPA and by the MAO inhibitors was associated with increased PIF activity in the hypothalamus. Kamberi 33 El. (1970) demonstrated that a single injection of dopamine into the third ventricle of rats increased PIF activity in the pituitary portal blood, whereas direct infusion of catecholamines into a single portal vessel had no effect on pituitary release of prolac- tin. From these and other related studies, the concept evolved that hypothalamic catecholamines inhibit pituitary prolactin release by increasing synthesis and release of PIF by the hypothalamus. In view of the finding that L-DOPA inhibited mammary tumor growth in the rat (Quadri and Meites, unpublished), and induced cessation of persistent lactation and caused resumption of menstral cycles in patients with Forbes- Albright syndrome (Turkington, 1971), it can be assumed that L-DOPA acts mainly via a central mechanism to inhibit prolactin and stimulate gonadotrOpin release by the pitui- tary. Evidence is presented in this thesis that a single injection of methyl-DOPA, d-amphetamine, reserpine, chlor- promazine, and tyrosine analogs, drugs known to reduce hypothalamic catecholamine levels, evoked a rapid release 195 of prolactin by the pituitary. Other workers have reported that reserpine (Ratner and Meites, 1965) and perphenazine (Danon 33 51., 1963), a compound related to chlorpromazine, decreased hypothalamic PIF activity. It seems probable that methyl-DOPA, d-amphetamine and the two tyrosine analogs used here can reduce PIF activity in the hypothalamus. It is noteworthy that hypothalamic catecholamines inhibit prolactin but stimulate gonadotropin release. Prolactin secretion often goes in an opposite direction to gonadotropin secretion under many physiological states. Prolactin is relatively high during late pregnancy, in post- partum lactation, and in old rats, whereas LH and FSH are relatively low during these conditions. In cycling female rats, prolactin and gonadotropins reach a peak at approxi- mately the same time, on the late afternoon of proestrus (Wuttke and Meites, 1970; Gay 3: 31., 1970). It remains to be determined, however, to what extent hypothalamic cate- cholamines participate in the control of these hormonal peaks, since catecholamines are stimulatory to pituitary release of gonadotrOpins but inhibitory to prolactin release. It is possible that the direct stimulatory action of estro- gen on the pituitary may override catecholamine inhibition of prolactin release, while catecholamines stimulate the release of gonadotrOpins. Another possible explanation is that the rise of these three hormones is the result of direct stimulation by estrogen on the pituitary without 196 participation of catecholamines. Estradiol has been shown to directly induce LH release by the rat pituitary in_zi££g (Piacsek and Meites, 1966). It would be interesting to see whether an injection of L-DOPA prior to the time of the pro- lactin surge on the late afternoon of proestrus can reduce or prevent the prolactin rise. There is also the possibil- ity that serotonin may participate in this process. This thesis presents evidence that serotonin and its product, metatonin, have stimulatory effects on pitui- tary release of prolactin. A single intraperitoneal injec- tion of S-hydroxytryptophan, the immediate precursor of serotonin, evoked a rapid and profound elevation in serum prolactin in cycling female rats. S-HydroxytryptOphan also increased prolactin release in hyp0physectomized, pituitary- grafted rats. Serotonin given by systemic injection was ineffective on prolactin release. Kamberi 32 21° (1971a) reported that a single injection of serotonin or melatonin into the third ventricle of rats increased prolactin in the blood, but they did not measure changes in PIF activity in the pituitary portal blood. This thesis presents evidence that injection of S-hydroxytryptophan does not change PIF nor induce PRF activity in the hypothalamus or in the sys- temic blood by our standard in vitro method. It is possible, however, that this in_vi£33_method may not be adequate to detect the appearance of the presumed PRF in the hypothalamus and its release into the systemic circulation. It would be 197 of interest to see whether the serum from these drug injected rats could increase prolactin release in vivo. Results presented in this thesis provide no proof that synthetic thyrotrOpin-releasing hormone (TRH) is a specific releaser for prolactin in the rat. TRH injection has no effect on serum prolactin in intact and thyro- parathyroidectomized male rats. TRH increased prolactin concentration in the pituitary when it was injected daily in relatively high dose for 6 days into intact male rats. This apparent stimulation of prolactin secretion by TRH was due to activation of the TSH-thyroid system, since no effect was observed in thyro-parathyroidectomized rats. TRH did not increase prolactin release when incubated with normal rat pituitary or rat pituitary tumor tissue in vitgg. TRH slightly increased prolactin release by about 30% by pitui- tary tissue from long-term hypothyroid rats. The relation of these observations in the rat to reports that a single injection of TRH can produce a rapid rise in blood prolactin concentration in humans (Bowers et 31,, 1971; Jacobs 33 31., 1971), monkeys (Knobil, personal communication), and cattle (Convey gt al., 1972) is not clear. In view of the rapidity with which an injection of TRH elevates blood prolactin levels in these mammalian species, and the evidence that TRH is even more effective in raising blood prolactin in hypothyroid than in euthyroid humans, it appears doubtful that TRH acts via the TSH-thyroid system to elevate blood 198 prolactin in human subjects. It long has been known that the rat is relatively hyperthyroid as compared to other species (Meites, 1949, 1950), and thyroid hormones can increase pituitary prolactin levels in the rat. The observa- tions that hypothyroid human subjects are more responsive to TRH to increase prolactin than hyperthroid humans may be analogous to what occurs in the relatively hyperthyroid rat. Thus, the failure of TRH to stimulate prolactin release by the rat pituitary may be due to its relative hyperthyroid state. It remains to be determined how TRH evokes a rapid rise in blood prolactin in primates and in cattle. Synthetic TRH may be related chemically to the presumed PRF molecule. The relation of biogenic monoamines, ergot drugs, and estrogen to pituitary release of prolactin can be sum- marized in a diagram as shown in Figure 17. 199 HYPOTHALAMUS CA Stimulators: L-DOPA MAO Inhibitors + , ~ ———a- CA Depressors: ' Reserpine : Chlorpromazine Catechol- Indole- {m i ...... 5-°“'§:§::232:“} Tyrosine Analogs - . 1H1;- L- .......... L ......... .a‘. Estrogen _ ' ———> ' +? I 9 Ergocornine + I PIF ' PRF . TRH I I r + +? PIF PRF Portal Vessels V Anterior V Ergocornine — Pituitary I+ Estrogen Systemic Blood + PROLACTIN \ Figure 17. Diagram Showing the Relation of Biogenic Monoamines, Ergot Drugs, and Estrogen to Pituitary Prolactin Release in Rats. LIST OF REFERENCES LIST OF REFERENCES Adams, J. H., P. M. Daniel and M. M. L. Prichard. 1965. Observations on the portal circulation of the pitu- itary gland. Neuroendocrinology, 1:193-213. Amenomori, Y. and J. Meites. 1970. Effect of a hypotha- lamic extract on serum prolactin levels during the estrous cycle and lactation. Pro. Soc. Exp. Biol. M39: 134:492-495. Amenomori, Y., C. L. Chen and J. Meites. 1970. Serum pro- lactin levels in rats during different reproductive states. Endocrinology, 86:506-510. Anden, N. E., A. Dahlstrom, K. Fuxe and K. Larsson. 1965. Mapping out of catecholamine and SHT neurons inner- vating the telencephalon and diencephalon. Life Sci. 5:1275-1279. Anden, N. E., A. Carlsson, A. Dahlstrom, K. Fuxe, N. A. Hillarp and K. Larsson. 1964. Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sci. 3:523-530. Anton, A. H. and D. F. Sayre. 1964. The distribution of dopamine and DOPA in various animals, and a method of their determination in diverse biological mater- ial. J. Pharmacol. Exp. Ther. 145:326-336. Anton-Tay, F. and R. J. Wurtman. 1968. Norepinephrine: turnover in rat brains after gonadectomy. Science. 159:1245. Anton-Tay, F. and R. J. Wurtman. 1971. Brain monoamines and endocrine function. Ip_Frontiers in Neuroendo- crinology, 1971, edited by L. Martini and W. F. Ganong, 45-66. Oxford University Press, New York. Anton-Tay, F., S. M. Anton and R. J. Wurtman. 1970. Mech- anism of changes in brain norepinephrine metabolism after ovariectomy. Neuroendocrinology, 6:265-273. 200 201 Anton-Tay, F., R. W. Pelham and R. J. Wurtman. 1969. In- creased turnover of H3-norepinephrine in rat brain following castration or treatment with ovine follicle- stimulating hormone. Endocrinology, 84:1489-1492. Asano, Y. 1971. The maturation of the circadian rhythm of brain norepinephrine and serotonin in the rat. Life Sci. 10:883-894. Averill, R. L. W. 1969. Failure of luteotrophic function due to pituitary grafts in the rat hypothalamus. Neuroendocrinolggy, 5:121-131. Bargmann, W. and E. Scharrer. 1951. The site of origin of the hormones of the posterior pituitary. Am. Scien- tist. 39:255. Barraclough, C. A. and C. H. Sawyer. 1957. Blockade of the release of pituitary ovulating hormone in the rat by chlorpromazine and reserpine: possible mechanisms of action. Endocrinology, 61:341-351. Benson, G. K. and S. J. Folley. 1956. Oxytocin as stimu- lator for the release of prolactin from the anter- ior pituitary. Nature. 117:700. Birge, C. A., L. S. Jacobs, C. T. Hammer and W. H. Daughaday. 1970. Catecholamine inhibition of prolactin secre- tion by isolated rat adenohypophyses. Endocrinol- ogy, 86:120—130. Bjorklund, A., B. Falck, F. Hromek, C. mean and K. A. West. 1970. Identification and terminal distribution of the tubero-hypophyseal monoamine fibre systems in the rat by means of stereotaxic and microspectro- fluorometric techniques. Brain Res. 17:1-24. Bowers, C. Y. 1971. Studies on the role of cyclic AMP in the release of anterior pituitary hormones. Annals New York Acad. Sci. 185:263-290. Bowers, C. Y., H. G. Friesen, P. Hwang, H. J. Guyda and K. Folkers. 1971. Prolactin and thyrotropin release in man by synthetic pyroglutamyl-histidyl- prolinamide. Biochem. Biophys. Res. Commun. 45: 1033-1041. Bowers, C. Y., A. V. Schally, F. Enzmann, J. Boler and K. Folkers. 1970. Porcine thyrotropin releasing fac— tor is (Pyro) Glu-His-Pro (NHZ). Endocrinology. 86:1143-1153. 202 Brodie, B. B., S. Spector and P. A. Shore. 1959. Interac- tion of drugs with norepinephrine in the brain. Pharmacol. Rev. 11:548-864. Bryant, G. D. and F. C. Greenwood. 1972. The concentra- tions of human prolactin in plasma measured by ra- dioimmunoassay: experimental and physiological mod- ifications. In Ciba Foundation Symposium: Lacto- genic Hormones: edited by G. E. W. Wolstenholme and J. Knight. 197-206. Churchill Livingstone, Edinburgh and London. Burgus, R., T. F. Dunn, D. Desiderio and R. Guillemin. 1969. Structure moleculaire du facteru hypothalamique hy- pophysiotrope TRF d'origine ovine: mise en evidence par spectrometric de masse de la sequence PCA-His- Pro-NHZ. Compt. Rend. Acad. Sci. (Paris) 269:1870- 18 3. Burgus, R., M. Butcher, N. Ling, M. Monahan, J. Rivier, R. Fellows, M. Amoss, R. Blackwell, W. Vale and R. Guillemin. 1972. Primary structure of the ovine hypothalamic luteinizing hormone-releasing factor (LRF). Proc. Nat. Acad. Sci. U.S. 69:278. Carlsen, R. A., G. H. Zeilmaker and M. C. Shelesnyak. 1961. Termination of early pregnancy in the mouse by sin- gle injection of ergocornine methanesulfonate. J; Reprod. Fertil. 2:369-373. Chen, C. L. 1969. Effects of hypothalamic extract, pitu— itary hormones and ovarian hormones on pituitary prolactin secretion. Ph.D. Dissertation, Michigan State University. Chen, C. L. and J. Meites. 1969. Effects of thyroxine and thiouracil on hypothalamic PIF and pituitary prolac- tin levels. Proc. Soc. Exp. Biol. Med. 131:576- 578. Chen, C. L. and J. Meites. 1970. Effects of estrogen and progesterone on serum and pituitary prolactin lev- els in ovariectomized rats. Endocrinology. 86: 503-505. Chen, C. L., S. L. King, M. L. Pattison and M. R. Fedde. 1972. Locations of hypothalamic area controlling prolactin release. Fed. Proc. 31:211. Chen, C. L., H. Minaguchi and J. Meites. 1967. Effects of transplanted pituitary tumors on host pituitary pro— lactin secretion. Proc. Soc. Exp. Biol. Med. 126: 317-320. 203 Chen, C. L., J. L. Voogt and J. Meites. 1968. Effect of median eminence implants of prolactin, LH and FSH on luteal function in the rat. Endocrinology, 83: 1273-1276. Chen, C. L., E. J. Bixler, A. I. Weber and J. Meites. 1968. Hypothalamic stimulation of prolactin release from the pituitary of turkey hens and poults. Gen. Comp. Endocrinology, 11:489-494. Chen, C. L., Y. Amenori, K. H. Lu, J. L. Voogt and J. Meites. 1970. Serum prolactin levels in rats with pituitary transplants or hypothalamic lesions. Neuroendocrin- ology. 6:220-227. Clark, R. H. and B. L. Baker, 1964. Circadian periodicity in the concentration of prolactin in the rat hypo- physis. Science. 143:375-376. Clemens, J. A. and J. Meites. 1968. Inhibition by hypotha- lamic prolactin implants of prolactin secretion, mammary growth and luteal function. Endocrinology, 82:878-881. Clemens, J. A. and J. Meites. 1972. Hypothalamic control of prolactin secretion. Hormones and Vitamins. Clemens, J. A., H. Minaguchi, R. Storey, J. L. Voogt and J. Meites. 1969. Induction of precocious puberty in female rats by prolactin. Neuroendocrinology. 4: 150-156. Clemens, J. A., M. Sar and J. Meites. 1969a. Termination of pregnancy in rats by a prolactin implant in me- dian eminence. Proc. Soc. Exp, Biol. Med. 130: 628-630. Clemens, J. A., M. Sar and J. Meites. 1969b. Inhibition of lactation and luteal function in postpartum rats by hypothalamic implantation of prolactin. Endo- crinology. 85:868-872. Convey, E. M., H. A. Tucker, V. G. Smith and J. Zolman. 1972. Prolactin, thyroxine and corticoid after TRH. J. Anim. Sci. 35:258. Coppola, J. A. 1968. The apparent involvement of the sym- pathetic nervous system in the gonadotropin secre- tion of female rats. J. Reprod. Fert. Suppl. 4: 35-45. 204 Coppola, J. A. 1969. Turnover of hypothalamic catechol- amines during various states of gonadotropin secre- tion. Neuroendocrinology, 5:75-80. Coppola, J. A. 1971. Brain catecholamines and gonadotropin secretion. In Frontiers in Neuroendocrinology, 1971, edited by L._Martini and W. F. Ganong. 129-143. Oxford University Press, New York. Coppola, J. A., R. G. Leonardi and W. Lippmann. 1966. Ovu- latory failure in rats after treatment with brain norepinephrine depletors. Endocrinology. 78:225- 228. Coppola, J. A., R. G. Leonardi, W. Lippmann, J. W. Perrine and I. Ringler. 1965. Induction of pseudopregnancy in rats by depletors of endogenous catecholamines. Endocrinolggy, 77:485-490. Costa, E. and N. H. Neff. 1970. Estimation of turnover rates to study the metabolic regulation of the steady-state level of neuronal monoamines. IE_Hand- book of Neurochemistry, edited by A. Lajtha. 4:45- 90. Plenum Press, New York. Creveling, C. R., J. Daly, T. Tokuyama and B. Witkop. 1968. The combined use of a-methyltyrosine and threo- dihydroxyphenylserine: selective reduction of dopa- mine levels in the central nervous system. Biochem. Pharmac. 17:65-70. Dahlstrom, A. and K. Fuxe. 1965. Evidence for the existence of monoamine neurons in the central nervous system. Acta. Physiol. Scand. 64; Suppl. 247:1-87. Daniel, P. M. 1966. The anatomy of the hypothalamus and pituitary gland. In Neuroendocrinology, edited by L. Martini and W. FT'Ganong, 1:15-80. Academic Press, New York. Daniel, P. M. and M. M. L. Prichard. 1956. Anterior pitu- itary necrosis. Quart. J. Exp. Physiol. 41:215- 229. Danon, A., S. Dikstein and F. G. Sulman. 1963. Stimulation of prolactin by perphenazine in pituitary-hypothal- amus organ culture. Proc. Soc. Exp, Biol. Med. 114:366-368. De Groot, J. 1959. The Rat Forebrain in Stereotaxic Coor- dinates. N. V. Noord-Hollandsche Maatschappiu, Amsterdam. 205 Dempsey, E. W. and U. U. Uotila. 1940. The effect of pitu- itary stalk section upon reproductive phenomena in the female rat. Endocrinology, 27:573-579. Desclin, L. 1956. Hypothalamus et liberation d'hormone luteotrophique. Experiences de graffe hypophysaire chez le rat hypophysectomise. Action luteotrophique de l'oxytocine. Ann. Endocrinol. (Paris). 17:586- 595. Desclin, L. and J. Flament-Durand. 1963. Presence de neuro- secretat dans le lobe posterieur d'hypophyses greffes dans l'hypothalamus chez le rat hypophysec- tomise. Ann. Endocrinol. (Paris). 24:131-135. Dickerman, S., J. Clark, E. Dickerman and J. Meites. 1972. Effects of haloperidol on serum and pituitary pro- lactin and on hypothalamic PIF in rats. Neuroendo- crinology. 9:332-340. Donoso, A. O. and F. J. E. Stefano. 1967. Sex hormones and concentration of noradrenaline and dopamine in the hypothalamus of castrated rats. Experentia. 23: 665-666. Donoso, A. O., F. J. E. Stefano, A. M. Biscardi and J. Cukier. 1967. Effects of castration of hypothalamic catecholamines. Am. J. Physiol. 212:737-739. Douglas, W. W. 1971. Histamine and antihistamine; S- Hydroxytryptamine and antigonists. Ig_The Pharma- cological Basis of Therapeutics, edited by L. S. Goofiman and A. Gilman, 621-662. Mcmillan Co., New Yor . Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics. 11:1-42. Dunn, J. D., A. Arimura and L. E. Scheving. 1972. Effect of stress on circadian periodicity in serum LH and prolactin concentration. Endocrinology, 90:29-33. Everett, J. W. 1954. Luteotrophic function of autografts of the rat hypophysis. Endocrinology, 54:685-690. Everett, J. W. 1956. Functional corpora lutea maintained for months by autografts of the rat hypophysis. Endocrinology. 58:786-796. 206 Everett, J. W. and D. L. Quinn. 1966. Differential hypo- thalamic mechanisms inciting ovulatiOn and pseudo- pregnancy in the rat. Endocrinology, 78:141-150. Everett, J. W., C. H. Sawyer and J. E. Markee. 1949. A neurogenic timing factor in control of the ovulat- ing discharge of luteinizing hormone in the cyclic rat. Endocrinology, 44:234-250. Fernstrom, J. D. and R. J. Wurtman. 1971. Brain serotonin content: Physiological dependence on plasm trypto- phan levels. Science. 173:149-152. Flament-Durand, J. 1964. Morphologie et fontion de trans- plants hypophysaires dans l'hypothalamus chez le rat. Compt. Rend. Acad. Sci. 259:4376-4378. Flament-Durand, J. 1965. Observations on pituitary trans- plants into the hypothalamus of the rat. Endocrin- ology. 77:446-454. Fuller, R. W. 19683. Influence of substrate in the inhibi- tion of rat liver and brain monoamine oxidase. Arch. int. Pharmacodyn. 174:32-36. Fuller, R. W. 1968b. Kinetic studies and effects in vivo of a new monoamine oxidase inhibitor, N-(Z-(Ol chlorophenoxy)-ethyl)-cyclopropylamine. Biochem. Pharmac. 17:2097-2106. Fuller, R. W., C. W. Hines and J. Mills. 1965. Lowering of brain serotonin level by chloramphetamines. Biochem. Pharmac. 14:483-488. Furth, J. 1961. Vistas in the etiology and pathogenesis of tumors. Fed. Proc. 20:865-873. Fuxe, K. and T. Hofkelt. 1969. Catecholamines in the hypo- thalamus and the pituitary gland. In Frontiers in Neuroendocrinology, 1969, edited by—E, Martini and W. F. Ganong, 47-96. Oxford University Press, New York. Fuxe, K. and O. Nilsson. 1967. Activity changes in the tuber-infundibular dopamine neurons of the rat dur- ing various states of the reproductive cycle. Life S51, 6:2057-2061. 207 Fuxe, K. and T. Hokfelt.r 1970. Central monoaminergic sys- tems and hypothalamic function. In The Hypothalamus, edited by L. Martini, M. Motta and—F. F. Flaschini, 123-138. Academic Press, New York. Gala, R. R. and R. P. Reece. 1965. Influence of neurohor- mones on anterior pituitary lactogen production lg; vitro. Proc. Soc. Exp. Biol. Med. 120:220-222. Gala, R. R., P. B. Markarian and O'Neill. 1970. The in- fluence of neural blocking agents implanted into the hypothalamus of the rat on induced deciduomata for- mation. Life Sci. 9:1055-1064. Gay, V. L., A. R. Midgley, Jr. and G. D. Niswender. 1970. Patterns of gonadotropin secretion associated with ovulation. Fed. Proc. 29:1880-1887. Gersten, B. E. and B. L. Baker. 1970. Local action of in- trahypophyseal implants of estrogen as revealed by staining with peroxidase-labeled antibody. Am. J. Anat. 128:1-20. Glowinski, J., I. J. Kopin and J. Axelrod. 1965. Metabol- ism of [H3] norepinephrine in the rat brain. J, Neurochem. 12:25-30. Gourdji, D. and A. Tixier-Vidal. 1966. Miss en evidence d'un control hypothalamique stimulant de la prolac- tine hypophysaire chez le canard. C. R. Acad. Sci. 263:162-165. Green, J. D. 1947. Vessels and nerves of amphibian hypothy- ses. A study of the living circulation of the hypo- physial vessels and nerves. Anat. Record. 99:21-53. Green, J. D. 1966. The comparative anatomy of the portal vascular system and of the innervation of the hy- pophysis. In The Pituitary Gland, edited by G. W. Harris and B7 T. Donovan, 1:127-146. University of California Press, Berkeley and Los Angeles. Green, J. D. and G. W. Harris. 1949. Observation of the hypophysio-portal vessels of the living rat. J, Physiol. 108:359-361. Grosvenor, C. E., S. M. McCann and M. D. Nallar. 1964. In- hibition of suckling-induced release of prolactin in rats injected with acid extract of bovine hypothal- amus. Program 46th Meeting Endocrine Soc., San Francisco, p. 96. 208 Guillemin, R. and A. V. Schally. 1963. Recent advances in the chemistry of neuroendocrine mediatros originat- ing in the central nervous system. Ig_Advances in Neuroendocrinology, edited by A. V. Nalbandov. 314-328. University of Illinois Press, Urbana. Gurdjian, E. S. 1927. Studies on the brain of the rat. II. The diencephalon of the albino rat. J. Comp. Neurol. 43-114. Halasz, B. and J. Szentagothai. 1962. The trophic depen- dence of the anterior pituitary on the hypothalamus. In Hypothalamic Control of the Anterior Pituitary, Edited by J. Szentagothai et a1. 266-276. Publ. House Hung. Acad. Sci., BudEpEEt. Halasz, B., L. Pupp and S. Uhlarik. 1962. Hypophysiotropic area in the hypothalamus. J. Endocrinol. 25:147- 154. Halasz, B., L. Pupp, S. Uhlarik and L. Tima. 1965. Further studies on the hormone secretion of the anterior pituitary transplanted into the hypophysiotropic area of the rat hypothalamus. Endocrinology. 77: 343-355. Harris, G. W. 1955. Neural Control of the Pituitary Gland. Arnold, London. Harris, G. W. 1970. Unsolved problems in the portal vessel- chemotransmitter hypothesis. In Hypophysiotropic Hormones of the Hypothalamus: _Assay and Chemistry, edited by J. Meites. 1-14. The Williams and Wilkins Co., Baltimore. Harris, G. W. and D. Jacobsohn. 1952. Functional grafts of the anterior pituitary gland. Proc. Roy. Soc. (London) Ser. B. 139:263-279. Hillarp, N. A., K. Fuxe and A. Dahlstrom. 1966. Central monoamine neurons. I§_Mechanisms of Release of Biogenic Amines, edited by U.S. von Euler, S. Rosell and B. Uvnas. 31-37. Pergamon Press, New York. Hopkins, T. F. and G. Pincus. 1963. Effects of reserpine on gonadotropin induced ovulation in immature rats. Endocrinology, 73:775-780. Houssay, B. A., A. Biasotti and R. Sammartino. 1935. Mod— ifications fonctionelles de l'hypophyse apres les lesions infundibulo-tuberiemes ches 1e crapaud. Compt. Rend. Soc. Biol. 120:725-726. 209 Hwang, P., H. J. Guyda and H. G. Friesen. 1971. Human-pro- lactin (HPr): purification and clinical studies. Clin. Res. 19:772. Innes, I. R. and M. Nickerson. 1971. Drugs acting on post- ganglionic adrenergic nerve endings and structures innervated by them (Sympathomimetic drugs). In The Pharmacological Basis of Therapeutics, edited—by L. S. Goodman and A. Gilman, 478-523. The Macmillan Co., New York. Jacobs, L. S., C. A. Birge, C. Hammer and W. H. Daughaday. 1968. The effect of epinephrine on synthesis and release of rat pituitary growth hormone and prolac- tin lg vitro. Clin. Res. 16:441. Jacobs, L. S., P. J. Snyder, J. F. Wilber, R. D. Utiger and W. H. Daughaday. 1971. Increased serum prolactin after administration of synthetic thyrotropin re- leasing hormone (TRH) in man. J. Clin. Endocr. 33:996-998. Jenkins, T. W. 1972. Functional Mammalian Neuroanatomy. Lea and Febriger, Philadelphia. Kamberi, I. A., R. S. Mical and J. C. Porter. 1969. Lu- teninizing hormone: Releasing activity in hyp0phy- sial stalk blood and elevation by dopamine. Science. 166:388-389. Kamberi, I. A., R. S. Mical and J. C. Porter. 1970a. Ef- fect of anterior pituitary erfusion and intraven- tricular injection of catec olamines and indole- amines on LH release. Endocrinology. 87:1-12. Kamberi, I. A., R. S. Mical and J. C. Porter. 1970b. In- traventricular injection or pituitary perfusion of catecholamines and prolactin release. Fed. Proc. 29:378. Kamberi, I. A., R. S. Mical and J. C. Porter. 1971a. Ef- fects of melatonin and serotonin on the release of FSH and prolactin. Endocrinology, 88:1288-1293. Kamberi, I. A., R. S. Mical and J. C. Porter. 1971b. Pi- tuitary portal vessel infusion of hypothalamic ex- tract and release of LH, FSH and prolactin. Endo crinology. 88:1294-1299. 210 Kamberi, I. A., H. P. G. Schneider and S. M. McCann. 1970c. Action of dopamine to induce release of FSH-releas- ing factor (FRF) from hypothalamic tissue ig vitro. Endocrinology, 86:278-284. Kanematsu, S., J. Hilliard and C. H. Sawyer. 1963. Effect of reserpine and chlorpromazine on pituitary prolac- tin content and its hypothalamic site of action in rabbits. Acta Endocrinol. 44:467-474. Knigge, K. M. 1962. Gonadotropic activity of neonatal pi- tuitary glands implanted in the rat brain. Am. J. Physiol. 202:387-391. Kobayashi, T., J. Kato and H. Minaguchi. 1964. Fluctuation in monoamine oxidase activity in the hypothalamus of rats during estrous cycle and after castration. Endocrinol. Jap. 11:283-290. Kobayashi, T., J. Kato and H. Minaguchi. 1966. Cholinergic and adrenergic mechanisms in the female rat hypo- thalamus with reference to feedback of ovarian ster- oid hormones. £2 Steroid Dynamics, edited by G. Pincus, T. Nakao and J. F. Tait, 303-339. Academic Press, New York. Koch, Y., Y. F. Chow and J. Meites. 1971. Metabolic clear- ance and secretion rates of prolactin in the rat. Endocrinology, 89:1303-1308. Koch, Y., K. H. Lu and J. Meites. 1970. Biphasic effects of catecholamines on pituitary prolactin release ;n_vitro. Endocrinology. 87:673-675. Koe, B. K. and A. Weissman. 1966. p-Chlorophenylalanine: A specific depletor of brain serotonin. J. Pharm- acol. Exp. Ther. 154:499-516. Koelle, G. B. 1971. Drugs acting at synaptic and neuro- effector junctional sites. In The Pharmacological Basis of Therapeutics, edited—by L. S. Goodman and A. Gilman, 402-441. The Macmillan Co., New York. Kordon, C. 1966. Recherches sur le controle hypothamamique des fonctions gonadotropes femelles du rat. Ph.D. dissertation, University of Paris. Kordon, C. and J. Glowinski. 1969. Selective inhibition of super-ovulation by blockade of dopamine synthe- sis during the "critical period" in the immature rat. Endocrinology, 85:924—931. 211 Kragt, C. L. and J. Meites. 1965. Stimulation of igeon pituitary prolactin release by pigeon hypot alamic extract in vitro. Endocrinology. 76:1169-1176. Kragt, C. L. and J. Meites. 1967. Dose-response relation- ships between hypothalamic PIF and prolactin re- lease by rat pituitary tissue i2 vitro. Endocrin- ology. 80:1170-1173. Krulich, L., M. Quijada and P. Illner. 1971. Localization of prolactin-inhibiting factor, p-releasing factor (PRF), growth hormone-RF (GRF) and GIF activities in the hypothalamus of the rat. Program 53rd Meet- ing, Endocrine Society, San Francisco, Calif., p. Kuroshima, A., A. Arimura, C. Y. Bowers and A. V. Schally. 1966. Inhibition by pig hypothalamic extracts of depletion of pituitary prolactin in rats following cervical stimulation. Endocrinology. 78:216-217. Kwa, H. G. and F. Verhofstad. 1967. Prolactin levels in the plasma of female mice. J. Endocrinol. 38:81- 84. LaBella, F. S. and S. R. Vivian. 1971. Effect of synthe- tic TRF on hormone release from bovine anterior pi- tuitary.ig_vitro. Endocrinology. 88:787-789. Landsmeer, L. M. F. 1963. A survey of the analysis of hy- pophyseal vascularity. In Advances in Neuroendo- crinology, edited by A. VT Nalbandov, 29-57. Uni- versity of Illinois Press, Urbana. Laverty, R. and D. F. Sharman. 1965. The estimation of small quantities of 3,4-dihydroxyphenylethylamine (Dopamine) in tissues. Brit. J. Pharmacol. 24:538- 548. Lichtensteiger, W. 1969. Cyclic variations of catechol- amines content in hypothalamic nerve cells during the estrous cycle of the rat, with a concomitant study of the substantia nigra. J. Pharmacol. Exp. Ther. 165:204-215. Lippmann, W. 1968. Relationship between hypothalamic nore- pinephrine and gonadotrophin secretion in the ham- ster. Nature. 218:173-174. 212 Lippmann, W., R. G. Leonardi, J. Ball and J. A. Coppola. 1967. Relationship between hypothalamic catechol- amines and gonadotropin secretion in rats. J. Pharmacol. Exp. Ther. 156:258-266. _ Lu, K. H. and J. Meites. 1971. Inhibition by L-DOPA and monoamine oxidase inhibitors of pituitary prolactin release; stimulation by methyldopa and d-amphetamine. Proc. Soc. Exp. Biol. Med. 137:480-483. Lu, K. H. and J. Meites. 1972. Effects of L-DOPA on serum prolactin and PIF in intact and hypophysectomized, pituitary-grafted rats. Endocrinology. 91:868-872. Lu, K. H., Y. Koch and J. Meites. 1971. Direct inhibition by ergocornine of pituitary prolactin release. Eo- docrinology. 89:229-233. Lu, K. H., Y. Amenomori, C. L. Chen and J. Meites. 1970b. Effects of central acting drugs on serum and pitu- itary prolactin levels in rats. Endocrinology, 87:667-672. Lu, K. H., Y. Koch, Y. Amenomori, C. L. Chen and J. Meites. 1970a. Ig_vivo and in vitro effects of drugs on prolactin release by the rat pituitary. Fed. Proc. 29:579. Lu, K. H., C. J. Shaar, K. H. Kortright and J. Meites. 1972. Effects of synthetic TRH on in vitro and in vivo prolactin release in the ratT—'Endocrinology, 91: (December). MacLeod, R. M. 1969. Influence of norepinephrine and cate- cholamine-depleting agents on the synthesis and re- lease of prolactin and growth hormone. Endocrino- logy. 85:916-923. MacLeod, R. M., G. W. DeWitt and M. C. Smith. 1968. Sup- pression of pituitary gland hormone content by pi- tuitary tumor hormones. Endocrinology, 82:889-894. MacLeod, R. M., M. C. Smith and G. W. DeWitt. 1966. Hor- monal properties of transplanted pituitary tumors and their relation to the pituitary gland. Endo- crinology. 79:1149-1156. Manshardt, J. and R. J. Wurtman. 1968. Daily rhythm in the noradrenalin content of the rat hypothalamus. Nature. 217:574-575. 213 Markee, J. E., J. W. Everett and C. H. Sawyer. 1952. The relationship of the nervous system to the release of gonadotropin and the regulation of the sex cycle. Recent Progp, Hormone Res. 7:139-163. Markee, J. E., C. H. Sawyer and W. H. Hollinshead. 1948. Adrenergic control of the release of luteinizing hormone from the hypophysis of the rabbit. Recent Pgogr. Hormone Res. 2:117-131. Matsuo, H., Y. Baba, R. M. G. Nair, A. Arimura and A. V. Schally. 1971. Structure of the porcine LH- and FSH- releasing hormone. I. The proposed amino acid sequence. Biochem. Biophys. Res. Commun. 43:1334- 1339. McCann, S. M. 1971. Mechanism of action of hypothalamic hypophyseal stimulating and inhibiting hormones. In Frontiers in Neuroendocrinology, 209-235. Oxford University, New York. McGeer, P. L. 1971. The chemistry of Mind. Am. Scientist. 59:221-229. McKenzie, J. M. 1958. The bioassay of thyrotropin in serum. Endocrinology. 63:372-382. McQueens-Williams, M. 1935. Decreased mammotropin in pi- tuitaries of thyroidectomized (Maternalized) male rats. Proc. Soc. Exp. Biol. Med. 33:406-407. Meites, J. 1949. Effects of starvation in rats and mice on thyroid secretion rate as indicated by uptake of radioactive iodine and thiouracil action. J. Ani. S51, 8:647. Meites, J. 1950. Effects of vitamin B12 on normal thyroid function in rats. Proc. Soc. Exp, Biol. Med. 75: 195. Meites, J. 1957. Induction of lactation in rabbits with reserpine. Pgoc. Soc. Exp. Biol. Med. 96:728-730. Meites, J. 1958. Effect of reserpine on prolactin content of rabbit pituitary. Proc. Soc. Exp. Biol. Med. 97:742-744. Meites, J. 1959. Induction and maintenance of mammary growth and lactation in rats with acetylcholine or epinephrine. Proc. Soc. Exp, Biol. Med. 100:750- 7S4. Meites, Meites, Meites, Meites, Meites, Meites, Meites, Meites, Meites, Meites, 214 J. 1962. Pharmacological control of prolactin se- cretion and lactation. Ip_Pharmacological Control of Release of Hormones Including Antidiabetic Drugs, Ediged by R. Guillemin, 151-181. Perganon Press, on on. J. 1966. Control of mammary growth and lactation. 12 Neuroendocrinology, edited by L. Martini and W. F. Ganong, 669-707. Academic Press, New York and London. J. 1967. Control of prolactin secretion. Archives D'Anatomie Microscopique et de Morphologie Exper- imentale.*’563516-529. J. 1970. Direct studies of the secretion of the hypothalamic hypophysiotropic hormones (HHH). Lg Hypophysiotropic Hormones of the Hypothalamus: Assay and Chemistry, edited by J. Meites, 261-278. The Williams and Wilkins Co., Baltimore. J., R. H. Kahn and C. S. Nicoll. 1961. Prolactin production by rat pituitary ip_vitro. Proc. Soc. Exp, Biol. Med. 108:440-443. J. and C. L. Kragt. 1964. Effects of a pituitary homotransplant and thyroxine on body and mammary growth in immature hypophysectomized rats. Endo- crinology. 75:565-570. J., K. H. Lu, W. Wuttke, C. W. Welsch, H. Nagasawa and S. K. Quadri. 1972. Recent studies on func- tions and control of prolactin secretion in rats. Recent Progr. Hormone Res. 28:471-516. J., C. S. Nicoll and P. K. Talwalker. 1963. The central nervous system and the secretion and re- lease of prolactin. In Advances in Neuroendocrin- ology, edited by A. VT‘Nalbandor. 238-277. Uni- versity of Illinois Press, Urbana. J., P. K. Talwalker and C. S. Nicoll. 1960. Init- iation of lactation in rats with hypothalamic or cerebral tissue. Proc. Soc. Exp. Biol. Med. 103: 298-300. J. and C. W. Turner. 1947. Effect of thiouracil and estrogen on lactogenic hormone and weight of pituitaries of rats. Proc. Soc. Exp. Biol. Med. 64:488-492. 215 Minaguchi, H. and J. Meites. 1967a. Effects of suckling on hypothalamic LH- releasing factor and prolactin- inhibiting factor, and on pituitary LH and prolac- tin. Endocrinology, 80:603-607. Minaguchi, H. and J. Meites. 1967b. Effects of a norethy- nodrelmestranol combination (Enovid) on hypothalam- ic and pituitary hormones in rats. Endocrinology, 81:826-834. Mischkinsky, J., K. Khazan and F. G. Sulman. 1968. Prolac- tin releasing activity of the hypothalamus of post- partum rats. Endocrinology. 82:611-613. Mittler, J. C. and J. Meites. 1967. Effects of epinephrine and acetylcholine on hypothalamic content of pro- lactin-inhibiting factor. Proc. Soc. Exp. Biol. Mod, 124:310-311. Motta, M., F. Fraschini and L. Martini. 1969. Short feed- back mechanisms in the control of anterior pituitary function. In Frontiers in Neuroendocrinology, 1969, edited by LT_Martini and W. F. Ganong, 211-253. Oxford University Press, New York. Nandi, S. 1959. Hormonal control of mammogenesis and lac- togenesis in the C3H/He Crgl Mouse. University of California Publication Zoology. 65:4. Nagasawa, H. and J. Meites. 1970. Suppression by ergocor- nine and iproniazid of carcinogen-induced mammary tumors in rats; Effects on pituitary and serum pro- lactin levels. Proc. Soc. Exp. Biol. Med. 135:469- 72. Nagasaws, H., C. L. Chen and J. Meites. 1969. Effects of estrogen implant in median eminence on serum and pituitary prolactin levels in the rat. Proc. Soc. Exp. Biol. Med. 132:859-861. Neill, J. D. 1972. Comparison of plasma prolactin levels in cannulated and decapitated rats. Endocrinology. 90:568-572. Neill, J. D., M. B. Freeman and S. A. Tillson. 1971. Con- trol of the proestrus surge of prolactin and lutein- izing hormone secretion by estrogens in the rat. Endocrinology, 89:1448-1453. 216 Netter, F. H. 1967. The Hypothalamus, supp. to Vol. I, Nervous System, The Ciba Collection of Medical Il- lustrations. Ciba Pharmacentical Products, Inc., Summit, N.J. ' Nicoll, C. S. 1965. Neural regulation of adenophypophy- seal prolactin secretion in tetrapods: indications from £2 vitro studies. J. Exp. 2001. 158:203-210. Nicoll, C. S. 1971. Aspects of the neural control of pro- lactin secretion. Ip_Frontiers in Neuroendocrino- logy, edited by L. Martini and W. F. Ganong, 291- 330. Oxford University Press, New York. Nicoll, C. S. and J. Meites. 1962. Estrogen stimulation of prolactin production by rat adenohypophysis lg vitro. Endocrinology, 70:272-277. Nicoll, C. S. and J. Meites. 1963. Prolactin secretion ip_vitro: effects of thyroid hormones and insulin. Endocrinology. 72:544-551. Nicoll, C. S. and J. Meites. 1964. Prolactin secretion lg vitro: effects of gonadal and adrenal cortical steroids. Proc. Soc. Exp. Biol. Med. 117:579-583. Nicoll, C. S., R. P. Fiorindo, C. T. McKennee and J. A. Parsons. 1970. Assay of hypothalamic factor which regulate prolactin secretion. In Hypophysiotropic Hormones of the Hypothalamus: Assay and Chemistry, edited by J. Meites, 115-144. The Williams and Wilkins Co., Baltimore. Nikitovitch-Winer, M. B. and J. W. Everett. 1958. Func- tional restitution of pituitary grafts re-trans- planted from kidney to median eminence. Endocrin- ol gy. 63:916-930. Nikitovitch-Winer, M. B. and J. W. Everett. 1959. Histo- cytologic changes in grafts of rat pituitary on the kidney and upon re-transplantation under the dien- cephalon. Endocrinology, 65:357-368. Niswender, G. D., C. L. Chen, A. R. Midgley, Jr., J. Meites and S. Ellis. 1969. Radioimmunoassay for rat pro- lactin. Proc. Soc. Exp. Biol. Med. 130:793-797. Pasteels, J. L. 1961. Secretion de prolactine par l'hypo- physe en culture de tissues. Compt. Rend. Soc. Biol. 253:2140-2142. 217 Peterson, W. E. 1942. New developments in the physiology and biochemistry of lactation: A review. J. Dairy Sci. 25:71-96. ' Piacsek, B. E. and J. Meites. 1966. Effects of castration and gonadal hormones on hypothalamic content of luteinizing hormone-releasing factor (LRF). Endo crinology. 79:432-439. Piezzi, R. S., F. Larin and R. J. Wurtman. 1970. Serotonin 5-hydroxyindoleacetic acid (S-HIAA) and monoamine oxidase in the bovine median eminence and pituitary gland. Endocrinology, 86:1460-1462. Pohorecky, L. A., M. J. Zigmond, H. Karten and R. J. Wurtman. 1969. Enzymatic conversion of norepinephrine to epinephrine by the brain. J. Pharmacol. Exp. Ther. 165:190-195. Popa, G. T. and U. Fielding. 1930. A portal circulation from the pituitary to the hypothalamus. J. Anat. (London) 65:88-91. Potter, L. T. and J. Axelrod. 1963. Properties of nore- pinephrine storage particles of the rat heart. J, Pharmacol. Exp. Ther. 142:299-305. Quadri, S. K., K. H. Lu and J. Meites. 1972. Ergot- induced inhibition of pituitary tumor growth in rats. Science. 176:417-418. Rabinowitz, P. and I. S. Friedman. 1961. Drug induced lac- tation in uremia. J. Clin. Endocrinol. Metab. 21: 1489-1493. - Ratner, A. and J. Meites. 1962. Effect of estrogen admin- istration in vivo on prolactin release io vitro. Proc. Soc.’Exp. Biol. Med. 112:12-15. Ratner, A. and J. Meites. 1964. Depletion of prolactin- inhibiting activity of rat hypothalamus by estradiol or suckling stimulus. Endocrinology, 75:377-382. Ratner, A., P. K. Talwalker and J. Meites. 1965. Effect of reserpine on prolactin-inhibiting activity of rat hypothalamus. Endocrinology. 77:315-319. Reis, D. J. and R. J. Wurtman. 1968. Diurnal changes in brain noradrenaline. Life Sci. 7:91-98. 218 Rinne, U. K. and V. Sonninen. 1968. The occurrence of dopamine and norepinephrine in the tubero-hypophy- seal system. Experimentia. 24:177-178. Sanders-Bush, E. and F. Sulser. 1970. p-Chloroamphetamine: In vivo investigations on the mechanism of action a? the selective depletion of cerebral serotonin. J. Pharmacol. Exp. Ther. 175:419-426. Sandler, R. 1968. Concentration of norepinephrine in the hypothalamus of the rat in relation to the estrous cycle. Endocrinology, 83:1383-1386. Sar, M. and J. Meites. 1968. Effects of progesterone, testosterone, and cortisol on hypothalamic prolactin- inhibiting factor and pituitary prolactin content. Proc. Soc. Exp. Biol. Med. 127:426-429. Sar, M. and J. Meites. 1969. Effects of suckling on pitu- itary release of prolactin, GH, and TSH in postpar- tum lactating rats. Neuroendocrinology. 4:25-31. Steinman, A. M., S. E. Smerin and J. D. Barchas. 1970. Epinephrine metabolism in mammalian brain after in- travenous and intraventricular administration. Science. 165:616-617. Sawyer, C. H., J. E. Markee and B. F. Townsend. 1949. Cholinergic and adrenergic components in the neuro- humoral control of the release of LH in the rabbit. Endocrinology, 44:18-37. Schally, A. V., A. Kurochuma, Y. Ishido, T. W. Redding and C. Bowers. 1965. The presence of prolactin inhi- biting factor (PIF) in extracts of beef, sheep_ and pig hypothalamus. Proc. Soc. Exp. Biol. Med. 1182350-352. Schneider, H. P. G. and S. M. McCann. 1970. Mono- and in- doleamines and control of LH secretion. Endocrin- ology. 86:1127-1133. Schneider, H. P. G. and S. M. McCann. 1970. Release of LH-releasing factor (LRF) into the peripheral cir- culation of hypophysectomized rats by dopamine and its blockage by estradiol. Endocrinology. 87:249- 253. Sgouris, J. T. and J. Meites. 1953. Differential inacti- vation of prolactin by mammary tissue from pregnant and parturient rats. Am. J. Physiol. 175:319-321. 219 Shelesnyak, M. C. 1955. Disturbance of hormone balance in the female rat by single injection of ergotoxine ethanesulfonate. Am. J. Physiol. 180:47-49. Shelesnyak, M. C. 1958. Maintenance of gestation in ergo- toxine treated pregnant rats by exogenous prolac- tin. Acta Endocrinol. (Kobenhavn). 27:99-109. Sinha, Y. N. and H. A. Tucker. 1968. Pituitary prolactin content and mammary development after chronic ad- ministration of prolactin. Proc. Soc. Exp, Biol. Mod. 128:84-88. Smelik, P. G. and J. H. van Mannen. 1968. Role of the tubero-infundibular dopaminergic neurons in the control of prolactin secretion. Excerpta Medica Int. Coog, Ser. 157, p. 135. Snyder, S. H., J. Axelrod and M. Zweig. 1967. Circadian rhythm in the sertonin content of the rat pineal gland: regulating factors. J. Pharmacol. Exp. Ther. 158:206-213. Snyder, S. H., M. Zweig, J. Axelrod and J. E. Fisher. 1965. Control of the circadian rhythm in serotonin con- tent of the rat pineal gland. Proc. Natl. Acad. Sci. U.S. 53:301-305. Salman, F. G. and H. Z. Winnik. 1956. Hormonal effects of chlorpromazine. Lancet. 270:161-162. Szentagothai, J. 1962. Anatomical considerations.“ In Hypothalamic Control of the Anterior Pituitaryj' edited by J. Szentagothai et a1. 19-105. Publ. House Hung. Acad. Sci., Budapest. Szentagothai, J. 1964. The parvicellular neurosecretory system. Progr. Brain Res. 5:135-146. Szentagothai, J. and B. Halasz. 1964. Regulation des endokrinen systems uber hypothalamus. (Vortrag auf der Jahresversammlung Deut. Akad. Naturforscher Leopoldifia, Halle/Saale, 1963). Nova Acta Leopoldina. 28:227-2 8. Talwalker, P. K. and J. Meites. 1961. Mammary lobuloal- veolar growth induced by anterior pituitary hor- mones in adreno-ovariectomized and adreno-ovariec- tomized-hypophysectomized rats. Proc. Soc. Exp. Biol. Med. 107:880-883. 220 Talwalker, P. K., A. Ratner and J. Meites. 1963. In vitro inhibition of ituitary prolactin synthesis—aha re- lease by hypot alamic extract. Am. J. Physiol. 205:213-218. Tashjian, A. H., Jr., P. C. Bancraft and L. Levine. 1970. Production of both prolactin and growth hormone by clonal strains of rat pituitary tumor cells. J. Cell Biol. 47:61-70. ‘ Tashjian, A. H., Jr., N. J. Barowsky and D. K. Jensen. 1971. Thyrotropin releasing hormone: direct evi- dence for stimulation of prolactin production by pituitary cells in culture. Biochem. Biophys. Res. Commun. 43:516-523. Taubenhaus, M. and S. Soskin. 1941. Release of luteini- zing hormone from anterior hypophysis by an acetyl- choline-like substance from the hypothalamic region. Endocrinology, 29:958-964. Torok, B. 1954. Lebendbeobachtung des hypOphysenk- reislaufes an hunden. Acta Morphol. Acad. Sci. H g. 4:83-89. Truex, R. C. and M. B. Carpenter. 1969. Human Neuroana- tom . Sixth edition. The Williams and’Wilkins Co., a timore. Turkington, R. W. 1971. Inhibition of prolactin secretion and successful therapy of the Forbes-Albright syn- drome with L-DOPA. Proc. Cent. Soc. Clin. Res. 44:44. Valverde, C. and V. Chieffo. 1971. Prolactin releasing factors in porcine hypothalamic extracts. Program 53rd Meeting, Endocrine Society, San Francisco, Calif., p. 84. Van Mannen, J. H. and P. G. Smelik. 1968. Induction of pseudopregnancy in rats following local depletion of monoamines in the hypothalamus. Neuroendocrin- ology. 3:177-186. Vogt, M. 1954. The concentration of synpathin in differ- ent parts of the central nervous system under nor- mal conditions and after the administration of drugs. J. Physiol. (London). 123:451-481. Voogt, J. L.and J. Meites. 1971. Effects of an implant of prolactin in median eminence of pseudopregnant rats on serum and pituitary LH, FSH and prolactin. Endocrinology, 88:286-292. FWJ. SOT-[II .‘J arm- 221 Voogt, J. L., J. A. Clemens and J. Meites. 1969. Stimula- tion of pituitary FSH release in immature female rats by prolactin implant in median eminence. Neuroendocrinology, 4:157-163. Watson, J. T., L. Krulich and S. M. McCann. 1971. Effect of crude rat hypothalamic extract on serum gonado- trOpin and prolactin levels in normal and orchidec- tomized male rats. Endocrinology, 89:1412-1418. Welsch, C. W., A. Negro-Vilar and J. Meites. 1968. Effects of pituitary homografts on host pituitary prolactin and hypothalamic PIF levels. Neuroendocrinology. 3:238-245. Welsch, C. W., M. D. Squiers, E. Cassell, C. L. Chen and J. Meites. 1971. Median eminence lesions and ser- um prolactin: influence of ovariectomy and ergo- cornine. Am. J. Physiol. 221:1714-1717. White, A., P. Handler and E. L. Smith. 1968. Principles of Biochemistry, 612-616. McGraw-Hill Co., New York. Wislocki, G. B. and L. S. King. 1936. The permeability of the hypophysis and hypothalamus to vital dyes, with a study of the hypophyseal vascular supply. Am. J. Anat. 58:421-427. Worthington, W. C., Jr. 1955. Some observations on the hypophyseal portal system in the living mouse. Bull. Johns Hopkins Hosp. 97:343-357. Wurtman, R. J. 1966. Catecholamines. Little, Brown and Co., Boston. Wurtman, R. J. 1970. Control of the synthesis of melaton- in and other methoxyindoles in the mammalian pineal organ. Io Neurochemical Aspects of Hypothalamic Function, edited by L. Martini and J. Meites, 135- 140. Academic Press, New York. Wurtman, R. J. 1970. Brain catecholamines and the control of secretion from the anterior pituitary gland. In Hypophysiotropic Hormones of the Hypothalamus: —— Assay and Chemistry, edited by J. Meites, 184-189. The Williams and Wilking Co., Baltimore. Wurtman, R. J., F. Anton-Tay and S. Anton. 1969. On the use of synthesis inhibitors to estimate brain nore- pinephrine synthesis in gonadectomized rats. Life Sol. 8:1015-1022. 222 WUrtman, R. J., C. M. Rose, C. Chou and P. Larin. 1968. Daily rhythms in the concentrations of various amino acids in human plasma. New Engl. J. Med. 270:171-175. wuttke, W. and J. Meites. 1970. Effects of ether and pen- tobarbital on serum prolactin and LH levels in pro- estrous rats. Proc. Soc. Exp. Biol. Med. 135:648- 652. Wuttke, W., E. Cassell and J. Meites. 1971. Effects of ergocornine on serum prolactin and LH, and on hy- pothalamic content of PIF and LRF. Endocrinology. 88:737-741. wuttke, W., M. Gelato and J. Meites. 1971. Mechanisms of pentobarbital actions on prolactin release. Endo crinology. 89:1191-1194. Yanai, R. and H. Nagasawa. 1970. Effects of ergocornine and 2-Br-a-ergocryptine (CB-154) on the formation of mammary hyperplastic alveolar nodules and the pi- tuitary prolactin levels in mice. Experientia. 26:649-650. Zarrow, M. X. and K. Brown-Grant. 1964. Inhibition of ovu- lation in rats by chlorpromazine. J. Endocrinol. 30:87-95. Zeilmaker, G. H. and R. A. Carlsen. 1962. Experimental studies on the effect of ergocornine methanesulfonate on the luteotrophic function of the rat pituitary gland. Acta Endocrinol. (Kobenhavn). 41:321-335. M-, p. APPENDIX CURRICULUM VITAE AND LIST OF PUBLICATIONS NAME: DATE OF BIRTH: PLACE OF BIRTH: NATIONALITY: MARITAL STATUS: PRESENT ADDRESS: FUTURE ADDRESS: HOME ADDRESS: EDUCATION: Degree Year B.S. 1956-1962 M.S. 1965-1967 None 1967-1968 Ph.D. 1968-1972 CURRICULUM VITAE LU, Kuew-Hsiung (John) September 16, 1937 Miaoli, Taiwan, China Chinese (Permanent resident of U.S.A.) Married SEX: Male Department of Physiology Michigan State University East Lansing, Michigan 48823 Department of Physiology School of Medicine The University of Pittsburgh Pittsburgh, Pennsylvania 15213 144 Wen-Fa Road Miaoli, Taiwan Republic of China Institution National Taiwan Normal University National Taiwan University Purdue University Michigan State University 223 Major Field of Study Biology Physiology Endocrinology Neuroendrocinology HONORS: (a) (b) 224 Recipient of the National Fellowship for Natural Science Students (Taiwan), 1957-1961. Recipient of the NCSD Research Fellowship, the National Council on Science Development (Taiwan), 1965-1967. Elected Associated Member of Sigma Xi, 1970. Sigma Xi Graduate Student Research Merit Award, 1971. Elected Full Member of Sigma Xi, 1972. POSITIONS HELD: (a) (b) (c) (d) (e) (f) Post-doctoral Fellow, The University of Pittsburgh, October, 1972. Teaching Assistant in Physiology and Research Assistant in Neuroendocrinology, Michigan State University, 1969-1972. Teaching Assistant in Biological Sciences, Michigan State University, 1968-1969. Research Assistant in Endocrinology, Purdue University, 1967-1968. Teaching Assistant in Zoology and Physiology, National Taiwan Normal University, 1963-1965. High School Teacher in Biology, Taiwan, 1961-1962. TALKS PRESENTED AT SCIENTIFIC MEETINGS: Meetings Year TOpic 56th Annual Meeting of 1972 TSH-releasing hormone effects Federation of American on prolactin release by rat Societies for Experimental pituitary in vivo and in Biology. vitro. __' _— Atlantic City, N.J. 76th Annual Meeting of 1972 Inhibition of prolactin Michigan Academy of secretion and stimulation Science. of PIF release by L-DOPA in East Lansing, Michigan. rats. 55th Annual Meeting of 1971 Direct inhibition by ergo- Federation of American cornine of pituitary pro- Societies for Experimental lactin secretion. Biology. Chicago, Illinois. 225 54th Annual Meeting of- 1970 In vivo and in vitro effects Federation of Amerlcan 3? drugs on EFOIacfln Societies for Experimental release by the rat pituitary. Biology. Atlantic City, N.J. RESEARCH PUBLICATIONS: 1. Lu, K. H. 1967. Force-feeding in the hypOphysectomized immature rat. Thesis for the Degree of Master of Science. National Taiwan University. 2. Philpott, J. E., M. X. Zarrow, H. Deneberg, K. H. Lu, R. W. Fuller and J. M. Hunt. 1969. Phenethanolamine N-methyl transferase and adrenal activity in the neo- natal rat. Life Sciences 8(1):367-371. 3. Lu, K. H., Y. Koch, Y. Amenomori, C. L. Chen, and J. Meites. 1970. In vivo and in vitro effects of drugs on prolactin releas§_by the rat—pitultary. Federation Proceedings 29(2):579. 4. Lu, K. H., Y. Amenomori, C. L. Chen and J. Meites. 1970. Effects of central acting drugs on serum and pituitary prolactin levels in rats. Endocrinology 87:667-672. 5. Koch, Y., K. H. Lu and J. Meites. 1970. Biphasic effects of catecholamines on pituitary prolactin release lo vitro. Endocrinology 87:673-675. 6. Chen, C. L., Y. Amenomori, K. H. Lu, J. L. Voogt and J. Meites. 1970. Serum prolactin levels in rats with pituitary transplants or hypothalamic lesions. Neuro- endocrinology 6:220-227. 7. Lu, K. H. and J. Meites. 1971. Inhibition by L-DOPA and monoamine oxidase inhibitors of pituitary prolactin release: Stimulation by methyldopa and d-amphetamine. Proc. Soc. Expt. Biol. Med. 137:480-483. 8. Lu, K. H., Y. Koch and J. Meites. 1971. Direct inhibi- tion by ergocornine of pituitary prolactin secretion. Federation Proceedings 30(2):474. 9. Lu, K. H., Y. Koch and J. Meites. 1971. Direct inhibi- tion of ergocornine of pituitary prolactin release. Endocrinology 89:229-233. 10. 11. 12. 13. 14. 15. 226 Quadri, S. K., K. H. Lu and J. Meites. 1972. Ergot- induced inhibition of pituitary tumor growth in rats. Science 176:417-418. Gelato, M. C., K. H. Lu and J. Meites. 1972. Inhibi- tion of luteolysis by iproniazid during the estrous cycle in rats. Program 5th Annual Meeting, The Society for the Study of Reproduction, East Lansing, pp. 80. Meites, J., K. H. Lu, W. Wuttke, C. W. Welsch, H. Nagasawa, and S. K. Quadri. 1972. Recent studies on functions and control of prolactin secretion in rats. Recent Progress in Hormone Research. 28:471-516. Lu, K. H., K. H. Kortright, C. J. Shaar and J. Meites. 1972. TSH-releasing hormone effects on prolactin release by rat pituitary in vivo and in vitro. Federa- tion Proceedings, 31(2):22I. __ Lu, K. H. and J. Meites. 1972. Effects of L-DOPA on serum prolactin and prolactin inhibiting activity in intact and hypophysectomized, pituitary-grafted rats. Endocrinology, :868-872. Lu, K. H., C. J. Shaar, K. H. Kortright and J. Meites. 1972. Effects of synthetic TRH on io vitro and lo vivo prolactin release in the rat. EndocrinoIogy 91: (December). "I111111.11!11111111155