L1 14 2: A R Y . Michigan State . University . Ill 00 ll ll llllIIHlI “Hill“ 3 1293 llflllll‘l IHHI 2615 TH ECQB This is to certify that the thesis entitled CONTROL OF PROLACTIN RECEPTORS IN KIDNEYS AND ADRENALS OF RATS, AND LIVER OF MICE presented by Stephen Marshall has been accepted towards fulfillment of the requirements for Ph ' D' degree in PhYSiC’lOgY 1 V/I/ "A /. // , (1 Y *lV/A.) \‘ [jet -_',_, MaIgr professor Date October 124 1978 0-7639 r43" U ovmimE FINES ARE 25¢ PER DAY ‘ PER ITEM Return to book drop to remove this checkout from your record. CONTROL OF PROLACTIN RECEPTORS IN KIDNEYS AND ADRENALS OF RATS, AND LIVER OF MICE BY Stephen Marshall A DISSERATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1978 ABSTRACT CONTROL OF PROLACTIN RECEPTORS ACTIVITY IN RAT KIDNEYS AND ADRENALS OF RATS, AND LIVER OF MICE. BY Stephen Marshall 1. Serum PRL levels and PRL binding in kidneys and adrenals were measured in control, water-deprived, unilat- erally nephrectomized, and salt-loaded male rats. Unilat- eral nephrectomy and water deprivation increased serum PRL levels significantly. Unilateral nephrectomy did not alter PRL binding in the kidneys, but significantly increased it in the adrenal glands. Salt loading had no effect on serum PRL levels or PRL binding in the kidneys, but significantly increased PRL binding in the adrenal glands. Inhibition curves and tests of cross reactivity with LH, FSH, TSH, and GH showed that binding of PRL to its receptors in the kid- neys and adrenals was specific. 2. The effects of estradiol benzoate in the female rat, testosterone propionate in the male rat, and cas- tration in both sexes on specific PRL binding sites in kidneys and adrenals were studied. Castration resulted in a significant increase in PRL binding in the kidneys of both males and females, and in a significant increase in PRL binding activity in female adrenals. Progesterone administration to castrate females failed to alter PRL binding in both tissues. 3. Binding of PRL sites in kidney and adrenal was shown to be time-and temperature-dependent. Adrenalectomy increased concentrations of PRL receptors in the kidneys 49% above control values, whereas dexamethasone treatment reduced PRL receptors to below control levels. The latter effect was not mediated primarily through the pituitary because dexamethasone treatment of hypophysectomized rats also produced a significant decrease in specific receptor- binding from 2.85 i 9.20% - 0.74 i 0.08%. In adrenals, a reduction in specific PRL binding also was observed after hypophysectomy, from 12.72 i 0.63% - 5.73 i 0.33%, and this was decreased further to 3.18 i 0.45% after dexamethasone treatment. Hydrocortisone as well as dexamethasone were effec- tive in lowering PRL, receptor binding in kidney membrane preparations in intact male rats, and this effect was found to be dose-dependent by using a high and low dose of dexamethasone. Neither aldosterone, its antagonist, spironolac- tone, nor renin was effective in altering concentrations of PRL receptors in the kidneys or adrenals of intact rats, suggesting that mineralocorticoids have little, if any, effect on PRL receptors in these tissues. 4. The effect of thyroxine (T4) on specific PRL receptor binding in the kidneys and adrenals were measured in intact, hypophysectomized (HYPOX) and thyroidectomized (TX) male rats. PRL binding in the kidneys was reduced from 12.1 i 1.1 in the intact group to 4.5 i 0.5% after HYPOX; 4 days replacement with T returned PRL binding 4 to intact values. T4 administered to intact rats signifi- cantly increased PRL binding above intact values. TX reduced PRL binding in the kidneys from 14.9 i 1.2 to 7.0 i 0.6%, and T4 treatment restored PRL binding to intact values. PRL binding was measured at 2, 3, 5, 7 and 10 days after TX in kidneys and found to progressively decrease from 8.2 i 0.5% in the intact rats to 2.3 i 0.3% on day 10. A single injection of T4 doubled PRL receptor binding in kidneys of TX rats at 12 hrs. In contrast, adrenal PRL binding was not altered by TX or T4. 5. To determine if thyroid hormone effects on renal PRL receptors were mediated by increased RNA or protein synthesis, the effects of actinomycin-D and cycloheximide, on PRL receptors were measured. In intact rats actinomycin-D alone reduced renal PRL binding 25.2%, whereas in the adren- als binding was reduced by 88.6%. The differential effects of actinomycin in reducing PRL receptors in the kidneys and adrenals may reflect differences in the half life of mRNA in these two organs. Cycloheximide reduced binding in the kidneys by 79.2%, and by 94.2% in the adrenals of intact rats. In TX rats actinomycin-D and cycloheximide were given concomitantly with a single injection of T4. Actinomycin and cycloheximide were both effective in preventing the T4 induced increase in (1251)iodo-PRL binding in the kidneys of Tx male rats. Although thyroid hormones had no effect on adrenal PRL receptors actinomycin-D or cycloheximide administered together with T4, did significantly decrease PRL receptors below Tx controls. 6. Ontological changes were observed in (1251)iodo- PRL binding to kidney and adrenal membranes from male rats during days 10-60 of age. PRL binding progressively increased from 4.94 i 0.16 to 16.91 i 1.27% in the kidneys, whereas in adrenals, PRL receptors decreased from 22.89 i 1.24 at day 10 of age to 4.98 i 0.48 at day 60. To determine PRL binding association and dissociation times in 3139, a single mg i.v. oPRL injection was given, the kidneys and prostates removed at various times there- after, and in vitrg (1251)iodo-PRL binding measured. Renal PRL binding sites were maximally reduced at 5 min post— injection from 9.98 i 0.75%, in the controls, to 0.40%, and a similar significant reduction in PRL binding (Pf9.9l) was found in prostatic tissue. PRL binding sites returned to control values 8 hrs. post—PRL injection in the kidneys, and at 1 hr in the prostate. When a single sc injection of 20 ug EB was given to OVX rats, PRL receptors increased in the liver, and de- creased in the adrenals. In both organs significant changes in PRL binding occurred on day 3, with maximal changes observable on day 7. On day 21, PRL receptors in the liver and adrenals were not significantly different from their respective control values. LIST OF TABLES LIST OF FIGURE TABLE OF CONTENTS S . . . . . . . . . . . . . . . . . . . INTRODUCTION. . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . I. Hypothalamic Regulation of Anterior Pituitary Secretion . . . . . . . . A. Role of the Hypothalamus . . . . . B. Hypothalamic-Hypophysial Vascular Connections. . . . . . . . . . . C. General Anatomy of the Pituitary Gland . . . . . . . . . . . . . D. Releasing and Inhibiting Hormones from the Hypothalamus and their Control of Anterior Pituitary Function . . . . . . . . . . . . II. Prolactin Secretion and Regulation . A. Prolactin Inhibiting Releasing Hormone(s) . . . . . . . . . . . B. Prolactin Releasing Hormone(s). . . C. Biogenic Amine Control of Pro— lactin Secretion . . . . . . . . D. Ergot Derivatives and their Action on the Pituitary . . . . . . . . E. Short-LOOp Feedback . . . . . . . . III. Interactions Between Prolactin and Other Hormones and Steroids . . . . A. Testosterone, Estrogen and Pro- gesterone . . . . . . . . . . . B. Glucocorticoids . . . . . . . . . . C. GonodotrOpins . . . . . . . . . . . D. Thyroid Hormones . . . . . . . . . IV. Role of Prolactin in Reproduction . . . A. Actions on the Mammary Gland and Pigeon CrOp Sac . . . . . . . . B. Actions on the Ovary . . . . . . . C. Actions on the Testis and Secondary Sex Accessory Organs . . . . . . ii Page vi vii 13 l6 19 23 28 31 34 34 37 40 42 45 45 49 55 VI. MATERIALS AND I. II. III. IV. V. EXPERIMENTAL. I. II. Page Other Functions of Prolactin. . . . . . . A. Osmoregulation . . . . . . . . . . . B. Adrenal Gland . . . . . . . . . . . . C. Liver . . . . . . . . . . . . . . . . Regulation of Prolactin Receptors in Several Target Organs . . . . . . . . A. Early Studies . . . . . . . . . . . . B 0 Liver I O O O O O O O O O o O O O O D C. Prostate. . . . . . . . . . . . . . . D. Mammary Gland . . . . . . . . . . . . E. Mammary Tumors. . . . . . . . . . . . METHODS . . . . . . . . . . . . . . . . . Animals . . . . . . . . . . . . . . . . . Radioimmunoassays . . . . . . . . . . . . Prolactin Receptor Determinations . . . . A. Preparation of Particulate Membranes B. Enzymatic Radioiodination of Prolactin . . . . . . . . . . . . C. Assay Procedure . . . . . . . . . . . Steroids, Hormones and Drugs. . . . . . . Statistical Analysis. . . . . . . . . . . Demonstration of Specific Prolactin Binding to Particulate Membrane Preparations from Kidneys and Adrenals of Rats. . . . . . . . . . . . . . . . Objectives. . . . . . . . . . . . . . Material and Methods. . . . . . . . . Results . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . 00w? Serum Prolactin Levels and Prolactin Binding Activity in Adrenals and Kidneys and Male Rats After Dehydra- tion Salt Loading, and Unilateral Nephrectomy. . . . . . . . . . . . . . Objectives. . . . . . . . . . . . . . Material and Methods. . . . . . . . . Results . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . 'UOwW iii 58 58 64 65 65 68 70 71 71 73 73 73 74 74 75 76 77 77 78 78 78 78 79 81 81 81 82 83 83 III. IV. VI. VII. Page Effects of Estrogen and Testosterone on Specific Prolactin Binding in the Kidneys and Adrenals of Rats . . . . . 85 A. Objectives. . . . . . . . . . . . . 85 B. Material and Methods. . . . . . . . . 86 C. Results . . . . . . . . . . . . . . . 87 D. Conclusions . . . . . . . . . . . . . 91 Glucocorticoid Regulation of Prolactin Receptors in Kidneys and Adrenals of Male Rats. . . . . . . . . . . . . . 93 A. Objectives. . . . . . . . . . . 93 B. Material and Methods. . . . . . . . 95 C. Results . . . . . . . . . . . . . . 96 D. Conclusions . . . . . . . . . . . . 105 Effects of HypOphysectomy, Thyroidectomy and Thyroxine on Specific Prolactin Receptor Sites in Kidneys and Adrenals of Male Rats . . . . . . . . . . . . .109 Objectives. . . . . . . . . . . . . .109 Material and Methods. . . . . . . . .110 Results . . . . . . . . . . . . . . .111 Conclusions . . . . . . . . . . . . .118 UOUJD' Inhibition of the Thyroid Hormone Induced Increase in Renal PRL Recep- tors by Actinomycin—D and Cycloheximide. . . . . . . . . . . . .122 A. Objectives. . . . . . . . . . . .122 B. Material and Methods. . . . . . . . .124 C. Results . . . . . . . . . . . . . . .125 D. Conclusions . . . . . . . . . . . . .129 Prolactin Receptors in Kidneys and Adrenals During Development and After Injection of Prolactin, Testosterone or Estrogen. . . . . . . . . . . . . .131 Objectives. . . . . . . . . . . . . .131 Material and Methods. . . . . . . . .133 Results . . . . . . . . . . . . . . .134 Conclusions . . . . . . . . . . . . .144 DOW? iv VIII. A. B. C. D GENERAL DISCUSSION REFERENCES . . . CURRICULUM VITAE Prolactin Receptors in Mouse Liver; Species Differences in Response to Estrogenic Stimulation . Objectives . Material and Methods Results Conclusions Page 148 148 149 151 155 158 164 197 LIST OF TABLES Table Page 1. Prolactin Binding Activity in Kidney Particulate Membranes of Dehydrated, Salt-Loaded (1.5% NaCl in Drinking Water), and Unilaterally Nephrectom- ized Male Rats . . . . . . . . . . . . . . . . 84 2. Prolactin Binding Activity in Adrenal Particulate Membranes of Dehydrated, Salt-Loaded (1.5% NaCl in Drinking Water), and Unilaterally Nephrectom- ized Male Rats. . . . . . . . . . . . . . . . . 84 3. Serum Prolactin Levels in Dehydrated, Salt-Loaded (1.5% Saline as Drinking Water), and Unilaterally Nephrectom- ized Male Rats. . . . . . . . . . . . . . . . . 84 4. Prolactin Binding in Kidney Membrane Preparations of 30 and 62 Day Old Intact (Control) and Testosterone PrOpionate (TP) Treated Male Rats. . . . . . . . . . . . . 88 5. Prolactin Binding Activity in Adrenal and Kidney Membrane Preparations of Intact (Controls), Castrated and Castrated + Testosterone PrOpionate (TP) Treated Male Rats. . . . . . . . . . . . . . . . . . . . . . 89 6. Prolactin Binding Activity in Adrenal and Kidney Membrane Preparations of Intact (Controls), Ovariectomized (OVX) OVX+ Progesterone (Prog). and OVX+ Estradiol Benzoate (EB) Treated Female Rats . . . . . . . 90 7. Effect of Aldosterone, Renin or Spirono- lactone on Serum Prolactin Levels and Prolactin Receptor Binding in the Kid- neys and Adrenals of Male Rats. . . . . . . . .104 vi Figure 1. 10. LIST OF FIGURES Page . . 125 . Compet1ve Displacement of ( I)1odo-PRL Binding to Kidney Membranes by Various Con- centrations of Unlabeled Hormones . . . . . . . 80 Competitive Displacement of (125I)iodo-PRL Binding to Adrenal Membranes by Various Con- centrations of Unlabeled Hormones . . . . . . . 80 Relationship Between The Concentration of Male Rat Kidney Membrane Protein and Spe- cific Binding of (125I)iodo-PRL . . . . . . . . 99 Time Course of Specific Binding of (1251)iodo- PRL at 4, 24, and 36 C to Rat Kidney Membranes.100 The Effects of ADX, ADX with Dexamethasone (DEX) Treatment, and ADX with DEX and ACTH Treatment on the Specific Binding of Ovine (125I)iodo-PRL to Kidney Membranes of Male Rats. . . . . . . .101 Effects of Hypophysectomy (HYPOX), HYPOX and Dexamethasone (DEX), and HYPOX and ACTH on Specific PRL Binding in Kidney and Adrenal Membrane Preparations From Male Rats. . . . . . . . . . . . . . . . . . . . . .102 Effects of Hydrocortisone (H.C., 0.1 mg) a High (100 ug/100 g BW) and a Low (10 ug/ 100 9 BW) Dose of Dexamethasone (DEX) for 5 Days, and A Low Dose of DEX for 10 and 20 Days on Specific PRL Binding in Kidney Mem- branes from Intact Male Rats. . . . . . . . . .103 Effects of Thyroidectomy and Tx With T Treatment in Male Rats on Specific Binding of (125I)iodo-PRL to Kidney and Adrenal Membranes . . . . . . . . . . . . . . . . . . .113 Effects of T , Hypophysectomy and Hypophysec- tomy with T on Specific Binding of (1 5I)iodo- PRL to Kidney Membranes from Adult Male Rats. .114 Specific PRL Binding Sites in Kidney and Adrenal Membranes, and Serum TSH values at 2, 3, 5, 7 and 10 days After TX . . . . . . . .115 vii Figure Page 11. 12. 13. 14. 15. l6. 17. 18. 19. 20. 21. 22. Effects of a Single 10 ug (P 1009 BW Injection on Specific Binding of ( 5I iodo—PRL to Kid— neys and Adrenal Membranes from Intact and Tx Male Rats . . . . . . . . . . . . . . . . . . . .116 Effects of Dexamethasone and Dexamethasone with T on Specific PRL Binding Sites on the Kidneyg of Male Rats. . . . . . . . . . . . . . .117 Effects of T , Actinomycin-D and Cyclohexi- mide on ( I)iodo-PRL Binding to Kidney and Adrenal Membranes from Intact Male Rats . . . . .127 Effects of T I T + Cyclohegimi e 03 (125I)iodo-PRL Binding t3 Kidney and Adrenal Membranes from Tx Male Rats. . . . . . . . . . . . . . . . . . . . . . .128 + lActinomycin-D and Effects of Age on Specific (1251)iodo—PRL Binding to Kidney and Adrenal Membranes from Male Rats O Q I O O O I O O O O O O O O O O O I O 1'37 Serum GH, PRL and TSH in the Male Rat During Development From Day 10 to Day 60 . . . . . . . .138 Time Course Effects of a Intravenous In'ection of 1 mg. oPRL on In Vitro Binding of (1 5I)iodo— PRL to Male Kidney Membranes. . . . . . . . . . .139 Time Course Effects of a Single Intravenous Injection2 of 1 mg. of oPRL on In Vitro Bind- ing of (1 251)iodo-PRL Binding to Male Prosta- tic Membranes . . . . . . . . . . . . . . . . . .140 Specific (1251)iodo-PRL Binding to Ventral Prostates at Various Times After a Single sc Injection of TP. . . . . . . . . . . . . . . .141 Changes in (lZSI)iodo-PRL Binding to Liver and Adrenal Membranes From OVX Rats at Various Times After a Single so 20 ug EB Injections . . .142 Serum LH and PRL (ng/ml) in OVX Rats at Various Times After a Single so 20 ug EB Injection 0 O O O O O O O O O O O O O O O O O O O 143 Effects of OVX and OVX With EB Replacement on Specific (1251)iodo-PRL Binding to Liver Membrane Preparations From Female Rats. . . . . .152 viii Figure 23. 24. Page Serum PRL Levels and (lzsl)iodo-PRL binding to Liver Membranes From Intact, OVX and OVX Mice Given Daily Injections of Different Doses of EB . . . . . . . . . . . . . . . . . . . 153 SpecifflzBinding of (1251)iodo—PRL to Liver Membranes From Male Mice 7 Days After a Single Injection of 2 ug EB . . . . . . . . . . . 154 ix INTRODUCTION Endocrinology as an investigative science began in the late nineteenth century. This was a time when very abnormal hormonal states were being causally related to growth and sexual function, and the importance of the pituitary gland in pathological as well as physiological states was being recognized. Interest continued into the early 1900's with many attempts made at extirpation of the pituitary gland from dogs and cats. A successful method for hypophysectomy was developed by E.P. Smith in the 1920's. Use of this technique lead to the dis— covery of growth hormone by Evans and Long (1921), pituitary gonadotrOpins by E.P. Smith (1933), lactogenic activity in the pituitary by Stricker and Grueter (1928), TSH and ACTH. From this time until the early 1960's much progress was made in laying the foundations of pituitary endocrinology. During this period it was rec- ognized that dependable assay systems were needed to quantitate the amount of any one hormone present in the pituitary so that a better understanding could be obtained of its functions during different physiological and path- ological states. After a great deal of hard work and ingenuity, 99 3939 and £9 39339 bioassays were developed for all the anterior pituitary hormones. However, assay sensitivity was very low. In the late 1950's and early 1960's a new technique that was to revolutionize the field of endocrinology was introduced by Solomon Berson and Rosalyn Yalow, namely the radioimmunoassay. This specific competitive binding radioassay, utilizing antibodies to hormones, allowed measurement of hormones in nanogram and picogramg amounts, levels normally found in the blood. Because of the ease, sensitivity and rapidity of this technique, a flood of data has since appeared in the literature that has extended our knowledge of hormone function and regulation; during normal physiological states, and increased our awareness of the complexity and integration of the endocrine system. Another technique has more recently become avail- able that can further extend our understanding of the complexity and integration of the endocrine system. This has been the development of a specific radioreceptor assay for prolactin (and other peptide hormones) that makes it possible to measure binding sites in prolactin target tis- sues (Shiu 99 93., 1973). It is now known that for a poly- peptide hormone to have an action on a target tissue, it must first bind to a specific receptor on the plasma mem- brane before any subsequent intracellular event can take place. Receptors for prolactin, growth hormone, luteinizing hormone,follic1e stimulating hormone, thyroid stimulating hormone and adrenocorticotropic hormone have recently been identified on their respective target tissues. Thus, it appears that hormone action is regulated not only by the levels of endogenous hormone but also by receptor alterations on the plasma membrane. With this technique studies eluci- dating receptor control in peripheral target organs are possible. The studies presented here were designed to inves- tigate the control of prolactin in the kidneys and adrenals of rats, and their possible role in salt and water regula— tion through measurements of prolactin receptors and endo- genous prolactin levels. Since no research has been done in this area of prolactin receptors, experiments were de- signed to establish the following: (1) The existence of specific binding sites for prolactin in the kidneys and adrenals of male and female rats which do not cross-react with other anterior pituitary hormones (2) The relationship between factors that alter salt and water regulation such as dehydration and salt loading and changes in prolactin receptors in the kidney and adrenal target tissues (3) Control of prolactin receptors in kidneys and adrenals by steroids and other hormones LITERATURE REVIEW I. Hypothalamic Regulation of Anterior Pituitary Secretion A. Role of the Hypothalamus and its Anatomy The function of the hypothalamus is to maintain a steady state in body structure and function known as homeostasis. It accomplishes this be receiving information from the external environment via sensory inputs, as well as neural and chemical inputs from the brain and the rest of the body. From this barrage of incoming information, the hypothalamus performs a high degree of integration and differentiation resulting in an output of both neural and chemical messages to control various bodily functions. These functions include cardiovascular dynamics such as heart rate and blood pressure, regulation of body temp- erature and body water, and gastrointestinal and feeding regulation (Guyton, 1976). Therefore, the hypothalamus is involved in a multitude of regulatory functions, but the most important role of the hypothalamus, at least for neuro- endocrinologists, is the regulation of the anterior pit- uitary gland. Understanding the anatomy of the hypothalamus is nec- essary to gain an appreciation of its role in pituitary reg- ulation. The hypothalamus forms the medial part of the floor of the diencephalon and is located on the wall of the third ventricle from the posterior margin of the mammil- lary bodies to the anterior margin of the Optic chiasm. Laterally it is bounded by the subthalamus and internal capsule and dorsally by the thalamus (Netter, 1968; Jacobson, 1972). Within the hypothalamus are numerous nuclei which can be divided into three regions reflecting their general loca- tion. The anterior region includes the preOptic and supra- optic nuclei; the middle zone comprises the dorsal medial, ventral medial, lateral and tuberal nuclei; the posterior region contains the posterior hypothalamic nuclei and the mammillary nuclei. In addition, there are periventricular nuclei next to the third ventricle in all the regions. Connecting these hypothalamic nuclei is a dense matrix of fine nerve fibers. In keeping with the hypothalamus as an integrating center, numerous afferent tracts synapse in this area. Of major importance is the medial forebrain bundle serving as a bidirectional link between the mesencephalic reticular formation structures and the limbic forebrain. Other major imputs come from the frontal associational cortex (corticohypothalamic fibers), amygdala (stria terminalis), cingulate cortex and the hippocampus (fornix). Efferents from the hypothalamus include the following major tracts: (1) the medial forebrain bundle as previously mentioned; (2) the mammillotegmental tracts connecting the medial mam- millary nuclei with the midbrain reticular nuclei; (3) mam- millothalamic tract connecting the medial mammillary nucleus with the anterior thalamic nucleus, (4) the paraventricular system from the supraoptic, tuberal, and posterior nuclei in the hypothalamus supplying the medial thalamic nucleus. Lastly, of major importance is the hypothalamic-hypophysial tract which originates in the paraventricular and supra- optic nuclei and converges on the pituitary stalk to supply the neural hypophysis. This tract is unique in that it is neurosecretioy and can transmit both nerve impulses and neurosecretory down its axons (Donovan, 1970; Knigge 93 93., 1975; Netter, 1968; Jacobson, 1972). B. Hypothalamic-HypOphysial Vascular Connection Between the hypothalamus and the anterior pituitary there is no direct neural connection, however, there is a vascular link known as the hypothalamic-hypophysial portal system. From the internal carotid and posterior communi- cating arteries the superior hypophysial arteries branch and terminate in the median eminence area where they form a capillary network. The venous blood is then drained into sinusoids of the adenohypophysis by the "long" portal ves- sels. A second source of blood supply to the anterior pituitary comes from the inferior hypOphyseal arteries which distribute to the posterior pituitary, form a cap- illary bed and then drain into the anterior pituitary by "short" portal vessels. The capillary 100ps of the median eminence are unique in that many neurosecretory neurons terminate on these vessels and discharge both releasing and inhibiting factors which traverse the portal circulation to control the secretion of hormones from the anterior pituitary (Turner and Bagnara, 1976; Jackson, 1972; Netter, 1968). What is known today about the hypothalamic—hypo- physial vascular connection is a culmination of research efforts started in 1930 by Popa and Fielding. These inves- tigators gave the first detailed description of this ana- tomical arrangement, but mistakenly thought blood flow was from the pituitary to the hypothalamus. Later studies sug- gested that blood flowed in the opposite direction from the hypothalamus to the pituitary (Wislock 99 99., 1936; Houssay, 1935). Then Green and Harris (1947) expanded this idea by preposing the neurovascular hypothesis stating that blood flowed from the median eminence to sinusoids of the anterior pituitary, and substances released into portal ves— sels released hormones from pituitary cells. Later, Harris (1955) proposed the chemotransmitter hypothesis suggesting that neural regulation of anterior pituitary function is mediated by substances carried from capillaries of the hypophyseal portal system to the pituitary. Subsequent studies have confirmed this functional arrangement by removal of the pituitary from hypothalamic control or interrupting portal vessels and demonstrating changes in histological appearance and secretory capacity. When the pituitary was grafted under the median eminence, histological and func- tional characteristics were partially restored (Harris and Jacobson, 1952; Nikitovitch-Winer and Everett, 1958; Halasz 99 99., 1962). 8 C. General Anatomy of the Pituitary Gland The pituitary is a dual endocrine gland that lies in the sella turcica at the base of the brain and encap- sulated by the dura mater. This gland can be divided func- tionally into two distinct portions: the anterior pitui- tary or adenohyp0physis and the posterior pituitary or neurohypophysis. Situated between these two glands is a relatively avascular region known as the pars intermedia. Connecting the pituitary gland to the median eminence por- tion of the hypothalamus is the hypophyseal stalk. The entire pituitary is derived from ectodermal tissue, but the anterior and posterior portions arise from differ- ent sources. While the anterior pituitary originates from Rathke's pouch, an embryonic invagination of the pharyn- geal epithelium, the posterior pituitary is formed from the infundibulum of the brain by an outgrowth of the hypo- thalamus. Because of differing origins of the two por— tions of the pituitary, the nature of its cellular struc- ture is also different. The neurohypOphysis is composed mostly of glial-like cells called pituicytes which serve as supporting structures for the terminal nerve fibers and endings that originate in the paraventricular and supra- optic nuclei in the hypothalamus. It is in the nerve end- ings that posterior hormones vas0pressin and oxytocin are stored until their release by nerve impulses traveling down the same fibers which originate in the two hypothalamic nuclei (Turner and Bagnara, 1976; Guyton, 1976). The diversity of cell types in the anterior pituitary was first recorded by Schonemann (1892). Since that time various methods have been devised for identifying the cells that secrete the six adenohypophyseal hormones. Special cell staining techniques have been employed to identify the major cell types into three groups: acidOphils, bas- ophils or chromOphobes depending on whether the cells could be stained with acidic or basic dyes. AcidOphils produce prolactin and growth hormone; basophils secrete lutein- izing, follicle and thyroid stimulating hormones and chrom- Ophobes secrete adrenocorticotropin. More recently the electron microsc0pe has been used for identifying hormone secreting cells more specifically by differences in cyto— plasmic granules, cell size, shape, etc. Furthermore, immunohistochemical techniques have been used for hormone localization in the anterior pituitary gland. Separate cells for prolactin, ACTH, growth hormone, and TSH have been found using this technique (Nakane, 1970). FSH and LH were frequently found in the same cell (Ganong, 1975; Guyton, 1976; Turner 99 99., 1976). D. Releasing and Inhibiting Hormones from the Hypothalamus and Their Control of Anterior Pituitary Function Hypothalamic regulation of anterior pituitary secretion is accomplished by trOphic hormones being syn- thesized in the hypothalamus, transported to nerve end- ings terminating on the median eminence portal vessels, 10 and then released into portal blood to influence adeno- hyp0physis secretions (Harris, 1955). The region of the hypothalamus that contains the cells that synthesize and control the releasing hormones has been identified by Halasz 99 99. (1962) and termed the "hypophysiotrOpic area". Since the chemotransmitter hypOphysis was proposed in 1955 much progress has been made in identifying the various hyp0physiotr0phic hormones as well as determining their structure and function. The first step in this research, however, called for an assay of releasing factors and this was originated by Saffran and Schally (1955). This 99 39999 short-term incubation system involved incubation of rat anterior pituitary glands in small beakers or flasks at 37° C with continuous oxygenation and shaking. When crude acid extracts of hypothalamus was added to the incu- bation media it caused the release of biologically detect- able ACTH. This releasing factor was called "cortico- trOpin releasing factor" (CRF). Thus far the chemistry of CRF has not been determined. In contrast, much more is known about thyrotrOpic releasing hormone (TRH). The existence of a TRH was pro- vided by Shibusawa 99 99. (1959), Schreiber (1963), and Guillemin 99 99. (1962). Later, Boler 99 99. (1969) and Burgus 99 99. (1969) identified TRH as a tripeptide (pyro Glu-His-Pro-NHZ). Recently, the highest concentration of TRH was found in the enriched synaptosomal cell fraction of the hypothalamus (Barnes 99 99., 1975). Furthermore, 11 high concentrations of TRH have also been found in various classes of vertebrates including the rat, chicken, snake, frog, tadpole and salmon (Jackson and Reichlin, 1974). Significant concentrations of TRH have been detected out“ side the hypothalamus of the rat brain and can account for as much as 80% of total brain TRH. A possible function of extrahypothalamic TRH was demonstrated by Keller 99 99. (1974) showing an enhancement of cerebral noradrenalin turnover by thyrotrOpin releasing hormone. TRH may also serve2as al‘phySidogical regulator of prolactin and is dis- cussed in the section on prolactin releasing hormone. Based on a bioassay method, McCann 99 99. (1960) provided the first evidence for the presence of a lutein- izing releasing hormone (LRH) in the hypothalamus. This was followed by the isolation and structural identifica- tion of LRH from porcine and ovine hypothalami, which was shown to be a linear peptide of ten amino acids (Schally 99 99., 1973; Burgus 99 99., 1972). Barraclough (1967) suggested that there are two LRH producing areas in the hypothalamus, one in the arcuate and ventromedial nuclei controlling basal secretion of LH, and the other in the preoptic suprachiasmatic nuclei regulating cyclic LH release. LRH has been shown to release FSH in many mam- malian species (Schally 99 99., 1973) suggesting that LRH is also the physiological regulator of FSH secretion. However, this idea is still controversial and there is some evidence for a separate FSH-RH (Shin 99 99., 1974). The greatest amount of LRH is in the median eminence in the 12 rat with smaller quantities in the arcuate nucleus. But again, extrahypothalamic LRH has been detected (Piacsek and Meites, 1966) and mating behavioral effects in rats have been induced by LRH (Moss and McCann, 1973). It is possible that LRH can act as a neurotransmitter with effects on the sex drive. A great deal of interest has been aroused by the find- ing that somatostatin (GH release inhibitory factor or GIF) not only controls anterior pituitary GH secretion, but can also influence glucose metabolism through its influence on glucagon secretion from the pancreas. The possible existence of a GIF was first reported by Krulich 99 99. (1968) who reported decreased GH release from rat pituitaries co-incubated with hypothalamic extracts. In 1973 Guillemin's group (Burgus 99 99., 1973) reported the primary structure of somatostatin which was obtained from purified ovine hypothalamic extracts. This hypothalamic hormone was found to be a tetradecapeptide, and was able to suppress both basal GH secretion and GH response to all known stimuli. In addition, GIF was found to in- hibit the TRH induced rise in TSH in the rat, mouse and man (Vale 99 99., 1974; Siler 99 99., 1974), but not basal or TRH stimulated prolactin secretion. Furthermore, soma- tostatin was found to have a direct effect on rat pancreas perfused 99 9999 by inhibiting glucagon and insulin secre- tion (Johnson 99 99., 1975). The use of somatostatin in human diabetes mellitus patients to improve glucose metab- olism and reduce insulin requirements is showing some 13 promise (Gerlich 99 99., 1974). An inhibitory effect of somatostatin on gastrin release has also been reported in man (Bloom 99 99., 1974). The other hypothalamic hormones reported to control anterior pituitary function are GRH (Deuben and Meites, 1963), MIF (Nair 99 99., 1971) and MSF (Taleisnik 99 99., 1965). II. Prolactin Secretion and Regulation In mammals, prolactin secretion from the pituitary is predominantly controlled by a prolactin inhibiting factor from the hypothalamus, believed to be dopamine by some workers, and probably by a prolactin releasing factor also of hypothalamic origin. However, the complexity of this regulatory system is now becoming evident as new data appears in the literature identifying various other agents that interact to regulate prolactin release. Among these substances that may modulate prolactin secretion are biogenic amines, other neurotransmitters, corticoids, thyroxine, cyclic AMP, prostaglandins, osmolarity, ions, steroids, and some metals. For some of these substances, their role in the regulation of prolactin secretion is still controversial and in a state of considerable con- fusion. Cyclic AMP has been reported to inhibit, stimulate or have no effect on prolactin secretion. Ojeda 99 99. (1974) demonstrated that dibutryle CAMP was able to reduce plasma prolactin levels in anesthetized estrogen-primed rats. 14 Stimulation of prolactin secretion by cyclic AMP was obtained when male and female rat pituitaries were incu- bated 99 39999 (Cehovic 99 99., 1973). Also, incubated GH3 pituitary tumor cells increased both synthesis and release of prolactin with the addition of cyclic AMP (Hinkle, 1974). Lemay 99 99. (1974) found no effect of cyclic AMP on release of prolactin from bovine secretory granules. Similar controversy exists on the role of prolac- tin secretion. Administration of prostaglandins E2, F1 alpha by any route was not able to stimulate prolactin secretion (Ojeda 99 99., 1974). However, in heifers PGF2 alpha was able to markedly elevate prolactin, growth hor- mone, glucocorticoids and insulin within 5-15 minutes when given intravenously (Louis, Stellflug, Tucker 99 99., 1974). Prostaglandin E1 given to rats on day 21 of preg- nancy caused a significant rise in maternal TSH, LH, and prolactin (D'Angelo 99 99., 1975). Much work needs to be done before we can fully understand the role of cyclic AMP and prostaglandins on prolactin regulation. Osmotic effects on prolactin release have been demon- strated 99 39999 by La Bella 99 99. (1974, 1975). Bovine pituitaries released prolactin inversely to the osmolarity of the incubation medium. Reducing the osmolality in- creased prolactin release, while increasing it reduced the secretion of both prolactin and ACTH; LH, TSH and growth hormone were also reduced but to a lesser estent. In fish pituitary, release of prolactin was also inversely related 15 to the medium osmolarity (Zambrano 99 99., 1974). In addition to osmolarity changes, metal ion concentrations 99 39999 have similarly been reported to alter prolactin secretion. Nickel has been reported to inhibit prolactin secretion, while copper and lithium stimulate release (Horrobin, 1975). Calcium 99 39999 was reported to inhib- it prolactin synthesis in low concentrations but to stim- ulate it in high concentrations, however, no change was found on the release of prolactin (Hinkle, 1974). The reported effects of osmotic and ionic alterations on pro- lactin secretion and their physiological significance 99 3939 awaits further study and confirmation. The effects of various hormones on modulating pro- lactin secretion is better established. Estrogen is known to stimulate prolactin release by a direct effect on the pituitary both 99 3939 (Chen 99 99., 1970) and 99 39999 (Nicoll and Meites, 1962), as well as having a hypothalamic action to decrease PIF activity (Chen 99 99., 1970). Sim- ilarly, thyroxine or trtkflothyronine have been reported to have a direct stimulatory effect on prolactin secretion when incubated with rat anterior pituitaries (Nicoll 99 99., 1963). However, other investigators have found that tri— iodothyronine could reduce prolactin secretion when added to cultured pituitary cells (Tsai 99 99., 1974). Cor- ticoids appear to inhibit prolactin release since adrenal- ectomy produces an increase in prolactin in rats (Ben-David 99 99., 1971; Euker 99 99., 1975). Furthermore, dexameth- asone, a potent glucocorticoid, has been shown to reduce 16 the acute release of prolactin during ether stress in a dose responsive manner (Euker 99 99., 1975). Stress induced prolactin release is inhibited by glucorticoids. In human subjects, similar results have been attained by Vanhaelst 99 99. (1974). Dexamethasone treatment in this study suppressed both basal prolactin and the prolactin rise in response to TRH. From all of the studies mentioned in the preceding section, it can be concluded that various factors such as hormones, cyclic AMP, etc. can alter pituitary or hypo- thalamic functions and modify the synthesis or release of prolactin. Moreover, these modifying factors probably set the background in which prolactin inhibiting and releasing factors regulate prolactin secretion from the pituitary. A. Prolactin Inhibiting Hormone(s) Removal of hypothalamic control from the pitui- tary by transplanting it beneath the kidney capsule was shown to cause a sustained release of prolactin concomi- tant with a decrease in all other pituitary hormones (Everett, 1954). This was confirmed by Nikitovitch-Winer and Everett (1958) and Chen 99 99. (1970) who measured serum prolactin levels with the recently develOped radioimmuno- assay for prolactin. Further support of hypothalamic inhi- bition was provided by the demonstration that a lesion in the median eminence or hypophysiotropic area could increase prolactin release (Chen 99 99., 1970; Welsh 99 99., 1971). Among the first to demonstrate the presence of a prolactin 17 inhibiting factor (PIF) 99 39999 was Talwalker 99 99. (1963) and Pasteels (1963) who used acidic extracts of hypothalamic tissue to inhibit the secretion of prolactin. Moreover, a negative dose-response relationship was ob- served between the quantity of hypothalamic extract added and the amount of prolactin released when tested 99 39999 (Kragt and Meites, 1967; Chen 99 99., 1969). Numerous 99 3939 studies were also reported sub- stantiating the inhibitory hypothalamic effects on prolactin release. Kamberi 99 99. (1971) provided supportive evi- dence that PIF from the hypothalamus had a primary effect directly on the pituitary. Cortical hypothalamic extracts when infused into the hypophysial portal vessels had no effect on prolactin secretion, but hypothalamic extracts inhibited prolactin release in a dose dependent manner. Additional 99 3939 experiments demonstrated that hypo- thalamic extracts could inhibit prolactin release in re- sponse to cervical stimulation (Kuroshima 99 99., 1966), following suckling in post-partum lactating rats (Gros- venor 99 99., 1964), in cycling and lactating rats (Amenomori 99 99., 1970), and in normal and orchidectomized male rats (Watson 99 99., 1971). A possible mechanism by which PIF inhibits prolactin was suggested by Nicoll (1971), who suggested that PIF acts to prevent Ca++ entry into the lactrotrophs, thereby inhibiting spontaneous release of prolactin secretory granules. The chemical nature of PIP has been the focus of intensive investigation for many years. MacLeod (1969) 18 and Birge 99 99. (1970) provided evidence to support the hypothesis that catecholamines can directly inhibit the release of prolactin from the pituitary. In contradiction to this idea, Kamberi 99 99. (1971) demonstrated no effect on prolactin release when depamine was infused into the pituitary stalk vessels. The other possibility was that PIF was a small polypeptide similar to the other hypothal- amic hypophysiotropic hormones (Meites 99 99., 1972). Schally and colleagues, after finding that many PIF prep- arations had high catecholamine content, decided to repeat the infusion experiments of Kamberi (Takahara 99 99., 1974; Schally 99 99., 1974). This time they were able to demon- strate a potent inhibitory effect of depamine on prolactin release and explain the failure of past studies to find this effect. They suggested that the negative results with cat- echolamines were due to rapid oxidation when infused with saline alone. Furthermore, when 5% glucose was added to prevent oxidation 60 and 600 ng of dopamine reduced pro— lactin secretion in a dose responsive fashion. The pos- sibility of dopamine as a PIF was strengthened by the work of Shaar and Clemens (1974). In these studies rat hypo- thalamic extracts containing PIF activity were treated with albumin to absorb catecholamines or enzymatically inacti- vated with monoamine oxidase. When again tested for PIF activity, the extracts were no longer able to inhibit pro- lactin release, suggesting that all PIF activity was due to catecholamines. Moreover, extracts treated with pepsin still retained PIF activity Opposing the idea that PIF is 19 a polypeptide. While it has been convincingly demon- strated that dopamine can inhibit prolactin release, the possibility of the existance of other PIFs cannot be ig- nored. Thus, when the dopamine blockers haloperidol (Ojeda 99 99., 1974) or pimozide (Vale 99 99., 1973) were added to a co-incubation system of pituitary and hypothalamic tissue, a hypothalamic inhibitory effect was still observable on pituitary prolactin release. B. Prolactin Releasing Hormone(s) Prolactin secretion in birds is regulated pre- dominantly by a hypothalamic releasing factor in contrast to inhibitory control in mammals. Therefore, when chicken pituitaries are transplanted to remove avian hypothalamic factors, no increase in prolactin release was seen (Ma and Nalbandov, 1963). Prolactin releasing factor (PRF) in birds has been demonstrated by Kragt and Meites (1965) who transplanted the pigeon pituitary to the crop sac without discernable stimulation of the crOp sac epithelium. Kragt 99 99. (1965) further showed that an avian hypothalamic extract was able to stimulate prolactin release 99 39999 from the pigeon pituitary. 99 39999 stimulation of prolactin release by hypothalamic extracts have similarly been reported for the turkey, duck, tricolored blackbird and Japanese quail (Meites, 1967). Though PRF is the primary regulator of prolactin release in avian species, there is the possi- bility that a PIF exists in birds as well. 20 In mammals, convincing evidence has been accumulating that the tripeptide thyrotrOpin releasing hormone (TRH) can stimulate prolactin release from the pituitary both 99 3939 and 99 39999 in a number of species including man. The first of such reports came from Tashjian 99 99. (1971) who showed that TRH can stimulate prolactin secretion 99 39999 when added to incubation medium containing cells from rat pituitary tumors. This was confirmed 99 3939 for the rat by Mueller 99 99. (1973) when an increase in prolactin was measured after injecting TRH. Porteus and Malven (1974) extended this work by confirming that TRH acts directly on the pituitary and that its action is not mediated by a hypothalamic mechanism. In these experiments TRH increased serum levels of prolactin in hypOphysectomized female rats with AP transplants under the kidney capsule or in animals with lesions in the median eminence. Similar reports on the stimulatory action of TRH on prolactin and TSH re- lease have been demonstrated in the cow (Convey 99 99., 1972; Kelly 99 99., 1973; Smith and Convey, 1975), monkey (Josimovich 99 99., 1974) and man (Bowers 99 99., 1971; Jacobs 99 99., 1971). In human subjects, Bowers 99 99. (1973) could find no TRH dose that would stimulate TSH but not prolactin secretion. It is now clear that TRH can stimulate prolactin as well as TSH release, but the physiological implications remain in question. There are numerous physiological con— ditions where prolactin and TSH are released differentially. In rats, the suckling stimulus in lactating females 21 increases serum prolactin to a much greater extent than serum TSH, and in proestrous female rats, ether stress elevates prolactin but not TSH. Moreover, Mueller 99 99. (1974) have convincingly demonstrated a dichotomy in the secretion of prolactin and TSH. Exposure to moderate heat of 40°C for 30 to 60 minutes significantly decreased serum TSH while increasing serum prolactin 5-10 fold. Similar findings in cattle on the effects of heat have been reported by Wettemann and Tucker (1974). In man, insulin hypoglycemia has been reported to raise prolactin levels with no sig- nificant changes in TSH (L'Hermite 99 99., 1973). Suck— ling also has no effect on TSH but can elevate prolactin levels in man (Gautvik 99 99., 1974). A tentative explanation of these data suggests that TRH is a physiological regulator of both prolactin and TSH, but a selective response may be attained through an inhibitory factor for each of these two hormones. However, no inhib— itory factor for TSH release has yet been found in the hypothalamus. Experimental evidence is now appearing to support this hypothesis. When L-dOpa is given to rats it can increase hypothalamic PIF (Lu and Meites, 1972) and reduce or abolish the effect of TRH on prolactin release without altering the TRH induced rise in TSH (Chen and Meites, 1975; Maeda 99 99., 1975). Conversely, recent reports dem- onstrate that somatostatin (growth hormone release inhib- iting factor (GIF) )can selectively inhibit TRH induced TSH secretion in man without altering the TRH stimulated pro- lactin secretion (Hall 99 99., 1973; Siler 99 99., 1974). 22 An inhibitory action of somatostatin has also been dem- onstrated on TSH secretion after administration of TRH (Vale 99 99., 1974). Consequently, it can be surmised that the pituitary secretion of the anterior pituitary hormones are determined by the summation of hypothalamic inhibitory and releasing factors as well as the pituitary milieu in- fluenced by circulating hormonal and steroidal levels and other modifying factors. The possibility of a PRF separate from TRH is also being investigated. Earlier studies looked at mammary secretions in estrogen-primed female rats after injections of crude hypothalamic extracts and suggested the existence of a PRF (Meites 99 99., 1960; Mishkinsky 99 99.,.l968). However, at that time endogenous prolactin levels could not be measured and stimulation by CRF, also found in hypo- thalamic extracts, was effective in initiating mammary secretion (Meties, 1962). Later reports of a PRF by Nicoll 99 99. (1970) and Valverde 99 99. (1971) were not substan- tiated. A more recent report by Dular 99 99. (1974) showed that a fraction isolated from bovine median eminence could stimulate prolactin release 99 39999 from bovine pitui- taries. This fraction appeared to be more potent than TRH and had no effect on TSH secretion. Voogt 99 99. (1975) also suggested that their studies provided possible evidence for PRF. Lactating rats were treated with a-methyl-p-tyrosine to inhibit catecholamine synthesis and then given the suckling stimulus by their pups. An increase in prolactin secretion was seen which was greater than the 23 rise in prolactin due to catecholamine inhibition alone. Since TSH was not elevated, prolactin increases due to TRH were assumed not to be the cause of this rise in prolactin. Therefore, the increase may have depended at least in part on a PRF. Only more research will determine the existence of PRF distinct from TRH. C. Biogenic Amine Control of Prolactin Secretion For biogenic amines to act as neurotransmitters to control the release of the hypothalamic hypOphysio- tropic hormones, which in turn can regulate anterior pi- tuitary secretions, they must first be shown to be present in the median eminence or hypothalamic area. Early studies did in fact demonstrate that there were high concentrations of norepinephrine and serotonin in the hypothalamus (Vogt, 1954; Brodie 99 99., 1959). Later studies by Piezzi 99 99. (1970) confirmed that serotonin was contained in high con; centrations in the bovine median eminence, and Bjorkland 99 99. (1970) found noradrenergic terminals in the median eminence. The presence of dOpaminergic nerve terminals was similarly demonstrated in the median eminence region (Anden 99 99., 1964; Fuxe and Hokfelt, 1969; Carlsson 99 99., 1962). Moreover, in an anatomical study, Hokfelt (1967) showed that these dOpaminergic neurons terminate in the median eminence adjacent to the portal blood vessels flow- ing into the anterior pituitary. Acetylcholine has been reported in the hypothalamus (Shute, 1969) and GABA has been proposed as a possible central neurotransmitter (Krnjevic, 1974; Defeudis, 1975). These studies provided 24 strong anatomical support for the concept of neurotrans- mitter control of releasing factors. At about the same time that the various neurotrans- mitters were being identified in the hypothalamus, physio- logical data was also appearing suggesting that biogenic amines may influence the release of pituitary hormones. The earliest experiments indicated that adrenergic and cho- linergic drugs induced ovulation in rats or rabbits (Markee 99 99., 1948; Sawyer 99 99., 1949). Drugs that depleted hypothalamic catecholamines such as reserpine and chlor— promazine were found to induce pseudopregnancy and lactation in rats, suggesting catecholamine control of prolactin se- cretion (Barraclough 99 99., 1959). This was confirmed by COppola 99 99. (1965) who extended this work by demonstrating that reserpine-induced pseudopregnancy in the rat could be blocked by monoamine oxidase inhibitors which increase cat— echolamines by inhibiting the enzyme that catabolized the amines. These and related experiments were repeated using radioimmunoassays to measure prolactin levels in serum (Donosa 99 99., 1971; Lu and Meites, 1971; Lu 99 99., 1970), and together with the earlier studies showed an inverse relationship between prolactin secretion and dOpamine levels, suggesting that dOpamine could stimulate PIF and thus de- crease prolactin secretion or act directly on the pitui— tary to inhibit prolactin release. D0pamine has been widely demonstrated to have an inhibitory role in prolactin secretion. Much of the data 25 substantiating this has come from the use of neurophar- macological drugs. Thus, the catecholamine receptor blockers which block the action of dOpamine on its recep- tor, have been reported to increase serum prolactin. Chlorpromazine and perphenazine both block dopamine, norepinephrine and histamine receptors, and consequently increase prolactin secretion (Lu 99 99., 1970; Ben-David 99 99., 1971; MacLeod 99 99., 1970). Similarly, haloperidol, a blocker of dopamine (and norepinephrine) receptors, and pimozide a specific dopaminergic receptor blocker over a limited dose range, have also been demonstrated to increase serum prolactin release (Dickerman 99 99., 1972; Clemens 99 99., 1974). The catecholamine synthesis inhibitors, alpha-methyl-para-tyrosine and alpha-methyldopa which de- crease dOpamine, have likewise been shown to increase pro- lactin release (Lu 99 99., 1970; Chen and Meites, 1975; Donoso 99 99., 1971; Lu and Meites, 1971). In contrast, a decrease in prolactin has been reported with the use of the dopamine agonist, apomorphine, in the rat (Smalstig and Clemens, 1974; MacLeod 99 99., 1974) and in human sub— jects (Martin 99 99., 1974). Furthermore, L-dopa, the depa- mine precursor and the monoamine oxidase inhibitors both decrease prolactin release from the pituitary by increas- ing hypothalamic dopamine (Lu 99 99., 1971). Therefore, it has been clearly demonstrated that dopamine has an inhibitory role in control of prolactin secretion, but whether this action is mediated by stimulation of a PIF or by a direct effect of dopamine on the pituitary or both, 26 remains to be conclusively demonstrated. Serotonin and serotonin precursors appear to be important in elevating prolactin secretion. Kamberi 99 99. (1971) injected serotonin into the third ventricle of rats and observed a significant rise in serum prolactin; however, when serotonin was injected into the pituitary no such in- crease was detected. Serotonin is unable to penetrate the blood-brain barrier (Goodman and Gilman, 1976) whereas ser- otonin precursors can pass through this barrier. Thus, when Lu and Meites (1973) injected systematically trypto- phan and 5-hydroxytryptophan, both serotonin precursors, into rats and increase in serum prolactin was found. In humans an oral dose of 5-hydroxytryptophan could cause a rise in prolactin that could be significantly suppressed but not abolished by the serotonin antagonist, cyprohepta- dine. Additional studies by Kordon 99 99. (1973) using lactating rats showed that the prolactin response to suck- ling could be blocked by p—chlorophenylanine, an inhib- itor of serotonin synthesis. In a more recent study Mueller 99 99. (1976) demonstrated an increase in hypo- thalamic turnover and concentration of serotonin and its metabolite 5 HIAA when male rats were subjected to re- straint stress. Stress induces an elevation of prolac- tin secretion which was also found in this study, together with a decrease in TSH. These studies therefore suggest that serotonin may be involved in increases in prolactin secretion such as those encountered during suckling-or stress . 27 A role for the neurotransmitter acetylcholine was suggested by the findings of Grandison 99 99. (1974) when a decrease in serum prolactin was observed after injec- tion of acetylcholine into the lateral ventricles. In the same study the cholinergic agonist, pilocarpine, and the anticholinesterase, physostigmine, administered system- ically, similarly decreased serum prolactin. The pilocar— pine effect on reducing prolactin secretion was confirmed by Chen and Meites and shown to have no effect on TSH levels. Lawson and Gala (1973) gave atropine, which blocks the action of acetylcholine, and reported a decrease in prolactin. The effects of GABA and norepinephrine in the control of prolactin secretion is still in a state of confusion. GABA injected into the lateral ventricles of rats increased serum prolactin (Mioduszewski 99 99., 1973), and GABA induced prolactin rises also were reported by Ondo and Pass (1976). However, Schally 99 99. (1976) reported that GABA could inhibit prolactin release 99 39999 and 99 3939. A similar situation exists for noradrenergic control of pro- lactin release. Increases in prolactin release have been reported with the administration of a precursor of norepine- phrine, L-DOPA (Donoso 99 99., 1971). In contrast, neither alpha nor beta adrenergic blockers were able to prevent the stress induced release of prolactin (Meltzer 99 99., 1976). Therefore, more data will be needed to resolve this conflict for these transmitters. 28 D. Ergot Derivatives and their Action on the Pituitary The present use of ergot alkaloids in medicine, endocrinology and pharmacology is the culmination of very successful research efforts on a drug that has been recog- nized for over 2000 years. Though ergot was first used by physicians almost 400 years ago as an oxtocic agent, its full useful potential was not realized until the 20th century (Goodman 99 99., 1976). Ergot is a fungus that grows mainly on rye and other grains, but it can be grown 99 39999 by fermentation tech- niques. The pharmacologically active compounds can be divided into two main groups: the first is the ergot alka- loids and the second consists of several amines including histamine, tyramine, and acetylcholine. Because of toxicity of these drugs and the propensity of the fungus to grow on edible grains, the history of ergot and ergot poisoning has been reported as early as 600 B.C. This is interestingly outlined in Goodman and Gilman (1976). The pharmacological effects of the ergot alkaloids are varied and complex with actions on the uterus to increase motor activity, on the cardiovascular system causing vasoconstriction, and on the endocrine system to inhibit the release of prolactin from the pituitary. Prolactin inhibition was identified indirectly by Dodart as early as 1676 when it was observed that women with ergotism failed to lactate (Floss 99 99., 1973). Since that time ergot alkaloids have been shown to be potent inhibitors of pro- lactin by measurement of serum prolactin in the rat 29 (Wuttke 99 99., 1971; Nagasawa and Meites, 1970) as well as in the cow, sheep, dogs, and in humans (Clemens 99 99., 1975). Wuttke 99 99. (1971) reported that ergot drugs can act on the hypothalamus to increase PIF activity, thus sug- gesting a possible mechanism of action. This idea was supported by the findings of Corrodi 99 99. (1973) who provided evidence that ergocornine and bromocriptine can activate brain dopamine receptors, acting as selective dopamine agonists. In addition to hypothalamic actions of the ergolines, investigators have shown that ergocornine can act directly on the pituitary to inhibit prolactin release (Malven 99 99., 1971; Lu and Meites, 1971). 99 39999, bromocriptine was able to inhibit prolactin release from the pituitary and this could be prevented by per- phenazine or haloperidol indicating that bromocriptine was acting through catecholamine receptors (MacLeod 99 99., 1974). This was confirmed and extended by Clemens 99 99. (1975) by demonstrating that lergotrile mesylate, a chlorinated analog, or ergoline, was able to block pitui— tary secretion of prolactin both 99 3939 and 99 39999, These effects could be opposed by the dopamine blocker, pimozide, suggesting a mechanism of action through pitui- tary dopamine receptors. The ability of the ergot derivatives to selectively inhibit prolactin release makes it an ideal tool for inves- tigating the role and functions of prolactin. Prolactin has been reported by many investigators to stimulate 30 growth of DMBA induced mammary tumors in rats, and pro- lactin suppression with ergot drugs has been shown to inhibit tumor growth (Meites and Clemens, 1972; Sinha 99 99., 1974). Furthermore, when ergot drugs were adminis- tered prior to giving the carcinogen DMBA, this was able to prevent the induction of mammary tumors (Clemens and Shaar, 1972). Other studies on rats showed that brom- ocriptine could inhibit PMS-induced ovulation in immature rats and inhibit spontaneous ovulation in adult females (Marko and Fluckiger, 1974). The suckling induced re- lease of prolactin hilactating rats could also be inhib- ited by bromocriptine (Fluckiger 99 99., 1974) as well as the proestrus surge of prolactin release. Clemens 99 99. (1972) reported that ergot alkaloids suppressed lactation in rats as indicated by loss of litter weight when compared to litter weight of controls. Dependence on high prolactin for lactating may be species specific since different re- sults were reported for the goat. Bromoergocryptine was effective in lowering prolactin levels in lactating goats with no effect on milk yield (Hart, 1973). One last rat study should be mentioned questioning the Specificity of bromocriptine on prolactin release. Seki 99 99. (1974) using estrogen-progesterone primed ovariectomized rats, showed that bromocriptine had no effect on basal LH levels, however, it did reduce the LH response to LHRF. Clinical studies have demonstrated the usefulness of the ergot drugs in treating a variety of human conditions and disorders. Bromoergocryptine has been used 31 successfully to inhibit puerperal lactation, and found effective both in reducing plasma prolactin levels and caus- ing a cessation of lactation in all patients who required lactational suppression (Del P020 99 99., 1975). Simi- larly, ergot drugs were found to be effective in treating patients with galactorrhea and amenorrhea (Lloyd 99 99., 1975; Schulz 99 99., 1975). Often galactorrhea is accom- panied by infertility and bromocriptine appears useful in inducing fertility (Thorner 99 99., 1975). In another report by Thorner 99 99. (1975) bromocriptine was used in the treatment of acromegaly and was found to be effective in lowering GH levels in a majority of the patients tested. Further testing and longer term studies will be needed to assess the effectiveness of ergot drugs in the treatment of acromegaly. A more standard therapeutic use of ergot alkaloids is in the treatment of migraine head- aches where iUSefficacious in 90% of all treated patients (Goodman 99 99., 1976). E. Short-Loop Feedback Regulation of the pituitary secretion of TSH, ACTH, FSH and LH is controlled primarily by the hormones from the target organ for which they stimulate, and this is termed "long-loop" or "end organ hormone feedback". In contrast, prolactin and growth hormone do not cause hormone or ster- oid release from specific target organs and therefore lack this "long-100p negative" feedback control. To explain how prolactin might be regulated, Sgouris and Meites (1953) were the first to hypothesize that circulating prolactin may 32 regulate prolactin secretion, thus inhibiting release through a "short-100p" feedback system. Several approaches were used to demonstrate autoreg- ulation of prolactin secretion, the earliest utilized pitui- tary tumor transplants which secrete large quantities of prolactin. MacLeod 99 99. (1966) reported that these tumors reduced the weight of pituitary gland and decreased prolac- tin and growth hormone content. This was confirmed and ex- tended by Chen 99 99. (1967) who showed that the high cir- culating levels of prolactin in tumor-bearing rats could increase PIF activity in addition to decreasing pituitary content and weight. More recent confirmation using radio- immunoassays have also shown that the pituitaries taken from rats with prolactin secreting tumors secreted much less prolactin and much more growth hormone than normal (MacLeod 99 99., 1974). Similar findings were reported when circulating prolac- tin levels were increased by injecting prolactin or trans- planting additional prolactin secreting pituitaries under the kidney capsule. Injections of 10 mg of prolactin into female rats was able to reduce pituitary prolactin content by about 40% (Sinha and Tucker, 1968). In cows, the suck- ling induced rise in prolactin was significantly reduced during infusion of exogenous bovine prolactin (Tucker 99 99., 1973). When pituitaries were transplanted under the kidney capsule of rats to raise prolactin titers, the accumulation of prolactin in the pituitary gland, which normally follows suckling, was inhibited (Mena 99 99., 1968). In addition, 33 a decrease was reported in pituitary prolactin content in mice (MacLeod, 1970) and rats (Mena 99 99., 1968) with elevated prolactin levels from transplanted pituitaries. Additional evidence for autoregulation of prolactin secretion was provided by experiments in which prolactin was implanted into the median eminence. Both serum (Niswender 99 99., 1969; Voogt and Meites, 1971) and pit- uitary levels (Clemens and Meites, 1968) were suppressed by these implants. Moreover, prolactin implants were demon- strated to shorten the duration of pseudopregnancy (Chen 99 99., 1968) reduce mammary gland lubulo-alveolar develop- ment (Clemens 99 99., 1968), inhibit milk secretion during lactation (Clemens 99 99., 1969) and induce abortion during pregnancy (Clemens 99 99., 1969), all suggestive of dim- inished prolactin secretion. The mechanism of action whereby prolactin is able to inhibit its own secretion is believed to be mediated by increases in dopaminergic activity in the hypothalamus resulting in an increased release of a PIF (dopamine ?) into the portal vessels to suppress prolactin release. Fuxe and Hokfelt (1971) supported this hypothesis by demon— strating that prolactin injections into rats markedly activated the tuberoinfundibular dOpaminergic neurons. This increased dopaminergic activity could also explain why prolactin is capable of inhibiting LH and FSH release (Grandison and Meites, 1977). The same group (Hgkfelt and Fuxe, 1972) in a later study observed activation of depamine meurons in pregnant and lactating rats with a 34 rapid decrease in dopamine turnover after injection Of ergocornine or bromoergocryptine. These studies are not as conclusive as they appear because another report from the same laboratory (Olson, Fuxe and HOkfelt, 1972) demonstrated that estrogen and testosterone, which stimu- late prolactin release, also were able to increase dopamine turnover. Consequently, this hypothesis is still Open to question. Voogt and Ganong (1974) performed experiments tO determine if prolactin can autoregulate its own secretion at the pituitary level. Rat pituitaries were incubated 99 39999 in the presence Of high and low quantities Of prolactin in the medium, and no difference in the amount Of prolactin secreted by these pituitaries was Observed. It was con- cluded that the autofeedback mechanism probably Operates at the hypothalamic rather than the pituitary level. However, Frantz 99 99. (1975) has reported that prolactin receptors could be found on normal rat pituitary cells. Their pres- ence,if not for autofeedback, remains to be determined. III. Interactions Between Prolactin and Other Hormones and Steroids A. Testosterone, Estrogen and Progesterone Estrogen has clearly been shown to increase pro- lactin secretion both 99 3939 and 99 39999. Nicoll and Meites (1962, 1964) provided early evidence that estradiol added to incubation media containing rat anterior pitui- taries could significantly increase prolactin release. This was confirmed by Lu 99 99. (1971) in a 12 hour 35 incubation study, measuring prolactin in the medium by radioimmunoassay. 99 3939 injections of estradiol benzoate into hypophysectomized rats with ectOpic pituitaries under the kidney capsule also were able tO increase serum prolactin levels (Chen 99 99., 1970), suggesting a direct effect on the pituitary. In another study Chen and Meites (1970) injected low, medium and high doses Of estrogen into ovari- ectomized female rats and reported that low and medium doses Of estrogen were more effective in raising serum and pitui— tary prolactin than the high dose. Estrogen also appears to stimulate the hypothalamus directly to increase prolactin secretion. This was sup- ported by experiments Of Ramirez 99 99. (1964) demon- strating than when estrogen was implanted in the median eminence, it induced pseudOpregnancy and development Of the mammary gland. However, the authors concede that estrogen from the implant may have entered into the portal system tO stimulate the pituitary directly. More Specific evi- dence for a hypothalamic action Of estrogen was provided by Fuxe 99 99. (1969). They reported that dopamine turn- over in the hypothalamus was increased by ovariectomy, and restored by estrogen or testosterone treatment. In the female rat during the estrous cycle, a large increase in prolactin release occurs on the afternoon Of proestrum (Sar and Meites, 1967; Niswender 99 99., 1969). This estrogen appears to regulate the prolactin surge center located in the anterior hypothalamus. Therefore, when estrogen anti- serum was given on the second day of diestrus, it abolished 36 the expected proestrus surge, but when diethylstilbestrol was given concomitantly it was able to overcome this inhi- bition (Neil 99 99., 1971; Jordon 99 99., 1975). It is clear from all these studies that estrogens have a dual action in the stimulation Of prolactin secretion, a dir- ect effect on the pituitary and an indirect action on the hypothalamus. Ojeda and McCann (1974) in a develOpmental study reported that estradiol in female rats could stimulate pro- lactin release beginning about the 24th day Of age and reaching a peak value greater than in adults, at the time Of vaginal Opening. In contrast, pimozide stimulated pro- lactin release by day three. The time Of day estrogen is injected was also demonstrated tO influence the prolactin response. Cheng 99 99. (1974) Observed that estrogen injected into immature female rats early in the dark period elicited a more rapid secretory response than when injected early in the light period. In castrate male and female rats single injections Of estrogen or testosterone increased serum prolactin with a peak effect between 48 - 72 hours (Kalra 99 99., 1973). Testosterone has been shown tO have nO effect on pitui- tary prolactin secretion 99 39999 (Nicoll and Meites, 1964). It does appear, however, that it can have an effect on the hypothalamus to increase prolactin release. In- creases in serum prolactin were demonstrated in castrated male and female rats after injecting testosterone prOpio- nate in doses from 0.5 tO 2.0 mg (Kalra 99 99., 1973). 37 The 0.5 and 1 mg doses were more effective in increasing prolactin in male rats. Similar findings were reported for male rats by Krulich 99 99. (1975). Progesterone in large doses, given 99 3939, have gen- erally been shown to slightly increase the release Of pit- uitary prolactin. The first Of such reports was by Meites (1959) who Observed increased mammary growth and elevated piutitary prolactin content. Later, Kalra 99 99. (1973) stimulated prolactin release in ovariectomized by admin- istering 5, 10 and 25 mg Of progesterone, but a lower dose Of 1.5 mg was without effect. This study would explain the lack Of effect Of progesterone reported earlier when lower doses were tried in castrate female rats (Blake 99 99., 1972). When 5 mg Of progesterone was given at 11:00 A.M. on proestrus it advanced and increased the surge of prolac- tin in the afternoon Of proestrus (Uchida 99 99., 1972). B. Glucorticoids The relationship between glucocorticoids and pro- lactin secretion is still a controversial area among inves- tigators, but the preponderence Of data suggests that glu- corticoids suppress prolactin secretion. 99 39999, cor- tisol and corticosterone were reported tO have no direct effect on pituitary prolactin release (Nicoll and Meites, 1964). However, more recent results have shown that incu- bated rat pituitary cells do secrete significantly less pro- lactin in the presence Of cortisol (Dannies and Tashjian, 1973). Better agreement has been attained with 99 vivo 38 experiments. Thus, Sar and Meites (1968) demonstrated that cortisol injections increased pituitary prolactin content in rats, presumably by inhibiting prolactin release. Fur- thermore, it was demonstrated that increasing amounts Of dexamethasone given to intact male rats could inhibit the stress-induced prolactin release in a dose dependent man— ner (Euker 99 99., 1975; Harms 99 99., 1975). In ring doves dexamethasone injected systemically was able to block prolactin release as judged by inhibition Of crop growth (Silver 99 99., 1973). Human studies have demonstrated that dexamethasone could suppress both basal prolactin levels and the prolactin response to insulin hypoglycemia on the following day (COpinshchi 99 99., 1975). Negative results were reported by Schrams 99 99. (1973) who found nO effect on prolactin secretion by cortisol infusions in cows. Ad- ditional evidence for a modulating role Of prolactin on glucorticoid secretion was provided by studies involving removal Of corticoids through adrenalectomy. Ben-David 99 99. (1971) showed that adrenalectomy resulted in elevated pituitary and serum prolactin levels in male rats, together with an increased responsiveness to the prolactin releasing effects Of perphenazine. This was confirmed by Harms 99 99. (1975) who further demonstrated that adrenalectomy could also potentiate prolactin release due to stress. A re- ciprocal effect was suggested by Endroczi 99 99. (1972) when they reported that prolactin inhibits the release Of stress-induced corticotrophin releasing factors (CRF). 39 Similarly, Schlein 99 99. (1973) found a reduced plasma corticosterone response to stress in lactating and prolac- tin treated rats as compared to normal female rats. Both prolactin and ACTH-corticosterone are secreted together in a variety Of physiological states including suckling (Voogt 99 99., 1969; Amenomori 99 99., 1970), stress (Grosvenor 99 99., 1965; Ajika 99 99., 1972; Euker 99 99., 1975; Harms 99 99., 1975) and parturition (Meites 99 99. 1972). In addition, the late afternoon-evening surge Of prolactin during proestrus occurs when corti— costerone secretion is at its daily peak (Critchlow 99 99., 1963). These findings could suggest that there may be a close temporal or biogenic relationship between prolactin and ACTH. In fact, this has led Harms 99 99. (1975) to speculate that a common pathway or mechanism may be in- volved in pituitary release Of ACTH and prolactin, and to suggest that dexamethasone may interfere with the release of a CRF which also acts as a PRF to release prolactin. However, these investigators may be unaware Of an earlier study by Stern and Voogt (1974) demonstrating the limi- tations Of this relationship. The magnitude and mainten- ance Of the secretion Of prolactin and corticosterone were shown to differ depending upon the physiological state Of the animal, the type of stimulus applied, and the duration Of the stimulus. In a recent review Of the role Of biogenic amines in the control Of AP hormones (Meites 99 99., 1975) it was 4O concluded that the serotonergic system could increase both prolactin and “CTH release from the pituitary. This may represent a common mechanism for the release Of both hormones, but it still leaves unexplained how phenothia- zines induce secretion Of both prolactin (Ben-David 99 99., 1970) and ACTH-corticosterone (DeWied, 1967). In summary, it appears that there is a relationship between prolactin and ACTH-corticosterone secretion, however, the control mechanisms regulating secretion is complex and not com- pletely understood at this time. C. Gonadotropins Prolactin and the gonadotropins LH and FSH are released from the pituitary in a reciprocal manner during various physiological states including suckling (Amenomori 99 99., 1970), puberal state (Voogt 99 99., 1970; Kragt 99 99., 1972) and after castration or estrogen treatment (Chen and Meites, 1970; Kalra 99 99., 1973). However, these two hormones are secreted in parallel during proestrus in the female (Gay 99 99., 1970) and with stress in the male (Euker 99 99., 1975). Prolactin may act at three different sites to alter gonadotrOpin secretion; the hypothalamus, pituitary or the corpus luteum. An action Of prolactin on the corpus luteum to inhibit gonadotropin secretion was suggested by Rothchild (1965) and Everett (1969). Possible evidence for inter- action at the pituitary level was provided by Nakane (1970) in male rats. He showed a close anatomical arrangement between prolactin and gonadotropin cells in the pituitary 41 which may have a physiological purpose. Many investigators have reported a relationship between prolactin and gonado- tropins via hypothalamic mediation. Meites and Clemens (1972) postulated that the short lOOp feedback Of prolactin on its own secretion can influence the secretion Of LH and FSH by acting on the hypothalamus. In support Of this, it was reported that minute prolactin implants in the median eminence could increase FSH levels (Voogt 99 99., 1969). In another experiment Voogt and Meites (1971) implanted minute amounts Of prolactin into the median eminence of pseudOpregnant rats and doubled serum levels Of LH and FSH. These data provided an explanation Of earlier findings that implants Of prolactin into the median eminence or pro- lactin injections were able tO advance puberty in rats. Ojeda and McCann (1974) found an increase in prolactin se- cretion with reduced LH release when the dopamine receptor blocker, pimozide, was given either subcutaneously or into the median eminence-arcuate region. Therefore, it might be concluded that hypothalamic dopamine content or turn- over could mediate this reciprocal relationship. However, inhibition Of catecholamine biosynthesis with alpha-methyl- p-tyrosine in ovariectomized rats produced an elevation Of prolactin without altering LH. Moreover, Fuxe 99 99. (1971) reported that prolactin can increase dOpamine turnover, but when bromocriptine was given to rats to lower endogenous pro- lactin, no changes were noted in basal LH levels. TO recon- cile these differences the most plausable explanation would be that there are many neural components regulating LH and 42 prolactin through biogenic amines. Thus, when Kalra 99 99. (1973) stimulated the basal median hypothalamus, they found an increase in serum LH and prolactin, but when the preoptic hypothalamic area was stimulated an increase in LH with a decrease in prolactin levels were found. In general, I would conclude that LH and prolactin can be regulated independently, but in most circumstances a re- ciprocal relationship between prolactin and gonadotrOpins exists. Control, in normal physiological conditions, is mediated through the hypothalamus to Obtain an endocrine state necessary to achieve a certain physiological state. Thus, during lactation elevated levels Of prolactin are needed for milk synthesis with LH and FSH low to prevent resumption Of,cycling, whereas during the estrous cycle, prolactin and the gonadotropins are all increased on the afternoon Of proestrus in the rat. D. Thyroid Hormones The relationship between prolactin and thyroid hormones is another controversial area Of endocrinology. Early studies demonstrated that thyroxine can alter pitui- tary prolactin content (McQueen-Williams, 1935; Meites and Nicoll, 1966). However, interpretations Of studies involv- ing thyroid hormone-prolactin interactions became more complicated when Tashjian 99 99. (1971) reported that TRH, in addition to stimulating TSH, could also stimulate pitui- tary prolactin secretion. Confirmation Of the stimulatory action Of TRH on prolactin was reported in the rat (Mueller 99 99., 1973), cow (Convey 99 99., 1972; Kelly 99 99., 1973), 43 monkey (Josimovich 99 99., 1974) and man (Bowers 99 99., 1971; Jacobs 99 99., 1971; Huang 99 99., 1971). Further- more, a direct effect Of TRH on the pituitary was demon- strated in hypophysectomized rats with pituitary trans- plants under the kidney capsule (Porteus 99 99., 1974) and in humans with a pituitary stalk section with a plate insertion to isolate the pituitary from hypothalamic influ- ences (Lister 99 99., 1974). Consequently, demonstrating a direct effect Of thyroid hormones 99 3939 on the pitui- tary would be difficult because of confounding changes by TRH on prolactin secretion. 99 39999 effects Of both thy- roxine and triiodotyronine were demonstrated to increase prolactin secretion, suggesting a direct action on the pit- uitary to release prolactin (Nicoll and Meites, 1963). However, conflicting results were reported by Tsai 99 99. (1974) who showed that thyroid hormones added to cultured pituitary cells reduced prolactin secretion and stimulated growth hormone release. Therefore, the question Of whether thyroid hormones can directly alter prolactin secretion from the pituitary has not been adequately answered. In contrast, peripheral interactions between prolactin and thyroxine have been demonstrated by numerous investi- gators in a variety Of species. The metamorphosis Of amphib- ians and salamanders from the larval to the adult stage is dependent on the effects Of thyroid hormones. Prolactin is able tO block the metamorphic-induced effects Of thyroid hormones by an antagonistic action at the level Of the 44 peripheral tissue, and perhaps at the thyroid (Nicoll, 1974). However, a synergistic action Of prolactin and thyroxine occurs in the newt in stimulating limb regen- eration and promoting molting (Nicoll, 1974). Peripheral interaction between these two hormones have also been demonstrated in mammals. An early report by Smithcors 99 99. (1942) showed that thyroidectomy in the male caused development Of mammary gland ducts and lobulo- alveolar growth. This was confirmed by Meites and Kragt (1964) who demonstrated that thyroxine injections, in im- mature hypophysectomized rats with pituitary transplants, reduced the mammary growth promoting effects Of the pro- lactin from the ectopic pituitaries. Singh and Bern (1969) similarly Observed that high concentrations Of thyroxine inhibited the 99 39999 growth promoting effects Of insulin, aldosterone and prolactin on mouse mammary glands in organ culture. However, low levels had a synergistic effect. A more recent report by Mittra (1974) showed that thyroid- ectomy in estrogen-treated rats was followed by stimulation Of mammary develOpment. Furthermore, this growth could be suppressed by bromoergocryptine or thyroxine, and enhanced by increasing prolactin with perphenazine treatment. Syn- ergism between prolactin and thyroxine was reported by Nejad 99 99. (1962) who restored lipogenesis in the liver Of hypo- physectomized rats. In the hypophysectomized pigeon pro- lactin and thyroxine acted together to stimulate body and visceral growth (Bates 99 99., 1962). 45 IV. Role Of Prolactin in Reproduction A. Actions on the Mammary Gland and the Pigeon CrOp Sac The reproductive action of prolactin on the mammary gland is the most widely known action Of this hormone and the most extensively studied. It was Stricker 99 99. (1928) who first recognized the actions Of the pituitary on the mammary gland in initiating lactation. After these investigators injected a crude pituitary extract into pseudOpregnant rab- bits, it was noted on autOpsy that the mammary glands con- tained an abundance Of milk. A few years later Riddle 99 99. (1931, 1932) discovered that the production Of pigeon crop milk was similarly controlled by a hormone from the anterior pituitary. Later, Riddle 99 99. (1933) purified this AP hormone and found it produced milk secretion both in pigeons on the crop sac and in mammals. In the same publication, they named this hormone prolactin and described the first prolactin bioassay--the systemic pigeon crOp-sac test. It was not until 1958 that the factors controlling mammary development and milk secretion were clearly demon- strated by Lyons 99 99. (1958). In this classic experiment it was shown that hypOphysectomized, adrenalectomized, ovar- iectomized rats, lacking all pituitary and steroid hormones, could develOp mammary duct growth when injected with estro- gen, growth hormone and adrenal steroids. However, for lubulo-alveolar development, progesterone and prolactin were also necessary. Similar results were reported for mammary growth in mice (Nandi 99 99., 1958, 1959). In rats, when estrogen and progesterone were given alone in hypophysec- tomized animals, they were unable to stimulate mammary 46 development (Lyons 99 99., 1958; Meites and HOpkins, 1961). However, Talwalker and Meites (1967) were able to induce moderate lObulo-alveolar growth without ovarian or adrenal steroids with injections Of prolactin and growth hormone. Therefore it was suggested that ovarian steroids have a synergistic effect with the pituitary hormones to sensitize the mammary tissue to these hormones in addition to stimu- lating their release (Meites, 1966). 99 39999 studies have confirmed the 99 3939 findings on steroidal and pituitary hormonal requirements for mammary gland growth (Riveria, 1964). The minimal hormonal requirements for initiation and maintenance Of lactation after full lobulo-alveolar develOp- ment varies among different species. In the rabbit, prolac- tin alone is sufficient tO induce lactation, but adrenal corticoids can improve lactogenesis (Cowie, 1969). The rat requires both prolactin and glucocorticoids as the minimal requirements to induce lactation (Folley, 1956; Lyons 99 99., 1958), whereas hypophysectomized sheep and goats require pro- lactin GH, glucorticoids and thyroxine for initiation and maintenance Of lactation (Cowie 99 99., 1966; Denamur, 1969). Prior to parturition in the rat there is an increase in estrogen secretion (Mayven, 1969) followed by an elevation Of both prolactin (Meites, 1966) and ACTH and serum corti- costerone (Voogt 99 99., 1969) on the day Of parturition. These hormonal increases are believed to be responsible for the initiation Of lactation (Meites, 1966). However, other reports suggest that the decrease in progesterone following 47 parturition is also partially responsible for lactogenesis. In the rat is has been shown that progesterone is the hor- mone responsible for the suppression Of alpha-lactalbumin synthesis, and that after parturition the fall in proges- terone secretion allows the stimulatory action Of prolac- tin tO be expressed (Kuhn, 1969a, 1969b, 1973; Herrenkahn, 1974; Murphy 99 99., 1973). After the initiation Of lacta- tion, milk secretion is maintained by frequent suckling by the young which in turn results in reflexive release Of pro- lactin and ACTH from the pituitary via the hypothalamus (Meites, 1966). In addition, the suckling stimulus acts tO inhibit LH and FSH secretion (Meites, 1966; Minaguchi and Meites, 1967) and tO delay resumption Of cyclic ovarian function. It has been amply demonstrated that prolactin release and synthesis is stimulated by suckling, and that prolactin stimulates synthesis Of milk proteins, lipid and carbohydrates. However, the exact relationship between pro- lactin secretion, lactation and milk yield requires clar- ification. The surge Of prolactin in response to suckling is greatest just after parturition, but decreases as lac- tation continues although the lactational response remains normal. This has been demonstrated in the goat (Buttle 99 99., 1971, 1972), sheep (Lamming 99 99., 1972), rat (Simpson 99 99., 1973), cow (Ingalls 99 99., 1973; Koprowski 99 99., 1973), and humans (Tyson 99 99., 1972). Moreover, late in lactation in humans and cattle their was very little response tO suckling or milking, but milk production contin— ued (Tyson 99 99., 1973; Ingalls 99 99., 1973). Another 48 aspect Of the same question is the lack Of correlation be- tween prolactin levels and milk yields. Hart (1973) was unable to inhibit milk secretion in mid—gestation in goats by eliminating the suckling induced surge Of prolactin by bromoergokryptine. Similarly, Smith 99 99. (1974) demon- strated a lack of effect of ergocryptine on milk yield in the cow. In contrast, rats injected with prolactin anti- serum did show a reduced milk yield (Shani 99 99., 1975). Other reports showed that bromocryptine can effectively terminate lactation in women (Del P020 99 99., 1975) and decrease milk yield 10-30% in dairy cows when given at parturiation and during early lactation (Fell 99 99., 1974). From such studies it appears that the role Of pro— lactin in lactation differs among species, and that other factors such as synergisms and antagonisms with other hor- mones as well as prolactin receptors changes may modify the lactogenic action Of prolactin. Prolactin has a similar function in pigeons and doves in the formation of crop milk within the crop sac. In response to this hormone the mucosal epithelium Of the crop sac proliferates and the cells hypertrophy while accum- 1ating lipids. These cells are desquamated into the large chambered crOp sac tO form the crop milk that is used to feed the hatchlings (Nicoll, 1974). This prolactin-induced stimulation Of the crop sac mucosal epithelium is mediated by stimulation Of both RNA and protein synthesis (Nicoll 99 99., 1967). Prolactin alone can cause crop sac growth in hypophysectomized pigeons, however, an augmented response 49 can be attained by combination injections of thyroxine, GH, and adrenocorticoids (Bates 99 99., 1962). Since prolactin actions on the crop sac proved tO be specific, it was develOped by Riddle 99 99. (1933) into the first bioassay for prolactin. This systemic assay was based on weight increases Of the crop sac after 4 daily injections Of prolactin or the material tO be assayed. Later, this was modified by Lyons 99 99. (1935) into a local crOp-sac or mic- romethod assay. This method has great sensitivity and in- volved intracutaneous injections over the crOp-sac. Today, prolactin is measured by radioimmunoassay (RIA) which is much easier and quicker to perform with greater sensitivity than the bioassays (Niswender 99 99., 1969). Bioassays, though, will always be needed to confirm and test these RIA's. B. Actions on the Ovary In addition to the traditional role Of prolactin in mammary gland development and milk secretion, it was also found that prolactin had a role tO play in some species on ovarian function, by influencing the corpus luteum. The concept Of a luteotrOpic hormone from the pituitary gland in the rat was first formulated by Astwood (1941), and the identification Of this luteotrOpin as prolactin was tO fol- low (Evans 99 99., 1941). Subsequent studies confirmed that prolactin could maintain functional corpora lutea for pro- longed periods Of time in hypophysectomized rats. In these experiments serum prolactin was elevated by transplanting pituitaries beneath the kidney capsule (Everett, 1956; Nikitovitch-Winer and Everett, 1958; Hausler 99 99., 1971) 50 or by prolactin injections (Astwood, 1941; Hausler 99 99., 1971). More recent work in rats, hamsters, ferrets and mice indicates that prolactin is involved in the maintenance Of progesterone production by the ovary. Moreover, for full progesterone secretion a minimal luteotropic complex is necessary consisting of prolactin and one or both gonado— tropins (Nalbandov, 1973). In the rat and ferret this con- sists Of prolactin and LH, whereas in the mouse and hamster prolactin and FSH are needed for maximal progesterone se- cretion (Greenwald, 1969, 1973; Choudary 99 99., 1969). Prolactin actions on luteal function has been inves- tigated during various physiological states in the rat in- cluding pseudOpregnancy, pregnancy and lactation. In the female rat, ovulation occurs every 4—5 days and the corpora lutea formed after ovulation secretes progesterone for 2-3 days Of each cycle (Hashimoto 99 99., 1968). However, this luteal phase can be extended to 12-14 days if cervical stim- ulation is provided during proestrus or estrus by sterile mating or manual stimulation with a glass rod (Long and Evans, 1922). This prolonged period Of luteal function is known as "pseudOpregnancy" and is thought to be initiated by increasing levels of prolactin which act on the ovary tO stimulate progesterone secretion. Support for this hypo- thesis was provided by experiments demonstrating that in- creasing prolactin levels, without cervical stimulation, could induce pseudopregnancy. Thus, prolactin injections on the day of estrus in intact and hysterectomized females 51 were able tO induce pseudopregnancy (Anderson, 1968). Also, pseudOpregnancy was produced in cycling female rats when they were allowed to nurse foster pups (Selye 99 99., 1935), and in hypophysectomized rats induced tO ovulate with PMS and HCG (Saito 99 99., 1970). Mclean and Nikitovitch-Winer (1973) provided further evidence by demonstrating that injections of prolactin antiserum could block pseudepregnancy induced by cervical stimula- tion during proestrus. Prolactin anti-serum was only effective when given between stimulation and 8 hours post stimulation. Many investigators have reported that prolac- tin is elevated after cervical stimulation and remains high throughout pseudopregnancy. Within 20 minutes after cer- vical stimulation prolactin was shown tO be increased (Spies 99 99., 1971) and thereafter remained elevated with daily biphasic surges, one during the day and the other nocturnal (Deis 99 99., 1973; Alonso and Deis, 1973). These studies suggest that prolactin can prolong the func- tional corpora lutea and stimulate the secretion Of pro— gesterone. Other investigators have reported that prolactin has similar effects on the corpora lutea and progesterone secre- tion during pregnancy. DOhler and Wuttke (1974) gave brom- oergokryptine during the first two days after fertile mat- ing and Observed subnormal levels Of progesterone indicating failure Of luteal function. After 4 days Of treatment no animal became pregnant. This was confirmed by Morishige and Rothchild (1974) who reported a sharp decrease in 52 progesterone secretion with ergocornine when given during the first 7 days Of pregnancy. NO effects on progesterone secretion were seen after day 7, possibly because Of the increasing titers Of rat placental lactogen. Earlier studies by Cutuly (1941) showed that rats hypOphysectomized and treated with prolactin shortly after mating were able to implant and maintain pregnancy. Meites 99 99. (1957) Observed a lengthening Of pregnancy when large doses Of pro- lactin were given, but this was due to contamination with LH. Again, studies during the pregnant state indicate that pro- lactin is luteotropic and stimulatory on progesterone secre- tion. Studies in mice indicate that prolactin is part Of a luteotropic complex along with FSH. When pregnant mice are hypophysectomized on day 6, pregnancy can be maintained by administering prolactin and FSH (Choudary and Greenwald, 1969). Bartke (1973) working on dwarf mice with a genetic prolactin deficiency also explored the role Of prolactin in pregnancy. When these mice were treated for eight days after mating with prolactin, pregnancy could be maintained. When prolactin was decreased by bromocryptine in normal mice after mating, pregnancy was terminated (Zumpe 99 99., 1974). In the pregnant hamster prolactin and FSH given after hypo— physectomy can increase vascularity Of the corpora lutea and maintain pregnancy (Greenwald, 1967). A role for prolac- tin in the secretion Of progesterone from the ovaries Of other species such as the cow, sheep, pig and human has not been demonstrated. Some reports claim an action in these 53 species, but other investigators Often report conflicting results. Part Of the prolactin action on the ovary to stimulate progesterone secretion appears to be mediated by changes in enzyme activity, which effect progesterone metabolism, and in maintaining an adequate cholesterol pOOl on which LH can act. In the rat ovary prolactin can prevent the induction Of enzymes that catabolize progesterone to progestationally inactive derivatives. These enzymes include 36- and 208 - hydroxysteroid dehydrogenases (20 HD) and 5cc -reductase (Wiest 99 99., 1968, 1969; Zmigrod 99 99., 1972). Lamprecht 99 99. (1969) reported a large increase in 20 HD in the ovary when serum prolactin was lowered with ergot alkaloids, and this increase could be prevented by exogenous prolactin. Hypophysectomy also increases 20 HD and prolactin replace- ment reverses this effect (Farmer, 1970). Increasing pro— lactin levels in the rat by perphenazine injections were found tO increase progesterone levels progressively over 5 days (Chartterton 99 99., 1975). Part Of this increase was reported tO be due tO decreased conversion tO 20cc —hydroxy- progesterone. Prolactin administration has similarly been reported to decrease 5a=-reductase and 3 B—hydrooxysteroid dehydrogenase (Zmigrod 99 99., 1972). In addition tO influ- encing progesterone metabolism, prolactin can also modify cholesterol accumulation and turnover. Behrman 99 99. (1970) reported that prolactin increases the levels Of the enzymes sterol acyl tranferase and sterol esterase in the rat corpus luteum. These enzymes function in the metabolism Of 54 cholesterol esters. Accumulation Of cholesterol in both interstitial and luteal compartments Of the ovary with pro- lactin treatment in intact and hypophysectomized immature rats has also been reported (Zarrow 99 99., 1969). Similar effect Of prolactin on the cholesterol stores Of the ovarian interstitial tissue of hypOphysectomized rabbits and in the testes Of prolactin-deficient dwarf mice have been shown (Hillard 99 99., 1969). It is possible that prolactin has a general luteotrophic effect in most mammals by maintaining progesterone precursor pools which would be important espec- ially during LH stimulation where cholesterol is more rap- idly converted intO steroids. However, in some species such as the rat and mouse, prolactins greater than luteotrophic action could be accounted for by a dual effect on both pro- gesterone and cholesterol metabolism. I In contrast to the prolactin luteotrophic effect in some species, Malven 99 99. (1969) presented evidence that prolactin has still another reproductive function at least in the rat and mouse, and that is a luteolytic effect on the corpus luteum. It was demonstrated that prolactin injec- tions in hypophysectomized rats could be either luteotro— phic or luteolytic depending on the time Of injection after hypOphysectomy. The early effect Of prolactin on the corpora lutea of hypophysectomized rats is luteotrophic, but beyond 80 hours an Opposite effect is seen with destruction Of cor- pora lutea. This luteolytic effects seems to be exerted when the corpora lutea lost their capacity tO secrete pro— gesterone (Lam and Rothchild, 1973). In the rat it appears 55 that the surge Of prolactin on the afternoon Of proestrus serves tO destroy the Old corpora lutea which are no longer functional in order to make room in the ovary for a new set. SO, when ergot drugs were given tO reduce endogenous prolac— tin the Old corpora lutea failed to degenerate, and after several cycles many corpora lutea accumulated from pre- vious cycles (Wuttke and Meites, 1971; Billeter 99 99., 1971 Richardson, 1973). Grandison and Meites (1972) showed a similar luteolytic action Of prolactin during the estrous cycle Of the mouse. C. Actions on the Testis and Secondary Sex Organs The role Of prolactin in the male has been a mystery for decades tO those studying endocrinology and reproduction. Significant amounts Of prolactin are found in both the pituitary and blood Of males Of several species, but no physiological role for prolactin could be found. Recent evidence, however, suggest a possible role for pro— lactin on the testes and male accessory sex organs. Con— vincing evidence for an action Of prolactin on the testes was reported by Bartke (1966) who investigated strains Of infertile dwarf male mice that have a genetic deficiency in prolactin and growth hormone. Injections Of prolactin in these dwarf males rendered them fertile when mated with normal mice. It was later found that prolactin replacement resulted in a significant increase in the yield Of sperm— atozoa in the testes Of these dwarf males (Bartke 99 99., 1970). Similar effects Of spermatogenesis were reported in hypOphysectomized genetically normal mice (Bartke, 1971), 56 and a synergism between LH and prolactin was noted. Pro- lactin appears to influence testicular function and sperm— atogenesis by increasing testosterone secretion from the Leydig cells. One mechanism by which prolactin elevated testosterone is by maintaining levels Of esterified choles- terol in the Leydig cells thus providing ample precursor for testosterone secretion. Bartke (1969, 1971) provided evidence that prolactin does have a direct effect in in- creasing cholesterol esters in the Leydig cells both in dwarf and hypophysectomized normal mice. Furthermore, in hypophysectomized mice, LH reduced cholesterol levels in the testis and prolactin was able to block the LH induced depletion. Another mechanism by which prolactin can synergize with LH in increasing androgen secretion is through changes in LH receptors in the testis. In a recent study by Bex and Bartke (1977) atrOphy Of the reproductive system Of the hamster was reversed by treatment with prolactin and sig- nificant increases in testicular weight and peripheral tes- tosterone levels were measured. Prolactin was able to med- iate this effect by increasing LH receptor levels in the testis. Similar studies have also demonstrated that pro- lactin can increase LH receptor activity in the ovary (Holt 99 99., 1976) and in the mouse testis (Bohnet 99 99., 1976). Consequently, prolactin also appears tO stimulate testicular function through increased binding of endogen- ously produced LH. In addition tO the other actions Of prolactin on the 57 testis, it may alter testicular function by enzymatic acti- vation Of steroidogenic pathways leading tO androgen bio- synthesis. Prolactin administration was reported to in- crease 3- B-hydroxysteroid dehydrogenase and l7-B-hydroxy— steroid dehydrogenase activity in the testis Of dwarf mice (Hafez 99 99., 1971; Musto 99 99., 1972). From all these data, it has been convincingly demonstrated that prolactin does play a role in testicular funciton, at least in the mouse, and that the prolactin mechanisms Of actions are varied. However, the importance Of prolactin in other species in the maintenance Of testicular funciton is less certain. A synergistic action Of prolactin and testosterone on prostatic growth and secretory activity has been demonstrated for the rat (Chase 99 99., 1957; Grayheck 99 99., 1967). Similar synergism was demonstrated 99 39999 in the mainten- ance Of rat prostate tissue in organ culture (Lasnitzki, 1972). Moreover, other evidence has appeared indicating that endogenous prolactin has a rOle in the function Of the prostate in the rat and other species. Peyre 99 99. (1968) was able to greatly augment the stimulatory effect Of tes- tosterone on the prostates Of castrated male rats with con- current administration Of reserpine. Since reserpine is a potent stimulator Of prolactin, this stimulatory effect was attributed tO increases in endogenous prolactin. In rab- bits, when endogenous prolactin was neutralized by injections of prolactin antiserum, prostatic atrophy was Observed (Asano 99 99., 1965).‘ More recently, Moger and Geschwind (1972) 58 have shown that in castrated male rats prolactin alone can increase prostatic activity as measured by increased uptake Of zinc. The mechanism by which prolactin synergizes with andro- gens on male sex accessories is unknown, but Nicoll (1974) has speculated that it may function through alterations in androgen receptor activity in these target organs. This is supported by findings that prolactin can augment the uptake Of tritiated testosterone in incubated human prostate tis- sue and in organ culture Of rat ventral prostate tissue (Farnsworth, 1972; Boynes, 1972). A similar synergism between prolactin and androgen in increasing weight and secretory activity has also been de- scribed for the seminal vesicles Of the mouse (Bartke 99 99., 1970), rat (Pasqualini, 1953) and guinea pig (Antliff 99 99., 1960). V. Other Functions Of Prolactin A. Osmoregulation In sub-mammalian vertebrates prolactin plays an im- portant part in the regulation Of fluid and electrolyte balance. For various euryhaline teleosts prolactin exerts an effect on the gills, gut kidney, urinary bladder and skin to facilitate their adaption to a fresh water habitat. Both in amphibians and lizards hypophysectomy results in ab- normal plasma Na levels which can be corrected with prolac- tin and corticosterone treatment (Nicoll, 1974). Recent work suggests that prolactin is involved in the functioning Of nasal salt glands Of certain avian species such as 59 seagulls and ducks. These glands serve to remove excess salt from these animals and thus function to reduce sodium levels in plasma to normal. Thus, the osmoregulatory role Of prolactin in sub-mammalian species is well established. Many studies have now been reported suggesting prolac- tin may have important osmoregulatory functions in mammals. The first such report Of a renal effect Of prolactin in mammals appeared in 1965 (Lockett). Using a heart-lung- kidney preparation on spinal cats, a renal retention Of water and sodium were Observed when prolactin or growth hormone was administered. In the same year it was demon- strated in rats that prolactin or growth hormone given in a single I.P. injection could reduce urinary water and sod- ium excretion (Lockett 99 99., 1965). Similar reports on prolactin‘s action on the kidney to reduce sodium and water excretion have been reported for the rabbit and human (Burstyn 99 99., 1974; Horrobin 99 99., 1971). When en— dogenous levels Of prolactin are reduced in the rat by 2- bromo-alpha-ergo-kryptine, an inhibitor Of prolactin release, an increase in urinary excretion Of sodium and potassium was Observed (Richardson, 1973). TO determine where in the nephron prolactin was acting Donatsch and Richardson (1974) attempted to trace the dis- tribution Of prolactin in the rat kidney at varying inter- vals Of time. The method they used was a fluorescein- labelled double antibody technqiue for micrOSCOpial locali- zation Of injected ovine prolactin. The results Of this study demonstrated that prolactin was localized exclusively 60 on the proximal tubule epithelium. Furthermore, binding was rapid with a large concentration on the brush border Of the proximal tubule 3 minutes after ovine prolactin was injected. In a similar study Rajaniemi 99 99. (1974) in- jected 125I-Ovine prolactin intravenously into male rats. Animals were then killed after various times and radio- activity located by whOle body microautoradiography. At two minutes there was large amounts Of radioactive prolactin on the apical border Of the proximal tubular cells in agree- ment with Donatsch 99 99. (1974). The mechanism Of action by which prolactin alters NaCl and water movement in the proximal tubule are still specu- lative. One possibility is that prolactin binding results in a change in Na/K ATPase. High concentrations Of ATPase have been found in the brush border Of proximal tubules Of the rat (Binkley 99 99., 1968) and this enzyme is consid- ered to be primarily involved in the transport Of sodium and potassium across cell membranes. Along this line, Linzell and Peaker (1971) suggested that a function Of pro- lactin in mammary cells may be that Of inhibiting Na/K ATPase at the apical surface Of the epithelial cells. Moreover, in teleosts, prolactin has been reported to stimulate this enzyme in the kidney (Pickford 99 99., 1970) and gill (Kamiya, 1972). Therefore, in the rat kidney proximal tub— ules, prolactin may stimulate reabsorption through a mech- anism involving Na/K ATPase. ‘Another possible way that pro- lactin may alter proximal tubular reabsorption was suggested by Lucci 99 99. (1975). In studying the anatomy Of the 61 proximal lateral intercellular spaces in control and prolac- tin treated rats infused with either saline or blood they found that prolactin virtually abolished the dilation Of the spaces seen in the saline infused rats. Therefore, it is possible that prolactin may act primarily be making "tight" junctions Of the proximal tubule tighter, and may be inacé~ tjvein situations where the lateral intercellular spaces are already constricted. Further complicating the role of prolactin in osmoreg- ulation in mammals are the findings Of numerous investiga- tors On interactions between prolactin and aldosterone, ADH, and cortisol. Burstyn 99 99. (1972) found that aldoster- one injections caused the expected renal retention Of sodium in sheep on a low salt load (80 mEq/day). However, this effect Of aldosterone was altered to a saluretic action when the sheep were given a high salt load (400 mEq/day). In- jections Of ovine prolactin was able to restore the sod- ium retaining properties Of aldosterone in the sheep on the high salt load. In another study using sheep on a low NaCl load Horrobin 99 99. (1973) reported that cortisol injec- tions could block the actions Of ADH on the kidney and cause diuresis. Treatment with prolactin in these cortisol treated sheep restored the expected water retaining effects Of ADH. In lithium treated rats no significant changes in urinary output Of either sodium or water occurred with treatment Of ADH, aldosterone or prolactin given alone. But, when pro- lactin and aldosterone were given together they produced sodium retention, and when prolactin and ADH were 62 administered they produced water retention (Mtabaji 99 99., 1975). Collectively, these experiments suggest that prolac- tin exerts its effect on salt and water metabolism partly or fully through interaction with other hormones. Another approach in the investigation of the role Of prolactin in salt and water regulation would be to measure changes in serum prolactin after changes in blood osmolar- ity. This was investigated in rats which received an intra- venous infusion Over a period Of thirty minutes Of either 0.45%, 0.9%, or 3% saline (Relkin, 1974). The hypotonic infusion decreased serum prolactin, the hypertonic solution raised serum prolactin and the isotonic infusion produced no changes. Thus, osmolarity was directly related to serum levels Of prolactin. Similar results on blOOd Na levels and serum prolactin concentrations were Obtained in man (Buckman and Peake, 1973). More recently, a report by Adler 99 99. (1975) refuted the earlier work by Buckman 99 99. in humans. These investigators could find nO reduction in plasma prolactin levels after oral water loading or intra- venous loading with hypotonic saline, and suggested the earlier findings were artifacts due tO high initial pro- lactin at the start Of the experiment. This high prolactin probably was due to stress which later fell to basal levels during the experiment. In 99 39999 experiments, Labella 99 99. (1974) using incubated bovine pituitaries demon- strated that the osmolarity Of the medium was inversely related to prolactin release. It is Obvious that more re- search is necessary tO clarify this situation. 63 In other experiments physiological conditions such as dehydration and sodium deficient diets have also been shown to alter prolactin levels. Rats placed on a sodium defi- cient diet for three weeks showed significant rises in serum and pituitary prolactin levels and aldosterone secre- tion without any change in corticosterone, growth hormone or TSH levels (Relkin 99 99., 1973). In male rats Marshall 99 99. (1975) demonstrated that two days Of water depriva— tion could significantly increase serum prolactin. In addition tO a prolactin effect on the kidney, other studies indicate prolactin also acts on the mammalian intes- tine On transport of fluid and sodium chloride (Mainoya 99 99., 1972; Ramsey 99 99., 1972; Mainoya 99 99., 1974). In these experiments 99 3939 treatment with ovine prolactin significantly increased fluid and NaCl absorption in rat, guinea pig and hamster jejunum when tested 99 39999 in everted sacs Of the small intestine. B. Adrenal Gland The recent finding Of prolactin receptors on adrenal cell membrane preparations Of mammals such as the rat, rab- bit, and sheep (Marshall 99 99., 1975; Posner 99 99., 1974) has provided evidence that prolactin may influence adrenal- cortical function. Earlier studies by Witersch and Kitay (1972) demonstrated that prolactin can increase corticosterone secretion in male and female rats by decreaSing the activity Of the adrenal enzyme Sa—reductase, which metabolizes cor- ticosterone to an inactive form. 99 39999 using isolated rat adrenal cells Lis and workers (1973) confirmed this data 64 by showing synergism between ACTH and prolactin in the stim- ulation Of corticosterone production. Gustafsson (1975) also reported that prolactin could decrease 5<=-reductase activity by 20-30% in the adrenals Of male and female rats. Prolactin has been reported to have other effects on the adrenals in addition tO increasing adrenal corticoster— one release in the rat. In the adrenals Of rats ornithine decarboxylase activity was increased 65 fold above controls after prolactin injections (Richards, 1975). Additionally, prolactin has been reported tO have a stimulatory effect on aldosterone production by rat adrenal glands (Lichtenstein 99 99., 1976), and selectively increase progesterone release (Piva 99 99., 1973). A more recent report by Carter 99 99. (1977) reported that hyperprolactinemia in man selectively stimulated adrenocortical androgen production. Overall, the reported findings on the function Of prolactin on ad- renal function are contradictory, since prolactin cannot selectively stimulate adrenal androgens, progesterones, corticosterone and aldosterone. Therefore, it remains to be determined whether prolactin can selectively release dif- ferent adrenal hormones under varying circumstances, or possibly that prolactin does not selectively release ad- renal steroids, but rather causes increased release Of all adrenal hormones. C. Liver Prolactin has been shown tO have numerous effects on liver functions in several species Of mammals. In gen- eral, these actions include effects on carbohydrate, lipid 65 and protein metabolism (Bern and Nicoll, 1968). Thus, in mice prolactin has been reported to induce liver glycogen- cylsis (Elghamry 99 99., 1966), and to stimulate hepatic RNA and protein synthesis in dwarf mice (Chen 99 99., 1972). In rats with prolactin secreting pituitary tumors, fatty acid and protein synthesis were increased, and in prolactin treated rats ornithine decarboxylase activity in the liver Of rats was enhanced. Prolactin treatment has also been reported to increase somatomedin release from rat liver (Francis and Hill, 1975). In the dog, prolactin has been shown tO regulate hepatic free fatty acid synthesis (Winkler 99 99., 1971). Although the physiological sig- nificance Of these actions are not understood at this time, the finding Of specific prolactin receptors in the liver Of rats (Gelate 99 99., 1975) further supports the hypothesis that prolactin has important metabolic functions through effects on the liver. VI. Regulation of Prolactin Receptors in Several Target Organs A. Early Studies Numerous studies Of hormonal effects led tO the con- ception that a single primary event, the binding tO a spe- cific recognition site, initiates a sequence Of steps cul- minating in the hormonal reSponse (Hechter, 1955). A sur- face location for the sites Of initial interation was sug— gested by the Observation that hormones are rapidly fixed tO tissue (Crofford, 1968), but can still interact with specific antibody (Pastan 99 99., 1966). Further support 66 for a plasma membrane location was provided by the demon- strations that ACTH, insulin and glucagon when covalently linked to inert polymers still retained biological activity (Schimmer 99, 99., 1968; Cuatrecases, 1969; Johnson 99 99., 1972). Research on polypeptide hormone-receptor interactions was greatly facilitated by three technical achievements. First, the development Of methods for the separation and characterization Of cellular components (DePierre and Karnovsky, 1973), and Of equal importance, the preparation Of highly purified polypeptide hormones. The availability Of a simple procedure fOr producing radio-iodinated hormones Of high specific activity either oxidatively (Hunter and Greenwood, 1962) or enzymatically (Marchalonis, 1969), made direct study of hormone-receptor interactions feasable. Using (1251)-labelled PRL injected intravenously into rabbits, Birkinshaw and Falconer (1972) reported the highest uptake ratio Of tissue: plasma was in the kidney and mammary gland. A similar study Of Rajaniemi 99 99. (1974) reported tissue distribution Of (125I)iodo-PRL in mice and rats using whole-body and microautoradiography. The labeled hormone rapidly accumulated in liver and kidney, with less labeling noted in the ventral prostate, seminal vesicals and mammary glands. In the ovary the labeling was weak and mainly lo— calized over the corpus luteum. A different approach to the study Of PRL binding to target tissue was used by Midgley (1973) who applied radioiodinated PRL tOpically tO rat ovar- ian sections, and through autoradiographic methods Observed 67 differences in the localization Of radioactivity to corpora lutea between newly formed and Old corpora. Among the first to study 99 39999 (125I)iodo-Prl bind- ing to subcellular particals were Turkington and Frantz (1972) using a crude, low speed membrane pellet, and Posner and colleagues (1974) who quantitated PRL binding in a more purified microsomal membrane pellet. Both studies reported PRL binding in various tissues Of the rat including the mam- mary gland, liver, kidney and prostate. However, the stud— ies Of Posner 99 99. extended these findings by including a survey Of (1251)-PRL binding tO various tissues Of the monkey, rat, guinea pig, rabbit, sheep, pigeon and frog. It was apparent from this study that the degree Of prolac- tin binding tO various organs differed between species. Identifying PRL receptors in various target organs and their quantification is but one application Of membrane bound receptors. The availability Of a convenient prep- aration Of receptors (cell, tissue fractions, etc.) and radiOlabelled PRL with a high specific activity led naturally to the development Of a radioreceptor assay (RRA) for PRL. Previously, Lefkowitz and associates (1970) demonstrate the feasibility Of using tissue fractions to construct a RRA when they reported the first RRA for a polypeptide hormone, ACTH. Thus Shiu 99 99. (1973) using the mammary gland Of mid-pregnant rabbits as a source Of PRL receptors, reported a RRA for prolactin and other lactogenic hormones. Since the RRA measures substances on the basis of bio- activity it has, like the bioassay, an advantage over the 68 radioimmunoassay in being able to measure a variety Of dif- ferent materials with comparable biological activity but different structural, and immunologic properties. More- over, RRA's are easier to perform, less time consuming, and less costly than previous PRL bioassays. However, since the RRA is based on PRL binding tO its receptor, which may or may not be related to the biological response Of PRL, radio- receptor assays for PRL shoudl be used as an adjunct to the bioassay not as a replacement. An excellent example Of the application Of RRA has been the employment Of PRL RRA's for measuring lactogen levels during pregnancy. Radioimmunoassays measure only PRL of pituitary origin, while the RRA PRL values are subtracted from RRA levels, placental lactogen levels can be measured. Such hormone determinations were performed on the serum Of rats and two peaks Of placental lactogen were found in the pregnant female, the first at day 12 and the second around day 18 (Kelly 99 99., 1975). The PRL RRA has also been used to monitor the hormonal activities during the purification and characterization Of ovine and rat placental lactogens (Chan 99 99., 1976; Robertson 99 99., 1975). B. Liver Specific binding sites for PRL have been identified in particulate membrane fractions Of liver tissue, and have been shown to be both time and temperature dependent. More- over, these binding sites are specific for PRL and do not cross react with Other anterior pituitary hormones, and have an affinity constant Of sufficient magnitude to bind 69 circulating levels Of PRL (GelatO 99 99., 1975; Posner, 1976; Sherman 99 99., 1977). Early studies suggested that PRL receptors in the liver may be regulated by endocrine factors. In liver membranes from female rats binding was low in immature rats, increased 3.5 fold between days 20 and 40 Of age and increased further in mid and late pregnancy. Binding to male liver membranes was significantly lower at all stages Of development (Kelly 99 99., 1974). The possibility that ovarian hormones mod- ulate hepatic PRL receptor levels was substantiated by GelatO 99_99. (1975) who demonstrated that ovariectomy de- creases and estrogen replacement enhances PRL binding in the liver Of female rats. Similarly, in the liver Of male rats, PRL binding sites can be induced by estrogen treatment (Pos- ner 99 99., 1974) and this effect blocked by prior adminis— tration Of anti-estrogen compounds (Kelly 99 99., 1975). Later studies reported that PRL can regulate its own receptor activity in rat liver, and that part Of the induc— tive effect Of estrogen is through estrogenic stimulated pituitary PRL release (Posner 99 99., 1975). Thus, in male rat liver hypophysectomy resulted in a decrease in hepatic PRL receptors which culd not be induced withestrogen treat- ment. However, a kidney pituitary implant partially pre- vented the decrease in PRL binding sites in the liver fol- lowing hypophysectomy. Moreover, estrogen treatment in hypo— physectomized rats with a pituitary implant increased cir— culating PRL levels and further enhanced hepatic PRL recep- tors. Autoregulation Of PRL receptors in the liver was 70 substantiated by Costlow 99 99. (1975) when it was shown that a single 2 mg PRL injection could increase PRL bind- ing in hypophysectomized female rats. PRL receptor levels in the liver were also found to be elevated in rats bearing prolactin-secreting tumors (Lis 99 99., 1975). Other hormones which have been reported to modulate PRL binding sites in the liver include testosterone and thy- roid hormones. Although PRL receptors in the liver Of male rats are low, binding can be increased by castration. Tes- tosterone administration completely prevented the increased PRL binding which followed castration (Aragona 99 99., 1976). When female rats were thyroidectomized, PRL binding activ- ity in liber tissue significantly decreased (Gelato 99 99., 1975). Doses Of 2.5 ug or 10 ug T4/100 g bw daily returned PRL binding in the liver Of TX rats to intact control levels. C. Prostate Several reports have suggested a role for PRL in controlling prostate functions (Huggins 99 99., 1946; Van- derLaan, 1953; Grayhack, 1963; AsanO 99 99., 1971). There- fore, the finding Of specific PRL binding sites in the ven- tral prostate Of male rats is not surprising. As in the liver Of female rats, PRL receptors in the prostate has been re- ported tO be modulated by sex steroids. When male rats were castrated PRL binding sites were decreased, and subsequent treatment with testosterone was able to increase binding (Kledzik 99 99., 1976; AragOna and Friesen, 1976). Estrogen treatment Of male rats however significantly reduced PRL re- ceptors in the ventral prostate in both studies. Specific 71 binding of (1251)ido-PRL was highest in prostates of 20 day old rats and was significantly reduced at day 270 (Aragona 99 99., 1976). D. Mammary Gland Properties of specific PRL receptors from mammary glands of the rabbit and lactating mouse have been described (Shiu 99 99., 1974; Sakai 99 99., 1975). Hormonal regulation in lactating and non-lactating rat mammary gland have also been investigated and the data indicated that mammary gland PRL receptors are increased by PRL and thus exhibit the same autoregulation noted previously in the liver (Bohnet 99 99., 1977). Estrogens, which increase hepatic PRL binding, exert an opposite effect on mammary gland PRL receptors. It ap- pears likely that PRL receptors in the rabbit mammary gland are similarly regulated since Djiane 99 99. (1977) reported that PRL binding increases during pregnancy with a further increase in the number of receptors during lactation. E. Mammary Tumors The binding of (1251)iodo-PRL membrane preparations of carcinogen-induced rat mammary cancers correlate well with the growth response of the tumor to the stimulatory actions of PRL (Kelly 99 99., 1974). Other investigators have ob- served the presence of high affinity PRL receptors in mam- mary tumors that are responsive to prolactin, and little re- ceptor activity in PRL-independent tumors (Costlow 99 99., 1974, 1975; Turkington, 1974). More recently, the measure- ment of both estrogen and prolactin receptor concentrations in carcinogen-induced rat mammary tumors indicated that a 72 better correlation of tumor response to encorine ablation resulted from a combination of the 2 receptor levels than from either estrogen or PRL receptor concentration alone. (DeSombre 99 99., 1976). Asselin 99 99. (1977) reported that estrogen, PRL and progesterone receptors are all in- volved in the control of growth of DMBA-induced mammary tumors, and that the presence of high levels of estrogen receptors were a good index of the hormonal dependency of the tumors, although the presence of sufficient levels of PRL and progesterone were essential for an effect of these hormones to be observed. 73 MATERIALS AND METHODS I. Animals Rats for all studies were housed in a temperature con- trolled (25: 1 C) and artificially illuminated room (lights on from 5:00 A.M. until 7:00 P.M. daily), and received a diet of Purina Rat Chow (Ralston Purina Co., St. Louis, Missouri) and tap water 99 libitum except in Experiment II where drinking water was controlled in the treatment groups. Male rats used in Experiment VII were of the Long-Evans strain obtained from Blue Spruce Farms (Altamont, New York). Hypophysectomized rats were purchased from Hormone Assay Labs., Chicago, together with appropriate intact control animals. All other rats were purchased from Spartan Re- search Animals, Inc. (Haslett, Michigan). All surgical op— erations and intravenous injections were performed under ether anesthesia. II. Radioimmunoassays Luteinizing hormone (LH) and prolactin were measured in the serum of rats uring NIAMDD rat LH and prolactin radioim- munoassay (RIA) kits. For prolactin the antigen for iodin- ation was NIAMDD-Rat Prolactin-I-l, biological potency 30 I.U./mg. Antisera to rat prolactin was Anti—Rat Prolactin- Serum-B diluted to 1:2,500. The antigen used in the LH assay was NIAMDD-Rat LH-I-3, biological potency 1.0 x NIH- LH-Sl. Antisera to rat LH was Anti-Rat LH-Serum-S-3 diluted to 1:10,000. Purified hormones used in these assays were 74 iodinated by the chloramine-T method of Greenwood 99 99. (1963). Serum values are expressed in ng/ml compared with the reference preparations distributed by NIAMDD, LH-RP-l and PRL-RP-l . III. Prolactin Receptor Determinations A. Preparation of Particulate Membranes Rats were killed by decapitation after each exper- iment and their kidneys, liver, or adrenals removed immedi- ately, wrapped in tin foil and placed on dry ice. Tissue was then stored frozen at -50° C for no longer than 6 weeks, until assayed for PRL binding activity. This storage did not affect PRL binding activity as judged by comparisons of fresh tissue versus frozen. Both kidneys from each rat were homogenized with a Brinkman Polytron PT-lO (setting of 8) for 20 seconds. The homogenate was then centrifuged at 15,000 g for 20 minutes and the pellet discarded. The supernatant was again centri- fuged on a Sorvall OTD ultracentrifuge for 90 minutes at 105,000 g to obtain the particulate membrane pellet. This pellet was then resuspended in Tris-Ca++ buffer (0.025 M Tris, pH 7.6, 10 mM CaClz) so that 100 ug of membrane pro- tein, as determined by the method of Lowry 99 99., (1955), was contained in 100 pl. Each kidney sample was assayed using 100 pl of membrane preparation containing 1000 Hg or protein. The adrenal glands from two rats were pooled to make one sample and particulate membrane pellets simi- larly obtained. Adrenal samples were assayed using 100 pl of membrane preparation containing 200 pg of protein. 75 Liver particulate membranes were diluted to a final concen- tration of 500 ug protein/100 ul. See Figure l for flow diagram of membrane isolation procedure. B. Enzymatic Radioiodination of Prolactin Radioiodination of ovine prolactin (NIH S-lO, 25.6 I.U./mg) was accomplished by a lactOperoxidase method reported by Thorell and Johansson (1971). This lactOper— oxidase method of labeling PRL has previously been demon- strated to retain its biological activity (Frantz 99 99., 1972). A comparison of the chloramine-T method and the lactoperoxidase method of labeling PRL in our laboratory indicated that the chloramine-T labeled PRL was unable to bind specifically to particulate membrane fractions whereas the lactoperoxidase labeled PRL did bind specifically to membrane protein. Other investigators (Shiu 99 99., 1973) have demonstrated that chloramine-T labeled PRL can bind specifically to PRL receptors, but that the lactoperoxidase method is superior for detecting specific PRL binding activ- ity. With the availability of a more purified lactoperoxi- dase enzyme this method was modified for use in our labora- tory and a description of the iodination procedure and the reagents used are given in the appendix. When the iodin-- ation reaction was completed the entire contents of the reaction vial was layered on a Sephedex G-50 column (0.9 cm x 20 cm) and eluted with Tris buffer. Two peaks of radio- activity were seen after column chromatography, the first was the radiolabeled prolactin and the second peak the free 76 iodide. Since some of the 125I—oPRL was often damaged in the iodination procedure a second repurification was done using a Sephadex G-100 column (0.9 cm x 50 cm). The first peak tube from the G-50 column was then carefully layered on the G-100, 1.0 ml fractions collected, and the major peak from this filtration diluted and used in the assay. 1251- oPRL was diluted in Tris-Bovine Serum Albumin (BSA) buffer (0.025 M Tris, pH 7.6, 10 mM CaCl 1% BSA) to give approx- 2, imately 100,000 cpm/100 p1. C. Assay Procedures Incubations were done in quadruplicate in dispos- able culture tubes with 100 pl of 125I-PRL and 100 p1 of mem- brane preparation in final volume of 0.5 ml containing Tris- Ca++ buffer. For each determination a parallel incubation was performed in quadruplicate with 100 p1 of 125I-PRL, 100 p1 of membrane preparation plus an excess of unlabeled PRL (1 pg) in 100 pl for a total volume of 0.5 ml containing Tris-Ca++ buffer. After an incubation time of 48 hours at 4° C 3 ml of Tris—Ca++ buffer was added and the tubes were centrifuged at low speed to yield a discerable pellet. Pellets were then counted in a Nuclear-Chicago 1185 auto- matic gamma counter for 1 minute. Total binding (TB) refers to the counts bound to the pellets in the presence of mem- 125 brane protein and I—PRL, whereas non-specific binding (NSB) refers to binding to the pellets in the presence of 125 membrane protein, I-PRL plus an excess of unlabeded PRL. Since the unlabeled PRL in the NSB tubes is occupying the PRL receptor sites the additional binding seen without the 77 excess PRL (TB tubes) represents specific PRL binding. The specific binding counts are sometimes expressed as a percent of the total counts added for ease of repre- sentation. Thus, a 1% change in specific binding would represent approximately 1000 cpm. IV. Steroids, Hormones and Drugs The following hormones and drugs were used: ovine prolactin (NIH-S-lO, 25.6IU/mg); ovine GH (NIH-S-ll, 0.56 IU/mg);ovine LH (NIH-S-lS, 0.99 NIH-LH-S—l units/mg); ovine FSH (NIH-S-7, 1.15 NIH-FSH-S-l units/mg); ovine TSH (NIH-S-6, 2.47 USP units/mg); estradiol benzonate and tes- tosterone propionate (Nutritional Biochemicals Corp., Cleveland, Ohio); ergocorinine methane-sulfonate (Sandoz Pharmaceuticals, Hanover, New Jersey); Dexamethasone, Hydrocortisone, Actinomycin—D, Cycloheximide, T3 and T4 (Sigma Chemical Co., St. Louis, Missouri). V. Statistical Analysis Unless otherwise stated, all data were statistically evaluated by analysis of variance and individual means compared by the Student-Newman-Keuls test at 5% level of significance. 78 EXPERIMENTAL I. Demonstration of Specific Prolactin Binding to Par- ticulate Membrane Preparations from the Kidneys of Rats A. Objectives Numerous reports have appeared in the literature implicating prolactin in salt and water regulations (see Re- view of Literature, VI. Other functions of Prolactin, A. Salt and Water Regulation). In addition, other investiga- tors have demonstrated prolactin receptor binding in a var- iety of target organs including the kidneys and adrenals of various species (Turkington 99 99., 1972; Posner 99 99., 1974). However, the presence of specific prolactin recep- tors in the kidneys and adrenals of rats has not been con- vincingly demonstrated. Thus, the first step in the inves- tigation of the prolactin role in salt and water regulation in the rat was to determine if prolactin receptors do in fact exist in the kidney and adrenals, and to demonstrate that these receptors are specific for prolactin and do not cross-react with other anterior pituitary hormones. B. Materials and Methods Intact male and female Sprague-Dawley rats weigh- ing 225-250 gms were obtained from Spartan Research Animals (Haslett, Michigan). The rats were housed in a temperature controlled 25: 1° C) and artificially illuminated room (lights on from 5:00 A.M. until 7:00 P.M. daily), and re- ceived food and water ad lib. On the morning of the second 79 day all rats were killed by decapitation and their adrenals and kidneys were removed immediately, placed on dry ice and stored frozen. The tissues were thawed at a later date and the kidneys from the male rats pooled before homogenization; the male adrenals, female adrenals and female kidneys were similarly pooled. From these homogenized organs a particu- late membrane preparation was obtained as previously describ- ed (See Materials and Methods, III. Prolactin Receptor De- terminations). Using these particulate membranes from the kidneys and adrenals the presence of specific prolactin re- ceptors were tested for by adding unlabelled prolactin in the range of 0.5 - 1,000 nanograms to tubes containing 1251- ovine prolactin and membranes followed by an incubation per- iod at 48 hr. at 4° C. If prolactin receptors were present on the membranes and could bind the radioactive prolactin, then this binding would be inhibited in a dose-response man- ner when increasing amounts of unlabelled prolactin was added to compete for binding sites. In addition, LH, FSH, TSH and GH were also tested to determine if these hormones would inhibit prolactin binding, thus testing for specific- ity of receptors. C. Results Figures 1 and 2 show that unlabelled prolactin can competitively inhibit the binding of 125I—ovine Prolactin in a dose-response fashion in both the kidneys and adrenals of rats. The inhibition curves for kidneys were identical for both male and female rats as were the curves for the partic- ulate membranes obtained from the adrenals of males and Figure 1. Figure 2. 80 100 1 :2 ' fl H '5" 2 so a O ' o J : 00 o . 2 6H ' 4o .- z u u a 1’ so oz 1 a a no so so so too Iooo UNlAIIlLID HORMONESmanograms Binding of 125I-labeled PRL to rat kidney membranes with LH, TSH, FSH, and CH. The abscissa denotes the concentration of unlabeled o-PRL and the ordinate represents the amount of radioactivity bound to the membranes expressed as a percentage of the control incubations in which no unlabeled hormone was present. FSH 75“ 00 GH BOUND 60 . PIICENI l'" 0 PI“ 20 0.5 I 1 C IO 20 3° 3° ’00 1°00 UNLAIELL‘D HORMONES nanograms Binding of 125I-labeled PRL to rat adrenal membranes with LH, FSH, and CH. The abscissa denotes the con- centration of unlabeled o-PRL and the ordinate re- presents the amount of radioactivity bound to the membranes expressed as a percentage of the control incubations in which no unlabeled hormone was present. 81 females. LH, FSH, and TSH were unable to displace the labeled prolactin from either adrenal or kidney membrane preparations during the incubation period. Ovine growth hormone did show some cross-reactivity at the 100 ng. dose, but this is believed to be due to prolactin contamination as stated by NIH. D. Conclusions These results demonstrate that prolactin binding in particulate membrane preparations obtained from the kid— neys and adrenals of rats is specific, and does not cross- react with other anterior pituitary hormones. These data also indicate that the sensitivity of the binding sites to displacement by unlabelled prolactin is within the range of the endogenous levels of prolactin found in the rat. Thus, levels of 0.5 ng or more of unlabelled ovine prolactin read- ily displaces 125I-ovine prolactin (Figures 1 and 2). II. Serum Prolactin Levels and Prolactin Binding Activity in Adrenals and Kidneys of Male Rats After Dehydration, Salt Loading and Unilateral Nephrectomy A. Objectives The previous study concluded that there are specific prolactin binding sites in both the kidneys and adrenals. Therefore, it was of interest to alter the salt and water regulatory system in the rat to determine if this would in- fluence prolactin receptors or alter blood prolactin levels. Other investigators have found that dehydration in female rats for 12 hours produces a decrease in pituitary prolactin (Ensor 99 99., 1972), which may reflect an increase in 82 release of prolactin. Also, plasma osmolar changes were shown to influence secretion of prolactin with serum pro- lactin changes paralleling changes in serum osmolarity (Buckman 99 99., 1973). B. Materials and Methods Intact male rats weighing 225-250 gms were allowed to acclimate to their surroundings for two days. On the morning of the third day after their arrival, treatments were begun as follows: the first group was given 1.5% NaCl ad lib in place of tap water. No water was given to the second group. The third group was unilaterally nephrec- tomized under light ether anesthesia and given tap water ad lib. Intact control rats were permitted access to tap water ad lib. All groups had free access to Purine Lab. Chow. At the end of 48 hours all rats were bled via cardiac puncture under light ether anesthesia to obtain samples for determin- ation of serum prolactin. The rats were then killed by de- capitation and their adrenals and kidneys were removed im— mediately, placed on dry ice and stored frozen until assayed for prolactin binding activity. Both kidneys from each rat were homogenized in 0.3 M sucrose and the microsomal membranes prepared as previously described. In the nephrectomized group, the remaining kidney was homogenized. Serum prolactin concentration was determined by the double-antibody method described by Niswender 99 99. (1969). The standard reference preparation was NIAMDD-rat prolactin RP-l. One way analysis of variance was used and the means 83 compared by Duncan's Multiple Range Test (Duncan, 1955). In a subsequent experiment salt loading was again tried in intact male rats using 2.5% saline as drinking water rath- er than l.5%, and the treatment duration was lengthened to 4 days. C. Results Prolactin binding activity in the kidney particu- late membrane preparation (Table l) was significantly re- duced after 2 days of water deprevation, whereas a 1.5% sa- line drinking water and unilateral nephrectomy were with- out effect on binding. In Table 2 it can be seen that both dehydration and salt loading resulted in a significant in- crease in prolactin binding to adrenal membranes when com- pared to controls. Again, unilateral nephrectomy did not alter prolactin binding in the adrenal glands. Serum pro- lactin in these rats (Table 3) was 250% higher in the dehy- dration group and 50% higher in the unilateral nephrectomy group compared with controls, both statistically signifi— cant. D. Conclusions The salient point of these experiments is that physiological conditions that alter salt and water regula- tion or osmoregulation in the rat are also able to change prolactin receptor binding in the kidney and adrenal, as well as endogenous prolactin levels. Furthermore, these data lend further support to the hypothesis that prolactin is involved in salt and water regulation by its action on the kidneys (Lockett 99 99., 1965; Horrobin 99 99., 1971). 84 Table 1. PRL binding activity in kidney homogenates of dehydrated, salt-loaded (1.5% NaCl in drinking water), and unilaterally nephrectomized male rats. ‘7. Specific binding Treatment and no. of rats/group of u"I-oPRL 1 Controls (10) 5.47 :1: 0.37“ 2 Dehydration (8) 4.13 :1: 0.38” 3 Salt load (9) 6.0) d; 0.45 4 Unilateral nephrectomy (12) 5.12 :1: 0.50 ° Standard error of mean. t P < 0.05 as compared to controls. Table 2. PRL binding activity in adrenal homogenates of dehydrated, salt-loaded (1.5% NaCl in drinking water), and unilaterally nephrectomized male rats. No. of oled Treatment and no. samples/ % Specific binding of rats/group group of "‘I-oPRL 1 Controls (10) 5 4.78 :1: 0.43‘ 2 Dehydration (10) 5 6.78 :t 0.42” 3 Salt load (10) 5 6.09 :1: 0.23c 4 Unilateral nephrec- 6 4.99 :1; 0.24 tomy (12) ° Standard error of mean. ‘ P < 0.05 as compared to controls. ' P < 0.01 as compared to controls. Table 3. Serum PRL levels in dehydrated, salt-loaded (1.5% NaCl in drinking water), and unilaterally nephrect- omized male rats. Plasma PRL levels Treatment and no. of rats/group (ng/ml) 1 Controls (10) 12.93 :1: 1.170 2 Dehydration (10) 31.04 :1: 0.43‘ 3 Salt load (10) 15.01 i 1.91 4 Unilateral nephrectomy 19.58 :1: 0.90‘ (13) ‘ Standard error of mean. 5 P < 0.05 as compared to controls. c P < 0.01 as compared to controls. 85 The changes in prolactin receptor binding in the ad- renals may reflect alterations in osmoregulation by the aldosterone system. A report by Relkin and Adachi (1973) indicates that prolactin may enhance aldosterone secretion in the Na-deprived rat. In addition, prolactin has also been reported to increase corticosterone secretion from the rat adrenal (Witorsch 99 99., 1972) as well as proges- terone (Piva 99 99., 1973). Increased serum prolactin levels measured after uni- lateral nephrectomy agree with previous reports. In human patients with chronic renal failure, elevated levels of serum prolactin were observed (Turkington 99 99., 1972; Frantz 99 99., 1972), which is similar to the rise in serum prolactin found after unilateral nephrectomy in male rats. It is possible that these increases in prolactin may be due to decreased metabolic clearance due to loss of some kidney function. III. Effects of Estrogen and Testosterone on Specific Pro— lactin Binding in the Kidneys and Adrenals of Rats A. Objectives Thus far it has been demonstrated that specific prolactin receptors exist in the kidneys and adrenals of rats and that various physiological conditions such as de— hydration can alter these receptors. The remainder of the experimental section will be devoted mostly to control of prolactin receptors in the kidneys and adrenals by hormones and steroids. Other investigators have shown that the sex 86 steroids can alter renal function. Trachewsky (1972) de- monstrated that in the rat renal cortex, estradiol-17B de- creased the capacity of ribosomes to incorporate phenyl— anine into polyphenylalanine, an effect Opposite to that exerted by aldosterone. Testosterone was shown to be [important in maintaining kidney weight (Huang 99 99., 1955), and both estrogen and adrogen administration in man were reported to produce salt and water retention (Astwood, 1970). The role of the adrenals in maintaining salt and water metabolism is well established, and both estrogen and androgen have been reported to influence adrenal cortical function (Astwood, 1972). In View of these actions of es- trogen and androgen on the kidneys and adrenals, and more recent work showing that estrogen in the male and female rat can increase specific prolactin receptors in the liver (Posner 99 99., 1975; Gelato 99 99., 1975), the present study was undertaken to determine the effects of the sex steroids on specific prolactin binding sites in the kidneys and adrenals of male and female rats. B. Materials and Methods Intact male rats 30 or 60 days of age were injected s.c. with either 1 mg testosterone propionate (TP) in 0.1 m1 corn oil or with the vehicle alone (controls) for 10 days. On the 11th day all rats were killed, and their kidneys and adrenals were removed and frozen. In a 2nd experiment, 60 day old male rats, 200-255 gms, were castrated and treatment was begun on the following day. The castrated rats were injected s.c. for 10 days as follows: 87 (1) controls, 0.1 cc corn oil, (2) 1 mg progesterone/0.1 cc corn oil, (3) 5 ug estradiol benzoate-3B (EB) 0.1 cc corn oil, and (4) intact controls, 0.1 cc corn oil. All rats were killed 26 hrs. after the last injection. Prior to decapitation, all groups received 100 ug ergocor- nine/100 gm BW in 0.87% saline 24 and 4 hrs. before decap- itation in order to reduce endogenous levels of prolactin. Blood was collected from the decapitated trunk, allowed to clot at 4° C, and the serum was separated and stored at -20°C until assayed. Serum prolactin levels were determined by the double antibody radioimmunoassay method of Niswender 99 99. (1969). The reference standard was NIAMDD PRL-RP-l (11 IU/mg). Rats were killed by decapitation after each experiment and their kidneys and adrenals removed immediately, wrapped in tin foil and placed on dry ice. Tissue was then stored frozen at -50° C for no longer than 6 weeks, until assayed for PRL binding activity. This storage did not affect PRL binding activity as judged by comparisons of fresh tissue versus frozen. Data was analyzed by analysis of variance for unequal sample size (Sokal 99 99., 1969) followed by Duncan's multi- ple range test for comparisons of means among groups (Duncan, 1955). C. Results In Exp. 1 (Table 4), TP treatment administered to immature 30 day old males reduced PRL receptor binding in the kidneys to 60% of immature controls. In the 60 day old ani- mals TP treatment reduced PRL receptor binding to 54% of the 88 Table 4. Prolactin binding in kidney membrane preparations of 30 and 62 day old intact (control) and test- osterone prOpionate (TP) treated male rats. Treatment and 2 Specific Binding no. of rats/group of 125I-oPRL 30 day old controls (4) 7.65 t 0.40 Ibid. + 1 mg TP (4) 4.6]. :l: 0.522 60 day old controls (4) 8.92 t 0.77 Ibid. + 1 mg TP (4) 4.81 1- 0.582 1 Means 1 Standard Error of the Means 2 P>0.01 as compared to its controls 89 Table 5. Prolactin binding activity in adrenal and kidney membrane preparations of intact (controls), castrated and castrated plus testosterone propionate (TP) treated male rats. Treatment and Z Speiégic Ringing no. of samples/group of I-oPRL Adrenals Controls (4) 4.96 1 0.802 Castrated (4) 5.78 t 0.462 Castrated + 0.5 mg TP (5) 2.83 t 0.293 Castrated + 1.0 mg TP (5) 2.45 t 0.183 Kidneys Controls (10) 3.07 i 0.212 Castrated (6) 4.23 t 0.41 3 Castrated + 0.5 mg TP (6) 1.07 t 0.154 Castrated + 1.0 mg TP (6) 1.32 f 0.254 1Means f Standard Error of the Means 2’B’I‘Means with different superscripts for each tissue are significantly different from each other at P>0.0l. 90 Table 6. Prolactin binding activity in adrenal and kidney membrane preparations of intact (controls), ovariectomized (OVX) OVX + progesterone (Frog) and OVX + estradiol Benzoate (EB) treated female rats. Treatment and Z Speci§§c Binding no. of samples/group of I-oPRL Adrenals Controls (5) 2.42 i 0.512 Ovariectomized (5) 4.47 t 0.273 ovx + 1 mg Frog (5) 4.99 i 0.523 ovx + 5 ug BB (5) 3.45 3 0.274 Kidneys Controls (10) 1.25 3 0.072 Ovariectomized (10) 1.56 t 0.153 OVX + 1 mg Prog (10) 1.50 1 0.133 ovx + 5 ug an (10) 0.86 i 0.074 1 Means 1 Standard Error of the Means 2’3’6 Means with different superscripts for each tissue are significantly different from each other at P>0.01. 91 60 day old control group. In Exp. 2 (Table 5), castration of mature male rats in- creased PRL binding in the adrenal glands to 110% of controls, but this difference was not significant. When 0.5 mg or 1.0 mg TP was given to the castrated animals, specific binding was significantly reduced to 51% and 58% of the castrated control group value. Similar results were obtained in the kidneys of the same animals. Castration significantly in— creased PRL binding to 138% of control values then 0.5 mg or 1.0 mg TP were given In Exp. 3 (Table 6), ovariectomy of the mature female rats increased PRL binding in the adrenal glands to 185% of control values. A dose of 1 mg of progesterone did not sig- nificantly alter binding in ovariectomized rats, whereas 5 ug EB significantly reduced PRL binding to 77% of castrate control values. Ovariectomy significantly increased PRL binding to 125% of control levels in the kidneys, and injec- tions of 1 mg progesterone did not significantly alter this binding. However, 5 ug EB reduced PRL binding to 55% of ovar- iectomized control values. Since estrogen is known to stim- ulate PRL release in the rat, and elevated PRL levels might interfere with PRL binding values, ergocornine was given 24 and 4 hrs. prior to decapitation. PRL levels in all ergocor- nine-treated groups were less than 5 ng/ml as measured by RIA. D. Conclusions These results indicate that estrogen in the female rat and androgen in the male rat can decrease specific PRL binding activity in the kidneys and adrenal glands. 92 Castration resulted in a significant rise in PRL binding in the kidneys of both sexes and in the adrenals of the females. This negative feedback system was Operative in immature as well as in mature male rats. Progesterone had no effect on PRL binding activity on either tissue of the OVX female rat in the dose administered. Changes in PRL binding in these two organs have previously been shown to occur during various physiological states such as dehydra- tion which increased PRL binding in the adrenal and de- creased it in the kidney, and salt loading which increased PRL receptors in the adrenal (Marshall 99 99., 1975). These findings suggest that PRL receptors are involved in salt and water regulation in the rat. Estrogen has been observed to increase H20 transfer by the rat intestinal mucosa (Crocker, 1971), decrease p-amino- hippuric acid uptake by rat kidney slices (Huang 99 99., 1955) and influence ribosomal function in the kidney (Trachewsky 99 99., 1972). Other studies have shown that urine flow, urinary Na concentration and Na excretion are lower during estrus than at diestrus when estrogen activity is low (Crocker 99 99., 1973). Covelli 99 99., (1973) re- ported an increase in NaCl appetite in wild rabbits from the 11th to the 25th day after initiation of pseudopregnancy and during treatment with 12.5,25 or 250 ug of l7B-estradiol diprOpionate/day, whereas progesterone was without effect on NaCl appetite. In male rats, orchidectomy decreased kid- ney weight whereas testosterone elicited renal hypertrophy in castrated males (Huang 99 99., 1955). Compensatory renal 93 hypertrophy was decreased after castration in male rats for 7 days and this deficit was corrected by testosterone re- placement (Basinger 99 99., 1973). In humans, retention of water and NaCl was produced by androgen treatment (Astwood, 1970). The literature suggests that both estrogen and andro- gen can alter kidney and adrenal function and salt and water regulation. It is possible that part of the action of these steroids on PRL binding is mediated via the pituitary, since both steroids have been reported to increase PRL release (Meites 99 99., 1972). The present study further implicates estrogen and testosterone in salt and water regulation by their ability to decrease PRL binding in the kidneys and ad- renals, effects Opposite to those produced by these steroids in the liver (Posner 99_99., 1975). The precise role of PRL receptors in the kidneys and adrenals remains to be estab- lished. IV. Glucocorticoid Regulation of Prolactin Receptors in Kidneys and Adrenals of Male Rats A. Objectives The ability of glucocorticoids to decrease pitui- tary prolactin (PRL) secretion was suggested by a report that cortisol injections increased pituitary PRL content in rats (Sar 99 99., 1968), presumably by inhibiting PRL release. This was later confirmed by a demonstration that adrenalec- tomy produced an increase in serum PRL in rats (Ben-David 99 99., 1971; Euker 99 99., 1975). More recent work has 94 indicated that glucocorticoids also can decrease the acute release of pituitary PRL during ether stress in a dose de- pendent manner (Harms 99 99., 1975). In addition to a glu- cocorticoid effect on PRL release, there also is evidence that PRL may alter corticosterone secretion in rats. Witorsch 99 99. (1972) demonstrated that PRL can increase corticosterone secretion in male and female rats by decreas- ing the adrenal enzyme 5 cc-reductase, which metabolized cor- ticosterone to its inactive form. This was confirmed by Gustafsson (1976) who showed that PRL decreased 5 -reduc- tase 20-30% in the adrenals of male and female rats. In order for PRL to have effect on the adrenals it must first interact with a specific receptor site on the target cell before subsequent intracellular events take place, such as a reduction of adrenal enzymes. A change in PRL recep- tors could represent an additional mechanism for modifying adrenal responsiveness to circulating PRL. In the present study, the effects of glucocorticoids on PRL receptors in the adrenal gland were investigated. Glucocorticoids also have been reported to have an effect on the kidneys by increasing glomerular filtration rate, thus increasing Na excretion (Bartter 99 99., 1974). In addition, cortisol can decrease distal tubular reabsorp- tion of water. Therefore, glucocorticoid effects on PRL receptors in the kidney were also examined. Since salt loading and dehydration have been shown to alter PRL recep— tors in the kidney and adrenal (Experiment II) a possible mineralcorticoid involvement similarly was investigated. 95 B. Materials and Methods In the first experiment, hypophysectomized male rats, 225-250 gms, were injected subcutaneously (SC) twice daily for four days as follows: controls, 0.1 m1 of 0.9% NaCl, 5 g dexamethasone per 100 gm b.w. in 0.1 ml saline. An intact control group also was added which received the same treatment as the hypophysectomized controls. On the fifth day all rats were killed and their kidneys and adren- als were removed and frozen. In the second experiment, male rats 225-250 gms, were adrenalectomized and maintained on 0.9% NaCl as drinking water. Two days after surgery, rats were injected (SC) twice daily for four days as follows: 1) adx. controls, 0.9% NaCl/0.1 ml, 2) Bug per 100 gm b.w. dexamethasone/0.1 m1 saline, 3) 6.75 I.U. porcine ACTH per 100 gm b.w./0.1 ml saline plus 541g per 100 gm b.w. dexamethasone, 4) intact controls, 0.9% NaCl/0.1 ml. On the fifth dayafllgrats were killed and their kidneys removed and frozen. In a third experiment, male rats, 225-250 gms. were injected twice daily (SC) for five days as follows: 1) controls, 0.1 m1 corn Oil, 2) 6 units renin/0.1 ml corn oil, 3) 50 ug aldosterone/0.1 m1 corn Oil, 4) 60 ug spiron- olactone/0.1 ml corn oil. In addition to the five day in- jection regime for the aldosterone and spironolactone groups, a ten-day treatment group also was included. On the day after treatment, the rats were killed and the kid- neys and adrenals removed and frozen. In the final experiment male rats, 225-250 gms, were 96 injected once daily (SC) for five days as follows: 1) con- trols, 0.1 ml corn oil, 2) 1 mg hydrocortisone acetate/0.1 ml, 3) 10 ug dexamethasone per 100 gm b.w./0.1 saline, 4) 100 ug dexamethasone per 100 gm b.w./0.1 ml saline. In addition to the five day regime for the low dose of dex- amethasone, a ten and twenty day treatment group were also included. The day after treatment, the rats were killed and their kidneys were removed and frozen. C. Results In preliminary studies prolactin receptors were identified in kidney and adrenal membrane preparations from male rats. The binding of (1251)-iodo PRL to these membranes could be inhibited in a dose-related manner by addition of 0.5—100 mg unlabeled PRL, while 100 mg of LH, FSH, TSH and GH were without effect (Marshall 99 99., 1976). This demon- strates that binding was specific for prolactin. A later study reported an interesting sex difference in PRL binding in the kidneys and adrenals of male and female rats (Gelato 99 99., 1975). In males, kidney and adrenal binding was 7.65 i 0.64 and 4.96 9 0.8%, respectively, while in the fe- male, PRL binding was significantly lower, 1.25 i 0.07% in the kidney and 2.42 i 0.51% in the adrenals (data not shown). Therefore, in the following experiments, males were chosen because decreases in specific PRL binding could be detected more easily. Figure 3 depicts a linear relationship between (1251)_ iodo PRL and rat kidney membrane prolactin concentrations up to about 1 mg protein per tube. Further increases in 97 protein concentration resulted in smaller rises in specific binding. Non—specific binding was 7.1 to 8.9% of total counts, which was not significantly changed by increases in protein concentration. Shown in Figure 4 is the time course of specific bind- ing of (125 I)-iodoPRL to kidney membranes after incubation at 37, 24 and 4 C. At 37 C maximal binding occurred after 6 h incubation, followed by a rapid decrease in binding accompanied by a large increase in non-specific binding. Prolactin binding at room temperature (24 C) was maximal at 12 h, with some loss of specific binding at 24 h. Incu- bation of membranes at 4 C resulted in a plateau of maximal binding between 2 and 5 days, with no significant increases in non-specific binding. The incubations for the studies reported were conducted at 4 C for 2 days. The minimal de- gradation of hormones and tissue at this temperature, and plateauing of binding at 2 days, indicative of a steady bind- ing state, were the reasons these conditions were chosen. Figure 5 shows the effects Of adrenalectomy, replace- ment with dexamethasone and combined treatment with both ACTH and dexamethasone on specific PRL receptors in membrane preparations from the kidneys of male rats. Adrenalectomy significantly increased specific PRL binding in kidney mem— branes in the control group from 9.98 i 0.32% to 14.8 9 0.51%. When adrenalectomized rats were treated with dexamethasone, a marked decrease to 7.06 i 0.40% was seen in PRL-receptor binding, which is significantly lower than in the adrenalec- tomized and control groups. Since the decrease in PRL 98 receptors seen with dexamethasone treatment can be explained either by an increase in total glucocorticoids or be a de- crease in ACTH due to glucocortioid feedback, both dexametha- sone and ACTH were administered concomitantly. It can be seen that ACTH did not increase PRL receptors, but in fact resulted in a further decrease in binding to 4.23 i 0.27%. This decrease may be explained by an effect of ACTH on in- creasing glucocorticoid turnover time (Urquhart, 1974). The effects of hypophysectomy alone or with dexametha- sone or ACTH on specific PRL receptors in kidneys and the adrenals of male rats are shown in Figure 6. PRL receptor binding in the kidneys was reduced from 9.98 i 0.32% in the control group to 2.85 i 0.20% in the hypophysectomized group. Dexamethasone treatment of hypophysectomized rats resulted in a further significant reduction to 0.74 i 0.08% specific binding in kidney membrane preparations, demonstrat- ing that glucocorticoid action is not mediated mainly through the pituitary gland. ACTH administration to hypOphysectom- ized rats produced a decrease (to 1.28 i 0.11%) as compared to hypOphysectomy alone. Scatchard analysis of kidney mem- 8 g for the different groups in the above experiments, indicating brane preparations gave an aparent Kd of 6.4 x 10— that the changes in specific binding reflect changes in binding capacity rather than in binding affinity. Specific PRL binding to adrenal membranes is also shown in Figure 6. Intact controls had 12.72 i 0.64% spe- cific binding, which was reduced to 5.37 9 0.33% after hypo- physectomy. A further significant reduction in binding to 125| - oPRL ,PERCENT SPECIFIC BINDING 99 Figure 3. .25 5:0 1..“ 2.0 MEMBRANE PROTEIN cue/runs) _‘ ,_ _ , _ r1 __..f7 Relationship between the concentration of male rat kidney membrane protein and specific binding of (125I)lodo-PRL. 100 37°C 111 I23455789fl00ll$ 125I- oPRL , PERCENT SPECIFIC BINDING N\“ d N I— w e. on — a , I‘ 00 Figure 4. Time course of specific binding of (1251)iodo-PRL at 4, 24, and 36 C to rat kidney membranes. 16,000 125 SPECIFIC BINDING or I-oPRL (0PM) Figure 5. 101 14,000 I— KIDNEY CONTROL ADX ADX APX DEX DEX ACTH The effects of ADX, ADX with dexamethasone (DEX) treatment, and ADX with DEX and ACTH treatment on the specific binding of ovine (125I)iodo-PRL to kidney membranes of male rats. 12000 10000 0000 0000 @000 SPECIFIC BINDING or 125I-oPIzI. (0PM) 2000 Figure 6. 102 7T .KIDNEY % aADRENAL / / / / / ¢ ¢ / g % I 3 / I ‘° 8 .' a CONTROL HYPOX HYPOX HYPOX + + DEX ACTH Q Effects of hypOphysectomy (HYPOX), HYPOX and dexamethasone (DEX), and HYPOX and ACTH on specific PRL binding in kidney and adrenal membrane prep- arations from male rats. 103 12 — KIDNEY 125 PERCENT SPECIFIC BINDING OF I-DPRL 01 I ‘ 6 121 11 CONTROLS H.C. HIGH LOW DEx DEx TREATMENT I J DURATION 5 DAYS 10 LOW DEX 10 DAYS 12 8 LOW CONTROLS DEX l J 20 DAYS Figure 7. Effects of hydrocortisone (B.C., 0.1 mg), a high (100 ug/100 g BW) and a low (10 ug/lOO 3 BW) dose of dexamethasone for 5 days, and a low dose of DEX for 10 and 20 days on specific PRL binding in kidney membranes from intact male rats. 104 .uonasaION .z.m.m H sum:H Anzac m \%NO x ~\w: omv Amy c.o H o.m AmV o.o H 0.0 va m.o H q.m moououmoca< Aommc m I u I \soo x ~\ms soc xkv o.o + m.s Amv m.o + m.s fihv m.o + s.w osooooaososaom I Amhmv m\hmc N N Ame m.o H s.m Any o.o + s.s Ame s.o H w.s \oosss so sssom NANHV m.o H m.m Nfiov no.0 H m.q Namav n.o H m.m maouuooo Haaa\wov Hmoouv< Hoavam mao>oq gum asuom HAMmIOOOHHHmNHH mo wchon afimwooom N pooauoouH mumm oaoz mo mamoouc< can whootwm Ono OH MOHOOHm noudooom cHuOmHoum can mHo>oA oHuumHoum aouom :O ocouooaoooufimm no uofioomiaooououmova< mo uoowmm .n manna 105 3.18 i 0.45% was seen in hypophysectomized rats given dex- amethasone. No significant differences from hypophysecto- mized controls was observed after ACTH treatment. The effect of a mineralcorticoid on specific PRL re- ceptors in both the kidney and adrenals of male rats was tested using aldosterone, its antagonist spironolactone, and renin. No significant differences were found in spe- cific PRL binding in either kidneys or adrenals, and no differences were found in endogenous PRL levels as measured by radioimmunoassay (not shown). The results of the last experiment, shown in Figure 7, point out 3 different aspects of glucocorticoid inhibition on PRL receptors. First, other glucocorticoids can reduce PRL receptors in the kidney. In this experiment hydrocor- tisone significantly reduced PRL receptors from 11.54 i 0.83% in the control group to 6.86 i 1.41%. Secondly, the glucocorticoid effect was dose-dependent with a low dose of dexamethasone reducing PRL receptors to 3.95 i 0.27% and a high dose reducing PRL receptors to 0.88 i 0.12%. Third, reduction of PRL receptors in the kidneys was time-depen- dent with the 1ow dose of dexamethasone: 5, 10, and 20 day PRL receptor values were 3.95 i 0.27%, 1.94 i 0.33%, and 1.52 i 0.13%, respectively. This demonstrates a progres- sive decrease in PRL receptors in the kidney with increas- ing treatment time. D. Conclusions These results indicate that specific PRL binding to kidney membranes is both time- and temperature-dependent. 106 They further demonstrate that glucocorticoids can reduce the concentration of PRL receptors in the 100,000 x g pellets from both the kidneys and adrenals of male rats. Dexamethasone was effective in reducing PRL binding in a dose—dependent manner, while a single dose of hydrocortisone also reduced binding. When the site of production of cor- ticoids was removed by adrenalectomy, receptor binding in- creased in the kidneys. Dexamethasone replacement therapy reduced PRL binding to below control levels. HypOphysec- tomy resulted in a decrease in PRL binding to below control levels. Hypophysectomy resulted in a decrease in PRL bind- ing in the kidneys. Other investigators found that hypophy- sectomy produced a reduction in PRL binding in the liver of female rats (Posner 99 99., 1975). A further significant reduction in PRL receptors in the kidneys of hypophysecto— mized rats was achieved with dexamethasone treatment, indicating that the glucocorticoid effect on PRL receptors does not require or is not primarily mediated by pituitary factors. The effects of adrenalectomy in elevating PRL bind- ing in the kidneys is not believed to be due toa general metabolic action, since previous investigators have shown that adrenalectomy does not significantly alter PRL bind- ing in liver membranes from adult female rats (Kelly 99 99., 1975). ACTH also may play a role in reducing PRL receptors in the kidneys, since in adrenalectomized-dexamethasone treated rats, ACTH was able to further reduce PRL receptor binding. Similarly, a reduction in PRL receptors in the 107 kidneys of hypophysectomized male rats was seen when ACTH was given alone. Again, this effect could be indirect through glucocorticoid release by the intact adrenal glands. Scatchard analysis revealed that changes in kidney mem- brane preparations reflected changes in the binding capac— ity of the tissue rather than a change in binding affinity. In the adrenal glands, hypophysectomy resulted in a decrease in PRL binding as compared to intact rats. Fur— thermore, dexamethasone administration to hypOphysectomized rats caused an additional significant decrease in adrenal binding indicating that the glucocorticoids also can act on the adrenal glands independently of the pituitary. Neither aldosterone nor its antagonist, spironolac- tone, proved effective in the doses given in altering PRL receptors in the kidneys or adrenals. Renin, an enzyme from the kidneys which regulates aldosterone secretion, also was ineffective. These results suggest that the glucocorticoids, in addition to their other myriad effects in the body, also are regulators of PRL receptors in the kidneys and adrenals, with the mineralocorticoids having little, if any, effect on PRL receptors. There is considerable evidence suggesting a functional relationship between PRL and the glucocorticoids. Schlein 99 99., 1973, found a reduced plasma corticosterone response to stress in lactating and PRL-treated rats as compared to normal female rats, indicating that PRL can influence ACTH secretion from the pituitary. Glucocorticoids can 108 inhibit PRL secretion from the pituitary and modify acute PRL release induced by either stress in a dose-dependent manner (Euker 99 99., 1975; Harms 99 99., 1975). PRL also has been reported to act directly on the adrenals to in— crease corticosterone secretion by reducing adrenal 5a- reductase activity (Witorsch 99 99., 1972). The present data suggest that an additional level of control may be exerted at the PRL receptor level, by modifying adrenal responsiveness to circulating levels of PRL. Thus, by changing the concentration of PRL receptors in the target organ tissues, peripheral control could be exerted in con- junction with circulating levels of endogenous PRL. The glucocorticoids exert an effect on the kidneys and adrenals by reducing the level of PRL receptors. We have previously shown that the binding of PRL to its receptors is altered by dehydration and salt loading (Marshall 99 99., 1975), and by estrogen and testosterone treatment (Marshall 99 99., 1976). Thus, PRL may have a role in salt and water regulation in rats. PRL has been reported to decrease Na excretion in rats, rabbits, and cats (Lockett 99 99., 1965; Burstyne 99 99., 1974; Lockett, 1965), and osmolar changes were observed to influence se- cretion of PRL in many experiments (Relkin, 1974; LaBella 99 99., 1975). If prolactin does alter salt and water metabolism in the kidney or rats, then changes in prolac- tin receptors in this target organ may modulate the re- sponse to circulating prolactin. 109 V. Effects of HypOphysectomy, Thyroidectomy and Thyroxine on Specific Prolactin Receptor Sites in Kidneys and Adrenals of Male Rats A. Objectives Data from several studies suggest that thyroid hor- mones may alter the actions of prolactin (PRL) on various target organs, through effects on PRL receptors. Thus in the newt (Connelly 99 99., 1968), thyroxine and PRL act synergistically to stimulate limb regeneration, and pro- mote molting in the lizard and newt (Chiu 99 99., 1972; Cent 99 99., 1973). In salamanders, Gona 99 99.(1973) re- ported that the PRL induced water drive can be blocked by high levels of thyroxine, whereas low levels of thyroid hormones are required for this response to PRL. More re- cently, Vonderharr (1977) convincingly demonstrated that triiodothyronine (T3) added 99 99999 to cultures of mouse mammary gland explants resulted in a 3-5 fold increase in lactalbumin synthesis. Thyroid hormones appeared to act by altering the responsiveness of the mammary gland explants to PRL. The hypothesis that thyroid hormones can alter PRL receptors in different tissues was strengthened by data demonstrating that thyroidectomy (Tx) in female rats sig— nificantly reduced specific PRL receptors in the liver, and T replacement restored PRL binding sites to intact 4 control levels (Gelato 99 99., 1975). 5I)iodo-PRL binding to kidney membranes Specific (12 from male and female rats has been demonstrated, and this binding can be regulated by estrogen, androgen and 110 glucocorticoid steroid hormones (Marshall 99_99., 1976; Marshall 99 99., 1978). Moreover, specific nuclear T3 receptors have been identified in the nuclei of rat kid- neys (Surks 99 99., 1973; Oppenheimer 99 99., 1974; Oppenheimer 99 99., 1972; Samuels 99 99., 1974). Since thyroid hormones can influence kidney growth, morphology and function (Katz 99 99., 1977), it was therefore of in- terest to investigate the actions of thyroid hormones on PRL receptors in the kidneys of male rats. In these stud- ies PRL binding sites in the adrenals were also measured. B. Materials and Methods In the lst experiment male rats (225-250g) were thyroidectomized (Tx) 2 days prior to treatment and main- tained on 1.0% calcium lactate in drinking water. Rats were injected sc daily for 4 days as follows: 1) intact controls, 0.1 ml 0.9% NaCl, 2) Tx, 0.1 ml 0.9% NaCl, 3) Tx, 10 ug T4/100 g bw in 0.1 ml 0.9% NaCl. On the 5th day, all rats were killed and their kidneys and adrenals were removed and frozen. In the 2nd experiment male rats (225-250 g) were hypo- physectomized (HYPOX) 4 days prior to treatment, these and intact male rats were injected sc for 4 days with 10 ug T4/100 g bw in 0.1 ml 0.9% NaCl. HYPOX and intact controls were injected with vehicle alone. On the 5th day all rats were killed and their kidneys and adrenals were removed and frozen. In the 3rd experiment male rats (225-250 g) were Tx and maintained on 1.0% calcium lactate in drinking water. 111 Rats were killed 2, 3, 5, 7 and 10 days after Tx. The last Tx group received 10 ug T4/100 g bw in 0.1 ml 0.9% NaCl 2 days prior to killing. Control rats were killed on the first day of treatment. After decapitation, kid- neys and adrenals were removed and frozen. Trunk blood was collected and the serum was separated and frozen. In the 4th experiment male rats (225-250 g) were Tx 8 days prior to treatment and maintained on 1.0% calcium lactate in drinking water. On the 9th day a single sc injection of 10 ug T4/100 g bw was given and the rats were killed 0, 6, 12, 24 and 48 h later. Intact rats were similarly injected and killed 0 and 24 h after T4 injec- tion. Kidneys and adrenals were removed from all animals and frozen. Serum was separated from trunk blood and also frozen. In the 5th experiment male rats (225-250 g) were injec- ted sc for 20 days as follows: 1) controls, 0.1 ml 0.9% NaCl, 2) 5 ug DEX/100 g bw in 0.1 ml 0.9% NaCl, 3) 5 ug DEX/100 g bw in 0.1 ml 0.9% NaCl and 10 ug T4/100 g bw in 0.1 ml 0.9% NaCl for the last 5 days of the treatment. On the 21st day the kidneys were removed and frozen. B. Results Figure 8 shows the effects of Tx and Tx with T4 replacement on specific PRL binding in membrane prepar— ations from kidneys and adrenals of male rats. Tx sig- nificantly reduced specific PRL binding in kidney mem- branes from 14.7 i 14% in the intact control group to 7.0 i 0.6%. When Tx rats were treated with T4 PRL 112 binding sites increased to 16.2 i 1.1%, which was not sig- nificantly different from intact controls. No significant changes were noted in adrenal membrane preparations with either treatment. Adrenal binding was 7.6 i 0.8%, 7.6 i 0.7% and 9.6 i 0.3%, respectively, in intact, Tx and T4 -treated rats. Figure 9 shows the effect of T 4 (1251)iodo-PRL to kidney membranes of intact and HYPOX on specific binding of male rats. T4 significantly increased PRL binding in the kidneys of intact rats from 12.1 9 1.1 to 18.9 i 0.9%. HYPOX reduced binding to 28% of intact values, in agree- ment with a similar decrease previously reported for kid- ney membranes (Marshall 99 99., 1978). After T4 treat- ment, the renal PRL receptors of HYPOX rats in this study returned to intact control values, from 3.4 i 0.3 to 12.8 i 1.4%. Specific PRL binding sites on kidney and adrenal mem- branes and serum TSH measurements at various times after Tx are depicted in Figure 10. Tx significantly reduced PRL binding sites in the kidneys from 8.2 i 0.5% in the intact control group to 6.6 + 0.7% by day 2 and progres- sively decreased to 2.3 i 0.3& by day 10. T replacement 4 on day 10 returned specific PRL binding sites in the kid- neys to intact control values. Serum TSH increased con— tinually during the 10 day Tx period from 102.5 to 2642 ng/ml, indicating a reduction in the negative feedback by T4 on TSH secretion. The correlation between renal PRL 12— 10- SPECIFIC PRL BINDING (96) Figure 8. 113 é "TE: é Illlllll|||||||||l||||I||||||||||||||||||||||||h 4 on 4 BIIITIIIIL TIIYIIIIIIIECTIIIV IIIYIIIIIIIECTIIIY 4 —. Effects of thyroidectomy and Tx with T ment in male rats on specific binding 0 iodo-PRL to kidney and adrenal membranes. treat- (1251): 114 20 _ t. KIDNEY 18 - 16— (96) 14— 12- 10— PRL BINDING *0 SPECIFIC 10 I 8 7 9 OOIIIIOL I, IIIPOX III-:OX TI Figure 9. Effects of T4, hypophysectomy and hypophysectomy with T4 on specific binding of (125I)iodo-PRL to kidney membranes from adult male rats. Figure 10. A 6 69 g 4 I. 0 EL 2 h I '6 (ONIIOI l 2 3 5 7 1O 2 o E O z i 0 E 5 ||l II. ,, In / A . - “”"lol 2 3 5 7 10 2 D"! "I" IX DAYS "III I. III ,,oo_ SERUM TSH A 1000 2 \\ O 2 1mm coo 3 5 7 10 i. ms mu Ix nmumI Specific PRL binding sites in kidney and adrenal membranes, and serum TSH values at 2,3,5,7 and 10 days after thyroidectomy. 115 KIDNEY I. IV///////////. .-.iw////////// 8 8 lo 3 1 6 12 24 48 116 at nu wmm11 TEE/gs nm on ' u 6m... Tx male T4/100 g BW injection ng of (125I)iodo-PRL to kidne from intact and single 10 ug of a ure 11. Effects 117 E 12— .J E a 10— L "3 a— KIDNEY Mb 0 o 6- E O .2. m 4— (J E 2— 0 El 0. “I CONTROL OEX Figure 12. Effects of dexamethasone and dexamethasone with T4 on specific PRL binding sites on kidneys of male rats. 118 binding and TSH levels was -0.96. Although PRL receptors in the adrenals tended to decrease, no significant dif- ferences were found among any groups. The effects of a single injection of T4 in intact and Tx male rats are shown in Figure 11. PRL receptors in the kidneys of intact rats increased from 9.3 9 0.8 to 15.6 9 1.9% 24 h after T4 administration. The reduced binding observed after 8 days of Tx was increased significantly by T from 3.4 9 0.4 to 7.9 9 1.0% by 2 h post-injection. 4 Binding remained at intact control levels for at least 48 In the adrenals there 4. was a slight decrease in the PRL binding of (125I)iodo-PRL h after the single injection of T after Tx which increased with T4. However these differ- ences were not significant. 25I)iodo-PRL to kid- In Figure 12 specific binding of (1 ney membranes of intact control rats decreased after DEX treatment from 10.1 9 0.9% to 1.5 9 0.1%, as demonstrated previously (Marshall 99 99., 1978). T4 treatment increased binding to 2.93 9 0.2% which was still significantly below intact control values, but not significantly different from DEX treatment. D. Conclusions These results indicate that specific (1251)iodo- PRL binding to kidney membranes are in part regulated by thyroid status, with hypothyroidism reducing and hyper- thyroidism increasing PRL receptors. This effect is be- lieved not to be a general metabolic effect on PRL recep- tors since altered thyroid status failed to influence 119 receptor sites in the adrenals. HYPOX reduced receptors in the kidneys, and binding was restored to intact con- trol levels with T4 replacement. Other investigators noted a marked decrease in PRL receptor binding in the livers of rats after HYPOX. However, binding in the liver could not be restored by treatment with T (Costlow 99 99., 3 1975). In the kidneys, the decrease in PRL binding after Tx was restored rapidly to control values by T A single 4. T injection in Tx rats increased binding to intact con- 4 trol values by 12 h and remained elevated for at least 48 h. The mechanism of action of thyroid hormones is not com- pletely understood, but T has been shown to bind to spe- 3 cific nuclear binding sites in rat liver and kidneys (Surks 99 99., 1973; Oppenheimer 99 99., 1972). It also is known that T3 can induce RNA and RNA polymerase activ- ity in rat liver nuclei within 8 h after hormone admin- istration (DeGroot 99 99., 1977). Therefore, it is pos- sible that T may alter protein synthesis through mRNA 4 to increase PRL receptor sites in the kidneys. Use of PRL receptor changes in the kidneys may prove to be a good method to study the mechanism of action of thyroid hormones, since of the end products of T stimulation appears to be 4 an increase in PRL receptors. PRL receptors are easy to quantitate and the response is rapid and large in magnitude. T increased renal PRL receptor sites in HYPOX rats, 4 suggesting a direct action on the kidney not mediated through other pituitary hormones. However, since TSH 120 stimulates T4 release and synthesis, it probably acts in- directly via the thyroid to modulate PRL receptors in the kidney. Other anterior pituitary hormones also appear to influence PRL receptors in the kidneys since DEX prevented the T induced increase in receptors. This suggests pos- 4 sible ACTH adrenal influence on PRL receptors in the kid- neys. Previous reports also have established a glucocor- ticoid influence on PRL receptors in the kidney (Marshall 99 99., 1978). Several studies (Lockett 99 99., 1965; Marshall 99 99., 1975) have reported that one of the functions of PRL is to regulate salt and water metabolism through actions of the kidneys of rats. If this is true, then changes in PRL re- ceptors induced by altered thyroid function also should in- fluence Na transport. Such effects were demonstrated in hypothyroid rats, who were defective in conserving Na when given a Na deficient diet, and showed an exaggerated natriuretic response to saline loading (Michael 99 99., 1972; Fregly 99 99., 1962). One postulated mechanism whereby PRL may influence salt and water metabolism is through changes in Na-K- ATPase in several target organs. PRL has been reported to stimulate Na-K-ATPase in the gill filaments Of the Jap- anese eel, and thus function in adaptive osmoregulation (Kamiya 99 99., 1972). In the mammary gland of rabbits, PRL has a significant influence upon the Na+ and K+ con- tent of mammary alveolar tissue, as well as the Na+, K+ concentrations of normal milk (Falconer 99 99., 1977; 121 Falconer 99 99., 1975; Taylor 99 99., 1975). Oubain, a specific inhibitor of the Na/K pump, reversed the PRL induced changes in ionic composition in mammary alveolar tissue, suggesting that PRL modulates these changes in intracellular ions via changes in Na-K-ATPase (Falconer 99 99., 1977). In kidney homogenates from hypothyroid rats, Na-K- ATPase was found to be low, but could be increased when rats were treated with T3 (Katz 99 99., 1973; Edelman 99 99., 1975). Thus, thyroid hormones can act on the kid- neys to increase renal Na transport and Na-K-ATPase activ- ity. In addition, PRL also has been reported to stimulate Na-K-ATPase in the kidney of certain teleost (Pickford 99 99., 1970). Therefore, it is possible that part of the action of thyroid hormones may be mediated by changes in PRL receptor concentrations with altered thyroid states. In the adrenal glands PRL binding sites were not chang- ed by hypo- or hyper-thyroidism, strongly suggesting that the effects of T on the kidney is not a non-specific met- 4 abolic effect, but rather a specific target organ action. However, although PRL binding sites in the adrenals were not changed by Tx or T adrenal function can be influenced 4! by PRL. Witorsch and Kitay (1972) have shown that PRL decreased the adrenal enzyme, 5s~rneductase, thereby in- creasing circulating corticosterone in the rat. In ad- dition, selective stimulation of progesterone secretion from rat adrenals (Piva 99 99., 1973), and a PRL induced increase in adrenal androgen release in humans has been 122 reported (Carter 99 99., 1977). Thyroxine has been reported to influence the actions of PRL at the tissue level in lower vertebrates (Nicoll, 1974), or the mammary gland and liver of mammals (Vonder- harr 99 99., 1977; Gelato 99 99., 1975), and as the pre- sent data suggests in the kidneys of rats. An additional interaction between thyroid hormones and PRL also may occur in mammary tumors, since mice made hypothyroid by propylthiouracil treatment resulted in inhibition of growth of established transplanted mouSe mammary tumors, and enhanced survival (Shoemaker 99 99., 1976). In conclusion, the results of this study clearly demon- strate that thyroid hormones can modulate PRL receptors in the kidneys, but not in the adrenals of rats. Since PRL has been implicated in renal Na and water metabolism in the rat, it appears reasonable to assume that PRL and T4 may interact at the renal level to influence salt and water regulation. Thus at least in the rat, Na and water metabolism may be associated with the prevailing general metabolic state, by the effect of thyroid hormones on both the basal metabolic rate and PRL receptors in the kidneys. VI. Inhibition of Thyroid Hormone Induced Increase in Renal PRL Receptors by Actinomycin-D and Cycloheximide B. Objectives Thyroid hormones play an important role in kidney growth and morphology, and in maintaining renal function by effects on kidney hemodynamics, tubular function, electro- lyte excretion, and renal Na-K+ ATPase (Kutz 99 99., 1977). 123 More recently, thyroxine (T4) has been shown to have an additional effect on the kidney by regulating specific pro- lactin (PRL) receptors (Marshall 99 99., 1978). In these experiments, thyroidectomy (Tx) decreased PRL receptors in the kidneys of male rats, and the lowered binding was re- stored to control values with T4 treatment. The effects of T4 on renal PRL binding sites was very rapid, significantly increasing at 12 h after a single T4 injection in Tx rats. This effect was believed to be direct, and not mediated by pituitary hormones, since T4 was able to increase PRL bind- ing sites in the kidneys of hypophysectomized male rats. The presence of specific PRL receptors also have been identified in the adrenals (Marshall 99 99., 1975) and liver (Gelato 99 99., 1975) of rats. In the liver of female rats hypothyroidism decreased hepatic PRL receptors, and hyper- thyroidism increased PRL binding sites (Gelato 99 99., 1975) (4), whereas altered thyroid status was without effect on PRL receptors in adrenals of rats (Marshall 99 99., 1978). These reports strongly suggest that the effects of T4 on PRL receptors in the liver and kidney is a specific target organ effect, rather than a non-specific general metabolic action, since Tx and T4 failed to alter adrenal PRL receptors. Several tissues, including the kidney and liver, have been shown to contain specific high affinity nuclear binding sites for T3 (Samuels 99 99., 1972; Oppenheimer 99 99., 1972, 1974; Surks 99 99., 1973) which were identified as protein with a M.W. of 50,000 daltons (Latham 99 99., 1976). 124 Although the exact mechanism of action of thyroid hormones is not known, evidence supports the idea that thyroid hor— mones act by binding to nuclear receptors to activate gene transcription, enhance RNA polymerase activity, augment hrRNA and mRNA, and stimulate protein synthesis, which in turn influences intracellular metabolic events. Thus, T3 increased nuclear RNA polymerase activity in livers from Tx rats (Viaredigo 99 99., 1975; Tata 99 99., 1966) and stimu- lated RNA and protein synthesis within 8 h Tata 99 99., 1966; DeGroot 99 99., 1977; Bernal 99 99., 1978). The T3 induced stimulation of RNA was shown to be inhibited by compounds which prevent mRNA and protein synthesis (DeGroot 99 99., 1977). Moreover, augmented levels of specific mRNA for 2 - globulin was found in the liver of hypothyroid animals after thyroid hormone treatment (Kurtz 99 99., 1976; Roy 99 99., 1976) whereas in rat pituitary tumor cells T3 stimulated growth hormone mRNA (Seo 99 99., 1977). In view of these findings, it was of interest to investigate the mechanism of action by which thyroid hor- mones increase PRL receptors in the kidneys of male rats. The present results suggest that thyroid hormones increase PRL binding sites by influencing mRNA translation, which then could stimulate protein synthesis, culminating in in- creased PRL binding sites. B. Materials and Methods Male Sprague-Dawley rats, weighing 225-250 g each were obtained from Spartan Research Animals (Haslett, MI). 125 The rats were housed in a temperature controlled (25 9 1°C) and artificially illuminated room (lights on from 0500 to 1900 h daily), and received food and water 99 libitum. In the first experiment, intact rats were given a single 0.1 ml s.c. injection of 10 g/100 g B.W. 3,5,3- triiodithyronine (T3) in 0.9% NaCl, 80 g 100 g B.W. acti- nomycin-D, or 250 g B.W. cycloheximide in 0.9% NaCl. Con- trols received a single s.c. injection of vehicle alone. In a second experiment, male rats, 225-250 g were para- thyroidectomized 8 days prior to treatment, and maintained on 1% calcium lactate in the drinking water. Tx rats were divided into groups and given a single 0.1 m1 s.c. injection as follows: 1) Tx controls, 0.9% NaCl, 2) 10 g/100 g B.W. T3, 3) 10 g B.W. tetraiodothyronine (T4), 4) 10 g/100 g B.W. T and 80 g/100 g B.W. actinomycin-D, S) 10 g/100 g 4 B.W. T and 250 g/100 g B.W. cycloheximide. Rats were 4 killed by decapitation in both experiments 15 h after in- jections. Both kidneys and adrenals were removed immediate- ly, wrapped in tin foil, and placed on dry ice. Tissues were stored at -50°C for less than 6 wks until assayed for [1251]iodo-PRL binding activity. C. Results As can be seen in Fig. 13 a single injection of T3 increased the binding of [1251]iodo-PRL to kidney membranes from intact males from 12.78 9 0.68% in the control group to 20.74 9 1.30%, whereas in the adrenals T3 had no effect on PRL binding. Actinomycin-D, a RNA transcription inhibitor, 126 decreased (P<0.01) PRL binding in the kidneys by 25.2% from 12.78 + 0.68 to 9.56 9 0.57%. In the adrenals a much great- er % decrease was found in specific PRL binding when com- pared to controls (88.6%) from 13.00 9 0.85 to 1.48 9 0.29%. The protein synthesis inhibitor, cycloheximide, produced a decrease in PRL binding in both the kidneys and adrenals when compared to actinomycin-D. However, in the adrenals this further decrease was not significant, whereas in the kidneys, PRL binding was significantly reduced (P<0.01). Compared to intact controls, cyclohexamide treatment re- duced PRL receptors in the kidneys by 79.2%, and by 94.2% in the adrenals. The results in the second experiment are depicted in Fig. 14. A single injection of T3 and T4 increased (P<0.01) PRL binding in the kidneys at 15 h from 5.87 9 0.68% in Tx controls, to 12.28 9 0.81 and 10.45 9 0.74%, respectively. However, when actinomycin-D or cycloheximide was given con- comitantly with T4, the T4 induced increase in PRL binding was completely blocked. Not only was the T4 induced increase in binding inhibited, it was significantly reduced below Tx controls by 35.8% with actinomycin treatment, and 86.7% with cycloheximide. In the adrenals, PRL binding was not significantly altered by either T3 or T4, but actinomycin-D or cycloheximide given with T4 resulted in a reduction in PRL binding from 16.57 9 1.43% (controls) to 3.28 9 0.51% (Actinomycin-D) and 0.97 9 0.43% (Cycloheximide), a decrease in PRL binding from controls of 80.2% and 94.1%. SPECIFIC BINDING DF "’I-oPRLI‘IIII Figure 13. d n N O O 9’ T l l a N 1 “5| -oPRI. ,PERCENT SPECIFIC BINDING 127 KIDNEY CONTROL T3 MTINOIYCIN - D (YCLONIXIIIOI ADRENAL \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"‘~ W \\\ \\ fl . ,2. (ONT! 3 KTIIDIYCIN - O CYCLONIRIIIOI. \\ O . r- 4 Effects of T3, actinomycin-D and cycloheximide on (1251)iodo-PRL binding to kidney and adrenal membranes from Tx male rats. 128 ‘° ' KIDNEY "’I- um. (96) i. SPECIFIC DINDI ND 0' a. I TX TX-T. TX-T‘ TX-T‘ TX‘T‘ CD'TIDL + + “TIN“YCII'D CYCLDNIXIIID! ADRENAL mm. '\\\ WW: SPECIFIC BINDING 0F "’I-oPRu‘lt.) MW. \\ '§\\\.: Ix -, —1 u- I, u- I, comm: + + AtTIIIoomiI-II trttoutrINInr c. . Figure 14. Effects of T3, T4, T4 4+-§§tinomycin-D and T4 + cycloheximide on (1 I)iodo-PRL binding to kidney and adrenal membranes from.Tx male rats. 129 D. Conclusions 3 and T4 can rapidly (15 h) increase PRL receptors in the kidneys of It is clear from the date presented that T Tx rats, whereas no significant changes were observed in adrenal PRL binding sites. Rapid changes were also obtained in intact males in which T3 increased binding in the kidneys, but not adrenals. Since T3 has been reported to be 5 x more potent than T in the rat (Sterling 99 99., 1977) the higher 4 PRL binding seen with T3 is not unusual, and probably re- flects the greater bioactivity of T3. It is possible that T4 exerts an effect on PRL receptors indirectly by peripher- al conversion to T3. Actinomycin-D and cycloheximide were both effective in preventing the T4 induced increase in PRL receptors in the kidneys of male rats. Not only was the stimulatory action of T4 blocked, but a significant decrease below Tx controls was observed. A similar decrease in PRL binding sites in the kidneys of intact rats was seen when actinomycin or cycloheximide was given alone. Therefore, as suggested by these data, part of the mechanism of aflfibn by which thyroid hormones induce PRL receptors in the kidney is probably through increased RNA transcription, ultimately resulting in an increase in RNA, protein synthesis and PRL binding sites. The finding of specific thyroid hormone binding sites in the kidney (Oppenheimer 99 99., 1974, 1972) and reports showing T3 can increase RNA polymerase activity (Viarengo 99 99., 1975, Tuta 99 99., 19 6) RNA synthesis 130 (11,12) as well as increasing specific mRNA in the liver (Kurtz 99 99., 1976; Roy 99 99., 1976) and tumor cells (Sec 99 99., 1977) give further support to the present data. Although Tx or thyroid hormones did not alter PRL re- ceptors in the adrenals, actinomycin-D and cycloheximide had a profound effect on adrenal PRL receptors in both Tx and intact male rats. Therefore, it is possible that mRNA may also regulate PRL receptors in the adrenals of rats. Previous reports from our laboratory have shown that estro- gen, testosterone and glucocorticoids decrease adrenal PRL binding sites (Marshall 99 99., 1976, 1978). These ster- oids may effect mRNA in a manner similar to thyroid hormones to regulate adrenal PRL binding sites. Results obtained using RNA and protein synthesis inhib- itors suggest that there may be differences in the half life of the mRNA which regulates the synthesis of PRL receptors. Thus, actinomycin-D reduced adrenal binding sites by 88.6% in the intact and 80.2% in Tx-T4 treated rats, suggesting a relatively short mRNA half life. However, in the kidney PRL binding sites were reduced only by 25.2% and 35.8% in intact and Tx-T4 treated animals. Other investigators have suggested that mRNA, involved in the synthesis of PRL bind- ing sites for lactogenic hormones may have a relatively long half life (Kelly 99 99., 1975). Their conclusion was based on the finding that actinomycin-D failed to signifi- cantly reduce PRL receptors in the liver of estrogen treated male rats, while cycloheximide had a potent inhibitory 131 effect on PRL binding. Differences in mRNA half-life would represent still another level of control in regulating the effect of PRL in different target organs. Among vertebrates the reported actions of PRL include 85 distinct and diverse effects (Nicoll 99 99., 1972) in- cluding effects on the liver, kidney, adrenal, mammary gland, ovaries and prostate. It is becoming increasingly evident that control can be regulated at the level of the target organ, as it is doubtful that the basal circulating level of PRL cannot possibly regulate so many sich diverse effects. The data presented here indicate that thyroid hormones have a very selective effect on the regulation of PRL receptors in different tissues, and that protein synthesis is integ- ral in this response. Moreover, the data also suggests that the half life of mRNA involved in the maintenance of PRL binding sites may differ in various organs. Peripheral con- trol systems, such as these may help in understanding the diverse actions of PRL. VII. Prolactin Receptors in the Rat During Development and After a single Injection of Prolactin, Testosterone or Estrogen A. Objectives In previous studies specific prolactin (PRL) bind- ing sites were identified in livers of male and female rats and rabbit: (Gelato 99 99., 1975; Posner 99 99., 1974, 1975; Kelly 99 99., 1974). Moreover PRL binding sites in the liver were shown to change with age (Kelly 99 99., 1974). Although 132 specific PRL receptors have been demonstrated in both the kidneys and adrenals of male and female rats (Marshall 99 99., 1975) no information is available on the ontogenesis of these binding sites. Therefore in the present study PRL receptors changes in the kidneys and adrenals of male rats were determined during develOpment. Several investigators have reported association and 25I]iodo-PRL binding to membrane dissociation times of [1 preparations of liver, kidneys, mammary gland, and prostatic tissue (Sherman 99 99., 1977; Shin 99 99., 1974; Marshall 99 99., 1978). These 99 99999 measurements were important in determining Optimum incubation times in order to accur— ately quantitate PRL receptors, but may not reflect actual 99 9999 binding kinetics. This was tested 99 9999 by measuring association and dissociation times after a single injection of oPRL given to male rats. PRL binding sites were then measured 99 99999 at various times from 5 min to 8 days. Determining 99 9999 binding of PRL to its receptor may have a physiological correlate in the PRL rise during stress, the proestrous surge of PRL and suckling induced PRL release. In the third part of these studies, the time sequence for the inductive and reductive effects of estrogen and 125I]iodo-PRL binding to liver, adrenal and androgen on [ prostatic membranes were determined. Previous reports have shown that estrogen can induce PRL binding sites in the livers of male and female rats (Gelato 99 99., 1974; 133 Aragona 99 99., 1976) whereas PRL binding sites in the ad- renals were reduced (Marshall 99 99., 1976). In the male, specific PRL binding sites have been identified in the pro- state, and PRL binding was reduced by castration and in- creased by testosterone treatment (Aragona 99 99., 1975; Kledzik 99 99., 1976). These finding were substantiated and extended by monitoring the time necessary to modulate PRL receptors in the liver, adrenals and prostate. B. Materials and Methods Male and female Sprague-Dawley rats were obtained from Spartan Research Animals (Haslett, MI). The rats were housed in a temperature controlled (25° 9 1°C) and arti- ficially illuminated room (lights on from 0500 to 1900 h dailYI. and received food and water 99 libitum. Experiment 1: Male rats 10, 20, 25, 30, 40 and 60 days of age were decapitated and their kidneys and adrenals were removed and frozen. Serum was separated from trunk blood and frozen at -20° for later hormone determinations. Experiment 2: Male rats 225-250 g were given a single intravenous injection of 1 mg oPRL (NIH-PRL S-10, 25.6 units/mg) dis- solved in 0.9% NaCl at pH 7.8 or 0.9% NaCl alone (controls). Control rats were killed by decapitation either 5 min or 8 days after injection. PRL injected rats were killed at various time intervals from 5 min to 8 days. Kidneys, 134 adrenals and ventral prostates were removed and frozen for further determination of PRL binding sites. Experiment 3: Male rats 225-250 g were injected so with 1 mg testos— terone propionate (TP) in 0.1 ml corn oil and killed at var- ious times from 12.5 to 20 days after injection. Control rats were killed either 5 min or 20 days after a single injection of corn oil alone. Ventral prostates were ex- cised, frozen and later assayed for PRL receptor activity. Experiment 4: Female rats, ovariectomized (OVX) 14 days prior to treatment, were given a single sc injection of 20 pg estra- diol benzoate (BB) in 0.1 m1 corn oil. At 1, 3, 5, 7, 9, 12, 16 and 21 days after injection, rats were decapitated and their liver and adrenals were removed and frozen. Trunk blood was collected and the serum separated and frozen at -20° C for subsequent hormone assay. OVX controls were killed on days 1 and 21, and liver, adrenals and serum were similarly obtained and frozen. C. Results Experiment 1: 12 I 5I]iodo-PRL binding to kidney and adrenal membranes from male rats varied according to age (Figure 15). PRL binding sites on kidney membranes progressively increased from 4.94 9 0.16 to 16.19 9 1.27% during days 10 to 60 of development, whereas in the adrenals a decrease in PRL re- ceptors was observed from 22.89 9 1.24 at day 10 to 4.98 135 9 0.48 at day 60. These binding changes apparently were not related to serum levels of PRL, GH, or TSH (Figure 16). Experiment 2: When a single i.v. injection of 1 mg oPRL was given to male rats 99 vivo, the binding of (125 I)iodo-PRL to kidney and prostatic membranes measured 99 99999 was inhibited in a time related manner in both the kidneys (Figure 17) and prostate (Figure 18). Renal PRL binding sites were de- creased 5 min after PRL injection from 9.98 9 0.75 (controls) to 0.40 9 0.46%. During the next hr binding remained low, then increased to control values at 8 h post-injection. Ventral prostate PRL binding sites were also significantly reduced (P 0.01) at 5 min after PRL injection. However, in contrast to the renal data, PRL binding sites in the prostate were not significantly different from controls 1 h after injection, or any time thereafter. Experiment 3: Shown in Figure 19 is specific (125I)iodo-PRL binding to ventral prostates at various times after a single so in- jection of TP. At 3 days after injection prostatic PRL binding sites were significantly increased (P <0.01) from control values, and remained high on days 4 and 5. By day 6, and at all subsequent times up to day 20 PRL binding was not significantly different from controls. Experiment 4: In a similar experiment OVX rats were given a single 136 20 pg sc injection of EB and PRL binding were measured in the liver and adrenals (Figure 20). Serum levels of PRL and LH also were determined (Figure 21). No significant differences in PRL binding were found in the liver until day 3, when PRL receptor activity nearly doubled from 10.7 9 0.56% in the controls to 19.28 9 0.66%. There was a continual increase in binding up to day 7, at which time PRL binding reached a peak of 29.33 9 0.28%; thereafter PRL receptors gradually decreased and by day 21 were not signif- icantly different from control values. In contrast, PRL binding sites in the adrenals were decreased after a single EB injection (Figure 20). On day 1 no difference in PRL binding was observed. However by day 3, a significant reduction from controls was obtained (12.41 9 0.96 to 8.63 9 0.10%). PRL binding reached its nadir on days 7 and 9 with binding activity at 6.8 9 0.65 and 6.73 9 0.30, respectively. By day 21 PRL binding was not significantly different from day 21 controls. Since LH secretion is inhibited by estrogen, LH levels were used to monitor estrogenic activity. Maximal LH in- hibition corresponded to the greatest changes in PRL binding in both the liver and adrenals. LH levels gradually retunred to control values from days 9-21 concomitant with a return of PRL binding to control levels. Although serum PRL increased on day 1 from 10.2 to 32.8 ng/ml, binding sites for PRL in the liver and adrenals were not altered as compared to controls. 137 22 — 201— ‘¢ADRENAL 5 18 - s 16 — 14 - 1o _ KIDNEY ¢ mi- oPRL , PERCENT SPECIFIC BINDING l I l 1 l l 10 20 25 30 40 60 DAYS OF AGE Figure 15. Effects of age on specific (1251)iodo-PRL binding to kidney and adrenal membranes from.ma1e rats. 138 100 Is 9" so Il/Il — - P b _ _ PRL 15- oglul 10- 240 1'5" 200 I./II 1.0 ss‘ 4 I l l l 1 10 20 25 30 40 00 DAYS OF AGE Figure 16. Serum GH, PRL and TSH in the male rat during development from day 10 to day 60. CPU BOUND (thousands) 139 d O O O 2 .4 a O P < > F C III III I I 8— 5... 4 _ 2 - v I I I 4 l 1 1 5 Min 15 Min 30 Min 1 Hr 2 Hr 4 Hr 8 Hr TIME I log cello) Figure 17. Time course effects of a intravenmjlgsinjection of 1 mg oPRL on 99 vitro binding of ( I)iodo-PRL to male kidney membranes. 125 SPECIFIC BINDING 0F l-oPRL (0PM) 140 " " Is" 30” III 2h 41: 8h 12): Id 2d 4d ad ad 30941.5 com TIME AFTER INJECTION Figure 18. Time coarse effects of a single intravenous ipggction of 1 mg oPRL on 99 vitro binding of ( I)iodo-PRL binding to male prostatic membranes. 19J E 17-« o. . 9 151 _; 4 a: a. 13— o | ‘1 g} 11< .. .1 u. C) 9‘ o 4 E 71 C: E d m 5: 2 J L'- 3‘ U . Lu o. 1‘ (I) Figure l 19. 141 1 ‘I 5" 125h1d 2d 3d 4d 5d 6d 8d 0d 12d 15d 20d CONT. TIME AFTER INJECTION Specific (1251)iodo-PRL binding to ventral prostates at various times after a single sc injection of testosterone propionate. mu CONY 142 (thousands) CPM BOUND OVX l1 3 5 7 9 12 16 CONTROL (thousands) CPI BOUND 3 5 7 9 12 16 21 21 ovx ovx ‘°"'°‘ oAvs If!!! A SIIGII 20.. II IIJI(IIOI ‘°""°l Figure 20. Changes in (1251)iodo-PRL binding to liver and adrenal membranes from OVX rats at various times after a single sc 20 ug EB injection. 143 12001 a C \ a u 900- c v 10 I " 000« I a C II 300- a a a a a on 1 a s 7 9 12 10 21 21 CONTROL l - Jeanna 8 30 — 3 § 30 — 3 v E 24 — .- 3 .3 18 —- g a “L I a II 6 - o l on 1 3 s 7 9 12 10 21 21 (0111101 I J on 0111 11m 1 1111611 20.. 11 1111101011 (0111101 Figure 21. Serum LH and PRL (ng/ml) in OVX rats at various times after a single sc 20 ug EB injection. 144 D. Discussion 25I)iodo—PRL These results indicate that specific (1 binding to kidney and adrenal membranes of male rats under— go rapid changes during development. From 10 to 60 days of age PRL receptors in the kidneys progressively increased, whereas PRL binding sites in the adrenals decreased. Changes in hormone concentrations observed during develOp- ment are in agreement with other reports in the literature (Negro-Vilar gt al., 1973; Dussault gt al., 1974). However, serum levels of GH, PRL or TSH could not be correlated to PRL receptor changes in the developing rat. It is possi- ble that the observed differences in PRL binding are modu- lated by other hormones not measured in this experiment, or are the result of genetic maturational changes. Other inves- 25I)iodo-PRL tigators reported changes in the binding of (1 to livers of male and female rabbits during develOpment, with binding increasing significantly between days 10 and 30, but not between days 30 and 60 (Posner 2E §£., 1974). These ontogenetic changes in binding differ from our data since significant differences were still observed in PRL binding sites between days 30 and 60 in both kidneys and adrenals. In prostatic tissue it was demonstrated that PRL receptors progressively decreased from days 20-270, although binding was not significantly reduced from the 20 day values until days 100 and 270 (Aragona gt al., 1975). Thus PRL re- ceptor content in the prostate appears to be more slowly altered during develOpment than for liver, kidney and adrenal tissues. Therefore data from the present studies and those 145 of others suggest that PRL binding sites change during devel- Opment, and that the rapidity, direction and magnitude of change differs for each target organ of PRL, probably re- flecting differential control of PRL receptors. After a single intravenous PRL injection was given to male rats i2 xtzg, the t2 gtttg binding of (1251)iodo-PRL to kidney and prostatic membrane preparations was maximally inhibited in organs removed 5 min post-injection. The data thus suggest that PRL binding sites were occupied by exo- genous PRL, and this i2 ytgg binding of PRL to receptors in the kidney and prostate occurred very rapidly. In contrast, (1251)iodo-PRL binding to kidney and prostatic membraned in- cubated EB ztttg at 37°C did not reach equilibrium for at least 3-4 h (Marshall gt gt., 1978; Arrauona gt gt., 1975), demonstrating an important difference between £2.2i22 and 12 ztttg binding of PRL to its own receptors. The $2 gtgg data is supported by an earlier report in which (1251)iodo- PRL was injected systemically, and found by autoradiographic methods to localize in the proximal tubules of the kidney within 2 min (Rataniemi gt gt., 1974). In the present studies, the reduction in prostatic PRL binding sites after PRL injection returned to contrOl values by l h, while in the kidney PRL binding returned to control levels at 8 h. The relatively rapid 12 ztzg dissociation time in kidney and prostatic tissues is at variance with the data of Birk- 12 5I )iodo-PRL inshaw and Falconer (1972), who reported that ( injected into rabbits localized in the mammary tissue, and remained there with a half-life of radioactivity of 52.4 h. 146 In this study PRL was iodinated using the chloromine-T method, which has since been shown to reduce the ability of PRL to bind to its receptor sites (Shiu gt gt., 1974). Consequently, damaged PRL may have given Spurious t2 vivo dissociation times, and possibly explain the different re- sults obtained in the 2 studies. Data obtained using OVX rats given a single injection of EB demonstrates similarities between the increase in PRL binding sites in the liver, and the decrease in binding ov- served in the adrenals of the same animals. In both organs significant changes in PRL binding occurred on day 3, with maximal changes observable on day 7. On day 21, PRL recep- tors in the liver and adrenals were not significantly dif— ferent from control values. Such similarities suggest that a common mechanism of action may be Operative in both in- creasing and decreasing PRL binding sites in these organs. Male rats given a single TP injection also significantly increased prostatic PRL binding sites at day 3, but binding remained elevated only on days 4 and 5 before returning to control levels. The fact that estrogen increased probably related to differences in the amounts given and the potency of the 2 steroids. However, testosterone did induce PRL binding sites on day 3 similar to the estrogenic effects on hepatic and adrenal PRL binding. Therefore these 2 steroids may share a common mechanism of action. The mechanism of action of estrogen has been reviewed (Gorski gt gt., 1976) and the data suggested that estrogen enters a target cell to bind to a cytOplasmic receptor which 147 then translocates to the nucleus to alter gene expression and subsequent protein synthesis. Therefore it is assumed that in the liver and adrenals of rats estrOgen also affects nuclear transcription and subsequent protein formation. However, the mechanism by which the resulting protein modu- late PRL receptors remains to be elucidated. Furthermore, cellular events occuring between the administration of es- trOgen and the appearance of PRL receptors 3 days later also require investigation. The relatively slow changes in PRL receptors after steroid treatment differs markedly from rapid increases elicited by thyroxine on renal PRL binding sites in which a doubling of binding was measured as quick- ly as 12 h after a single T injection (unpublished). It 4 appears likely that PRL receptors can be regulated by dif- ferent hormones through different mechanisms of action. The physiological significance of this would reflect both short and long term control of PRL receptors, which in turn should alter the sensitivity of PRL target organs to the actions of circulating PRL. Both short and long term regu- lation abound in physiological control systems. In the same experiment changes in serum LH levels were found to follow a time course similar to changes in PRL receptors in both liver and adrenals. Both of these effects are believed to be due to the direct action of estrogen. However, it is possible that PRL receptor changes were medi- ated in part by estrogen-stimulated PRL release. Different investigators have shown that PRL can increase its own PRL binding sites in the liver of both male and female rats 148 (Posner gt gt., 1975; Castlow gt gt., 1975). An auto—reg- ulatory action of PRL on its receptors in the kidney and adrenals is currently being investigated. In conclusion, these results demonstrate that PRL bind— ing sites progressively increased in the kidney during de- velOpment of the male rat, whereas in the adrenals PRL bind- ing sites decreased. The $2 3129 binding of PRL to receptor sites in the kidney and prostate occurred rapidly within 5 min, and subsequently dissociated by 8 h. In the last part of these studies it was shown that PRL binding sites in the kidneys and adrenals were modulated in opposite directions by estrogenic stimulation. The minimal time required to observe a significant change in PRL receptors, the duration of effect and the time required to return to control values were similar, suggesting a common mechanism of action. VIII. Prolactin Receptors in Mouse Liver: Species Dif— ferences in Response to Estrogenic Stimulation A. Objectives Specific prolactin (PRL) receptors have been demonstrated in the liver of many species, including rats and mice (Gelato gt gt., 1975; Posner gt gt., 1974, 1975). Ovariectomy (OVX) decreased and estrogen replacement in- creased PRL binding sites. The inductive effects of estro- gen on PRL binding in the liver was dose related to OVX rats, and anti—estrogens reduced PRL receptors in the liver of female rats (Kelly gt gt., 1976). One mechanism whereby estrogen induced PRL receptors is by stimulation of 149 pituitary PRL release, resulting in induction of hepatic PRL binding sites in the liver (Posner gt gt., 1975; Costlow gt gt., 1975). However, since very low doses of estrogen increased PRL binding in the liver without altering serum PRL levels (Kelly gt gt., 1976) and since the PRL-inhib- iting ergot drug CB-154 did not decrease the estrogen in- duced increase in hepatic PRL binding sites (Kelly gt gt., 1976), it is possible that estrogen may act directly on the liver to increase PRL receptors. All of the above studies were performed in rats. To determine whether the estrogen effect on PRL receptors was observable in other species, we examined the effects of estrogen on PRL receptors in the liver of mice. The re- sults indicate that estrogen inhibits induction of PRL re- ceptors in the liver of female mice, in contrast to its stimulation of PRL receptors in the liver of male and female rats. B. Materials and Methods Adult male and female Swiss-Webster mice were ob- tained from Spartan Research Animals, Haslett, MI. Mice were housed in a temperature controlled (25° :1° C) and artifically illuminated room (lights on from 0500 to 1900 h daily), and received food and water gg_libitum. Exp. 1: Female mice were OVX on day l and were injected s.c. , daily with 2 pg estradiol benzoate (EB) in 50 pl corn oil on days 8-14. On the 15th day all OVX were killed together with a group of intact females which were similarly injected daily on days 8-14 with vehicle alone. 150 Exp. 2: Female mice, OVX 14 days prior to estrOgen treat- ment, were given daily s.c. injections of either l, 10, 20 or 50‘ug EB in 50 ul corn oil. Mice were then killed after 12 days of treatment, together with groups of intact and OVX controls which were injected with vehicle alone. Additional treatment groups, given daily injections of 20 ug EB were killed after 6 or 9 days of treatment. Exp. 3: Male mice were given a single 2 ug EB s.c. injec- tion in 50 1 corn oil and killed 7 days later. Controls were injected with vehicle alone. At the end of each experiment the mice were anesthe— tized with ether, decapitated, and the blood obtained from the cervical wound was allowed to clot at 4°C. The serum was deparated by centrifugation and stored at -20°C for later serum PRL measurements. Livers were removed and a microsomal membrane fraction was obtained by differential centrifugation as described previously (Gelato gt gt., 1975). PRL was iodinated by a lactoperoxidase method (Gelato gt gt., 1975) and the binding of (1251)iodo-PRL to liver membranes was determined. Incubations with membrane 25I)iodo-PRL were performed at 4°C for 60 h, protein and (l in the presence of excess ( 1 pg) unlabeled PRL and in its absence. Livers from female mice were assayed for PRL binding, using 300119 membrane protein per tube, whereas for male livers 1000119 per tube was used. Specific bind- ing refers to the difference in radioactivity bound to membranes after incubations with and without unlabeled PRL, and for ease of representation is expressed as % of total 151 counts added. PRL binding to liver membranes from mice has been shown to be both time and temperature dependent, and specific for lactogenic hormones (Posner gt gt., 1976). Mouse PRL was measured by a double antibody radioimmuno- assay using the materials and methods of Sinha gt gt. (1972). The biological potency of the mouse PRL standard was 25.0 IU/mg. Data in Exp. 1 and 2 were treated by analysis of variance for unequal sample size, followed by Student-Neuman-Kuels test for comparison of means among groups. Student's "t" test was used to determine signif- icance in Exp. 3. P 0‘05 was considered to be significant. C. Results Figure 22 shows that OVX significantly increased (P <0.01) (1251)iodo-PRL binding to mouse liver membranes, and that this enhanced binding could be decreased to in- tact control values by estrogen replacement. When this ex- periment was repeated (Figure 23) with various doses of EB and longer treatment times, similar results were obtained. OVX increased (P< 0.05) specific (1251)iodo-PRL binding from 14.48 i 0.85% in the intact controls to 19.93 i 0.60%. Replacement by injecting l and 10 9 EB for 12 days reduced PRL binding to 11.84 i 0.53% and 10.90 i 0.81%, respectively, which were not significantly different from intact control values, whereas 20 and 50119 EB significantly reduced bind- ing to below intact levels. Serum PRL was reduced from 12.0 i 1.5 ng/ml (intact controls) to 8.09 i 2.0 ng/ml in the OVX rats. All estrogen treated groups had serum PRL values significantly higher than in intact controls. 152 3; 1 MOUSE g 20— o. o .1. 5 16— IL C) o 12— E C) E In (3-— 2 I1- 9. 4— -s—. 8 10 10 (01111101 01111 0:1! 209 El Figure 22. Effects of OVX and OVX with EB replacement on specific (1251)iodo-PRL binding to liver mem- brane preparations from female rats. 153 0 g 22 L 1101- _ _ .1 n 20 - MOUSE 2 18 — - h a - LIVER .. 1s — - a ;, " 1- 14“ - l 5 - - i - It 3 12- = = I I *"' I l n t 10— I I - - i - I _; - - I I I I - I I: 8 " - - - - - - - - .'. SJ“ I - = - - - - - a: f [E a E] 111110 ovx '7”qu 11 ‘ “1" “1" ”1" '9 1 10 501 11 L4 l—l l__l '— _P 9 I2 I2 DAYS TREATMEN‘I’ 5 110 SERUM PRL 90— 70- NG/ML 9 5 [1 5 INIACI OVX P - on on on 4' 20 II * 0 4- OVX " I I0 50" El L—l I—l H L 6 9 I2 12 DAYS IMF Figure 23. Serum PRL levels and (1251)iodo-PRL binding to Liver membranes from intact, OVX and OVX mice given daily injections of different doses of EB. 154 ; MOUSE E 40 _ LIVER O. O 1 "3 30 — IL 0 (1 EE 1: :2(>*- 2! ii S! u _ ('1 10 III 0. U) MAI! CONTROI Figure 24. Specific binding of (1251)iodo-PRL to liver membranes from male mice 7 days after a single injection of 2 ug EB. 155 Figure 24 demonstrates the effects of a single in- jection of 2 pg EB on specific PRL binding sites in liver membranes obtained from male mice. PRL binding increased (P <0.01) from 22.61 i 1.16 to 33.72 i 1.29% at 7 days post-injection. Since PRL binding sites on male liver membranes were measured using 1000 pg membrane protein rather than 300 pg membrane protein (as used in quanti- tating PRL receptors in the liver of females), specific binding is higher in the livers of females than in the livers of males when compared on a mg of protein basis. This is in agreement with the data of Posner gt gt., 1976. D. Discussion The presence of specific PRL receptors in liver membranes of female mice agrees with the findings of other investigators (Posner gt gt., 1976; Knazek gt gt., 1977). However, our results indicate that OVX results in an in- crease of hepatic PRL receptors in female mice, whereas estrogen treatment over a large dose range reduced PRL binding to intact or below intact values. These data in female mice represent a striking contrast to the effects of OVX and estrogen replacement on PRL receptors in liver of female and male rats. In female rats the effect of estrogen on increasing hepatic PRL receptors was demonstrated to be mediated through stimulation of pituitary PRL release (Fortz gt gt., 1977; Winkler gt gt., 1971). However, other data suggest a direct effect of estrogen on the liver to modulate PRL bind- ing sites (Kelly gt gt., 1976). In the present study, all 156 doses of estrogen significantly increased serum PRL levels in female mice. The increase in PRL, however, is not be— lieved to have altered hepatic PRL receptors since other in- vestigators have reported that neither the high levels of endogenous PRL during pregnancy, nor exogenous oPRL in— jections to female mice, influenced PRL binding sites in the liver (Posner gt gt., 1976; Knazek gt gt., 1977). Therefore, a direct effect on the liver appears likely, although an indirect effect of estrogen cannot be excluded. In male mice a single injection of 211g EB was able to significantly increase PRL binding sites in the liver. thxaestradiol valerate has been reported to stimulate PRL binding sites in the liver of male rats (Posner gt gt., 1975), it is apparent that both male rats and male mice re- spond similarly to the stimulatory action of estrogen on hepatic PRL receptors. This is in contrast to the Opposite effects of estrogen on hepatic PRL binding sites of female rats and mice. Although the physiological significance of these re- sults is not known at this time, PRL has been shown to have numerous effects on liver function of various Species. Thus PRL was reported to regulate free fatty acid synthesis in dog and rat (Macleod gt gt., 1965) livers, stimulate hepatic RNA synthesis in dwarf mice (Chen gt gt., 1972), modulate ornithine decarboxylase activity in the liver of rats (Richards, 1975), and increase somatomedin release from rat livers (Francis gt gt., 1975). However, in order for PRL to exert an effect on a target cell, it must first bind to a 157 stereospecific plasma membrane receptor to induce intra- cellular changes. Consequently, receptor modulation could provide a mechanism for altering the sensitivity of target organs to circulating PRL. Therefore, determining which hormones can alter PRL receptors and the direction of these changes are important for clarifying the physiological actions of PRL on liver function. The present data clearly demonstrate an important spe- cies difference between female rats and mice in estrogenic control of hepatic PRL receptors, and may have several im— plications. Thus, the use of the rat as a model for inves- tigating factors modulating PRL receptors in the liver can- not be considered valid for other species. Moreover, the functions of PRL on liver function may be different be- tween males and females of even the same species, since con- trol of PRL receptors in liver of male and female mice are different. Our data indicate that estrogen inhibits PRL binding sites in the female, whereas in the male, binding is stimulated. Thus the response of hepatic PRL receptors to estrogen is both species and sex dependent. The mechan- isms of action by which these effects are mediated remain to be clarified. The differential findings in these 2 species need to be considered when designing and interpreting stud- ies on the effects of PRL on liver function. 158 GENERAL DISCUSSION There is now abundant evidence that peptide hormonesaud transmitter molecules such as catecholamines and acetylcho- line exert their primary actions on target cells by binding to specific high-affinity receptor sites in the plasma mem- brane. The existence of such receptors, previously implied in theories of drug and hormone action, has recently been demonstrated in numberous target cells for peptide hormones and transmitters by direct binding studies with radioactive ligands. Utilizing the relatively new techniques for quan- titating binding, the data presented in this thesis demon- strate specific high-affinity receptor sites for prolactin in the kidney and adrenal of male and female rats. Inhi- bition curves and tests of cross reactivity with LH, FSH, TSH and GH showed that binding of PRL to receptors in the kidneys and adrenals was specific. Moreover, binding of PRL to specific binding sites was shown to be time- and temperature dependent, a characteristic of membrane bound receptors. The binding of (1251)iodo-PRL to kidney prepar- ations increased with increasing amounts of membrane protein. Lastly, after a single intravenous PRL injection was given 25I)iodo-PRL to male rats tg vivo, the t2 vitro binding of (l to kidney membrane preparations was maximally inhibited when kidneys were removed 5 minutes post-injection. These data 159 suggest that PRL binding sites were occupied by exogenous PRL, and the 12 2129 binding of PRL to receptors in the kidney occurred very rapidly. Dissociation of bound PRL was also observed 12 3129 and found to return to control values within 8 hours. All of these studies strongly sug- gest, but do not prove, that the kidneys and adrenal glands are target organs for the action of prolactin. Although PRL receptors were identified in the kidneys and adrenal, studies demonstrating that binding of PRL to its receptors is followed by activation leading to subse- quent biochemical events, have not been performed for other hormones. Occupancy of the angio-tensin II recep- tors of the adrenal glomerulosa cell is correlated with increased steroidogenesis and aldosterone production over the whole range of hormone binding (Douglas gt gt., 1976). In another tissue, the interstitial cells of the testis, gonadotrOpin binding to receptor sites is accom- panied by a progressive increase in production and release of cAMP. However, gonadotropin binding can increase CAMP in excess of what is required for complete activation of steroidogenesis and testosterone production. Thus, only 1% occupancy of the total receptor pOpulation is necessary for a full steroidogenic response (Mendelson gt gt., 1975; Catt gt gt., 1973). The problems encountered in demonstrating biochemical events that should follow the binding of PRL to cellular receptor sites are twofold. First, the mechanism of action of prolactin is poorly understood in comparison to the 160 sequence of events that are now known to follow the bind- ing of epinephrine to its cellular binding sites. In this classical example the actions of epinephrine are mediated by the second messenger cyclic AMP via the adenyl cyclase system, CAMP in turn activates protein kinases which cause a cascading of events culminating in the release of glucose from hepatocytes (Sutherland, gt gt., 1965). Prolactin, however, is thought not to work by the stimulation of cAMP, but possibly through stimulating Na/K ATPase, inducing pro- tein kinase or by stimulating prostaglandin synthesis. Another difficulty encountered in relating PRL re- ceptor occupancy to a physiological response in kidneys and adrenals is the lack of information as to the role of prolactin in these target organs and the nature of the specific target organ responses. In mammals, PRL has been reported to influence salt and water regulation by an action on the renal tubules. Thus, PRL injections reduce salt and water excretion while reducing PRL through ergot drugs in- creases excretion of salt and water (see Literature Review, V, Other Functions Of Prolactin). However, the explanations as to how PRL accomplishes these functions are only specu— lative, and involve changes in Na/K ATPase or the altera- tion of proximal tubule "tight junctions". With so little known about the specifics of how prolactin alters renal function, it is very difficult to link receptor binding to biological function in the kidney. In the adrenal glands, linking the binding of prolactin to specific cellular functions should be easier, since PRL has been demonstrated 161 to alter the adrenal enzyme 5a -reductase as well as corti- costerone synthesis and release (Witorsch and Kitay, 1972). Studies associating PRL binding with specific cellular changes need to be performed to get a better understanding of the function of prolactin in the kidneys and adrenals. Experiments were not performed linking binding with subsequent biochemical events, however, evidence supporting a physiological role of PRL and PRL receptors in the kidneys and adrenals were given by demonstrating changes in serum PRL and PRL receptors with dehydration, salt loading and during early ontogenetical develOpment. Again, while such experiments do not prove that PRL receptors have a physio- logical role, they do strongly suggest a function which may be modified through changes in PRL receptor activity. One approach to the problem of ascribing a function to PRL receptors in the kidneys and adrenals is by investi- gating under which conditions PRL binding sites change, and how these changes are mediated. The data presented in this thesis indicated that PRL receptors in the kidneys and adre- nal can be influenced by many different hormones, and under some conditions are independently regulated. Thus, estrogen, testosterone and gluooxniicoids decreased PRL receptors in the kidneys and adrenals, while thyroxine or T3 increased PRL binding sites in the kidneys of rat, but not in the ad- renal glands of rat. Progesterone, aldosterone and renin treatment were without effect. The implications of hormon- al control are discussed in the Conclusions of the Experi— mental section. Nevertheless, it can be generally 162 concluded from the data presented that PRL binding sites in the kidneys and adrenals are regulated through the inter- action of many hormones of the endocrine system. Any field of scientific knowledge goes through three general stages: collection, classification, and explanation. Having gathered together as many examples as possible of the phenomenon under consideration, the scientist arranges them in some sort of rational system, taking account of both their similarities and their differences, and finally, de- vises a rationale that explains, or at least elucidate, the arrangement. Data presented in this thesis represents only the first stage, collection of data relating to PRL receptor changes in the kidneys and adrenals of rats, and the expla- nation of these results at this stage are highly speculative at most. However, it is noteworthy that in every species studied including man, serum PRL dramatically increases con- comitantly with ACTH in stressful situations, but the reason for this stress induced PRL release or the functions of PRL in stress is not understood. Moreover, PRL can increase cor- ticosterone secretion, while glucorticoids have been shown to alter PRL receptors in the adrenal glands. Therefore, it can be speculated that PRL has an adaptive function during stress by interacting with ACTH to stimulate glucocorticoid secretion from the adrenal gland. Protracted periods of high circulating glucorticoids may then feed back on the adrenals to modify PRL receptors, and the subsequent cellular events associated with increased receptor occupancy. Prolactin, in stressful situations, may also act on the proximal tubules 163 of the kidney to conserve salt and water, and thus augment the effects of increased ADH and aldosterone observed dur- ing stress. Speculating further, the action of PRL on kid- ney and adrenal target organs may be influenoaiby the pre- vailing general metabolic state, as regulated by the thy— roid hormones. In addition, it can be visualized that the reproductive state would further alter prolactin's response on its target organs through steroid hormones. The function of prolactin in the non-stressed state remain to be elucidated. The data presented in this thesis, while Opening up a new and exciting area of prolactin physiology, leave many questions unanswered. Dehydration, salt loading and onto- genetical development altered PRL receptors in the kidneys and adrenals of rats; but how were these changes mediated? PRL receptor activity in the kidneys and adrenals were found to be different between male and female rats as well as be! tween species. What are the physiological reasons for these differences? Are there any pathologies of PRL receptors as- sociated with physiological abnormalities such as reported for insulin, where insulin resistance in Obesity was, in some cases, found to be due to a reduction in the number of insulin receptors? 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The permeability of the hypo- physis and hypothalamus to vital dyes with a study of the hypo- physeal vascular system. Am. J. Anat. 58:421-427. Witorsch, R.J. and J.I. Kitay. 1972. Pituitary hormones affecting adrenal 5 u-reductase activity: ACTH, growth hormone and pro- lactin. Endocrinol. 91:764-769. Wuttke, W. and J. Meites. 1971. Luteolytic role of prolactin during the estrous cycle of the rat. Proc. Soc. Exptl. Biol. Med. 137: 988-995. Wuttke, W., E. Cassell and J. Meites. 1971b. Effects of ergocornine on serum prolactin and LH, and on hypothalamic content of PIF and LRF. Endocrinol. 88:737-741. Zambrano, D., W.G. Clarke, A. Hajek, M. Sage and H.A. Bern. 1974. Influence of medium concentration on prolactin and growth hormone cells during short term incubation of pituitary glands from Tilapia mossambica. Acta ZooL 55:205-216. 196 Zarrow, M.X. and J.H. Clark. 1969. Gonadotropin regulation of ovarian cholesterol levels in the rat. Endocrinol. 84:340-346. Zmigrod, A., H.R. Lindner and S.A. Lamprecht. Reductive path- ways of progesterone metabolism in the rat ovary. Acta Endocrinol. 69:141-152. 197 CURRICULUM VITAE NAME: STEPHEN MARSHALL ADDRESS: Office: Department of Medicine, B151 University of Colorado Medical Center 4200 East Ninth Avenue Denver, Colorado 80262 Phone: (303) 394-8443 Home: Phone: PERSONAL: Date of Birth: May 10, 1947 Place of Birth: Brooklyn, New York Marital Status: Married, wife Henri Kruse Military Status: Veteran, U.S. Air Force 1965-69 Honorable Discharge EDUCATION: Degree Year Institution Major Field of Study B.S. 1969-1973 Michigan Statelkuv; Psychology Ph.D. 1973-1978 Michigan State Univ. NeuroendOcrinology HONORS: Date a) Nominated and accepted to Honors College, MSU 1971 b) Invited and accepted member to National Honor Society of Phi Kappa Phi, MSU Chapter 1971 c) Graduated with a B.S. degree from MSU 1973 (Very High Honors) ACADEMIC POSITIONS AND ACCOMPLISHMENTS: a) Technician in Neuroendocrinology lab, MSU 1972-1973 b) Research Assistant in Physiology, MSU 1973-1978 c) Supervised two technicians in maintaining 1973-1978 and supplying reagents for five radio- immunoassays for general laboratory use . d) Established prolactin radioreceptor assay 1973-1978 and conducted research on the control of prolactin receptors in the kidneys and adrenals of rats 198 Stephen Marshall Curriculum Vitae e) Visiting scientist, National Institute Summer, 1976 of Health, Dept. of Biochemistry, Budapest, Hungary. Successfully established the first RIAs in Hungary for rat LH, FSH, prolactin and human GH. f) Attended the 6th Training Course on March, 1977 Radioassay Techniques, sponsored by the Endocrine Society, Bethesda, MD RESEARCH PUBLICATIONS: (Abstracts not included) 1. Marshall, 8., M. Gelato and J. Meites: Serum prolactin binding activity in adrenals and kidneys of male rats after dehydration, salt loading and unilateral nephrectomy. Proc Soc. Exp. Biol. Med. 149: 185, 1975. 2. Gelato, M., S. Marshall, M. Boudreau, J. Bruni, G.A. Campbell and J. Meites: Effects of thyroid and ovaries on prolactin binding activity in rat liver. Endocrinology 96:1292, 1975. 3. Bruni, J.F., S. Marshall, J.A. Dibbet and J. Meites: Effects of hyper- and hypothyroidism on serum LH and FSH levels in intact and gon- adectomized male and female rats. Endocrinology 97:558, 1975. 4. Kledzik, G., S. Marshall, M. Gelato, G.A. Campbell and J. Meites. Prolactin binding activity in the crOp sacs of juvenile, mature, parent and prolactin injected pigeons. Endocrine Research Communications 2: 345, 1975. 5. Campbell, G.A., M. Kurcz, S. Marshall and J. Meites: Anterior pituitary function and response to synthetic LRH-TRH during acute and chronic starvation. Symposium of the International Society of Psychoneuroendo- crinolOgy, Visegrad, 1975. 6. DeSombre, E.R., G. Kledzik, S. Marshall and J. Meites: Estrogen and prolactin receptor concentra- tions in rat mammary tumors and response to endocrine ablation. Cancer Research 30:354, 1976. 7. Huang, H.H., S. Marshall and J. Meites. Induction of estrogen cycles in old non-cylic rats by progesterone, ACTH, ether stress of L-dopa. Neuroendocrinology 20: 21-34, 1976. 8. Bruni, J.F., S. Marshall, H.H. Huang, H.J. Chen and J. Meites: Serum prolactin, LH and FSH in old and young male rats. IRCS Medical Services 4:265, 1976. 199 Stephen Marshall Curriculum Vitae 10. 11. 12. 13. 14. 15. 16. 17. Kledzik, G.S., C.J. Bradley, S. Marshall, G.A. Campbell and J. Meites: Effects of high doses of estrogen on prolactin binding activity and growth of carcinogen induced mammary can- cers in rats. Cancer Research 36: 3265- 3268, 1976. Gelato, M.J. Dibett, S. Marshall, J. Meites and W. Wuttke: Prolactin-adrenal interactions in the immature female rat. Ann. Biol. Anim. Bioch. Biophys. 16: 395-397, 1976. Kledzik, G.S., S. Marshall, G.A. Campbell, M. Gelato and J. Meites. Effects of castration, testosterone, estradiol and prolactin on specific prolactin binding activity in ventral prostate on female rats. Endo- crinology 98: 373, 1976. Huang, H.H., 8. Marshall and J. Meites. Capacity of old versus young female rats to secrete LH, FSH and prolactin. Biol. of Reprod. 14: 538-543, 1976. Marshall, 8., G.S. Kledzik, M. Gelato, G.A. Campbell and J. Meites. Effects of estrogen and testosterone on specific prolactin bind- ing in the kidneys and adrenals of rats. Steroids 27:187, 1976. Lu, K.H., H.T. Chen, H.H. Huang, L. Grandison, S. Marshall and J. Meites: Relation between prolactin and gonadotrOphin secretion in post-partum lactating rats. J. Endocrin- ology 68:241, 1976. Lu, K.H., L. Grandison, H.H. Huang, S. Marshall and J. Meites: Relation of gonadotropin secre- tion by pituitary grafts to spermatogen- esis in hypophysectomized male rats. EndocrinolOgy 100:380, 1977. Campbell, G.A., M. Kurcz, S. Marshall and J. Meites: Effects of starvation in rats on serum levels of FSH, LH, TSH, GH and PRL: ReSponse to LRH-TRH. Endocrinology 106: 508, 1977. Bruni, J.F., D. Van Vugt, S. Marshall and J. Meites: Effects of naloxone, morphine and meth- ionine enkephalin on serum prolactin, luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone and growth hormone. Life Sciences 21:461-466, 1977. 200 Stephen Marshall Curriculum Vitae 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. Bruni, J.F., H.H. Huang, S. Marshall and J. Meites. Effects of single and multiple injections of synthetic GnRH on serum LH, FSH and testosterone in young and old male rats. Bio. of Reprod. 17:309-312, 1977. Marshall, 8., H.H. Huang, G.S. Kledzik, G.A. Camp bell and J. Meites. Glucocorticoid regulation of prolactin receptors in the adrenal and kidney of male rats. Endocrinology 102:869-875, 1978. Marshall, 8., J.F. Bruni and J. Meites. Prolactin receptors in mouse liver: species differ- ences in the response to estrogen stimu- lation. Proc. Soc. Exp. Biol. Med. (in pressL Marshall, 8., J.F. Bruni and J. Meites. Effects of hypophysectomy, thyroidectomy and thyrox- ine on specific prolactin receptors in the kidneys and adrenals, Endocrinology (in press). Marshall, 5., G.S. Kledzik, J.F. Bruni and J. Meites. Prolactin receptor changes during develOp- ment and after a single injection of estro- gen or prolactin (Submitted to Endocrinology). Marshall, 8., J.F. Bruni and J. Meites. Inhibition of thyroid hormone induced increase in renal PRL receptors by actinomycin-D and cyclo- heximide (Submitted to Proc. Soc. Exp. Biol. Med.). Marshall, 5., J.F. Bruni and J. Meites. Changes in PRL receptors and serum PRL after salt loading in the rat: Lack of effect in the mouse. (In preparation). Marshall, 8., J.F. Bruni and J. Meites. Direct estro— gen inhibition of prolactin receptors in the kidneys of male rats. (In preparation). Lu, K.H., H.T. Chen, J.F. Bruni, S. Marshall and J. Meites. Relation of thyroid to LRF induced release of LH in the rat. (Submitted to Neuroendocrinology). Kledzik, G.S., S. Marshall, H.H. Huang, J.F. Bruni and J. Meites. Serum prolactin, testosterone and prolactin receptor activity in ventral prostate of old and young male rats (Sub- mitted to Neuroendocrinology). Huang, H.H., S. Marshall and J. Meites. Induction of ovulation by GnRH and the responsiveness of the pituitary in the old constant estrous rat (In preparation). W- . n _..<. Jen's-m . " 7‘ 201 Stephen Marshall Curriculum Vitae 29. Huang, H.H., S. Marshall and J. Meites. Positive feedback of ovarian steroids on LH release in old and young female rats (In preparation). HICHIGRN STRTE UN LI! mm “W IV. LIBRARIES "ll?WWWIIWIWI 1 293 063261 5