Inhibition Of Growth Hormone Release Research Articles

Direct actions of serotonin on gonadotropin-II and growth hormone release from goldfish pituitary cells: interactions with gonadotropin-releasing hormone and dopamine and further evaluation of serotonin receptor specificity

In this study, the direct actions of serotonin (5HT) on gonadotropin (GTH)-II and growth hormone (GH) release in the goldfish were tested at the pituitary cell level. 5HT (10 nM - 10 µM) stimulated GTH-II but inhibited GH release from perifused goldfish pituitary cells in a dose-dependent manner. The minimal effective dose of 5HT tested to suppress basal GH secretion (10 nM) was 10-fold lower than that to stimulate GTH-II release (100 nM). The GTH-II releasing effect of 5HT was abolished by repeated 5HT treatment (10 µM) whereas the corresponding inhibition on GH release was unaffected. These results suggest that 5HT receptors on goldfish gonadotrophs and somatotrophs exhibit intrinsic differences in terms of sensitivity to stimulation and resistance to desensitization. Salmon GTH-releasing hormone (sGnRH, 100 nM) stimulated GTH-II and GH release from goldfish pituitary cells. The GTH-II releasing action of sGnRH was unaffected by simultaneous treatment of 5HT (1 µM). However, the corresponding GH response to sGnRH (100 nM) was inhibited. In the goldfish, dopamine is known to stimulate GH release through activation of pituitary D1 receptors. In the present study, the GH-releasing action of dopamine (1 µM) and the D1 agonist SKF38393 (1 µM) was significantly reduced by 5HT (1 µM). To examine the receptor specificity of 5HT action, the effects of 5HT1 and 5HT2 analogs on GTH-II and GH release were tested in goldfish pituitary cells. The 5HT1 agonist 8OH DPAT (0.1 and 1µM) and 5HT2 agonist α methyl 5HT (0.1 1µM) mimicked the GTH-II releasing effect of 5HT. The 5HT1 agonist 8OH DPAT (0.1 and 1µM) also stimulated GH release but the 5HT2 agonist α methyl 5HT (0.1 and 1µM) was inhibitory to basal GH secretion. In addition, 5HT (1µM) -stimulated GTH-II release was abolished by the 5HT1 antagonist methiothepin (10µM) and 5HT2 antagonist mianserin (10µM). Similarly, the inhibitory action of 5HT (1µM) on basal GH release was blocked by the 5HT2 antagonist mianserin (10µM). The 5HT1 antagonist methiothepin (10µM) was not effective in this regard. These results, taken together, indicate that 5HT exerts its regulatory actions on GTH-II and GH release in the goldfish directly at the pituitary cell level, probably through interactions with other regulators including sGnRH and dopamine. The GTH-II releasing action of 5HT is mediated through 5HT2 and possibly 5HT1 receptors. The inhibition of 5HT on basal GH release is mediated through 5HT2 receptors only. Apparently, 5HT1 receptors are not involved in this inhibitory action. In this study, a paradoxical stimulatory component of 5HT on GH release by activating 5HT1 receptors is also implicated.

Effects of Neonatal Administration of Monosodium Glutamate on Bone in Rats.

It has been known that monosodium glutamate (MSG) treatment to neonatal rodents affects hypothalamic arcuate nucleus, the primary site of growth hormone releasing hormone, and consequently inhibits growth hormone release. However, there have been few reports on bone changes with aging after MSG treatment to neonatal rodents. Male and female neonatal F344 rats were subcutaneously given 4,000 mg/kg of MSG on days 0, 2, 4, 6, and 8 after birth, and the changes of the femurs were examined at 12 months of age. As a result, the bone length, diameters of the femur diaphysis and bone mineral density, especially the metaphysis region, were decreased by the treatment. Histologically, the amount of trabecular bone in the epiphysis and metaphysis regions also decreased in MSG-treated rats, together with an increase of adipose tissue in the bone marrow.

Somatostatin Inhibition of Growth Hormone Release in Goldfish: Possible Targets of Intracellular Mechanisms of Action

Previous studies have demonstrated that growth hormone (GH) release in goldfish is under the stimulatory control of gonadotropin-releasing hormone (GnRH) and dopamine and the inhibitory control of somatostatin (SRIF). GnRH stimulation is mediated through protein kinase C (PKC)- and calcium-dependent mechanisms, whereas dopamine D1 receptor activation increases GH secretion through cyclic (c) AMP-dependent intracellular signal transduction pathways. In this study, the mechanisms of SRIF inhibition on GH secretion were examined using primary cultures of dispersed goldfish pituitary cells in static incubation. Application of 1 μMSRIF inhibited the GH-release responses to 100 nMsalmon GnRH, 100 nMchicken GnRH-II, and 1 μMSKF38393, a D1 agonist. These results indicate that inhibitory action of SRIF on stimulated GH release is direct, at the level of the pituitary cells. Addition of SRIF reduced the GH release responses to two activators of PKC (100 μMdioctanoyl glycerol and 100 nMtetradecanoyl phorbol acetate) and to two ionophores (10 μMA23187 and 10 μMionomycin). Similarly, SRIF abolished the GH responses to an activator of adenylate cyclase (10 μMforskolin), a membrane-permeant cAMP analog (1 mM8-bromo-cAMP), and a voltage-sensitive calcium channel agonist (1 μMBay K 8644). Taken together, these observations indicate that the inhibitory actions of SRIF on D1- and GnRH-stimulated GH release can be exerted at sites distal to cAMP production and PKC activation, respectively. SRIF also exerts its effect at sites distal to calcium mobilization. Since SRIF inhibition was more effective against Bay K 8644-induced response than against ionophore-induced GH response, an inhibitory action at the level of extracellular calcium entry through voltage-sensitive channels is also possible.

Somatostatin plays a dual, stimulatory/inhibitory role in the control of growth hormone secretion by two somatotrope subpopulations from porcine pituitary.

Previous results from our laboratory demonstrated the existence of two subpopulations of porcine somatotropes of low- (LD) and high density (HD) that exhibit differences in ultrastructure and respond in an opposite manner to somatostatin (SRIF) in vitro. In LD cells, SRIF did not affect basal growth hormone (GH) release but partially blocked the stimulatory effect induced by GH-releasing factor (GRF). Conversely, SRIF paradoxically stimulated the secretory activity of HD somatotropes. Here, we have analysed in detail the basic parameters that characterize this differential response. To this end, the time- and dose-dependent effects of SRIF-14 were evaluated on separate monolayer cultures of both subpopulations. Likewise, the direct effect of the peptide on individual somatotropes from each subset was assessed by cell immunoblot assay. Finally, we compared the effects of SRIF-14 and SRIF-28 on cultures of LD and HD cells. SRIF-14 (10(-7) M) induced a rapid (30 min) and sustained (4 h) 2-fold increase in GH release from HD cells, whereas it did not affect GH secretion from LD somatotropes. Surprisingly, a low dose of SRIF (10(-15) M) stimulated GH release from both LD (154.1 +/- 8.2% of basal, P < 0.05) and HD (337.2 +/- 55.5% of basal, P < 0.05) subpopulations, even more effectively than higher doses of the peptide. Results from cell blotting showed that SRIF stimulatory effects were exerted directly upon individual somatotropes. Finally, SRIF-28 elicited similar responses to those observed for SRIF-14 in both somatotrope subpopulations, yet 10(-15) M SRIF-28 was less potent than the same dose of SRIF-14 in stimulating GH release from HD cells. Our present findings demonstrate that SRIF can function as a true GH-releasing factor in cultures of porcine pituitary cells by acting specifically and directly upon somatotropes. Furthermore, together with previous observations, these results strongly suggest that SRIF is not merely an inhibitor of GH release in pigs, but might play a dual modulatory role. Heterogeneity of the somatotrope population contributes greatly to this divergent effect of SRIF.

Estradiol Inhibits Plasma Somatostatin 14 (SRIF-14) Levels and Inhibits the Response of Somatotrophic Cells to SRIF-14 Challengein Vitroin Rainbow Trout,Oncorhynchus mykiss

In the present study, the effects of 17β-estradiol (E2) treatment on plasma growth hormone (GH) and somatostatin 14 (SRIF-14) concentrations were investigated, as well as the effect ofin vivoE2treatment on thein vitroGH response to SRIF-14 challenge in sexually immature rainbow trout (Oncorhynchus mykiss). Two weeks after receiving a steroid hormone implant, plasma E2and GH levels were significantly (P<0.05) elevated, and plasma SRIF levels were significantly (P<0.05) lowered relative to the control. Pituitary glands were taken from E2-primed and control fish and challenged with a single pulse of SRIF-14 (10−8M) in a perifusion unit to evaluate the effect of E2on the response of somatotrophs to the effect of SRIF-14. Whereas SRIF-14 challenge significantly (P<0.01) inhibited GH release from pituitary fragments taken from control fish, there was no such response in E2-primed fish. Furthermore, GH release following SRIF-14 administration (at the point of maximal inhibition) was significantly depressed in control fish with respect to the E2treatment group. These data suggest that E2treatment may increase plasma GH concentrations by altered somatotroph responsiveness to SRIF-14 inhibition. Furthermore, E2may increase plasma GH by suppressing plasma SRIF-14 levels, although the role of circulating SRIF-14 on the regulation of GH release has not been fully determined in teleosts.

Inhibitory effects of galanin on growth hormone (GH) release in cultured GH-secreting adenoma cells: Comparative study with octreotide, GH-releasing hormone, and thyrotropin-releasing hormone

The aim of the present study was to characterize in a large series (N = 12) of cultured somatotrope adenomas the in vitro effects of the neuropeptide galanin on growth hormone (GH) secretion. This was contrasted with two peptides known to be GH secretagogues (GH-releasing hormone [GHRH] and thyrotropin-releasing hormone [TRH]) and a peptide with a known GH-inhibitory effect (the somatostatin analog octreotide). Groups of three wells were incubated for 4 hours with growth medium alone (control incubation), galanin, GHRH(1–29)NH 2, TRH, or octreotide. Galanin and octreotide were applied at concentrations of 0.1, 1, and 10 μmol/L, and GHRH and TRH at concentrations of 0.01, 0.1, and 1 μmol/L. Galanin was able to inhibit GH release in nine of 12 cultured somatotrope adenoma cells. This inhibitory effect was clearly dose-dependent in five adenomas. Overall, the mean GH nadir after galanin was −36.1% in nine responder adenoma cultures versus control wells. Octreotide inhibited GH release in five of eight cultured somatotrope adenoma cells. The mean GH nadir after octreotide was −32.7% in five responder adenoma cultures compared with control wells. GHRH and TRH were able to stimulate GH release, respectively, in seven of 11 and in six of seven cultured somatotrope adenoma cells. The mean GH peaks after either GHRH or TRH in responder adenoma cultures were, respectively, +71.5% and +143.7% compared with levels in the control wells. In conclusion, the consistency and potency of the in vitro GH-inhibitory effect of galanin in a large series of somatotrope adenomas are at least similar to those of the most effective available GH-lowering agent, the somatostatin analog octreotide.

Activation of the hypothalamo-pituitary-adrenal axis by the growth hormone (GH) secretagogue, GH-releasing peptide-6, in rats.

GH-releasing hexapeptide (GHRP-6) is a synthetic secretagogue that stimulates the release of GH by acting at both hypothalamic and pituitary sites. GHRPs also consistently elicit small, but significant, increases in plasma concentrations of ACTH and adrenal steroids. As these secretagogues do not release ACTH directly, they probably interact with the hypothalamic peptidergic systems controlling ACTH release, such as CRH and arginine vasopressin (AVP). We have now examined the activation of the hypothalamo-pituitary-adrenal axis by GHRP-6 in conscious rats. In a series of experiments, rats were injected i.v. with 10 microg GHRP-6, 2 microg CRH, 0.5 microg AVP, or saline, alone or in combination, and serial plasma samples withdrawn and assayed for ACTH, corticosterone, and GH. CRH and AVP increased plasma ACTH levels in all rats, whereas ACTH and corticosterone responses to GHRP-6 were variable and were dependent on the prevailing activity of the hypothalamo-pituitary-adrenal axis. GHRP-6 stimulated the largest ACTH responses in rats that had the lowest basal plasma ACTH and corticosterone levels before GHRP-6 administration. GHRP-6 given in combination with CRH did not increase ACTH levels beyond the response to CRH alone (change in ACTH, 1570 +/- 207 vs. 1714 +/- 245 pg/ml), whereas the combination of GHRP-6 and AVP markedly increased ACTH levels compared with the effects of AVP alone (change in ACTH, 5587 +/- 669 vs. 2338 +/- 451 pg/ml; P < 0.05). The GH responses to GHRP-6 were significantly greater in rats with low basal plasma ACTH and corticosterone levels than in rats with elevated ACTH and corticosterone levels (change in GH response, 119 +/- 27 vs. 29 +/- 7 ng/ml; P < 0.01). CRH alone significantly inhibited GH release (pre- vs. 40 min post-CRH, 11.9 +/- 3.8 vs. 1.7 +/- 0.4 ng/ml; P < 0.05), whereas AVP alone had no effect on GH levels. Neither CRH nor AVP had any effect on the GH response to GHRP-6. We suggest that GHRP-6 acts via the hypothalamus to mediate the release of ACTH, and that these effects are probably mediated at least in part via the release of endogenous CRH and are subject to regulation by circulating glucocorticoids.

Effect of somatostatin on the release of gonadotropins in male rats.

Since somatostatin, the growth hormone release-inhibiting hormone, has inhibitory actions in many cell types and is delivered to the anterior pituitary gland via the hypophysial portal vessels, as well as being synthesized by cells within the gland, we tested the hypothesis that it might inhibit the release of gonadotropins from anterior pituitaries in vitro. Consequently, the effect of somatostatin on gonadotropin release from incubated anterior pituitaries of male rats with and without the stimulatory action of luteinizing hormone-releasing hormone (LHRH) was studied. After a preincubation period of 1 hr, hemipituitaries from adult male rats were incubated in fresh Krebs-Ringer bicarbonate (KRB) buffer in a Dubnoff incubator with an atmosphere of 95% O(2)-5% CO2 at 37 degrees C for 3 hr. Incubation with somatostatin (10(-6), 10(-7), and 10(-8) M) had no effect on basal release of either LH or follicle-stimulating hormone (FSH). However, somatostatin (10(-6)-10(-8) M) suppressed LHRH (1.7 x 10(-8) M)-induced release of LH (P < 0.01 to P < 0.0001), but not FSH. Furthermore, somatostatin antiserum (1:1000) had no significant effect on basal LH or FSH release, whereas incubation with the antiserum plus LHRH (1.7 x 10(-9) or 1.7 x 10(-8) M) increased LH (P = 0.015 and P=.005, respectively), but not FSH release. In summary, our results suggest that somatostatin exerts a physiologically significant inhibitory effect on LH but not FSH release in the presence of LHRH in vitro. Presumably, somatostatin is secreted in vitro by pituitary cells since not only have anterior pituitaries of rats been shown to contain somatostatin, but also somatostatin mRNA. Somatostatin then diffuses to the LH gonadotropes, where it exerts its inhibitory action. However, the release of somatostatin is insufficient to alter basal in vitro release. On the other hand, at least at the concentrations employed, there was no significant effect either of somatostatin or the antiserum to alter basal or stimulated FSH release.

Heterogeneity of growth hormone (GH)-producing cells in aging male rats: ultrastructure and GH gene expression in somatotrope subpopulations

Mammalian aging is characterized by a decline in the content and release of pituitary growth hormone (GH). However, few studies on the age-related changes in the population of GH-producing cells (somatotropes) have been carried out. We have investigated whether changes in number, ultrastructure and GH gene expression in subpopulations of somatotropes could explain the reduced GH release in aged rats. Three representative ages were studied: adult (5-month-old), old (19-month-old), and senescent (26-month-old) male rats. The total number of immunoreactive-GH cells per pituitary gland remained invariable to age. The separation of dispersed pituitary cells on a density gradient yielded two somatotrope subpopulations, of low density (LD) and high density (HD). Both subpopulations were equally represented in adults, whereas in old and senescent rats a predominance of LD-somatotropes was observed. Morphometric analysis showed that subpopulations exhibited storage and biosynthetic features inversely related. In LD-somatotropes, rough endoplasmic reticulum (RER) was more prominent but secretory granules (SG) were less abundant than in HD somatotropes. Concurrently, in situ hybridization for GH mRNA showed that GH gene expression was higher in LD-cells. Differences between subpopulations were essentially retained through the animals' lifespan, but small-sized SG, reduced RER, and low GH mRNA levels were inherent to aging both in LD- and in HD-somatotropes. The present findings demonstrate that the reduced content of pituitary GH in aged male rats is not due to a diminished number of GH-producing cells, but to the numerical predominance of scarcely granulated LD-somatotropes, combined with the decline in GH biosynthetic capacity observed in both subpopulations. In addition, age-related changes in ultrastructure and GH gene expression suggest a chronic inhibition of GH release and/or a weak stimulation of GH biosynthesis affecting both subpopulations.

Somatostatin analogues for somatostatin-receptor-mediated radiotherapy of cancer.

The aim of the present study was to selectively target a beta-emitter-labelled octreotide analogue to somatostatin (SRIF)-receptor-expressing tumours and to evaluate the feasibility of SRIF-receptor-mediated radiotherapy by delivering a lethal dose of radiation to the tumour. The most promising compound in a series of DTPA-coupled octreotide analogues was DTPA-benzyl-acetamido-D-Phe1, Tyr3-octreotide (SDZ413). In vitro, SDZ413 binds with nanomolar affinity to SRIF-receptors (IC50 = 4.0 nM) and inhibits growth hormone release from primary cultures of rat pituitary cells with an IC50 of 7.2 nM. Biodistribution studies with [90Y]SDZ413 demonstrated a fast and significant SRIF-receptor-specific accumulation of the labelled conjugate (tumour/muscle ratio after 24 h: 52/1). [90Y]SDZ413 was effective in the radiotherapy of SRIF-receptor-positive tumours in a nude mouse model. A single treatment with [90Y]SDZ413 led to a significant decrease (25%) of tumour mass. This effect was mediated by the intact radioligand, since treatment with [90Y]SDZ978, a derivative of SDZ413 which does not bind with high affinity to SRIF-receptors or with the unlabelled SDZ413 alone, failed to affect tumour growth. These results suggest that receptor-targeted radiotherapy with a 90Y-labelled octreotide analogue represents a new strategy for the treatment of SRIF-receptor-positive tumours that have been previously diagnosed with OctreoScan111 (pentetreotide).

Involvement of brain catecholamines and acetylcholine in growth hormone hypersecretory states. Pathophysiological, diagnostic and therapeutic implications.

Secretion of growth hormone (GH) is excessive in acromegaly, but also in a number of other pathological states such as anorexia nervosa, insulin-dependent diabetes mellitus (IDDM), liver cirrhosis, depression, renal failure and GH-insensitivity syndrome. Abnormalities in the neuroendocrine control of GH secretion and/or a state of insensitivity to GH contribute to hypersecretion of GH in these states, with the possible exception of acromegaly, which appears to be a primary pituitary disease. GH hypersecretion may also occur in neonates or adolescents with tall stature, thus reflecting particular physiological or paraphysiological conditions. In the cohort of brain neurotransmitters, catecholamines and acetylcholine reportedly play a major role in the control of neurosecretory GH-releasing hormone (GHRH) and somatostatin (SS)-producing neurons, and hence GH secretion. Activation of alpha 2-adrenoceptors or of muscarinic cholinergic receptors in the hypothalamus stimulates GH release, probably through stimulation of GHRH and inhibition of SS release, respectively. Activation of dopamine receptors likewise stimulates GH release, while activation of beta-receptors inhibits GH release through stimulation of hypothalamic SS function. This review discusses the involvement of brain catecholamines and acetylcholine in GH hypersecretory states, including anorexia nervosa, acromegaly, IDDM, liver cirrhosis, depression, renal failure and GH insensitivity syndrome, with a view to providing a fuller understanding of their pathophysiology and, whenever possible, diagnostic and therapeutic implications.

Psycholeine, a natural alkaloid extracted from Psychotria oleoides, acts as a weak antagonist of somatostatin

The effects of psycholeine, a plant alkaloid, were investigated on binding of radiolabelled somatostatin ([ 125I] N-Tyr-SRIF) and on somatostatin (SRIF)-induced inhibition of adenylate cyclase activity and growth hormone (GH) secretion by rat anterior pituitary cells. Psycholeine was shown to displace specific binding of [ 125I] N-Tyr-SRIF to pituitary membrane preparations, with an IC 50 of 10 −5 M. At this concentration, psycholeine was also effective in significantly reducing the SRIF-induced inhibition of adenylate cyclase activity previously stimulated by growth hormone releasing factor (GRF). In parallel, it reduced the SRIF-induced inhibition of GH release stimulated by GRF in primary pituitary cell cultures in a dose-dependent manner. At a moderate concentration, the alkaloid affected neither adenylate cyclase activity nor GH release when applied in the absence of SRIF. These data suggest that psycholeine has antagonistic properties at the SRIF receptor. Quadrigemine C, a precursor of psycholeine, has a similar action.

Effect of long-acting antagonists of growth hormone (GH)-releasing hormone on GH and cyclic adenosine 3',5'-monophosphate release in superfused rat pituitary cells.

In a recent study, investigating the time course of both GH and cAMP secretion induced by GH-releasing hormone (GHRH) in the superfusion system, we found that the amount of cAMP liberated from the cells was not proportional to GH release and that cAMP discharged after a GHRH pulse alone cannot maintain the release of GH. In the present study, two potent antagonists of GHRH, MZ-4-71 ([Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]human GHRH-(1-28)Agm) and MZ-4-243 ([Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]human GHRH-(1-28)Agm) were evaluated for their long term effect in the superfusion system and for their ability to influence the release of GH and cAMP. Our present findings showed that after a 9-min preincubation, antagonist MZ-4-71 and MZ-4-243 at 3 and 1 nM, respectively, caused an inhibition of GH release, stimulated by 1 nM GHRH, similar to that caused by the 100-nM dose of the standard antagonist ([Ac-Tyr1,D-Arg2]human GHRH-(1-29)NH2). The standard antagonist at 100 nM did not influence the GHRH-induced GH response 30 min later, and the inhibition caused by MZ-4-71 at 30 nM decreased gradually to 30% 120 min after the treatment, but the 30-nM dose of MZ-4-243 reduced the GH response by more than 90% even 270 min after its administration. During a 2-h incubation with 1 nM GHRH in combination with a 30-min infusion of the standard antagonist, MZ-4-71, MZ-4-243, or somatostatin, from the 30th to the 60th min, the decrease in GH discharge preceded the inhibition of cAMP release. After infusion of the antagonists or somatostatin was stopped, GH release resumed sooner than that of cAMP. Simultaneous determinations of cAMP and GH in the samples showed that changes in GH levels were never preceded by a rise or decrease in cAMP release, in contrast to existing information. The participation of more than one signal transduction mechanism in the mediation of the effect of GHRH is very likely, and the balance of these mechanisms may vary with the dose and duration of stimulation.

Regulation of hypothalamic somatostatin by glucocorticoids.

Glucocorticoids (GCs) play a key role in the physiology of the hypothalamic-somatotroph axis, since these steroids enhance growth hormone (GH) gene transcription and increase GHRH receptor synthesis. However, GC excess inhibits normal growth in all species studied. This is mainly due to the impaired GH secretion observed during hypercortisolism, a situation in which GH responses to a number of stimuli, including GHRH, are blunted. The inhibitory effect of GCs on GH secretion seems to be dependent on enhanced hypothalamic SS secretion. Since SS release is stimulated by beta-adrenergic agonism we tested the possibility that GC inhibition of GH secretion would depend on increased beta-adrenoceptor activity in SS-producing neurons. The experimental design consisted in evaluating the GH response to GHRH in normal subjects after having induced hypercortisolism, with DEX, and blocking beta-adrenoceptors with propranolol (PRO). Moreover, to investigate the specificity of this mechanism, GHRH-induced GH release was tested after inducing hypercortisolism and enhancing alpha 2-adrenergic or muscarinic cholinergic tone, by giving clonidine (CLO) or pyridostigmine (PD), respectively. As expected, nocturnal DEX administration inhibited the GH response to GHRH. In this situation of hypercortisolism, both PRO and CLO, but not PD, were able to reverse the inhibitory effect of DEX on GHRH-elicited release. However, the potentiating effect of these drugs on the GHRH-induced GH secretion was only observed for PRO. These data confirm that GC excess inhibits GH release by increasing hypothalamic SS secretion, and that the mechanism is mediated by GC-induced enhanced beta-adrenergic responsiveness. Therefore, the defective GHRH secretion observed in chronic hypercortisolism must be a consequence of the continuous blockade that SS excess exerts on GHRH-producing neurons. Our postulate agrees with other data in the literature showing that GCs modulate the secretion of some hypothalamic peptides by changing the responsiveness of the producing neurons from alpha 2-adrenoceptors to that of beta-adrenoceptors.

Inhibitory effect of amylin on growth hormone responsiveness to growth-hormone-releasing hormone in the rat.

The effects of intracerebroventricular (i.c.v.) or intracarotid (i.a.) administration of amylin (AMY) on growth hormone (GH) release induced by GH-releasing hormone (GHRH) were examined in conscious male rats. Amylin (25 ng-5 micrograms/rat, i.c.v. or 10 micrograms/rat, i.a.) was administered 10 min before GHRH (2 micrograms/kg, i.a.). I.c.v. administration of AMY dose-dependently inhibited GH secretion induced by GHRH but when given peripherally, AMY did not modify the GH response to GHRH. Amylin (10(-8)-10(-6) M) had no direct effect on the rat anterior pituitary in vitro either alone or when incubated with GHRH. To characterize the mechanism(s) involved in vivo in the inhibition of GH by AMY, we examined, at first, the effects of AMY on GHRH-induced GH release in rats depleted of somatostatin by pretreatment (4 h before) with cysteamine (300 mg/kg s.c.). The inhibitory activity of AMY on GH secretion elicited by GHRH seems to be independent of hypothalamic somatostatin; in fact, AMY was still active in rats treated with cysteamine. In addition, we examined the effects of i.c.v. AMY administration on clonidine (CLO)-induced GH secretion and on dopamine and noradrenaline content in the brain, since it is known that GHRH is a stimulus sensitive to changes in central catecholaminergic activity. The failure of AMY to affect GH secretion induced by activation of postsynaptic alpha 2-receptors by CLO and the finding that the peptide decreased noradrenaline content in the hypothalamus and striatum, indicates that AMY may inhibit GH release by interfering with the facilitatory influence of the catecholaminergic system on GH secretion.

Hypothalamic and Thyroidal Regulation of Growth Hormone in Tilapia

A radioimmunoassay (RIA) for recombinant tilapia growth hormone (GH) was established and validated. The ability of various hypothalamic factors to regulate GH secretion in the tilapia hybrid ( Oreochromis niloticus × Oreochromis aureus) was studied. Somatostatin 1-14 (SRIF 1-14; 10-100 μg/kg) was found to reduce circulating GH levels in a dose-dependent manner. SRIF 1-14 (0.1-1000 n M) inhibited GH release from perifused pituitary fragments (ED 50 0.83 n M). Human growth hormone-releasing hormone fragment 1-29 (hGHRH 1-29; 100 μg/kg) doubled circulating GH levels and modestly stimulated GH secretion in vitro. Carp growth hormone-releasing hormone (cGHRH) stimulated GH secretion in vitro to a similar degree at the same dose (1 μ M). Injection of salmon gonadotropin-releasing hormone (sGnRH) superactive analog (10-100 μg/kg) increased plasma GH levels sixfold. sGnRH also stimulated GH release in vitro (ED 50 142.56 n M). Dopamine (0.1-10 μ M) and the D 1 DA receptor agonist SKF 38393 increased GH secretion from perifused pituitary fragments dose-relatedly. Thyrotropin-releasing hormone (TRH) had no effect on GH secretion from perifused pituitary fragments, but increased plasma GH levels, as did bovine thyroid stimulating hormone (bTSH). The increased plasma GH in the bTSH-treated fish coincided with a dramatic increase in T 4; however, TRH increased GH without changing T 4 levels. T 3 increased the synthesis of GH by isolated pituitaries (incorporation of [ 3H]leucine). SRIF 1-14 seems to be a most potent hypothalamic regulator of GH secretion in tilapia; sGnRH and DA both increased GH secretion, although sGnRH elicited considerably greater responses at lower doses. Two forms of GHRH increased GH levels, although the unavailability of the homologous peptide prevented an accurate evaluation of its importance in regulating GH secretion. The thyroid axis (TRH, TSH, and T 3) stimulates both synthesis and release of GH, although TRH did not appear to have a direct effect on the level of the pituitary.

Inhibition of growth hormone release after the combined administration of GHRH and GHRP-6 in patients with Cushing's syndrome.

In patients with Cushing's syndrome there is a blunted GH response to all types of stimuli. Although inferential data point towards a direct perturbation in the pituitary exerted by glucocorticoids, the basic mechanism is unknown. His-D-TRP-ALA-TRP-D-Phe-Lys-NH2 (GHRP-6) is a synthetic hexapeptide which releases GH by a direct pituitary effect through receptors other than GHRH receptors. Furthermore, the combined administration of GHRH and GHRP-6 is able to induce a large GH discharge even in some pathological states such as obesity, associated with GH blockade. To gain further insight into the disrupted mechanisms of GH secretion, Cushing's syndrome patients were challenged with either GHRH, GHRP-6 or GHRH together with GHRP-6. A group of normal subjects was included for control purposes. Three different tests were undertaken: (a) GHRH 100 micrograms i.v.; (b) GHRP-6 100 micrograms i.v. and (c) GHRH plus GHRP-6 100 micrograms i.v. of each; administered to each subject on different days, at least 4 days apart. Ten patients (8 women, 2 men) with untreated Cushing's syndrome, 9 Cushing's disease and 1 adrenal adenoma. Five healthy volunteers (3 women, 2 men) of similar ages served as a control group. Plasma GH levels were measured by immunoradiometric assay. The areas under the curve (AUC) of GH secretion (mean +/- SEM in mU/I/120 min) in the control subjects after each test were: GHRH, 1420 +/- 330; GHRP-6, 2278 +/- 290 and GHRH plus GHRP-6, 7332 +/- 592 (P < 0.05 vs each compound alone). The AUCs for Cushing's syndrome patients were: GHRH, 248 +/- 165; GHRP-6 530 +/- 170 and for GHRH plus GHRP-6, 870 +/- 258 (P < 0.05 vs GHRH alone). After the combined stimulus only one out of the ten patients with hypercortisolism showed a GH peak over 20 mU/I, while all the controls had a peak over 84 mU/I. GHRP-6 induced GH secretion as well as the GH discharge elicited by GHRH and GHRP-6 are considerably reduced in Cushing's syndrome patients. This suggests that the main impairment of GH secretion in that pathological state resides at pituitary level.

Biological Characterisation of [67Ga] or [68Ga] Labelled DFO-Octreotide (SDZ 216-927) for PET Studies of Somatostatin Receptor Positive Tumors

Radiolabelled analogues of Somatostatin (SRIF) were demonstrated to be useful for conventional gamma-camera imaging of SRIF receptor-positive tumors and their metastases. To evaluate the feasibility of positron emission tomography (PET) or SRIF receptor-positive tumors deferoxamine (DFO) was conjugated to octreotide via a succinyl linker to form a stable conjugate with the gallium isotopes 67Ga and 68Ga. This new octreotide analog, SDZ 216-927, binds specifically and with high affinity to SRIF receptors in vitro (pKi = 8.94 +/- 0.06) and exhibits SRIF like biological properties as demonstrated by the inhibition of growth hormone (GH) release from cultured pituitary cells. SDZ 216-927 was efficiently labelled with 67Ga without affecting high affinity binding to SRIF receptors. Biodistribution studies revealed that [67Ga]SDZ 216-927 was stable in vivo and rapidly cleared from the circulation, as indicated by the low amount of 67Ga detected in the blood four hours post injection (p.i.). SRIF receptor-positive tumors were clearly visualized 10 minutes p.i. in tumor bearing rats. The specificity of ligand binding in vivo was demonstrated i) by the high tumor/non-tumor ratio 4 hours p.i. (tumor/blood 22.3:1, tumor/muscle 64.5:1, tumor/liver 4.0:1, tumor/spleen 16.8:1) and ii) by a significantly lower uptake of radioactivity in the tumor after pretreatment of tumor bearing animals with an excess of unlabelled SDZ 216-927. SDZ 216-927, when labelled with the positron emitting isotope 68Ga, clearly imaged SRIF receptor-positive tumors using positron emission tomography (PET). Therefore quantitative SRIF receptor imaging with PET seems to be possible using this new radiopharmaceutical.

(2-[18F]fluoropropionyl-(d)phe1)-octreotide, a potential radiopharmaceutical for quantitative somatostatin receptor imaging with PET: Synthesis, radiolabeling, in vitro validation and biodistribution in mice

Octreotide is labeled with fluorine-18 as a potential radiopharmaceutical for quantitative in vivo mapping of somatostatin receptors. [18F]-fluoroacylation is achieved with n.c.a. 2-[18F]fluoropropionic acid 4-nitrophenylester which is reacted with ϵ-Boc-Lys5-octreotide. After deprotection the desired Nα-[18F]fluoropropionylated octreotide ([18F]SDZ 223–228) is obtained. Final HPLC purification gives rise to radiochemical yields of 65 ± 5% based on the fluoroacylation agent. Binding experiments using rat cortex membranes indicate an affinity for somatostatin receptors of pKi = 8.6 ± 0.2. The biological activity of this SRIF analog is demonstrated by the inhibition of growth hormone release from cultured pituitary cells. The pIC50 in this test system is 8.75, indicating full biological activity. Biodistribution studies with NMRI mice show predominantly renal excretion, rapid blood clearance and only negligible bone activity, i.e. formation of free fluoride.

Androgen receptor immunoreactivity in somatostatin neurons of the periventricular nucleus but not in the bed nucleus of the stria terminalis in male rats

Somatostatin (SS) is a neuropeptide responsible for the inhibition of growth hormone (GH) release from the anterior pituitary. The existence of sexual dimorphism in the pattern of GH secretion in several mammalian species strongly suggests a role of sex steroids in the regulation of GH release. It has been recently demonstrated that SS mRNA levels are decreased by gonadectomy in male rats while administration of testosterone reversed the postgonadectomy decrease in hypothalamic SS mRNA levels. The mechanism(s) by which androgens regulate somatostatin neurons are unknown at this time. Both direct and indirect pathways have been suggested. In this study, double-labeling immunocytochemistry was used to address the question of whether SS neurons have androgen receptors in adult male rats. About 70–75% of SS-immunoreactive (-ir) neurons in the periventricular hypothalamic nucleus possess androgen receptors and this co-localization persists throughout the rostrocaudal extent of this nucleus. In the bed nucleus of the stria terminalis, no SS-ir neurons were shown to have androgen receptors. These results suggest that androgens may directly exert their effect upon SS neurons in the periventricular nucleus.