Thyrotrophin-releasing hormone (TRH)-induced growth hormone secretion in fowl: binding of TRH to pituitary membranes

Heat-labile specific binding sites for [3H]3-methylhistidine2-TRH [( 3H]Me-TRH) were demonstrated on chicken adenohypophysial membranes. These binding sites were of both high affinity (dissociation constant, Kd = 15.53 nM) and low capacity (maximum binding capacity, Bmax = 8.73 pmol/g tissue) and of low affinity (Kd = 104.5 nM) and high capacity (Bmax = 32.41 pmol/g). Binding of [3H]Me-TRH to the pituitary membranes was greater at 4 degrees C than at 39 degrees C and occurred with rate constants (k1) of 1.6 x 10(6) and 3.39 x 10(6) M-1 min-1 respectively. Dissociation of [3H]Me-TRH binding at 4 degrees C occurred with a rate constant (k-1) of 0.125 min-1. Binding sites for [3H]Me-TRH were found in the cephalic lobe of the pituitary gland (the location of most lactotrophs and thyrotrophs) and in the caudal lobe (the location of most somatotrophs). The number of binding sites was greater in the caudal lobe than in the cephalic lobe, although the affinity of [3H]Me-TRH binding did not differ. The binding of [3H]Me-TRH to caudal lobe membranes was displaced by Me-TRH, TRH, pGlu-His-Pro-Gly-NH2 and [Glu1]-TRH, with half-maximal effective doses of 33 nM, 70.7 nM, 1.23 microM and 22 microM respectively, but not by [Phe2]-TRH, TRH free acid or His-Pro-diketopiperazine. The number of caudal lobe binding sites for [3H]Me-TRH in old birds was less than that in young ones, and the number of binding sites was increased in birds deprived of food for 48 h. TRH-induced GH secretion in birds would thus appear to be mediated by specific receptors on caudal lobe somatotrophs, and these results suggest that physiological changes in GH secretion (during growth and periods of fasting) are causally related to the abundance of TRH-binding sites.     

Homologous and heterologous regulation of somatostatin-binding sites on chicken adenohypophysial membranes

Binding of 125I-labelled [Tyr1]-somatostatin (125I-[Tyr1]-SRIF) to pituitary caudal lobe membranes was suppressed in immature chickens 1 and 2 h after i.v. administration of unlabelled SRIF at concentrations of 1-100 micrograms/kg. In-vitro preincubation of chicken pituitary glands for 0.5-4.0 h with 0.1 mumol SRIF/l similarly reduced the binding of 125I-[Tyr1]-SRIF to caudal lobe membrane preparations. After a 4-h incubation in 0.1 mmol SRIF/l, the withdrawal of SRIF from the incubation media was accompanied 4 h later by a partial recovery in the binding of 125I-[Tyr1]-SRIF to pituitary membranes. Passive immunoneutralization of endogenous SRIF resulted in a prompt (within 1 h) and sustained (for at least 24 h) suppression of 125I-[Tyr1]-SRIF binding to pituitary membranes. The i.m. administration of cysteamine (300 mg/kg) to 12-week-old birds depleted hypothalamic SRIF stores and decreased the density of 125I-[Tyr1]-SRIF-binding sites in the caudal and cephalic lobes of the chicken pituitary gland. The reduction in SRIF content and in SRIF-binding sites occurred within 1 h of cysteamine administration and was maintained for at least 24 h. In 6-week-old birds, cysteamine (300 mg/kg) administration suppressed pituitary binding of 125I-[Tyr1]-SRIF for at least 5 days. Circulating concentrations of GH were markedly decreased 1 and 4 h after cysteamine injection, but not after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

The somatostatin receptor is directly coupled to adenylate cyclase in GH4C1 pituitary cell membranes

Somatostatin (SRIF) inhibits vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation in the GH4C1 strain of rat pituitary tumor cells, and this effect is responsible for SRIF inhibition of VIP-stimulated hormone release. In this study we examined the interaction between the SRIF receptor and adenylate cyclase in GH4C1 cell membranes. Maximal concentrations of VIP (50 nM) increased membrane adenylate cyclase activity 4.2-fold; half-maximal stimulation was observed with 0.75 nM VIP. SRIF noncompetitively inhibited the stimulatory effect of VIP, but it did not alter basal adenylate cyclase activity. The relative potencies of SRIF and two SRIF analogs as inhibitors of VIP-stimulated adenylate cyclase activity in membranes and of VIP-stimulated cAMP accumulation in intact cells were similar. Furthermore, the concentration of SRIF that caused half-maximal inhibition of adenylate cyclase activity (ED50 = 2.3 nM) was close to the equilibrium dissociation constant for SRIF (Kd = 0.40 nM) measured in membrane preparations in the presence of GTP. Therefore, SRIF inhibition of adenylate cyclase appears to be receptor mediated. As with receptors known to regulate adenylate cyclase by interaction with a guanine nucleotide regulatory subunit, SRIF receptor binding was decreased in the presence of guanine nucleotides. Addition of GTP (150 microM) or the nonhydrolyzable GTP analog guanyl-5'-yl-imidodiphosphate (100 microM) decreased the specific binding of [125I-Tyr1]SRIF to 31% and 13% of the control value, respectively. This decrease in specific binding was due entirely to decreased receptor affinity for SRIF. GTP (150 microM) increased the equilibrium dissociation constant for SRIF from 0.11 to 0.40 nM, whereas the number of binding sites was unaffected by the nucleotide (Bmax = 0.2 pmol/mg protein). Analysis of dissociation kinetics demonstrated that in the absence of guanyl nucleotides, the rate of [125I-Tyr1]SRIF dissociation was first order (t 1/2 = 180 min). However, in the presence of a half-maximal concentration of guanyl-5'-yl-imidodiphosphate (0.3 microM), [125I-Tyr1]SRIF dissociation occurred with biphasic kinetics. Fifty percent of the specifically bound peptide dissociated at the same rate as that observed in the absence of nucleotide, whereas the remainder dissociated 15 times more rapidly (t 1/2 = 9.6 min).(ABSTRACT TRUNCATED AT 400 WORDS)     

Reciprocal interactions of somatostatin with thyrotropin-releasing hormone and vasoactive intestinal peptide on prolactin and growth hormone secretion in vitro

Reciprocal interactions of somatostatin (SRIF) and vasoactive intestinal peptide (VIP) or TRH on in vitro PRL and GH release from male rats hemipituitaries were investigated. SRIF did not modify basal PRL release, but TRH- or VIP-induced release was inhibited by SRIF in a dose-dependent manner [effective concentration-fifty (EC50) = 1.7 +/- 0.9 nM for SRIF inhibition of TRH stimulation and EC50 = 0.8 +/- 0.5 nM for SRIF inhibition of VIP stimulation]. VIP and TRH did not affect GH release by themselves, but reduced the inhibition of GH secretion elicited by SRIF (EC50 = 7.6 +/- 3.4 nM for TRH blockade of SRIF inhibition and EC50 = 4.6 +/- 3.1 nM for VIP blockade of SRIF inhibition). Secretin, a partial structural analog of VIP, also blocked SRIF-induced inhibition of GH and stimulated PRL release. Secretin stimulation of PRL release was also prevented by SRIF. [D-Trp8,D-Cys14]SRIF, a potent analog of SRIF, antagonized VIP stimulation of PRL secretion with the same apparent affinity as the native peptide. The maximal stimulation, but not the apparent affinity of VIP action on prolactin release was reduced by SRIF, suggesting that the interaction is of a noncompetitive nature. This conclusion as further substantiated by the observation that neither TRH nor VIP were able to displace specific 125I-labeled [Tyr1] SRIF high affinity binding to pituitary membranes. The three peptides tested thus appear to exhibit reciprocal interactions mediated by independent receptor sites on GH as well as on PRL-producing cells.     

Feedback-inhibition of growth hormone (GH) secretion in fowl: GH-induced down-regulation of thyrotrophin-releasing hormone binding to pituitary membranes

Administration of ovine GH to immature domestic fowl blunted their subsequent GH response to thyrotrophin-releasing hormone (TRH), a GH secretagogue in birds. The in-vivo administration of GH also reduced the ability of radiolabelled TRH to bind to plasma membranes of the pituitary caudal lobe, in which GH cells predominate. These inhibitory effects of GH were mediated by extrapituitary actions, since GH had no direct inhibitory effects on TRH-induced GH release or on pituitary TRH binding in vitro. GH inhibition of GH secretion and TRH binding would not appear to be mediated by hypothalamic somatostatin (SRIF) or peripheral somatomedin (IGF-I), since SRIF and IGF-I had no direct effects in vitro.     

The interrelationship between the effects of somatostatin and human pancreatic growth hormone-releasing factor on growth hormone release by cultured pituitary tumor cells from patients with acromegaly

The effects of somatostatin (SRIF) and human pancreatic tumor GRF on GH release by cultured pituitary tumor cells obtained during transsphenoidal operation from 15 acromegalic patients were investigated. In a study of the sensitivity of pathological GH release to SRIF, 1-10 nM SRIF induced maximal inhibition of hormone release in 3 consecutive tumors. In 12 of 15 tumor cell cultures, 10 nM SRIF produced statistically significant inhibition of basal GH release by 39 +/- 3% (mean +/- SEM). In 2 of the 3 other tumors, SRIF inhibited GRF-stimulated GH release, while this was not investigated in the third tumor. A dose-response study of the effect of GRF on GH release by cultured pituitary tumor cells showed that doses of 0.1, 1, 10, and 100 nM induced similar maximal (35%) stimulation of hormone secretion. In four of five consecutive tumor cell suspensions, 1 and 10 nM GRF induced statistically significant GH stimulation by 18-300%. Preincubation of the tumor cells with 5 nM dexamethasone greatly increased the sensitivity and the maximal stimulation in response to GRF and made one tumor cell suspension, which did not react to GRF initially, sensitive to GRF. In the tumors of four patients, the interrelationship between the effects of SRIF and GRF on GH release were also studied. SRIF (10 nM) inhibited the stimulatory effects of GRF on GH release virtually completely. In conclusion, GH release by in vitro cell cultures of GH-secreting pituitary adenomas was inhibited by SRIF and stimulated by GRF. The interaction of GRF and SRIF on GH release by these pituitary tumor cells was similar to that in normal rat GH cells, as SRIF virtually completely overcame the GRF-induced GH release.     

Modulation of somatostatin binding to rat pituitary membranes by exogenously administered growth hormone

The effect of exogenously administered GH on somatostatin (SRIF) receptor regulation was studied in rat anterior pituitary membranes. A single class, high affinity specific receptor for SRIF was identified by binding studies with [125I-Tyr11]SRIF [binding capacity (mean +/- SD), 129.4 +/- 23.3 fmol/mg protein; binding affinity, 2.8 +/- 0.6 X 10(10) M-1]. A single injection of rat GH (150 micrograms) caused a significant reduction in capacity, but not in affinity, of SRIF receptors 2 and 6 h after injection (mean decrease, 23% and 24%, respectively) from that in controls. In contrast, mean SRIF binding capacity 24 h after a single injection of rat GH was increased 48% above control values, but affinity was unaffected. Measurement of membrane SRIF content indicated that these changes could not be explained by alterations in receptor occupancy. When rat GH was injected repeatedly for 3 days (150 micrograms/rat X day), the mean binding capacity, though not the affinity, of SRIF receptors was decreased 23% from that in controls 24 h after the last injection. The results can be explained by stimulation of SRIF release from the hypothalamus by GH and somatomedins, with subsequent internalization of the pituitary plasma membrane SRIF receptor. They suggest yet another level of neuroendocrine regulation of GH secretion.     

The effect of age on somatostatin suppression of basal, growth hormone (GH)-releasing factor-stimulated, and dibutyryl adenosine 3',5'-monophosphate-stimulated GH release from rat pituitary cells in monolayer culture

To test the hypothesis that relative resistance of the somatotroph to somatostatin (SRIF) contributes to elevated circulating levels of GH in the newborn rat, we examined the effects of SRIF (0.1, 0.33, and 1 nM) on basal, human pancreatic GH-releasing factor-40 (hpGRF-40; 1 nM)-stimulated, and (Bu)2cAMP (0.5 mM)-stimulated GH release from pituitary cells of 2-day-old, 15-day-old, and adult Sprague-Dawley rats in monolayer culture. The effect of SRIF on basal GH release varied markedly with age. SRIF, in the doses studied, did not inhibit basal GH release (nanograms of GH per 10(5) cells/3 h) from pituitary cultures of 2-day-old rats. In those of 15-day-old rats, only the two higher doses of SRIF (0.33 and 1 nM) suppressed GH release. By contrast, in pituitary cell cultures of adult male and female rats, all doses of SRIF significantly inhibited basal GH release (P less than 0.001). Similarly, the degree of SRIF suppression of both hpGRF-40- and (Bu)2cAMP-stimulated GH release differed among the age groups. In pituitary cultures of 2-day-old rats, SRIF did not significantly inhibit stimulated GH release. In 15-day-old rat pituitary cells, SRIF inhibited GH release, but did not eradicate the stimulatory effect of hpGRF-40 or (Bu)2cAMP. By contrast, in pituitary cell cultures of adult male and female rats, SRIF completely abolished the stimulatory effect of both hpGRF-40 and (Bu)2cAMP. When expressed as a percentage of the control (or stimulated) value, GH release at each SRIF dose varied markedly with age (P less than 0.001). Furthermore, a similar age-associated trend was evident when, in a separate series of experiments (n = 37), we examined the suppressive effect of a single concentration of SRIF (0.33 nM) on (Bu)2cAMP (0.5 mM)-stimulated GH release in cultured pituitary cells of rats ranging in age from -1 (day 20 of gestation) to 78 days. The degree of suppression increased progressively with advancing age; GH release decreased from 82 +/- 2% (+/- SE) of stimulated values in cultured cells of perinatal rats to 20 +/- 1% of stimulated values in cultured cells of 78-day-old rats. There was a significant negative correlation between age and SRIF-inhibited GH release (r = -0.89; P less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)     

Short-loop inhibition of thyrotrophin-releasing hormone-induced growth hormone secretion in fowl

The i.c.v. administration of 0.1 or 10 micrograms ovine (o)GH to 12- to 16-week-old hypothyroid chickens of a sex-linked dwarf (SLD) strain suppressed the basal plasma GH concentrations, measured 24 h afterwards. The GH response of the oGH-injected SLDs to TRH was suppressed, in a dose-related way, in comparison with that induced by TRH in birds given control injections (10 micrograms) of bovine serum albumin (BSA). Basal circulating concentrations of GH in euthyroid K strain birds of the same age were even lower than in the SLDs following injection of 10 micrograms oGH, and were not further reduced by oGH administration. The GH response to TRH in the K strain birds injected i.c.v. with 0.1 or 10 micrograms oGH was, nevertheless, suppressed in comparison with the BSA-injected K strain controls. The i.c.v. administration of oGH also suppressed circulating concentrations of LH and the LH response to TRH in the K strain birds. Twenty-four hours after i.c.v. administration of oGH (10 micrograms), the somatostatin (SRIF) content in the medial basal hypothalamus of 8-week-old euthyroid cockerels was greater than that in BSA (10 micrograms)-injected controls. At the same time, the binding of [3H]3-methyl-histidine2-TRH to the pituitary caudal and cephalic lobes of GH-injected birds was less than that in the controls. These results suggest that GH regulation in avian species is partly mediated by an inhibitory short-loop mechanism (mediated by hypothalamic SRIF and a down-regulation of pituitary TRH-binding sites) that suppresses basal and secretagogue-induced GH release.     

Differential binding of somatostatin agonists to somatostatin receptors in brain and adenohypophysis

In order to improve our understanding of the ligand specificity of somatostatin (SRIF) receptors in pituitary and brain, and to identify analogs that bind to one type exclusively, we compared several new SRIF analogs for competitive binding to pituitary and cerebral cortex membranes. Binding of [125I-Tyr11]SRIF to hypophysis and brain was of high affinity [KD = 0.76 nM (0.2-1.3) and 0.37 nM(0.1-0.8), mean (95% confidence limits)] and was characteristic of binding to one class of sites in both tissues. Competition by several SRIF analogs for such radioligand binding demonstrated that ligand specificity of adenohypophysial receptors was distinctly different from that of cerebral cortex. Two cyclic octapeptides (sequence; see text) bound to pituitary SRIF receptors with high affinity [Ki = 0.85 nM (0.5-1.2) and 0.35 nM (-0.3-0.9)] and were potent inhibitors of GH secretion from primary cultured pituitary cells [EC50 = 0.009 nM (0-0.02) and 0.017 nM (0.01-0.02), respectively]. However, these selective peptides did not compete (Ki much greater than 1 microM) for radioligand binding to brain. Amidation of the C-terminal end appeared to strikingly alter brain SRIF receptor recognition of the substituted ligand. Indeed, such amidation of the parent peptide, (sequence; see text) resulted in a reduced ability to displace labeled ligand from brain sites [Ki = 165.3 nM (47.6-282.9) to 842.2 nM (603.9-1,081)] but did not affect competition for pituitary receptors. Our results indicate that anterior pituitary SRIF receptors (SRIFa) have ligand specificities which are clearly different from those of their brain counterparts (SRIFb).(ABSTRACT TRUNCATED AT 250 WORDS)     

Desensitization of thyrotrophin-releasing hormone (TRH)-induced growth hormone secretion in chickens: coincident down-regulation of TRH binding to pituitary membranes

Release of GH is stimulated by TRH in chickens. However, for 60 min following a priming injection of TRH, a second injection of TRH is unable to provoke further GH release. TRH binds to the plasma membranes of the pituitary caudal lobe, in which somatotrophs predominate, although the magnitude of [3H]3-methyl-histidine2-TRH ([3H]Me-TRH) binding was reduced (by 25-50%) 30 min after the i.v. administration of a dose of TRH (5 micrograms/kg) maximally effective in provoking GH release. A significant reduction in [3H]Me-TRH binding to caudal lobe membranes was also observed within 15 min of TRH administration and was maintained for at least 60 min. Control levels of [3H]Me-TRH binding were restored 2 h after TRH injection, coincident with the restoration of GH responsiveness to TRH challenge. The suppression of [3H]Me-TRH binding to pituitary membranes 30 min after in-vivo TRH administration was dose related, whereas the maximal (10 min) GH response to TRH was biphasic. The suppression of [3H]Me-TRH binding to chicken pituitary membranes was due to direct pituitary actions of TRH and could be induced by a 30-min exposure to 100 nM TRH in vitro. These results demonstrate that avian pituitary TRH-binding sites differ greatly from mammalian ones in the timing of the onset and duration of down-regulation. Pituitary TRH-binding sites in birds are rapidly and transiently down-regulated following TRH administration in vivo, coincident with the period of GH refractoriness to TRH challenge.     

17 beta-estradiol induces somatostatin (SRIF) inhibition of prolactin release and regulates SRIF receptors in rat anterior pituitary cells

The role of 17 beta-estradiol (E2) in the induction of an inhibitory effect of somatostatin (SRIF) on PRL release and in the regulation of SRIF receptors was analyzed in rat anterior pituitary cells. SRIF exerts a moderate inhibitory effect on basal PRL release from cultured pituitary cells prepared from normal rats. The inhibitory effect of SRIF was weakened when the cells were prepared from castrated rats, but was strengthened in cells from E2-treated rats. When cells from ovariectomized rats were cultured with charcoal-treated sera in the absence of E2, SRIF affected neither basal PRL release nor the increased PRL release elicited by TRH, (Bu)2cAMP, or vasoactive intestinal peptide. However, in cells cultured with E2 (10(-9) M) for more than 12 h, SRIF decreased basal as well as secretagogue-stimulated PRL release. Enhancement of both the sensitivity and magnitude of the inhibitory effect of SRIF appeared dependent upon the length of the preincubation and the E2 concentration. On the other hand, an inhibitory effect of SRIF on GH release was unaffected by castration, E2 administration in vivo, or E2 treatment in vitro. Dihydrotestosterone (10(-6) M) produced an effect similar to that of E2, but other steroids tested did not. Binding studies demonstrated that the specific binding of [125I-Tyr11]SRIF was saturable, with a Kd of 99 pM and a maximum binding capacity of 89.4 fmol/mg protein in pituitary membranes from control rats, and a Kd of 45 pM and a maximum binding capacity of 305.2 fmol/mg protein in membranes from rats primed with E2 for 4 weeks. Apparent Ki values for native SRIF in both membranes were similar. Treatment of pituitary cells with E2 for 8 days in vitro in culture produced a 2-fold increase in the number of binding sites. These results demonstrate that E2 acts on mammotrophs at the pituitary level, rendering them sensitive to SRIF, probably by increasing the number of SRIF receptor sites.     

Somatostatin analogs: correlation between receptor binding affinity and biological potency in GH pituitary cells

Somatostatin (SRIF) is a hypothalamic tetradecapeptide which acts on several different types of pituitary cells to inhibit hormone release both in vivo and in vitro. We have previously shown that the GH4C1 clonal strain of rat pituitary tumor cells contains a single class of specific high-affinity SRIF receptors and that SRIF is a potent inhibitor of GH and PRL release by these cells. In this study, we have determined the relationship between the apparent binding affinity and biological potency of 19 SRIF analogs in GH4C1 cells. Receptor binding and biological activity were assayed under identical conditions. A good correlation (r = 0.96) was observed over a 10,000-fold range between the receptor binding affinities and biological potencies of SRIF analogs. Modifications at the C- and N-terminal regions of the SRIF molecule had minimal effects on binding to the receptor or potency to inhibit PRL release. However, substitution of residues 6 through 10 or reduction of the disulfide bond resulted in a 100-fold or greater decrease in both activities. The N-terminal extended SRIF analogs, SRIF-28, [D-Trp22]SRIF-28, and SRIF-25, were all somewhat less potent than SRIF. These results strongly support the involvement of the characterized SRIF receptor in initiating the biological actions of SRIF in GH4C1 cells and define the structural features of the SRIF molecule required for both receptor binding and activation.     

Somatostatin inhibits basal and vasoactive intestinal peptide-stimulated hormone release by different mechanisms in GH pituitary cells

Somatostatin (SRIF) inhibits both basal and vasoactive intestinal peptide (VIP)-stimulated hormone secretion by the GH4C1 clonal strain of rat pituitary tumor cells. We have previously shown that SRIF inhibits cAMP accumulation stimulated by VIP but does not alter basal cAMP levels in this cell line. To determine the importance of changes in cAMP accumulation in the mechanism of SRIF action, we have compared the effect of SRIF on hormone release stimulated by VIP and two other secretagogues which increase effective intracellular cAMP concentrations: forskolin and 8-Bromo-cAMP (8-Br-cAMP). VIP stimulated GH and PRL secretion to the same maximal extent (220% of control) with similar ED50 values (0.37 +/- 0.03 and 0.43 +/- 0.08 nM, mean +/- SE, respectively). SRIF (100 nM) reduced maximal VIP-stimulation of both GH and PRL release from 220 to 140% of control; however, it did not significantly change the ED50 values for VIP. The effect of SRIF on VIP-stimulated hormone release parallels its action on VIP-stimulated cAMP accumulation. Furthermore, the concentrations of SRIF required to produce half-maximal inhibition of VIP-stimulated GH and PRL release (0.8 +/- 0.2 nM and 0.7 +/- 0.1 nM, respectively) were similar to its potency to inhibit VIP-stimulated cAMP accumulation (1.2 +/- 0.1 nM). These data indicate that changes in cAMP levels mediate inhibition of VIP-stimulated hormone secretion by SRIF. Forskolin increased cAMP accumulation with an ED50 value of 2.4 +/- 0.5 microM. A maximal concentration of forskolin (100 microM) stimulated cAMP accumulation to a greater extent than 100 nM VIP (34 +/- 4-fold vs. 9 +/- 1-fold). Together, forskolin (100 microM) and VIP (100 nM) stimulated cAMP accumulation by more than 50-fold. However, PRL secretion in response to maximal concentrations of VIP or forskolin individually or together were the same (approximately 200% of control). These results support the conclusion that both compounds stimulate PRL secretion by a cAMP-mediated mechanism which can be fully activated by either one alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thyroidal inhibition of growth hormone secretion in fowl: tri-iodothyronine-induced down-regulation of thyrotrophin-releasing hormone-binding sites on pituitary membranes

The number, but not affinity, of binding sites for [3H]3-methyl-histidine2-TRH ([3H]Me-TRH) on chicken adenohypophysial plasma membranes was increased in chickens made hypothyroid by goitrogen (methimazole) treatment (50 mg/kg per day for 7 days), which also increased circulating GH concentrations. Daily i.p. injection of thyroxine (T4; 100 micrograms/kg for 7 days) had no effect on [3H]Me-TRH binding to pituitary membranes, although it suppressed endogenous GH secretion. Binding of [3H]Me-TRH to pituitary caudal lobe membranes was, however, suppressed by tri-iodothyronine (T3) injected chronically (100 micrograms/kg per day, i.p., for 7 days) or acutely (100 micrograms/kg, 2 h before being killed). The suppression of [3H]Me-TRH binding and inhibition of GH secretion following T3 administration was dose related. Binding of [3H]Me-TRH to caudal lobe membranes was also suppressed following the incubation of pituitary glands with T3 in vitro, and the response was both dose and time related. These results suggest that T3 inhibits GH secretion in fowl by a down-regulation of pituitary TRH receptors. However, other mechanisms are involved in thyroidal inhibition of GH release in birds, since T4 had no effects on [3H]Me-TRH binding yet suppressed GH secretion in vivo.     

Inhibitory effect of platelet-activating factor (PAF) on luteinizing hormone-releasing hormone and somatostatin release from rat median eminence in vitro correlated with the characterization of specific PAF receptor sites in rat hypothalamus

Platelet-activating factor (PAF) exhibits a wide range of biological activities, including the stimulation of secretory processes in various cell types. However, little is known regarding its possible influence on the release of brain neuropeptides. In the present study we have examined the effect of PAF on the release of three hypothalamic releasing hormones in adult male rats, and have characterized the presence of specific PAF binding sites in rat hypothalamic membranes. PAF decreased LHRH and somatostatin (SRIF) release from the median eminence with a maximal inhibition at 10(-14) M for both neuropeptides, whereas GRF release was not significantly altered. Moreover, PAF strongly counteracted the Ca2+ ionophore A 23187-stimulated release of LHRH and SRIF from median eminence and medial basal hypothalamus (greater than 50% inhibition). These results suggest an involvement of Ca2+ dependent events in PAF action. This inhibitory effect was specifically exerted at a hypothalamic site because PAF failed to depress LH and GH release from the anterior pituitary. A specific, reversible and saturable binding of [3H]PAF to membrane preparations of rat hypothalamus was demonstrated and two classes of binding sites were characterized. The affinity (KD) of each binding class was 2.14 +/- 0.32 nM and 61.63 +/- 16.4 nM, respectively, and the corresponding maximal number of each binding class was 25.41 +/- 3.2 fmol/mg protein and 146.2 +/- 47.5 fmol/mg protein. In the same conditions no specific binding was observed using rat pituitary membranes. The specificity of PAF analogs for these binding sites was well correlated to their relative effectiveness in altering LHRH and SRIF release (order of potency: L-652,731, kadsurenone greater than BN 52021 greater than Lyso-PAF). These data suggest that the binding sites identified in the hypothalamus have the characteristics expected of a specific PAF receptor and that PAF effect on neuropeptides release is a receptor-mediated process.     

Growth hormone secretion from the Arctic charr (Salvelinus alpinus) pituitary gland in vitro: effects of somatostatin-14, insulin-like growth factor-I, and nutritional status

This study investigated the influence of nutritional status on the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis in Arctic charr (Salvelinus alpinus). The objectives were to study the regulation of GH secretion in vitro by somatostatin-14 (SRIF) and hIGF-I, and to determine whether pituitary sensitivity to these factors is dependent upon nutritional status. Arctic charr were fed at three different ration levels (0, 0.35, and 0.70% BWd(-1)), and pituitary glands were harvested at 1, 2, and 5 weeks for in vitro study. Both SRIF and hIGF-I inhibited GH secretion from Arctic charr pituitary tissue in long-term (18 h) static hemipituitary culture, as well as after acute exposure in a pituitary fragment perifusion system. This response appeared to be dose-dependent for SRIF in static culture over the range of 0.01-1 nM, but not for hIGF-I. The acute inhibitory action of hIGF-I on GH release in the perifusion system suggests an action that is initially independent of any effects on GH gene expression or protein synthesis. Nutritional status did not affect the sensitivity of Arctic charr pituitary tissue to either SRIF or hIGF-I in vitro, indicating that changes in abundance of pituitary SRIF or IGF-I receptors may not explain the alterations in plasma GH levels found during dietary restriction.     

Characterization of somatostatin receptors which mediate inhibition of insulin secretion in RINm5F insulinoma cells

Somatostatin (SRIF) is a neuropeptide which inhibits secretion from a variety of target cells including pancreatic beta-cells. In this study we have used the RINm5F rat insulinoma cell line to characterize high affinity receptors for SRIF. The binding of 0.03 nM [125I-Tyr11]SRIF to RINm5F cells reached a plateau level within 4 h at 37 C at which time 80% of the total binding could be displaced by 100 nM unlabeled SRIF. In contrast, 100 nM concentrations of eight structurally unrelated peptides did not inhibit [125I-Tyr11]SRIF binding. Scatchard analysis indicated that RINm5F cells contained a single class of noninteracting binding sites (910 +/- 190 sites per cell) with high affinity for [125I-Tyr11]SRIF [equilibrium dissociation constant (Kd) = 0.04 +/- 0.01 nM]. Competition experiments with SRIF analogs showed that the binding affinity for [I-Tyr11]SRIF (Kd = 0.03 +/- 0.02 nM) was higher than that for either SRIF (0.24 +/- 0.04 nM) or [Tyr11]SRIF (0.27 +/- 0.04 nM) and that reduced SRIF analogs bound poorly (Kd greater than 50 nM). These results demonstrate that RINm5F cells possess specific, high affinity binding sites for SRIF. Insulin release stimulated by 20 mM leucine or 15 mM glyceraldehyde was inhibited as much as 80% by maximal concentrations (100 nM) of SRIF. The IC50 for SRIF inhibition of leucine-stimulated insulin secretion was 0.43 +/- 0.15 nM, in good agreement with the apparent Kd for binding. In fact, this close correlation between binding affinity and potency to inhibit insulin release was observed for six SRIF analogs, indicating that the characterized binding sites are the receptors which mediate the biological actions of SRIF in RINm5F cells.     

The inhibition of growth hormone release by gastrin-releasing peptide involves somatostatin release

Injection of gastrin-releasing peptide-27 (GRP) into the third ventricle (IVT) has been shown previously to lower plasma GH levels and block the GH release induced by GRF, suggesting that GRP might act via stimulation of the release of somatostatin (SRIF) into hypophysial portal vessels. Several experiments were performed to test this hypothesis. In the first experiment rat median eminence (ME) fragments were incubated in medium containing concentrations of GRP ranging from 1 pM to 1 microM, and SRIF levels were measured after the 30-min incubation period. GRP significantly stimulated SRIF release at doses of 0.1 nM to 1 microM. Microinjection of SRIF antiserum (3 microliters) IVT prevented GRP (2 micrograms, IVT) from inhibiting the GH surge induced by GRF (1 microgram/kg, iv). A slight but significant decrease in basal plasma GH levels was observed after GRP administration even in the presence of SRIF antiserum. Finally, to rule out a GRP-GRF interaction at the pituitary level, tubes containing dispersed rat pituitary cells (2.5 x 10(5) cells/tube) were incubated for 1.5 h in medium containing various concentrations of GRF (0.4-40 nM) alone or with 0.1 microM GRP. The addition of GRP to the medium had no significant effect on the dose-dependent stimulation of GH release by GRF. The results of these studies demonstrate that GRP can directly stimulate SRIF release in vitro. They further suggest that SRIF is a component of the mechanism whereby GRP inhibits GH release in vivo. Finally, the possibility that GRP acts at the pituitary level to inhibit GH release by blocking GRF receptors on somatotrophs has been ruled out.     

Regulation of growth hormone (GH) secretion by GH-releasing factor, somatostatin, and insulin-like growth factor I in ovine fetal and neonatal pituitary cells in vitro

Serum GH concentrations in the ovine fetus are much higher than those in the neonate, and the maximal GH response induced by GRF is 5-fold greater in the fetus than in the neonate. To clarify these in vivo observations further, we studied the effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on primary cultures of fetal and neonatal ovine pituitary cells. GH secretion from fetal ovine pituitary cells increased from 148 +/- 34 to 950 +/- 130 ng/10(5) cells.3 h in response to 1 nM GRF, whereas GH secretion from neonatal pituitary cells rose from 113 +/- 26 to 1221 +/- 129 ng/10(5) cells.3 h, a significantly greater response (P less than 0.001). This greater GRF-induced GH response in neonatal than fetal cells differs from the response in vivo and suggests that the increased in vivo response in the fetus is not due to inherently increased sensitivity of pituitary cells to GRF. SRIF (10 nM) decreased maximal GRF-induced GH secretion by 37 +/- 3% in fetal cells compared with 59 +/- 8% in neonatal cells (P less than 0.01). This may explain in part the decreased in vivo sensitivity to SRIF in the ovine fetus compared to that in the neonatal lamb. In fetal pituitary cells, 10 nM GRF increased ovine (o) GH mRNA from 100 +/- 14% to 145 +/- 40%, SRIF decreased oGH mRNA to 84 +/- 3%, and GRF and SRIF in combination increased fetal oGH mRNA to 126 +/- 24%. Values in neonatal pituitary cell cultures were similar (control, 100 +/- 17%; GRF, 132 +/- 6%, SRIF, 85 +/- 15%; GRF plus SRIF, 105 +/- 26%). Pretreating fetal cells with 100 nM IGF-I for 3 days reduced GRF-stimulated GH secretion from 1049 +/- 38 to 232 +/- 8 ng/10(5) cells.3 h (P less than 0.001). Similarly, IGF-I pretreatment of neonatal cells reduced GRF-stimulated GH secretion from 810 +/- 18 to 419 +/- 16 ng/10(5) cells.3 h (P less than 0.001). The mean secreted IGF-I was 0.58 U/ml (36 nM) in culture medium from neonatal cells and was unchanged by incubation for 3 days with 5 micrograms/ml hGH. Secreted IGF-I in medium from fetal cells was 0.87 U/ml (54 nM) without GH and 0.81 U/ml (51 nM) after incubation with human GH. IGF-I mRNA was present in neonatal pituitary and brain.(ABSTRACT TRUNCATED AT 400 WORDS)