is -. , I/L/////,//L/£{[{//ll’////////////////I//l//////////l//I 1? “7137*: 513:; 3r 10529 8982 A€#_'v.,wasi a. 5:33:33: agate ‘ . fimivcraity This is to certify that the thesis entitled INVOLVEMENT OF ENDOGENOUS OPIOID PEPTIDES IN REGULATION OF PROLACTIN AND LUTEINIZING HORMONE SECRETION presented by Dean Alan Van Vugt has been accepted towards fulfillment of the requirements for M__degree in Lhysiology Date 0,7//rél/LI/,?/ 0-7 639 MSU LIBRARIES RETURNING MATERIALS: PIace in book drOp to I remove this checkout from your record. FINES wil] be charged if book is returned after the date stamped below. INVOLVEMENT 0F ENDOGENOUS OPIOID PEPTIDES IN REGULATION OF PROLACTIN AND LUTEINIZING HORMONE SECRETION By Dean Alan Van Vugt A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 198l ABSTRACT IIQVTDLVEMENT OF ENDOGENOUS OPIOID PEPTIDES IN REGULATION OF PROLACTIN AND LUTEINIZING HORMONE SECRETION by Dean Alan Van Vugt 1. Serum prolactin (PRL) concentrations in adult Inale rats were measured after acute administration of Inorphine (MOR), beta-endorphin (B-END), methionine- enkephalin (MET-ENK), dynorphin, or naloxone (NAL). Intraperitoneal injection of MOR significantly in- creased serum PRL concentrations. Similarly, serum PRL concentrations were significantly increased by intraventricular injection of B-END, MET-ENK, or dynorphin. In all cases, the stimulatory action of these opiates was blocked by concurrent injection of NAL, a specific opiate antagonist. Moreover, when NAL was administered alone, serum PRL concentrations were significantly reduced. These results suggest that endogenous opioid peptides (EOP) are involved in determining the basal secretion rate of PRL in adult male rats. Dean Alan Van Vugt 2. The effect of the opiate antagonist, NAL, on stress-induced PRL release was determined. Serum PRL levels were increased by ether, heat, or immobilization stzreess. Pretreatment of stressed rats with NAL signif- iczaxttly inhibited the release of PRL in response to all 3 strresses tested. The ability of NAL to block stress- induced PRL release suggests that EOP are involved in tire process by which stress increases PRL secretion. 3. Treatment of male rats with L-DOPA, the pre- c1irsor of dopamine (DA), piribedil, a DA agonist, or zamineptine, a DA reuptake inhibitor, all decreased serum PRL concentrations and blocked the stimulatory action of MOR on PRL release. In a second experiment, the effect of intraventricular injection of B-END on alpha-methyl-para-tyrosine (a—mpt) depletion of median eminence (ME) DA was determined. B-END completely blocked a-mpt depletion of ME DA and significantly in- creased serum PRL concentration. It is concluded that opiates stimulate PRL release by decreasing tuberoin- fundibular DA activity. 4. Injection of NAL increased serum luteinizing hormone (LH) concentration approximately 4-fold. Pre- treatment with a-mpt, diethyldithiocarbamate, or phen- oxybenzamine, all anti—noradrenergic drugs, inhibited Dean Alan Van Vugt the stimulatory action of NAL. These results suggest that NAL-induced LH release is produced by activation of hypothalamic noradrenergic neurons. 5. Male rats were castrated and injected once daily with testosterone propionate or twice daily with MOR for 12 days. Blood samples were collected at 2 day intervals, and the hypothalamus was removed after decap- itation on the twelfth day. The post-castration rise in serum LH was inhibited by either chronic testosterone or MOR administration. The reduction in hypothalamic LH- releasing hormone (LHRH) concentration in nontreated castrated rats was completely blocked by either testos- terone or MOR. It is concluded that both MOR and testosterone inhibited the post-castration rise of LH by inhibiting the release of LHRH from the hypothalamus. 6. Administration of estradiol benzoate (EB) alone or together with progesterone reduced serum LH levels in long-term ovariectomized rats. This inhibitory action of ovarian steroids was reversed by a single in- jection of NAL. Similarly, NAL blocked the negative feed- back action of testosterone propionate on LH release in acutely castrated male rats. These results indicate that EOP may at least partially mediate gonadal steroid in- hibition of LH release in male and female rats. DEDICATION This thesis is dedicated to my wife, Jann. Her continual support and understanding were of invaluable assistance to the completion of this project. I also am very grateful to my parents, Ernest and Phyllis Van Vugt. Their words of encouragement and expressions of interest in my work were more important to me than perhaps they realized. ii ACKNOWLEDGEMENTS I wish to thank all members of the Neuroendocrine Research Laboratory. The constant exchange of new ideas between fellow members contributed much to my education at Michigan State University. Additionally, I would like to thank Charles F. Aylsworth, John F. Bruni, Frederick Leung, and Paul N. Sylvester for their help in performing the actual experiments of this thesis, and Eugenia M. Dayton for her secretarial help in preparing this dissertation. I would like to express my deepest appreciation to Professor Joseph Meites for his unselfish investment of time and energy in the development of my career during the past five years. His exemplary leadership is rivaled only by his vast knowledge of endocrinology, both of which contributed greatly to my education. TABLE OF CONTENTS LIST OF TABLESOOOOOOOOCCOOOOOOOOOCOIOOOOOOOO LIST OF FIGURESOOOOOOOOOOOOOOOOOOOOOOOOOOOOO INTRODUCTIONOOOOOOOO0.0... ...... .0. ....... O ....... LITERATURE REVIEW......... ..... ..... .............. I. Hypothalamic Control of Anterior II. III. IV. VI. Pituitary Hormone Secretion..... . Classical Observations............ . Hypothalamic Anatomy.............. Hypophysiotropic Hormones......... . Hypothalamo-Hypophyseal Portal Vessels and Neurosecretion........ Ufim> Localization of Biogenic Amines and Opiates A. DopamineOOOOOOIOOOOOOOOOOOOOOIOOOO BO Norepinephrine..OOOOIOOOOOOOOOOOOO C. serotoninOOOOOOOO0.00...... 000000 O D. OpiateSOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO Biogenic Amine Metabolism........ ........... A. Synthesis and Release............. B. Monoamine Inactivation........ ....... . Opioid Peptide Metabolism............. A. syntheSiSOO000000000000000.000.000.000 BO Releaseooobooooo 000000000000000 000.00. C. Inactivationooooooooooooooooooooooooooo Hypothalamic Control of Prolactin Secretion. A. Hypothalamic Inhibition of Prolactin secretion.OOOOOOOOOOOOOOOOO00.0.0.0... B. Hypothalamic Stimulation of Prolactin secretion.0.0.0.0....OOOOOOOOOOOIOOOOO Control of Luteinizing Hormone Secretion.. A. Inhibition of Luteinizing Hormone Secretion by Gonadal Steroids......... B. Stimulation of Luteinizing Hormone Secretion by Ovarian Steroids......... C. Monoaminergic Effects on Luteinizing HormoneOOOOOOOOOOOO0.0.... ...... 0.0... D. Opiate Effects on Luteinizing Hormone secretionOOOOOOOOOOOOOO ...... 0.... Page vii viii 10 I3 16 19 19 22 23 29 29 32 34 37 39 43 43 46 53 53 55 59 70 MATERIALS AND METHODS....,........ ..... ...... I. Animals, Treatment, and Blood Collection ..... II. Radioimmunoassays of Hormones............... III. Assay of Dopamine, Norepinephrine, and Luteinizing Hormone Releasing Hormone in A. B. C. Brain.0.0.0....OOOOOOOOOOOOOOOOOOOOOOOO Isolation and Preparation of Brain Page Tissue..0.00000000000000000000000 ........ Radioenzymatic Assay of Dopamine and Norepinephrine............... ....... Radioimmunoassay of Luteinizing Hormone Releasing Hormone..... ....... .. EXPERIMENTAL..................................... I. II. III. Initial Studies on Opiate Stimulation and Naloxone Inhibition of Prolactin Release.. A. B. C. D Objectives............................ Materials and Methods................. Results............................... Discussion............................ Role of Endogenous Opioid Peptides in Stress-Induced Prolactin Release.... ...... Objectives...OOOOOOOOOOOOOOOOOOOOOOO ..... A. com Effect of Opiates on Hypothalamic Dopamine ~ Activity; Evidence That Opiates Stimulate Materials and Methods................. Resu1ts...0..COO...OOOOOOOOOOOOOOOOOOOO Discuss1on.0.0000000000000000000000000 Prolactin Release Via a Dopaminergic MeChanismooooooooooo00000000000900.000 ...... A. B. C. D ObjeCtiveSOOOOOOOOOOOOOOOOOOOOOOOOOOOO Materials and Methods.................. Resu1ts...I0.000IOOOOOOOOOOOOIOO....0..0 DiSCUSSi-onoooooooooooooooooooooooooooo 73 73 75 76 76 77 78 80 80 80 82 85 IV. Evidence That Stimulation of Luteinizing Hormone Release by Naloxone is Mediated by Norepinephrine............................ . Objectives............................. Materials and Methods.................... ReSUItSOOOOOOOOOOOOOOOOO ...... O ......... O O DisCUSSionOOOOOOOOOOO00.0.00...00......O. coma: V. Morphine Exerts a Testosterone-Like Effect on Luteinizing Hormone Release; Involvement of Luteinizing Hormone Releasing Hormone........ A. Objectives......................... ..... . B. Materials and Methods........... ..... .... C. Results...................... ....... ..... D. Discussion.................... ..... ...... VI. Evidence That Brain Opiates Mediate Gonadal Steroid Inhibition of Luteinizing Hormone Releasel.0..0......OOOOOOOOOOOOOOOOOOOOO ..... A. ObjectiveSOO0.0.0.0000COOOOOOOOOOOOOOOOO. B. Materials and Methods............. ..... .. C. ReSUIts.OOOOOOOOOOOOOOOOOOOOOOOOIOOOOOOOI D. DisCUSSion. OOOOOOOOOOOO 000...... ..... 0... GENERAL DISCUSSION .................... ...... ...... BIBLIOGRAPHY O O O O O O O O ....... O O O ....... O O O O O O O ...... APPENDICES...... ............................. ..... CURRICULUM VITAE vi Page 109 109 110 114 121 124 124 125 128 133 136 136 137 139 143 147 157 197 204 LIST OF TABLES TABLE Page 1. Dose-Response Effects of Morphine, Methionine- Enkephalin, and Naloxone on Serum Prolactin.... 83 2. Effect of DAMME on Serum Prolactin concentration (ng/mI)IOOOIOOOOOOOOOOOO.00....O. 84 3. Acute Effects of Dynorphin and B-Endorphin on PrOIactin ReleaseOOOOOOOIOOO0.00.00.00.00... 86 4. Effects of Naloxone on Basal Serum Prolactin Concentrations...OOOOOOIOOOOOOOOOOO0.0.0.000... 9] 5. Effects of Naloxone on Stress-Induced Prolactin ReleaseOI00......OOOOOOOOOOOOOOOOOI.0.0.0.0.... 92 6. Effects of Dopaminergic Drugs on Morphine Induced Increase in Serum Prolactin......................100 7. Effects of PCPA on Morphine and Methionine- Enkephalin Stimulation of Prolactin Release......104 8. Effects of Naloxone, Morphine, and EB-Endorphin on the Post-Castration Rise of Serum LH..........130 LIST OF FIGURES FIGURE 1. Efiict of naloxone (NAL) on restraint stress- fluwced Prolactin (PRL)re1ease (n = 4)........ 2. Effects of haloperidol (HAL) and morphine (MS) on serum prolactin (PRL) concentration. 3. EffeCtS Of B-END On ME DA TUI‘UOVCI‘........... 4. Effects of PCPA on MOR and B-END stimulation and NAL inhibition of PRL release............ 5. Effects ofa-methyl—para-tyrosine (a-mpt) on NAL-induced release of LH.................... 6. Effect of wmethyl-para-tyrosine (a-mpt) and phenoxybenzamine hydrochloride (PBH) on NAL- 1nduced release or LB...OIOOOOOOOOCOOOIOOOOOO 7. Effects of diethyldithiocarbamate (DDC) on NAL-induced release of LH.................... 8. Effects of NAL, DDC, or NAL plus DDC on anterior hypothalamic (AH) NE and DA concentratiODSOOOO0......IOOOOOOOOOOOO ....... 9. Effects of NAL, DDC, or DDC plus NAL on medial basal hypothalamic (MBH) NE and DA concentrationSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 10. The effects of morphine sulfate (MS) and NAL on the post-castration rise of serum LH...... 11. Inhibition of the post-castration rise of serum LH by chronic administration of testosterone or MOR.......................... 12. Inhibition of the castration—induced reduction of hypothalamic LHRH concentration by testosterone propionate (TP) or morphine sulfate (MS) administration......... ........ . viii Page 93 101 103 105 116 117 119 120 129 131 132 13. 14. 15. Page Effects of NAL on EB or TP inhibition of LH release in ovariectomized rats...............140 Effect of NAL on the combined inhibitory action of estradiol benzoate (EB) and progesterone (P) on LH release in ovari- ectomized rats............................... 142 Effect of NAL on testosterone propionate (TP) inhibition of LH release in acutely castrated male rats..................................... 43 ix INTRODUCTION The number of hypothalamic factors which are known presently to influence anterior pituitary (AP) hormone secretion, either by a direct action on the pituitary or via the hypothalamic hypophysiotropic hormones, has grown to a list of over 20. The latest addition to this list of factors is a group of peptides which exhibit morphinomimetic properties. Presently, this group of peptides consists of the 2 enkephalins (methionine- enkephalin and leucine-enkephalin), dynorphin, and the 3 endorphins (alpha-, beta-, and gamma-endorphin). When referring to these brain opioid peptides as a group, it has become acceptable to use the term endogenous opioid peptides (EOP). The discovery' of' the EOP was by no means a serendipitous process. Rather, it was a systematic effort by several groups to isolate and characterize an opioid ligand which they were certain existed in the brain. The idea that an endogenous opioid ligand existed in the brain was derived from work showing the presence of specific opiate receptors in brain tissue (Pert and Synder, 1973; Terenius, 1973). This hypo- thesis was given greater impetus by the finding that analgesia could be produced by electrical stimulation of the mesencephalic grey area of the brain (Reynolds, 1969). Hughes _<_e_t_ g_1_. (1975) successfully isolated from porcine brain, sequenced, and synthesized 2 opioid peptides in 1975, both pentapeptides with identical structures with the exception of the C terminal amino acid. One of the peptides contains methionine at the C terminus, whereas the other peptide contains leucine. These were named methionine-enkephalin (MET-ENK) and leucine-enkephalin (LEU-ENK), respectively. A third opioid peptide, beta—endorphin (B-END) was isolated from camel pituitary and its structure identified in 1976 by C.H. Li and co-workers (Li and Chung, 1976). B-END contains 31 amino acids and is identical to B-lipotropin6l_91 ( B-LPH61_91). In addition, the first 5 amino acids of B-END are identical to MET-ENK. Guillemin and co-workers (1976) reported the structures cfl‘ 2 additional endorphins. They were alpha- and gamma-endorphin and are identical in structure to e-LPH'61_76 and B-LPH61_77. reapectively. Most recently, a sixth opioid peptide has been partially identified by Goldstein 3} El- (1979). This peptide was named dynorphin and contains within its sequence the 1.13 structure of LEU-ENK. The guinea pig ileum and mouse vas deferens were commonly used for bioassay during the isolation pro- cedure in order to determine opioid activity of the preparations. Like morphine (MOR), the» EOP all in- hibited smooth muscle contraction induced by electrical stimulation, and this effect was reversed by the spe- cific opiate antagonist, naloxone (NAL). The EOP also mimicked other actions of MOR. These properties include analgesia, miosis, constipation, and catatonia. This demonstration that EOP exhibited MOR-like properties resulted in efforts by several laboratories to dis- cover physiological roles for the EOP. Since MOR pre- viously had been shown to influence anterior pituitary (AP) hormone secretion (Barraclough and Sawyer, 1955: George and Hay, 1955; Meites, 1962; Lomax gt 31., 1970; Martin 35 31., 1975), the EOP also were tested for their effects on pituitary hormone secretion. Prior to the discovery of the EOP, it was demonstrated that opiates such as MOR and methadone could stimulate secretion of prolactin (PRL, Meites, 1962; Clemens and Sawyer, 1974), growth hormone (GH, Martin ‘_2 ‘21,, 1975), and adrenocorticotropin (ACTH, George and Way, 1951), and inhibit secretion of luteinizing hormone (LH, Barraclough and Sawyer, 1955). and thyroid stimulating hormone (TSH, Lomax gt 31., 1970). Therefore it was logical to determine if the EOP had similar effects on AP hormone secretion. In recent years, it was shown by us and others that acute admin- istration of MET-ENK and (FEM3could elicit changes in AP hormone secretion that are similar to those produced by MOR. These effects were reversed by NAL. Somewhat surprising was our observation that NAL given alone decreased basal serum PRL and GH levels and increased LH and follicle stimulating hormone (FSH) levels ix: the blood of mature male rats (Bruni 33 33., 1977). These results suggested to us that EOP may be involved in regulating the secretion rates of certain AP hormones during basal conditions. Experiments also were done to determine if EOP were involved in the PRL response during non-basal physiological states. The previous finding that stress could produce analgesia, presumably by stimulating opioid neuronal activity in the brain, was a compelling reason for studying the role of EOP in stress-induced PRL release. Several stresses have been shown to produce a prompt and significant release of PRL (Euker _£ 33., 1973; Krulich 33 33., 1974). Evidence will be presented in this thesis that EOP are involved in stress-induced PRL release. The EOP do not alter hormone secretion by a direct action on the AP. Thus, addition of opiates or NAL to hemipituitaries or pituitary cell cultures does not produce the hormonal response one observes 33 3313. The logical alternative is that EOP alter .AP hormone secretion via :3 hypothalamic mechanism. Hypothalamic factors important in the regulation of PRL secretion are numerous, and include tuberoinfundibular dopamine (DA), serotonin (5—HT), and possibly other presently unidentified PRL releasing and inhibiting factors. LH secretion is directly controlled by hypothalamic LH— releasing hormone (LHRH) that is released into the portal circulation. Additional factors which influence LH secretion indirectly include the biogenic amines, of which norepinephrine (NE) is stimulatory and appears to be the most important in the control of LH release. DA and 5-HT have been reported to either stimulate or inhibit LH (Meites 33 3_l_., 1977), and hence may be of lesser importance» ‘We have studied the interaction of opiates and hypothalamic catecholamines, and will pre- sent evidence in this thesis that opiates stimulate PRL release by inhibiting tuberoinfundibular DA ‘turnover, and inhibit LH release by decreasing hypothalamic NE activity. It. is well known that testosterone tonically inhibits LH secretion in the male by a negative feedback action on the hypothalamus and pituitary. This is also true of cwarian steroids in the female, except immedi- ately prior to ovulation at which time ovarian steroids exert a positive feedback action on the hypothalamo- pituitary-gonadotropic axis. The stimulatory action of NAL on LH release suggested that it may be tonically inhibited by EOP. It. was therefore of interest to determine if the EOP mediate the negative feedback action of gonadal steroids in male and female rats. To test this hypothesis, we determined the effects of NAL on the inhibitory action of gonadal steroids on LH release in castrated male and female rats. Presumably, if gonadal steroids inhibit LH release by activating opiate neurons, NAL should be able to block this action of gonadal steroids. In addition, we compared the effects of MOR and testosterone on the release of LHRH from the hypothalamus of castrated male rats. The results of these experiments suggest that EOP mediate gonadal steroid inhibition of IJl release ix: male and female rats. LITERATURE REVIEW I. Hypothalamic Control of Anterior Pituitary Hormone Secretion A. Classical Observations It has been known for many years that the pituitary is intimately involved :hn many important physiological functions and is necessary for the maintenance of normal life. The pituitary was shown to contain hormones that are necessary for body growth (Evans and Long, 1921; 1922), for growth and maturation of the ovaries and testes (Smith, 1926; Zondek and Ascheim, 1926), for normal thyroid (Allen, 1919; Smith and Smith, 1922) and adrenal cortical function (Allen, 1922: Smith, 1926), for milk production (Stricker and Grueter, 1928), and for many other functions. These observations led to the pituitary being called the "master gland" of the body. Subsequent to these studies, it was demonstrated that the central nervous system (CNS), particularly the hypothalamus, exerts a pmofound control over pituitary function. Although this finding does not diminish the importance of pituitary function, it does make questionable the appropriateness of referring to the pituitary as the "master gland" since it is itself "mastered" by the hypothalamus. The importance of the CNS in the control of AP hormone secretion was apparent from experiments in which the hypothalamus was either lesioned the supraoptic nucleus. The supraoptic and paraventricular nuclei contain the cell bodies which give rise to the supraopticohypophyseal tract which transports antidiuretic hormone and oxytocin to the neurohypophysis. The tuberal cu“ intermediate hypothalamus lies between the optic chiasm and the mammillary bodies. 3T! Q. Q n\.b 11 Situated at the base of the third ventricle are the arcuate and periventricular nuclei which contain the cell bodies of the tuberoinfundibular DA neurons. Slightly dorsolateral to these nuclei are the ventro- medial and dorsomedial nuclei. These nuclei make up the medial border of the lateral hypothalamic nucleus which is bordered laterally by the optic tract and subthalamus. The ventral surface of the tuberal hypo— thalamus contains the median eminence (ME). The ME can be divided into the following 3 zones: 3) inner ependymal zone which contains ependymal cells which line the third ventricle; t0 inner palisade layer which contains the hypothalamo-hypophyseal neurons; 0) outer palisade layer which contains the Junction between the tuberohypophyseal neurons and the capillary plexus (Knigge and Scott, 1970). The mammillary or caudal hypo- thalamic area contains the caudal hypothalamic nucleus. The hypothalamus contains both afferent and efferent nerve tracts (Jenkins, 1972). Postcommissural fibers of the fornix terminate in the lateral portion of the mammillary body. A portion of the medial forebrain bundle which originates in the septal area of the olfactoryr stria innervates several. hypothalamic nuclei before continuing (Hi to the midbrain tegmentum. Hypo- thalamic nuclei receive input from the thalamus via the 12 periventricular fibers. The stria terminalis originates in the amygdaloid complex and arches caudodorsally prior to terminating mainly 1J1 the supraoptic area cd‘ the hypothalamus. Two efferent tracts originate 1J1 the mammillary body. The mammillothalamic tract passes rostrodorsally and terminates in the thalamus, whereas the mammillo- tegmental tract passes caudally and terminates in the brain stem tegmentum. There are lightly myelinated fibers which course from the periventricular nucleus to the dorsomedial thalamic nucleus and a caudal tract which innervates the brain stem via the dorsal longi- tudinal bundle. The supraoptic and paraventricular nuclei give rise tx> the supraoptico-hypophyseal tract which is joined in the ME by the tuberohypophyseal tract of the tubercinereum. Upon joining, these 2 tracts make up the hypothalamo-hypophyseal tract which terminates in the posterior lobe of the pituitary. It is this tract which delivers oxytocin and antidiuretic hormone from the paraventricular and supraoptic nuclei respectiveb/to the posterior pituitary for storage. 13 C. Hypothalamo-Hypophyseal Portal Vessels and Neurosecretion Unlike the neurohypophysis. the pars distalis has no neural connections with the hypothalamus. Rather, the connection between hypothalamus and AP is vascular. Pope and Fielding (1930) first described a portal system which connected the sinusoids of the AP with a capillary plexus in the ME. Based on morpho1ogica1 evidence alone, they proposed that blood flowed from the AP to the hypothalamus. Houssay _3 _3. (1935) were the first to correctly propose that blood flow was from hypothal- amus to AP. Definite proof of this proposal was obtain- ed with the use of vital dyes. Systemic injection of vital dyes into the toad resulted in their appearance in the capillary plexus of the ME prior to their appearance in the AP (Wislocki and King, 1936). Green and Harris (1949) subsequently showed that portal blood flow in the mammal was from hypothalamus to AP. The total blood flow to the AP is by way of the portal vessels. The only exception is 1J1 the rabbit (Harris, 1947; Goldman and Saperstein, 1962). The hypophyseal portal system is comprised of long and short portal vessels. The superior hypophyseal artery gives rise to the long portal vessels which travel along the lateral and anterior aspects of the infundibulum to the 14 sinusoids of the AP (Netter, 1965). The long portal vessels provide 70-901 of the total blood to the AP (Adams 33 33., 1963; Porter 33 33., 1967). The remaining blood to the AP is supplied by the short portal vessels which originate in the distal portion of the infundibulum, and travel deep within the infundib- ulum to the AP (Netter, 1965). Thus, although a neural connection between the hypothalamus and AP does not exist, the hypothalamo-hypophyseal portal vessels provide a means by which CNS activity can infuence AP function. The concept of neurosecretion was proposed in the late 1930's and early 1940's shortly after the hypo— physeal portal vessels and the direction cu‘ its blood flow was correctly defined. Haterius (1937) and Hensey (1937) first proposed that CNS activity resulted in the release of factors into the portal vasculature *which upon reaching the AP elicited a response. Scharrer and Scharrer (1940) first proposed the existence of neuro- secretion into the general circulation. The Scharrers, together with Bargman, demonstrated that antidiuretic hormone and oxytocin were synthesized in the supraoptic and paraventricular nuclei, and were transported by axonal flow to the posterior pituitary where they were stored until released lJHH) the general circulation 15 (Bargman and Scharrer, 1951; Scharrer, 1952; Scharrer and Scharrer, 1954). During this period in which the concept of neurosecretion was beginning to be established, Harris introduced the chemotransmitter hypothesis. Based on the earlier morphological work on the portal circulation and the pioneering work of Bargman and the Scharrers, Harris suggested that hypothalamic activity resulted 1J1 the secretion of hormones into the portal circulation, which upon reaching the AP, regulated the secretion of AP hormones (Harris, 1948). The chemotransmitter hypothesis provided a mechanism to explain how exteroceptive stimuli such as light, temperature, olfaction and sound affect hormone secretion (Marshall, 1942; Harris, 1955). The neuro- secretory cells of the hypothalamus are now viewed as transducer cells which convert the electrical signals of the CNS into chemical messages which can be perceived by the AP cells. This function of the neurosecretory cells is necessary for the coordination of the CNS with the endocrine system and for the maintenance of homeostasis (Hurtman, 1973). 16 D. Hypohysiotropic Hormones Shortly after the chemotransmitter hypothesis was proposed, several laboratories attempted tx> show that hypothalamic releasing and inhibiting factors did indeed exist. These studies were done in the 1950's and 1960's. The first releasing factor to be discovered was corticotropin releasing factor (GR?) in 1955 (Saffran and Schally, 1955; Guillemin and Rosenberg, 1955). They showed that the addition of NE to a hypothalamic- pituitary coincubation system resulted in the release of ACTH. This effect of NE was not observed in the absence of hypothalamic tissue, suggesting that NE released an ACTH-releasing factor from the hypothalamus. Subsequently, releasing factors for thyrotropin (Shibusawa, 1956), PRL (Meites 33 33., 1960), LH (McCann _3 _3., 1960), FSH (Igarashi and McCann, 1964; Mittler and Meites, 1964), and GH (Deuben and Meites, 1964) were demonstrated. In addition, hypothalamic inhibiting factors were reported for PRL (Pasteels, 1961; Talwalker 33 33, 1963), and GH (Krulich 33 33., 1968). Thus far, only 3 of the 8 hypothalamic releasing and inhibiting factors have been sequenced and synthe- sized. The sequence and synthesis of thyrotropin releasing hormone (TRH) was performed independently by the laboratories of Guillemin (Burgus 33 33., 1969) and 17 Schally (Boler 33 33., 1969) and shown to be a tripeptide. Luteinizing hormone releasing hormone (LHRH), a decapeptide, was sequenced and synthesized 2 years later tut Schally's group (Matsuo 33 33., 1971). In 1973. CH inhibiting hormone or somatostatin was sequenced (Brazeau 33 33., 1973) and synthesized (Rivier _3 33., 1973) by Guillemin and coworkers and shown to be a tetradecapeptide. .At present, the structures of the other hypothalamic factors have not been discovered, although partial sequences for CRF and GH releasing factor have been reported. The structural identification and synthesis of these 3 hypothalamic hormones has made their local- ization possible through immunohistochemical studies and radioimmunoassay (RIA) of discrete areas of the brain. All 3 peptides are located in the ME in high concen- trations (Brownstein ‘_3 .33., 1976b). This would be expected since the ME is the final common pathway all factors must take in their transport to the capillary plexus prior to diffusing into the portal vessels. Lower concentrations of LHRH are found in the preoptic- suprachiasmatic area, and LHRH perikarya have been visualized 1J1 this region. Electrical stimulation of the preoptic area was shown to increase LHRH concen- tration in portal blood (Eskay 33 33., 1977). Deaffer- 18 entation of the medial basal hypothalamus (MBH) resulted in decreased LHRH concentration in the MBH (Weiner 33 33., 1975; Kalra 33 33., 1977; Brownstein, 1977). Efforts to visualize LHRH cell bodies in additional brain areas of the rat have been unsuccessful. Therefore, i1; is now believed that LHRH neurons which originate in the preoptic area are the only source of ME LHRH in the rat (Baker _3 33., 1975; Weiner 33 33., 1975). As with LHRH, THHl and somatostatin have high concentrations in the external layer of the ME. TRH and somatostatin also have been localized in brain regions other than the hypothalamus (Hgkfelt _3; 33., 1975a; Jackson and Reichlin, 1979). TRH and somatostatin also are distributed outside the brain including gut and pancreas (Hgkfelt 33 33., 1975a; Jackson and Reichlin, 1979) and substantia gelatinosa (Hgkfelt 33 33., 1975b). Although LHRH, TRH and somatostatin alter the release of their respective AP hormones in a dose dependent manner (Schally, 1973), they are not without effect on other hormones. TRH stimulates PRL secretion in humans in addition to TSH (Jacobs _3 33., 1971). Furthermore, TRH antiserum was reported to decrease both serum TSH and PRL levels in the rat (Koch 33 33., 1977). However, it is unlikely that TRH is a: major physio- 19 logical PRL-releasing factor since there is a poor correlation between PRL and TSH secretion during many physiological conditions. Somatostatin, in addition to inhibiting GH release, has been reported to inhibit TRH induced TSH, but not TRH-induced PRL secretion (Vale 33 33., 1974), whereas somatostatin antiserum increased both GH and TSH levels (Ferland 33 _3., 1976b). LHRH stimulates LH and FSH secretion. However, the secretory patterns of LH and FSH are different, which has led to the speculation of different gonadotropin releasing hormones for 1J1 and FSH. Alternatively, i1; has been shown that the different secretory profiles of LH and FSH may be due to their different half-lives (Gay 33 33., 1970), modulation tn! the steroid environment (Schally 33 33., 1973: Yen 33 33., 1975), or a specific inhibitory action of inhibin from the gonads on FSH secretion, II. Localization of Biogenic Amines and Opiates A. Dopamine The presence of DA in the mammalian brain was first demonstrated 1J1 1957 (Montagu, 1957). The observation 'that DA distribution was distinct from that of NE which 20 had previously been isolated, suggested that DA may be a neurotransmitter rather than merely the precursor of NE (Bertler and Rosengren, 1959). The distribution of DA in the brain can be divided into 3 distinct systems. The nigrostriatal system contains approximately 80% of the total DA within the brain. Cell bodies within the zona compacta of the substantia nigra (A9) combine with a cell group (A8) in the adjacent ventral tegmental area. Together, these give rise to dopaminergic neurons which course rostrally and terminate in the putamen and caudate nucleus (striatum; Anden 33 _3., 1964; 1966c). The nigrostriatal dopaminergic system is involved in the normal control of extrapyramidal upper motor neurons. A deficiency in this system has been shown to be associated with ‘Parkinson's disease (Hornykiewicz, 1963). The mesolimbic dopaminergic system originates in the A cell. group and. passes rostrally' in close 10 association with the nigrostriatal DA neurons before terminating ix) the nucleus accubens and olfactory tubercle (Andeh 33 al., 1966; Ungerstedt, 1971; Weiner 33 _3., 1972b). The third dopaminergic division is the tubero- infundibular DA system (ruxe’ and H6kfelt, 1966). Immunohistofluorescent studies show that the tubero- .infundibular DA system is located completely within the 21 hypothalamus. Its cell bodies are in the arcuate and periventricular nuclei (Fuxg, 1963). Tuberoinfundibular DA neurons terminate in the external layer of the ME (Fuxé and Hgkfelt, 1966; Ungerstedt, 1971), while others continue on and terminate in the neural and intermediate lobes (tuberohypophyseal DA system). Deafferentation of the hypothalamus does not result in a reduction of hypo- thalamic DA concentration (Weiner 33 33.. 1972b), which further supports the view that tuberoinfundibular DA cell bodies are restricted to the hypothalamus. B. Norepinephrine The presence of NE in the brain was demonstrated in 1939 (Holtz, 1939). The cell bodies of NE neurons are located in the pens-medulla (A1, A2, A5, and A7) and locus coeruleus (A6). These cell groups give rise to axons which enter the medial forebrain bundle and dorsal bundle which innervate the lower brainstem, limbic system, cerebral cortex, hippocampus and hypothalamus (Fuxg, 1965a, 1965b; Ungerstedt, 1971). In the hypo- thalamus, NE is concentrated in the retrochiasmatic area of the anterior hypothalamus, the supraoptic, para— Ventricular, and periventricular nuclei, and 1J1 the ME (Fuxe', 1965a, 1965b). NE in the ME is primarily associated with the internal layer in contrast to DA 22 which is found primarily in the external layer (Jonsson _33 33., 1972). Lesioning cfi‘ the locus coeruleus (Loizou, 1969; Korf‘ et '33., 1973a; 1973b) not necessarily have identical physiological functions. 29 III. Biogenic Amine Metabolism A. Synthesis and Release The synthesis of DA and NE begins with active uptake of tyrosine into catecholamine neurons (Iverson, 1971). Tyrosine is converted to DA and NE by a chain of enzy- matic reactions which occurs at the nerve terminals. The enzymes involved in these conversions are synthe- sized :h1 catecholamine cell bodies and transported to the nerve terminals (McClure, 1972). Tyrosine is hydroxylated to dihydroxyphenylalanine (DOPA) by the rate limiting enzyme tyrosine hydroxylase (Levitt '33 33.. 1965). Tyrosine hydroxylase is active only when in its reduced form which is accomplished by the co-factor tetrahydropteridine. DOPA is decarboxylated by aromatic-L-amino acid decarboxylase to DA. Unlike tyrosine hydroxylase, aromatic-L-amino acid decar- boxylase is a nonspecific enzyme (Carlsson 33 a1., 1972), which is common to all tissues and metabolizes neutral amino acids (Goldstein 33 33., 1974). DA is hydroxylated to NE by the hydroxylating enzyme, DA- g-hydroxylase. DA-B—hydroxylase is a tetrameric glyco- +2 protein which contains four moles of Cu (Goldstein 33 33., 1965). It is localized (Hi the membrane storage 30 vesicles of NE neurons only (Potter and Axelrod, 1963; Friedman and Kaufman, 1965), unlike tyrosine hydroxylase which is found in all catecholamine neurons. Epine- phrine is the final product of the enzymatic reactions which begin with the hydroxylation of tyrosine. Epine- phrine is the product of NE methylation by phenyl- ethanolamine-N-methyltransferase. This enzyme is found in high concentrations in the adrenal medulla, but has a low concentration in the brain (Axelrod, 1962). There- fore, the importance of epinephrine as :3 brain neuro- transmitter is not well established. Steady state DA and NE concentrations are main- tained by product inhibition of tyrosine hydroxylase. Decreased activity cu‘ catecholamine neurons results in increased concentrations of intraneuronal DA and NE. DA and NE compete with oxidized tyrosine hydroxylase for binding to tetrahydropteridine and thus decrease the amount cfi‘ reduced tyrosine hydroxylase (Udenfriend 33 33., 1965; Costa and Neff, 1966). The end result is a decrease in active tyrosine hydroxylase and reduced synthesis of catecholamines. Conversely, increased release of DA or NE increases tyrosine hydroxylase activity resulting in increased DA or NE synthesis (Sedvall et 1., 1968). Thus, acute changes in cate- cholaminergic activity (M) not alter catecholamine 31 stores. 5—HT is synthesized from tryptophan in 2 enzymatic steps. Tryptophan is actively taken up into the nerve terminal and hydroxylated to 5-hydroxytryptophan (5-HTP) by the rate limiting enzyme, tryptophan hydroxylase (Udenfriend, 1959; Ichiyama 33 33. 1970). 5-HTP is decarboxylated by aromatic-L-amino acid decarboxylase to 5-HT (Udenfriend, 1959). This enzyme also catalyzes the conversion of DOPA to DA. Thus administration of DOPA in order to increase DA synthesis is somewhat non- specific since 5-HT neurons also convert DOPA to DA, and DA inside 5-HT neurons can displace 5-HT (Bartholini 33 33., 1968). The synthesis of 5-HT is not as finely controlled as that of DA and NE. The rate limiting enzyme, trypto- phan hydroxylase, normally is not saturated (Lovenberg _3 __1.., 1968; Fernstrom and Wurtman, 1971). In addi- tion, product inhibition of tryptophan hydroxylase does not exist under normal conditions (Lin _3 33., 1969; Macon 33 33., 1971), although product inhibition can be demonstrated when 5-HT levels are elevated by pharmaco- logical methods (Macon 33 33., 1971). Monoamines are stored within vesicles at the nerve terminals. Unlike the monoamines which are synthesized at the teminals, the vesicles are synthesized in the nerve cell body and transported to the terminal by axonal flow (Dahlstrgm 33 33, 1973). Vesicular storage of the monoamines serves 2 functions: a) prevents enzymatic degradation (Hgkfelt, 1968); b) provides a mechanism for quantal release. The existence of 2 pools of monoamines has been suggested (Axelrod, 1974). There is a readily releasable pool characterized by vesicles closely associated with the nerve terminal membrane and believed to be made up of newly synthesized neurotrans- mitters. There also is thought to be a larger storage pool more distant from the membrane which may serve as a reservoir. Nerve activity results in release of newly synthesized monoamines (Kopin 33 33., 1968; Glowinski, 1972). The neurotransmitter is released into the extra- cellular space by the process of exocytosis in which the vesicular membrane fuses with the nerve terminal mem- brane. This event is calcium dependent (DeRobertes and Vas Ferreira, 1957). B. Monoamine Inactivation The action of monoamines is terminated by 3 differ- ent means. They are reuptake by the nerve terminal, enzyamtic metabolism, and diffusion from the receptor sites. Reuptake is the most important means of inacti- vating CNS DA, NE, and 5-HT (Glowinski 33 33., 1965; 33 Iverson, 1967; Coyle and Synder, 1969). Reuptake of monoamines, in addition to terminating neurotrans- mitter action, is also a method for recycling amines and thus is metabolically practical. The reuptake process is saturable, stereospecific and NA+ dependent (Iverson, 1974). However, the reuptake systems are run; totally specific. Reuptake of 5-HT by catecholamine neurons has been demonstrated (Lichtensteiger 33 33. 1967; Bartholini 33 33., 1968). Monoaminer oxidase (MAO) and catechol-o-methyl transferase (COMT) are the 2 primary enzymes involved in enzymatic degradation of DA, NE, and 5-HT. MAO is intraneuronal and associated with the mitochondria (Nukada 33 33., 1963). MAO deaminates DA and NE to 3,4- dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenyl- glycolaldehyde, respectively. Extraneuronally, COMT o- methylates DA to 3-methoxytyramine and NE to normetane- phrine (Alberici _3 33. 1955; Axelrod 33 33., 1959). The primary metabolic product of 5-HT is 5-hydroxy- indoleacetic acid and is accomplished by MAO and an aldehyde-dehydrogenase (Sjoerdsma 33 33., 1955). Inhibition of catecholamine uptake by cocaine, phenoxybenzamine or imipramine can greatly potentiate adrenergic stimulation (Iversen, 1971), whereas in- hibitors of MAO and COMT only slightly potentiate 34 sympathetic stimulation (Pletscher, 1973). These observations suggest that reuptake of monoamines, and not degradation, is the primary means of monoamine inactivation. IV. Opioid Peptide Metabolism A. Synthesis Although presently incomplete, the area cH‘ EOP metabolism has been expanded significantly since their discovery. Synthesis of enkephalins and endorphins appear to be separate processes, and the proposal that enkephalins are synthesized from B-LPH cn' merely metabolites of the endorphins is no longer accepted. Rejection of this proposal is based on the following observations: The distribution cM‘ enkephalins is distinctly different from the distribution of endorphins (Bloom 33 33., 1976), An endopeptidase capable of processing MET- ENK from either B-LPH or B-END has not been isolated (Smyth, 1980). Furthermore, an Opioid peptide of greater molecular weight than the enkephalins has been shown to produce a peptide upon trypsin degradation that is identicle to MET—ENK in terms of immunoreactivity and receptor binding activity (Lewis 33 35 33., 1978; Yang 33 33., 1978). This putative precursor of MET-ENK was shown not to be B-LPH or a fragment of B-LPH. A potential precursor of LEU-ENK has been identified and was named c-neo-endorphin (Kangawa 33 33., 1979). The N-terminus contains the structure of LEU-ENK and has a total of 15aa. Dynorphin may also be a precursor of LEU-ENK. Dynorphin has been partially sequenced (first 13aa), and 'its N—terminus also is identicle to the structure of LEU-ENK (Goldstein 33 33., 1979). Dynorphin and a-neo-endorphin exhibit signi- ficantly greater opioid activity than LEU-ENK, and is thought to be due to increased resistance to enzymatic degradation. The structure of a MET-ENK precursor has not yet been identified. However, the precursor has been given the provisional name cfi‘ B-neo-endorphin (Smyth, 1980). It is speculated that a-neo-endorphin and gg-neo- endorphin may in turn have a common precursor. This hypothesis is based on the observation that the distributions of LEU- and MET-ENK are identical and may be synthesized in the same cells (Watson 33 33., 1978; Smyth 33 _3., 1980). The amino acid sequence of B-END is identical to B- LPH61_91, and B-LPH is believed to be the precursor of 36 B-END. B-LPH also is a cleavage product of pro- opiocortin, a: 31K precursor molecule. Pro-opiocortin, as demonstrated by the pulse-chase experiments of Mains 33 33. (1977) gives rise to one molecule of B-LPH, which is cleaved to B-END ( 8-LPH61_91) and B-melanocyte stimulating hormone ( B -LPH38_58). In addition, pro- opiocortin is the precursor molecule of ACTH. That pro-opiocortin is in fact the precursor of both ACTH and B-END is further supported by immunohistochemical data demonstrating the presence of ACTH, B-LPH and EFEND in the same cells of the AP (Bloom 33 33., 1977). Further- more, ACTH and B-END are released concommitantly from the pituitary. Thus, acute stress, adrenalectomy, and CR? stimulated ACTH and B-END release, whereas dexa- methasone inhibited both ACTH and B-END release (Guillemin 33 33., 1977: Vale 33 33., 1979). It has not been firmly established that c-END (8- ) and‘Y-END ( B-LPH LPH ) are naturally occur- 61-76 61-77 ring EOP. There is speculation that they may be artifacts of the procedures used to extract B-END (Rossier 33 33., 1977). When brain tissues were boiled prior to extraction in order to limit enzymatic break- down of B-END, a-END was undetectable in the tissue. However, when steps were not taken to limit B-END degradation, c-END was detectable, but at the expense of 37 reduced B-END concentration (Rossier 33 33., 1977). B. Release Increased nerve electrical activity is believed to release EOP. Thus depolarization of enkephalin neurons by K+ was reported to increase enkephalin release 33 33333. K+-induced enkephalin release was Ca++-dependent (Smith 33 33., 1976; Osborne 33 33., 1980). There are numerous reports containing indirect evidence that opiate release is coupled in) neuronal electrical activity. Reynolds (1969) first reported that electri- cal stimulation of the mesencepalic grey region produced analgesia. Subsequently, Akil (personal communication) showed that B-END concentration in the cerebral spinal fluid was increased by stimulation of the mesencephalic grey region. Stress also has been shown to produce analgesia. Akil 33 _3. (1976) reported that intermittent footshock evoked analgesia as measured by the tailflick test, and this effect of stress was reversed by NAL. Further- more, opioid activity in the brain as measured by bioassay was increased by stress. Stress-induced analgesia has been confirmed by others, and is thought to be due to the release of EOP (Madden 33 33., 1977; Fried and Singer, 1979). 38 Because cfi' the inherent problems associated with interpreting concentrations of substances in the brain, a number of investigators have studied the effects of different treatments on B-END concentrations ill the blood and pituitary in order to evaluate release. The concentration of B-END in the blood was increased by intermittent footshock or immobilization stress (Guillemin 33 33., 1977; Rossier 33 33., 1979; Sapun 33 33., 1981). Stress also was reported to decrease B-END concentration 1J1 the pituitary (Baizman 33 33,, 1979), indicating that stress stimulates the release of B-END from the pituitary. Plasma B-END concentration was reported increased after adrenalectomy, in addition to elevated ACTH levels, and this effect of adrenalectomy was reversed by dexamethesone (Guillemin 33 33., 1977; Akil 33 33., 1979; Rossier 33 _3., 1979). Adrenalectomy also was reported to increase B-END concentrations in the pituitary, hypothalamus and midbrain (Lee 33 33., 1980). Blood levels of B-END were reported elevated during pregnancy and parturition (Akil 33 33., 1979). There is evidence that hypothalamic factors may be involved in the control of B-END release from the pituitary. Purified CRF and NE were reported to stimulate B-END release from AP cells 33 vitro, whereas DA and apomorphine inhibited 33 vitro release of B-END 39 from the intermediate lobe (Vale 33 _a_l., 1979). 5-HT has been reported to stimulate B-END release from the pituitary. Administration of 5-HTP or quipazine, a 5-HT agonist, elevated plasma B-END levels, whereas 5,7- dihydroxytryptamine significantly reduced the stress- induced rise of plasma g-END levels (Sapun _3; 33., 1981). It is not known presently what function circulating B-END serves. Nor is it known whether the factors in- volved ix) the regulation cfi' pituitary B-END secretion apply tn) endorphin and enkephalin neuronal systems in the brain. The answer to this latter question depends on the development of new techniques for measuring opioid activity in the CNS. C. Inactivation The enkephalins have extremely short half-lives ( ). When 3H-MET-ENK was injected iv , 74: of the t1/2 radioactivity measured 15 sec later migrated with tyrosine, suggesting that the TYR-GLY bond is cleaved very rapidly (Dupont 33 33., 1977). The tetrapeptide produced by removal of the N-terminal tyrosine was shown to be completely devoid of opioid activity (Hambrook 33 33., 1976; Terenius 33 1., 1976). Replacement of GLY2 with D-alanine2 results in a peptide with increased 40 activity, and is believed to result from increased resistance’ to cleavage cM‘ the N-terminal tyrosine (Terenius 33 33., 1976). There is evidence for a membrane bound amino- peptidase in rat brain that is capable of inactivating MET- and LEU-ENK. Incubation of MET- or LEU-ENK with washed rat brain membranes resulted in rapid inactiv- ation. Substitution of D-alanime for GLY2 produced a peptide that was resistant to degradation, whereas substitutions at the carboxyl terminal failed to protect the peptide from enzymatic degradation (Pert t: al., 1976). These results further substantiate that cleavage of the TYR-GLY2 bond by an aminopeptidase is the major means of enzymatic inactivation of the enkephalins (Meek _3 _3., 1977). There is recent evidence that additional membrane bound enzymes are involved in enkephalin inactivation: a dipeptidyl carboxypeptidase (enkephalinase A) which cleaves the GLY-PHE bond and a dipeptidyl aminopeptidase (enkephalinase B) *which cleaves the GLY-GLY bond (Gorenstein and Synder, 1980). There also is 1 report that the action of MET-ENK may be terminated by a high affinity uptake system (George and Van Loon, 1981). The endorphins exhibit prolonged opioid activity compared to the enkephalins. The 151/2 of B-END when ~ —-‘.—.-- _. 41 injected into the cerebral spinal fluid was calculated to be 60 min (Bloom 33 33., 1978). A similar t was 1/2 reported for B-END 33 33333, whereas the tl/2 of MET- and LEU-ENK under the same conditions was less than 1 min (Hambrook 33 33., 1976). Exposure of B-END to brain homogenates dramatically reduced the tl/2 of B-END. The calculated t1/2 was approximately 4 min (Bloom 33 33., 1976), and believed to be due to enzymatic degradation. Heat inactivation of enzymes by microwave irridiation or boiling prior to homogenization was reported to prevent enzymatic destruction of B-END (Rossier 33 33., 1977). At least 3 products of B-END inactivation have been isolated which exhibit immunoreactivity, but are devoid of Opioid activity. The C'-Fragment results from enzymatic cleavage of B-END at position 88-89 of B-LPH. The other 2 products of B-END inactivation are the a-N acetyl derivatives of B-END and the C'-Fragment (Zakarian and Smyth, 1980). These investigators observed that the distribution of B—END and its inactive derivatives was not the same in all regions. They reported that the predominant peptide in the hypo- thalamus was B-END, whereas all 4 peptides were present in the midbrain. Ihi the porcine pituitary, the tx-N- acetyl derivatives of B-END and the C'-Fragment were reported to predominate (Smyth 33 33., 1979). In 42 contrast, it was shown that the majority of the endor- phin immunoreactivity ill the neurointermediate lobe cM‘ the rat was actually a-N-acetylated derivatives, whereas the anterior lobe was authentic s-END (Akil, personal communication). 43 V. Hypothalamic Control of Prolactin Secretion A. Hypothalamic Inhibition of Prolactin Secretion PRL secretion is inhibited tur the hypothalamus during basal conditions. This was clearly demonstrated by the observations that ectOpic transplantation of the AP underneath the kidney capsule (Everett, 1954; Chen 33 33., 1970), sectioning cn’ the pituitary stalk, or lesioning of the ME resulted in enhanced PRL secretion (Meites 33 33., 1963). Subsequently, the hypothalamus was shown to contain ea PRL-release inhibiting factor (PIE) as evidenced by the ability of hypothalamic extracts to inhibit PRL release (Pasteels, 1961; Talwalker 33 _3., 1963). This PIF was believed to be a small polypeptide. However its structure has not been elucidated. Measurement of hypothalamic PIF activity by bioassay methods showed that PIF activity was decreased during states of elevated PRL secreton, such as suckling and estrogen treatment (Ratner and Meites, 1964), and increased after administration of ergot drugs, L-DOPA, or iproniazid, all of which decrease PRL secretion (Meites £3 33.. 1963; Meites and Clemens, 1972). There is growing evidence that the majority of hypothalamic PIF activity is due to DA. DA was shown to 44 be a potent inhibitor of PRL release. Intraventricular (ivt) injection of DA decreased PRL release (Kamberi 33 333, 1971) as did systemic injection cn‘ L-DOPA, the precursor of DA (Lu and Meites, 1972). Apomorphine, a potent DA agonist, decreased PRL in rats (MacLeod and Lehmeyer, 1974; Smalstig 33 _3., 1974) and in humans (Martin 33 .33., 1974). Conversely, circulating PRL levels were increased by the DA antagonists, pimozide, sulpiride, and haloperidol (Meites. and Clemens, 1972; Meites 33 33., 1972; Clemens 33 33,, 1974; Mueller 33 33. 1976b). These findings indicated that hypothalamic DA tonically inhibits PRL secretion during basal conditions. The role of NE in the regulation of PRL secretion is less clear than DA. NE has been reported to stimu- late or inhibit PRL release. Administration of L-DOPS, a precursor of NE (Donoso 33 33., 1971), or clonidine, an at-agonist (Lawson and Gala, 1975) was reported to stimulate PRL release. Disulfram, a NE synthesis in- hibitor, decreased PRL release (Meites and Clemens, 1972), which is in agreement with the above evidence that NE is stimulatory to PRL release. Conversely, there is evidence that NE inhibits PRL release. High doses of NE were reported to inhibit PRL release 33 vitro (Koch 33 1., 1970; Shaar and Clemens, 45 1974; Labrie 33 33., 1978), as did clonidine 33 3333 (Mueller, unpublished). Unlike DA, NE concentration in portal blood is quite low (Ben-Jonathon 33 33., 1980). Furthermore, NE is approximately 1/10 as effective as DA in reducing PRL release 33 33333 (Labrie 33 33., 1979). Therefore the importance of NE in the control of PRL release is presently questionable. Hypothalamic PIF activity can not be attributed solely to hypothalamic catecholamines. Although Shaar and Clemens (1974) reported that absorption cM' cate- cholamines onto alumina removed all PIF activity from hypothalamic extracts, it can not be concluded from this study that other PIFs were not altered by this pro- cedure. Evidence for a non-catecholamine PIF has been reported (Takahara 33 _3., 1974: Schally 33 33., 1977). In addition, it was reported that PIF activity of hypo- thalamic extracts could not be blocked by the DA antagonists, pimozide (Vale 33 33., 1973) or haloperidol (Ojeda 33 33., 1974). Thus, although DA is a potent inhibitor of PRL release, there are certainly additional hypothalamic factors which exert inhibitory actions on AP PRL release. Acetylcholine is present in the hypo- thalamus and can inhibit PRL release (Grandison 33 33., 1974; Kuhn and Lens, 1974), and there is increasing evi- dence that acetylcholine has a physiological role in PRL 46 control. B. Hypothalamic Stimulation of Prolactin Secretion The hypothalamus, in addition to inhibiting PRL release, also is capable of stimulating PRL release. PRL releasing activity of hypothalamic extracts was reported in 1960 by Meites 33 33. when such extracts were shown to initiate lactation in estrogen-primed female rats. The factor believed to be responsible for releasing PRL was termed PRL-releasing factor (PRF). Although the structure of PRF is not known, the evidence for a PRF entity has received further validation (Nicoll _3 _3., 1970; Valverde 33 33., 1972). TRH, the first hypophysiotropic hormone to be sequenced and synthesized, was shown to have PRF activity. TRH stimulated PRL release 33 33333 from a pituitary tumor cell line (Tashjian 33 33., 1971), and i vivo in humans, rats and cows (Jacobs t 1., 1971; Meites 33 33., 1973; Mueller 33 33., 1973). It also was reported that injection of TRH antiserum decreased both circulating TSH and PRL levels (Koch _3 33., 1977). although these results have not been confirmed. It has been suggested that TRH may be the primary PRF. However, this postulate has been viewed skeptically, primarily because cM’ the poor correlation 47 between TSH and PRL secretion during many physiological states. TSH secretion is increased during exposure to cold temperatures, whereas PRL is decreased (Mueller 33 33., 197“). Stresses such as ether, restraint, and heat have been shown to decrease TSH levels, whereas PRL levels are increased (Krulich 33 33., 197R; Mueller 33 33., 197"). Thus the different responses of TSH and PRL to the same physiological stimulus argues against TRH as the primary PRF. There is convincing evidence that hypothalamic S-HT stimulates PRL release. Kamberi 33 33., (1970) demon- strated that S-HT, which does not cross the blood brain barrier, stimulated PRL release when injected ivt. Systemic injection cfl‘ S-HT precursors, tryptophan and S-HTP, which increase brain 5-HT concentrations, pro- duced a: similar blood PRL rise (Meites and Clemens, 1972).. Clemens 33 33., (1977) minimized the non- specific effects of high doses of S-HTP by injecting subeffective doses of S—HTP, together with fluoxetine, a 5-HT reuptake blocker. These low doses of S-HTP, which had no effect on PRL levels when given alone, signifi- cantly increased PRL levels when injected together with fluoxetine. This suggested that the stimulatory action of S-HTP on PRL release is due to its conversion to S-HT rather than tn) a non-specific effect. Reduction of 48 hypothalamic S-HT concentrations decreased blood PRL concentrations. Depletion of 5—HT by injection of PCPA, or lesioning of the raphé'nucleus, were shown to reduce PRL levels. This effect was reversed by S-HTP admin- istration (Caligaris and Taleisnik, 197”; Barophy and Harney, 1975). There is good evidence that S-HT is involved in suckling-induced PRL release. Suckling-induced PRL release was blocked by pretreatment with PCPA or methysergide, a 5-HT antagonist (Kordon 33 _a_l., 1971:; Gallo 33 _3., 1975). In addition, the suckling stimulus was shown to increase hypothalamic S-HT turnover (Mena 33l‘33., 1976). Similarly, Mueller ._3 '33., (1976a) showed that stress-induced PRL release was associated with an increase in hypothalamic S—HT turnover. Together, the above results indicate that S-HT can stimulate PRL release and may very well function physiologically to increase PRL release, probably by enhancing the release of PRF (Clemens 33 33., 1978). The EOP are a recent addition to the list of putative hypothalamic PRFs. It was shown prior to the discovery of EOP, that MOR administration stimulated PRL secretion. Meites (1962) reported that MOR initiated lactation in estrogen-primed female rats, presumably by stimulating PRL release. This stimulatory action of an 49 opiate on PRL release was confirmed by RIA measurement of PRL after MOR (McCann 33 _a__1_., 19714) or methadone administration (Clemens and Sawyer, 1974). The stimulatory action of EOP on PRL release has been demonstrated by our laboratory (Bruni 33 33., 1977) and others, and is a topic of this thesis. Opiates do not stimulate PRL release by a direct action on the AP (Grandison and Guidotti, 1977; Rivier _3 _3., 1977; Shaar 33 _3., 1977). Rather, hypothalamic mechanisms are involved. Evidence that opiates stimu— late .PRL via a decrease in tuberoinfundibular DA turnover will be presented in this thesis. Estrogens are very important regulators of PRL synthesis and release. They are probably the most important non-hypothalamic agents involved in PRL regulation. Because estrogens regulate PRL secretion via hypothalamic mechanisms, in addition to a direct action on the AP, it is appropriate to discuss the role of estrogens in this section on hypothalamic control of PRL. Reece and Turner (1937) were the first to report that estrogens could stimulate PRL. They reported that estrogens increased pituitary PRL content and induced lactation in rats and guinea pigs. They correctly concluded that estrogens stimulated INN. synthesis and release. These results were confirmed in rabbits by 50 bioassay (Meites and Turner, 19u2), as well as in rats using RIA to measure PRL concentrations in the blood and pituitary (Chen and Meites, 1970). Blood estrogen and INN. levels are often directly correlated during different endocrine states. Estrogens and PRL levels are low prior to puberty and begin to rise immediately before the onset of puberty (Brown- Grant 33 33., 1970). There is a distinct surge of PRL on the afternoon of proestrus in rats, mice, hamsters, sheep, goats and cows, but not in primates. This surge is preceeded by increased estrogen titers (Meites and Clemens, 1972). On diestrus, PRL and estrogen levels are both low (Meites _3 _3., 1972). Evidence that the coinciding levels of estrogen evoke, at least in part, the proestrous surge of PRL is that administration of estrogen antiserum blocked the PRL surge (Neil 33 33., 1971).. Furthermore, removal of estrogens by ovariectomy on the morning of proestrus or before, also blocked the PRL surge (Meites 33 33., 1972). It has been conclusively demonstrated that estrogens stimulate PRL secretion by a direct action on AP lactotrophs. Addition of estradiol to AP cultures (Nicoll and Meites, 1962) or hemipituitaries (Lu 33 33., 1971), increased 33 vitro PRL release. Administration of estrogen to hypophysectomized rats bearing a 5] pituitary graft underneath the kidney capsule (Chen 33 3_l_., 1970), or a so pituitary tumor (Mizuno _3 33., 196“), significantly increased PRL levels further. Also, uptake of estrogen by AP cells and the presence of estrogen receptors in AP tissue has been demonstrated (Vertes 33 ‘33., 1973). These observations provide convincing evidence that estrogens stimulate PRL secretion by a direct action on the AP. The hypothalamic mechanism involved in estrogen stimulation of PRL is less well defined. Implantation of small amounts of estrogen into the ME increased serum PRL concentration 3-fold (Nagasawa 33 33., 1969), suggesting that estrogens can stimulate PRL secretion via the hypothalamus. However, one can not rule out the possibility that ME estrogen diffused to the AP and acted directly on the AP. Estrogen administration was reported to decrease hypothalamic PIP activity (Ratner and Meites, 1961!), which is in agreement with a hypo- thalamic site of action. Since PIF activity was measured by bioassay, a decrease in PIF activity may also involve an increase in PRF activity, or a combination of both. The effect of estrogens on hypo- thalamic TRH, 5-HT, and EOP, all of which stimulate PRL release, remains to be thoroughly studied. A complicating factor in determining the hypo- 52 thalamic mechanisms by which estrogens increase PRL secretion is the PRL short-100p feedback. Elevated blood PRL levels have been shown to inhibit PRL release (Advis 33 33., 1977), and probably involves tuberoin- fundibular DA. Tuberoinfundibular DA turnover (Eikenbers £3 _3., 1977) and release into the portal vasculature (Gudelsky 33 3_l_., 1981) were shown to be increased during states of elevated PRL levels. Additional hypothalamic PIFs and PRFs also may be involved 1J1 PRL autoregulation. Therefore, it. is difficult to determine whether effects of estrogen on PRL secretion are mediated via the hypothalamus, or by a direct action on the AP, or via both actions. 53 VI. Control of Luteinizing Hormone (LH) Secretion A. Inhibition of Luteinizing‘Hormone Secretion by Gonadal Steroids Feedback inhibition of 1J1 release by gonadal steroids occurs in male and female mammals. Androgen inhibition of LH release in the male is tonic (Bogdanove, 1967; Turner, 19?”), whereas ovarian steroids in the female are stimulatory for a short period prior to ovulation, and inhibitory during the luteal phase of the cycle. Removal of steroid negative feedback by ovariectomy or orchidectomy results in a persistent elevation of circulating LH levels (Gay and Midgley, 1969: Yamamoto ‘_3 .33., 1970). Sex steroid replacement returns 1J1 concentration in) pre-castration levels (Ramirez 33 33., l96u; Chowers and McCann, 1967; Ferland 33 33., 1976a). Steroid inhibition of IJi secretion appears to involve both a hypothalamic and AP site of action. This conclusion is based primarily on studies in which small amounts of steroids were implanted either in the hypo— thalamus or pituitary. AP implantation of estrogen reduced LH secretion (Rose and Nelson, 1957: Bogdanove, 1963). However, estrogen was shown to be more effective 54 when implanted in the hypothalamus (Davidson, 1969; Sawyer and Hilliard, 1972). LH levels in the male rat also were reduced by medial basal hypothalamic (MBH) implantation of testosterone (Simpkins 33 33., 1980). It is doubtful that inhibition of LH release by hypo- thalamic implants of a steroid is due simply to the steroid being transported to the AP where it exerts a direct action, as has been suggested (Bogdanove, 196”). Although steroids implanted in the hypothalamus do reach the AP (Bogdanove, 196”), steroid inhibition of LH release can be observed prior to delivery of significant amounts of the steroid to the AP (Turner and Simpkins, unpublished). Furthermore, the inhibitory action of estrogen on the AP lasts for only a few hours, and estrogen becomes facilitatory after approximately 8 hours of exposure. However, the inhibitory action of estrogen 'is maintained 1J1 the female. Therefore inhibition is probably maintained by a hypothalamic site of action. The hypothalamic area thought to be most important in steroid negative feedback is the MBH. Deaffer- entation of the MBH did not alter ovarian steroid inhibition of LH secretion (Blake, 1977). Furthermore, MBH implants of testosterone were as effective as so administration of testosterone in reducing post- 55 castration LH levels (Simpkins 33 33., 1980). Thus, the present view of gonadal steroid feedback inhibition of LH secretion is that steroids inhibit LH secretion init- ially by a dual action on the AP and MBH, but the long- term inhibitory action is restricted in) a hypothalamic site (Blake 33 33., 1974; Blake, 1977). B. Stimulation of Luteinizing Hormone Secretion by Ovarian Steroids Ovarian steroids can stimulate LH secretion in female mammals (McCann, 1974). The stimulatory action of estrogen on LH secretion was first demonstrated by its ability to cause ovulation, as evidenced by the formation cfi‘ corpora lutea (Hohlweg, 193”). Sub- sequently, Everett (1948) and Brown-Grant (1969) showed that estrogen and progesterone could advance ovulation when administered during diestrus. In cycling female rats, basal LH levels are interrupted every “-5 days by a surge of LH which typically occurs between 1600—1800 hours on proestrus (Monroe 33 33., 19693 Butcher 33 33., 197“). Estrogen levels begin to rise on diestrous day 2 and reach a peak level on proestrous morning (Hori 33 33., 1968). Elevated estrogen levels were shown to be absolutely necessary for the LH surge to occur since administration of antibodies to estrogen blocked the 56 proestrous LH surge and ovulation in rats (Ferin 33 33., 1969) and the LH surge induced by estrogen and progesterone in monkeys (Knobil, 197R). Progesterone by itself has little effect on LH secretion, but potentiates the stimulatory action of estrogen on LH secretion (Dgcke and Dgrner, 1966; Kalra and Kalra, 1974). There is a small surge of proges— terone on the morning of proestrus which is probably adrenal in origin (Barraclough 33 33., 1971). A second larger progesterone surge occurs which i4; ovarian in origin, and coincides with the LH surge (Barraclough 33 33., 1971; Feder 33 __l_., 1971; Freeman 33 3_l_., 1976). Therefore, both estrogen and progesterone levels are elevated during proestrus and probably stimulate LH secretion in a facilitatory manner. Estrogen and progesterone can produce phasic release of LH in ovariectomized rats which is similar to the proestrous IJ! surge. In fact, the estrogen- progesterone-primed ovariectomized rat has been used frequently to study the hypothalamic mechanisms involved in ovarian steroid positive feedback. Three different models have been used. One model involves ovariectomy and estrogen administration on the day of diestrus, followed by progesterone on the early afternoon of the next, day (expected proestrus). This steroid regimen 57 produces an LH surge similar to that. observed in proestrous rats (Aiyer and Fink, 197“). A second model involves administration of estrogen to long-term ovari- ectomized rats, followed 72 hours later by progesterone. The LH surge produced is identical in timing to the proestrous surge, but is slightly greater in magnitude (Caligaris ._3 .33., 1971). The third model involves lmugtenn ovariectomized rats in which estrogen is admin- istered either by' daily injections CH‘ sc capsule implants. Unlike the other 2 models, this model is characterized by a daily LH surge between 1600-1800 hours (Legan 33 33., 1975), and demonstrated the need for progesterone to block the daily neural signal produced by estrogen (Freeman 33 33., 1976). The site of the positive feedback by estrogen appears tx> be the hypothalamus and AP. More specif- ically, the hypothalamic site is probably the preoptic- suprachiasmatic area (Flerko, 1966). Lesioning the afferents to the MBH from the preoptic-suprachiasmatic area blocked ovulation (Hillard, 19M9; Halgsz and Gorski, 1967; Halasz, 1972) and the gonadotropin surge (Palka 33 33., 1969; Blake 33 33., 1972; Weiner 33 333, 1972). Similarly, MBH deafferentation blocked the estrogen-progesterone induced LH surge (Taleisnik 33 ‘33., 1970). Goodman (1978) showed that estradiol 58 implants in the preOptic area were more effective than MBH implants in producing a LH surge. Furthermore, by measuring pituitary estradiol concentrations, he con- cluded that eXposure of the pituitary to estradiol was not enough tn) cause LH release, since the pituitary estradiol concentration in the MBH implanted rats was higher than in the preoptic implanted rats. In agree— ment with a preoptic site of action is the report that estradiol enhanced LHRH release produced by preOptic electrical stimulation, but not ME stimulation. Lastly, the preoptic-anterior hypothalamic area receives sub- stantial innervation from serotonergic and noradrenergic neurons (Ungerstedt, 1971: Brownstein 33 33., 1976), and there is considerable evidence for a serotonergic and noradrenergic role in phasic release of LH (Kalra and McCann, 1972; Coen and MacKinnon, 1976). The AP also is most probably a site of estrogen positive feedback. The [Al response to [Anni adminis- tration was increased by prior exposure of the AP to estrogen. This was shown 33 3333 (Arimura and Schally, 1971) and 33 33333 (Labrie 33 33., 1976; Drouin 33 33., 1976). These results are in agreement with the finding that the responsiveness of the AP to LHRH was greatest on the afternoon of proestrus, when estrogen levels are high (Aiyer 33 33., 1974; Zeballos and McCann, 1975). 59 Thus, estrogen positive feedback at the AP level appears to enhance the action of LHRH, and may do so by in- creasing LHRH receptor number or affinity (Kyringza 33 33., 1975; Park 33 33., 1975). To summarize the events leading to the preovulatory surge, the blood estradiol level begins to rise on late diestrus/early proestrus. Estradiol increases LHRH release into the portal blood and at the same time increases AP sensitivity to LHRH, resulting in LH release. Increased blood LH stimulates the secretion of progesterone which by as facilitatory action with estradiol, further increases AP responsiveness. The responsiveness of the AP, together with the self-priming action of LHRH, produces a LH surge of high enough magnitude to cause ovulation. C. Monoaminergjc Effects on Luteinizing Hormone Secretion The biogenic amines have become well established as important regulators of LH secretion. They are localized in close association with LHRH cell bodies in the preoptic-suprachiasmatic area and with LHRH terminals in the ME (Cuello, 1978; Weiner and Ganong, 1978). Drugs which inhibit biogenic amine synthesis or 60 deplete their stores were shown to produce dramatic changes in LH secretion. Reserpine, which depletes hypothalamic biogenic amine stores (Dahlstrgm ._3 33., 1965), blocked normal ovulation 5J1 adult female) rats (Brown, 1967) and pregnant mare serum (PMS) induced ovulation in immature female rats (Barraclough and Sawyer, 1957). Alpha-methylparatyrosine (armpt), which blocks catecholamine synthesis by competing with the rate limiting enzyme, tyrosine hydroxylase (Spector 33 33., 1965; Corrodi and Hansen, 1966), was shown to inhibit LH secretion during several different endocrine states. Thus, administration of a-mpt. on diestrus blocked the proestrous LH surge (Kalra and McCann, 1973; Kalra and McCann, 197”) and ovulation in rats (Brown, 1967; Lippman 33 33., 1967). Similarly, a-mpt blocked the LH surge in ovariectomized rats primed with estradiol benzoate and progesterone (EB-P) (Kalra .33 33., 1972). These results indicate that biogenic amines are important neuromodulators cd‘ the hypothalamo- pituitary-LH axis. The above studies are important since they established the biogenic amines as regulators of LH secretion. However, the action of a particular biogenic amine on 1J1 secretion cannot be determined from these experiments. Subsequent studies were designed to 61 differentiate the effects of NE, DA, and 5—HT on LH secretion and to determine their physiological signifi- cance as it pertained to LH secretion. It has been firmly established that NE stimulates LH secretion. Intraventricular infusion of NE increased circulating LH levels in the rat and rabbit (Krieg and Sawyer, 1976) and induced ovulation in the rat (Rubinstein and Sawyer, 1970). Results of experiments in which the noradrenergic system was disrupted sub- stantiate a stimulatory action of NE on LH release, and strongly suggest that NE is involved in the phasic release of LH. Thus, deafferentation of the MBH eliminated the proestrous LH surge and produced rats that were anovulatory (Palka 33 33., 1969; Blake 33 33., 1972; Weiner 33 33., 1972a). This surgical procedure depleted hypothalamic NE content by 60% and had no effect on DA content, suggesting that the antigonado- tropic effect of MBH deafferentation was due to disruption of the hypothalamic noradrenergic system. Similarly, chemical lesioning of catecholamine axons and terminals by injecting; 6-hydroxydopamine (6-OH-DA), a neurotoxin (Thoenen and Tranzer, 1968), into the third ventricle, blocked the proestrous and EB-P induced LH surge (Kalra, 1975). Implantation of 6-OH-DA into the ventral noradrenergic tract, a major source of hypo- 62 thalamic NE (Fuxg'l965b; Ungerstedt, 1971) or into the anterior hypothalamus, dramatically reduced hypothalamic NE content without affecting DA. This route of admin- istration also blocked the proestrous and EB-P induced LH surge (Martinovic and McCann, 1977; Simpkins 33 33., 1979a), suggesting that the inhibitory effects of 6-OH-DA on LH secretion are due to its toxic effect on NE axons and terminals. Kalra and McCann (1972) provided convincing evidence that NE was involved in the phasic release of LH. They showed that inhibition of DA-B-hydroxylase, the enzyme that converts DA to NE, by administration of diethyldithiocarbamate (DDC) or l-phenyl-3—(2-thiozolyl) thiourea (U-lu,624) (Goldstein and Nakajima, 1967), blocked the EB-P induced LH surge in ovariectomized rats. Furthermore, injection of dihydroxyphenylserine (DOPS), which is converted to NE independently of DA- s-hydroxylase, partially reversed the inhibitory effects of DDC on the proestrous LH surge and ovulation in the rat (Terasawa 33 33., 1975). FLA-63, also a NE synthesis inhibitor, was reported to block the pulsatile release of [AI in long-term ovariectomized rats (Drouva and Gallo, 1976), as did phenoxybenzamine, an a -antag- onist, in ovariectomized monkeys (Bhattacharya 33 33., 1972). 63 Studies in which hypothalamic NE activity was measured during different LH secretory states provided additional evidence that NE plays an important role in LH regulation. Hypothalamic NE turnvoer, as determined by the cx-mpt depletion method (Coppola, 1969) or synthesis of 3H-NE after 3H-tyrosine administration (Anton-Tay 33 33., 1970; Bapna 33 33., 1971), was increased by castration. This effect of castration was reversed by gonadal steroid replacement. Consistent with these observations are the reports that tyrosine hydroxylase activity was increased in ovariectomized rats (Beattie 33 33., 1972), and that 0-mpt, phenoxy- benzamine, and DDC each blocked the post-castration rise of LH (Ojeda and McCann, 1973). An increase in NE turnover 5J1 the rostral hypothalamus (Stefano and Donoso, 1967: Coppola, 1969) and ME (Selmanoff 33 33., 1976; Rance ._3 .33., 1981) was reported to occur in association with the proestrous LH surge. Similarly, Simpkins 33 33. (1979b) observed that anterior hypothalamic NE turnover was increased immediately prior to the EB-P induced LH surge. Hypothalamic NE turnover on the first proestrous day of young female rats also was reported to be increased (Advis 33_ 33., 1978). Conversely, hypothalamic NE turnover was reported to be decreased in hyperprolactinemic rats which had reduced 64 LH levels (Aylsworth and Meites, unpublished). These reported changes in NE activity associated with different LH secretion rates, together with the drug studies, are convincing evidence that hypothalamic noradrenergic neurons are involved in the regulation of LH secretion. The role of DA in the regulation of LH secretion presently is not well established, mainly due to the contradictory reports on its effects. DA has been reported to stimulate and inhibit LH secretion, while others have reported no effect. It has been shown that the steroid status or steroid environment of the animal is an important factor in determining the response of LH to dopaminergic compounds, although not all discrep- ancies can be accounted for on this basis alone (Vijayan and McCann, 19788; 1978b). Intraventricular injection of DA or apomorphine, a DA agonist, was reported to stimulate LH secretion in ovariectomized EB-P primed rats. This stimulatory action of DA or apomorphine was not. observed i1! unprimed ovariectomized rats (Vijayan and McCann, 19788), suggesting that estrogen and/or progesterone must be present for DA tx> stimulate LH release. Similarly, DA stimulated LH secretion when injected on diestrous day 2 or proestrus when circulating 65 estrogen and progesterone levels are increased, but was ineffective when injected on diestrous day 1 when estrogen and progesterone levels had run: yet begun to rise (Schneider and McCann, 1970). Systemic injection of low doses of DA were reported to increase LH levels in EB-P primed ovariectomized rats, whereas high doses actually inhibited LH release in unprimed ovariectomized rats (Vijayan and McCann, 1978b). In agreement with this latter observation is the report that injection of either apomorphine or CB—154, both DA agonists, blocked pulsatile LH release in ovariectomized rats (Drouva and Gallo, 1976), and reduced the post-castration rise of LH in female rats (Beck and Wuttke, 1977). It should be noted that in both of these studies, estrogen was very low due to ovariectomy, and may be the reason why apomorphine and CB-154 inhibited LH release. This tentative conclusion is supported by the observation that apomorphine or piribedil, a DA agonist, were unable to block the proestrous LH surge (Beck 33 33., 1978) or the EB-P induced LH surge (Simpkins .33 33,, 1979a). Rats in both of these latter 2 models have elevated estrogen and progesterone levels and may be the reason why no inhibitory effect was observed. Further evidence that DA is stimulatory to LH secretion is that HAL, a DA antagonist, decreased basal 66 LH levels and decreased hypothalamic LRF activity (Dickerman 33 33., 1974). It also was reported that HAL blocked ovulation in the rat (Boris 33 a_1_., 1970) and that pimozide, also :3 DA antagonist, reduced the preovulatory LH surge (Beattie 33 33., 1976). DA does not influence LH secretion by a direct action on the AP. Addition of DA to a hypothalamo- pituitary coincubation system was reported to stimulate LH release 33 33333, presumably by stimulting LHRH release into the medium (Schneider and McCann, 1969). This effect of DA on LHRH was confirmed 33 3333 by Kamberi 33 33. (1969) who reported that ivt injection of DA increased LRF' activity 1J1 portal blood. More recently, Rotsztejn 33 33. (1976) reported that DA stimulated LHRH release from incubated MBH of ovari- ectomized EB-P primed rats, but not from MBH of unprimed ovariectomized rats. Still others have reported that DA either inhibited (Miyachi 33 33., 1973) or had no effect (Quijada 33 33., 1974) on LHRH release 33 33333. The inverse correlation between 1J1 secretion and hypothalamic DA turnover strongly suggests that DA is inhibitory to IA! secretion. DA turnover is reduced after castration (Fuxefi 1965b) and on proestrous after- noon (Fuxé’33 33,, 1967; Rance 33 33., 1981), whereas circulating 1J1 levels are increased. Conversely, LH 67 secretion is reduced and DA turnover is increased during lactation (Ben-Jonathan and Porter, 1976). It has been suggested that the LH stimulatory effect of DA administration may be due to the conversion of DA to NE upon its uptake into NE neurons (Fux; and Hgkfelt, 1970). This hypothesis is suported by the finding that a-antagonists can block the stimulatory action of DA (Schneider and McCann, 1969). However, this hypothesis can not account for the stimulatory effect of apomorphine. Furthermore, DDC, which should block the conversion of DA to NE, only partially reduced the stimulatory action of DA (Vijayan and McCann, 1978a). Lastly, one should consider the possibility that DA is both stimulatory and inhibitory to LH secretion. A possible explanation may tn: that 2 functionally different dopaminergic neuronal systems are involved in LH control. There is some evidence that the incerto- hypothalamic DA neurons in the preoptic area are stimulatory (Kawakanfi. _3 .33., 1975), whereas the tuberoinfundibular DA system is inhibitory (Ojeda 33 33., 1974). Until recently, the effect of 5-HT on LH secretion was believed tx> be strictly inhibitory. Systemic injection of 5-HTP, the precursor of 5—HT, blocked cyclic ovulation, whereas parachlorophenylalanine 68 (PCPA), a 5-HT synthesis inhibitor, facilitated ovulation in PMS treated immature rats (Kordon 33 33., 1968). Furthermore, electrical stimulation of the raphé: which increases S-HT turnover, blocked ovulation (Carrer and Taleisnik, 1970). Different methods of increasing hypothalamic S-HT content, such as systemic injection of S—HTP or ivt injection of 5-HT decreased circulating LH levels, blocked the proestrous surge of LH, and inhibited ovulation (Kamberi 33 33., 1970; Kamberi, 1973). S-HT concentration in the ME was reported to be decreased prior to the LH surge in sheep, and may reflect decreased S-HT synthesis which may serve to facilitate LH secretion (Nheaton 33 33., 1972). S-HT turnover as determined by the ratio of the S-HT metabo- lite, S-hydroxyindoleacteic acid (S-HIAA), to 5-HT was reported to be increased during suckling and may account for the reduced LH levels observed during suckling (Mena _3 33., 1976). More recently, there is increased evidence that under certain conditions S-HT may be stimulatory to LH secretion. Kordon showed, in contrast to his earlier report (Kordon 33 33., 1968), that injection of PCPA 20 hours before the critical period was able to block PMS- induced ovulation (Kordon and Glowinski, 1972). Injection of either parachloroamphetamine (PCA) or 69 5,6-dihydroxytryptamine, both S-HT neurotoxins, or PCPA, blocked the daily LH surge in estrogen-primed, ovari- ectomized rats (Coen and MacKinnon, 1976). Similarly, I Hery t l. (1976) reported that inhibition of 5-HT by injection of' either PCPA cn- methiothepin, a 5-HT antagonist, also inhibited the estrogen-induced LH surge in ovariectomized rats, and that the inhibitory effect of PCPA was reversed by S-HTP. Chen _3 33. (1981) published similar results using PCPA and PCA, and reported that S-HTP not only reversed the effects of PCPA and PCA, but greatly potentiated the 1J1 surge. Lastly, Fajor 33 33. (1970) reported that PCPA administration delayed the onset of puberty. These results indicate that S-HT may play a stimulatory, as well as an inhibitory role. The stimulatory role of 5-HT may be limited to situations where LH is released in a phasic manner, and may mediate the stimulatory feedback action of estrogen (Kalra and McCann, 1972). Estrogen was reported to increase S-HT turnover in ovariectomized rats (Fuxé _3 33., 1974). S-HT does not alter LH secretion by a direct effect on the AP. Infusion of 5-HT into the portal vessels had no effect on LH secretion, whereas ivt injection of 5-HT reduced circulating 1J1 levels (Kamberi 33 33,, 1971). Thus, S-HT is believed to influence LH secretion via 70 hypothalamic mechanisms, presumably by altering LHRH release into the portal blood. S-HT neuron terminals are closely associated with LHRH cell bodies in the preoptic-suprachiasmatic area and LHRH terminals in the arcuate-ME region (Dahlstrgm and Fuxec 1964; Aghajanian, 1969; Ungerstedt, 1971; Kuhar 33 33., 1972; Saavedra 33 a3,, 1974), which. gives anatomical support. for a hypothalamic mechanism. The inhibitory S-HT input to the hypothalamus appears to be limited to the arcuate-ME region. This conclusion is based on the observation that this region was the only hypothalamic area in which 5—HT implan- tation inhibited LH secretion and blocked ovulation (Kordon, 1969). The hypothalamic region involved in S-HT stimulation of LH release is believed to be the suprachiasmatic nucleus. The integrity of this nucleus is essential for phasic release of LH (Clemens 33 33., 1976; Coen and MacKinnon, 1971). Furthermore lesioning the raphé'eliminated S-HT input into this area (Bjorklund 33 33., 1973), blocked PMS-induced ovulation (Meyer, 1978), and eliminated the estrogen-induced LH surge in ovariectomized rats (Coen and MacKinnon, 1976). D. Opiate Effects on Luteinizing Hormone Secretion Barraclough 33 33. (1955) reported that MOR administration during the critical period of proestrus 71 prevented ovulation in rats. More recently, Pang 33 33. (1977) demonstrated this action of MOR was due to inhibition of the proestrous LH surge, and that NAL counteracted the inhibitory effects of MOR on ovulation and IJi release. MOR cu“ methadone administration was shown to reduce basal LH and testosterone levels in the male rat, and reduce seminal vesicle and ventral pro- state weight (Cicero 33 _a__1_., 1976). We also have observed that MOR reduced basal LH levels in male rats. In addition, MET-ENK similarly reduced serum LH concentration (Bruni 33 33., 1977). Stubbs 33 33. (1978) reported similar observation in tflue human after administration of DAMME, a MET-ENK analog. Concurrent injection of NAL was shown to reverse the inhibitory effects of opiates on LH release (Bruni 33 33., 1977: Pang 33 33., 1977: Stubbs 33 33., 1978), and when given alone, NAL significantly increased serum LH levels (Bruni 33 33., 1977; Cicero 33 a3., 1979). This effect of NAL suggested that basal LH secretion may be tonically inhibited by EOP. Experiments subsequent to the studies described above have further established the EOP as important regulators of LH secretion. Included in this thesis are results of experiments in which hypothalamic mechanisms 72 involved in opiate inhibition and NAL stimulation of LH secretion were studied. Results indicating that EOP may mediate the negative feedback of gonadal steroids on LH secretion also are included. Pertinent findings from other laboratories will be discussed in each of these sections. MATERIALS AND METHODS I. Animals, Treatment, and Blood Collection Mature male and female Sprague-Dawley rats were purchased from either Spartan Research Animals (Haslett, MI) or Harlan Industries (Indianapolis, IN), and housed in our facility for at least 1 week prior to experi- mentation. Rats were kept in temperature (25°C) and light (14 h light/10 h dark) controlled rooms. Ralston Purina Rat Chow (Ralston Purina Co., St. Louis, MO) and water were provided 33 libitum throughout the periods of acclimatization and experimentation. Drugs and steroids were administered by several different routes. The diluent and route of admin- istration are stated i1: the Materials and Methods section of each experiment. B-END and dynorphin were injected ivt via the right lateral ventricle by a slightly modified procedure of deBalbian Verster 33 33. (1971). Lateral ventricular cannulae were made from PE-20 tubing (inside dia. = .015", outside dia. =.043"). A wire was inserted into the tubing in order to keep the 73 74 tubing patent while it was heated and compressed to form a bulb approximately .08" in diameter. A beveled tip was cut 4 mm from the bulb. The total length of the cannula was 20 mm and had a dead space volume of 3 ul. Rats were anesthetized with chloral hydrate (8% solution, 1 ml/’200 grams BW), and the cranium was exposed by making a 2 cm longitudinal out. A hole was drilled 2 mm lateral to the sagital suture and 1 mm caudal to the coronal suture. The beveled cannula was inserted to a depth of 4 mm and secured in place with dental cement and one anchoring screw. The incision was closed with wound clips. Rats were allowed to recover for at least 1 week. Injections were performed without anesthesia. Rats were gently held while a 20 ul Glenco micro syringe was attached to the cannula. A volume of 13 H1 was injected and the cannula was immediately heat sealed. Blood was collected by decapitation, orbital sinus puncture under light ether anesthesia, or from a chronic right atrial cannula. Cardiac cannulae were made from silastic tubing (Dow Corning, Midland, MI) having an inside dia. of .025" and an outside dia. of .0H7". The saline filled cannula was inserted into the right atrium via a small incision in the right external jugular vein approximately 32 mm above the right atrium. The cannula 75 was secured in place by sutures above and below the incision. The free end was passed underneath the skin and exited approximately 2 cm posterior to the base of the skull. Rats were housed in individual cages and allowed to recover for at least 2 days. Cannulae were flushed daily with sterile saline. 0n the day of the experiment, a 1 ml syringe with a 30 cm extension of silastic tubing was attached to the cannula and exited outside the cage. Rats were able to move freely about their cage while injections and blood samples were accomplished without disturbing the animals. Blood was stored overnight at 4°C and serum ‘was separated and frozen at -20°C until assayed. II. Radioimmunoassay of Hormones Serum PRL and LH were measured by a double antibody technique described in the NIAMDD RIA kits. These assays were nonequilibrium assays which. used specific antibodies to rat-PRL and rat-LH. Rat PRL and LH were iodinated using chloramine-t, followed by separation on a P-60 bio-gel column (Bio—Rad Labs, Richmond, CA). Antibody-antigen complexes were precipitated by addition of rabbit gamma globulin antiserum produced in sheep. 76 Serum samples were run in either triplicate or quadruplicate. Only those volumes which gave hormone values which corresponded to the linear portion of the standard curve were used. Hormone concentrations were expressed as the mean 3 standard error of the mean (SEM). Differences between group means were determined by one—way analysis of variance and Student-Newman- Keuls’ test when multiple comparisons were made. Students "t" test was used only when comparing the means of 2 groups alone. The level of significance chosen in all experiments was pi<0.05. III. Assay of DOpaminel Norepinephrine and LHRH in Brain A. Isolation and Preparation of Brain Tissue Brains were removed from the cranium after decapitation and laid dorsal side down. The hypo- thalamus was removed by cutting at the following land- marks: a) anterior, 2 mm rostral to the optic chiasm; b) posterior, immediately rostral to the mammillary bodies; c) lateral, the lateral hypothalamic sulci; d) dorsal, at the level of the anterior commissure (3-4 mm). The hypothalamus was divided into the anterior 77 hypothalamic-preoptrc area (AH) and medial basal hypo- thalamic (MBH) area by cutting immediately caudal to the optic chiasm (EXperiments IV and V). The stalk-ME (Experiment III) was dissected using fine iris scissors with the aid of a dissecting microscope. Cuts were made at a 20° angle from the ventral surface along the lateral aspects of the tuber- cinereum beginning at the posterior border of the infun- dibulum. The stalk-ME contained approximately 20 ug protein as determined by a micro-protein assay (Lowry 33 33., 1951). The ME was homogenized in 30 ul perchloric acid containing 10 mg% EDTA. The AH and MBH regions were homogenized in 10 ul perchloric acid plus 10 mg 1 EDTA /mg wet tissue weight. 1 N acetic acid was used to extract hypothalamic tissue for LHRH measurements. All tissues were sonified in a Cell Disruptor (Branson Sonic Power Co., Plainview, NY) and centrifuged at 2000 )( g for 15 min at 4°C. B. Radioenzymatic Assay of Dopamine and Norepinephrine Tissue DA and NE were assayed by a modification of the method of Coyle and Henry (1976) (see appendix A). Ten ul aliquots of supernatant or standard DA and NE (Sigma Chemical Co., St. Louis, M0)hmre incubated in the 78 presence of buffered COMT and 3 H-S-adenosyl- methionine (New England Nuclear, Boston, MA), a methyl donor. COMT was partially purified from rat liver by the method of Nikodejevic 33 33. (1970). The DA and NE metabolites, methoxytyramine and normetanephrine respectively, were separated by solvent extraction and thin layer chromatography. Amine content was determined by counting the chromatographic spots containing 3H-labeled metabolites in glass scintillation vials containing 10 ml aqueous counting scintillant (Amersham Corp., Arlington Heights, IL). Samples were counted in a: Beckman LS-lOO liquid scintillation counter (Beckman Instruments, Palo Alto, CA). C. Radioimmunoassay of Luteinizing Hormone Releasing Hormone Tissue supernatant was diluted 1 to 20 in 0.02 M borate buffer containing 0.11 gel (pH 8.4) and assayed in quadruplicate. LHRH antiserum (R-42 pool) was generously supplied by Dr. G.D. Niswender (Colorado State Univ., Ft. Collins, CO). Synthetic LHRH (Beckman) was iodinated using the lactoperoxidase-glucose method (Tower _3 33., 1977) and purified on a 0.5 x 30-CM (CM-22, Whatman Inc., Clifton, NJ) column. The specific activity of the tracer was 1800 uCi/ug (Marshall and 79 Odell, 1975). Tracer-antibody binding was inhibited in a dose-related manner by synthetic LHRH. The minimum detectable dose was 1.0 pg/tube, and 50% inhibition of tracer binding was achieved with 16 pg/tube. Unknowns were parallel to the standard curve and expressed as pg/mg wet tissue weight. EXPERIMENTAL I. Initial Studies on Opiate Stimulation and Naloxone Inhibition of Prolactin Release A. Objectives The discovery of the EOP has generated great inter- est in their possible physiological functions. Although much research has centered on the effects of these com- pounds on pain perception, behavior, and psychiatric disease (Frederickson, 1977), their relatively high con- centrations in the hypothalamus prompted us to investi- gate their endocrine effects. PK”! and methadone were shown previously to stimulate PRL secretion (Meites, 1962; Clemens and Sawyer, 1974). Therefore, it was of interest to determine the effects of several different EOP on PRL secretion. B. Materials and Methods Male Sprague—Dawley rats (200-225 g, Spartan Research Animals, Haslett, M1) were injected with MOR (Mallinkrodt Labs., St. Louis, MO), MET-ENK (Bachem, Marina Del Ray, CA), or NAL (Endo Labs., Garden City, NY) alone or together with tuna or MET-ENK. The drugs 80 81 were given ip in 0.1 m1 saline/100 g EN. The rats were injected in a randomized block design and decapitated 20 min after injection. Trunk blood was collected, and serum was separated and frozen at -20°C until assayed for PRL. Because MET-ENK is metabolized very rapidly, MET- ENK was injected ivt or the MET-ENK analog, [D-Alaz, MePheu, Met(O)-01] enekphaline (DAMME, Sandoz Inc., East Hanover, NJ) was injected ip in a second experiment in order to determine the effect of MET-ENK on PRL release. The lateral ventricle cu? male Sprague Dawley rats was cannulated as described in Materials and Methods, and rats were allowed to recover for 10 days. Rats were injected with 150 ug MET-ENK dissolved in saline or an equivalent volume (13 ul) of saline. Blood was collect— ed upon decapitation 10 min after injection. Alterna- tively, rats were injected ip with the enkephalin analog, DAMME. Doses of DAMME ranged from 1 ug to 1 mg/kg BN. Rats injected ip with saline served as con- trols. Rats were bled by orbital sinus puncture under light ether anesthesia 30, 60 and 120 min after injection. In a third experiment, the effects of B-END and dynorphin on PRL release were tested. The right lateral ventricle was cannulated. Ten days later, 23 silastic 82 cannula for withdrawing blood was placed into the right atrium, and rats were allowed to recover for 2 days. Rats were divided into 6 groups with 8 rats in each group and injected ivt with either 1 or 10 ug dynorphin (US Biochemical Corp., Cleveland, OH), 1 or 10 ug B-END (provided by Dr. C.H. Li, Univ. of Calif., San Francisco, CA), 10 ug dynorphin plus 20 ug NAL, or 13 ul saline. One ml of blood was withdrawn via the atrial cannula 10, 30, 60, and 120 min after injection. One ml of saline was replaced immediately after each bleeding in order to minimize extracellular fluid changes. Blood was allowed to clot overnight at 4°C, and serum was separated and frozen at -20°C until assayed for PRL. C. Results The results of the first experiment are shown in Table 14 The 2 higher doses of MOR significantly in- creased serum PRL concentrations 20 min after injection, whereas MET-ENK had no effect. NAL completely blocked the stimulatory action of PM”? when injected together with MOR. Injection of NAL alone significantly reduced serum PRL concentrations at all doses tested. MET-ENK, when injected ivt, significantly increased serum PRL concentration. The mean serum PRL concentra— tion 10 min after injection of MET-ENK was 44.0:2.5 83 ng/ml, and was significantly greater than the saline injected controls (10.6:l.2 ng/ml). Table l. Dose-Response Effects of Morphine, Methionine— Enkephalin, and Naloxone on Serum Prolactin Group Prolactin Controls 9.0 g 0.48 NAL (0.2 mg/kg) 4.6 : 0.nb NAL (2.0 mg/kg) 4.2 1 0.3b NAL (5.0 mg/kg) u.0 : 0.3b MOR (2.0 mg/kg) 10.1 1 0.8 MOR (10.0 mg/kg) 20.2 1 2.3b MOR (15.0 mg/kg) 18.5 3 1.2b MET - ENK (5.0 mg/kg) 11.5 3 0.6 NAL and MOR (0.2+2.0 mg/kg) 3.6 : 0.ub NAL and MOR (0.2+10.0 mg/kg) 9.0 i 1.1 NAL & MET-ENK (0.2+5.0 mg/kg) 5.4 : 0.8b : x + SEM; all data are expressed in ng/ml serum. NAL n : p<0.05 compared with controls. MOR = morphine; = naloxone; MET-ENK = methionine-enkephalin. 10 animals per group. The effects of the MET-ENK analog, DAMME, on serum PRL levels is shown in Table 2. The 2 lower doses of DAMME did not significantly increase serum PRL levels. However, injection of DAMME at doses of either 0.5 or 84 l.0 mg/kg Bw significantly increased serum PRL concentrations 30 and 60 min after injection. The stimulatory effect of DAMME was 1“) longer evident 120 min after injection. Table 2. Effect of DAMME on Serum Prolactin Concentrations (ng/ml) Groug Time in Min After Injection 30 60 120 Control 11.6: 6.43 23.7: 8.5 7.0:3.2 DAMME: 0.001 mg/kg 22.8:10.4 12.7: 3.0 ll.4:4.2 0.1 mg/kg 27.2: 6.2 10.1: 4.3 6.013.0 0.5 mg/kg 76.3: 6.1b 71.5:15.0b 6.8:3.7 1.0 mg/kg 92.5:18.2b eu.9123.3b 12.039.u a x + SEM; b p<0.05 compared with controls. n = 8 animals per group. The effects of dynorphin and B—END on PRL release are shown in Table 3. Both 1 and 10 ug doses of dynorphin significantly elevated serum 1%“. concentra- tions 10 min post-injection as compared to control values. Serum PRL concentrations in the rats given 1 ug dynorphin returned to control values by 30 min, whereas the mean serum PRL level of the group given 10 ug dynorphin was approximately 3 times greater than con- 85 trols at 30 Inin. However, this difference was not statistically significant. Serunl PRL levels 1J1 both dynorphin treated groups at 60 and 120 min post- injection were run; different from the controls. Both doses of s-END significantly increased serum PRL concentrations at 10 min, although the higher dose appeared to be less effective than the lower dose. PRL levels in the B—END treated rats remained significantly elevated 30 and 60 min after injection in contrast to the loss of effect of dynorphin at these 2 periods. Injection of 20 ug NAL together with 10 ug dynorphin, completely blocked the stimulatory action of dynorphin, and resulted in a reduction in serum PRL values. D. Discussion These results show that the EOP have in common with MOR the ability to stimulate PRL release in the rat. MET-ENKI is inactivated very rapidly’ when administered systemically, and this could account for its inability to increase serum PRL levels when injected ip. However, when MET-ENK was injected ivt or the MET-ENK analog was injected ip, serum PRL levels were significantly elevated. These results are in agreement with those of Ferland et .33. (1977) and Stubbs et .33. (1978) who reported that MET-ENK and DAMME significantly increased 86 PRL levels in the blood. Table 3. Acute Effects of Dynorphin and B—Endorphin on Prolactin Release Treatment 10 min 30 min 60 min 120 min Saline 16.1: 1.98 12.5: 1.8 10.9: 2.2 7.1:0.9 (13 ul) DYNOR 52.4: 8.4b 16.5: 3.6 5.9: 1.8 4.8:l.3 (1 pg) DYNOR 82.6:15.2b 34.3: 6.3 10.8+ 1.8 5.1+0.7 (10 ug) - - b b b B-END 76.1:12.6 63.3315.4 39.8: 7.2 6.110.8 (1 ug) b b b B-END 42.2: 4.4 S6.6:12.2 63.4:15.9 8.7:1.2 (10 ug) DYNOR+NAL 6.2: 1.8 2.0: 1.1 1.7: 0.5 4.3:O.6 (1035+20353 a x + SEM; b p<0.05 compared with controls. DYNOE = dynorphin; B-END = B-endorphin. n = 8 animals per group. Serum 1”“. concentrations also were significantly increased by B—END and (dynorphin. The stimulatory effect of B-END on PRL secretion has been reported pre- viously (Rivier 33 1., 1977; Dupont 33 33., 1979; Van Vugt 33 al., 1979), whereas this is the first report that dynorphin stimulates PRL secretion. Furthermore, this action was specific since NAL completely blocked dynorphin—induced PRL release. 87 Dynorphin was reported to be 30 times more potent than B.,-END in the guinea pig ileum bioassay (Goldstein 33 1.. 1979). It is interesting that this rank order of potency was not observed by us 33 vivo. A possible explanation is that fi-END may be more resistant to peptidase degradation by brain tissue than dynorphin. The reduction of serum PRL concentrations produced by NAL at 3 different doses in the first experiment is of great interest. This effect of NAL has been observed by others (Grandison and Guidotti, 1977; Shaar al., 2.2 1977: Guidotti and Grandison, 1978; Meltzer ‘33 a H 1978; Gold 33 33., 1979: Blank _3 33., 1980), and suggests that basal PRL levels are under the tonic stimulatory action of EOP. There also are reports that NAL had no effect on basal PRL levels and thus disagree with this conclusion (Martin 33 33., 1979; Blankstein 33 33., 1979). A possible explanation may involve stress due to bleeding since different methods of blood collection were used. 88 II. Role of Endogenous Opioid Peptides in Stress- Induced Prolactin Release A. Objectives EOP have been shown to be potent stimulators of PRL secretion (Cusan t; al., 1977; Rivier et Egg, 1977; Shaar 33 33., 1977; Dupont 33 33.. 1979). The inhibi- tory effect of NAL (n1 PRL secretion suggests that EOP may regulate PRL secretion during basal conditions (Bruni 33 al., 1977: Grandison and Guidotti, 1977; Shaar 33 al., 1977; Guidotti and Grandison, 1978; Meltzer 33 33., 1978; Blank 33 33.. 1980). It also was of interest to determine if EOP are involved in PRL secretion during non-basal states. Several different stresses have been shown to stimulate PRL secretion (Euker 33 33., 1973: Krulich 33 33., 1974; Mueller 33 33., 1974). More recently, stress was shown to produce analgesia and increase opioid concentrations in the brain (Akil 33 33., 1976; Madden et 33., 1977: Fried and Singer, 1979; Wesche and Frederickson, 1979). Thus, activation of opioid neurons in the brain by stress may be part of the mechanism by which stress increases PRL secretion. In order to test this hypothesis, we determined if pretreatment with NAL 89 could block stress-induced PRL secretion. B. Materials and Methods The effect of NAL on stress-induced PRL release was studied in the first experiment in which the stress used was exposure to ether. Male Sprague Dawley rats weigh- ing 250-300 g were divided into 2 groups of 20 each. One group was injected with 0.2 mg NAL/kg BW in 0.2 ml saline/100 g EN. The other group was injected with an equivalent volume of saline. Thirty min after injec- tions, half of the rats in the 2 groups were bled by de- capitation, and the remaining rats were bled by orbital sinus puncture under light ether anesthesia. In a second experiment, we investigated the effect of NAL on PRL release in male Sprague-Dawley rats stressed by immobilization. Thirty rats were randomly divided into 3 groups of 10 each. One group was in- jected ip with 0.2 mg NAL/kg BN and immediately sub- jected to 30 min restraint stress. The 2 remaining groups were injected with the saline vehicle, and l of these 2 groups was stressed for 30 min. Trunk blood was collected by decapitation 30 min after the time of injection. The effect of NAL on PRL release in heat-stressed rats was tested in a third experiment. Two groups of 10 90 rats were injected with either 0.2 mg NAL/kg Bw or an equivalent volume of saline and placed in an oven at 40°:2°C for 20 nun. A third group was injected with saline and returned to their cage and served as ambient temperature controls. Blood was collected by decapi- tation 20 min after the saline or NAL injection. In a fourth experiment, we determined the effect of NAL on the time response of PRL release in male rats stressed by immobilization. Male Sprague Dawley rats, 250—300 g each, were fitted with an intra-atria] cannula and allowed to recover from surgery for 3 days. Rats were randomly divided into 1% uniform groups of ii rats each. Two groups were given 0.2 mg NAL/kg Bw by ip injection. The other 2 groups were injected with an equivalent volume of the saline vehicle. At the time of the injections, a 0.3 ml blood sample was collected via the cannula from each rat. The rats were then bled a second time, 5 min after the initial blood sample was collected. At this time, restraint stress was imposed on 1 group injected with NAL and on another group given saline. Blood samples were collected from all 4 groups at 5 min intervals during the next 30 min. The 2 restrained groups remained immobilized during the 30 min sampling period while the unrestrained animals were allowed to move freely in their cages. 91 In all 4 experiments, blood was allowed to clot at 0C, and the serum was separated and frozen at -20°C u until assayed for PRL. The data were expressed as ng/ml in terms of NIAMDD-PRL-RP-l. Analysis of variance and Student—Newman-Keuls' test for multiple comparisons between groups were used to analyze the data. Differ- ences were considered significant at p<0.05. C. Results The effects of ether exposure and NAL on serum PRL concentrations are shown in Table 4. Ether exposure elevated PRL levels approximately 2-fold when compared to decapitated controls. NAL administration signifi- cantly reduced basal PRL levels and partially blocked the stress-induced increase in serum PRL concentration. Table 4. Effects of Naloxone on Basal Serum Prolactin Concentrations Serum Prolactin (ng/ml) Method of Bleedin3_ Saline Naloxone Decapitation 16.3 1 4.0a 3.7 : 0.6b Orbital Sinus 35.1 : 4.3b 23.9 : 3.2c x + SEM. b p<0.05 compared to decapitated controls. p<0.05 compared to orbital sinus bled controls. 92 Table 5. Effects of Naloxone on Stress-Induced Prolactin Release Serum Prolactin (ng/ml) Treatment Non-Stressed Stressed Stressed + NAL Restraint 9.8:l.6a 55.0:14.0b ll.6:2.4c Stress Heat Stress 18.812.1 91.0: 8.8b 38.6:l.4c a b c x + SEM. p<0.05 compared to non-stressed controls. p<0.05 compared to stressed controls. NAL = naloxone; n = 10 animals per groups. Table 5 shows that both restraint and heat stress produced about a 5-fold elevation in serum PRL levels. Injection of NAL prior to each of these stresses com- pletely prevented the rise in serum PRL induced by restraint stress, and significantly reduced the increase in serum PRL evoked by heat stress. The results of the experiment in which chronic cannulas were used to take blood samples (Figure 1), indicate that restraint stress increased serum PRL con- centrations above control values. These PRL levels remained elevated throughout the experiment. NAL given prior to restraint stress depressed the stimulatory effect of restraint on PRL release. When administered to unstressed rats, NAL consistently reduced serum PRL concentrations below control levels. 93 200 3 150 E a 5 _J 100 STRESS (I 0. 00" \ I “\ “‘ g [’1 I“\ [III 1 ‘l (I 50 1’ ‘Y’ NAL.& STRESS 3i ,, . CONTROL ”1 “'7‘1°»«....,.........,.-~"‘"'W'”w‘ NALOXONE O 5 1O 15 20 25 30 35 f M I N FIGURE 1. Effect of naloxone (NAL) on restraint stress-induced prolactin (PRL) release (n = 4). Arrow indicates time of immobilization, and vertical bars indicate SEM. D. Discussion These results demonstrate that ether, restraint and heat stress each increased PRL release, and are in agreement with previous reports (Euker t; 33., 1973: Krulich 33 33., 1974; Mueller 33 33., 1974). They also 94 show that NAL significantly inhibited the increase in PRL release induced by stress, in addition to reducing PRL levels in non-stressed rats. These results indicate that stress may increase Opioid neuronal activity in the brain. Increased opioid activity could explain stress-induced PRL release, in addition to stress- induced analgesia. The ability of NAL to block stress-induced PRL release has been confirmed (Grandison and Guidotti, 1977; Dupont 33 _3., 1979; Ragavan, 1981). In addition, ivt injection cu’ s-END antiserum was reported to par- tially block the response of PRL to stress, suggesting that stress-induced PRL release is due in part to activation of a-END neurons (Ragavan, 1981). Similar experiments using antiserum in: the other EOP have not been done, although stress has been reported to increase MET-ENE concentrations in the brain of rats (Wesche and Frederickson, 1979). Results of NAL administration during other states of elevated PRL secretion further suggest that EOP participate in the regulation of PRL secretion during non-basal conditions. NAL was reported to reduce the suckling—induced release of PRL in post-partum lactating rats (Dupont 33 33.. 1979; Miki and Meites, unpublished observation). NAL also was reported to block the 95 nocturnal PRL surge in humans (Dupont et _a_l., 1979). Multiple injections of NAL were shown to completely block the PRL surge on the afternoon of proestrus (Ieiri _3 _3., 1980). In this regard, hypothalamic MET-ENK concentrations were reported to be increased on pro- estrous morning and may be involved in producing the proestrous afternoon PRL surge (Kumar 33 33., 1979). III. Effects of Opiates on Hypothalamic Dopamine Activity; Evidence That Opiates Stimulate Prolactin Release Via a Dopaminer§3c Mechanism A. Objectives Opioid stimulation of PRL release is not due to a direct action on the AP. Thus, incubation of opiates or opiate antagonists with hemi-pituitaries or pituitary cell cultures does not elicit the response observed 33 3333 (Grandison and Guidotti, 1977; Rivier 33_33., 1977: Shaar 33_33., 1977). If a direct action on the AP is not the mechanism by which Opiates stimulate PRL release, the logical alternative is that opiates stimulate PRL release via hypothalamic mechanisms. In agreement with this conclusion is the report by Grandison and Guidotti (1977) that B-END and MOR stimulated PRL release in a NAL reversible manner in 96 rats with a deafferentated hypothalamus. Basal secretion appears to be under the tonic inhibitory influence of tuberoinfundibular DA. Administration of DA antagonists increased serum PRL levels above basal PRL levels (Meites and Clemens, 1972; Mueller _33 _3., 1976a), whereas DA agonists signifi- cantly decreased PRL levels (IA: and Meites, 1971; Mueller _3 33., 1976b). Since DA can inhibit PRL release by a direct action on the AP lactotrophs (Koch and Meites, 1970: Smalstig 33 33., 1974), a possible mechanism by which opiates stimulate PRL release could be by decreasing tuberoinfundibular DA activity. We investigated this problem by utilizing central acting drugs, as well as measuring tuberoinfundibular DA turnover. B. Materials and Methods In the first experiment, male Sprague-Dawley rats weighing 200—250 g, were given a single so injection of 50 mg IL-DOPA/kg (Hoffman-LaRoche, Nutley, NJ), 3 mg piribedil/kg (Les Laboratories Servier, Neuilly—sur- Seine, France), 20 mg amineptine/kg (Les Labs. Servier), 1 mg haloperidol (HAL)/kg (McNeil Labs., Ft. Washington, PA), or 10 mg MOR/kg (Mallinkrodt Labs., St. Louis, MO) alone or in combination with the above drugs. One ml of 97 blood was collected by orbital sinus puncture under light ether anesthesia at time 0, 1, and 2 hrs after drug administration. In a second experiment, male Sprague-Dawley rats, 225-250 g, were injected so with 0.025 mg HAL/kg, 0.05 mg HAL/kg or 5 mg MOR/kg alone or in combination with the 2 doses of HAL. A sixth group was injected so with saline and served as controls. Rats were decapitated 1 hr after injection and trunk blood was collected. The effect of B-END on ME DA turnover was investi- gated 1J1 a third experiment. A cannula was placed in the right lateral ventricle of male Sprague—Dawley rats by the method of deBalbian—Verster 33 1. (1971). They were allowed to recover for 10 days. During the recovery period, rats were handled daily tx> minimize stress from injection without anesthesia. Rats were randomly divided into 14 groups. Two groups were injected ip with 250 mg a-mpt/kg (Sigma Chemical Co., St. Louis, MO). At the same time, these rats were injected ivt with either 20 ug B-END (provided by Dr. A.A. Manian, Dept. of Psychopharmacology, NIH) in 13 ul of saline or with the vehicle. The ivt injection was repeated 30 min later. The 2 remaining groups were treated similarly except that saline was injected ip instead of o-mpt. Rats were decapitated 1 hr after the 98 initial injections and blood was collected from the trunk. The ME were removed with fine iris scissors and immediately homogenized in 30 ul of 0.4 N perchloric acid containing 10 mg EDTA/100 m1. Opiate stimulation of 5—HT activity could be an alternative mechanism by ‘which opiates stimulate PRL release. To test this hypothesis, the following 2 eXperiments were done. A cannula was placed in the right lateral ventricle of male Sprague Dawley rats, and rats were allowed to recover for 10 days. In the first experiment, 1/2 of the rats were injected ip with 300 mg PCPA (Sigma Chemical Co., St. Louis, M0)/kg BW. The remaining rats were injected with an equivalent volume of saline at this time. Rats were divided into 3 groups (n = 14) consisting of 7 PCPA pretreated and 7 saline pretreated rats and injected ivt with either 10 ug MOR, 150 ug MET-ENK, or 13 ul saline. Rats were decapitated 20 min after ivt injection and trunk blood was collected for PRL determinations. In a second experiment, the same protocol as above was followed except rats were injected ivt with either 20 ug MOR, 20 ug B-END, 20 ug NAL, or 13 ul saline. Blood was collected by decapitated 20 HHJI after ivt injection, approximately 48 hours after PCPA injection. Serum PRL was assayed by a standard RIA pro- 99 cedure using the NIAMDD kit provided by A.F. Parlow (Harbor General Hospital, Torrance, CA). DA was assayed by the radioenzymatic method of Coyle and Henry (1973), using COMT isolated from the rat liver by the method of Nikodijevic 33 33. (1970). Methoxytyramine was separ- ated utilizing the solvent extracticn: and thin layer chromatography method of Ben-Jonathan and Porter (1976; Appendix BO. The protein content of the ME was deter- mined by the method of Lowry 33 33. (1951). Results were expressed as ng DA per mg protein. Analysis of variance and Student-Newman-Keuls' test for multiple comparisons between groups, or Students "t" test when appropriate, were used to analyze the data. The results were considered to be significant at p<0.05. C. Results L-DOPA, piribedil, and amineptine each signifi- cantly decreased serum PRL concentrations 1 and 2 hrs after injection (Table 6). HAL and MOR each sig- nificantly increased serum PRL levels at the 1 hr sampling period. PRL remained elevated 2 hrs in the HAL treated, but not in the MOR treated rats. When MOR was injected in combination with each of the above drugs, the inhibitory effects of L-DOPA and piribedil on serum PRL levels were not altered. MOR prevented amineptine 100 from lowering serum PRL levels at 1 hr, hr sampling period. but not at the 2 It should be noted that MOR given alone had no effect on serum PRL at 2 hrs. MOR in com- bination with HAL did not increase PRL levels above that produced by HAL alone. Table 6. Effects of D3paminergic Drugs on Morphine Induced Increase in Serum Prolactin 0 hr 1 hr 2 hr Controls 11 3 1a 17 3 l3 3 3 saline L-DOPA 15 3 4 3 3 6 3 2b 50 mg/kg PIR 15 3 3 3 3 8 3 1b 3 ms/ks AMIN 16 3 4 6 3 5 3 1b 20 mg/kg HAL 14 3 3 37 3 33 3 3b 1 mg/kg MOR l8 3 3 36 3 l7 1 7 10 mg/kg L-DOPA+MOR 13 3 3 6 3 7 3 2b PIR + MOR l6 3 2 4 3 4 3 2b AMIN 3 MOR 16 3 4 15 3 5 3 1b HAL 3 MOR 15 3 2 36 3 34 3 2b a x 3 SEM; b p<0.05 compared to saline controls; PIR = pipiribedil; AMIN = amineptine: HAL = haloperidol; MOR morphine; n = 6 animals per group. 101 ‘40 M: 8/9roup SERUM PRL (nu/ml) 2C) O SAL HAL HAL HS HAL HAL .025 .05 5 .035 .0: MS MS FIGURE 2. Effects of haloperidol (HAL) and morphine (MS) on serum prolactin (PRL) concentrations. vertical bars represent SEM. “represents p‘<0.05 as compared to controls. Number below each treatment indicates dose of drug (mg/kg). When subeffective doses (0.025 or 0.05 mg) of HAL/ kg were injected, serum PRL levels were not altered 1 hr later (Figure 2). A subeffective dose (5 mg/kg) of MOR 102 also did run; increase serum PRL concentrations. How- ever, when doses of these two drugs were combined, serum PRL levels were significantly elevated. In the third experiment a—mpt, B-END, and B-END to- gether with a-mpt each significantly increased serum PRL concentrations over control values (controls = 9.7:0.5 ng/ml, a- mpt = 72.13103 ng/ml; B-END = 109.7:lO.2 ng/ ml;B-END +a-mpt = 126.93u.8 ng/ml). DA content in the ME of rats treated with B-END was less than 2% depleted 1 hr aftercrmpt, but DA was depleted approximately 31% 1 hr after administration of a-mpt alone (Figure 3). The results of PCPA pretreatment on MOR and MET-ENK stimulation CM‘ PRL release are shown 1J1 Table 7. Intraventricular injection of either 10 ug MOR or 150 ug MET-ENK significantly increased serum PRL levels 20 min after injection. Blockade of 5-HT synthesis by pre- treatment with PCPA had no effect on the stimulatory action of these 2 opiates, nor did it effect basal PRL levels. 112‘) 103 100 CD (3 DA CONC. ('7. or cownou) 20 FIGURE 3. Effects of e-END on ME DA turnover: Since there was no significant difference in the DA concentration of rats treated with saline and rats treated with B-END (131.4 312.7 vs 118.3318.l ng/mg protein), these values were combined. The mean was set at 1001 and is represented by the solid horizontal line. The dotted horizontal lines represent 1 1 SEM. The solid verticle bar repre- sents ME DA concentration (122.237.4 ng/mg protein) 1 hr after Chmpt and B-END administration. The hatched bar represents DA concentration (85.8:2.7 ng/mg protein) in the ME 1 hr after<+mpt injection. Both values are pre- sented as 1 of controls. The vertical lines represent 3 1 SEM based on six determinations. DA concentrations in the ME of rats treated with a-mpt and rats treated with a-mpt together with B-END were significantly different (p<0.01) from each other, as determined by Students "t" test for unpaired data. 104 Table 7. Effects of PCPA on Morphine and Methionine- Enkephalin Stimulation of Prolactin Release Treatment Serum Prolactin (ng/ml) Saline + Saline 10.4 3 1.0a Saline + PCPA 7.8 3 0.9 MOR + Saline 3H.” 3 8.9b MOR + PCPA 36.0 3 7.9b MET-ENK + SALINE 39.9 3 2.7b MET—ENK + PCPA 36.4 3 3.6b goses were PC A = 300 mg/kg; MOR=10 ug; MET-ENK=150 ug. = x 3 SEM; p<0.05 vs control. The effects of PCPA pretreatment on serum PRL concentrations 20 min after injection of either saline, MOR, B—END, or NAL are shown in Figure 4. Intraven- tricular injection of 20 ug MOR or 20 ug B—END resulted in a ”-5 fold increase in serum PRL concentrations 20 min after injection. Conversely, ivt injection of 20 ug NAL significantly reduced serum PRL concentration. Pretreatment with PCPA had no effect on the stimulatory action of these 2 opiates, nor on the inhibitory action of the opiate antagonist, NAL. Once again, PCPA pre- treatment did not alter basal PRL levels. 105 120 100 80 SERUM FRI. (fig/ml) 60 40 20 SAI. MOR NAL END 20 2O 20 FIGURE 4. Effects of PCPA cu) MOR and B-END stimulation and NAL inhibition cn‘ PRL release. Rats were pretreated with either PCPA (hatched bars) CH' saline (solid bars), ”8 hrs prior to ivt injection of MOR, NAL, and B-END. ' p<0.05 vs controls; n = 7. 106 D. Discussion In general, these reSults indicate that opiates increase PRL release by reducing tuberoinfundibular DA activity. MOR stimulation of PRL release was blocked by L-DOPA. and piribedil, both dopaminergic agents. MOR prevented amineptine, a DA reuptake inhibitor, from re- ducing serum PRL levels, presumably by decreasing the amount of DA available at the amineptine site of action. MOR had no effect on PRL levels when given together with a large dose of HAL. HAL alone may have inhibited maximally DA receptors, and thus further inhibition of tuberoinfundibular DA by MOR had no effect. When sub- effective doses of MOR or HAL were injected, no increases in serum PRL were observed, but when the 2 drugs were administered concurrently, serum PRL levels were increased significantly. This effect is believed to indicate that the combination of the 2 drugs was able to reduce DA activity sufficiently to elevate serum PRL values. A direct demonstration of the ability of opiates to reduce hypothalamic DA activity was provided in the experiment with B-END which decreased DA turnover in the ME and increased serum PRL levels approximately lO-fold. These results are in agreement with those recently re- ported by Ferland 23 El. (1977), who showed that MET-ENK 107 infusion reduced DA activity in the ME as measured fluorimetrically, and also increased serum PRL in rats. The effect of opiates on tuberoinfundibular DA activity also has been confirmed by several other laboratories (Deyo 35 al., 1979; Van Loon and Kim, 1980). Further- more, Gudelsky and Porter (1979) reported that DA in pituitary stalk plasma was significantly reduced by MOR, &END or an enkephalin analog. Together, these results strongly suggest that the stimulatory action of Opiates on PRL secretion is mediated via a reduction of tubero- infundibular DA activity. Additional hypothalamic neurotransmitters, most notably S-HT, also may mediate the stimulatory action of opiates on PRL secretion. B-END has been reported to stimulate hypothalamic S-HT turnover (Van Loon and de Souza, 1978). However, we failed to observe any effect of 5-HT synthesis blockade by PCPA on opiate stimulation of PRL release. Cusan £3 3;. (1977) also observed no effect of PCPA on opiate—induced PRL release, whereas Spampinato 33 El- (1979) reported that PCPA potentiated the response of PRL to MET-ENK. In contrast to these results which suggest S-HT does not mediate the PRL stimulatory action of opiates, it was reported that metergoline and methysergide, both S-HT antagonists, or 5,6-dihydroxytryptamine, a 5-HT neurotoxin, blocked the 108 stimulatory action of MET-ENK on PRL release (Spampinato EB 1" 19793 Koenig £5 23.. 1979). Although inferen- tial, data suggesting that EOP are involved in stress- induced (Grandison and Guidotti, 1978: Van Vugt g; 31.. 1979) and suckling-induced PRL release (DUPONt 9.1; fl-a1979) are consonant with the proposal that S-HT mediates the stimulatory action of mfiates on PRL release, since S-HT activity appears tn) be increased during both of these states (Mena £3 31., 1976; Mueller 3; 31., 1976a). Thus, in addition to reducing tuberoinfundibular DA activity, opiates may increase PRL release by stimulating hypo- thalamic S-HT activity. An interesting observation by Demarest and Moore (1981) suggests that these opposite effects of opiates on DA and 5-HT activity may in fact be related. They reported that disruption of 5-HT neuronal activity by injection cM‘ either metergoline