ovmua FINES ARE 25¢ “R D” ‘ pan ITEM Return to 500k d this Checkout from top to remove your record. NEUROTRANSMITTER REGULATION OF GROWTH HORMONE SECRETION BY John Frederick Bruni A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1978 ABSTRACT Neurotransmitter Regulation of Growth Hormone Secretion BY John Frederick Bruni 1. Sequential plasma samples collected throughout the day indicated that plasma growth hormone (GH) was released in large episodes every 3 - 3 1/2 hours. Basal GH concentra- tions were approrimately 20 - 30 ng/ml plasma, and during an episode of GH release, plasma concentrations reached upwards of 200 - 300 ng/ml. These major episodes occured at 0930 - 1030, 1200 - 1300, and 1500 - 1600 hours. 2. The effects of specific adrenergic drugs on GH re- lease were studied $2,!l22 and in zitrg. Clonidine, an alpha adrenergic agonist, stimulated GH release in 2332, This in- crease was prevented by concurrent injections of phentolamine, an a-adrenergic receptor blocker, but not by propranalol, a B-adrenergic receptor blocker. Chlorpromazine, a catechola- mine blocker, also reduced GH release. These observations were confirmed in yitgg using a pituitary-hypothalamus co- incubation system. In such a system, the hypothalamus inhibited GH release by the pituitary. Norepinephrine (NE) did not act directly on the pituitary to stimulate GH release, but removed the inhibitory influence of the hypothalamus on John Frederick Bruni GH release. The effects of NE in yitgg were reversed by con- current treatment with phentolamine, but not by propranalol or pimozide, the latter a dopamine receptor blocker. This suggests that GH is regulated at least in part by the a-adrenergic receptor for NE. 3. Para-chlor0phenylalanine (PCPA), a tryptophan hy- droxylase inhibitor, also significantly decreased serum GH concentrations 2 - 8 days after injection. Another serotonin antagonist, methsergide, significantly decreased serum GH concentrations 10, 30 and 60 minutes after injection in a dose related manner. A specific neurotoxin for serotonergic neutons,. 5, 7-dihydroxytryptamine injected intraventricu— larly significantly reduced serum GH within two days after injection. Serum GH was maximally depleted ten days after the initial injection. When 5, 7-dihydroxytryptamine was injected 45 - 60 min after desmethylimipramine, serum GH con- centrations were significantly reduced by 6 days and maximally reduced by 12 days after injections. in yitrg co-incubation of anterior pituitary halves with hypothalamic fragments re- leased less GH than anterior pituitary halves alone. When varying doses of serotonin were added to the incubation med- ium, serotonin removed the hypothalamic inhibition of GH release. Methysergide added to the incubation medium reversed the effects of serotonin in a dose related manner, indicating John Frederick Bruni that serotonin stimulated GH release. 4. Acetylcholine and the cholinergic agonists, pilo- carpine and physostigmine, increased GH release in vivo. The increase in GH release by pilocarpine was reversed by con- current administration of the cholinergic receptor blocker, atropine, whereas atropine alone decreased basal GH concen- trations. Cholinergic stimulation of GH release appears to be partially mediated through a catecholaminergic system since the response was partially inhibited by pimozide, a dopamine receptor blocker, or by phentolamine, an a-adrener- gic receptor blocker. The increase in GH release produced by pilocarpine, was also prevented by a-methyl-paratyrosine, an inhibitor of catecholamine synthesis. Atropine similarly prevented GH release induced by pilocarpine or acetylcholine in a co-incubation system. Monoaminergic blocking drugs such as para-chloroamphetamine, haloperidol, pimozide and methy- sergide, did not alter GH release induced by pilocarpine, indicating that this action is a specific function of acetylcholine. 5. Intraventricular injections of gamma-aminobutyric acid (GABA) or parenteral injection of amino-oxyacetic acid, a GABA agonist significantly decreased GH release. Con- versely, intraventricular injection of bicuculline methyl- iodide or systemic injections of bicuculline or picrotoxin, John Frederick Bruni GABA antagonists, significantly increased serum GH concentra- tions and reversed the effects of GABA injections. The effects of bicuculline appear to be mediated through the catecholamines, since alpha-methyl-para-tyrosine prevented the increase in GE produced by bicuculline injections. Sim- ilarly, bicuculline injections increased the hypothalamic NE turnover index. GABA appears to decrease GH via a neuronal mechanism since GABA does not act directly on the pituitary in yiyg or ifl,XiE£2- When GABA was co-incubated with anterior pituitary and hypothalamus, GABA further reduced GH release produced by the presence of the hypothalamus. 6. Systemic injections of morphine or the morphino- mimetic peptide, methionine enkephalin, increased GH release. Injections of naloxone, a specific opioid antagonist, de- creased GH release. Concurrent injections of naloxone with either morphone or methionine enkephalin, partially prevent- ed the increase in serum GH produced by either drug. It is possible that the endogenous opioid eptides participate in the stress induced decrease in GH release, since morphine injection prevented the stress induced decrease in GB release. TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . I. Hypothalamic Control of Anterior Pituitary Hormone Secretion . . . . . . . . . . . . . Early Observations . . . . . . . . . . . . II. III. Hypothalamic Anatomy . . . . . . . . . . . Pituitary Anatomy . . . . . . . . . . . . . Hypothalamic Hormones and Neurotransmitters Hormone Releasing Factors . . . . . . . . . Putative Hypothalamic Neurotransmitters Norepinephrine (NE) . . . . . . . . . . . . Dopamine . . . . . . . . . . . . . . . . Serotonin (5-HT) . . . . . . . . . . . . Y-Aminobutyric acid (GABA). . . . . . . . . Endorphins and Enkephalins . . . . . . . AcetYlChOIj-ne (ACh) o o o o o o o o o o o 0 Control Of Growth Hormone Secretion . . Growth Hormone Releasing Factor . . . . . Growth Hormone Release Inhibiting Hormone - Somatostatin . . . . . . . . . . . . . ii Page vi 10 13 13 17 18 20 21 21 22 25 27 27 28 IV. Noradrenergic Control of GH . . . . . . . . . Dopaminergic Control of GH . . . . . . . Serotonergic Control of GH . . . . . . . Other Putative Neurotransmitters . . Hypothalamic and Extrahypothalamic Centers for Control of GH . . . . . . . . . . . . . . ShorthOp Feedback of GH . . . . . . . . . . Hormone effects on GH . . . . . . . . . . . . Age, Nutrition and Stress Effects on GR . . . Metabolic Effects of GH . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . I. Animals and Blood Collection . . . . . . . . II. Cannulation of the Right Atria . . . . . . . III. Cannulation of the Lateral Cerebral Ventricle of the Rat . . . . . . . . . . . . . . . . . IV. Radioimmunoassay for Serum GH . . . . . . . . V. Assay of Norepinephrine and Depamine in Hypothalamic Tissue . . . . . . . . . . . . . VI. EMCo-Incubation . . .' . . . . . . . . VII. Statistical Methods . . . . . . . . . . . . . EXPERIMENTAL DATA . . . . . . . . . . . . . . . . . . I. Pulsatile Secretory Pattern of Plasma GH . . Introduction . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . II. Adrenergic Control of GH Release in ziyg and in vitro . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . iii Page 32 34 36 38 4O 42 42 48 51 54 S4 55 56 58 59 60 61 62 62 62 62 63 63 63 63 66 TABLES ll. 12. 13. 14. 15. 16. 17. LIST OF TABLES Effects of Chlorpromazine on serum GH . ........... Dose response effects of clonidine on serum GH ... Time related effects of clonidine on serum GH .... Effects of and B-adrenergic blocking drugs on serum GH ...... ............ ........... ............ Effects of clonidine and adrenergic blocking agents on serum GH ........... ........ ............ Effects of PCPA on serum GH ..................... . Effects of methysergide on GH release in vivo .... Effects of desmethylimipramine and 5,7-dihydroxy- tryptamine on serum GH ......... .................. In vitro stimulation of GH by 5-HT ...... ......... In vitro effects of methysergide on serotonin F —""‘_ o induced GH secretion . ........ .................... Effects of cholinergic drugs on serum GH ......... Effects of pilocarpine on stress induced decrease in serum GH ...................................... In vitro stimulation of GH by acetylcholine ...... In vitro stimulation of GH by pilocarpine ........ In_vitro effects of monoaminergic receptor block- ers on pilocarpine induced GH secretion .......... Direct effects of GABA on serum GH in vivo ....... Effects of naloxone, morphine, and methionine on serum GH .. ..... . ................................. Page 69 70 71 72 74 83 84 85 87 88 96 103 105 106 120 TABLES 18. 19. LIST OF TABLES Effects of naloxone, morphine, and methionine enkephalin on serum GH . .......... ..... ..... ....... 129 Effect of morphine sulfate on stress induced decrease in serum GH .............................. 130 FIGURE 11. 12. 13. 14. 15. 16. 17. LIST OF FIGURES Page Metabolic pathway for gamma-aminobutyric acid .... 23 Metabolic pathway for acetylcholine ........ . ..... 26 Normal secretory pattern of GH in four male rats . 64 Average periodicity of GH secretion in ten male rats ............................................. 65 In vitro effects of NE on GH release ............. 75 In vitro effects of PIM and NE on GH release ..... 76 In vitro effects of phentolamine and NE on GH reIease ... ....................................... 77 In vitro effects of propranolol and NE on GH release ........ ... ...... ......OOOOOOOOCOOOO ..... O 78 Effects of acetylcholine bromide on serum GH ..... 94 Time course effects of cholinergic drugs on serum GH ......................................... 95 Effects of atropine sulfate on Plasma GH ......... 98 Effects of pilocarpine, physostigmine and atropine on serum GH ............. . ............... 99 Effects of armethyl-paratyrosine on pilocarpine induced GH release ........................ ....... 100 Effects of catecholamine blocking drugs on pilocarpine induced GH relaease ......... ....... .. 101 Dose response effects of GABA on serum GH ........ 111 Effects of intraventricular injections of bicuculline methyliodide on serum GH ............. 113 Effects of GABA and bicuculline methyliodide on serum GH ...... . .................................. 114 FIGURE 18. 19. 20. 21. 22. 23. 24. 25. LIST OF FIGURES Page Time course effects amino-oxyacetic acid (AOAA) serum GH ................................... . ..... 115 Effects of GABA antagonists on serum GH .......... 116 Effects of bicuculline and a-methyl-paratyrosine on serum GH ............................. .. ....... 117 Effects of bicuculline on hypothalamic NE and DA . 119 In vitro effects of GABA on GH release .... ....... 121 In vitro effects of GABA and picrotoxin on GH reIease in co-incubation .......... ............... 122 In vitro effects of central monoaminergic blocking drugs and GABA on GH release in co-incubation .... 123 Effects of morphine on serum GH . ................. 131 vii INTRODUCTION ~The importance of the hypothalamus to the functional integrity of the pituitary gland is well established. The synthesis and release of anterior pituitary (AP) hormones are regulated in part by hypophysiotropic hormones (releasing factors) produced by peptidergic neurons contained within the hypothalamus. These peptidergic neurons participate as a transducer neuron. The peptidergic neurons receive afferent signals from the autonomic nervous system and external environment. The signal is then transferred (transduced) from a neuronal response to an endocrine response by the re- lease of an AP hormone. Since there is no direct neural connection between the AP and the hypothalamus, these releasing factors are carried to the AP through the portal vessels which connect the AP with the hypothalamus. Recent observations indicate that the hypothalamus not only sends its releasing factors to the pi- tuitary, but the hormones released by the pituitary also are transported in small amounts, retrograde, to the hypothalamus. This supports the concept that the hypothalamus not only governs pituitary secretion, but the pituitary also influ- ences the secretion of hypothalamic hormones ("short-loop" feedback). In addition to the hypothalamic hormones which alter AP hormone secretion, the hypothalamus contains high concentra- tions of neurotransmitters. These neurotransmitter sub- stances mediate the transmission of nerve impulses between neurons. Among the neurotransmitters which have been shown to be in high concentrations in the hypothalamus are dopamine, norepinephrine, serotonin, acetylcholine, gamma-aminobutyric acid and histamine. Methionine and leucine enkephalin have been isolated from the hypothalamus, but the" role in neural transmission is not clearly understood. Most of these neuro- transmitters were previously shown to alter AP hormone secretion, either by a direct action on the pituitary or through altering the release of hypothalamic releasing factors (for review see Meites 33 31., 1977). It is not within the scepe of this thesis to consider the effects of neurotransmitters on all AP hormones. I shall consider primarily the neurotransmitter control of growth hormone (GH) secretion. Generally, GB is primarily controlled by neural mechanisms in the rat. Thus hypoglycemia, glucose infusion, insulin and carbohydrates do not alter GH release in rats as in man and several. other Species. Stress and sleep are among the primary neural stimuli for CH release in human subjects but not in rats. Dopamine appears to be a potent stimulus for OH release both in man and rats. However, the role of serotonin and norepinephrine in the control of GH release are not clearly understood. A portion of this thesis is devoted to determining the role of serotonin and norepinephrine on GH secretion ig_zizg and in yitrg. Gamma-aminobutyric acid (GABA) has been shown to alter prolactin secretion, and to stimulate leutinizing hormone (LH) release, and to inhibit adrenocorticotrOpic hormone (ACTH) and melanocyte stimulating hormone (MSH) release. A portion of this thesis is devoted to determining the effects of GABA on GH release in ziyg and in yitgg. Also, several mechanisms whereby GABA alters GH release are reported. Acetylcholine (Ach) has been shown to increase LH re- lease and to inhibit prolactin release. Two preliminary reports have suggested that Ach may increase GH release. One report by Cehovic 35 51. (1972) claimed that paraoxon, a cholinesterase inhibitor, increased pituitary GH concentra- tions in the rat. However there was no evidence that paraoxon increased or decreased GH release into the blood. In human subjects,B—methylcholine, a cholinergic agonist, was reported to stimulate GH release, but inadequate controls were used. (Soulairac gt 31., 1968). In this thesis the effects of Ach, a cholinergic agonist and a cholinergic antagonist on GH re- lease in yigg and in gitgg were studied. Also, the mechanism whereby these drugs altered GH release was investigated. Lastly, the effects of morphine, an endogenous opioid peptide, methionine-enkephalin, and naloxone, an opioid an- tagonist, were investigated for their effects on GH release and their possible role in regulating GH secretion. LITERATURE REVIEW I. Hypothalamic Control of Anterior Pituitary Hormone Secretion Early Observations The earliest observations suggesting that the central nervous system (CNS) participated in pituitary function were observed by the 2nd century Greek physician and medical scientist, Claudius Galenasi (Galen). He theorized that "Spirits" from the brain were conveyed to the body, but the wastes were conveyed down the pituitary stalk, through the pituitary and voided to the exterior via the nose. This philosophy persisted for nearly 1500 years when Lower in 1670 disproved the original hypothesis. He surmised that these substances were reabsorbed into the vasculature and voided elsewhere (see Harris, 1972). Early in the 20th century the pituitary was recognized to have an important role in physiology. Among the earliest observations that the pituitary was necessary for main— tainence of homeostasis were those by Cushing and co-workers (Crowe 3E 31., 1910). The pituitary was found to produce substances necessary for growth (Evans and Long 1921, 1922), thyroid function (Allen 1919; Smith 33 31,, 1922), adrenal 4 cortical function (Allen, 1920; Smith, 1926a), gonadal function (Zondeck and Asc'zheim, 1926; Smith 1926b), and milk production (Stricker and Grueter, 1928). One of the first recorded observations that the CNS influenced anterior pituitary function was made by Haighton (1792), who observed that several hours after coitus, the female rabbit ovulated and formed corpora lutea. The precise mechanism was inexplicit, but appeared to be of neurological origin rather than due to the presence of sperm near the ovary. This phenomenon was later referred to as ”a neuroendocrine reflex“; sensory impulses from the genitoeurital tract are relayed to the brain and cause the release of luteinizing hormone (LH), and subsequently, ovulation. Today, many additional exteroceptive stimuli are known to alter pituitary hormone secretion Ceg. light, temperature, smell, taste, and touch) by action through the CNS (for reviews see Marshall, 1936; 1942; Harris, 1955). Similar studies on the structural and functional relationship of the pituitary with the brain have been expanded into the field of neuroendocrinology. Early observations suggesting that the pituitary' is regulated by the hypothalamus include those of Aschner (1912). He showed that lesions in the anterior hypothalamus resulted in gonada1 atrophy in dogs. These observations were confirmed in the dog (Nestman and Jacobson, 1940), in rats (Camus and Roussy, 1920), and in guinea pigs (Dey, 1943). Hypothalamic lesions also were reported to result in atrophy of the adrenal cortex (deGroot and Harris, 1950), and the thyroid gland (Cahane and Cahane, 1938; Greer, 1952; Bogdanove and Halmi 1953; Harris and Woods, 1958). Cahane and Cahane (1938) also observed reduced growth rates of rats bearing hypothalamic lesions. In human patients, (Armstrong and Durh(1922)andFrazier, (1936) observed that tumors of the infundibulum and hypophyseal stalk resulted in growth retardation. Further information on hypothalamic control of pituitary function came from experiments in which various hypothalamic regions were stimulated. Stimulation of the anterior hypothalamus and the median eminence induced ovulation in the rabbit (Harris, 1937; Haterius and Derbyshire, 1937), whereas stimulation of the medial basal hypothalamus enhanced thyroid function (Harris, 1948a). Stimulation of the posterior hypothalamus inhibited adrenal cortical function (deGroot and Harris, 1950). By contrast, stimulation of the anterior pituitary failed to induce physiological changes in these target organs (Markee 3E 31., 1946; Harris, 1948b). In addition to the observations that manipulation of the hypothalamus had an effect on target organ physiology, the indirect hypothalamic control of target organ physiology also was indicated after removal of the pituitary gland (Harris, 1948a; 1955; Everett, 1954; 1956). Removal of the pituitary or stalk section yielded very profound .7 effects on the body and target organs (Dott, 1923; Smith 1926a; Harris, 1937i. Harris C1948al demonstrated that the hypophysial stalk must be intact for the hypothalamus to regulate pituitary function, and following stalk section rapid regeneration of the portal vessels occurred (Harris, 1948a; Harris and Jacobson, 1950). Insertion of a small wax plate between the pituitary and hypothalamus prevented vascular regeneration, and inhibited pituitary function. Removal of the pituitary from the sella turcica and transplantation to an ectopic location resulted in maintenance of corpora lutea (Harris, 1948a; 1955; Everett, 1954; 1956), and the mammary gland (Meites, 1967). Atrophy of the gonads, thyroid and adrenals also was observed in animals bearing ectopic pituitary transplants (Harris, 1937; 1948; and 19501. When the pituitary was transplanted to its 13 3133 location, normal endocrine physiology resumed (Nikitovichsweiner and Everett, 1958). These observations demonstrated that the hypothalamus has both a stimulatory and inhibitory influence on pituitary function. Hypothalamic Anatomy- Before one can understand the hormonal and humoral influence of the brain or more specifically, the hypo« thalamus, on endocrine function, it is necessary to have an understanding of the anatomical relationship of these structures. The hypothalamus is the most ventral portion of the diencephalon (for general anatomical reviews consult DeGroot, 1959; Netter, 1968; Szentagothai, 1968; Jenkins, 1972; Knigge, 1974: C. Martin, 1976). Viewing the ventral side of the brain, rostral, medial and caudal landmarks of the hypothalamus can be ovserved: the optic chiasma, tubercinereum, and mammillary bodies, respectively. The rostral border of the hypothalamus extends from the optic chiasm dorsally, following the lamina terminalis to the anterior commisure. Dorsally, the hypothalamus is separated from the thalamus by the hypothalamic sulcus. The caudal border follows the interpenduncular fossa to the hypothalamic sulcus. The lateral border which lies between the hypothalamus and subthalamus is less discernable. However, the hypothalamus is separated from the subthalamus in part by the internal capsule, the optic tracts and the subthalamic nuclei. Histologically, one may observe 3 major gray regions in a rostral-caudal sequence. These are the anterior (surpaoptic), intermediate (tuberal), and posterior (mammilary) hypothalamic regions. Except for the arcuate nucleus and the median eminence, specific hypothalamic nuclei which are distributed bilaterally on each side of the third ventrical include these three hypothalamic regions. The anterior and tuberal regions of the hypothalamus appear to be more important in pituitary function than the posterior hypothalamic nuclei (Mayer, 1953; Halasz and Pupp, 1965; Gorski, 1966; Krey, 1975; C. Martin, 1976). The hypothalamic nuclei in a rostral- caudal direction are as follows: The preoptic nucleus lies anterior to the supraoptic nucleus which lies dorsal to the optic chiasm. Also contained within the supraOptic re- gion is the paraventricular nucleus which is responsible for antidiuretic hormone (ADH) synthesis (Bargman and Sharrer, 1951). The tuberal region consists of the arcuate nucleus (AN), the ventromedial nucleus (VMN), the dorsal medial nucleus (DMN), the lateral hypothalamus (LHA) and the dorsal hypothalamic nuclei (DHN). Finally, the caudal hypothalamus is composed primarily of mammillary nuclei and the posterior hypothalamic nucleus. The hypothalamus receives its major afferent nerve tracts from the median forebrain bundle (MFB) which conveys impulses from the hippocampus and amygdaloid formations to the anter- ior and ventromedial hypothalamus. The stria terminals sends its major imputs into the medial hypothalamus and is named the medial cortical hypothalamic tract. Additionally, the hypothalamus receives afferent fibers from the fornix, the thalamus and the mammillary peduncle. Three major efferent systems originate in the hypothal- amus. These are (1) an ascending tract to the basal forebrain area, (2) a descending tract to the brain stem which controls autonomic functions, and (3) the hypothalamo-hypophysical tract which transports the neural peptides of the posterior pituitary from their site of production in the hypothalamus to the posterior pituitary where they are stored and released. 10 PituitaryiAnatomy The pituitary gland is attached to the most ventral portion of the diencephalon, the hypothalamus, by the infundibular or hypOpohysial stalk. The hypophysial stalk consists primarily of (1) blood vessels which communicate between the hypothalamus and pituitary gland (2) nerve tracts which transport oxytocin and antidiuretic hormone from their sites of production in the paraventricular nucleus and supraoptic nucleus, respectively, to the posterior pituitary via the hypothalamo-hypoPhyseal tract, and (3) structural elements consisting of pituicytes and connective tissue. . The pituitary gland (hypophysis) is composed of primarily two types of tissue. The anterior pituitary (adenohypi- physis; pars distalis; AP), and the intermediate lobes (pars intermedia) are composed of glandular epithelial tissue. The posterior pituitary (neural hypophysis; pars nervosa, PP) is composed of axons, terminal buttons, and glial cells (pituicytes) (Buoy, 1932). Both the AP and the PP are enclosed in a fossa within the sphenoid bone, the sella turcica, (Atwell, 1926) and are covered by a specialized extension of the dura mater, the diaphragma sella. The AP is formed by an evagination of the buccal ectoderm, Rathke's pouch, whereas the PP is formed by a diverticulum of the third cerebral ventricle. As these structures converge the AP envelops the PP near the hypo- thalamus forming the tuber cinereum (see Netter, 1974). ll Histological examination of the AP discloses 3 types of epithelial tissue (1) 35% acidOphils (2) 15% baSOphils, and (3) 50% chromophobes (see Purves and Griesbach, 1956; Netter, 1974; Baker, 1974; and Rodin, 1974). The acidophils secrete the protein hormones; growth hormone (GH), and prolactin (PRL). The glycoproteins which consiSt of luteinizing hormone (LH), thyrotroPin (TSH), and follicle stimulating hormone (FSH) are produced by the basophils. Additionally, the polypeptide hormone adrenocorticotropin (ACTH) from the AP and B melanocyte stimulating hormone (MSH) from the intermediate lobe are believed to be produced by the baso- philis (see Netter, 1974; Rodin, 1974; and Baker, 1974). Recently these cells have been characterized by immunohisto- chemical staining techniques (see Nakame, 1970). The AP is perfused by the inferior hypophyseal artery which branches from the posterior communicating artery of the Circle of Willis, whereas the PP is perfused by the superior hypophysial artery which arises from the internal carotid artery. Popa and Fielding (1930, 1933) first observed the hyp0physial vasculature to be a true portal sys- tem which connected the capillary bed in the median eminence with the sinusoids of the AP. However, Popa and Fielding surmised that the blood flowed from the pituitary to the hypothalamus. Similar observations were made in living amphibians by Houssay 33 31. (1035), except they reported that the blood flowed from the hypothalamus to the pituitary. One year later, Wislocki and co-workers (1936, 1937, 1938) 12 using systemic injected dyes, confirmed the observations of Houssay in mammalian animals. Furthermore, these results were confirmed by Green and Harris (1947, 1949) in the rat, by Torok (1954) in the dog, and by Worthington (1955) in mice. Recently, Page 33 31.(1976) and Oliver 33 31(1977) observed that a small percentage of the portal blood ascends from the subependymal plexus through the pituitary and perfuses the medial basal hypothalamus, supporting at least part of the early observation of Popa and Fielding (1930, 1933) that the blood flowed up the pitaitary stalk. Closer examination of the hypophysial portal system reveals two distinct types of portal vessels; long portal vessels which arise from the capillary plexus of the median eminence which traverses the anterio-lateral portion of the infundibular stalk, and short portal vessels which arise toward the caudal infundibular region (see Netter, 1974; Adams 31;: g. 1963, 1965; Daniel 33 31., 1956, 1966). 13 II. Hypgthalamic Hormones and Neurotransmitters Hypothalamic Releasing Factors In the late 1930's several investigators postulated that the neurons of the central nervous system secreted substances into the hypOphysial portal circulation (Hensey, 1937) or directly into the general circulation (Scharrer and Scharrer, 1940). This hypothesis was further examined by Bargman and Scharrer (1951) and Scharrer and Scharrer (1954) who demonstrated that the cell bodies in the pariven- tricular and supraoptic nuclei synthesized oxytocin and antidiurectic hormone, respectively. These hormones are transported down the axons of the hypothalamo-hypophysial tract and are stored in the terminal buttons located in the posterior pituitary. This neurosecretory hypothesis was confirmed and widely accepted. Harris (1947) suggested that the AP was controlled by endocrine-like substances produced in the hypothalamus, secreted into the hypOphysial portal circulation and transported to the AP where they controlled the release of AP hormones. This "chemotransmitter hypothesis" was formulated entirely on the anatomical relationships ob- served between the pituitary and hypothalamus. Inasmuch as the "chemotransmitter hypothesis" was for- mulated to explain the control of AP function, many ques- tions remained unanswered. First, investigators had to determine whether the hypothalamus produced hormone-like 14 substances to modulate pituitary function. Working independently, Saffran and Schally (1955), and Guillemin and Rosenberg (1955) demonstrated that hypothalamic extracts 13,31333_caused adrenocorticotrOpin (ACTH) release from the AP as measured by the adrenal ascorbic acid depletion assay. Further 13_31333 experiments revealed that there were other active factors in the hypothalamus which released thyroid stimulating hormone (TSH) (Shibusawa 33 31., 1956), luteinizing hormone (LH) (McCann 33 31., 1960), prolactin (PRL) (Meites 33 31., 1960), growth hormone (GH) (Franz 33 31., 1962, Deuben and Meites, 1964, 1965; Krulich 33 31., 1965), and follicle stimulating hormone (FSH) (Mittler and Meites, 1964, Igarashi and McCann, 1964). Additionally, 2 hypothalamic release inhibiting factors were demonstrated for PRL (Pasteel, 1961: Talwalker 33 31., 1961, 1963) and GH (Krulich 33 31., 1968). Many attempts have been made to isolate and characterize the releasing and inhibiting factors. However the relative success in this area has been limited. CorticotrOphin releasing factor (CRF) was first hypothalamic hypophysio- tropic hormone located in hypothalamic tissue (Saffran and Schally, 1955; Saffran 33 31., 1955; Guillemin 33 31,, 1957). Saffran and Schally named this active principle a "releasing factor" because the compound was a chemically uncharacterized hypophysiotropic agent that released ACTH. The term "releasing hormone" applies only to those agents which have been fully characterized. Although this was the 15 first hypothalamic hormone to be isolated, and physiological significance has been attributed to this "factor", the chem- ical structure of the hormone has not been determined to date. Royce and Sayers (1958) partially purified CRF and in 1964, Schally and Bowers proposed that the structure of CRF was a tridecapeptide. Additionally an extrahypothalamic or tissue CRF was identified and was believed to function under severe stress (Witorsch and Brodish, 1972; Lymangrover and Brodish, 1973.) The second hypothalamic factor, luteinizing hormone re- leasing factor (LHRH), was first identified by McCann 33 31. (1960). Eleven years later, Schally and collquues reported the structure of LRF (Matso 33 31., 1971a,b; and Schally 33 31., 1971a). The structure of luteininzing hormone releas- ing hormone (LHRH) was determined to be a decapeptide with the following amino acid sequence: Pyro-Glu-His-Trp-Ser-Tyr-Gly- Leu-Arg-Pro-Gly-NH LHRH not only stimulated the release of 2. LE from the pituitary but also FSH 13_3133 and 13 31333 (Schally 33 31., 1971b). Therefore this releasing hormone also was named gonadotropin releasing hormone (GnRH) by some. However, the existence of a single GnRH does ont preclude the possibility of an individual LHRH and FSHRH which remain to be identified. Also in 1960, a prolactin releasing factor (PRF) was identified by Meites 33_31. PRF appears to be a peptide in nature and is separate from thyrotropin releasing hormone which has been reported to release PRL in addition to TSH (Boyd 3; 31., 1976). 16 TRF was first reported to be present in many tissues and urine in 1956 by Shibushawa 33 31. and later was claimed to be confirmed by Schreiber 33 21. (1961) who used a non- specific TSH assay. Schally 33 31. (1966) first reported that TRF from porcine hypothalami contained three amino acids: glutamic acid, proline and histidine in equimolar concentra- tion. In 1969, Burgus 33. 31., Folkers 33. 31., and Boler 33. 31. reported the sequence of these amino acids in the tripeptide hormone, which appears to be identical in all species tested. Even though releasing hormones were origin- ally thought to release only one AP hormone, TRH has been shown to release PRL 13 3133_and 13_31333 (Jacobs 33 31., 1971; Bowers 33. 31., 1971; Convey 33. 31., 1972, 1973; Mueller 33. 31., 1973; Takahara 33. 31., 1974a,b; Dibbet 33. 31., 1974; Smith and Convey, 1975). TRH also has been demonstrated to release GH in acromegalic human subjects (Faglia 33. 31., 1973; Liuzzi 33. 31., 1974) and in male and female rats (Panerai 33. 31., 1977; and Ojeda 33. 31., 1977). The existence of GH inhibiting factor (GIF) or somato- statin was first reported by Krulich 33. 31. in 1968. In 1973, Brazeau and Guillemin isolated and characterized GIF which they renamed somatostatin. Somatostatin is a tetra- decapeptide containing the following amino acid sequence: H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thy-Phe-Thr-Sey-Cys-OH, either in a cyclic or linear conformation. Somatostatin de- creased GH secretion 13 3133 and 13,31333 (for review see Vale, 1975 and Martin, 1976). However, somatostatin not only 17 decreased GH but also decreased TRH induced TSH release (Hall 33. 31., 1973; Yen 33. 31., 1974). In addition to its high concentrations in the hypothal- amus, somatostatin also is located in the D-cells of the pan- creas (Dubois, 1975). The functional significance of somatostatin in the pancreas is not clear. Somatostatin inhibits both glucagon from the a-cells and insulin from the B-cells of the pancreas (Gerich 33.31., 1974; Efendic 33. 31., 1976). These observations offer the possible use of somato- statin as a potential therapeutic agent in controlling dia- betes mellitus, since somatostatin is approximately 20 times as effective in suppressing glucagon as compared to insulin (Gerich 33. 31., 1971a, b). Somatostatin also inhibited gastrin production by the human stomach (Bloom 33. 31., 1974). (Further discussion under Somatostatin.) Aside from TRH, GnRH and somatostatin, the remaining releasing or inhibiting factors have not been chemically characterized. These include the prolactin inhibiting and releasing factors, growth hormone releasing factor, melancyte stimulating hormone releasing and inhibiting factors, or separate releasing factors for LH and FSH. Putative Hypothalamic Neurotransmitters Chemically active substances which mediate the trans- mission of nerve impulses across synapses, are neurotrans- mitters. I will refer to these chemical substances as pu- tative neurotransmitters (PN) because all criteria necessary 18 for a substance to be classified as a neurotransmitter have not been fulfilled. These criteria are: 1) The chemical must be released by the presynaptic nerve ending upon appropriate stimulation. After release, the PN must be chemically identified. 2) The transmitter must be localized to the synapse and be present in the synaptic cleft after stimulation. 3) The PN when applied microiontophoretically must mimic the post-synaptic response to stimulation of the pre- synaptic neuron. 4) The post-synaptic membrane potential should elicite similar responses when agonists to the PN are applied. 5) The postsynaptic membrane should be inhibited when blocking agents are applied (Cooper 33 31., 1974). The putative neurotransmitters to which I will limit my discussion are: the catecholamines, dopamine (DA) and nore- pinephrine (NE); the indolamine, serotonin (SHT); the neural- ly active amino acid y-amino-butyric acid (GABA); the neurally active peptides, B-endorphin and methionine-enke- phalin; and acetylcholine. Norepinephrine (NE) NE was first demonstrated to be present in the hypo- thalamus of dogs and cats (Bogt, 1954). Later, using a histo- fluorescent technique (Falck 33. 31., 1962), NE was found in high concentrations in the anterior hypothalamus and the in- ternal layer of the median eminence of rats (Carlsson 33. 31., 19 1962; Dahlstrom and Fuxe, 1964, 1965). Additionally, small quantities of NE are found in the posterior hypothalamus (Hgkfelt 33. 31., 1978). Employing the same technique, Ungerstedt (1971) showed that the noradrenergic cell bodies were localized in the locus coeruleus (also Anden 33 31., 1966a; Kobayashi 33 31., 1974), and mesencephalic reticular formation. These nuclei send their axons into the medial forebrain bundle which innervates the paraventricular nuc- 1eus, pre- and supra-optic nuclei and the ventromediabexcuaie complex (Ungerstedt, 1971). Two additional techniques which provided valuable know- ledge regarding the localization of hypothalamic NE were: the micro-punch technique (Palkovits, 1973) and a sensitive radioenzymatic assay for DA and NE (Cuello 33 31., 1973; Coyle and Henry, 1973). Palkovits 33 31. (1974a) found NE unevenly distributed throughout the hypothalamus. The anterior and medial basal hypothalamus contained the highest concentra- tions of NE. Hypothalamic NE appears to be derived entirely from extra hypothalamic noradrenergic cell bodies. Hypothalamic deafferentation results in a dramatic reduction in hypo- thalamic NE and total loss of depamine - 8 -hydroxy1ase, the enzyme which converts DA to NE (Brownstein 33 31., 1976). However, Iversen (1974) found the hypothalamus to contain small concentrations of NE in neuroglia which are refractory to extrahypothalamic lesions. 20 Dopamine The central dopamine stores are different from those of norepinephrine (Carlsson, 1959). The dopaminergic system is divided into three separate distinct systems. First the nigro-striatal dOpaminergic system which arises from the mid- brain and terminates in the basal ganglia (Andén 33. 31., 1964, 1965; Hgkfelt and Ungerstedt, 1969). The second dopa- minergic system also originates in the midbrain and termin- ates in the olfactory tubercles and nucleus acumbens (Andén, 1966b; Ungerstedt, 1971). The final dopaminergic system is confined almost entirely to the hypothalamus and is referred to as the tuberoinfundibular DA system (Fuxe and kufelt, 1966; Weiner 33. 31,, 1972). The hypothalamic dOpaminergic cell bodies consist of two major gourps: a dorsal gourp, and a ventral group (ngrklund 33. 31., 1975a; Hgkfelt 33. 31., 1978). Both nuc- lei lie lateral to the third ventricle. However, the dorsal nuclear group has not been implicated in endocrine function. Total deafferentation did not result in any detectable change in the hypothalamic DA content (Weiner 33. 31., 1972) indi- cating this DA system was confined to the hypothalamus. The ventral tuberoinfundibular system arises from the arcuate nucleus (A12) and terminates primarily in the external layer of the median eminence (Fuxe, 1963; Palkovits 33. 31,, 1974b, Browstein 33. 31., 1974; Hokfelt 33. 31., 1978). 21 Serotonin (S—HT) Regional distribution of 5—HT was first reported in the dog (Amin 33. 31., 1954). The hypothalamus, mid-rain, cere- bellum and limbic system contained varying concentrations of 5-HT (Welsch, 1969). Histofluorescence of the hypothalamus disclosed that the suprachiasmatic nucleus contained the highest concentration of 5-HT (Fuxe, 1965; Loizou, 1972). More recently, Saavedra 33. 31. (1974) employing the micro- disection method (Palkovits, 1973) confirmed this observa- tion. In addition to the suprachiasmatic nucleus, they found high concentrations of 5-HT in the median eminence and arcu- ate nucleus. Several other hypothalamic nuclei contained minuscule concentrations of 5-HT. The hypothalamus receives its major S-HT input from the pontine raphae nucleus in the brainstem. These axons unite with the medial forebrain bundle to supply S-HT to the hypothalamus and median eminence (Dahlstrom 33. 31., 1964, Ungerstedt, 1971; Baumgarten, 1972). Baumgarten 33. 31. (1974) also observed S-HT terminals in the median eminence using electron microscope and histofluor- escence methods. y-Aminobutyric acid (GABA) Like the monoamines, GABA is widely and unevenly dis- tributed in the mammalian central nervous system. In the rat, GABA is present in high concentrations in the substan- tia nigra, basal ganglia, hypothalamus and brainstem (10—15 IJmoles/g brain tissue, Okada 33. 31., 1971). Similarly, high 22 concentrations were found in these structures in humans (Perry 33, 31., 1971) and the rhesus monkey (Fahn 33. 31., 1968; Cote 33. 31., 1969). The relative neural concentrations of GABA correlate well with the enzyme glutamate decarboxy- lase (GAD), the major anabolic enzyme in GABA synthesis (Maller 33. 31., 1962; McGeer 33. 31,, 1971) (See Figure 1). Likewise, GABA-transaminase (GABA-T) has been shown to be present in the hypothalamus (Salvador 33. 31., 1959; Sheridan 33, 31., 1967). GABA was detected in high concentrations in the lateral hypothalamus and ventral medial nucleus of the hypothalamus (Kimura and Kuriyama, 1975). GAD was shown to be present in the anterior hypothalamus, suprachiasmatic nucleus, para- ventricular nucleus and dorsomedial nucleus (Tappaz 33. 31,, 1976). Positive GAD terminals also are found in the internal and external layers of the median eminence which appear to have their origin within the hypothalamus (Hgkfelt 33. 31., 1978). Nonetheless, this does not account for all the GABA contained within the hypothalamus. Therefore the hypothala- mus must receive extra-hypothalamic GABA-ergic innervation. Endorphins and Enkephalins Recently, two classes of morphinomimetic peptides have been isolated from the pituitary and brain tissue. The first of these were methionine and leucine enkephalin (Hughes 33. 31., 1975). Methionine-enkephalin (Met-Enk) is in much higher concentrations in the rat brain than leucine enkephalin 23 Glugose Acetyl CoA l‘Oxsloacetic Acid —‘-—-——I Citric Acid- a-Oxoglutaric (a-OA) Acid L . f + \ . Glutamine ------ ~\- ———————————————————— p. H \\ 3 \ in. *- Glutamic Acid 2 a-Aminobutyric Acid +aOA 1 Succinic Semialdehyde + Glutamic Acid 1 GABA a-oxoglutaric acid transaminase. Glutanic acid decarboxylase. Figure 1. Metabolic pathway of gamma-aminobutyric acid. 24 (Leu-Enk) (Yang 33. 31., 1977). Using autoradiography, immunohistochemistry and the radioreceptor assay, the dis- tribution of the enkephalins in the CNS paralleled the rela- tive distribution of the opiate receptors. (see Pert 33. 31., 1976; Simantov 33. 31., 1976, 1977; Johansson 33. 31., 1978). The enkephalins are contained in several hypothalamic nuclei involved in neuroendocrine regulation. Among these are: the preoptic, paraventricular, ventromedial, and arcuate nuclei (see Elde and Hgkfelt, 1978). Many enkephalin positive ter- minals were also found in other areas of the brain (Pert fi- a_1_., 1976; Simantov e_t_. g” 1977; Atweh 33. 31., 1977a,b). The second class of morphinomimetic peptides was iso- lated from the camel pituitary (Cox 33. 31., 1976). This compound was referred to as B-endorphin or C-fragment. Closer examination of the sequence of B-endorphin revealed that it contained the same amino acid sequence, 61 to 91, of B-lipotropin. This raised the question whether B-lipotropin was a prohormone for B-endorphin. Concurrently, Lazarus 33. 31. (1976) isolated a-, B - and y-endorphin which also possess morphinomimetic properties, all which were contained in the B-lipotropin molecule. High concentrations of B-end- orphin was also reported to be present in the hypothalamus (490 ng/g), septum (234 ng/g), and the midbrain (207 ng/g) (see Bloom 33, 31., 1978). To my knowledge, there have been no reports regarding the precise location of B-endorphin in the hypothalamus. 25 Acetylcholine (Ach) Inasmuch as Ach was one of the first neurotransmitter discovered, the distribution of cholinergic neurons has not been well documented. Early observations suggested that neural concentrations of Ach should parallel the concentra- tions of acetylcholinesterases (ACE), the major catabolic enzyme for Ach (Shute and Lewis, 1961, 1967, 1969). Some of the cholinergic nerve tracts, like those of DA, appear to be contained entirely within the hypothalamus (Brownstein 33. 31., 1976), since hypothalamic deafferentation does not alter choline acetyltransferase in the median eminence. Intrahypothalamic cholinergic nerve tracts arise from the preoptic area, and amygdala and extend to the supraoptic nucleus (Shute and Lewis, 1967). Since acetylcholine cannot be measured at the cellular level (McGeer 33. 31., 1974), the relative distribution of Ach has been correlated with the distribution of the major catabolic enzyme, acetylcholinesterase (ACE) or the major anabolic enzyme choline acetyltransferase (CAT) (see Figure 2). CAT has been measured in theexternal and internal lay- ers of the median eminence (Kizer 33. 31., 1976 a,b; Brownstein 33. 31., 1975). Similarly, ACE has been reported to be present in high concentrations in the posterior hypo- thalamic nucleus, (Uchimura 33. 31., 1975), the supraoptic nucleus, the pre-optic area, and the medial forebrain bundle (Palkovits and Jacobowitcz, 1974). Criteria for the choliner- gic system in the hypothalamus have been delineated by Hebb g. 31. , (1970). 26 Phosphetidyl-choline Fatty acids 1) Glycerophoephocholine Citrate Glucose Glycerophosphate Acetyl CoAT Choline Achetylcholine Phosphorylcholine Acetate 2 L.Choline Choline ‘ (Free 5 Lipid Bound) C Serine 1 Choline Acetylase. Acetylcholineeterese. Figure 2. Metabolic pathway for Acetylcho¢ine. Q 27 III. Control of GH Secretion Growth Hormone Releasing Factor Armstrong and Durh (1922) observed that infundibular and hypophysial tumors resulted in growth cessation in human patients. Subsequently other investigators tried different approaches to prove that pituitary growth hormone was con- trolled by the hypothalamus. Cahane and Cahane (1938) lesioned the hypothalamus; Uotila (1939) sectioned the infundibular stalk and pituitaries were transplanted from their 13_situ location to an ectopic site (Greep, 1936; Goldberg and Knobil, 1957) to assess whether the hypothalamus controlled growth hormone (GH) secretion. In all of these experiments and others, there was either a cessation or reduction in body growth in rats. In view of these results and the observa- tions of the portal circulation (Wistocki 33. 31., 1937, 1938), and the chemotransmitter hypothesis (Green and Harris, 1949) the presumption was made that the hypothalamus must contain a growth hormone releasing factor (GHRF). The first conclusive evidence that the hypothalamus contained a GEM?was reported by Deuben and Meites (1964, 1965). Using neutralized acid extracts of rat hypothalamus in a pituitary incubation system, they reported an approximate S-fold increase in GE release from a 6-day pituitary culture. Cerebral cortical extracts failed to alter GH release from pituitary culture. Thus the hypothalamus was believed to contain GHRF. These 13 31333 results were confirmed 13 31333 and 13 vivo (Dhariwal 33. 31., 1965, 1965/66; Ishida 33. 31., 28 et. 31., 1977), the cow (Convey 33. 31., 1973; Tucker 33. 31., 1975), and in human patients with acromegaly (Irie 33. 31., 1972), renal failure (Gonzalez-Barcena 33. 31., 1973), and depression (Maeda 33. 31., 1975). These effects apparently resulted from a direct action of TRH on the AP (Underschini 33. 31., 1976; Pamerai 33. 31., 1977). TRH may have mediated its effects through cyclic adenosine monophosphate (C-AMP) (Dannies 33. 31., 1976) or some other mechanism (Hinkle' 33. 31., 1977). Growth Hormone Release Inhibiting Hormone - Somatostatin While trying to isolate GHRF, Krulich 33. 31. with the use of chromatography, (1972) partially purified a peptide (GIF) that inhibited pituitary GH release 13 31333, One year later, Brazeau and co-workers (1973) isolated and characterized somatostatin (GIF) from ovine hypothalami as a tetradecapeptide. After the isolation and characterization of GIF, the hypothalamic localization of the substance became of interest. In 1975, Arimura 33. 31. developed a radioimmunoassay for GIF which would enable investigators to measure somatostatin by immunohistochemistry and radio- immunoassay in various hypothalamic regions. Radioimmuno- assay of somatostatin revealed that the median eminence contained the highest concentration of GIF in the hypothalamus (Brownstein 33. 31., 1975). High concentrations of GIF also were found in the ventromedial nucleus, arcuate nucleus, paraventricular nucleus, and to a lessor extent, in other hypothalamic areas (Brownstein 33. 31., 1975b; Palkovits 29 1965; Krulich 33. 31., 1965; Schally 33. 31., 1968; Dickerman 33. 31., 1969b; Sawano 33. 31,, 1968; as measured by tibia test for GE (Greenspan 33. 31., 1949). However, when crude hypothalamic extract was injected into animals or incubated with pituitaries there was no change in radioimmunoassayable GH (Daughaday 33. 31., 1968; Schalach and Reichlin, 1966). These discreggncies in radioimmunoassayable and bioassayable GH remain unclear to date. GHRF was purified from ovine hypothalami (Krulich, 33, 31., 1965), porcine and bovine hypothalami (Ishida 33. 31., 1965; Schally 33. 31., 1965, 1966, 1969). A GHRF was decapeptide was isolated which stimulated bioassayable, but not radioimmunoassayable GH. Later it was found to be a portion of the Schain of hemoglobin, which was considered to be an artifact of extraction pro- cedures (see Martin 33. 31., 1977). Vasopressin also has been demonstrated to release GH in the rat (Arimura 33. 31., 1967; Malacara 33. 31,, 1972; Undeschini 33. 31., 1976), in the monkey (Meyer and Knobil, 1966, 1967; Krey 33. 31., 1975), and in the human (Greenwood and Landon, 1966; Maller 33. 31., 1967; Heidingsfelder and Blackard, 1968). The effects of vasopressin (antidiuretic hormone - ADH) may be mediated directly on the pituitary since rats bearing an ectOpic pituitary under the kidney capsule released more GH when injected with ADH than their hypo- physectomized controls (Undeschini 33. 31,, 1976). Addition- ally TRH was reported to stimulate GH in the rat (Takahara 33. 31., 1974b; Kato 33, 31., 1975; Chihara 33. 31., 1976a, b; Undeschini 33. 31., 1976; Ojeda 33. 31., 1977; Panerai 30 33. 31., 1976). Immunohistochemical examination of the rat hypothalamus revealed that GIF was distributed in neuronal synaptsomes (Pelletier 33. 31., 1976; Styne 33. 31., 1977), and cell bodies (Elde 33. 31., 1975; Hgkfelt 2E- 31., 1976). GIF was located in high concentrations in the anterior hypo- thalamus; more specifically, the preoptic, anterior hypo- thalamus, and anterior ventromedial nucleus (Alpert 33. 31., 1976). Additionally, GIF was localized in very high concen- trations in the median eminence and tuberoinfundibular stalk (Pelletier 33, 31., 1974, 1975a; Setalo 33. 31., 1975). In the guinea pig, no somatostatin was present in the hippo- campus or parietal cortex (Hgkfelt 33, 31., 1974), but was measured in high concentrations in the hypothalamus. GIF is not confined to the limits of the CNS. GIF was reported to be present in the fetal (Dubois 33. 31., 1975a,b, 1976) and adult pancreas of man (Polak 33. 31., 1975; Dubois 1975 a,b; Pelletier 33. 31., 1975b; Orci 33, 31,,]976), and dog (Rufener 33. 31,, 1975). The cells of the pancreas which contained GIF are different from the and cells which secrete glucagon and insulin. These cells were designated as D cells. Immunohistochemical examination of the thyroid gland (Parsons 33. 31., 1976) and gastrointestinal tract (Polak 33. 31., 1975; Rufener 33. 31., 1975, and Dubois 33. 31., 1976) showed GIF containing cells in these organs also. The first reported physiological action of GIF was 31 depression of GH 13 3133 and 13 31333_(Brazeau 33. 31., 1973; Belanger 33. 31., 1974; Stachura, 1976). Treatment of rats with an anti-somatostatin elevated serum GH concen- trations without changing the intervals of episodic release (Ferland 33. 31., 1976; Arimura and Schally, 1976). Antisera to GIF also prevented the stress induced decrease in serum GH concentrations in rats (Arimura 33, 31., 1976). GIF inhibited the insulin induced hypoglycemia stimulation of serum GH in human patients (Hall 33. 31., 1973). These results suggest that GIF is a physiological regulator of GH release and synthesis. Electrical stimulation of the ventromedial nucleus and the basolateral amygdala resulted in an increase in serum GH concentrations. This increase was prevented by prior injections of GIF in the rat (Martin, 1974). The effects of morphine on GH release were also prevented by concurrent injections of GIF (Martin 33. 31., 1975). Similarly GIF prevented the increase in serum GH concentrations due to pentobarbital anesthesia in rats (Brazeau 33. 31., 1974). The insulin (Hall 33, 31,, 1973), and TRH (Carlson 33. 31., 1974) induced increase in serum GH concentrations was also attenuated by prior administration of GIF. Other effects of GIF are well documented ( see Martin 33, 31., 1977). The effects of GIF on the pituitary are not limited to the regulation of GH secretion. GIF inhibited ACTH in human subjects with hypersecretion of ACTH (Nelson's syndrome, Tyrrell 33. 31., 1975). GIF also inhibited TRH induced TSH secretion without altering basal serum concentrations of TSH 32 or prolactin concentrations (see Martin 33_31., 1977). Inasmuch as the highest concentrations of GIF are located in the hypothalamus and gastrointestinal tract, many investigators believed that GIF may participate in the regulation of blood glucose. GIF was reported to in- hibit glucagon and insulin secretion from the pancreas of cats (Koerker 33 31., 1974), rats (Koerker 33 31., 1974; Orci 33 31., 1976; Brown 33 31., 1976), and human subjects (Yen 33 31., 1974; Gerich 33 31., 1975 a,b). Presumably, GIF inhibited the actions of glucagon on liver by inhibiting C-AMP accumulation (Oliver 33 31., 1976; Vinicor 33 31., 1977), which was similar to the effects of GIF on pituitary cyclic nucleotides (Kaneko 33 31., 1973; or Boss 33 31., 1975). These studies revealed a potential application of somatostatin as an adjunct treatment with insulin in control of diabetis mellitus in juvenile and maturity onset diabetes. Noradrenergic Control of GH The most widely accepted hypothesis for the control of GH regulation is that which has been adopted through manipulation of brain neurotransmitters. Generally, neurotransmitter substances which enhance GH release. either stimulate the release of GRF or inhibit the se- cretion of GIF. However, GRF has not been isolated, and to my knowledge, measurement of GIF concentrations or turnover under various neurOpharmacological manipulation has not been pursued to date. GH appears to be under direct stimulatory action 33 of the central noradrenergic pathways. Several methods have been employed to study the role of NE in the regulation of GH in primate and sub-primate species. L—dopa, the immediate precursor to DA which is converted to NE by DA-B-hydroxylase, stimulates GH release in rats (Chen 33. 31., 1974) and in human patients (Martin, 1972). The effects of l-dopa appeared to be mediated through the a-adrenergic receptor because the effects of l-dopa were partially prevented by the a-adren- ergic blocking agent, phentolamine (in rats, Martin 33. 31., 1977, Kato 33. 31, 1973; in humans, Liuzz 33. 31., 1971; Heidingsfelder 33. 31., 1968) in baboons (Toivala 33. 31,, 1971). Sheep responded opposite compared to rats to G-receptor blocker phenoxybenzamine with an increase of serum PRL and GH concentrations (Davis and Borger, 1973). NE has opposite effects in urethane anesthetized rats (Kato 33, 31,, 1973 Collu 33. 31., 1972). NE decreased serum GH and this decrease was inhibited by prior admin- istration of the B-receptor blocker propranalol. There has long been this decrepency between urethane anesthetized rats and rats handled under ether, pentobarbital or merely through sampling without anesthesia (Martin, 1976). The B-adrenergic receptor appears to have little or no effect on GH secretion (Bruni, Ph.D. thesis). The secretion of GH via adrenergic agents appears to correlate well with the diurnal episodes of GH release. A good correlation appears to exist between central adrenergic and serotonergic activity and other neural transmitters modulating this effect 34 (J.B. Martin, personal communication). The direct effect of NE on GH release may be excuded since NE does not stimulate the pituitary directly (MacLeod, 1969; MacLeod 33. 31., 1970, (Bruni, Ph.D. thesis) but requires the presence of the hypo- thalamus (Bruni, Ph.D. thesis). Dopaminergic Control of GH The effects of DA and DA precursors on serum GH in hu- man subjects and in rats had been somewhat of a mystery until the past several years. Several methods have been used to determine the effects of DA on GH release in humans and in animals. One method of administration of monoamine pre- cursors in humans to treat depression (Carrol, 1971). After injections of L-dopa or tyrosine there is a marked increase in DA and NE in the brain. Another method is injection of apomorphine, a DA receptor stimulator (Andén 33. 31., 1967). or piribedil, another DA agonist. In general, DA agonists and precursors stimulate GH release in normal human patients. L-dOpa the immediate precursor to DA stimulated GH release in normal human patients (Boyd 33. 31., 1970; Parlow 33, 31., 1972; Kansal 33. 31., 1972; Millar 33. 31., 1973; Silver 33. 31., 1974). and Parkinsonian patients (Parlow 33. 31., 1972). However, in patients with pituitary insufficiency, l-dOpa stimulated GH to a lesser degree (Laron 33. 31., 1973). Apomorphine, a DA agonist, similarly elevated serum GH in human patients (Lal 33. 31., 1972; Brown 33. 31., 1973; Maany 33. 31., 1975; 35 Nilsson, 1975). However, in patients with acromegaly (Cryer and Daughaday, 1974; Ghiodini 33. 31., 1974), Huntington's Chorea (Podolsky and LeOpold, 1974) or obesity (Fingerhat and Krieger, 1974), l-dopa decreased serum GH concentrations. The reason for this pathological discrepency is not known. DA could possible be affected by other neurotransmitters like GABA or Ach (Perry 33, 31., 1973; McGeer 33. 31., 1973; Bird and Iverson, 1974). Injections of DA into the lateral ventricle of rats induced a depletion in pituitary GH content indicating that DA stimulated GH release in the rat (Maller 33. 31., 1968). Similarly, systemic injection of 1-dopa (Chen 33. 31., 1974; Smythe 33. 31. , 1975; Bruni, Ph.D. thesis) increased serum GH in rats, dogs (Lovinger 33. 31., 1974), and monkeys (Chambers and Brown, 1976). Other workers have reported either no change (Kato 33. 31., 1973) or inhibition (Maller 33. 31., 1973) of GH release in urethane anethetized rats. Martin (1976) attributes these differences to the methods of anesthesia.‘ Two other methods have been used to clarify these discrepencies. Mueller 33. 31. (1976), showed that injections of apomorphine or peribedil increased serum GH in unanesthe- tized rats. This increase was prevented by prior admin- istration of haloperidol, a DA receptor blocker. Similarly, Willoughby 33. 31.(1977) partially prevented the episodic ~release of GH by butaclamol, another DA receptor blocker. Simon and George (1975) observed that the diurnal variation in brain dopamine concentrations correlated with changes in 36 serum GH concentrations. From these results one may conclude that DA stimulates GH release in rats, dogs, monkeys and humans. The mechanism whereby DA stimulated GH release appears to lie within the hypothalamic since DA does not act directly on the pituitary to stimulate GH release or synthesis (MacLeod, 1969; MacLeod 33. 31., 1970). Serotonergic Control of GH The role of the indoleamine, S—HT, in the control of GH secretion is less conclusive than that of DA and NE. Several experimental models have been used to determine the role of 5-HT in control of GH Collu 33. 31. (1972) reported that intraventricular injections of 5HT into urethane anesthetized rats dramatically increased serum GH concen= trations. This increase was completely prevented by prior injection of phenoxybenzamine, which blocks the a-adrenergic receptor, as well as the serotonergic receptor. Similarly, systemic injections of S-hydroxytryptOphan (SHTP), the immediate precursor to»S-HT,increased serum GH concentrations in unanesthetized rats, (Smythe and Lazarus, 1973). The release of GH due to 5HTP injections was inhibited by prior injection of cyproheptadine, a proposed S-HTantagonist (Smythe 33. 31., 1975) or methysergide (Meites 33. 31., 1977,Bruni, Ph.D.. thesis). In contrast to these results, Maller 33, 31. (1973) showed that intraventricular injections of S-HTsignificantly decreased serum GH concentrations in rats. Systemic injections of 5HTP 37 which elevated hypothalamic 5+HT did not alter serum GH concentrations (Maller 33 31., 1973). However, systemic injections of para-chloroamphetamine, a drug which decreases brain serotonin, increased serum GH, indicating that the serotonergic system inhibits GH in the rat. In the monkey (Chambers and Brown, 1976), parenteral injections of 5HTP increased serum GH concentrations. Tryptophan (Maller 33 31., 1974) or 5HTP (Imura 33 31., 1973) increased serum GH concentrations in human subjects. The discrepencies in the above mentioned results may be due to the lack of specificity of the drugs used and the time when blood samples were taken. Butcher 33 31. (1972) and Wurtman and Fernstrgm (1972) showed that systemic injections of 5HTP resulted in an increase in hypothalamic serotonin. This increase, however, was not confined to serotonergic neurons. These investigators demonstrated that any neurons which contain l-aromatic amino acid decarboxylase (dOpa decarboxylase) are capable of decar- boxylating dopa to DA or 5HTP to S—HT. SsHTin a dopaminergic or adrenergic neuron will displace the endogenous neuro- transmitter initially and later act as a false transmitter. After specific neurotoxins were isolated, the effects of 5,7-dihydroxytryptamine (Baumgarten 33 31., 1972a, b; Baumgarten 33 31., 1974) on brain and hypothalamic 5HT content were studied. 5,7-dihydroxytryptamine when injected together with desmethylimipramine, resulted in a significant depletion of brain and hypothalamic 5HT. These drugs also inhibited the morning episodes of GH release without altering the 38 periodicity of GH release (Bruni, Ph.D. thesis). Also, methysergide attenuated the episode release of GH from the pituitary (Martin 33 31., 1978; Bruni, Ph.D. thesis). Collectively, these results suggest that S-HT is a positive modulator of GH secretion. Despite some of the earlier disagreement as to whether 5-HT is stimulatory or inhibitory to GH release, ablation of the serotonergic sys- tem results in reduced GH release. Therefore, one amay con- clude that S-HT is a stimulator of GH secretion. Other Putative Neurotransmitters Acetylcholine, GABA, met-enkephalin and B-endorphin all have been implicated in the control of pituitary function (see Maller 33 31., 1977). However, little information on the effects of these agents on the secretion of GH is yet available. Paraoxon, an anti-cholinesterase, was reported to increase pituitary GH concentrations in rats (Cehovic 33_31., 1972). However Cehovic 33 31., (1972) did not mention whether paraoxon increased the release or synthesis of GH. Additionally, Soulairac 33 31. (1968) reported that injections of 8-methylcholine, a cholinergic agonist, increased serum GH concentrations in humans. This study was not conclusive since he did not have adequate controls throughout his experiment. Ach pilocarpine, and physostigmine, all cholin- ergic agonists increased serum GH in rats, as will be shown by this thesis. These effects were inhibited by the muscarinic , _ it!!! 39 receptor blocker, atropine. Atropine also inhibited the morning episodic release of GH (Bruni, Ph.D. thesis). These results suggest that the cholinergic system may participate in the physiological control of GH secretion. The effects of Ach appear to be mediated through a neural mechanism since Ach did not stimulate GH release directly from the piruitary but required the presence of hypothalamic tissue (Bruni, Ph.D. thesis). GABA appears to inhibit GH secretion (Bruni 33 31., 1977a). Similarly the GABA agonist, aminooxyacetic acid, inhibited GH release. The GABA antagonists, bicuculline and picrotoxin, increased serum GH concentrations 13 vivo (Bruni 33 31. 1977a, Bruni, Ph.D. thesis). GABA did not act directly on the pituitary to inhibit GH secretion 13 3133 and 13 31333, as will be shown. The endogenous Opioid peptides also appear to modulate pituitary hormone secretion (Bruni 33 31., 1977b). B-endorphin was reported to increase serum GH concentration 13 3133_&Rivier 33 31., 1977). Similarly met-enkephalin increased serum GH 13 3133 (Bruni 33 31. 1977b; Shaar 33 31., 1977). These effects probably require a neural mechanism since met-enkephalin did not act directly on the pituitary to alter GH release 13 vitro (Bruni, unpublished). 4O Hypothalamic and Extrahypothalamic Centers for Control of CH The hypothalamic sites which primarily control GH secretion appear to be located in the medial basal hypo- thalamus. Isolation of the medial basal hypothalamus in- creased serum GH concentrations (Rice 33 31., 1976; Mitchell 33 31., 1973). The primary loci within the medial basal hypothalamus which control GH are the median eminence and ventromedialarcuate complex. Stimulation of the median em- inence in rats increased radioimmunoassayable serum GH con- centrations in rats (Bernardis and Frohman, 1971) and inhib- ited GH release in sheep (Malven, 1975). The inhibition in sheep was attributed to an increase in secretion of GIF even though GIF concentrations were not measured. Stimulation of the ventromedial nucleus also resulted in elevated serum GH concentrations (Bernardis and Frohman, 1971; Martin, 1972). Lesions of the ventromedial nucleus de- creased GH concentrations as measured by the tibia test (Bernardis 33 31. 1963; Frohman and Bernardis 1968) and body growth. The hypothalamus receives afferent neurons from the amygdala, hippocampus lucus coeruleus; and raphae nucleus (see section I). The ventromedial nucleus receives affer- ent neurons from both the amygdala and hippocampal form- ations. The corticomedial and basolateral amygdala send efferent fibers to the ventromedial nucleus, whereas the hippocampus sends it efferent fibers to both the ventro- medial nucleus and arcuate nucleus. 41 Lesions of the amygdala increased radioimmunoasayable GH in the rat (Newman et al., 1967). These observations were con- firmed by Martin (1972, 1974). However, Martin reported that stimulation of the basolateral amygdala and mesencephalic in- terpeduncular nucleus increased plasma GH concentrations where- as stimulation of the cortiomedial amygdala inhibited plasma GH concentrations in the rat. The difference in corticomedial amygdala stimulation and basolateral amygdala stimulations are not clearly understood since both of these areas send efferent fibers to the ventromedial. nucleus. Stimulation of the hippocampus also resulted in elevated plasma GH concentrations (Martin, 1972). These effects from hippocampal stimulation may have resulted from the hippocompal efferents which inner- vate the amygdala (Rosene and VanHoesen, 1977). The extrahypothalamic structures which send efferent neurons to the hypothalamus may participate in the stress in- duced increase in serum GH in humans (Greenwood and Landon, 1966) and monkeys (Mason 33 31., 1974) and the decrease in serum GH in rats (Collu 33 31., 1973). Also these structures may be partially responsible for the episodic release of GH in all species thus far examined (see Martin, 1976). Before one may conclude that these structures are involved in the physiological control of GH, one must first examine the neur- onal activity of these structures throughout the day and dur- ing stress and correlate the activity of these neurons with serum GH concentrations. 42 Shortloop Feedback of GH Since GH does not have a specific target organ which produced another hormone to control its secretion, a "short- loop" negative feedback mechanism for GH was prOposed by Voogt 33 31. (1971). Implantation of human GH pellets into the median eminence decreased anterior pituitary weight, and decreased serum and pituitary GH concentrations. These obser- vations were confirmed by implanting GH into the hypothalamus or GH secreting tumors systemically (Brown and Reichlin, 1972). In human beings and monkeys, administration of GH, prior to arginine infusion or insulin induced hypoglycemia, prevented the increase in plasma GH due to these stimuli (in humans, Abrams 33_31. 1971; in monkeys, Sakuma and Knobil, 1970). The recent observation of Oliver 33 31., (1977) that pituitary hormones can be collected from the descending portal vessels in very high concentrations (2000-3000 ng/ml) supports the concept of a "short loop" feedback control of AP secretion. If GH is secreted by the AP and transported back into the hypo- thalamus to stimulate GIF and inhibit GHRF secretion, this may explain in part the episodic release of GH from the AP. Hormone Effects on GH OH is controlled in part by hormones from other endocrine organs. Among these are oxytocin, antidiuretic hormone (ADH), thyroid hormone, adrenal hormones, insulin, and gonadal steroids. 43 Oxytocin (Malacana33 31,, 1972) stimulated GH in estrogen- progesterone primed male rats. The other posterior pituitary hormone ADH was once thought to be GRF (see Growth Hormone Releasing Factor). Pitressin and lysine-vaSOpressin stimulated GH release in the rhesus monkey (Meyer and Knobil, 1966,1967). In monkeys which have had their medial basal hypothalamus deafferentated, ADH elicited a larger increase in plasma GH concentrations (Krey 33 31., 1975). Similarly in human patients (Greenwood and Landon, 1966; Heidingsfelder and Blackard, 1968) and in rats (Arimura 33 31., 1967),ADH increased serum GH concentrations. Thyroidectomy in rats resulted in growth retardation (Koneff 33 31., 1949) and a decrease in pituitary GH concen- trations(Knigge 1958; Soloman and Greep 1950; Meites and Fiel 1967). This decrease in pituitary GH may have resulted from several effects of thyroid hormone. Purves and Griesbach (1956) observed that thyroidectomy caused degranulation of pituitary acidophils. This degranulation may be reversed by either thyroxine or cortisol (Meyer and Evans, 1964). Meites and Fiel (1967) demonstrated that thyroidectomy resulted in a decreased concentration of hypothalamic GHRF which could be replenished by thyroxine therapy. Presumably, pituitary GH concentration was decreased after thyroidectomy (Lewis 33 31., 1968; Hervas 33 31., 1975) or propylthiouracil treatment (Daughady 33 31., 1968; Peake 33_31., 1973). 13 31333 experiments revealed that pituitaries from thyroidectomized rats synthesized smaller amounts of GH as revealed by H3 44 leucine incorporation into GH (Augustine and MacLeod, 1975). In urethane anesthetized rats, serum GH concentrations are increased after propylthiouracil treatment (Chihara 33 31., 1976b). These contradictory results were probably a function of the method of anesthesia. The effects of the adrenal steroids on pituitary and serum GH are not well understood. Frantz and Rabkin (1964) observed that glucocorticoid therapy in human patients prevented the insulin induced hypoglycemia discharge of pituitary GH. These observations led investigators to believe that corticoid induced dwarfism was a result of suppression of pituitary GH secretion. However, if one considers some of the earlier work, (Marx 33 31., 1943), there appears to be a direct antagonism between the glucocorticoids and GH on the growth of the epiphysis in hypophysectomized rats. These results were later believed to be the result of competition of cortisol with the somatomedins (Mosier and Jansons, 1976), but cortisol did not alter somatomedin production from the liver. Therefore the decrease in body growth was attributed to a decrease in food intake in rats. Reichlin and Brown (1960) observed that adrenalectomy resulted in impaired growth but pituitary GH concentrations were unaltered and food intake was reduced. Other investigators thought that cortisol stimulated GH synthesis by the pituitary (Meyer and Evans 1964). Cortisol injected into propylthiouracil treated rats caused acidophil regranulation which was believed to indicate an increase in 45 pituitary GH concentration. However, quantitative measurements of GH failed to confirm this observation (Daughaday 33 31., 1968). The effects of the adrenal steroids on SE secretion have not been well documented. Several experiments which need to be done include: 1) the effects of adrenalectomy on the pulsatile release of GH, 2) the effects of glucocorticoid replacement on this pulsatile secretion, immediately following thearapy and several hours after replacement therapy 3) the diurnal rhythm of GH release in patients with Cushing's disease and Addison's disease. The study by Grimm 33_31. (1974) is instructive. These investigators reported on the effects of high serum cortisol in renin hypertensive patients. Plasma GH concentrations or GH secretory patterns were found to be unaltered in these patients. The first evidence that estrogen effected GH secretion in human subjects was reported by Frantz and Rabkin in 1964 (see Reichlin, 1974). They randomly sampled blood from women and men and found no change in basal GH concentrations. However in patients requiring bed rest, GH concentrations were higher in women. They also observed a decrease in serum GH concen- trations in post-menopausal women indicating that estrogens must stimulate GH secretion. However, estrogen treatment was known to ameliorate symptoms of acromegaly (see Reichlin, 1974) indicating that estrogens inhibit GH release in humans. These discrepencies could be explained if estrogen in some way inhibits systemic utilization of GH. 46 Estrogens in rats appears to inhibit GH release 13 vivo (Gaarestrom and Levie, 1939) via inhibition of body growth. Daughaday 33 31. (1969) reported similar results in ovariec- tomized rats given estrogen and observed an increase in serum GH as measured by the tibia test. These results were con- firmed in the mouse by radioimmunoassay (Sinha 33 31., 1972). The mechanism whereby estrogen inhibits GH release appears to be by acting directly on the pituitary (MacLeod and Lehmeyer, 1974; Dannies 33 31., 1977). Pituitaries, in- cubated in the presence of estradiol, decreased the synthesis of GH as measured by incorporation of tritiated leucine. (MacLeod and Lehmeyer, 1974). Dannies 33 31. (1977) ob— served that pituitary cells incubated with several antiestro- gens synthesised more GH and that these effects were reversed by estradiol. However, GH release 13 31333_is not affected by the stage of the estrous cycle and therefore estrogens probably sensitize the pituitary to the action of GIF or desensitize the pituitary to GRF. These experiments have not been performed. Progestational compounds appear to be inhibitory to GH secretion in human subjects (Lawrence and Kirsteins, 1970). Medroxyprogesterone decreased serum GH in normal and acrome- galic patients. These results were exemplified by Yen 33 31. (1970), who showed that plasma GH concentrations were lower in pregnant than in non-pregnant control women after insulin induced hypoglycemia. The male gonadal steroid, testosterone appears to increase plasma GH concentration in castrated male rats (Daughaday 3331., 47 1968). Also in human patients, testosterone enhanced the GH response-to insulin (Deller 33 31., 1966). The most dynamic hormonal effects on the regulation of GH secretion in humans are elicited by insulin. In 1963, Roth 33 31. first demonstrated that insulin induced hypoglycemia greatly increased GH secretion. These observations were subsequently confirmed by many other investigators (Slick 33 31., 1963; Frantz 33 31., 1964; Tchobroutsky 33 31., 1966; Millar 33 31., 1973; Hampshire 33 31., 1975). In search for a mechanism which may be responsible for the glucose depression of GH in monkeys (Blanco 33 31. 1966) or insulin induced hypoglycemia stimulations of GH in humans (see above), Oomura 33 31, (1969) reported the existence of glucose receptors in the ventromedial nucleus. Mayer (1953) showed that the neurons in the ventromedial nucleus increased their firing rate following increases in blood glucose. Injec- tions of gold thioglucose into mice specifically lesioned the ventromedial nucleus and decreased serum GH concentrations (Sinha 33 31., 1975). However, the ventromedial nucleus is not the only hypothalamic center responsive to changes in glucose concentrations. Himsworth 33 31. (1972) microinjected 2-deoxy-D-glucose, a compound which interferes with the metab- olism of glucose into several hypothalamic loci in monkeys. He reported that injections into the ventromedial nucleus resulted in decreased plasma concentrations of GH. GH hyper- secretion was produced in those monkeys in which 2—deoxy-D- glucose was infused into the lateral hypothalamic area. One may surmise that there is a reciprocal relationship between 48 the lateral hypothalamic and the ventromedial hypothalamus in the control of GH secretion (see Reichlin, 1974). Insulin induced hypoglycemia does not stimulate GH release in rats (see Martin 33 31., 1977; Reichlin, 1974, and Brown and Reichlin 1972). There was no correlation between the episodic release of GH and that of insulin in the rat (Tannenbaun 33 31., 1976). Therefore glucose does not appear to be a major regulator of GH in the rat. Age, Nutrition and Stress Effects on GH The relation of serum GH concentrations to the rate at which an animal grows is very poor. In the human subject. fetal GH was detectable early in gestation and reached a peak at about midgestation, and declined until delivery (Kaplan 33 31., 1972). However, the relative growth of the fetus was independent of plasma concentrations of GH (Reichlin, 1973). Following partuition, GH concentrations were relatively high and declined soon thereafter (Cornblath 33 31., 1965). CH concentrations remained relatively constant throughout childhood and adulthood (Reichlin, 1974). In the rat, pituitary GH was detected late in gestation and increased dramatically until the first week of neonatal life (Birge 33_31., 1967). Serum GH concentrations decreased steadily from gestation until day 12 of neonatal life (Walker 33 31., 1977). From day 12 until day 20 serum GH concen- trations increased steadily then another peak was observed on day 20 (Blazquez 33 31., 1974; Walker 33 31., 1977). Serum GH concentrations again declined, exhibiting several peaks, until 49 immediately after puberty when serum GH concentrations increased to another zenith at about 52 days of age (Walker 33 31., 1977). These changes in serum GH concentrations from birth to puberty negatively correlated with the hypothalamic concentrations of somatostatin (Walker 33 31., 1977). Subsequently, serum GH concentrations remained relatively unchanged until old age, at which time a slight, but insignif- icant decrease in serum GH concentrations occurred (Bruni, unpublished). Serum GH concentrations are alsoégffected by diet (for review see Reichlin, 1974). A decline in blood glucose con- centrations resulted in an increase in blood GH concentrations in human patients (Luft 33 31., 1966) and in monkeys and dogs (Tsushima 33 31., 1971). Opposite effects of insulin induced hypoglycemia were observed in the rat (see Martin 33 31., 1977; and Brown and Reichlin, 1972). Amino acids, particularly arginine, induced GH release in primates and inhibited GH release in the rat. Similarly, free fatty acids stimulated GH release in humans (Lucke 33 31,, 1972). Starvation in rats decreased serum GH concentrations (Dickerman 33 31, 1969a; Trenkle, 1970). Undernourishment during neonatal development similarly decreased plasma and pituitary GH concentrations in the rat (Sinha 33 31., 1973), and in mice (Sinha 33 31., 1975). These decreases resulted in a decrease in body growth. However, the decrease in serum GH concentrations did not alter the ability of the liver to produce somatomedin (Phillips and Young, 1976). Nevertheless, 50 a proper diet is needed for GH and somatomedin production. The effects of nutrition on GH regulation are probably mediated through the ability of the pituitary to synthesize GH and the hypothalamic activity during various nutritive states. The ventromedial nucleus and the lateral hypothalamus have been shown to effect appetite and feeding behavior (Ellison, 1968; Sorensen and Ellison, 1970). Stress in human subjects and monkeys has been reported to increase GH secretion (Greenwood and Landon, 1966; Meyer and Knobil, 1967; Mason 33 31., 1974; Reichlin, 1974). In sub- primate species (i.e., dog, pig, rat, moose), stresses have been shown to decrease GH secretion (Bellinger and Mendel, 1975; Martin, 1976). The mechanisms whereby stresses alters GH release are not clearly understood, but appear to be mediated through extrahypothalamic structures (Collu 33 31., 1973). Complete and incomplete hypothalamic deafferentation attenuated the inhibition of GH release by auditory and ether stress in rats. Ether stress was partially inhibited by a-methyl-para-tyrosine, indicating that the catecholamines mediate the effects of ether stress. Other PN may participate in stress induced changes in CH release since heat and cold stress had opposite effects on GH release (Mueller 33 31., 1974). These neuronal mechanisms remain to be investigated. 51 IV. Metabolic Effects of GH GH affects carbohydrate, protein, lipid, and calcium metabolism. These effects have been reviewed in mOSt text- books of endocringology (see Knobil and Greep, 1959; Cheek and Hill, 1974; Kostyo and Nutting, 1974; Goodman and Schwartz, 1974; Altszuler, 1974; Williams, 1974; C. Martin, 1976; Turner and Bagnara, 1976; Pecile and Maller, 1976). Evans and Long (1921) first reported that alkaline pit- uitary extracts injected into rats produced a profound increase in body mass. These observations led other investigators to believe that this pituitary extract promoted body growth through altering general body metabolism. This pituitary factor which is knomnas GH has a wide range of effects on protein metabolism. GH has been demonstrated to decrease ur- inary nitrogen excretions. These observations were confirmed by other observations that GH increased amino acid uptake into skeletal muscle, kidneys, livers and a variety of other tissues. Moreover, GH has been shown to counteract the glu- coneogenic effects of the glucocorticoids. However these observations did not explain the mechanism whereby GH promoted a positive nitrogen balance. In addition to promoting amino acid uptake, GH appears to stimulate pro- tein anabolism by increasing the cellular synthetic mechanisms for protein Synthesis. GH also has been shown to have both agonistic and antago- nistic prOperties to that of insulin. Initially after GH injections there was a rapid uptake of sugars into skeletal 52 muscle and adipose tissue. Later GH prevented this active , uptake of sugars into these tissues, demonstrating the dia- betogenic properties of GH. This action of GH is probably a protective mechanism to prevent the loss of plasma glucose to the brain while concurrently preventing a negative nitro- gen balance by retaining amino acids and stimulating protein synthesis. The effects of GH on fats was first demonstrated in the early 1900's. Pituitary extracts were shown to decrease total body fats and to increase total body proteins. GH stimulates lipolysis which leads to an increase in serum free fatty acids. However GH appears to have a permissive role in this process because thyroxine and glucocorticoids are also nec- essary for GH to promote 1ypolysis. These effects can prob- ably be mediated through a cyclic adenosine monophosphate (C-AMP) dependent lipase (C. Martin, 1976). Normal bone growth is also dependent on the presence of GH. However, GH does not stimulate the bone directly, but requires the presence of a factor produced in the liver. This factor was initially called sulfation factor because it promoted the uptake of sulfate into cartilage. Later the sulfation factor was renamed somatomedin. It mediates the effects of GH on bone growth. For normal growth to occur, one not only requires GH, but also thyroxine and the gluco- corticoids. While trying to isolate a single somatomedin, three somatomedins were isolated. Somatomedin A was found to be more potent as a "sulfation factor" than the other 53 somatomedins. On the other hand, somatomedin B exerted its effects primarily on thymidine incorporation into DNA. Finally, somatomedin C appears to bind to the placenta and act as a fetal growth factor. The precise role of the somatomedins is not fully understood and requires more investigation. GH also stimulated erythropoietin from the kidney (Crafts, 1953; Peschle 33 31,, 1972). CH was found to be necessary for lactation in cattle (Shaw 33 31., 1955), but not in rats-(Meites, 1957). The major metabolic effects of GH have been discussed. However the biochemical intermediate and changes in all cells have not been totally defined (for a more comprehensive re- view, see references cited above). MATERIALS AND METHODS I. Animals and Blood Collection Mature male Sprague-Dawley rats (Spartan Research Ani- mals, Haslett, MI) and male Wistar rats (Harlan Ind., Indian- apolis, IN), or hypophysectomized male Sprague Dawley rats (Hormone Assay Labs, Chicago, IL) were housed in temperature (25°31°C) and light controlled (14 h on 10 h off) rooms. Rats were provided with Purina Rat Chow (Ralston Purina Co., St. Louis, MO) and tap water 33_libitum. Hypophysectomized (HYPOX) rats and Wistar rats received a dietary supplement of fresh whole wheat bread and oranges 33 libitum. Surgically treated animals received 0.2 m1 Longicil-S (60,000 units of penicillin G (Fort Dodge Labs., Fort Dodge, IA). Blood samples were collected either by decapitation,via an indwelling atrial cannula or orbital sinus cannulation under light ether anesthesia (for details, see Experiments). Blood samples were allowed to clot for 24 h at 4°C and centri- fuged 3,000 x g for 20 min. The serum was separated and stored at -20°C until assayed for GH. 54 55 II. Cannulation of the Right Atria The right atrium was cannulated to remove serial blood samples in unanesthetized, unrestrained rats. Cannulae were prepared using Silastic Medical Grade Tubing (Dow Corning Co., Midland, MI). First a 6 in. segment of Silastic Tubing 0.030 in. inside diameter (ID), 0.065 in. outside diameter (OD), was dilated using a fine pair of hemostats. A smaller segment of tubing, 1.5 in. length, 0.020 in. ID, and 0.037 in. OD, was inserted into the dilated end, and then trimmed to 3 cm in length. Rats were anesthetized with Nembutal (Abbot Laboratories, North Chicago, IL; 35 mg/kg). After reaching a surgical level of anesthesia, a ventral sagittal incision, 0.75 in. long, was made rostral to the clavical midway between the sternum and the right shoulder. The jugular vein was isolated using blunt dissecting techniques to avoid injury to the vessel. After the jugular vein was isolated, the cephalic portion was ligated with silk suture, and a small nick was made in the vein 0.5 cm above the pectoralis major. The hole was held Open, and the Silastic tubing was inserted into the jugular vein until the end reached the right atria (3 cm). The vessel was ligated around the tubing caudal to where the cannulae entered the vessel and anchored to the pectoralis major. 56 The cannula was then routed subsutaneously to the dorsal side of the rat where it was exposed through a small incision placed in the skin in the cervicle region of the rat. The efficiency of the cannula was examined and the animal received an intravenous injection of 1600 units/kg Sodium heparin (Sigma Chemical Co., St. Louis, MO) prepared in .87% NaCl (saline). The cannula was sealed with a plug made of wire and the in- cisions were sutured. The animals were allowed to recover for several days postoperatively before experimentation began. On the night prior to the experiment, the animals were removed from the animal rooms and transported to the surgery room and housed in individual plexiglass cages. On the morning of the exper- iment a longer cannula made of the larger tubing was attached to the indwelling cannula for injections and serial blood sampling, after initially injecting the animals intravenously with 500 IU of heparinized saline. Blood samples were with- drawn with a 1 m1 tuberculine syringe at designated time intervals and volumes described in the Experimental Section. III. Cannulation of the Lateral Cerebral Ventricle of the Rat For injections of GABA, acetylcholine, bicuculline methyliodine, and 5,7-dihydroxytrptamine, cannulae were im- planted chronically into the skull. (Verster 33 31., 1971). Cannulae were constructed from polyethylene tubing #10 (Clay Adams, Parsippany, N.J.). A segment of wire 0.009" in diameter was threaded through the lumen of the tubing before heating 57 over an electric soldering iron. The tubing was allowed to soften and the ends were pushed toward the middle forming a bulb. The tubing was allowed to cool, and trimmed to the fol- lowing specification: the distance from the bulb to one end was 4 mm with a 1 mm bevel on the end, and the distance from the bulb to the other was 5-6 cm. Prior to cannulation, rats were anesthetized with ether or Nembutal (Abbott Laboratoreis, North Chicago, IL; 35 mg/kg). After reaching a surgical level of anesthesia, animals were placed in a stellar stereotaxic instrument (C.H. Stoelling, East Chicago, IL). The scalp was shaved, a longitudinal in- cision was made from the middle of the frontal bone to the beginning of the occipital bone following the sagittal suture. The skin and the underlying facia were retracted. One hole, 0.03 inch in diameter was drilled 2 mm lateral and 1 mm caudal to the intersection of the coronal and sagittal sutures, breg- ma. The dura mater was pierced with a hypodermic needle and the 4 mm end of the cannula was inserted through the hole into the lateral ventricle. The other end was sealed with heat. Two additional holes were drilled 3-5 mm caudal to the coronal suture. The skull was dried and metal screws (bowline anchor screws, Shuron/Continental, Rochester, N.Y.) were inserted into the later 2 holes to allow for support of the cannula. Dental cement (NuWeld Caulk, L.D., Caulk Co., Milford, DL) was used to anchor the cannulae to the anchoring screws. After the cement hardened completely, the incision was sutured. The animals were allowed to recover for three days postoperatively. 58 Two days prior to the experiments, the animals were gentled by handling and cannulae were checked by injecting 10 ul of 0.87% NaCl with a 10 ul microsyringe (Glenco Scientific, Ind., Houston, TX). Following injections of experimental drugs and blood col- lection, 20 ul of an aqueous solution of methylene blue dye was injected into the cannulae, and the brain was removed and sectioned along the coronal plane through the hypothalamus. Data was only used from rats showing stain in the medican em- inence and third ventricle. IV. Radioimmunoassay for Serum GH Serum concentrations of GH were determined using a standard double antibody ratioimmunoassay procedures as de- scribed in the NIAMDD-kit for rat GH. The second antibody was harvested from our goat and diluted to the appropriate anti- body concentration where maximal binding was observed. Serum hormone concentrations are expressed in terms of the standard reference preparation, NIAMDD-rat-GH-RP-l. All serum and incubation samples were assayed in duplicate or triplicate. Hormone concentrations were determined only from volumes which resulted in hormone values on the linear portion of the stan- dard curve. All samples from an individual experiment were tested in the same assay to avoid interassay variations. Also, a standard serum sample was used with each assay to monitor interassay variations. 59 V. Assay of Norepin3phrine and Dopamine in Hypothalamic Tissue Immediately following decapitation, brains were removed and placed dorsal side down in a pool of ice cold 0.89% NaCl. The hypothalamus was removed with find iris scissors using the following landmarks: anterior hypothalamus, immediately rostral to the optic chiasm following the lamina terminals 30° rostral to a perpendicular line of the horizontal axis; cau- dal hypothalamus, middle of the mammilary bodies perpendicu- lar to the horizontal axis; lateral hypothalamus following the hypothalamic sulci; and dorsal hypothalamus 2-3 mm to the dorsal hypothalamic surface. The median eminence (ME) was dissected from the hypo- thalamus using a dissecting microscope and fine iris scissors and the following landmarks: posterior border of the infundi- bular stalk, anterior border of the infundibular stalk and along the lateral aspects of the tubercinereum at an angle of 20° from the ventral hypothalamic horizontal surface yielding a piece of tissue containing 1032 mg protein (N=24) as assayed by the method of Lowry 33 31., 1951. Medial basal hypothalamus (MBH) was dissected using similar boundaries as the ME except cuts were made 45° from the horizontal plane following the ventral hypothalamic surface. These tissues contained 35:3mg protein (N = 24). Whole hypothalami were homogenized in 0.4 N perchloric acid containing 10% EDTA (1 mg tissue/ 10 ul homogenate) using microhomoginizers and centrifuged in a microcentrifuge (Coleman International, Oak Brook, IL) to separate the particulate 60 matter from the supernatant. ME were homogenized in 25 ul and MBH in 40 ul of the PCA solution. Dopamine (DA) and norephine- phrine (NE) tissue concentrations were determined in 10 ul aliquote of the supernatant assayed by the method of Coyle and Henry (1973). VI. 13 Vitro Co-Incubation Anterior pituitaries and hypothalami were removed immed- iately after decapitation and placed in 10 m1 scintillation vials containing 2 ml DIFCO medium 199 (DIFCO Labs, Detroit, MI), prepared as described in DIFCO supplimentary literature. The vials were placed in a Dubnoff metabolic shaker, 60 cpm at 37° 3 1°C and incubated under constant gassing 95% 0 and 2 5% CO2 for 1 h. After 1 h pre-incubation, the medium was dis- carded and 2 ml of fresh medium was added with or without drug treatment (see Experimental section for treatments), and incu- bation was resumed for 2 h. After 2 h incubation, the medium was removed and placed into 12 x 75 mm culture tubes, capped and frozen until assayed. Two m1 of fresh treatment medium was added to the pituitaries and hypothalami and incubation resumed. Upon completion of the incubation, the pituitary halves were weighed to the nearest tenth of a milligram and discarded. Later the medium was thawed, diluted 1:50 with 0.1% gelatin PBS and assayed in triplicate for GH. Results are expressed as ug GH/ml/h/mg AP. 61 VII. Statistical Methods Unless otherwise stated all 13 3133 experiments were analysed by analysis of variance and students - Newman-Keuls test for multiple comparisons between groups. 13 31333 incubations were analysed using the paired-t- test. EXPERIMENTAL DATA I. Pulsatile Secretory Pattern of Plasma GH Introduction OH is released in episodes throughout the day and night in experimental animals and in man (Finkelstein 33 31., 1972; Tannenbaum and Martin, 1976; Tannenbaum 33 31., 1976). These secretory patterns were not effected by insulin, hyperglycemia or hypoglycemia (Tannenbaum 33 31., 1976). A series of experiments were performed to determine when these pulsatile secretions of GH occurred so that experiments could be designed to either potentiate or inhibit the episodic release of GH. Materials and Methods The right atria of male Sprague-Dawley rats were can- nulated, as described in Materials and Methods, 2 hours prior to the experiment. 1600 units/kg of sodium heparin was injected intravenously to avoid coagulation of the blood in the cannula. 200 ul samples were withdrawn from the cannulae every 15 min for the duration of the experimental period. 200 N1 of 0.87% NaCl was reinjected intravenously into each animal fOllowing the sampling procedure. At the end of the exper- imerital period, blood samples were centrifuged, plasma was SeParated and stored for radioimmunoassay. These experiments 62 63 were performed at0900 to 1200, 1100 to 1400, and 1400 to 1600 hours (h). Results GH concentrations remained at approximately 30 ng/ml throughout the sampling period, except during episodes of GH release at 930-1030, 1200-1300, and 1500-1600 hours. Figure 3 represents the typical response of 4 animals sampled between 1100 and 1300 hours. Figure 4 represents the average response of 10 animals sampled at these times. Conclusions These observations confirm observations that GE is released in episodes every 3-3.5 hours throughout the day in the rat (Tannenbaum and Martin, 1976; Tannenbaum 33 31., 1976). These results helped to establish the times at which several of the experiments which follow were performed. If a change in GH concentration was expected, I investigated the effect of various drug treatments on plasma or serum GH concentrations during the morning or early afternoon espisodes of GH release. II. Adrenergic Control of GH Release 13 vivo and 13 vitro Introduction The hypothalamus contains catecholaminergic neurons which appear to modulate the release of all anterior pituitary hor- mones (Meites 33_31,, 1977). L-dopa, the immediate precursor to DA and NE increased serum GH concentration in rats 64 400- 300- :"Q/HH 3 ‘P ) fie...) .. A PLASMA GH a l : I 3 60 120 180 1fiOOam MINUTES Figure 3. Normal secretory pattern of GH in four male rats. 66 (Chen 33 31., 1974) and in human patients (Martin, 1973). These effects apparently were mediated through the a-adren- ergic receptor, since phentolamine, a a-adrenergic receptor blocker, partially inhibited this response (Liuzzi 33 31., 1971; Kato 33 31., 1973; Martin 33 31., 1977). In rats anesthetized with urethanep:- the B-adrenergic receptor blocker, propranalol, was most effective in stim- ulating GH release (Collu 33 31., 1972; Kato 33 31., 1973). This stimulation may have resulted from the anesthesia used. The present study shows the effects of several noradrenergic drugs on release of GH 13 vivo and 13_vitro. Materials and Methods Male Sprague-Dawley rats (225-2509) were used in all of these studies. In the first experiment, chlorpromazine (CPZ) (Smith, Klein and French Labs, Philadelphia, Penn.) a catecholamine reuptake inhibitor, was injected intraperitoneally (i.p.) to rats. Animals were bled under light ether anesthesia at 30, 60 and 120 min intervals after injection. The second experiment represents a dose response study on the effects of clonidine (Beohringer Ingelheim Ltd., Elmsford, N.Y.), an a-adrenergic receptor stimulator, on serum GH concentrations. Rats were injected with 0.01, 0.02, 0.05 and 1 mg/kg clonidine dissolved in 0.87% NaCl solution. Each rat received an injection of 0.2 ml/lOO g B.W. Blood samples were obtained 30 min and 60 min, after injection during the second A.M. surge of GH. All rats were decapitated between 1200 and 1300 hours. Blood from the cervical wound was 67 collected and treated as described in Materials and Methods. In the third experiment, rats bearing an indwelling carotid cannula were injected i.p. with clonidine. Blood samples of 0.5 ml each, were collected via the cannula at 0, 30, 60 and 120 min after the initial injection to deter- mine the time-related effects of clonidine on serum GH concentrations. In the fourth experiment, male rats were injected i.p. with prOpranalol (PROP), (Ayerst Labs., N.Y.), a B-adrenergic receptor blocker, phenoyxbenzamine (Smith, Klein and French Labs, Philadelphia, Penn.), and a-adrenergic receptor blocker, and several doses of phentolamine (PHEN), (CIBA Pharmaceutical Co., Summitt, N.J.), an a-adrenergic receptor blocker. After 1 h between 1200 and 1300 hours, animals were decapitated (Table 4). In the fifth experiment, PROP and PHEN were injected alone and simultaneously with clonidine to investigate the interactions of the<1and Breceptor blockers with theoo usouommao.o. ou pouneaoo mo.cvm A. .Hfich “.:.m.m + m m .. a: _ on A me one a am no H no oN + Hoe nnn\msm Nos. 1 a I 1 Amy ma + mw NH + om 6N + no NH + OOH mHOHucoo . HH. u u : I Amy so + 2 so + No as + H: m + 2: unimam use. I a a I Amy ON + ow «H + mm NH + COM w + m.cm mHouuaoo H one oo on. o .I cowuooficH nouu< mouacax mu asuom so oswuoaoumKoHsu mo nuuommm .H OHQMB 7O .mswmaaom oooao room an maouuaoo cu omumaaoo mo.ovm . I o .aspmm\w: u.z.m.m + m m we H mes om A one ms\wa o.H. one A non sees a Noe ws\ms mo.o. son H was one « oee ms\es no.o one w mom sen A New ws\ms Ho.o Aeonz Nem.oot on « em nos n we neonneoo sea oo sea on m i s mu Epsom so owuoucoao we muommmm .N oases oncommmm moon 71 . U l . mousoflfi ca oEHu u a wcaaoaom.ooOHn mo mafia room an mHOMusoo ou touresoo mo.ovm n_ .asuom\w: n.=.m.m H m m Ams\ms mo.ov. one A oem new H man one a one assessoeo . . Aws\wa No.ov sen + one one + one see a hen oeeoesoao ) u u Aaonz Nem.ov. em u was mm + me on + mud nos + an neonusoo can a oo 9 on e o e e n e mu asuom so endowsoao mo nuumwwm oouoaox mass .m «Home 72 Table 4. I. A ... Effects of a and B-Adrenergic Blocking Drugs on SerugTGH n - 10/group I II T Controls Controls (0.872 NaCl) 125 1 18a (0.872 NaCl) 106 i 26 Propranalol PrOpranalol (5.0 mg/kg) 136 t 26 (5.0 mg/kg) 98 f 29 Phenoxybenzamine Phenoxybenzamine (0.5 mg/kg) 87 f 16 (1 mg/kg) 76 t 12 Phentolamine Phentolamine (0.5 mg/kg) 68 t 12b (0.5 mg/kg) 68 i 15 Phentolamine Phentolamine (1 mg/kg) 52 t 8b (1 mg/kg) 62 1 9b Phentolamine b Phentolamine (5 mg/kg) 48 t 10 (5.0 mg/kg) 33 i 16b a i t S.E.M.; ng/serum. b_PfQ.O5 comp33edeith controls. 73 Injection of PROP (5 mg/kg) alone did not alter serum GH concentrations (Table 5). On the other hand, PHEN (5mg/kg) significantly decreased GH release; whereas CLON (0.02 mg/kg) significantly increased GH release. This increase was significantly attenuated by PHEN, but not PROP. 13 31333 co-incubation of pituitary halves with and without a hypothalamic fragment revealed that the presence of the hypothalamus significantly reduced GH release from the AP (Figure 5). Addition of 1, 10, and 100 ng NE/ml to incubation medium had no effect pn pituitary GH release. Moreover, addition of NE to tubes containing both an AP half and a hypothalamus significantly increased- GH release, as compared to AP halves and hypothalami incubated alone. Removal of this medium after 2 hrs and replacement with fresh medium not containing NE in the presence of a hypothalamus significantly reduced GH release for the AP. When PIM, a dopamine receptor blocker, was added to the incubation medium, GH release was not changed (Figure 6). Also, PIM did not alter the effects of 100 ng NE/ml media on hypothalamic stimulated GH release. PHEN alone did not alter GH release directly from the pituitary or hypothalamic inhibited GH release from AP halves co-incubated with hypothalamus (Figure 7)._ However, PHEN decreased the NE stimulated increase in CH rélease from the co-incubated pituitary halves. PROP did not alter GH releaSe or NE induced GH release from co-incubated pituitaries (Figure 8). 74 Table 5: ‘ 0‘--x Effects of C1pnidine andiAdrenergicjB1ockinEnggan_Qn Serum—5E n - 8 Q 60 min Controls 112 t 158 98 t 22 (0.872 NaCl) Propranalol 98 t 21 76 f 31 (5 mslks) Phentolamine 95 t 31 38 t 6b (5 ms/ks) Clonidine 113 1 18 323 3 36b (0.02 mg/kg) Propranalol 129 t 18 296 1 51b + Clonidine Phentolamine 108 r 12 126 t 31 + Clonidine S.E.M.} ng/serum. i <0.05 compared to controls. I O a- x b P 75 I AP/2 E AP/2 & HYP. 1.24 0.8- ugGH/ mI/hr/ I ‘Ing P _ 0.4— 199 ° 3,9 "‘2 ‘°° 199 O—2hrs 2-4hrs 4-6hrs Figure 5. 13 vitro effects of NE on GR release. Vertical bars represent SEM. N=6/group. AP/2=Anterior Pituitary half; Hyp=Hypotha1amus, 76 I AP/2 g AP/2 & HYP. 0.8 - usGH/ , Inl/hr I mg A 0.4 - 199 1ug 199 PIM PIM “3% "9 . . - O —2 hrs 2-4 hrs 4—6 hrs Figure 6. 13 vitro effects of PIM and NE on GH release. Vertical bars represent SEM. N=6/group. AP/2=Anterior Pituitary half; Hyp=Hypothalamus 77 'I AP/2 E AP/2 8: HYF‘. 12- (18- ugGH/ nfl/hr .nuaA 04s I 10000 0 10 10000 10 0 00 0 ° ns‘pne“)? +100 n9 NE 0-2 hrs q 2—4hts- 4—_6 hrs Figure 7. 13 vitro effects of phentolamine and NE on GH release. Vertical bars represent SEM. N=6/ group. AP/2=Anterior Pituitary half; Hyp= Hypothalamus. 1.2— Figure 8. 78 o 10 10000 nQPROP O —2 hrs fElease. I AP/2 J: '5' AP/2&HYP. 0 10 10000 0 10 10000 +100ng NE 2-4hts__ __ 4_-6 hrs . r In vitro effects of propranolol and NE on GH Vertical bars represent SEM. N=6/group. AP /2=Anterior Pituitary half; Hyp=Hypothalamus. \ 79 Conclusions GH release was blocked by the catecholamine receptor blocker, CPZ. These effects are believed to be the result 'of blocking both DA and NE. However, when a specific a-adrenergic receptor stimulator, CLON, was injected into male rats, there was a significant increase in serum GH concentrations. Phenoxybenzamine slightly reduced serum GH. These observations were clarified by using another aadrenergic blocker, PHEN. PROP, a Badrenergic receptor blocker, had no effect 13 3133_or 13 31333. These results were confirmed by using a pituitary-hypo- thalamus co-incubation system. I conclude from these results that the a-adrenergic receptors actively étimulate GH release. While this work was in progress, Durand 33 31., 1977 and Martin 33 31., 1978, published similar results. Phenoxybenzamine and PHEN both inhibited the pulsatile release of GH. PROP was without effect. CLON similarly enhanced the episodic release of GH. However, they did not examine the direct effects of these drugs on the pituitary and hypothalamus. NE appears to stimulate GH release by removing inhibition of the hypothalamic influence on OK release 13 31333. It is possible that NE through an a-adrenergic receptor, inhibits the release of GIF from the pituitary, thus increasing GH release 13 3133 and 13 31333, However, GIF was not measured in these experiments due to the unavailability of somatostatin radioimmunoassay. 80 III. Serotonergic Control of GH 13 vivo and 13_vitro Introduction Several experimental models have been used to determine the role of 5-HT in the control of GH. However, the results from these experiments have been inconclusive. Injections of 5-HT into the lateral cerebral ventricle of urethane anesthetized rats increased serum GH concentrations (Collu 33 31., 1975). Similarly, systemic injections of S-HTP, the immediate precursor to S-HT, increased serum GH con- centrations in unanesthetized rats. Injections of cypro- heptadine, a 5-HT receptor blocker, inhibited GH release in the neonatal rat (Stuart 33 31., 1976). Conversely, intraventricular injections of S-HT significatnly decreased serum GH concentrations, and systemic injections of S-HTP which elevated hypothalamic concentrations of 5-HT did not alter GH release. Injec- tions of parachloramphetamine, a drug which inhibits tryptophan hydroxylase, increased serum GH concentrations (Maller 33 31., 1973). In view of these discrepencies, the effects of 5, 7- dihydroxytryptamine, a specific neurotoxin for S-HT; methysergide, a serotonin receptor blocker; and para- chlorophenylalanine, a tryptophan hydroxyalase inhibitor, were studied on GH release 13 3133. Additionally, the direct effects of 5-HT and methysergide (METH) were studied in a pituitary-hypothalamus co-incubation system. 81 Materials and Methods Thirty male Sprague-Dawley rats were injectedisp.rx1day 0 with 300 mg/kg of paraclorophenylalanine (PCPA), (Aldrich Chem. Co., Milwaukee, Wis.). Thirty additional rats were injected with 0.87% NaCl (0.2 ml/100 g B.W.) and served as controls. On days 2, 4, 6 and 8, following injections, 6 animals were decapitated and blood was collected for GE assays. Twenty animals bearing an indwelling atrial cannula were used in this study. Beginning at 0930 hrs, 10 animals were injected with 1600 units of sodium heparin/kg. Immediately following heparin injections, 5 animals were injected intravenously with 10 mg/kg METH (Sandoz Phar- maceuticals, Hanover, N.J.). Five animals served as controls and were injected with equivalent volumes of 0.87% NaCl. Blood samples were obtained 10, 30, and 60 min after injections. On separate days during the same time interval, the experiment was repeated using 20 mg/kg and 40 mg/kg METH. In a third study, rats were injected intraventricularly (lateral ventrical) with 5, 7-dihydroxytryptamine (5,7-DHT), Regis Chemical Co., Morton Grove, Ill). Another group of rats was pre-treated with desmethylimipramine (DMI), (LSTV.Pharmaceuticals, Tuckahoe, N.Y.) 45 - 60 min before 5,7-DHT injections. One additional group of rats was injected with DMI; and another group which was injected with 0.87% NaCl served as controls. Rats injected with DMI 82 were killed on days 2 and 10 after injections. Rats given the other drug treatments were killed on days 2, 6, 10. and 14. Blood was collected from the cervical wound and stored for radioimmunoassay (RIA). The final study was performed to determine the direct effects of 5-HT (Sigma Chemical Co., St. Louis, MO), and METH on GH release in a co-incubation system (see Materials and Methods, and Tables 9 and 10 for doses of drugs and times at which blood samples were taken). Results The S-HT antagonist, PCPA, decreased serum GH concen- trations 2 days after injections (Table 6). Serum GH concentrations were not maximally reduced until 4 days after injections. There was no further change in serum GH concentrations 6 and 8 days following the initial injections. A dose of 10 mg/kg METH did not alter serum GH concentrations 10, 30, or 60 min after injections (Table 7). Rats injected i.v. with 20 mg/kg METH had lower serum GH concentrations 30 and 60 min after injections as compared to controls (p <0.05). Rats given the 40 mg dose of METH showed reduced serum GH concentrations reduced throughout the entire experimental period (p <0.05). Rats injected with DMI, the catecholamine reuptake inhibitor, had serum GH concentrations which were not significantly different from control concentrations (Table 8). 5,7-DHT alone significantly decreased serum GH, 2, 6, 10 and 14 days after intraventricular injections. 83 .mausmamaasonoouoanomume n C) a h s a e. .. < c Q < E '5 s a ° 3 o 0; E - E _ u 0 no '0 I. O , A B C D E Figure 12. Effects of pilocarpine, physostigmine and atropine on serum GH. Vertical bars rep- resent SEM. N=8 or 9/group. 100 1501 ' FIT )1 SERUM 1 N=8 GH .. NG ML / - "a m . m _z_ _, o. . .o m .l. 20 a; < ,_ o z 0 E 2 ‘ O :". 5 a O O- ' a a TREATMENT; DosE MG/KG Figure 13. Effects of a-methyl-paratyrosine on pilocarpine induced GH release. Vertical bars represent SEM. lOl 140- 100‘ f Nza/GROUP SERUM g on G" a u? NG/ML < e . ,3 N ... o 60« E g I a o. 8 CONTROLS PILOCARPINE ;5 PRORANOLOL; 5 PROP + Pl LO PHE N.+Pll.0. FINA-Pl LO. TREATMENT; DOSE MG/KG Figure 14. Effects of catecholamine blocking drugs on pilocarpine induced GH release. Vertical bars represent SEM. .mHouuaoo Ou vmuoquo no.0vm A .Ha\m= 1.2.6.. « m 6 nos R mm pm M EH 6 R mos HH As H as Ann M am as H mm “one masmumuosum Aosv Aosv, 2 wx\w5 m + moopum Wampum mHouuaoo 0 H l -.a'.6ll' :u Eauom mfi.ommouomn.pOO=ch moouum so Cpl. .mauoanme Ocfidumoodfim mo mucoumm 0- O n - 6 per group Hypothalamus l()3 Table 13. ug GH/ml/mg AP/hr In Vitro Stimulation of GH by Acethycholine 0-2 hrs 2-4 hrs 6-6 hrs 199 ACh 199 (100 ng/ml) . Pituitary 2.29 i .108 2.69 i .26 2.18 t .12 Pituitary + 1.66 t .15b c +2.36 1 .25 9 +1.55 t .16b Hypothalamus PCA PCA & ACh PCA (1 us/ml) Pituitary 1.96 t .22 2.05 i .16 2.00 i .17 Pituitary + 1.30 t .06b c+2.07 t .13 ° +1.62 t .25 Hypothalamus PIM PIM 6 ACh PIM (100 ng/ml) Pituitary 1.93 1 .26 1.83 t .18 1.96 t .16 Pituitary + 1.13 t .16b 1.95 t .12 c +1.66 1 .09b Hypothalamus ACh ACh 6 AIR ACh (100 ng/ml) Pituitary 2.29 t .16 2.17 i .17 2.32 t .31 Pituitary + 2.18 t .21 ° +1.71 t .21b 9 +2.09 t .18 ‘ Mean t 3.2.x. P<0.05 compared to control. c P<0.05 compared with preceeding 2 h period. ACh - acetylcholine; PCA - parachloramphetamine; PIM - pimozide; AIR I atrOpine. ‘ -- -_—. - 104 co-incubation system. The S-HT antagonist PCA or the DA receptor blocker PIM did not alter GH release directly on the AP or on the AP co-incubated with the hypothalamus. Also, these drugs did not prevent the Ach stimulated release of GH from co-incubated AP halves. ATRZ? completely prevented the cholinergic stimulated release of GH from co-incubated APs, without acting directly on the pituitary to alter GH release (Table 13). PIL, at doses of 1, 10 and 100 ng/ml similarly prevented hypothalamic inhibition of GH release from co-incubated APs (Table 14). ATRLE (100 ng/ml) completely prevented the increase in GH release from co-incubated APs without altering GH release directly in the pituitary (Table 14). Neither PIM, HAL, nor METH altered GH release from co-incubated AP halves which were treated with PIL (Table 15). Each of these drugs did.mm:effect GH release directly from the AP or from co-incubated AP halves. Conclusions These studies provide evidence that cholinergic drugs can stimulate release of GH. Ach, PIL, and PHYSOS each stimulated GH release £2 331g. Ach and PIL both stimulated GH release from co-incubated AP halves. Atropine, a mus- carinic receptor blocker prevented the action of Ach on GH release tg gtzg_and tg zttto, indicating that this is a Specific action of Ach. Since all the ttpgttg_experiments ‘were performed during an episodic surge Of GH, these results :Suggest that Ach may potentiate the episodic release of GH l()5 Table 14. In Vitro Stimulation of ca by Pi1ocatpiue n - 6 per group ‘Pituitary Pituitary + Hypothalamus Pituitary Pituitary + Hypothalamus Pituitary Pituitary + Hypothalamus Pituitary Pituitary + Hypothalamus ug Gfl/ml/mg AP/hr 0-2 hrs 19 1.85 t .218 1.27 t .16b P110. (1 n m1) 2.01 t .14 P110. (10 ng7m1) 1.77 t .23 1.98 t .31 P110. -(100_ng/m1) 1.96 t .21 1.98 t .32 P110. Pilo. Pilo. 2-4 hrs 99 1.63 t .11 1.31 t .18 1.95 g .22 t + 1.36 t .16b 1.85 + .14 1.61 i .20b 1.81 t .27 1.53 1 .29b (1 02/91) + Atropine (100 ng/ml) (10 ng/ml) + Atropine (100 ng/ml) (100 ng/ml) + Atropine (100 ng/ml) a Mean t S.E.M. P<0.05 compared to control AP half. c P<0.05 compared with preceeding 2 h period. Pilo.- pilocarpine. H l()6 TablenlS. n Vitro Effects of Monoaminergic Receptor Blockers On Pilocarpine Induced GH Secretion n I 6 per group Pituitary Pituitary + Hypothalamus Pituitary Pituitary + fiypotha1amus Pituitary Pituitary + Hypothalamus Pituitary Pituitary + Hypothalamus Pituitary Pituitary + ug GH/gljmg AP/hr 0+2 hrs PIM (1 n ml) 1.86 i .32‘ 1.35 g .18b PIM (100‘331u1) 1.75 t .26 1.25 1 .16b HAL (10 n ml) 1.98 i .23 1.38 t .33 HAL (10 ml) 1.97 t .16 1.26 1 .08b METH (10 ng7m1) 1.87 t .21 1.21 t .10b 2-4 hrs PIM (1 ng/ml) + Pilo. (100 ngfét) 2.01 t .16 c + 1.96 t .25 PIM (100 ngjgt)_+ Pilo. (100 ng/ml) 1.86 t .21 c + 1.75 t .19 HAL 10 n m1 + Pilo. (100 ng gt) 1.87 t .22 1.65 + .09 HAL (1 pg/ml) + Pilo. (100 ng/ml) 1.87 t .23 t + 1.93 g .31 unragj10 ngigl) + P110. (100 nng;) 1.75 t .31 1.88 t .21 a ”883' t Sega". b P4).05 compared to control. c P<0.05 compared to previous 2 h period. PIM I pimozide; HAL I haloperidol; METH I methysergide; Pilo. I polocarpine. ' 107 $3 2122- The dose of Ach (50‘ng) injected into the lateral ventricle, inducing GH release, may be pharmacological. However, the observation that PIL and PHYSOS also increased OH release, as well as the earlier observations on promotion of GH release by other cholinergic drugs (Cehovic gt gt., 1972; Soulairac gt gt., 1967) suggest that Ach may have a physiological role in regulating GH secretion. In addition, there is evidence that the hypothalamic cholinergic system may help regulate the secretion of LH, FSH (Everett gt gt., 1949a,b; Libertun and McCann, 1973) and PRL (Grandison gt gt., 1974; Grandison and Meites, 1976). The observation that cholinergic stimulation of GH release can be blocked by a dopamine receptor blocker, PIM or PHEN, an aadrenergic receptor blocker, suggesusthat the action of Ach on GR release is mediated via the catecholamin- ergic system. Grandison and Meites (1976) reported that cholinergic inhibition of PRL release in rats is similarly mediated via the catecholaminergic system. 3 Since PIL* did not alter the stress-induced decrease in serum GH, there appear to be other neuronal mechanisms which decrease GH release during stress, and probably also effect the diurnal variations in GH release. 108 V. A Possible Role of GABA in the Control of GH Release Introduction Neurotransmitters in the hypothalamus have been shown to alter the release of hormones from the pituitary (for reviews see Blackwell and Guillemin, 1973; Meites gt gt., 1977; Muller gt gt., 1978). The neurally active amino acid,GABA, also was found to be present in high concentra- tions in the hypothalamus (Kimura and Kuriyama, 1975; Tappaz gt gt., 1976). GABA was reported to inhibit ACTH (Burden gt gt., 1974; Markara and Stark, 1974) and MSH (Takeisnik gt gt., 1973/74), and to stimulate LH and PRL release (Ondo, 1974; Mioduszewski gt gt., 1976; Ondo and Pass, 1976). The present series of experiments were undertaken to examine the effects of GABA, and GABA agonists and antagonists on GH release. Materials and Methods GABA (Nutritional Biochemical Co., Cleveland, OH), and bicuculline(Pierce, Rockford, IL) and bicuculline methy- liodide (Pierce, Rockford, IL), both GABA receptor blockers, were dissolved in 0.87% NaCl and the pH of these solutions was adjusted to pH-7 by addition of 0.1N NaOH. Picrotoxin (PIC) (Nutritional Biochemical Corp., Cleveland, OH), a GABA receptor blocker, amino-oxyacetic acid, a GABA agonist (AOAA, Aldrich Chemical Co., Milwaukee, WI), a a-methyl- paratyrosine, PIM, MET, PROP, and PHEN were dissolved in 0.87% NaCl. 109 In the first three experiments, male rats were implanted with a polyethylene cannula in the lateral cerebral ven- tricle (see Materials and Methods; Verster 2E 21" 1971). GABA, bicuculline methyliodide(BIC MI) and combinations of the two drugs were injected into the lateral ventricle. Each drug was infused (60 sec) into the lateral ventricle in a volume of 8 n1. The cannulae were rinsed with 2 n1 of 0.87% NaCl. Control rats were injected with 10 pl of 0.87% NaCl. In the next two experiments, AOAA, a GABA agonist; picrotoxin, and bicuculline, both GABA receptor blockers were injected systemically. Rats injected with AOAA were bled via orbital sinus cannulation under light ether anes- thesia 1.5 and 6 h after injection. Rats injected with BIC and PIC were decapitated 3O min'after injections. The effects of BIC on hypothalamic and median eminence (ME) DA and NE concentrations, and turnover index were determined in a fifth experiment. Male rats were injected i.p. with BIC (2.5 mg/kg) alone and in combination with a-MPT (250 mg/kg). After 1 hr rats were decapitated, brains removed and the hfififi' thalamus and median eminence were dissected away from the brain using a dissecting microscope. The tissue was rapidly frozen on dry ice and transferred to a -40°C freezer until assayed for DA and NE concentrations by the methods of Coyle and Henry, 1973, and Cuello gt gt., 1973 (see Materials and Methods). Blood was collected from the cervical wound for CH assays. 110 blood was collected from the cervical wound for GH assays. In a sixth experiment, the direct effects of GABA on GH release $2 2122 were examined. Male hypOphysectomized (HYPOX) rats (Hormone Assay Labs, Chicago, IL) were transplanted with a single AP under the kidney capsule. Ten HYPOX rats served as controls. Rats given AP transplants were injected daily with 10, and 100 mg/kg GABA beginning on the second day after the APs were transplanted under the kidney capsule. Blood samples were taken via cardiac puncture under light ether anesthesia 1 hr after injection on days 2, 3, and 5 after initial GABA injections. GABA at doses of 1, 10, 100, and 1000 mg/ml were incubated with AP halves to determine the direct $2.!iEEE effects of GABA on GH release. Also GABA at several doses (Figure 23) was co-incubated with AP halves and a hypothalamus to determine whether GABA acted on the hypo- thalamus to alter GH release. GABA was also incubated with monoaminergic receptor blockers and PIC in medium containing hypothalamus and pituitary (for doses of drugs see Figure 24 and Methods and Materials). Results Intraventricular injections of GABA at doses of 0.5 uM or 1 uM.per rat slightly reduced serum GH concentrations 20 min after injection (Figure 15). The 5 and 10 uu_doses of GABA significantly reduced serum GH concentrations during the same time interval. Conversely, i.v. injections of BIC MI at doses of 0.2 and 0.4 ug slightly increased serum GH, lll 150- 9 9 no<fli 3 m perm! 75- o 6‘ 9 9 C SERUM ._ g + Z 2 o o o o I s E. 8 9 p a o 2 g -< < 3 1 2 :2 < 2 a: < 2 q o o a a - o ’ I . s g a g- s g 5. z 2 d 3‘ m 9 0 A s c o E F Figure 15. Dose response effects of GABA on serum GH. Vertical bars represent SEM. N=9/group. 112 concentrations whereas the 0.6mgg dose significantly increased serum GH concentrations 20 min after injections (Figure 16). In another experiment 10 UM of GABA significantly reduced serum GH concentrations, whereas 0.7 pg BIC MI significantly increased serum GH concentrations 20 min after injection (Figure 17). These two drugs injected together did mm:alter serum GH concentrations as compared to control rats injected with 0.87% NaCl. Intraperitoneal injections of AOAA, a GABA agonist, at a dose of 25 mg/kg significantly reduced serum GH concentrations 1.5 hr, but not 6 hr after injection (Figure 18). The 50 mg dose of AOAA significantly reduced serum GH concentrations 6 hr after injection, but not 1.5 hr after injection. This is a result of the large standard error observed 1.5 hr after injection. Systemic injections of BIC (1.25 and 2.5 mg/kg) or PIC (0.5 mg/kg) significantly increased serum GH con- centration above control values and those animals injected with 1011M GABA (Figure 19). The larger dose of picrotoxin did not alter serum GH concentrations. BIC,(2.5 mg/kg). i.p., significantly increased GH release (Figure 20), and LchPT alone decreased serum GH concentrations. WhenO;-MPT was injected concurrently with BIC,cx-MPT completely inhibited GH release produced by BIC. Measurement of the biogenic amines revealed that BIC significantly increased NE turnover index in the hypothalamus, as revealed by a greater NE depletion aftera -MPT treatment 200‘ SERUM ng/ml 100- Figure 16. 113 0 0.2 0.5 0.6 __DOSE pg - Effects of intraventricular injections of bicuculline methyliodide on serum GH. Vertical bars represent SEM. N=8/deter- mination. R=0.84. 114 200- 150- 8 *NSGH r_. per ml 100- SERUM y 9 9 3‘ 2' < 3 2 .. 2 g " a o I! z a. 3 . O h 1 0 ° 6 2 a 0 A 8 c 0 l l Effects of GABA and bicuculline methyliodide on serum GH. Vertical bars represent SEM. N=8 or 9/group. Figure 17. 115 am- 9 s -- II can 100- + s + muml 8 SERUM 9 s + g 3 3 9 0 ° ° C C C ‘ ‘ |_ i- ( < l- ‘ z :z < 1t z '< < o O O O O O O 0 o < ( 0 ‘ g d F 2 h g x < <5 .\ 2 <3 \. \ < - < o a 8 '2' E g 5 ‘5 E a - a n ‘- N 3 O 1.-.“ | HOURS Figure 18. Time course effects of amino-oxyacetic acid (AOAA)on serum GH. Vertical bars represent SEM. N=8 or 9/group. 116 d ¢ l I ? ' " i ‘ ‘ -— __ 500- 400-! 3001 "CG” pol-ml SERUM 5' 3' III "I 8 ' 2 E E j :3 g :2 Fl- 0 o o 3 4 ‘ 2 52 c1 0 2 3 a m E g 2 E E E E O a. a m 9 ° ‘” .- °5 3 A. 3 C, D E p Figure 19. Effects of GABA antagonists on.serum GH. Vertical bars represent SEM. N=8, 9 or lO/group. ll7 200— Q N=6/GROUP n9 GH perml 100— SERUM l OD .: ‘\. u: 2. '3 ...l :3 c: g . ‘3 g * 2 m 2 I- g’ m z *5; ea 0 “2 2 E C) OI a a Figure 20. Effects of bicuculline and a-methyl-paratyrosine on serum GH. Vertical bars represent SEM. N=6/group. a MPT= a-methyl-paratyrosine. 118 (Figure 21). BIC did not alter steady state concentrations of DA or NE. Additionally, BIC did not alter ME steady state or turnover index concentrations of DA or NE. Intraperitoneal injections of GABA into HYPOX rats bearing a single AP transplanted under the kidney capsule did not alter GH release from transplanted pituitaries (Table 16). £2.21EE2 incubation of GABA with AP halves did not alter GH release from the; AP (Figure 22). Co-incubation of GABA at several doses with AP halves and hypothalami increased the inhibition of GH release produced by the hypothalamus alone (Figure 23). This inhibition was overcome by addition of PIC to the incubation media. The DA receptor blocker, PIM; the 5-HT receptor blocker, MET; the GABA receptor blocker, PIC; the B-adrenergic receptor blocker, PRO; and the¢1-adrenergic receptor blocker, PHE, did not alter GH release directly from AP alone or from ‘co-incubated APs (Figure 24). Addition of 114g GABA/ml media did not effect GH release from these co—incubated AP halves. Conclusions An increase in central GABA, either by intraventricular injections of GABA or by parenteral injections of AOAA, resulted in decreased GH release. Even though AOAA elevates brain GABA for 6 hrs (Wallach, 1961; Perry et al., 1974), the effects on GH at this time were minimal.‘ This dis- crepency could be explained if other neural mechanisms 119 ..Né_._ _ .. 0‘ ~ 500» 2000 15 ' 26 26 " i 3.6 ‘ —3OO NG/GM 1ooo Foo ‘ —— ' -7O 19 12— _ 29 - 3 3 3° - so NG/MG _ 6- ~30 PROTEIN _ ‘ ~10 coNrROLS B|CUCULL|NE 2.5mg/k9 - 7... l-_____ -7, ,-_ ._N.=, 6(GROUP Figure 21. Effects of bicuculline on hypothalamic and median eminence NE and DA. Lower panel represents median eminence and the upper panel represents the remainder of the hyp- othalamus. Solid bars represent steady state concentrations; broken bars rep- resent concentrations 1 h after aMPT. Vertical bars represent SEM. Numbers above broken bars represent % depletion. 120 .msonm\oau3 onv;masmdmo umcpwx ecu naps: m< H nag: much xomwm cu poundaoo no.0vm ow .mHouuaou xomwm cu pmuaasou mc.ovm b .Ha\m: “.=.m.m w m a aa.o+H.aH am.o«m.m~ no.m«o.Hn o.mH~.mH unosumouu uumoa u: H m Nan b¢.©fim.m~ ao.H>_mm zo eauom so =. AoHv maouucou_ xomwz 121 I AP/2 I AP/2&GABA ..t i ..l. t...}.lu.‘(triniwnmim. ...... I .1“. I “.0 ....!wo.n.l.n,0 .H, 3”,! ..Ho filu‘b (i1..- w. .3“7&3“...,._¢,.H,.3...t.u.omlom.o ......... c 2!. 02!, , . , .. . . . . . . . . .3..1.v.I.!,?.l.l.1.n“5_nr ‘8 gm/ml 1...! . I: 2.3 will”? ..1...!..l HT “ium,-.. in I.“ tattfwnu t “ locoDQOQOAAIQO 1- . _n10§9§t.nrov.7..,o,xl20.”..-..lq!“oH.~mt.u_|...b 6,. 0.3.3202? 9.320.»! by I . 5,; ., t at... (at... 09.9.}.“390 H.0.H_6.”.D...,3m or'u0,”.l.u..0...“0...rlu fululwtm. 12!“; '6 .10.“.-.ng ..-..QOImTQIUT40,03... o mimimtov . D u 0 n I ..-...Obto or, 1. -7 DOSE GABA1Ox O—2hrs !..!..O..3.,.i ...-..T_H.I.x0..,!u, I “.0 h.Iota..-ml..!v-u£ulxt.n 0.2.1“? -8 t_urv,wc_”.c.wlxl..uo ”7&1“? :1“, in 3“. 05.15.- A, P o .h t h 1.37...- .. .. 1. a .. u ... .2. H37 ... o .. -10 I”.-. not,” I womin.!.i.n.1..i ..7 Hi ”3...?” 1.”? ”F H,2min}Q-..-.HLaOmHorxloUHO. C Q6- 02- // HMP Ug(3 ml/ mgA 2—4hr_s N=8/group. In vitro effects of GABA on GH release. Vertical bars represent SEM. AP/2=Anterior Pituitary half. Figure 22. “9(fl1/ ml/hr ["91\ Figure 23. 122 t2‘ (181 0.4- GABA 0109 gmlml O-2hrs- I AP/2 AP/2&HYP. GABA+ PIC. 1u9 2-4hrs -5 ”A. . -.. ..——- In vitro effects of GABA and picrotoxin on GH release in co- -incubation. Vertical bars represent SEM. N=8/group. AP/2=Anterior Pituitary half; Hyp=Hypothalamus. 123 " ~ ‘ I AP/2 t2 ._ .=. AP/2&HYP. ' 08 ug GH/ mI/hr mgA l 04— c PIMMEIPIC.PRO.PH ' + 10° 1 1 1 1 1 GABA ng us He us He “9 o—Zhrs 2-r4hrs Figure 24. In vitro effects of central monoaminergic block- 171g drugs and GABA on GH release in coaincubation. Vertical bars represent SEM. N=8/group, AP/2=Anterior Pituitary half; Hyp=Hypothalamus. 124 changed to compensate for this increase in brain GABA. BIC, BIC MI, and PIC all GABA receptor blockers (Curtis gt gt., 197l; Pong and Graham, 1972; Nistri gt gt., 1974; Shank gt gt., 1974) significantly increased serum GH concentrations 20 and 60 min after injections. Additionally, BIC MI was capable of reversing the decrease in GR release produced by GABA. GABA does not act directly on the pituitary to decrease GH release, but appears to increase the inhibitory effects of the hypothalamus on GH release in a co-incubation. NE was previously shown to decrease the hypothalamic inhib- itory influence on GH release £2 ztttg (Bruni, Ph.D. thesis). In view of these results, GABA could be decreasing NE activ- ity in the hypothalamus, thus resulting in greater inhibi- tion. of GH release. GABA also may stimulate the release of somatostatin from the hypothalamus, resulting in decreased GH release observed $2.21EEE- ”a-MPT decreased the stim- ulatory effects of BIC on GH release. These results suggest that BIC exerts its effects on GH release through the catecholamines. Examination of hypothalamic catecholamine activity showed that the NE turnover index (TI) was increased by treatment with BIC. NE was previously shown to stimulate GH release 12 ggzg (Luizzi gt gt., 1971; Durrand gt gt., 1977; Bruni, Ph.D. thesis) and £2.Z£E£2 in a co-incubation system (Bruni, Ph.D. thesis). Thus, GABA may inhibit NE turnover which can result in depression of GH release. Measurement of hypothalamic somatostatin concentrations following drug 125 treatment would help clarify the mechanism whereby these putative neurotransmitters affect GH release. VI. Effects of Methionine—Enkephalin, Naloxone and Morphine on GR Release Introduction Recently several morphinomimetic peptides have been isolated from the mammalian central nervous system, includ- ing methionine- (MET-) and leucine- (LEU-) enkephalin (ENK) (Hughes gt gt., 1975), and Brendorphin (Cox gt gt., 1976). These peptides share common amino acid sequences with the B-lipotropin molecule, which may infer a role for B-lipo- tropin as a prohormone for endorphins (Lazarus gt gt., 1976). The pars intermedia and isolated clusters of cells in the pars distalis were shown to contain high concentra- tions of B-lipotrOpin (Pelletier gt 21" 1977). These' same cells also contained abundant amounts of ACTH (Pelletier gt gt., 1977). Several labs recently reported that morphinomimetic peptides released PRL and GH (Lein gt gt., 1976; Rivier gt gt., l977b, Bruni gt gt., 1977b; Bruni, Ph.D. thesis). The stimulatory effects of B-endorphin and MET-ENK on GH and PRL release were shown to be reversed by concurrent treatment with the opiate antagonist, naloxone (NAL) (Rivier gt gt., 1977b, Bruni gt gt., 1977b,Bruni, Ph.D. thesis). Similarly, morphine has been shown to increase GH 126 release in rats (Kokka gt gt., 1972; Kokka gt gt., 1973; Martin gt gt., 1975; Rivier gt gt., 1977a, Bruni gt gt., 1977b, Bruni, Ph.D. thesis). This study was performed O to determine the effects of MET-ENK, morhpine (MS) and NAL on GR release in rats. Also the effects of MS on the stress induced decrease in serum GH concentrations were observed. Materials and Methods Male Sprague-Dawley rats (ZOO-2509 each) were injected with NAL (Endo Labs, Garden City, N.Y.), MS (Mallinkrodt Labs., St. Louis, MO) or MET-ENK (Bachem, Marina Del Ray, CA), each given individually, or combinations of these drugs injected simultaneously. The drugs~were injected i.pfl in 0.1 ml of 0.87% NaCl/lOO g B.W. (for doses see Tables 17 and 18). All rats were decapitated 20 min after injections. In a second experiment MS was injected i.v. 30 min after the initial blood sample was taken. Sequential blood samples (200 pl) were taken from an indwelling atrial cannula every 5 min for the next 30 min. Plasma was separated and assayed for GH. Forty male Sprague-Dawley rats were used in a third experiment. Ten rats served as controls and were decap- itated 30 min after i.p. injections of 0.87% NaCl. Ten of the remaining rats were injected with 0.87% NaCl and were restrained for 30 min. The remaining 2 groups of rats were injected i.p. with 2 mg/kg MS or 5 mg MS and were restrained for 30 min. After 30 min of restraint stress 127 rats were decapitated and blood was collected for CH radioimmunoassay. Results The effects of NAL, MS, and MET-ENK on serum GH con- centrations are shown in Tables 17 and 18. NAL at a dose of 0.2 and 5 mg/kg significantly reduced serum GH concen- trations 20 min after injections. The 2 mg dose was inef- fective due to the large standard errOr. MS at doses of 2, l0 and l5 mg/kg significantly increased serum GH con- centration 20 min after injection. Similarly, MET-ENK (5 mg/kg) significantly increased serum GH concentrations after injection. When MS or MET-ENK were injected con- currently with NAL, NAL attenuated the increase in serum GH concentrations produced by MS or MET-ENK. Intravenous injections of MS (5 mg/kg) significantly increased serum GH concentrations 10 min after injections (Figure 25). Serum GH continued to increase until 20 min after injection and thereafter plateaued and remained higher than controls for the continuation of the experi- .ments. Rats placed under restraint stress had significantly lower serum GH concentrations than the non-restrained con- trol rats (Table l9). MS (2 mg/kg) failed to increase serum GH concentrations 30 min after injections. However, MS (10 mg/kg) injected into restrained rats significantly increased serum GH concentrations above control values and prevented the decrease in serum.GH induced by restraint stress. 128 Table 17. b l ,,,_ EFFECTS OF NALOXONE, MORPHINE, AND METHIONINE ENKEPHALIN ON SERUM GH n . lO/GROUP ng/ml a CONTROLS - 148 t 17 0.872 NaCI NALOXONE 74 1 24b 0-2 mslkz MORPHINE 1622 1 129b 10.0 mg/kg MET-ERR 258 t 62b 5.0 mg/kg NAL & ME 149 t 16 0.2 + 5.0 , NAL & MOR 1155 i 56b 0.2 + 10.0 a i 3.2.x. .EgfiptOS compared with controls. 129 Table 18. EFFECTS OF NALOXONE, MORPHINE, AND METHIONINE ENKEPHALIN ON SERUM GB n - lO/GROUP ng/ml a CONTROLS 131 t 23 0.87: NaCl NALOXONE 77 I 10b 0.2 mg/kg NALOXONE 103 t 40 2.0 mg/kg NALOXONE 48 1 6b 5.0 mg/kg MORPHINE 839 1 172b 2.0 mg/kg MORPHINE 1211 1 185b 10.0 mg/kg MORPHINE 1775 1 172b 15.0 mg/kg MEI-aux 215 t 27b 5.0 mg/kg NAL & MOR 383 1 71b 0.2 + 2.0 NAL & MGR 902 3 168b 0.2 + 10.0 NAL & ME 138 t 37 0.2 + 5.0 a i t S.E.M. 5 P<0.05 compared with controls. aaomo 3:332. oh ESE—zoo modvafi 13o 2mm “....xuo 3.. 5 e; beam 333 STE . «1.8 «mama m: 3%... 3 ms. 9R9: « .5892" 2 wmmmbm mammhm mmmmhw mnemhzoo V e n N F :o Enema 2. mm