Cellular mechanisms controlling melanophore stimulating hormone (MSH) release.

Melanophore stimulating hormone (MSH) release from the vertebrate pars intermedia is under an inhibitory control by the hypothalamus. Catecholamines both inhibit and stimulate MSH release from the isolated frog neurointermediate lobe or rat (and mouse) pituitary. Classical pharmacological methods using specific adrenergic receptor agonists and antagonists reveal that inhibition of MSH release by catecholamines is mediated through either alpha adrenergic receptors and/or dopamine receptors whereas stimulation of MSH release by catecholamines is mediated through beta adrenergic receptors. These results provide the physiological correlate for the morphological evidence of pars intermedia regulation by direct neuronal innervation. Acetylcholine also stimulates MSH release from the frog neurointermediate lobe and this enhanced hormone secretion is mediated through cholinergic receptors (blocked by atrophine, but not by propranolol). The possible interactions of adrenergic and cholinergic receptor mechanisms in the control of MSH release is still to be determined.Neither boiled nor acid-treated hypothalamic extracts inhibit or stimulate MSH release in vitro thus failing to implicate (by these methods) hypothalamic inhibiting or stimulating factors, if present, in the control of MSH release. Our in vitro experiments also fail to support a so-called “auto-feedback” (mass action) regulation of MSH release in either the rat, the frog, or the toad. Numerous similarities between the mechanisms involved in the control of MSH and prolactin release are apparent.     

Acetylcholine stimulates alpha-melanocyte-stimulating hormone release from frog pituitary melanotrophs through activation of muscarinic and nicotinic receptors

The release of alpha MSH from the pars intermedia of amphibians is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In this study we have examined the possible involvement of acetylcholine (ACh) in the regulation of alpha MSH secretion from the pars intermedia of the frog (Rana ridibunda) using the perifusion technique. When intact neurointermediate lobes (NIL) were exposed to graded doses of ACh (3 X 10(-7) to 3 X 10(-4) M), a dose-dependent stimulation of alpha MSH release was observed. Repeated administration of ACh (10(-4) M) induced reproducible responses of NIL without any desensitization phenomenon. ACh was also capable of stimulating alpha MSH release from dispersed intermediate lobe cells, indicating that the neurotransmitter exerts its effect by acting directly on frog melanotrophs. Using the monoclonal antibody M-35 against calf muscarinic receptors we have visualized, by the immunofluorescence technique, the presence of muscarinic receptor-like immunoreactivity in the frog pars intermedia. The stimulatory action of ACh was mimicked by both nicotine and muscarine (10(-5) M each). Nicotine-induced stimulation of alpha MSH release was partially abolished by alpha-bungarotoxin (10(-6) M) and hexamethonium (10(-4) M). The stimulatory effect of muscarine was suppressed by atropine and the M1-muscarinic antagonist pirenzepine (10(-5) M), but not by the M2-muscarinic antagonist gallamine. We have investigated the effect of ACh during administration of specific nicotinic and muscarinic antagonists. While hexomethonium or atropine could block only part of the stimulatory effect of ACh, concomitant administration of these antagonists totally abolished the response of NIL to ACh. Finally, the stimulatory effect of ACh was not impaired during prolonged administration of the beta-adrenergic antagonist propranolol. These data show that ACh stimulates in vitro alpha MSH secretion by frog NIL. Our results also indicate that amphibian pars intermedia cells possess two types of cholinergic receptors, an M1-muscarinic receptor sensitive to pirenzepine and nicotinic receptors sensitive to hexamethonium and alpha-bungarotoxin.     

In vitro study of frog (Rana ridibunda Pallas) neurointermediate lobe secretion by use of a simplified perifusion system. IV. Interaction between dopamine and thyrotropin-releasing hormone on alpha-melanocyte-stimulating hormone secretion

The interaction between dopamine and TRH on alpha-melanocyte-stimulating hormone (MSH) release from the intermediate lobe of amphibian pituitary has been studied in vitro using the perifusion model. Dopamine (10(-10) to 10(-6) M) was responsible for a dose-related inhibition of alpha-MSH secretion. The inhibitory effect of dopamine (10(-8) and 3.16 X 10(-8) M) was completely abolished in the presence of haloperidol (10(-5) and 10(-6) M, respectively). It has been previously established that, in amphibians, TRH stimulated alpha-MSH release in vitro and that the action of TRH was not mediated via an inhibition of the release of endogenous dopamine (M. C. Tonon, P. Leroux, M. E. Stoeckel, S. Jégou, G. Pelletier, and H. Vaudry, 1986, Endocrinology 112, 133-141). In the present study we demonstrate that TRH (10(-7) M) reverses the inhibitory effect of dopamine (for concentrations ranging from 3.16 X 10(-8) to 10(-6) M) on alpha-MSH secretion and that the effects of TRH and dopamine are additive. Thus, these results indicate that the intracellular events associated with TRH-induced stimulation and dopamine-induced inhibition of alpha-MSH release are not linked together.     

The effects of dopamine on renin release in vitro.

To examine the direct effects of dopamine on renin release, the in vitro rat kidney slice system, devoid of hemodynamic and humoral effects, was chosen. In the presence of an antioxidant, ascorbic acid (6 X 10(-4)M), a significant dose-related stimulation of renin release was observed with addition of 10(-5)M and higher concentrations of dopamine. When the monoamine oxidase inhibitor, pheniprazine (1 X 10(-5)M) was added, significant, dose-related stimulation of renin release was observed with 10(-8)M and higher concentrations of dopamine. Dopamine-induced renin release was not inhibited by the presence of the alpha-adrenergic antagonist, phentolamine (9 X 10(-4)M), the dopaminergic antagonist, haloperidol (5 X 10(-5)M) or the neural uptake inhibitor, cocaine (1 X 10(-5)M). However, the presence of the beta-adrenergic antagonist, propranolol (2 X 10(-4)M) completely inhibited dopamine-induced renin release. These studies indicate that dopamine can directly stimulate renin release in the absence of effects of hemodynamic factors, alterations in sodium metabolism or release of endogenous adrenergic agents. Further, this direct effect of dopamine on renin release appears to be mediated by an agonistic effect on the juxtaglomerular beta receptor rather than by the presence of a specific dopaminergic receptor for renin release.     

Dopaminergic and beta-adrenergic receptor control of alpha-melanocyte-stimulating hormone secretion during stress

The relative roles of dopaminergic and beta-adrenergic receptors in mediating the stress-induced increase in the secretion of alpha-melanocyte-stimulating hormone (alpha-MSH) from the intermediate lobe of the pituitary were determined in the male rat. Thirty minutes of physical immobilization (restraint stress) increased the circulating concentrations of alpha-MSH and decreased the 3,4-dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratio in the intermediate lobe of the pituitary, reflecting a decrease in the tuberohypophysial dopaminergic neuronal activity. Pretreatment with the beta-adrenergic antagonist propranolol reduced the stress-induced increase in the circulating levels of alpha-MSH, but had no effect on the basal plasma concentrations of this hormone or the stress-induced decrease in DOPAC/DA in the intermediate lobe. If the dopaminergic tone during stress was maintained by administration of the DA agonist apomorphine, the stress-induced increase in alpha-MSH secretion was prevented. In nonstressed animals the administration of the beta 2-adrenergic agonist metaproterenol increased the plasma levels of alpha-MSH, and the effect of this drug was augmented if the inhibitory dopaminergic tone on alpha-MSH secretion was blocked by the administration of the DA antagonist haloperidol. Severing neurons in the retrochiasmatic region of the hypothalamus blocked the stress-induced decrease in DOPAC/DA in the intermediate lobe and attenuated the stress-induced increase in plasma concentrations of alpha-MSH. Taken together, these results indicate that a decrease in tuberohypophysial dopaminergic neuronal inhibitory tone and an increase in beta-adrenergic stimulation are both necessary for the full expression of the stress-induced increase in secretion of alpha-MSH from melanotrophs in the intermediate lobe of the rat pituitary.     

The alpha-1-adrenergic neuronal system is involved in the pulsatile release of luteinizing hormone-releasing hormone in the ovariectomized female rhesus monkey.

It has been postulated that catecholamines are integral to the control of LHRH release. In the present study, the roles of adrenergic and dopaminergic receptors in the control of pulsatile LHRH release are examined. The effects of prazosin (an alpha 1-antagonist), rauwolscine (an alpha 2-antagonist), propranolol (a beta-antagonist), haloperidol (a dopamine antagonist), SCH23390 (a D1 antagonist), and LY163502 (a D2 agonist), on in vivo LHRH release in the stalk-median eminence were tested in ovariectomized female rhesus monkeys using push-pull perfusion. Prazosin caused a significant suppression of the LHRH release. This was primarily due to a significant suppression of LHRH pulse amplitude, but not pulse frequency, i.e. the interpulse interval was not affected by the administration of prazosin. In contrast to prazosin, none of the other adrenergic or dopaminergic drugs had significant effects on LHRH release. We conclude from these results that (1) the stimulatory effects of norepinephrine (NE) and/or epinephrine on pulsatile LHRH release are mediated by alpha 1-adrenergic receptors but not alpha 2- or beta-adrenergic receptors, and (2) dopaminergic receptors do not appear to be involved in pulsatile LHRH release in ovariectomized rhesus monkeys.     

Evidence for alpha 1-adrenergic stimulatory control of in vitro release of immunoreactive thyrotropin-releasing hormone from rat median eminence: in vivo corroboration

The aim of this study was to investigate whether the alpha-adrenergic stimulation of TSH secretion may occur directly at the median eminence (ME) level by modulating the release of TRH. The effects of pharmacological manipulations of the two subtypes of central alpha-adrenergic receptors, alpha 1 and alpha 2, were tested on in vitro TRH release from medial basal hypothalami containing mainly the ME. Hypothalamic fragments were superfused with a modified Locke medium, and TRH was measured by RIA in samples collected every 10 min. After a preliminary period of 40 min to test TRH release during basal conditions, drug effects were checked for 20 min. Superfusion with norepinephrine (NE) (10(-10), 10(-8), 10(-6) M) induced a rapid and dose-dependent rise of TRH release; epinephrine (10(-8) M) induced an effect similar to that of NE 10(-8) M. Phentolamine (10(-7) M), an alpha-adrenergic antagonist, completely blocked the NE (10(-8) M)-induced release of TRH, which was not modified by the beta-adrenergic antagonist propranolol (10(-7) M). Neither antagonist had an effect on basal TRH release when added alone to the medium. The NE-induced release of TRH was completely suppressed by prazosin (10(-7) M), whereas yohimbine had no effect. Superfusion with clonidine (10(-9), 10(-8), 10(-7), 10(-6) M), an alpha 2-receptor agonist, did not alter basal TRH release. In contrast, phenylephrine (10(-8) and 10(-6) M), an alpha 1-receptor agonist, induced a significant (P less than 0.01) rise in TRH release. These results were corroborated in vivo in several unanesthetized rats bearing a push-pull cannula previously and stereotaxically implanted into the ME. Perfusion with artificial cerebrospinal fluid containing NE (10(-7), 10(-6) M) or phenylephrine (10(-7) M) elicited a rapid rise in TRH release, within 15 min after the onset of drug perfusion. Clonidine (10(-5) M), similarly perfused for 15 min, had no effect. Our data suggest a direct stimulatory influence of catecholamines on TRH release at the ME level that is mediated through alpha 1-adrenergic receptors.     

On the role of the central noradrenergic and dopaminergic systems in the regulation of TSH secretion in the rat.

Systemic administration of drugs affecting central noradrenergic and dopaminergic systems was used to evaluate their role in the regulation of TSH secretion in the rat. Alpha-methyl-p-tyrosine (alpha-MT) caused a depletion of brain norepinephrine and dopamine and a gradual decrease of serum TSH levels. Specific inhibitors of dopamine-beta-hydroxylase, diethyldithiocarbamate (DDC) and FLA 63, depleted central norepinephrine only and led to a simultaneous striking decrease of serum TSH. Blockade of alpha adrenergic receptors with phenoxybenzamine, but not with phentolamine, also depressed serum TSH. Blockade of beta receptors with propranolol had no effect. In contrast, the centrally and peripherally acting alpha receptor agonist, clonidine, increased serum TSH, whereas the peripherally acting methoxamine caused a decrease, probably due to non specific stress effect. A dose-related rapid inhibition of TSH secretion was observed following stimulation of dopamine receptors with apomorphine. Injection of L-Dopa had a similar effect. Blockade of the dopamine receptors with pimozide did not alter serum TSH, while blockade with spiroperidol led to a slight increase. The cold-induced surgeof TSH was abolished by pretreatment with DDC or phenoxybenzamine, reduced by apomorphine, but unaffected by pimozide or propranolol. The pituitary responsiveness to exogenous TRH was unaffected by administration of DDC or apomorphine. On the basis of these results, it is assumed that the central noradrenergic system has a stimulatory effect on the release of TRH from the hypothalamus, reflected in our experiments by the changes of serum TSH levels. It probably provides the drive for the tonic release of TRH in resting conditions and stimuli for the enhanced secretion during cold exposure. The effect is probably mediated by a central alpha-adrenergic mechanism. Activation of the dopaminergic system is inhibitory, but the physiological role of this effect remains to be established.     

Dopaminergic suppression of pancreatic somatostatin secretion

To characterize dopaminergic influences on pancreatic islet D cell function and its potential interaction with islet A and B cell function, the effect of dopamine (0.5-100 micro M) on immunoreactive somatostatin (IRS), insulin (IRI), and glucagon (IRG) release from rat islets incubated in vitro was studied. Dopamine significantly suppressed the release of IRS (P less than 0.001) and IRI (P less than 0.001) and augmented IRG release (P less than 0.001). Maximum suppression of IRS and IRI release was evident at 20 micro M dopamine with half-maximal suppression occurring at 0.5-1 micro M. Maximal stimulation of IRG release was observed at 100 micro M dopamine with a half-maximal response occurring at 5-10 micro M. Suppression of IRS secretion by dopamine (20 micro M) was completely reversed by the dopaminergic antagonists haloperidol (5 micro M) and pimozide (5 micro M) but was only partially reversed by the alpha adrenergic antagonist phentolamine (2 micro M), and was further suppressed by the beta adrenergic antagonist phentolamine (2 micro M). Suppression of IRI release by dopamine was completely reversed by propranolol, but was unaffected by haloperidol, pimozide, and phentolamine. There results indicate that dopamine directly affects pancreatic islet D cell function, and that islet B and D cells appear to be more sensitive to dopamine than are A cells. Dopamine suppresses IRS secretion predominantly through activation of dopaminergic receptors, whereas it suppresses IRI release through an alpha adrenergic mechanism and stimulates IRG release through a beta adrenergic mechanism.     

Stimulation by alpha-adrenergic mechanisms of the secretion of growth hormone-releasing factor (GRF) from perifused rat hypothalamus

The possible involvement of adrenergic mechanisms in regulating the secretion of growth hormone (GH)-releasing factor (GRF) from the rat hypothalamus was examined in vitro with a perifusion system. A high potassium concentration (56 mM) stimulated GRF release from the hypothalamus. The infusion of clonidine (10(-4) M), an alpha 2-adrenergic stimulant, resulted in an increase in the spontaneous release of GRF. In the presence of propranolol (10(-5) M), a beta-adrenergic blocking agent, clonidine (10(-5) and 10(-4) M) stimulated GRF release more prominently in a dose-related manner, whereas propranolol (10(-5) and 10(-4) M) by itself did not affect the spontaneous GRF release. The stimulatory effect of clonidine (10(-4) M) on GRF release in the presence of propranolol was inhibited by yohimbine (10(-4) M), an alpha 2-adrenergic blocking agent. These findings suggest that alpha 2-adrenergic mechanisms play a role in stimulating GRF release from the hypothalamus in rats.     

Central nervous system-mediated glucagon secretion is enhanced by alpha 2-adrenoreceptor activation

We assessed the response of the adrenergic receptor in pancreatic glucagon secretion to central nervous system stimulation. Injection of neostigmine (5 x 10(-8) mol) into the third cerebral ventricle in intact rats resulted in increased epinephrine and norepinephrine secretion associated with glucagon secretion. This glucagon secretion was still observed in bilateral adrenalectomized (ADX) rats, although its concentration was significantly lower than that in the intact rats. This glucagon rise was significantly inhibited by ip treatment of ganglionic blocker with hexamethonium. Intraperitoneal injection of alpha-adrenergic receptor antagonist phentolamine (5 x 10(-7) mol), but not of beta-adrenergic receptor antagonist propranolol (1 x 10(-6) mol), reduced the hyperglucagonemic effect of a subsequent neostigmine injection in intact and ADX rats, although these antagonists did not influence epinephrine or norepinephrine secretion in intact rats. In addition, ip injection of the selective alpha 2-receptor antagonist yohimbine (5 x 10(-7) mol), but not of the selective alpha 1-receptor antagonist prazosin (1 x 10(-6) mol), inhibited the neostigmine-induced glucagon secretion in intact and ADX rats. From this evidence it is suggested that central nervous system-mediated glucagon release is enhanced by alpha 2-adrenoreceptor stimulation by either catecholamines or the autonomic nervous system.     

The major immunoreactive alpha-melanocyte-stimulating hormone (alpha MSH)-like substance found in human fetal pituitary tissue is not alpha MSH but may be desacetyl alpha MSH (adrenocorticotropin1-13NH2)

Pituitary glands were obtained from human abortuses during the second half of gestation. Acid extracts were made from the anterior and neurointermediate lobes, and alpha MSH immunoreactivity (alpha MSHi) was quantified by RIA. alpha MSHi was found in both lobes of the pituitary gland, with 20-80% of the total pituitary alpha MSHi being present in extracts of the anterior lobe. Anterior and neurointermediate lobe extracts subjected to gel filtration on Sephadex G-50 revealed one peak of alpha MSHi having an elution profile identical to those of alpha MSH and desacetyl alpha MSH (ACTH1-13NH2). To characterize further the alpha MSHi extracts were subjected to high pressure liquid chromatography. No alpha MSH could be identified in extracts of the anterior lobe, and most of the alpha MSHi had an elution profile identical to that of desacetyl alpha MSH. Although small amounts of alpha MSH might be present in the neurointermediate lobe, most of the alpha MSHi in this lobe coeluted with desacetyl alpha MSH. Since alpha MSH was not converted to desacetyl alpha MSH during the extraction and chromatographic procedures, we hypothesize that the predominant form of alpha MSH-like material in the human fetal pituitary gland may be desacetyl alpha MSH.     

Adrenergic and dopaminergic regulation of gonadotropin-releasing hormone release from goldfish preoptic-anterior hypothalamus and pituitary in vitro.

The involvement of adrenergic and dopaminergic receptor subtypes on in vitro release of radioimmunoassayable gonadotropin-releasing hormone (GnRH) from incubated preoptic-anterior hypothalamic (P-AH) slices and pituitary fragments of sexually mature male goldfish was studied. Norepinephrine (NE) produced a dose-related stimulation of GnRH from P-AH slices, but not from pituitary fragments. The effects of some adrenergic receptor agonists (1 microM) on GnRH release from P-AH slices were tested: phenylephrine (alpha 1-agonist) significantly stimulated GnRH release; clonidine (alpha 2-agonist) and isoproterenol (beta-agonist) were ineffective. Incubation of P-AH slices with phentolamine (alpha 1/alpha 2-antagonist) and prazosin (alpha 1-antagonist), at a concentration of 1 microM, inhibited the release of GnRH induced by NE (60 microM); the alpha 2-antagonist yombibin and the beta-antagonist propanolol were ineffective. None of the adrenergic antagonists (1 microM) tested produced significant effects on spontaneous release of GnRH from both tissue preparations. Spontaneous release of GnRH from both P-AH slices and pituitary fragments was reduced by dopamine (DA) in a dose-related manner. The effects of some DA agonists (1 microM) were tested: apomorphine (D1/D2-agonist) and SKF 38398 (D1-agonist), but not bromocriptine and LY-171555 (D2-agonists) significantly reduced spontaneous GnRH release from P-AH slices in vitro. On the other hand, D2-agonists, but not D1-agonists, significantly reduced GnRH release from pituitary fragments. The effects of DA antagonists (1 microM) were also tested: in P-AH slices, addition of SKF-83566 (D1-antagonist) significantly reduced spontaneous GnRH release; pimozide and domperidone (D2-antagonist) were ineffective when tested alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of calcium in thyrotrophin-releasing hormone-stimulated release of melanocyte-stimulating hormone from frog neurointermediate lobe

The effect of modifications of extracellular calcium concentrations on alpha-MSH release has been studied using perifused frog neurointermediate lobes. Increasing concentrations of calcium (from 2 to 10 mmol/l) gave rise to a dose-related stimulation of alpha-MSH secretion, whereas reduction of Ca2+ from 2 to 1.5 mmol/l partially inhibited alpha-MSH release. The direct effect of extracellular Ca2+ on alpha-MSH secretion was confirmed by the dose-dependent stimulation of alpha-MSH release induced by the calcium ionophore A23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mmol Co2+/l both resulted in an inhibition of spontaneous and TRH-induced alpha-MSH release. Conversely, administration of verapamil or methoxyverapamil (10 mumol/l each) did not alter basal secretion and had no effect on the response of the glands to TRH. Nifedipine (10 mumol/l), which was able to block KCl (20 mmol/l)-evoked alpha-MSH release, induced a slight inhibition of basal alpha-MSH secretion, indicating that extracellular Ca2+ levels may regulate alpha-MSH release in part by Ca2+ influx through voltage-dependent Ca2+ channels. In contrast TRH-induced alpha-MSH release was not affected by nifedipine or dantrolene (10 mumol/l), and BAY-K-8644 (1 mumol/l) did not significantly modify the response of neurointermediate lobes to TRH. Taken together, these results suggest that TRH-induced alpha-MSH secretion is associated with calcium influx across the plasma membrane and that calcium entry caused by TRH may occur through nifedipine/verapamil-insensitive Ca2+ channels.     

Thyrotrophin-releasing hormone stimulates polyphosphoinositide metabolism in the frog neurointermediate lobe

Previous studies have demonstrated that TRH is a potent stimulator of alpha-MSH secretion from frog pituitary melanotrophs. In order to determine the intracellular events responsible for TRH-evoked alpha-MSH release, we have investigated the effect of TRH on polyphosphoinositide breakdown in frog pars intermedia. Neurointermediate lobes were labelled to isotopic equilibrium with myo-[3H]inositol. TRH stimulated the rate of incorporation of [3H]inositol into the phospholipid fraction. The effect of TRH was concentration-dependent; half-maximal stimulation of alpha-MSH release and inositol incorporation occurred at 12 and 28 nmol TRH/l respectively. In prelabelled neurointermediate lobes, lithium (10 mmol/l) enhanced the radioactivity in inositol monophosphate, bisphosphate (IP2) and trisphosphate (IP3). LiCl (10 mmol/l) induced a 38% inhibition of alpha-MSH release from perifused neurointermediate lobes but did not impair TRH-induced alpha-MSH secretion. In the presence of LiCl, TRH (1 mumol/l) induced a transient increase of the radioactivity in IP3, which was evident by 30 s and maximal by 1 min (+100%). TRH treatment also increased the radioactivity in IP2, which reached a plateau after 5 min (+100%). The increase in radioactivity in IP3 induced by TRH was closely paralleled by a rapid loss of [3H]phosphatidylinositol bisphosphate (PIP2), which was maximal by 1 min (-70%). These results indicate that, in frog pars intermedia, TRH-evoked alpha-MSH secretion is coupled to breakdown of PIP2. The data suggest that, in amphibian melanotrophs, as previously shown in GH3 tumour cells and in rat pituitary mammotrophs, TRH causes rapid stimulation of polyphosphoinositide-hydrolysing phospholipase C.     

Thyrotropin-releasing hormone stimulates growth hormone release from the anterior pituitary of hypothyroid rats in vitro

We have examined the interaction of thyroid hormone and TRH on GH release from rat pituitary monolayer cultures and perifused rat pituitary fragments. TRH (10(-9) and 10(-8)M) consistently stimulated the release of TSH and PRL, but not GH, in pituitary cell cultures of euthyroid male rats. Basal and TRH-stimulated TSH secretion were significantly increased in cells from thyroidectomized rats cultured in medium supplemented with hypothyroid serum, and a dose-related stimulation of GH release by 10(-9)-10(-8) M TRH was observed. The minimum duration of hypothyroidism required to demonstrate the onset of this GH stimulatory effect of TRH was 4 weeks, a period significantly longer than that required to cause intracellular GH depletion, decreased basal secretion of GH, elevated serum TSH, or increased basal secretion of TSH by cultured cells. In vivo T4 replacement of hypothyroid rats (20 micrograms/kg, ip, daily for 4 days) restored serum TSH, intracellular GH, and basal secretion of GH and TSH to normal levels, but suppressed only slightly the stimulatory effect of TRH on GH release. The GH response to TRH was maintained for up to 10 days of T4 replacement. In vitro addition of T3 (10(-6) M) during the 4-day primary culture period significantly stimulated basal GH release, but did not affect the GH response to TRH. A GH stimulatory effect of TRH was also demonstrated in cultured adenohypophyseal cells from rats rendered hypothyroid by oral administration of methimazole for 6 weeks. TRH stimulated GH secretion in perifused [3H]leucine-prelabeled anterior pituitary fragments from euthyroid rats. A 15-min pulse of 10(-8) M TRH stimulated the release of both immunoprecipitable [3H]rat GH and [3H]rat PRL. The GH release response was markedly enhanced in pituitary fragments from hypothyroid rats, and this enhanced response was significantly suppressed by T4 replacement for 4 days. The PRL response to TRH was enhanced to a lesser extent by thyroidectomy and was not affected by T4 replacement. These data suggest the existence of TRH receptors on somatotrophs which are suppressed by normal amounts of thyroid hormones and may provide an explanation for the TRH-stimulated GH secretion observed clinically in primary hypothyroidism.     

Effects of synthetic corticotropin-releasing factor and dopamine on the release of immunoreactive beta-endorphin/beta-lipotropin and alpha-melanocyte-stimulating hormone from human fetal pituitaries in vitro

The effects of synthetic corticotropin-releasing factor (CRF) and dopamine on immunoreactive beta-endorphin/beta-lipotropin (i beta-END/LPH) and alpha MSH release were studied in superfused human fetal pituitary glands. CRF (20 ng) stimulated the release of i beta-END/LPH in four anterior hemipituitaries from fetuses older than 20 weeks in gestation. There was no effect on three anterior hemipituitaries from fetuses of 19-20 weeks gestation. CRF had no effect on i beta-END/LPH or alpha MSH secretion from neurointermediate lobes regardless of fetal age. Dopamine (10(-6) M) had no effect on i beta-END/LPH or alpha MSH secretion from either anterior or neurointermediate lobes. The data suggest that anterior pituitary responsiveness to CRF develops at about 20 weeks gestation and that fetal neurointermediate lobe secretion of peptides is not regulated by CRF.     

Regulation of melanotropin release from the pars intermedia of the amphibian Xenopus laevis: evaluation of the involvement of serotonergic, cholinergic, or adrenergic receptor mechanisms

Melanophore-stimulating hormone (MSH) release from the pars intermedia of the pituitary gland is probably regulated by multiple factors of hypothalamic origin. We have examined a number of potential regulatory factors for their effects on MSH release from the amphibian Xenopus laevis. Serotonin and acetylcholine have no effect on MSH release. Both adrenaline and noradrenaline inhibit release of MSH in a dose-dependent manner. Studies with specific receptor agonists and antagonists reveal that these neurotransmitters exert their in vitro effects primarily through a dopamine D-2 receptor, although an alpha-adrenergic receptor could not be excluded. We further conclude that the pars intermedia of X. laevis lacks a beta-adrenergic receptor for the regulation of MSH secretion from the pars intermedia. In mammals, this receptor activates the adenylate cyclase system. Our studies reveal that despite the lack of beta-adrenergic receptors, cyclic-AMP is likely an intracellular factor involved in the stimulation of MSH release.     

In vitro study of frog (Rana radibunda Pallas) interrenal function by use of a simplified perifusion system. I. Influence of adrenocorticotropin upon corticosterone release.

In order to examine the dynamics of the steroidogenic response from amphibian interrenal glands to various stimulae, frog interrenal secretion was studied using a perifusion system technique, Rana ridibunda adrenal fragments were continuously perifused for 10 hr with amphibian culture medium (ACM). Corticosterone release was assayed using a sensitive competitive-binding radioassay. Inhibition curves using either standard corticosterone dissolved in ACM or serial dilutions of effluent perfusate were strictly parallel. When the temperature of the perifusing medium was raised from 5 to 30°, the corticosterone secretion rate increased eightfold, suggesting a possible direct effect of the ambient temperature on corticosterone secretion in the frog. Perifusion of frog interrenal fragments with homologous crude pituitary distal lobe extracts led to a linear log-dose response. When synthetic ^1–24ACTH was used as a reference standard, the perifusion technique proved useful as a semiquantitative assay system for the detection of biologically active pituitary ACTH. Since immunoreactive ACTH is also found in frog intermediate lobes, pars intermedia extracts were assayed for their ability to stimulate corticosterone release in vitro. At a dose as low as 0.006 intermediate lobe equivalent/ml a significant increase in corticosterone release was observed (1.8-fold; P < 0.001). At the lower doses, the effects of intermediate lobe extracts on corticosterone release were dose-related. The maximum effective dose is $\text{1}\text{40}$ intermediate lobe eq/ml. At higher doses (up to $\text{1}\text{5}$ eq/ml) no stronger stimulation could be elicited. Dibutyryl-cyclic-AMP at 10^?2M induced a 3.3-fold increase in corticosterone secretion. From these results, we conclude that the perifusion system technique will prove useful for the study of the mechanisms governing the regulation of frog steroidogenesis in vitro.     

Lack of effect of TRH on alpha-MSH release from the neurointermediate lobe of the lizard Lacerta vivipara.

Thyrotropin-releasing hormone (TRH) is a potent stimulator of melanotropin (alpha-MSH) release from pituitary melanotrophs in pig, frog, and fish. Concurrently, it has recently been shown that injection of TRH induces skin darkening in the lizard Anolis carolinensis (Licht and Denver, 1988). In the present study, we have thus investigated in vitro the possible effect of TRH on alpha-MSH release from the lizard (Lacerta vivipara) neurointermediate lobe, by means of the perifusion technique. Using our radioimmunoassay procedure, we found that serial dilutions of L. vivipara NIL extracts and synthetic alpha-MSH gave parallel binding curves. Administration of graded doses of TRH (10(-8)-10(-6) M) did not cause any modification of alpha-MSH release. In contrast, infusion of a depolarizing concentration of K+ induced a robust stimulation of alpha-MSH secretion. These results indicate that, in the lizard L. vivipara, the neuropeptide TRH does not stimulate pituitary melanotrophs.