”E‘HE EFFECTS OF CATECHOLAMENES ON ANTEREGR PHUETARY PROLACTIN RELEASE Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY CARL J. SHAAR 1975 This is to certify that the thesis entitled The Effects of Catecholamines on Anterior Pituitary Prolactin Release presented by Carl Joseph Shaar has been accepted towards fulfillment of the requirements for Ph . D. degree in Phys iOlogy Date 1/14/76 0-7639 g BINDING BY me & 80N8’ IBUUK BINDERY INC LIBRARY BINDERS ABSTRACT THE EFFECTS OF CATECHOLAMINES ON ANTERIOR PITUITARY PROLACTIN RELEASE BY Carl J. Shaar 1. Various concentrations of depamine, norepinephrine and epinephrine were incubated with rat anterior pituitary 9 tissue in vitro. DOpamine (5.26 x 10- to 5.26 x 10-7M), 8 to 4.85 x 10-7M) and high con- 8 norepinephrine (2.42 x 10‘ centrations of epinephrine (6.0 x 10‘ to 3.0 x 10-7M) significantly inhibited prolactin release in_zi££9, It is concluded that dopamine and norepinephrine in amounts less than those found in the hypothalamus can directly act on‘ the rat anterior pituitary gland to inhibit prolactin release in_vitro. 2. A single intraperitoneal injection of iproniazid, a monoamine oxidase enzyme inhibitor, significantly reduced serum prolactin levels in female rats as compared to saline- treated control rats. Concurrent with the decreased serum prolactin concentrations there was increased prolactin inhibiting activity in the hypothalamus along with in- creased hypothalamic content of dopamine and norepinephrine. 'r1 n) T) Carl J. Shaar The treatment of female rats with reserpine, a drug known to deplete catecholamines, resulted in increased serum prolactin levels as compared with saline-treated control rats. There was also an associated decrease in prolactin inhibiting activity and a decrease in hypothalamic dopamine and norepinephrine content in reserpine treated rats. It is concluded that the alterations in prolactin inhibiting activity of the hypothalamic extracts, as demonstrated by an in_gitrg technique, represent at least partially the effects of alteration of hypothalamic extract catecholamine content. 3. Rat hypothalamic extracts incubated with rat anterior pituitary tissue significantly inhibited pituitary prolactin release in_yitgg. When hypothalamic extracts were subjected to preincubation with a rat brain monoamine oxidase preparation or to aluminum oxide catecholamine adsorption, the extracts lost their ability to inhibit prolactin release ig_yit£g, Iproniazid a monoamine oxidase inhibitor, blocked the effect of the monoamine oxidase enzyme preparation on the hypothalamic extracts. The cate— cholamine containing acid eluate from aluminum oxide adsorption was able to inhibit prolactin release to the same degree as untreated hypothalamic extracts. The dop- amine and norepinephrine content of pretreated and untreated ,‘f‘f‘,’ Carl J. Shaar hypothalamic extracts was measured by a spectrophotofluoro- metric assay technique. It is concluded that the prolactin inhibiting activity of hypothalamic extracts can be account- ed for by the amount of endogenous catecholamine normally present in the hypothalamic extracts. 4. Lergotrile mesylate (2 chloro-6-methyl-ergoline-8- acetonitrile, methane salt) was incubated with rat anterior pituitary tissue in_vitrg. Lergotrile mesylate significant- ly inhibited prolactin release in_vitro. Pimozide, a specific dopaminergic receptor blocker, but not alpha and beta adrenergic receptor blockers, was able to antagonize the inhibitory action of lergotrile mesylate. It is con- cluded the ergolines such as lergotrile mesylate inhibit prolactin release from anterior pituitary tissue by activa- tion of an adenohypophyseal dopamine receptor. 5. Cycling female rats possessing permanent indwelling carotid-aortic arterial cannulas were administered small amounts of dopamine hydrochloride at various intervals dur— ing the afternoon of proestrus. At the end of each infu- sion period a serum sample was taken and infusion was restarted. Dopamine administered in this manner was effec- tive in preventing the naturally occurring rise in serum prolactin during the afternoon of proestrus. The serum prolactin levels of dopamine treated rats were compared to w {firms no?! . 34.1.3 .W. .. . 5.... . , . .u. e Carl J. Shaar serum prolactin concentrations of similarly prepared rats receiving a 5% glucose solution as a control substance. It is concluded that dopamine administered by this infusion technique can inhibit prolactin release and inhibit the rise in serum prolactin during the afternoon of proestrus in the rat. 6. Minute amounts of L-DOPA, the immediate precursor of dopamine and dOpamine were infused via an external jugu- lar vein cannula into female rats that had previously received bilateral electrolytic lesions in the median eminence of the hypothalamus. Infusions of L-DOPA and dopamine significantly lowered the already existing elevated serum prolactin levels when compared to rats receiving physiological saline. Because the median eminence was completely destroyed by the bilateral electrolytic lesions, no hypothalamic influence was present at the time of drug infusion. It is concluded that both L—DOPA and dopamine lowered serum prolactin levels by acting directly on the anterior pituitary gland to inhibit prolactin release. THE EFFECTS OF CATECHOLAMINES ON ANTERIOR PITUITARY PROLACTIN RELEASE BY «fl Carl JifShaar A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1975 Dedicated to my loving wife, Judy, my children, Jennifer and Andrew, and my parents, Harold and Miriam Shaar. ii ACKNOWLEDGMENTS My deepest appreciation must first be given to Dr. Joseph Meites who was so very willing to help and guide me during my stay at Michigan State University and during the completion of my research in Indianapolis. When I was faced with the possibility that ill health might terminate my graduate study, Dr. Meites immediately without hesitation proposed to my guidance committee an alternative plan which has allowed me to complete my study program. I will always be grateful to Dr. Meites for his interest in my well-being and for his guidance and understanding during these past years. I would also like to express my gratitude to Dr. James A. Clemens who so willingly accepted the responsibility for guiding my research during my absence from Michigan State University. Dr. Clemens first suggested that I attempt advanced graduate study and without his constant encourage- ment and friendship I would hever have completed the many individual tasks involved with such study. His success and ability as a researcher have always been an inspiration to me. iii E. u......’.ru..r .... I wish to thank the members of my guidance committee: Drs. W. D. Collings, G. D. Riegle, H. A. Tucker and C. W. Welsch who willingly counseled me in preparing this disser- tation and for their willingness to allow me to pursue my goal in spite of having to leave Michigan State University. Appreciation is also expressed to Dr. K. H. Lu, Dr. Marie Gelato, Mr. Greg Mueller, Mr. H. J. Chen, Mr. Gary Kledzik and Mrs. Claire Twohy from whom I received encourage- ment and assistance during my stay at Michigan State University, and Mr. Michael E. Roush, Mr. E. Barry Smalstig, Mr. Barry Sawyer, Mr. Michael Hanlin and Mr. A. Philip Yount for their invaluable technical assistance and friendship. Special thanks is given to Mrs. Amylou Davis for her willingness to type the many forms and letters necessary throughout the years and especially for her assistance over long distance phone. Also, a special thanks is due Mrs. Jeanie Fischer for typing many manuscripts and the rough copy of this dissertation. I wish also to thank Dr. Meites and the members of the Graduate Affairs Committee of the Physiology Department of Michigan State University for the Assistantship granted me from September, 1971, until December, 1972. Last, but by no means least, I wish to express my gratitude to my loving wife, Judy, who never lost faith in me and who stayed by my side during these arduous years. iv TABLE OF CONTENTS Page LIST OF TABLESOOOOOOOOOOOOOO ..... OOOOOOOOOOOOOIOOOOOO X LIST OF FIGURESOOOOOOOOOOOOOOOOOOOO ..... OOOOOOOOOOOOO Xii INTRODUCTIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0....O 1 REVIEWOF LITERATUREOOOOOOOOOOOOCOOO0.00000000000COO. 5 I. Functional Neuroanatomy of the Hypothalamo- hypophyseal SYStemoocooooooo00.000.000.000... 5 A. Functional Anatomy of the Hypothalamus.. 5 B. Hypothalamo-hypophyseal Portal System... 7 C. Effects of Removal of Hypothalamic Influence on Anterior Pituitary Prolac- tin Secretion and Histology............. 10 II. Hypothalamo Hypophysiotropic Hormones........ 14 A. BiOChemical NatureOOIOOOCOOOOOOO00...... 14 B. Site Of originOOOOOOOOOOOOOICOCOOIOOOOOO 16 C. The Influence of the Biogenic Amines on the Release of Gonadotropin Releasing Factor: An Example of Biogenic Amine- hypophysiotropic Hormone Interaction.... 19 III. Biogenic Amines in the Hypothalamus..... ..... 25 A. GeneraJ-OOOOOOOOOOOOOOOOOOOOOOOOCO0.0...I 25 B. Biosynthesis of Brain Monoamines........ 27 C. Physiological Disposition of Brain Mono- amineSOOOOOOOOOOOOOOOOOOOOOOOOOOOOIOOOO. 30 D. The Effects of Various Pharmacological Agents on Catecholamine Activity........ 32 TABLE OF CONTENTS--continued Page IV. Hypothalamic Control of Prolactin Secretion.. 34 A. Inhibition of Prolactin Secretion from Pituitary Tissue Incubated In Vitro with Hypothalamic Extracts.............. 34 B. Inhibition of Pituitary Prolactin Secre- tion In Vivo by Administration of Hypothalamic Extracts................... 37 C. Proposed Chemical Nature of PIF......... 38 D. Mechanism of Action of Prolactin Inhibiting Factor (PIF)................. 41 E. Prolactin Releasing Factor: Possible Presence in Mammalian Hypothalamus...... 43 V. The Role of Biogenic Amines in the Control of Prolactin Secretion....................... 47 A. Effect of Hypothalamic Catecholamines on Prolactin Secretion..................... 47 B. The Effects of Various Agents which Alter Catecholamine Activity on Prolac- tin Secretion In_Vivo................... 53 C. The Direct Effects of Catecholamines on Pituitary Prolactin Secretion........... 58 D. The Effects of Other Biogenic Amines on Prolactin Secretion In_Vivo............. 65 E. The Direct Effects of Other Biogenic Amines on Pituitary Prolactin Secretion. 69 VI. Effects of Ergot Derivatives on Prolactin secretiODOOOOOOOO0.00...OOOOOOOOOOOOCOOOCOOOC 70 A. The Effects of Ergot Administration In_Vivo: A General Review.............. 70 B. Indirect Effects of Ergots Administra- tion on Ovarian Weight.................. 72 C. Effect of Ergot Administration on Pituitary Weight........................ 73 D. Indirect Effects of Ergot Administration on Mammary Tissue....................... 74 E. Direct Effects of Ergots on Anterior Pituitary Prolactin Secretion: A Mechan— ism and Site of Action.................. 76 vi TABLE OF CONTENTS-~continued MATERIALS ANDMETHODSOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO I. II. III. IV. V. VI. VII. VIII. IX. X. AnimalSOCCOOOOCOOOCOOOOOQOOOOOCOOOOOOOCIOOOOO Method of Placing Electrolytic Lesions in median MinenceOOOOOIOOOOOOOIOOOOOOOOIOOOOOOO Blood Vessel Cannulation for Acute Studies... Blood Vessel Cannulation for Chronic Studies. In_Vitro Pituitary Tissue Incubation......... Preparation of Hypothalamic Extracts for I—rl-Vitro Incubation.OOOOOOOOOOOOOOOOOI..0.0.. Radioimmunoassay of Rat Prolactin............ A. B. C. Preparation of Serum for Radioimmuno- assay of Prolactin...................... Preparation of Incubation Medium........ Michigan State University Radioimmuno- assay for Rat Prolactin................. Rat Brain Monoamine Oxidase Enzyme Prepara- tionOOOOOOCOOOOOOOCOOOCOCOOOOOCOOOOOCOOOOOCOO Catecholamine Measurement.................... A. B. C. D. Preparation of Hypothalamic Extracts for Catecholamine Assay..................... Preparation and Activation of Aluminum Oxide (Alumina)......................... Catecholamine Adsorption Technique...... Fluorometric Catecholamine Measurement.. Methods of Statistical Analysis.............. EXPERIMENTALOCOCCOOOOOOOO...OOOOOOOOOOOOOOOOOOOCICOOO I. Direct Effects of Catecholamines on Prolactin Release -I_-rlVitrOOOOOOOOOOOOOOOOOOIOOOOOOOO0.0 A. DOW Objective............................... Materials and Methods................... Results................................. Conclusions............................. vii Page 79 79 80 81 83 87 9O 91 91 92 92 93 94 94 95 98 101 103 103 103 103 105 110 TABLE OF CONTENTS--continued Page II. Effects of Iproniazid and Reserpine Adminis— tration In Vivo on Hypothalamic Catecholamine Content and Prolactin Inhibiting Activity.... 112 A. Objectives.............................. 112 B. Materials and Methods................... 113 C. Results................................. 115 D. Conclusions............................. 117 III. Removal of the Prolactin Inhibiting Activity of Hypothalamic Extracts I2_Vitro by Pretreat- ment of the Extracts with Monoamine Oxidase and Aluminlm OXide O O O O O O O O O O I I O O O O O O I O O I O O O O O 118 A. Objective. 0 O O 0 O O O O O O O O O 0 O O O O I O O O O O O O O O O O 118 B. Materials and Methods................... 118 C. Results. 0 O O O O O O O O O O O O O O O O O O O O O I O O O O O O O O O 122 D. conCIuSionS O O O O 0 O O O O O O O O O O O O O O O O O O O O O O O O 129 IV. Direct Effects of an Ergoline Derivative on Anterior Pituitary Prolactin Release In Vitro: Blockade by Pimozide, a Specific Dopamine Receptor BlOCker O O O I O O O O O O O O O O O O O O O O O O O O O I O O O 132 A. Objective 0 I O O O O O O O O O O O O O O O O O O I O O O O O O O O 0 O 132 B. Methods and Materials................... 132 C. ResultSOOOO0.0...OOOOOOOOOOOOOOOOOOOOOOO 133 D. conCIuSions O O O O O O O O O O O O O O I O O O O O C O O O O O O O O 136 V. The Effects of Dopamine Infusion on the Proestrus Afternoon Rise in Serum Prolactin in Normal Cycling Female Rats: Chronic Cannulation Studies.......................... 137 A. ObjectiveSOOOOOOOOOOOOOOOOOOCOOOOOOCOOOO 137 B. Methods and Materials................... 138 C. Results................................. 140 D. conCIuSionSOOOOCOOOOOOOOOOOOOCOOCCOOOOOO 140 VI. The Effects of Systemic Infusion of Dopamine and L-DOPA on Serum Prolactin in Rats with Bilateral Median Eminence Lesions: Acute Cannulation Studies.......................... 142 A. Objectives.............................. 142 B. Materials and Methods................... 142 viii TABLE OF CONTENTS--continued Page C. Results.......... ....... ................ 144 D. Conclusion.............................. 144 GENERAL DISCUSSIONOOOOOOOOOOOO...OOOOOOOOOOOOOOOOOOOO 147 CURRICULUM VITAE ..... ............. ........ ........... 158 REFERENCES. ..... ..................................... 161 ix Table 10. LIST OF TABLES Effect of Dopamine Hydrochloride on Anterior Pituitary Prolactin Release In_Vitro............ Effect of Norepinephrine Hydrochloride on Anterior Pituitary Prolactin Release In_Vitro... Effect of Epinephrine Bitartrate on Anterior Pituitary Prolactin Release In Vitro............ The Effect of Catecholamines on the Measurement of a Standard Dose (20 ng/ml) of Rat Prolactin RP-IOOOOICOOOOOOOOOOOOOO.0...OOOOOOIOIOOOOOOOOOO Effect of Pretreatment of Female Rats with Iproniazid and Reserpine on the Prolactin Inhibi- ting Activity In Vitro and Catecholamine Content of Hypothalamic Extracts Prepared from those RatSOCOOOOOCCOCOOOOOO0.0.0.0...0....0.000.000... Effect of Rat Brain Monoamine Oxidase on the Ability of Rat Hypothalamic Extracts to Inhibit Pituitary Prolactin Release In Vitro............ The Effect of Monoamine Oxidase Treatment of Hypothalamic Extracts on Rat Pituitary Prolactin Release £1.VitrOOOOOOOOOCOOOOOOOOOOIOOOOOOOOOOCC Lack of Effect of Rat Brain Monoamine Oxidase of Anterior Pituitary Prolactin Release In Vitro... Effect of Blockade of Rat Brain Monoamine Oxi- dase with Iproniazid on the Ability of Hypothal- amic Extracts to Inhibit Pituitary Prolactin Release In_Vitro................................ Effect of Catecholamine Adsorption on the Ability of Hypothalamic Extracts to Inhibit Pituitary Prolactin Release I2_Vitro............ Page 106 107 108 109 116 123 124 126 127 128 LIST OF TABLES--continued TABLE 11. 12. 13. 14. Page The Effect of Pepsin Treated Hypothalamic Extracts and Pepsin Treated Aluminum Oxide Eluates on Prolactin Release In_Vitro........... 130 Effect of Receptor Blockers of the Ability of Lergotrile Mesylate to Inhibit Prolactin Release from Anterior Pituitary Tissue In Vitro......... 135 Effects of Dopamine Hydrochloride Infusion on Serum Prolactin Concentration During the After- noon of Proestrus in Unrestrained Chronically Cannulated Female Rats.......................... 141 Effects of Dopamine Hydrochloride and L-DOPA in Female Rats with Bilateral Median Eminence LeSionSOOOOOOOOOOOOO0.00...OOOOOOOOOOOOOOOOOOOO. 145 xi FIGURE 1. LIST OF FIGURES Page Biosynthetic pathway of catecholamines......... 28 Catabolic pathway of catecholamines............ 31 Photograph showing the completed chronic cannu- lation preparation............................. 88 Dose response curves for dopamine, norepine- phrine and epinephrine based on their ability to inhibit prolactin release from rat anterior pituitary tissue in_vitro...................... 111 In_vitro inhibition of prolactin release by lergotrile meSYIateOOOOIOOOOOOOIOOOOOOOOIOOOOOO 134 xii INTRODUCTION The demonstration of the influence of the hypothalamic hypophysiotropic hormones (HHH) on anterior pituitary func- tion and the recent biochemical characterization of at least three such polypeptide neurohormones have added conclusive proof to the "chemotransmitter" hypothesis forwarded by G. W. Harris (1955). Currently there are extensive efforts underway to characterize the remaining HHH including the elusive prolactin inhibiting factor (PIF). The catecholamines, dOpamine and norepinephrine, and the indoleamine, serotonin, are found in high concentration in the medial basal hypothalamus. In addition to their neurotransmitter functions, these monoamines have been strongly implicated as controllers of HHH synthesis and release. Pharmacological and physiological factors which alter the activity of the hypothalamic monoamines have been shown to also alter the synthesis and release of several anterior pituitary hormones presumably by altering the releaseand synthesis of the individual HHH. The relation- ship of hypothalamic catecholamine activity to the secretion of prolactin has been studied extensively in the past several years. These studies have indicated that factors which increase or decrease hypothalamic catecholamine activity cause inhibition and stimulation of prolactin release, respectively, primarily by increasing or decreasing the release of hypothalamic PIF into the hypophyseal portal vessels. The recent discovery that pharmacological amounts of dopamine and norepinephrine can affect prolactin release in_vitro by a direct action on anterior pituitary tissue has led some researchers to investigate the possibility that such a direct action may occur in_yiyg under normal physiological situations. In spite of their direct effects on prolactin release in_yit£g, the indirect and direct effects of the catecholamines in_yiyg have been very diffi- cult to demonstrate. Early work reported that neither catecholamine perfused directly into the hyp0physeal portal vessels (perfused directly into the anterior pituitary) nor catecholamine administered by systemic injection had any effect on pituitary prolactin release in_yiyg. Further, the fact that large pharmacological doses of the catechol- amines were needed to demonstrate the direct action of these amines on prolactin release in yiE£2_has therefore raised the question as to the physiological significance of such a direct action. The work presented in this thesis is devoted to the demonstration that small physiological amounts of both dopamine and norepinephrine cause marked inhibition of prolactin release in yiE£g_and that dopamine is by far the more potent inhibitor of prolactin release. This thesis also demonstrates that the endogenous dopamine and norepine- phrine content of hypothalamic extracts can account for the inhibitory activity of the hypothalamic extracts on pitui- tary prolactin release in vitro. Evidence is presented that supports the concept of the existence of dopaminergic receptors present on the anterior pituitary lactotrophs which are capable of mediating the release of prolactin from the anterior pituitary. Very recent evidence in the literature has suggested that doPamine and norepinephrine infused directly into the hypophyseal portal vessels can inhibit prolactin release by a direct action in yiyg. This work is in direct opposition to earlier research reports. In support of this more recent evidence, this thesis presents in_yiyg_data which suggest that small pharmacological doses of dopamine and its direct precursor, L-dihydroxyphenylalanine (L-DOPA) administered by intra-arterial perfusion can significantly inhibit prolactin release in yiyg. This evidence further indicates that dopamine administered in this manner in- hibits prolactin release in_yiyg_by both a direct and indirect action on the anterior pituitary tissue. In view of the evidence presented in this thesis and reports presently in the neuroendocrinological literature, it is becoming evident that in addition to a polypeptide PIF of hypothalamic origin, a catecholamine, presumably dopamine, possibly represents still another formidable agent for inhibiting prolactin secretion. REVIEW OF LI TE RATURE I. Functional Neuroanatomy of the Hypothalamo-hypophyseal System A. Functional Anatomy of the Hypothalamus Many books and reviews have been written concerning the neuroanatomy of the hypothalamus (DeGroot, 1959; Daniel, 1966; Netter, 1967; Jenkins, 1972). By using all these literature sources one can obtain a concise description of the anatomy of the hypothalamus. The hypothalamus is the ventral-most portion of the, diencephalon and is exposed on the central surface of the brain. The hypothalamus comprises the lateral walls of the third ventricle below the hypothalamic sulcus and the struc- tures of the ventricular floor. The structures making up the floor of the third ventricle are the optic chiasm, tuber cinerium and infundibulum, neurohypophysis and the mammillary bodies. The region of the brain just anterior to the optic chiasm extending to the lamina terminalis and the anterior commissure is identified as the preoptic area. A perpendicular line passing through the posterior edges of the mammillary bodies is considered to be the caudal boundary of the hypothalamus. The thalamus and subthalamus make up the dorsal and lateral boundaries of the hypothala- mus respectively and the third ventricle divides the hypothalamus medially into right and left halves. The pituitary gland is attached to the brain by the infundibulum or pituitary stalk. The upper portion of the infundibulum has a hollow center which represents the ventral extension of the third ventricle. The neurohypo— physis, the posterior lobe of the pituitary gland, is composed of tissue originating from neural ectoderm and is continuous with the basal portion of the hypothalamus. Three distinct regions can be identified when the hypo- thalamus is analyzed in a rostral to caudal sequence: the supraoptic, tuberal and mammillary areas. Within these rather distinct areas there are specific groups of nuclei. The supraoptic area lies above the optic chiasm and fuses rostrally with the preoptic area. The supraoptic region contains the supraoptic and paraventricular nuclei con- cerned primarily with the secretion of oxytocin and anti- diuretic hormone (ADH) respectively (Bargmann and Scharrer, 1951). The tuberal region of the hypothalamus refers to the area just dorsal to the tuber cinereum located on the central surface of the brain between the optic chiasm and mammillary bodies. The infundibulum attaches to the median eminence of the tuber cinereum. Near the infundibular attachment is located the periventricular nucleus or arcuate nucleus. Both names are used to describe the gray area enveloping the base of the third ventricle. Immediately lateral to the periventricular nuclear borders of the third ventricle are the dorsomedial and ventromedial nuclei. The mammillary or posterior hypothalamic region is named for the mammillary bodies, the most prominent structures in the region. The hypothalamus receives afferent information via the fornix, medial forebrain bundle, thalamohypothalamic fibers, mammillary peduncle and stria terminalis. The hypothalamo— hypophysial tract, periventricular fibers and mammillary efferents are the three major efferent fiber tracts of the hypothalamus. B. Hypothalamo-hypophyseal Portal System The importance of the hypothalamus in controlling the function of the anterior pituitary gland is now well- established because of the impetus set forth by Harris (1948, 1955, 1960). As there is no convincing evidence of a direct nerve supply to the secretory epithelial cells of the anterior pituitary, presumably stimuli reach the anterior lobe of the pituitary through the hypothalamo— hypophyseal nerve tract and thence through the hypothalamo- hypophyseal portal blood vessels. Popa and Fielding (1930) described a set of blood vessels running along the pituitary stalk which connected a capillary bed in the anterior pituitary and a capillary bed in the hypothalamus and therefore classified the blood vessels as a portal blood system. They suggested, based on morphological evidence, that the portal blood flow was directed from the pituitary gland toward the hypothalamus. Thereafter, other researchers described a similar hypo- physeal portal system but with a flow direction from the hypothalamus to the anterior pituitary (Houssay et_al., 1935; Wislocki and King, 1936; Green 1947; Green, 1948; and Xuereb §t_gl,, 1954). Green (1947) and Green and Harris (1949) observed the hypophyseal portal vessels in the liv- ing frog and rat respectively. Their research showed that the portal vessels originated in the median eminence of the hypothalamic tuberal cinereum and in the upper infundibular stem and observed blood flow leading to the anterior pitui- tary. Thereafter, similar observations were made in the living rat by Barrnett and Greep (1951), in the dog by Torok (1954) and in the mouse by Worthington (1963). While the infundibular process (posterior lobe) has a conventional arterial blood supply obtained from the in- ferior hypophyseal arteries, the anterior pituitary is not supplied directly by any artery but receives only portal venous blood (blood that has already passed through a first capillary bed). The exceptions to this phenomenon are the rabbit (Harris, 1947) and man (Stanfield, 1960). In these two species researchers believe that the epithelial cells of the anterior pituitary are supplied by a few arterial twigs as well as by portal vessels. The hypophyseal portal vessels which deliver the blood to the anterior pituitary tissue form two distinct groups, the long and short portal vessels. The long portal vessels run down the pituitary stalk and the short vessels lie at the lower level (Adams gt;al., 1966). Upon entering the second capillary bed, the sinusoids of the anterior lobe, the blood is carried past the parenchymal cells and is dis- charged through venules into venous sinuses which lie around the pituitary gland. The capillary bed in the median eminence and stalk feeding the long portal vessels is sup- plied by the superior hypophyseal arteries while the capil- lary bed which drains the short portal vessels is supplied mainly by the inferior hypophyseal arteries and by the artery of the trabecula, which is part of the superior hypo- physeal arteries. Each group of portal vessels, the long and the short, supplies a specific territory in the anterior pituitary. These specific areas and results of their vessel ablation are discussed in further detail for man (Xuereb §t_gl., 1954) and in the rat and the sheep (Daniel and Prichard 1956, 1957). More thorough discussion of the anatomy of the hypophyseal portal system is given by Adams §t_213 (1965/66), Daniel and Prichard (1956) and Green (1966). 10 C. Effects of Removal of Hypothalamic Influence on Anterior Pituitary Pr61actin Secretion and Histology l. The Effects of Stalk Transection When any tissue is deprived of its blood supply its cells die and an area of necrosis develops. The boundaries of this necrosis indicate the area supplied by the severed blood vessels. The histological changes which occur follow- ing complete pituitary stalk section has been thoroughly documented in the rat (Adams gt_al., 1963a), the goat (Adams §E_§l., 1964), the sheep (Adams e£_31., 1963b) and the monkey (Adams, 1963c). In their experiments complete stalk section interferred with the long portal vessels but did not interrupt the short portal vessels. They reported that within a few days after the operation a massive area of necrosis was found in the central and ventral region of the anterior pituitary gland. Small areas along the dorsal border regularly survived this operation indicating that these areas derive their blood supply from the short portal vessels (Adams et;gl,, 1965). There was also noted a thin superficial rim of surviving parenchymal tissue along the central and lateral borders of the anterior pituitary which they hypothesized was kept alive due to its close proximity to the vascular dura mater. It has been demonstrated that there are large concentrations of acidophilic cells capable of secreting prolactin and growth hormone located around 11 the peripheral areas of the anterior pituitary while the greater concentration of basophilic and chromophobic cells lies mainly within the central core of the anterior pitui- tary gland (Purves, 1961; Daniel et_al., 1964a, 1964b). Early hormonal studies indicated that the anterior pituitary deprived of its blood supply by stalk section was capable of autonomous secretion of prolactin (Everett and Nikitovich-Winer, 1963), while secretion of the other five anterior pituitary hormones was reduced to basal levels (Meites and Nicoll, 1966). 2. The Effects of Anterior Pituitary Transplantation Separation of the mammalian anterior pituitary from hypothalamic influence by transplantation beneath the kidney capsule results in increase prolactin release (Everett, 1954; Meites §£_§l,, 1963; Chen et_al,, 1970; Sud gt_gl., 1970; Shaar and Clemens, 1972) and reduced secretion of the other five anterior pituitary hormones (Meites e£_gl,, 1963). Early histological studies demonstrated that struc- tural changes in the normal anterior pituitary occur almost immediately following pituitary grafting procedure (Nikitovich-Winer and Everett, 1959). They found that as early as 24 hours after transplant the transplant was turgid, there was a central infarction and only a thin shell of healthy parenchymal cells remained functional. The. gonadotrophs and thyrotrophs at the periphery were reduced 12 in number and degranulated. In the next 3 days the shell of healthy parenchymal cells increased in thickness due mainly to cell proliferation and reduced size of the im— plant. By the end of 7 days they found that all cells of the established graft are much reduced in volume and the bulk are chromophobes and.acid0phils(Nikitovich-Winer and Everett, 1963). 3. The Effects of Electrolytic Lesions of’the Final Common Pathway The median eminence of the hypothalamus has been termed the final common pathway. This description has been used to designate the fact that all the various neural in- puts which influence anterior pituitary function must pass through the median eminence into the primary plexus of the hypophyseal portal vessels in the form of release or in- hibiting factors. Many investigators have used bilateral electrolytic lesions of the median eminence as means of eliminating hypothalamic influence on the pituitary (Chen §E_§l., 1970; Welsch et_al., 1971; Sud §t_al., 1970). It has been demonstrated that almost immediately following the placement of such lesions, serum prolactin levels increase drastically to very high levels and even though serum pro- lactin levels decline from their initial elevated levels, they remain significantly elevated above normal basal levels for at least 5 months (Welsch et al., 1971). 13 4. The Effects of In Vi ro Incubation of Anterior Pituitary Tissue Many investigators, using in 23:33 tissue culture and incubation techniques, have demonstrated the ability of anterior pituitary tissue to secrete substantial amounts of prolactin when completely removed from hypothalamic influ- ence (Meites et al., 1961; Talwalker et al., 1963; Meites and Nicoll, 1966). The anterior pituitary has been shown to be capable of releasing other hormones while under in_zi£rg conditions but will release increased amounts of the other hormones only when stimulated by releasing factors. In a very early experiment, it was demonstrated that histological examination of anterior pituitary tissue after 6-7 days of culture revealed that tissue exposed to air atmosphere was necrotic with only a thin margin of living cells remaining while tissue constantly gassed with 95% 02-5% CO2 consisted predominantly of viable cells and the tissue also compared favorably in appearance with fresh rat anterior pituitary tissue. The researchers stated that they had successfully cultured AP tissue for 21 days in an air atmosphere with continuous prolactin production (Meites et al., 1961). 14 II. Hypothalamo Hypophysiotropic Hormones A. Biochemical Nature To date three peptide hypophysiotrOpic hormones of hypothalamic origin have been isolated, biochemically char- acterized and synthesized: thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone (LRH), and growth hormone release-inhibiting hormone (Somatostatin). After several years of arduous work, principally in two laboratories, the structure of ovine TRH was established as the tripeptide pyro-glutamyl-histady1-proline-amide (Burgus §E_§l., 1969, 1970). The porcine hormone was shown to have the same structure (Nair et_al,, 1970). In 1971, LRH of porcine (Matsuo et_§l., 1971; Baba et_gl., 1971) and ovine (Burgus gE_§l., 1971) origin was characterized as the deca- peptide pro-glutamine—histidine-tryptophan-serine-tyrosine- glycine leucine-arginine-proline—glycine-amide. Another polypeptide of hypothalamic origin was recently character- ized. This tetradecapeptide of ovine origin called somato- statin has the following structure: H-alanine-glycine- cystine-lysine—asparagine-pheny1a1anine-pheny1alanine- tryptophan-lysine-threonine-serine-cystine-OH (Brazeau gt_§l., 1974). A somatostatin of porcine origin possessing the same biochemical structure has likewise been character- ized (Greibrokk et al., 1974). 15 TRH and LRH as well as many synthetic replicates and analogs have been shown to stimulate the release of radio- immunoassayable and bioassayable TSH and LH respectively. Natural and synthetic TRH, in addition to causing the release of TSH has been shown to stimulate prolactin release in_yit£g (Hill-Samli and MacLeod, 1974; Dibbet §E_gl., 1974; Smith and Convey, 1975) and in_yi!g_(Bowers §£;§l,, 1973; Mueller §£;Elf' 1973; Takahara e£;§l,, 1974) in several species including man, and to stimulate release of growth hormone in_yiyg in rats (Takahara gt_al., 1974). Likewise, Somatostatin has been shown to cause inhibition of growth hormone release ifl.!i££2.in rats (Carlson gt_§l,, 1974), in_!iyg_in rats (Chen et_al,, 1974) and in normal (Besser gt_gl., 1975) and acromegalic (Besser gt_gl., 1974) human subjects. Biological evidence demonstrating that crude hypothalamic extracts inhibit prolactin release in gi!2_(Dharwal et_gl., 1968; Amenomori and Meites, 1970), and in yitgg (Talwalker §E_al,, 1962) has prompted some investigators to attempt the isolation of prolactin inhibit- ing factor (PIF) (Greibrokk et_al., 1974; Schally gt_al., 1975). Both groups have reported isolation of peptide fractions free of catecholamines which significantly inhibit prolactin release in yitrg. Several laboratories have reported the isolation and characterization of other neurohormones which appear to influence other pituitary hormone release. It has been 16 reported that a tripeptide, H-Proline-Leucine-Glycine-NH2, from porcine hypothalamic extracts inhibits the release of melanocyte stimulating hormone (MSH) (Celis et_al., 1971; Nair gt_gl., 1971). This peptide, which corresponds to the C-terminal tripeptide sequence of oxytocin has been found in hypothalamic tissue incubated with oxytocin and may originate from the action of enzymes in hypothalamic tissue on oxytocin (Celis gt_31., 1971). It has been designated as MRIH—I and has been prOposed as the inhibitor of MSH (Hruby §E_§l,, 1972). Another peptide found in porcine hypothalamus, MRIH-II, H-Proline-Histidine-Phenylalanine- Arginine-Glycine—NHZ, has also been proposed as an MSH release-inhibiting factor (Nair et_al., 1972). Although corticotrophin-releasing factor (CRF), which releases adrenocorticotropic hormone (ACTH), was the first of the releasing factors to be studied, the chemical nature of hypothalamic CRF is elusive. It has been proposed by Chan gt_§l, (1969) that the factor is unstable. However sub- stances of posterior pituitary origin which have CRF activity, have been reported to be related, but not identi- cal, to MSH and vaSOpressin respectively (Burgus and Guillemin, 1970). B. Site of Origin Numerous experiments have demonstrated the role of the central nervous system and in particular the hypothalamus 17 in the control of anterior pituitary hormone secretion. Early investigations demonstrated that the anterior pitui- tary gland lost most of its histological characteristics when its vascular connection with the median eminence of the hypothalamus was interrupted (Harris and Jacobsohn, 1952; Nikitovich-Winer and Everett, 1959). Further, Halasz and Szentagothai (1962) introduced the term "hypophysiotropic area" to refer to the need for connection with that portion of the hypothalamus for the maintenance of normal anterior pituitary function and histology. The chemotransmitter hypothesis of Harris (1955) proposed that neurohormones released by the hypothalamus are conveyed to the anterior pituitary gland via the hypophyseal portal blood system. These hypothalamic neurohormones are responsible for regu- lating anterior pituitary function and hence have been given the name hypophysiotropic hormones. Early experiments demonstrated that transplanting anterior pituitary tissue fragments into the medial basal hypothalamus maintained their histological integrity and they remained functional (Halasz and Szentagothai, 1962; Halasz et al., 1965; Flament-Durant, 1965). This medial basal area included the arcuate nucleus and the medial parvicellular region of the retrochiasmatic area. In recent years several assay systems have been employed to verify the presence of the various hypOphysiotropic hormones in 18 specific hypothalamic nuclei. In 33559 incubation of minute hypothalamic sections has located LRH activity mainly in the medial tuberal region, an area which includes the median eminence, arcuate nucleus, and more rostrally in an area which includes the suprachiasmatic nucleus and all but the most rostral portion, the preoptic nucleus (Creighton gt_al., 1970). Immunohistochemical studies have recently also localized LRH in the nerve endings of the same medial basal hypothalamic area (Zimmerman §£_31,, 1974; Pelletier g£_§l,, 1974; Kordon et_al,, 1974). Guillemen et_§1. (1965), found equal concentrations of TRH activity measured separately in the median eminence and the rest of the hypothalamus in sheep. Other workers have determined that the TRH activity in the median eminence is twice as high as that found in the rest of the hypothalamus (Averill and Kennedy, 1967). Recently TRH activity distribution was determined by the thyroid stimulating hormone (TSH) releasing ability in yitgg' of minute histological sections of the hypothalamus (Krulich et_al., 1974). TRH activity was shown to be con- centrated in three separate locations: the median eminence, dorsomedial hypothalamic nucleus and the preoptic area. It was concluded that the dorsomedial nucleus and the pre- optic area are sites of biosynthesis from which TRH is conveyed to the median eminence presumably along axons. The finding of high concentration of TRH synthetase in the l9 dorsomedial hypothalamus substantiates this dorsomedial hypothalamic prosynthetic location (Reichlin and Mitnick, 1973). There are also presently reports of substantial extra hypothalamic concentrations of TRH in the rat brain (Oliver §E_al., 1974). Somatostatin activity has been located mostly concen- trated in the external layers of the posterior median eminence and in the ventral medial hypothalamic nucleus of the guinea pig (Hokfelt gt_al., 1974) and also of the rat (Pelletier e£_gl,, 1974). Both these groups of investiga- tors used immunofluorescence and peroxidase labelled anti- body to measure somatostatin in hypothalamic tissue sections. Prolactin inhibiting factor (PIF) has also been specifically located within the basal medial hypothalamus (see Section IV, A). C. The Influence of the Biogenic Amines on the Release of Gonadotropin Releasing Factor: An Example of Biogenic AmIHe-hypophysiotropic Hormone Interaction At least three peptide hypothalamo—hypophysiotropic hormones (HHH) have been chemically characterized and many other HHH activities have been demonstrated by in yitrg and in zizg_techniques. HHH biosynthesis and release into the hyp0physeal portal system appears to be under the control of the biogenic amines including the catecholamines, indole- amines and possibly other neurotransmitter substances such 20 as acetylcholine, histamine and Glutamic Amino Butyric Acid (GABA). The effects of the biogenic amines on the release and synthesis of prolactin inhibiting factor (PIF) are dis- cussed in another section (see Section V, A). Aside from their effects on hypothalamic PIF, the effects of the bio- genic amines on Gonadotropin Releasing Factor (LRH-FSHRF) have been most thoroughly studied. In early studies it was shown that an adrenergic blocking drug, dibenamine, could block ovulation (Sawyer §£;31., 1947). When various amines were incubated with anterior pituitary tissue in_yitgg, little effect was ob- served on release of luteinizing hormone (LH) and follicle stimulating hormone (FSH), but when a co-incubation system was used in which ventral hypothalamic fragments were incu- bated with anterior pituitaries, it was observed that the addition of dopamine to the incubation medium increased the release of both FSH (Kamberi gt_al., 1970b) and LH (Schneider and McCann, 1969). Since dopamine did not alter the action of added gonadotropin-releasing factors, it was concluded that it evoked the release of these factors from the ventral hypothelamic fragments. In these experiments the response to dopamine was blocked by the alpha adrenergic blocking agent phentolamine, but not by pronethalol, a beta blocker. The releasing action of dopamine was also blocked by estradiol (Schneider and McCann, 1970c) and the blockade 21 prevented by addition of puromycin and cyclohexamide, both inhibitors of protein synthesis. Dopamine, but not nor- epinephrine or epinephrine, injected directly into the third ventricle dramatically increased LRF levels in the peripheral plasma of hypophysectomized rats (Schneider and McCann, 1970c), and this stimulation was blocked by es- tradiol. Likewise, dopamine administered intraventricularly was shown to increase plasma LH and FSH and LRH activity in the hypophyseal portal blood (Schneider and McCann, 1970b; Kamberi gt_§l,, 1970a, 1970b). Norepinephrine has been reported by some workers to be less effective than dopamine in releasing LRH (Kamberi §E_§1., 1970a; Schneider and McCann, 19700), while other workers have demonstrated that intraventricular administra- tion of norepinephrine and not dopamine was highly effective in causing ovulation in rats (Rubinstein and Sawyer, 1970) and rabbit (Sawyer gt_al., 1974). Weiner et_§l. (1972) found that d0pamine was relatively ineffective in stimulat- ing electrical changes in the median eminence which were readily evoked by epinephrine or norepinephrine. Further, Quijada §£_al, (1973/74) failed to confirm the stimulatory effect of d0pamine, and Miyachi et_al. (1973) reported that dopamine actually blocked LH release in_yi££g. Recent reports (Kalra gt_gl,, 1972; Kalra and McCann, 1973; Ojeda and McCann, 1973; Kalra and McCann, 1974) have supported 22 norepinephrine as the catecholamine which facilitates LH release. It has recently been stated that much of the early work employing intraventricular injection and hypo- physeal portal vein perfusion techniques (Kamberi gt_al., 1969, 1970a, 1971a, 1971b, 1971c) have been diffi- cult to reproduce (Cramer and Porter, 1973). In their experiments, Cramer and Porter (1973), intraventricularly administered epinephrine stimulated LH release in estrogen- treated and in untreated rats and inhibited LH release in castrated male rats. Norepinephrine did not alter LH release in untreated or estrogen-treated rats but did inhibit LH release in castrated male rats. FSH was not affected by catecholamines injections. Receptor blockers have also been used during various physiological states to determine their effects on gonado- trOpin release. It has been shown that phentolamine, an alpha receptor blocker, can block the post castration rise in FSH and LH (Ojeda and McCann, 1973), and the pulsatile release of LH which occurs in ovariectomized monkeys (Bhattacharya §t_§l,, 1972). Pimozide, a dopamine receptor blocker, produced no effect and a small reduction in the post castration rise in LH and FSH respectively (Ojeda and McCann, 1973). Alpha methyl tyrosine, which inhibits tyrosine hydrox— ylase, can block the postcastration rise in serum LH but 23 not FSH and the blockade can be reversed by administration of L-dihydroxyphentlalanine (L-DOPA) and dihydroxyphenyl- serine (DOPS) to reinitiate the synthesis of dopamine and norepinephrine or only norepinephrine respectively (Ojeda and McCann, 1973). Alpha methyl tyrosine can also block the stimulatory effects on gonadotropin release of either estrogen or progesterone in estrogen-primed rats (Kalra gt_al., 1972; Kalra and McCann, 1973), and can block the preovulatory discharge of gonadotropins during the afternoon of proestrus in rats (Kalra and McCann, 1974). Selective impairment of norepinephrine synthesis has resulted in blockade of gonadotropin release in castrate, estrogen or estrogen-progesterone-primed rats and the normal preovula- tory release in rats (Ojeda and McCann, 1973; Kalra and McCann, 1974; Kalra §t_al., 1972). Assay of endogenous hypothalamic catecholamine content and turnover has given much insight into the relationship of the catecholamines and gonadotropin control. There is an increase in hypothalamic norepinephrine on the day of proestrus in rats (Donoso et_al., 1971) and also at the time of the ovulatory release of gonadotropin. Brain norepine- phrine turnover is also increased in castrated rats (Anton- Tay and Wurtman, 1968). During the proestrous period, dopamine turnover is reduced (Fuxe et_gl., 1973). Subcutaneous administration of atropine was shown to block ovulation (Markee et al., 1952). More recently, 24 atropine, administered either subcutaneously or intra- ventricularly, was shown to block gonadotropin release (Libertun and McCann, 1974a). The doses of atropine were rather large, being one-fourth the LD50 for intraventricular injections. A recent report was shown that large doses of acetylcholine can increase FSH release from a co-incubation system of ventral hypothalami and anterior pituitaries in_!itrg (Simonovic et_al., 1973), and systemic administra- tion of pilocarpine or eserine produce immediate inhibition of gonadotropin release followed by a delayed release in ovariectomized estrogen-primed rats (Libertun and McCann, 1974). Both serotonin and melatonin have been shown to inhibit gonadotropin release following injection into the third ventricle of rats (Schneider and McCann, 1970b; Kamberi et al., 1970b) but parachlorophenylalanine (PCPA), an in- hibitor of serotonin biosynthesis had little effect on gonadotropin release in male rats (Donoso et al., 1971). A more recent report states that serotonin administered intraventricularly stimulates LH release in untreated and estrogen-treated female rats and has no effect on FSH release (Cramer and Porter, 1973). Histamine has long been known to be concentrated in the basal tuberal region (Harris et al., 1952) and appears 25 to be found in synaptosomes there (Snyder e£_al., 1974). It has recently been shown that intraventricular histamine, at relatively high doses, can release gonadotropins in ovariectomized, estrogen-primed rats (Libertun and McCann, 1974a, 1974b). III. Biogenic Amines in the Hypothalamus A. General Substances generally accepted as serving as neurotrans- mitters in the brain include acetylcholine and three mono- amines: dopamine, norepinephrine and serotonin. Dopamine and norepinephrine are catecholamines whereas serotonin is an indoleamine. The use of histochemical fluorescence (Falck §E_gl., 1962), photospectrofluorometric (Laverty and Taylor, 1968) and enzymatic (Christensen, 1973) monoamine assays for the identification and quantitation of these amines has demonstrated that norepinephrine, dopamine and serotonin are in high concentration in the hypothalamus (Vogt, 1954; Brodie et_§1., 1959; Carlsson §£_§l., 1962; Fuxe and Hokfelt, 1966; Constantinidis, 1969; Kavanagh and Weisz, 1974). More specifically, it has been shown that the median eminence and the nigrostriatal system are rich in dopaminergic nerve terminals (Dahstrom and Fuxe, 1965; Anden et al., 1964). 26 Fluorescence produced in histochemically prepared rat median eminence tissue consistently has been reported to be predominantly due to dopamine with a small contribution from norepinephrine (Fuxe, 1964; Corrodi §E_§l,, 1970; Jonsson et al., 1971; Kavanagh and Weisz, 1974). The stalk median eminence of goat, sheep, cat, and rabbit contains predominantly dopamine (Laverty and Sherman, 1965) whereas in cattle, pig and man the concentration of norepinephrine equals or exceeds that of dopamine (Rinne and Sonninen, 1967). To clarify the spatial localization of dopamine and norepinephrine in the basal hypothalamus of the rat, Kavanagh and Weisz (1974) assayed the superficial, inter— mediate and deep portions of the hypothalamus for dopamine and norepinephrine. They demonstrated that the superficial portion comprised mainly of the median eminence had the highest concentration of dopamine whereas the deeper por- tions contained decreased amounts of dopamine. In contrast, norepinephrine concentrations did not differ in the three respective areas. The high content of dopamine in the median eminence appears to be due to this catecholamine localized in the nerve terminals surrounding the primary capillary bed of the hyp0physial portal blood system (Fuxe, 1964). These nerve terminals may represent one important site where biogenic monoamines exert their influence on anterior pituitary function (Fuxe and Hkaelt, 1969; Wurtman, 1970). 27 In addition to their function as neurotransmitters, dOpamine and norepinephrine may function as neurohormones. The cate- cholamines may be released into the hypothalamo-hypophyseal portal system from neurons which surround the primary capillary bed of the portal system (Fuxe et_§l,, 1967; Ben- Jonathan and Porter, 1956b; Van Maanen and Smelik, 1968). Some workers have speculated that dopamine or norepinephrine may represent hypophysiotropic hormone which act directly on the anterior pituitary gland to regulate hormone secre- tion (Van Maanen and Smelik, 1968; Shaar and Clemens, 1974; Ben-Jonathan §£_gl,, 1975b; Takahara et_31., 19740). There is now some evidence that at least norepinephrine (Ben- Jonathan §£_§l., 1975b) in free form and dopamine (Ben- Jonathan gt_§1., 1975a) in bound form are present in the hypophysial portal blood. B. Biosynthesis of Brain Monoamines (Figure l) The biosynthesis of catecholamines begins with tyrosine. The four enzymes involved in catecholamine biosynthesis do not have the same subcellular distribution and therefore there is subcellular migration of substrates for these enzymes as tyrosine is converted to norepinephrine within the central nervous system. The hydroxylation of tyrosine is catalyzed by L-tyrosine hydroxylase and results in the formation of the catechol-amino acid L-dihydrophenylalanine (L-DOPA). The enzyme tyrosine hydroxylase is distributed 28 EPINEPHRINE PHENYLE‘I’HANOL— AMI NE-N -METHYI. TRANSFERASE NOREPINEPHRINE ooumm E p-oxnoAse DOPAMINE DOPA DECARBOXYLASE DOPA TYROSINE HYDROXYLASE TYROSINE PHENYLALANINE monoxvuse PHENYLALANINE F‘ o u lgure 1. Biosynthetic pathway of catecholamines. s“ f: 29 in the cytoplasm and is believed to be the rate limiting enzyme in the catecholamine biosynthetic pathway (Costa and Neff, 1970; Levitt et_§l., 1965). L-DOPA is decarboxylated and transformed to dopamine by the non-specific cytOplasmic enzyme, L-aromatic amino acid decarboxylase (referred to in this case as DOPA-decarboxylase). Thus decarboxylation steps in the synthesis of the biogenic amines (catechol- amines, tyramine, serotonin and histamine) are all catalyzed by this enzyme. The transformation of dopamine to norepine- phrine is catalyzed by the enzyme dopamine-B- hydroxylase. This enzyme is localized within norepinephrine storage granules located in the presynaptic neuron (Livett g£_al., 1969). At the present time there is no clear evidence that epinephrine is synthesized by any part of the mammalian brain for use as a neurotransmitter. However, phenylethanol- amine N—methyltransferase (PNMT), an enzyme which catalyzes the conversion of norepinephrine to epinephrine, has been identified throughout the brain of the rat and rabbit (Axelrod, 1962). ' The biosynthesis of brain serotonin (5-hydroxytrypt- amine, - 5-HT) involves the conversion of the amino acid tryptophan to 5-hydroxytryptophan in the presence of the enzyme tryptophan hydroxylase. The conversion of 5-hydroxy— tryptophan to S-hydroxytryptamine is catalyzed by the enzyme aromatic L—amino acid decarboxylase (Wurtman, 1970). 30 C. Physiological Disposition of Brain Monoamines TFigure 2) The metabolism of catecholamines involves primarily two enzymes: catechol-o-methyl transferase (COMT) and monoamine oxidase (MAO). Both enzymes act on a wide variety of amines and each is fully active on the product of the other. The enzyme MAO is generally considered to be bound intracellularly to the mitochondria while COMT appears to have high activity within the synaptic cleft (Anton-Tay and Wurtman, 1971). Histochemical analysis techniques have demonstrated that the catecholamines are more concentrated within the nerve terminal synaptic vesicles than in other parts of the neuron (Dahlstrom and Fuxe, 1965; Potter and Axelrod, 1963). By contrast serotonin is homogenously distributed within the serotonin-containing neuron (Anden et al., 1965). Nerve stimulation causes release of mono- amine molecules from the synaptic vesicles. The monoamines traverse the synaptic cleft and interact with the post synaptic membrane receptors. COMT and MAO act on monoamines to produce physiologically inactive metabolic products, but neither of these enzymes play an important role in terminating the physiological actions of the neurotrans- mitters. Physiological activity of dopamine and norepine- phrine is terminated by the energy requiring active process of re-uptake into the nerve terminal. Most of the mono- amines present in the neuron except that present in vesicles 31 99.. uzmozSz eo< osmozIwO._< U_._mOZ<<< mZEImmZK—mEaOZJSqWI wZEImmZEmaOZ >XO¢O>IIVI>XOIhm<.mm mmmmHmm cauomHoum humuwsuwm uofluoucd co mowuoasoouoamnmcfladmoo mo uommmm .H manna 107 umou =u: omuamm on» mean: mocmuommao coma mo uouum oumocmum.fl oocmummmao cmmzo cmmZU mumuasuam uoaumucm u ado caUUMaoum aImmI024Hz on mm ommmmumxmm . .I . 3. I ma. E oa xmmi a5? ooa + “Ra + O O O ' Aaoo vmv vo mm om. Aao vmv vo +na am. v n mm< . .I . am. . .I . 8. E oaxvmi a5? ov + v22 Aaoo vmv mo +mm ov. Aaoo vmv ao +va vv. v wI mam . .I . mm. . .I . mm. E 3358 EB: om + $2 Aao vmv mo +aa av. Amo vmv mo +aa mv. v m wad I X. I 3.. E oaxmmi dime S + v22 0 0+ I 0 0+ 0 I Amo vmv no mo mv. Amo vmv no ma mm. v m wad . .I . mm. . .I . 3. E oaxmvé as}: o.m + «.34 Amo X: 8 +3 mm. 30 v.3 ao +8 3. v m: “Ra I. mm. .I ov. A2 oaxmm.vv aE\mc o.a + mam mg 0+ 0 ma 0+ 0 | A V vo mo pm. A v mo 50 mv. v m Nam I mm. I 3. E oaxmvé aexmc me + “Ea mg 0 0 mg 0 0 II A v vo +mo vm. A v No +mo av. v m xmd .I mm. .I mv. ocaamm + ram m: . . m: . . A V we +mo mm. A V poo +mo av. v wad o o N mxm a mxm a mxm Ammaozv aE\mc M mommae m4 mE\m1 ommmmawm cauomaoum mmfldfi whflfldflU MO muflmm mmoo 0cm macauapcou ouua>.mm mmmoamm aauomaoum humuasuam Hoaumucd :o mpauoagooupmmImsaunmmcammuoz mo uommwm .N manna 108 ummu :u: pmuamm osu mcams mocmummmao some umnu mo uonum pumpcmum.fl mucoummmao cmoz p cmmzo humuasuam uoauoucm u m.mm mmmmamm cauUMaoum humuasuam Hoaumcd co mumuuumuam mcansmmcamm mo uommmm .m wand a 109 Table 4. The Effect of Catecholamines on the Measurement of a Standard Dose (20 ng/ml) of Rat Prolactin RP-l Catecholamine Number of Amount of Rat (Dose/m1) Culture Tubes Prolactin ng/ml . a Dopamine 4 16.4:2.0 (100 ng/ml) Norepinephrine 4 l7.0:2.5 (100 ng/ml) Epinephrine 4 18.8:2.8 (100 ng/ml) Control (Medium 199) 4 17.4:l.9 aMean : standard error of the mean. 110 D. Conclusions The data presented from these experiments indicate that low concentrations of dopamine and norepinephrine can directly inhibit anterior pituitary release in yitrg. The inhibition reported is not due to the inactivation of radio- immunoassayable rat prolactin by the catecholamines. The smallest amounts of dopamine (1.0 ng/ml to 10 ng/ml) and norepinephrine (5 ng/ml to 20 ng/ml) are less than the concentrations of each reported to be present in the rat hypothalamus (Lippman, 1968; Donoso et_§l,, 1969; Coppola, 1969; Kavanagh and Weisz, 1973/74) and less than those con- centrations of each found in hypothalamic extracts (Shaar and Clemens, 1974). The data are in agreement with those previous observations that catecholamines can directly inhibit prolactin release in yi3£g_(MacLeod, 1969; MacLeod and Lehmeyer, 1973; Birge et_al., 1970; Schally et_gl., 1974; Dibbet _e_t_al_., 1974; Greibrokk M., 1974), and in_!iyg_(Takahara et_al., 1974; Schally et_al., 1974; Davis et_gl., 1975). These data differ from those of Koch et_§l. (1970) who reported that 10 or 20 ng of epinephrine or norepinephrine per ml of incubation medium significantly increased prolactin release while 200 to 1000 ng of epine- phrine or norepinephrine per ml of incubation medium sig— nificantly inhibited prolactin release in yitrg, They further reported that the addition of 80 to 640 ng of dop- amine per m1 of incubation medium was inhibitory, whereas, 111 CD =3 30 .JltOI E i o DOPAMINE g: .25. A NOREPINEPHRINE "*1" g g o EPINEPHRINE E § 20‘ 101.05 a a 2 .5. :5 E u_ E 121.021 ca‘g . 3 8 .10« MN 001.02 2 V E g .054 a “3"“: ~ . . 31.05 3 021.02 021.04 02.02, . . {011,03 ‘ 2 0 0.5 1.0 5.0 10.0 20.0 40.0 100.0 09 CATECHOLAMINE PER ml INCUBATION MEDIUM Figure 4. Dose response curves for dopamine, norepinephrine and epinephrine based on their ability to inhibit prolactin release from rat anterior pituitary tissue in_vitro. The abscissa is ng of catechol- amine per m1 and the ordinate is mean difference in prolactin release in pg/mg pituitary tissue. 112 lesser amounts had no effect on prolactin release ig_vitro. The results do not agree with the earlier in_yiyg_work by Kamberi et a1. (1971). The apparent causes of these differ- ences are discussed in the general discussion section of this dissertation. Similar in_vitro work using rat anterior pituitary tissue has pharmacologically characterized a dopaminergic receptor capable of mediating the inhibition of prolactin release (Smalstig and Clemens, 1974; Clemens et al., 1975). II. Effects of Iproniazid and Reserpine Administration 1n_yiyg on Hypothalamic Catecholamine Content and Prolactin Inhibiting ActiVity A. Objectives Many researchers have shown that agents which increase or decrease hypothalamic catecholamine activity lead to in- creased or decreased in_vitro prolactin inhibiting activity, respectively. However, no evidence has appeared in the neuroendocrinological literature which clearly demonstrates that the in_yiyg treatment with such pharmacological agents actually alters the catecholamine content of the subsequent hypothalamic extracts. The objectives of this experiment were to demonstrate that iproniazid and reserpine treatment in_yiyg_could alter the catecholamine content of hypothal- amic extracts made from the pretreated animals. Further, that the increased (n: decreased catecholamine content of 113 the extracts increased cm' decreased the prolactin inhibit- ing activity respectively when tested in_vitro. B. Materials and Methods 1. Animals Adult female Sprague Dawley rats weighing approximately 225 g were used for drug treatment and hypothalamic extract preparation. Adult male Sprague Dawley rats weighing approximately 225 g were used as anterior pituitary donors for the in_gitrg incubations. Preparations of hypothalamic extracts, the in_yit£g incubation technique, and the methods for hypothalamic extract catecholamine measurement have been described in detail in the Materials and Methods sec— tion. The estrous cycles of the female rats were followed, as determined by daily vaginal smear, for at least 2 full cycles prior to use in the study. 2-D_rr19: Iproniazid Phosphate (Sigma Chemical Company) was dis- solved and reserpine (Eli Lilly and Company) was suspended in physiological saline (0.9% NaCl). Physiological saline was used as a diluent control solution. 3. Experimental Procedure On the first day of diestrus the female rats were treated with iproniazid phosphate (50 mg/kg), reserpine (5 mg/kg) or physiological saline (1.0 ml/kg) by intraperi- toneal injection. Three hours after drug administration, 114 the female rats were killed to obtain hypothalamic tissue for extract preparation. There was always a double set of hypothalamic extracts prepared so that one set could be used for catecholamine measurement and the other set could be used for the incubation studies. The volume of extract equivalent to 0.5 of a hypothalamus (0.5 HE) was placed into each appropriate incubation tube containing half a male rat anterior pituitary gland. In this manner one-half of the rat male pituitary gland received hypothalamic extract and the prolactin released from that anterior pituitary gland was compared to the prolactin released by the corresponding pituitary half incubated in Medium 199 alone. A similar paired incubation system has been used by other researchers to demonstrate the prolactin inhibitory activity of hypothalamic extracts. The incubations lasted 4 hours; at which time the anterior pituitary tissue was removed and weighed. The medium was measured for prolac- tin in order to determine the amount of hormone released per mg of anterior pituitary tissue. 4. Prolactin Measurement and Statistical Analysis The prolactin released into the incubation medium of individual culture tubes was measured by radioimmunoassay. The prolactin values are expressed in terms of the reference preparation NIAMDD rat prolactin RP-l. The data were analyzed using Student's "t" test for paired observations 115 to compare the response of a hypothalamic extract treated anterior pituitary half with its corresponding half exposed to Medium 199 alone. C. Results The results of this experiment are shown in Table 5. It can be seen that hypothalamic extracts prepared from saline pretreated rats inhibited prolactin release in yitrg by approximately 50 percent. The mean dopamine and nor- epinephrine content of the saline pretreated 0.5 HES were 15.3 1.3-2 ng and 27.3 i 2.0 ng, respectively. The mean serum prolactin levels in saline treated rats was 28.9 i 5.8 ng/ml which was normal for this strain of rats on the morning of diestrus day 1. When iproniazid was administered the serum prolactin levels were significantly reduced compared with those of saline-treated rats. The mean dopamine and norepinephrine content of hypothalamic extracts prepared from iproniazid pretreated rats was significantly higher compared with the extracts prepared from saline treated rats and likewise the iproniazid extracts possessed greater prolactin inhibiting activity than hypothalamic extracts from saline-treated rats. Reserpine pretreated rats possessed significantly elevated serum prolactin levels and the hypothalamic ex- tracts prepared from these animals contained decreased amounts of dopamine and norepinephrine and decreased 116 .muomuuxm oasmamsuomws aMEacm owummuu maaamm 0cm omummuu mayo «0 ucmucoo ocaunmwcammuoc cam mcaEmmoo some ca mucoummwao on» no mocmoamacmamw .mumu owumwuu mcaaMm on omummfioo mm coaumuucmocoo cauOMaoum Esumm some ca mucouommao on» no mocmoawacmam p .mSmmau ma ooummuu pom aouucoo an pwmmmamu cauomaoum comspmn mucouommao some on» no mocmoamacmam wnu mucmmmumomo .coaumuucoocoo cauomaoum Edumm came mo .m.m.H cmmzo A.msmmau m4 coupon» cam aouucoo Eoum mmmmamu cauOMaoum ca mocmummmao mucmmmummmV .mocoummmao same no .m.m + mocwummmap :mmZm mAflm.vmV mAflm.vmV I. I. Amx\mE mV m.a+w.m v.m+m.m Aaoo.vme.0m+o.omm m.ma Asz mo.+mo. ma mcamummom oAmmeV 1668 I I Ami? omV m.m+m.mm m.m+o.hMaAao.vmV m.m +m.oa m.mm oAao.vmV vo.+vv. ma mammacouma I I I . I A935 : m.m+m.ma m.m+m.hm om.m +m.mm a.om oAao.vmV m~o.+mm. ma mcaamm I aouucou mammas mE\mn mm m.o mm m.o Aooummuu m> aouucooV mnamm \.mm wua>auo¢ mcauanancH GauUMaoum on» co mqamummmm 0cm wannacouma duwz mumm mamamm mo unmaumwuumum mo muommmm .m wanna 117 prolactin inhibiting activity in_vitro compared with saline pretreated rats. D. Conclusions This experimental evidence demonstrates that pretreat- ment of rats in yiyg_with two pharmacological agents capable of altering the catecholamine activity of the hypothalamus alters both the catecholamic content of hypothalamic extracts prepared from those rats and the prolactin inhibit- ing activity of the extracts in vitro. Iproniazid, a mono- amine oxidase inhibitor, caused increased catecholamine content and prolactin inhibiting activity; and reserpine, a depletor of catecholamines, caused reduction of catechol- amine content and prolactin inhibiting activity of hypothal- amic extracts. This experiment confirms those findings of other researchers who demonstrated similar findings while employ- ing various catecholamine altering agents but who never measured the actual changes in catecholamine content of their hypothalamic extracts (Ratner et_al., 1965; Dickerman et al., 1972; Lu and Meites, 1972). 118 III. Removal of the Prolactin Inhibiting Activity of Hypothalamic Extracts In.YiLLQ.by Pretreatment of the Extracts with Monoamine Oxidase and Aluminum Oxide A. Objective The objective of these experiments was to determine if the endogenous hypothalamic content of dopamine and nor- epinephrine could account for the inhibitory activity of hypothalamic extracts on pituitary prolactin release in vitro. B. Materials and Methods 1. Animals Adult male Sprague Dawley rats weighing approximately 225 g each were used as pituitary gland and hypothalamic extract donors. The brains of 5 male Sprague Dawley rats of varying body weights were used to make the monoamine oxidase preparation. The method of removal of anterior pituitary glands for all incubations and method of hypothal- amic extract preparation are discussed in detail under the general Materials and Methods section. 2. Rat Brain Monoamine Oxidase Preparation The procedure for the rat brain monoamine oxidase (MAO) preparation is discussed under the general Materials and Methods section. MAO was tested for activity according to the procedure of Wurtman and Axelrod (1963), diluted with 0.05 M sodium phosphate buffer pH 7.4 and frozen at -20°C 119 until used. The enzyme potency was 0.185 nMoles/min/20 p1 of MAO as tested against serotonin as a substrate. The MAO was further tested and found to possess no proteolytic activity: no trypsin-like activity was found using benzoyl- arginine p—nitroanilide as a substrate and no chymotrypsin- like activity using acetyl tyrosine ethyl ester as the sub- strate. Incubation of the MAO with Luteinizing Hormone Releasing Hormone (LHRH) did not reduce the potency of the neurohormone in its ability to cause the release of LH from anterior pituitary tissue in_gitrg. 3. Pre—incubation of Hypothalamic Extracts with MAO and Iproni- azid Each neutralized hypothalamic extract (HE) to be pre- incubated with MAO was adjusted to its final volume and placed in a 5-ml culture tube containing 200 pl of MAO plus 50 pl of 0.05 M sodium phosphate buffer pH 7.4 and was pre— incubated in room air at 37°C for 30 min. Each control HE was pre-incubated under similar conditions without MAO. When iproniazid was employed it was added to each HE at a concentration of 10 u9/ml of final incubation volume at least 5 min prior to the addition of MAO. Iproniazid was found to be unable to inhibit peptidases: iproniazid was unable to inhibit the activity of trypsin (20 pg iproniazid per 3.2 pg trypsin) or of chymotrypsin (20 pg iproniazid per 2.0 pg chymotrypsin). 120 4. Aluminum Oxide Adsorption of Hypothalamic Extract Dopamine and Norepinephrine and Incubation of Aluminum Oxide Pretreated Extracts With Pituitary Tissue Pooled neutralized hypothalamic extracts prepared as discussed under the Materials and Methods section were divided into three separate groups: 1) those hypothalamic extracts to be combined with aluminum oxide (alumina) to facilitate catecholamine adsorption, 2) those which remained as neutralized extracts, and 3) those extracts titrated to pH 8.35 (optimum pH for catecholamine adsorption), held there for 30 min and then once again neutralized. Within the first group single hypothalamic equivalents were treated with alumina separately to facilitate more complete catecholamine adsorption. Both HCl and HClO. extracts were subjected to all treatments and catecholamine adsorption was carried out according to the method already described in detail under the materials and methods section. At the termination of the adsorption period each HE was separated from the alumina by centrifugation at 500 x g for 10 min at 4°C and each HE was retitrated to pH 7.0 and added to cul- ture tubes containing anterior pituitary tissue. The alumina containing the catecholamines was applied to previ- ously prepared glass columns, was three times rinsed with distilled water and the catecholamines were eluted with 5 ml of 0.2 N acetic acid. After evaporation of the acid eluate the catecholamines were reconstituted in 0.2 m1 of 121 distilled water, the pH was adjusted to 7.0 and each hypo- thalamic equivalent of catecholamines was added to appro- priate incubation tubes. In each case a separate set of hypothalamic extracts was treated in precisely the same manner but subjected to catecholamine measurement pro- cedures. 5. Preparation of Aluminum Oxide and Measurement of Hypothalamic Extract Dppamine and Norepinephrine The aluminum oxide (alumina) was prepared and acti- vated as described in detail under the Materials and Methods section. For each hypothalamic equivalent incu- bated with anterior pituitary tissue, the dopamine and norepinephrine content of a hypothalamic equivalent chemical- ly manipulated in the same manner was measured according to the procedure previously described under the Materials and Methods section. 6. Pretreatment of Hypothalamic Extracts with Pepsin When pepsin (Worthington Biochemical Corp. 3000 U/mg) was pre-incubated with hypothalamic extracts, the extracts were maintained at pH 2.0 with HCl and combined with a 0.5% solution of the enzyme for 4 hrs in room air at 37°C. Neutralization of the extracts terminated the enzyme activ- ity of the pepsin. Medium 199 also was pre-incubated with a solution of pepsin under the same conditions. Upon termination of the pepsin pre—incubation, both the 122 hypothalamic extracts and the Medium 199 were titrated to pH 7.25 and placed in the appropriate incubation tubes containing anterior pituitary tissue. C. Results 1. The Effect of MAO on the Ability of Hypothalamic Extracts to Inhibit Pituitarprrolactin Release;n_yggg; The ability of rat brain MAO to remove prolactin in- hibiting activity from hypothalamic extracts is demon- strated by the results shown in Tables 6 and 7. In each case when MAO was pre-incubated with hypothalamic extracts, the extracts lost their ability to inhibit significantly pituitary prolactin release ig’yiggg, The dopamine and norepinephrine content of similarly MAO treated and nontreated hypothalamic extracts is shown in Table 6. It can be seen that the amount of each catechol- amine remaining after MAO treatment was below the minimal effective dose needed to inhibit prolactin release (see Tables 1 and 2). Furthermore, it can be noted that the MAO preparation was highly effective in inactivating both norepinephrine and dopamine in each hypothalamic equivalent. The data in Table 7 demonstrated more conclusively that the prolactin release from anterior pituitary halves incubated with nontreated hypothalamic extracts was sig— nificantly less than the prolactin released when the Mao treated hypothalamic extracts were incubated with the 123 OE 0.5a aampmfiaxoummm monoamz noa£3 no room mm mom on mm pmmmmumxm m coaumummmnm mmmoaxo mcaEMOGOE gamma you w Spas mm mo coaumnsocHIwum u O¢2m umou =u= pouamm on» mcam: moconommac come no uouuo oumocmum.H mocmuomwao cmmzo com: o ucoam>aswm anmamnuomhn u mmo wumuasuam u0aumucm u mdn aImm cauomaoum umu Q2.mm mmmoamm cauomaoum aumuasuam pananca ou mpomuuxm anmamnuomwm pom mo muaaand mzu co mommaxo anaemocoz camum umm mo uomwmm .o manna 124 COaumummmum mmmoaxo mcaEMOCOE Gamma you m sua3 mm mo ceaumndocaImum 0 cm: w ummu =u: oouamm on» mean: mocmuwmmao came mnu mo uouuo pumocmum o:u.H mocwummmao cmmzo cmm 2o ucmam>asvm anMamnuommz u mmo humuasuam HOHHmucm 0 man aImm cauUMaoum you Q2.mm mmmmamm caUUMaoum xumuasuam umm co muomuuxm andamnuomxm mo ucmsummue ommoaxo mcaemocoz mo uommmm one .5 manna 125 corresponding anterior pituitary halves. Two separate experiments were carried out to determine the effects of MAO, and the sodium phosphate buffer pH 7.4 used as its diluent, directly on anterior pituitary pro- 1actin release. As can be seen in Table 8, neither the MAO preparation nor its diluent had any effect on anterior pituitary prolactin release i2 vitro. 2. Blockade of MAO Activity by Iproniazid The results shown in Table 9 demonstrate that 10 pg of iproniazid per m1 of physiological medium inhibited the MAO effect. This dose of iproniazid had no effect on anterior pituitary prolactin release ig vitro. 3. The Effects of Aluminum Oxide Cate- cholamine Adsorption, and Treatment of Extracts with Pepsin on Their Ability to Inhibit Pituitary Prolactin Release In Vitro It can be seen in Table 10 that aluminum oxide (alumina) adsorption of catecholamines from hypothalamic extracts resulted in the loss of the inhibitory properties of the extracts on pituitary prolactin release i3 giggg. There was no effect on inhibition when the hypothalamic extracts in a second control group had their pH titrated to 8.35 (the pH for optimal catecholamine adsorption) for 30 min. It can be seen that hypothalamic extracts subjected to catechol- amine adsorption had dopamine and norepinephrine contents below the minimal effective amounts needed to inhibit 126 coaumummmum mmmpaxo mcaamocoe Gamma you n 042 m umwu :u: ompamm map mcam: mocmummwao came onu mo uouum Unmocmum.H mocmummmao cmwzm com 2o v.n mm “woman mumnmmonm asaoom E m.o a: omo humuasuam MOaumucm 0 man aImm cauomaoum umu Gadaz on mm owmmmnmxmm .I vm. I. vm. om: + mdm ms .+ . ms .+ . A V ao No mm. A V mo mo mm. v v w mdm I vm. I am. 8&3 + 9% m: . . mc . . o A V mo +vo mm. A Vomo +No mm. v v mdw U n N mxm a mxm m mxm a mxm QOaumndoca mmsmmae mm mE\m1 ommmmamm sauomaoum wonde musuasu mo muamm mo macauapcou ouua>.mm mmmoamm CauUMaoum >umuaspam Heaumuc4 co mmmoaxo mcaEmocoz namum pom mo uommmm mo xoma .m manna 127 coaumummmum mmmpaxo mcaEdocoa gamma you cua3 mm mo coaumndocamum u 042 ‘H ammo z»: omuamm on» mcams mocmummmao come gnu mo nouum Unmocmum.H wocmummmao cmme cmmz ucmam>asvm andamnuomxn a ()g '0 wumuasuam Hoaumucm 12% aImm cauUMaoum umuIQz¢Hz 01 mm ommmmumxm I'd Aaa\mn oaV canmacouma .I mm. I. av. oaz + mma + max m: . . ma . . A V mo +mo om. A V mo +vo «v. v v mma + max .I mm. .I mo. Aae\ma oaV cannacouma + m4» mg 0+ 0 ms 0+ I A V mo mo mm. A V mo mo as. v v may Aaexmn oaV vaumacouma .I om. I. mv. oaz + mma + may 0 0+ 0 0 0+ 0 Aao vmV mo mm vm. Aao vmV mo om on. v v m max .I mm. I. mm. oaz + mma + mam ma 0 0 ma 0 I A V mo +mo mm. A V mo +mo am. v v may I. «v. .I am. mma + may 0 O I O O O O Aao vmV oa +am mm. Aao vmeno +om am. v v may v n m mxm a 88 m mxm a mxm mammae ma mE\mn pmmmmamm caHUMaoum mmnde manuasu mo muamm GOHumndocH mo mcoauapcou M onua>.mm mmmmamm cauomaoum xumuasuam panancH ou muomuuxm anmamnuommm mo Auaaand ocu co cannacouma cua3 mmmoaxo mcaEdocoz camum and mo momeOam mo uommum .m magma 128 m8 o.ha Namumaax0pmmm omnmao3 noagz mo room mm mom on mm ommmmpmxmm ummu :u: omuamm may mcams oocmummmap come no uouum oumocmum.H wocmuwmmap com: com ucmam>asvm andamnuomhn u mmm Mumuasuam uoaumuam n mdw almm cauomaoum you QEdaz no mm mm oommmumxm ucmEumwuu sumo pom pom: mumz muamm was» musuaso usomm Amm.m on vmumuuau mmV I. I. I. ma. mma + dd» 0 + I O + O O m 0 + O AvV a v o om AvV m m o vm Aao v V mo oN mm. mdx coaumuom m modes 8 I. I. .I Nm. A . o mwa H WE“ soaOm AvV v.o+o.N AvV m.o+o.a Ach No.+mo. mm. Adm 2v.o Ca pmnacmmoson . I.. . I.. . .I. . ma. mma + mam muomuuxm + + + AvV N m 0 mm AvV N m N am Aao vmV No aN mv. Adm N ......................................................... IIIIIIIIIIIAmmum-NMIwmmmmmmfimaIIiI-IIII-I..II I. I. I. am. mma + m4» . + . . + . . .+ . AvV N o a Nm AvV N m m mN Aaoo vmV mo mv Nm. mdw AcoauQHOmnm mcaEdamV I I .II o + AvV N.H+m.m AvV m.o+m.A Ach vo.+oa. mm. mma “MM Hum zd.o CH Umuacomoson I. I. I. . + m omnuxm AvV m.h+m.vm AvV v.v+a.0m Aaoo.vmV mo.+vm. vm. omma mm» D m mam ommx a wcaunmocamwuoz wCaEmmoo mm\mm. nmsmmae dd ma\m1 Honadz war no acoucoo mcaemaonomumu oommmamm cauomaoum anneauaocou ucmEauwmxm ouua>.mm mmmwamm caUOMaoum mumuasuam pananca ou muomuuxm oasmamnuomxm mo Auaaana map so coauEHOmvm maaamaozomumo mo powwow .oa magma 129 prolactin release (see Tables 1 and 2). The results shown in Table 11 demonstrate that pre- treatment of each hypothalamic equivalent with pepsin (0.5%) solution in saline for 4 hours had no effect on the extracts ability to inhibit prolactin release ig_yi££g. Medium 199 pretreated with pepsin and then titrated to pH 7.25 had no effect on prolactin release. The acid eluates from the alumina, whether pretreated or not with pepsin were equally as effective as normal hypothalamic extracts in their abil- ity to inhibit prolactin release ig_yi3£g. Catecholamine measurement confirmed the fact that dopamine and norepine- phrine were removed from the hypothalamic extracts by alumina and were present in the acid eluates. D. Conclusions The data obtained from these experiments suggest that a monoamine, presumably dopamine or norepinephrine or both, in normal endogenous concentrations can totally account for the prolactin inhibiting capability of acidified hypothal- amic extracts ip_yiE£g, It has been generally assumed that prolactin inhibiting factor (PIF), like the other hypo- physiotropic hormones, is a polypeptide. To substantiate this assumption several workers have isolated hypothalamic peptide fractions free of catecholamine activity with PIF activity (Greibrokk ep_al., 1974; Schally e£_al,, 1975). These data do not discount the possibility that a peptide 130 coauduompm mcaamaosomumo mafiadam umuwm mmafiamaonooumo mo moum mmaw uamam>a=vm oaamamfiuomms u mmaw umou :u: omuamm mnu moan: mocmuowmav some ozu mo nouuo vumvamuw + mocmuowwao cmmzm Gmm humuasuam uoauouam u mamm AvV o.o&o.§ AvV m.v&m.mm Aao.vmV 8&3. om” A865 8&3. vmn Amaaasam mo 33am 363 B: + v22 ON ov mmd Amcaasam mo mumsao AvV N.o+¢.¢v AvV o.nHo.vN Aao.vmV aa.Ham. aN. ANo.vmV mo.Hva. mm. vaom woummuu cammmmv mma + Nm< mm mm. wag. AS m.o&a.~ A3 may; Ame 8&8. on” Ach 8&3. am. Asmfomvmécaaflswmi + “1.2 8 mm. mm... AC w..v& an A3 ~.m&a.a~ Sofie 8&2: mm” A863 8&3. Wm. $83303 5395 a: + Wm“ AS v.~& 2 A3 q.N&o.m~ Goofs 8&8. SH A863 3&8. om” one: + “1.2 mm mm .922 Amcv vo.H$o. vmn chv vo.HWo. 0v. mma adapoz pmummuu cammmm + wm< Nm mv. wm< Amcv mOaHNo. an oAmcv mOaHno. an” wm< av vow owm< a mean: oaamouoz ocaammoo :oaom zv.o no aom Za.o Amma\wq%. N dam a axm ca omnacmwoao: wuomuuxm a axm ucmucou maaamaozumumo nonmmaa m< wmwwm.vmmmmamm cauomaopm mcoaumosoca uo coauawsoo ouua>.mw mommamm cauomaoum so moumsam moaxo asaaasa< woummuH cammmm wow muomuuxm andamnuoamm vmummue camdmm mo uommwm 038 .aa manna 131 PIF exists. They do, however, suggest that the concentra- tion of a peptide PIF in one hypothalamic equivalent is less than that quantity needed to demonstrate inhibitory activity in the £3 31359 incubation system used in these experiments. The data further suggest that the catecholamines present in hypothalamic tissue represent a PIF in addition to the possible peptide PIF. The fact that iproniazid, a MAO inhibitor, inhibited the effect of MAO on the hypothalamic extracts eliminates the possibility that the catecholamines in the extracts were bound in some manner to protein in the enzyme preparation and suggests that the catecholamines alone were responsible for the PIF activity of the extracts. Catecholamines far below the amounts found in the hypo- thalamus have the ability to directly inhibit prolactin release ig_yi2£g_(5haar and Clemens, 1974a) and ig.yiyg' (Schally ep_al., 1974; Takahara eE_al., 1974c) and there are presently reports that norepinephrine (Ben-Jonathan e2_§l., 1975b) and dopamine (Ben-Jonathan eg_al., 1975a) are present in hypophyseal portal blood. A catecholamine could represent a PIF present in and released from the hypothal- amus. The data further suggest that future measurements of the prolactin inhibiting activity of hypothalamic extracts must first take into account the inhibitory activity of the catecholamines themselves before any conclusion on a pep- tide PIF is made. 132 IV. Direct Effects of an Ergoline Derivative on Anterior—Rituitary Prolactin Release ;g_VitrQ: Blockade by Pimozide, a Specific Dopamine Receptor Blocker A. Objective The objective of this experiment was to determine whether alpha-adrenergic or dopaminergic receptors or a combination of the two in the pituitary gland mediate the prolactin inhibiting action of the ergoline derivatives. B. Methods and Materials 1. Animals Adult male Sprague Dawley rats weighing approximately 225 g were used as pituitary gland donors for all the incubation. The methods of removal of anterior pituitary tissue from the rats is described under the Materials and Methods section. 2432128. Lergotrile mesylate (2—chloro-6-methylergoline—8—aceto- nitrile, methanesulphate salt) (Eli Lilly and Company, Indianapolis, Indiana) was used as the prolactin inhibitor. Pimozide (McNeil Laboratories), a dopamine receptor blocker, propranolol (Regis Chemical Company), a beta-adrenergic blocking agent, and phentolamine (CIBA Pharmaceutical Company), an alpha adrenergic receptor blocker were used to attempt blockade of the ergoline derivative. Physiological saline (0.9% NaCl) was used as a vehicle control in all 133 incubations except those incubations in which pimozide was used. A 0.9% NaCl solution containing 0.001 M tartaric acid was used as the control solution in the pimozide incu- bations. 3. Incubation and Prolactin Assay Techniques The incubation system used in this experiment is dis- cussed in detail under the Materials and Methods section. Each sample was assayed for prolactin by radioimmunoassay using 2 different dilutions. The prolactin values were averaged and expressed in terms of the reference prepara- tion NIAMDD rat prolactin RP-l. The data were analyzed using Student's "t" test for paired observations so as to compare the response of a treated pituitary half with its corresponding half exposed to Medium 199 alone or another control solution. C. Results £5.3iggg, 1.0 ng/ml of Lergotrile Mesylate (LM) was able to significantly inhibit prolactin release from anterior pituitary tissue. Figure 5 demonstrates the log dose response curve for this inhibitory effect. Table 12 shows the effects of some catecholamine antagonists on the ability of LM to inhibit prolactin release. The beta- adrenergic blocker, propranolol, and the alpha-adrenergic blocker, phentolamine, incubated along with LM in approxi— mately equimolar amounts had no effect on the ability of LM 134 0.5- E? It! .— = E I :I ~0."‘ -J m E ‘2 z 5 O u! u 8 -— 00- :: E a 2 g G E z 3 0.2- x z - ‘ I.“ a 0J~ I l f I 0.1 1.0 10.0 IND IIG lElIGlITIIIlE PER III. or IIIOIIBAIIIIII IEIIIIIII Figure 5. In vitro inhibition of prolactin release by fergotrile Mesylate. Each point represents the average mean difference (control vs treated) in prolactin release for at least 6 paired pitui- tary halves. 11} 135 .2 ca x v.m ma AzaV muma>mmz maauuomuma no coaumuucmocoo one m . n .Amo.vmV aouucoo onwapmoummm Eoum pcoumwmap waucmoamacmamm mum & 8? ma zmIoa x Aim 63...er m> n5 I. £5 + MN + vm + Na Emloa x m.m QCHEMaoucmzm m> 324 II Q5 + ma + av I Na Zmloa x 5.5 aoaocmumoum m> Qza .H I x . m> mca mm mmN mNN Na 2mIoa v m nza .a maouucoo .m> poummpe “owmmmaom mmaumuasuam Houananca mo moose omummue medB aouucoo caUOMaoum ca mocmummmao com: omuamm mo umnEsz coaumuucmocoo ucosummue ouua>.mm mommae >umuasuam Hoaumucd Scum mmmwamm cauomaoum uaoanca ou mumaxmwz maauuomuma mo auaaan< wow so mumeOam Moummomm mo muommmm .Na magma 136 to inhibit prolactin release. In contrast, pimozide, a specific dopaminergic receptor blocker was able to reverse the inhibitory effect of LM on prolactin release. The addi- tion of an equimolar amount of pimozide to LM resulted in the release of 60% more prolactin than was released from pituitary halves treated only with LM. D. Conclusions This study demonstrates the LM, an ergoline derivative, is a potent inhibitor of prolactin release ip_yipgg, Additional experiments have shown this compound to be also a potent inhibitor of prolactin release ig_yiyg_(Clemens e£;213, 1975; Clemens eg_§l., 1974). The most significant finding of this study is the observation that the direct action of LM on the anterior pituitary gland can be antagon- ized by the specific dopamine receptor blocker, pimozide. Neither the alpha-adrenergic blocking agent, phentolamine, nor the beta-adrenergic blocking agent, propranolol had any influence on the ability of lergotrile mesylate to inhibit prolactin release ig_ziE£g, The use of equimolar ratios of drugs in this study indicates inhibition of a specific nature since the incubation system was not overloaded with any of the receptor blockers. Stimulation of a pituitary dopamine receptor by the ergoline derivative seems to be the mechanism by which this drug inhibits prolactin release ip_vitro. There are 137 several lines of evidence which support this finding. The ergolines have been shown to be dopaminergic in negrostri- atal lesioned animals. Administration of ergolines to these lesioned animals produces rotational behavior indistinguish- able from that produced by known dopamine receptor stimulants (Corrodi eg_al., 1973). Dopamine itself can act directly on anterior pituitary tissue i2 yiggg to inhibit prolactin release (MacLeod, 1969; Shaar eE_§l,, 1973; Shaar and Clemens, 1974) and this effect can be blocked by dopamine receptor blockers (MacLeod and Lehmeyer, 1974). The dopamine receptor stimulant, apomorphine, can inhibit prolactin release by a direct action on the pituitary (Smalstig eE_al., 1973; MacLeod and Lehmeyer, 1974), and its action can be blocked only with dopamine receptor blockers (Smalstig and Clemens, 1974). It is therefore concluded that the ergo- line derivative, lergotrile mesylate, inhibits anterior pituitary prolactin release ip_yiE£g_by stimulation of a dopamine receptor which mediates the control of prolactin secretion. V. The Effects of Dopamine Infusion on the Proestrus Afternoon RiSe in Serum Prolactin—in Normal Cycling Female Rats: Chronic Cannulation Studies A. Objectives Systemic injection of dopamine has never produced an effect on prolactin release in vivo, yet some investigators 138 consider dopamine a possible PIF. It was the objective of this experiment to demonstrate that prolonged arterial in- fusion of minute pharmacological doses of dopamine could inhibit prolactin release in unrestrained chronically cannu— lated female rats. It was also the objective to see if dopamine could inhibit the normally occurring rise in serum prolactin on the afternoon of proestrus. B. Methods and Materials 1. Animals Mature female Sprague Dawley rats weighing approxi- mately 250 g were used for all cannulation procedures. Both the cannulation procedure and the subsequent housing in an acoustic isolation chamber have been described in detail in the Materials and Methods section. Vaginal smears were taken each day between 8:00 and 9:00 am to ascertain regularity of the cannulated rat's estrous cycles. Z-w Dopamine hydrochloride (Sigma Chemical Company, St. Louis) was used for infusion studies. The dopamine hydrochloride was dissolved in a 5% glucose solution. Con- trols received al$%glucose solution. Glucose was used to prevent the oxidation of the catecholamine in the reservoir of drug used to fill infusion syringes. 139 3. Experimental Procedure On the morning of proestrus (determined by vaginal smear) the free end of the permanent cannula was passed out of the chamber to an exterior sampling position. The rats were therefore not disturbed for 5 hours prior to sampling of blood. Blood sampling and drug infusion were accom— plished from this exterior position and the animal remained unstressed except for any physiological effect produced by the drug itself. Blood samples were taken and infusion was accomplished by a Harvard Apparatus Infusion/Withdrawal Pump (Harvard Apparatus, Mellis, Mass.). At 1345 hrs on the day of proestrus a preinfusion sample was taken. Thereafter infusion progressed at 30 min intervals: 1415, 1445 and 1515 hrs. Each infusion period was followed immediately by the withdrawal of a 0.3 ml blood sample. At 1515 hrs all infusion was stopped and at 1545 hrs a final blood sample was withdrawn. At the termination of the experiment all cannulas were flushed with a heparin— saline solution to insure the use of the cannula for another observation period on the following proestrous afternoon. The infusion rate in all experiments was set at 0.5 m1/30 min and the drug infusion dose was 1.1 pg/min. The drug treated animals exhibited the gnawing behavior character- istic of dopaminergic stimulation. This behaviour indicated that some of the dopamine was passing through the blood brain barrier into the central nervous system. 140 C. Results As can be seen in Table 13, dopamine hydrochloride infusion prevented the rise in serum prolactin on the after- noon of proestrous. When infusion was stopped at 1515 hrs and the animals were allowed to remain untreated for 30 min the serum prolactin levels of the previously dopamine-treated rats rebounded. By 1545 hrs there was no significant dif- ference between both non-treated and previously treated rats. D. Conclusions The data presented in this experiment indicate that prolactin release can be inhibited by dopamine administered ig.ziyg by arterial infusion. This is the first account of dopamine infusion being inhibitory to a normally occurring rise in serum prolactin on proestrous afternoon in the rat. There is presently a report in the literature concerning the inhibitory effect of dopamine on prolactin release in the sheep (Davis et al., 1975). In this report, minute amounts of dopamine were infused systemically and it was presumed that dopamine was acting via a direct action on AP tissue since dopamine could not pass through the blood brain barrier. Such a conclusion may be true; however, an indirect action via the hypothalamus and PIF release cannot be discounted. The evidence in this study cannot warrant a conclusion on the site of action but only suggest two sites of action: the hypothalamus and the anterior pituitary gland. 141 .aImm aauUMaoum um» oozdaz mo mayo» ca ommmmumxw come no .m.m.H aE\m: ca Gauomaoum Eduwmn .mumu mamEmm Goudasccmo ucmumwwap m mo moms macaum>nwmno ucmmmummu mumu Umummuu paw aonucoo,nuon mo mcoaum>ummno mzam CHE E . m: Ame ABOVE Amoowve Solve 5% A . on: m 3 8V o.ma+v.om o.a +m.aa o.~ +m.oa v.m +m.~m m.v+m.vm a wcHEMmoo Acae om \aEm.O\mmoosam me mNHm.maa v.aa&n.mma m.ma&m.am o.oa&a.mn n~.v&v.mm we aouucoo was mvma mug mama my; mvva mun mava mu: mvma Aoamamm coamsmcamumV mcoaum>ummoo AmmooV meaB oamaoomm um ceaumuucmocou cauUMaoum Edumm mo Honfidz ucmEumoHB mumm maMEom tonnasccmo waamoacouno omcamuumwuco ca msuummoum mo coocuwpmd on» mcanza coaumuucmocoo cauUMaoum Esuom co coamdmca oUHNOanooqum mcaemmoo mo muommmm .ma magma 142 VI. The Effects of Systemic Infusion of Dopamine and L—DOPA on Serum Prolactin in Rats with Biiateral Median Eminence Lesions: Acute Cannulation Studies A. Objectives The catecholamines have been shown to exert no effect on serum prolactin when administered by systemic injection. Likewise, the direct action of catecholamines on pituitary prolactin release i2 yiyg has been a controversial issue. It was the objective of this experiment to demonstrate that systemic infusion of minute pharmacological amounts of dopamine and L-DOPA could inhibit pituitary prolactin re- lease and that this inhibition could be accomplished by a direct action on the anterior pituitary gland. B. Materials and Methods 1. Animals Adult female Sprague Dawley rats weighing approximately 250 g were used for the study. The electrolytic bilateral median eminence lesion placement and acute external jugular vein cannulation procedures are discussed in detail under the Materials and Methods section. 2-Dr_usg The drugs used were L—dihydroxyphenylalanine (L-DOPA) and L-dopamine hydrochloride (both from Regis Chemical Com- pany). Both drugs were dissolved directly in physiological saline (0.9% NaCl) containing L-ascorbic acid (0.01% W/V) 143 as an antioxidant. 3. Experimental Procedure Seven days after placement of the bilateral median eminence lesions all rats were anesthetized with urethane and the right jugular vein of each animal was cannulated. The surgical procedure was complete in the shortest possible time (5 min/rat) to enable all rats to be used during the same experimental period. Following the withdrawal of a pre-infusion blood sample, dopamine hydrochloride, L-DOPA or physiological saline—ascorbic acid diluent—control were infused for 20 min into the jugular cannula by using a Harvard Apparatus infusion/withdrawal pump (Harvard Appara- tus, Mellis, Mass.). Following the infusion period (20 min), a post infusion blood sample was removed. Upon termination of the experiment the animals were perfused with a solution containing 6% potassium ferrocyanide and 6% potassium ferri- cyanide to develop the prussian blue reaction with the iron deposited by the lesioning electrode. The brains were examined to note the destruction of the median eminence. 4. Prolactin Assay Prolactin in individual serum samples were measured by radioimmunoassay. Each sample was measured in duplicate and the prolactin values were averaged and expressed in terms of the purified rat prolactin reference standard NIAMDD-rat prolactin RP-l. Student's "t" test for paired observations was used to determine the significance of 144 differences between pre-infusion and post infusion serum prolactin concentration. C. Results Dopamine hydrochloride and L-DOPA but not the physio- logical saline containing L-ascorbic acid significantly reduced the already elevated serum prolactin levels in the female rats with bilateral median eminence lesions (Table 14). D. Conclusion These results demonstrate that dopamine and L-DOPA administered by prolonged infusion inhibited prolactin release by acting directly on the anterior pituitary gland. L-DOPA administered by systemic injection has been demon- strated to be highly effective in reducing serum prolactin levels in laboratory animals ip_yiyg_(Lu and Meites, 1971; Chen and Meites, 1975; Smythe and Lazarus, 1973). All these reports attributed the prolactin inhibiting activity of L-DOPA to an increase in release of hypothalamic PIF. These results agree with those of Donoso etpal, (1973), who showed that L-DOPA administered to rats with median eminence lesions inhibited prolactin release. The infusion of dop- amine in this experiment is the first report that this cate- cholamine administered systemically can inhibit prolactin release in rats. A similar experiment has recently been reported in sheep (Davis et al., 1975). In their experiment 145 .mocouowmao same man mo Houum pumpamum.H mocmnmwmao saw: a .aImm cauomaoum umu ooz¢Hz mo mEpmu Ca ommmmumxw mm some may we uouum oumocwum.fl aE\ms ca cauomaoum Edumm cmozm aas\ae vao.o\ma m.m Aaoo.vmV m.m.&n.am N.om&o.mo m.mm&v.nva m «mooIq I. I. I. caa\ae vao.0\ma o.a Aao.vmV o.ma+o.mn m.m +m.mm o.mm+m.maa o aomImcaemaoo Acas\ae vao.oV aouucoo vaoa Asz gm.m +m.m o.Om+o.vma mm.mm+v.mva m oanuooma wao.o I mcaamm aE\mc mocmummmao AcaE 0N+V aE\mc mumm AmmooV cmoz GOamsmcaumom coamsmcaoum mo Honesz ucmfiummue macaumuucmocoo cauUMaoum Eduom chamma mococaem smaomz amumumaam spas mumm mamsmm ca mmooIq new ovauoanoouexm mcasmmoo «0 muommmm .va magma 146 the researchers explained their results as a direct inhibi- tory action on pituitary prolactin release because dopamine was reported not to cross the blood brain barrier. It is assumed that the destruction of the median eminence, the final common pathway, eliminates hypothalamic influence on the pituitary gland. Therefore it can be concluded that dopamine and L-DOPA both exerted their inhibitory effect on pituitary prolactin release by a direct action on pituitary prolactin-secreting cells. GENERAL DISCUSSION The data presented in this thesis indicate that low concentrations of dopamine (DA) and norepinephrine (NE) can directly inhibit anterior pituitary prolactin release ig_yitrg. The smaller amounts of the catecholamines, DA (1.0 ng/ml to 10 ng/ml) and NE (5 ng/ml to 20 ng/ml) are less than the concentrations reported to be present in the rat hypothalamus (Lippmann,l968; Donoso et_al., 1969; Shaar and Clemens, 1974b; Kavanaugh and Weisz, 1973/74). These data are in agreement with the early work of MacLeod (1969) and Birge et_§l. (1970) and has been confirmed by very recent work (Takahara et_al., 1974; Schally et;31,, 1974; Dibbet et_al,, 1974; Greibrokk et_§l., 1974). The results do differ from those of Koch et_§l. (1970) who reported that 10 or 20 ng epinephrine (EP) or NE per ml of incuba- tion medium significantly increased while 200 to 1000 ng of EP or NE per ml of incubation medium inhibited prolactin release iglyitrg, They further reported that addition of 80 to 640 ng of DA per ml of incubation medium was inhibi- tory; whereas, lesser amounts had no effect on prolactin release ig_gi§rg. It is difficult to explain the apparent disagreement and one can only speculate that the differences 147 148 are due to variation in experimental technique. The author attempted to gain sensitivity in the incubation system by using a paired hemipituitary method which allowed the use of the "t“ test for paired observation during statistical analysis. Refinements such as increasing the pre—incubation time from 0.5 to 1.0 hr and the maintenance of all medium and pituitary tissue to be used during the incubations in a gassed condition at 37°C may have added sensitivity and uniformity (Labella et_§l., 1973; Uehara et_§l., 1973). All drugs used in the incubations were diluted immediately prior to use to ensure their potency. The results differ from those reported by Kamberi M. (1971b). They perfused the anterior pituitary glands of rats directly via a hypo- physeal portal vessel with large pharmacological doses (up to 2.5 pg/rat/20 min infusion period) of DA, NE and EP. They found that none of the catecholamines infused directly into the anterior pituitary had any effect on plasma pro- lactin. DA but not NE or EP injected into the third ventri- cle of rats reduCed plasma prolactin levels. These re- searchers concluded that DA was acting on the hypothalamus to increase prolactin inhibiting factor (PIF) release and that none of the catecholamines could act directly on the anterior pituitary to inhibit prolactin release 12 yiyg, Because of the apparent disagreement between the ig_yiyg_ and ip_yitrg_work it has, until recently, been assumed that the ip_vitro work represented only a pharmacological 149 phenomenon and had no physiological significance. Recently, attempts have been undertaken to repeat the work of Kamberi et_al.(l97ltn. Since cannulation of the hypophyseal portal vessels is a very tedious technique, very few laboratories in the world can accomplish such a task. The infusion of DA and NE into the rat anterior pituitary via a hypophysial portal vessel for 30 min caused significant decreases in serum prolactin levels (Schally EEVélrr 1974; Takahara enggr, 1974a). The doses used in their study were equal to or smaller than those used by Kamberi et_al. (1971b). Two procedural modifications must be noted when compar- ing the original work by Kamberi 3L1]: (1971b) and the more recent work (Schally et_al., 1974; Takahara et_§l,, 1974a). The former workers infused catecholamines diluted in physio- logical saline into the anterior pituitaries of pentobar- bital anesthetized rats while that latter group used a 5% glucose solution as a diluent and urethane as their anesthe- sia. Further, the more recent work demonstrated that cate- cholamines dissolved in 0.9% saline exhibited less or no PIF activity. These two modifications could explain the apparent differences in the two groups of data: the cate- cholamines infused in physiological saline may have been already oxidized and inactive before infusion or the anterior pituitary of the rats to be infused was refractory to the inhibitory effect of the catecholamines as demon- strated ip_vitro by Stone and MacLeod (1973). 150 The possibility therefore exists that the 1p yitrg data on the direct inhibitory effects of DA and NE on pituitary prolactin release reported in this thesis and by other researchers (Shaar ep_gl., 1973; Dibbit eg_al., 1974; Greibrokk et_al,, 1974) represents a true physiological phenomenon. Data presented in this thesis suggest that monoamines, presumably DA and NE, in the hypothalamus at normal endo- genous concentrations can account totally for the anterior pituitary prolactin inhibitory activity of acidified rat hypothalamic extracts ig_yitrg. The adsorption of the cate- cholamines onto aluminum oxide (alumina) and the inactiva- tion of the endogenous catecholamines by a rat brain mono- amine oxidase (MAO) preparation resulted in the inability of hypothalamic extracts to inhibit prolactin release ig_yitrg. Subsequent incubation of pituitary tissue with catecholamines eluted from the alumina evoked prolactin inhibition characteristic of normal hypothalamic extracts. The MAO inhibitor, iproniazid, blocked the effect of MAO on hypothalamic extracts. The fact that iproniazid inhibited the effect of MAO eliminates the possibility that the cate- cholamines in the hypothalamic extracts were simply inacti- vated by being bound to a protein in the enzyme preparation and suggests that the catecholamines themselves were responsible for the PIF activity in the extracts. 151 The question arises once again as to the relationship between the catecholamines and a polypeptide PIF. It has been generally assumed that PIF, like the other hypophysio- tropic hormones, is a polypeptide derived from neurosecre- tory cells in the basal hypothalamus. Evidence presented here does not discount the possibility that a polypeptide PIF exists. There are presently two laboratories that claim to have isolated a polypeptide catecholamine-free substance of hypothalamic origin which has high PIF activity (Schally et_§l., 1975; Greibrokk ep_al., 1974). If such a peptide PIF exists, it is not present in sufficient quantities with- in a single hypothalamic equivalent to demonstrate inhibi- tion of prolactin release using the author's ip_yitrg.incu— bation system. For several years researchers have been using a similar ig_yitr9 incubation system to demonstrate changes in PIF activity produced by catecholamine-activity- altering-drugs. The evidence in this thesis suggests that the PIF activity of the endogenous hypothalamic catechol- amines must also be accounted for before any conclusion can be made concerning the alteration of a polypeptide PIF activity. In order to evaluate changes induced on a pos- sible polypeptide PIF, the catecholamines within the hypo- thalamic extracts will first have to be removed. A possible prolactin inhibiting system in the tuberal part of the hypothalamus, consisting mostly of dopaminergic fibers has been described using histochemical fluorescent 152 methods (Carlsson et_al., 1962; Fuxe and Hokfelt, 1964, 1966). The neurons of this system have their cell bodies in the region of the periventricular (arcuate) nucleus and send their axon ventrally to the median eminence ending in the vicinity of or on the endothelial walls of the primary capillary loops of the hypothalamo-hypophyseal portal sys- tem. Groups of these dopaminergic neurons differ from normal dopaminergic neurons by the fact that they are unaffected by apomorphine, a specific dopaminergic receptor stimulant (Anden et_§l., 1967) and that these neurons fail to participate in the uptake of 6-hydroxydopamine as other dopaminergic neurons do (Shoemaker, 1975). The fact that these neurons are unaffected by a receptor stimulant and possess no uptake mechanism suggests that they serve a neuro- secretory function. Both DA (Ben-Jonathan et_al., 1975a) and NE (Ben—Jonathan et_al., 1975b) have recently been detected in hypophyseal portal blood. While DA appears to be bound to a large unidentified protein, NE is found in the free form. The inhibitory effect of the ergot alkaloids on pro- lactin release is well-established (Linder and Shelesnyak, 1967; Nagasawa and Meites, 1970; Wuttke et_al., 1971; Lu and Meites, 1972; Shaar and Clemens, 1972). Although the naturally occurring ergots are potent inhibitors of prolac- tin release they exhibit several toxic side-effects which make them unfavorable for long term therapeutic use. 153 Simple ergoline derivatives without the complex sidechain characteristic of ergotoxins have been shown to also be potent inhibitors of prolactin secretion (Clemens etpgl,, 1974; Cassady eg_al., 1974; Clemens et_al., 1975). Lergotrile Mesylate (LM), an ergoline derivative has been shown to be a highly potent prolactin inhibitor (Clemens gt_gl., 1974). The hypothalamus and anterior pituitary gland have been proposed as sites of action (Malven and Hoge, 1971; Lu and Meites, 1972; Wuttke et_§l., 1971; Shaar and Clemens, 1972). The precise pharmacological characterization of an anterior pituitary dopaminergic receptor capable of medi- ating the inhibition of prolactin release, by Smalstig and Clemens (1974) and discovery that various ergot derivatives were able to stimulate central nervous system dopaminergic receptors (Corrodi et_gl., 1973; Stone, 1974) have had great bearing on the ergoline data reported in this thesis. Not only has LM been shown to be a potent inhibitor of pro- lactin release 12.21EE2.bUt its mechanism appears to be through activation of a dopaminergic receptor present in anterior pituitary tissue. The direct action of LM was antagonized by pimozide, a specific DA receptor blocker. Neither the alpha adrenergic blocking agent, phentolamine, nor the beta adrenergic blocking agent propranolol had any influence on the ability of LM to inhibit prolactin release. MacLeod and Lehmeyer (1974) found another ergot, 154 2-bromo—a-ergocryptine (C8154), capable of acting directly on pituitary tissue to inhibit prolactin release ig'yitrg. They found that the inhibitory effect of C8154 was antagon- ized by alpha-adrenergic and dopaminergic blocking agents and suggested that the ergots might be acting on alpha adrenergic or dopaminergic receptors to inhibit prolactin release. The specific dopamine receptor stimulant, apomor- phine, has also been shown to inhibit prolactin release both ip.yiyg_and ig_yitrg_(8malstig et_al., 1974; Shaar gt_§1., 1973; MacLeod and Lehmeyer, 1974). Because very minute doses of apomorphine were effective ig_yitrg, it was concluded that the inhibitory effect on prolactin release ig_yiyg_must at least partially be attributed to a direct action on the pituitary. In the past researchers have had a great deal of diffi- culty demonstrating the effects of systemically administered catecholamines on prolactin release ig_yiyg (Lu et_§l., 1970). The usual manner of administration has been intra- peritoneal or intravenous injection. The likelihood of a bolus intraperitoneal injection of catecholamine reaching the hypothalamus or anterior pituitary is very slim since by this route of administration the injected substance first passes through the hepatic portal system into the liver. It is well-known that the liver is an abundant source of the enzyme Catechol-o-methyl transferase (COMT) which would quite effectively inactivate the catecholamines (Hogeboom, 155 1955). An intravenous bolus injection would also meet with a similar enzymatic inactivation fate. It appears that the use of long term (20-30 min) intra- venous or intraarterial infusion of catecholamine dissolved in a 5% glucose or 0.1% C-ascorbic acid solution will allow sufficient amounts of drug to reach the anterior pituitary or the hypothalamus. By using this technique to infuse DA and L-DOPA, the author was able to effectively reduce the elevated serum prolactin levels in urethane anesthetized female rats bearing median eminence lesions. Likewise, the author was able to prevent the normal rise in serum prolac- tin during the afternoon of proestrus in unrestrained chron- ically-cannulated female rats. Recently it was reported that DA infused over a long period of time (30 min) into sheep effectively reduced serum prolactin levels (Davis e_t_a_1_., 1975). These workers concluded that since DA does not pass through the blood brain barrier, the action of the drug was due to a direct action on the anterior pituitary gland. The author is not aware of any literature available demonstrating that long term infusion of pharmacological doses of catechol- amines can alter hypothalamic content or turnover of those amines. However, such an elevation cannot be discounted and thus the catecholamine action on pituitary prolactin release might be in part due to an indirect influence via the release of a polypeptide PIF. It can be concluded, however, that a major portion of the inhibitory effect on 156 prolactin of both DA and L-DOPA, administered to the median eminence lesioned rats, is due to their direct action on the anterior pituitary. This conclusion would be in agree- ment with earlier work by several workers who observed similar effects but were reluctant to accept a "direct action" hypothesis (Donoso et_§l,, 1973; Lu and Meites, 1972). Now that a method of administering catecholamines has been developed, further research must be done to deter- mine their effects. There can be no doubt that the catecholamines influence prolactin release both ip_vitro and ip}yiyg. This thesis has demonstrated that dopamine and an ergoline derivative which is presumably a dopaminergic agonist, can in very small amounts significantly inhibit prolactin release i§_ vitro, and pharmacological doses of dopamine can inhibit prolactin release ig_yiyg. These data leave little doubt that a mechanism exists by which dopamine can directly influence prolactin release, probably by activating a dop— aminergic receptors present on the cell membranes of the pituitary lactotrophs. Norepinephrine, although not tested in yiyg, also significantly inhibited prolactin release i3 vitro. Some researchers believe that norepinephrine may also be inhibitory ip_yiyg_while others believe it to be stimulatory to prolactin release 12,2139. The physiological significance of the catecholamine influence on pituitary prolactin remains to be demonstrated. 157 Pharmacological analytical techniques are now available which allow neuroendocrinologists to broaden their measure- ment of hormones to include the neurotransmitters: especially in the hypophyseal portal blood system. There is evidence already cited that a polypeptide PIF exists in addition to a catecholamine. The data presented in this thesis do not exclude or contradict the possibility that a peptide PIF exists. The ability of the ip_yitrg_incubation system to demonstrate the polypeptide PIF activity of a single hypothalamic equivalent may be a factor since it is quite clear from the data reviewed above that the catechol- amine content of a single hypothalamic equivalent is more than adequate to directly inhibit prolactin release. It is suggested that when the catecholamines are removed, the remaining peptide PIF content in a single hypothalamic equivalent may be well below that needed to show PIF activ- ity in such an incubation system. The possibility that a polypeptide PIF became bound to the aluminum oxide does exist. However, in an experiment not reported in this thesis, luteinizing hormone-releasing hormone (LHRH) was demonstrated not to bind to aluminum oxide. The author believes that in addition to the existence of a polypeptide PIF a catecholamine neurotransmitter, presumably dopamine, must also be considered physiologically capable of regu- lating prolactin release. CURRICULUM VITAE NAME: Shaar, Carl Joseph DATE OF BIRTH: October 21, 1940 PLACE OF BIRTH: Lancaster, Pennsylvania, U.S.A. NATIONALITY: American SEX: Male MARITAL STATUS: Married PRESENT ADDRESS: Department of Physiological Research Lilly Research Laboratories Indianapolis, Indiana 46206 FUTURE ADDRESS: Same EDUCATION: Degree Year Institution Major Field of Study» 3.8. 1962 Michigan State University Biological Science M.S. 1967 Michigan State University Biological Science Ph.D. 1975 Michigan State University Physiology POSITIONS HELD: Communications Officer--U.S. Navy USS Sandoval APA194 1962-1965 Physiologist, Lilly Research Laboratories 1967-1970; l972-present Research Assistant, Department of Physiology Michigan State University 1970-1972 TALKS PRESENTED AT SCIENTIFIC MEETINGS: Meeting Date Topic 1. 55th Annual Meeting of 1971 Estrogen requirement for Fed. Am. Soc. Exptl. Biol. neural stimulation of Chicago, Illinois prolactin secretion. 2. Fall Meeting of 1973 The effects of catechol— Am. Soc. for Pharm. and amines apomorphine, and Exptl. Therap., monoamine oxidase on rat East Lansing, Michigan anterior pituitary prolactin release in vitro. 3. 58th Ann. Meeting of 1974 Effect of aluminum oxide Fed. Am. Soc. Exptl. Biol. catecholamine adsorption Atlantic City, New Jersey and monoamine oxidase on the inhibition of rat pitui— tary prolactin release by hypothalamic extracts in vivo. continued 158 159 RESEARCH PUBLICATIONS: 1. 10. 11. 12. Clemens, J. A., J. Kleber, C. Shaar and W. Tandy. (1970). Effect of electrochemical stimulation of the preoptic area on plasma LH and FSH in rats. Fed. Proc. 29: 382 (abstract). Clemens, J. A., C. J. Shaar, J. W. Kleber, and W. A. Tandy. (1971). Areas of the brain stimulatory to LH and FSH secretion. Endocrinology 88: 180-184. Clemens, J. A., C. J. Shaar, J. W. Kleber, and W. A. Tandy. (1971). Reciprocal control by the preoptic area of LH and prolactin. Exp. Brain Res. 12: 250-253. Clemens, J. A., C. J. Shaar, W. A. Tandy and M. E. Roush. (1971). Effects of hypothalamic stimulation on prolactin secretion in steroid treated rats. Endocrinology 89: 1317-1320. Smalstig, E. B., D. R. Bennett, C. J. Shaar and R. L. Cochrane. (1971). Effects of corpus luteum removal on ovarian cyclicity in rats. Endocrinology 89: 714-721. Shaar, C. J. and J. A. Clemens. (1971). Estrogen requirement for neural stimulation of prolactin secretion. Fed. Proc. 30: 185. Shaar, C. J. and J. A. Clemens. (1972). Inhibition of lactation and prolactin secretion in rats by ergot alkaloids. Endocrinology 90: 285-288. Lu, K. H., C. J. Shaar, K. H. Kortright, and J. Meites. (1972). Effects of synthetic TRH on i£_vitro and ig_vivo prolactin release in the rat. Endocrinology 91: 1540-1544. Clemens, J. A., C. J. Shaar and E. B. Smalstig. (1972). Effects of electrochemical stimulation of the hypothalamus on LH release in steroid-treated rats. Neuroendocrinology 10: 175- 182. Clemens, J. A. and C. J. Shaar. (1972). Inhibition by Ergocornine of initiation and growth of 7,12-Dimethylbenzanthracene- induced mammary tumors in rats: effect of tumor size. Proc. Soc. Exp. Biol. Med. 139: 659-662. Lu, K. H., K. H. Kortright and C. J. Shaar. (1972). TRH effects on prolactin release by rat pituitary i2 vivo and i2_vitro. Fed. Proc. 31: 320. Shaar, C. J., E. B. Smalstig and J. A. Clemens. (1973). The effect of catecholamines, apomorphine, and monoamine oxidase on rat anterior pituitary prolactin release ig_vitro. Pharmacologist 15: 15. 13. 14. 15. 16. 17. 18. 19. 20. 160 Clemens, J. A., C. J. Shaar, E. B. Smalstig and C. Matsumoto. (1974). Effects of some psychoactive agents on prolactin secretion in rats of different endocrine states. Horm. Metab. Res, 6: 187-190. Clemens, J. A., C. J. Shaar, E. B. Smalstig, N. J. Bach, and E. C. Kornfeld. (1974). Inhibition of prolactin secretion by ergolines. Endocrinology 94: 1171-1176. Shaar, C. J. and J. A. Clemens. (1974). Effect of aluminum oxide catecholamine adsorption and monoamine oxidase on the inhibi- tion of rat anterior pituitary prolactin release by hypothal- amic extracts £2 vitro. Fed. Proc. 33: 237. Shaar, C. J. and J. A. Clemens. (1974). The role of catecholamines in the release of anterior pituitary prolactin release ig_ vitro. Endocrinology_95: 1202-1212. Shaar, C. J., D. R. Bennett, J. G. Powell, Jr., E. B. Smalstig, F. C. Tinsley and R. L. Cochrane. (1975). The effects of 5-Bromo-2-thienyl—ethyl ketone thiosemicarbazone on ovarian cyclicity and ovulation in the rat. J. Reprod. Part. 44: 421-428. Clemens, J. A., E. B. Smalstig and C. J. Shaar. (1975). Inhibition of prolactin secretion by Lergotrile Mesylate: mechanism of action. Acta Endocr. 79: 230-237. Schally, A. V., A. Dupont, A. Arimura, J. Takahara, T. W. Redding, J. A. Clemens and C. J. Shaar. (1975). Purification of a catecholamine rich fraction with PIF activity from porcine hypothalami. Acta Endocr. (in press). Shaar, C. 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