A bicyclo-somatostatin analog,highly specific for the inhibition of growth hormone release

The tetradecapeptide somatostatin was cyclized by a combination of conventional and solid phase peptide synthesis methods, to a homodetic cyclic disulfide tetradecapeptide, Wy-40,391:

The analog inhibits the release of growth hormone (GH) $\text{in vivo}$ without affecting either insulin or glucagon secretion. A correlation between binding affinity to the receptors and specificity is suggested.     

Cyclic response of hypophysectomized rats to ovulation induced by LHRH agonist: mediation by prostaglandins

Further confirmation that the LHRH/LHRH agonist-induced ovulation in the hypophysectomized (hypx) rat is due to a direct ovarian effect and not mediated by LH release from residual pituitary tissue or other CNS sites is provided by the persistence of this effect despite concomitant median eminence lesion or passive immunization to LH. Adrenalectomy did not affect the ovulatory activity of the LHRH agonist, D-Trp6-N alpha MeLeu7-DesGly10-Pro9-NHEt-LHRH (Wy-40,972), in the hypx rat. Prior administration of a potent LHRH antagonist blocked ovulation induced in hypx proestrous rats by Wy-40,972 but not by LH-S19. Ovulation can be induced by Wy-40,972 one day earlier (e.g. metestrus) in the intact rat than it can in the hypx rat. Results in the hypx metestrous rat indicate that the ovulatory responsiveness of the intact rat at this stage of the cycle may occur by complementary action of Wy-40,972-stimulated endogenous LH release and a direct ovarian effect of the agonist. Prostaglandins (PG) are involved in the ovulatory mechanism of Wy-40,972 in the hypx proestrous rat as evidenced by the dose-dependent inhibition of this effect by PG synthetase inhibitors, indomethacin and Fentiazac. Moreover, there were significant increases in ovarian concentrations of PGF2 alpha and PGE2-PGE1 in response to Wy-40,972 that could be prevented by indomethacin. However, exogenous administration of either of these PG's was not effective in inducing ovulation in the hypx rat.     

Correlation between thyroid hormone status and hepatic hyperplasia and hypertrophy caused by the peroxisome proliferator-activated receptor alpha agonist Wy-14,643

BACKGROUND: The metabolic inhibitor rotenone inhibits hepatocellular proliferation and the incidence of liver cancer resulting from exposure to the PPARalpha agonist Wy-14,643, via unknown mechanisms. Since the absence of thyroid hormones diminishes hepatomegaly, an early biomarker for the hepatocarcinogenicity induced by PPARalpha agonists, this study was undertaken to investigate whether rotenone might interference with the ability of Wy-14,643 to alter the animal thyroid status. METHODS: Male B6C3F1 mice were given Wy-14,643 (100 ppm), rotenone (600 ppm) or a mixture of both, in the feed for 7 days. Bromodeoxyuridine (BrDU), marker of cell replication, was delivered through subcutaneously implanted osmotic mini-pumps. At the end of the experiment, sera were collected and corticosterone and thyroid hormone levels were measured by solid-phase radioimmunoassay kits. In addition, liver tissue samples were stained immunohistochemically for BrDU to determine percentages of labeled cells. Further, cell surface area was determined from images generated by a Zeiss Axioplan microscope equipped with a plan Neofluar x40 0.75 na objective. Tracings of individual hepatocyte perimeters were then analyzed and cell-surface areas were calculated using MicroMeasure FL-4000. RESULTS: Wy-14,643 caused a significant increase in liver weights, hepatocyte BrDU labeling index (LI), and hepatocyte surface area. In animals which received both Wy-14,643 and rotenone simultaneously, all of these effects were significantly less pronounced compared with mice that received Wy-14,643 alone. Rotenone alone decreased liver weights, LI and surface area. The Free Thyroid Index (FTI), which provides an accurate reflection of the animal's thyroid status, was 5.0 +/- 0.3 in control mice. In animals exposed to rotenone, these values decreased to 2.0 +/- 0.9, but in animals which received Wy-14,643, levels increased significantly to 7.7 +/- 0.9. FTI values decreased to 3.4 +/- 0.8 in mice receiving both rotenone and Wy-14,643. CONCLUSION: A strong correlation was observed between the animal thyroid status and both, hepatocyte proliferation (r2 = 0.62), and hepatocyte surface area (r2 = 0.83). These results support the hypothesis that the thyroid status of the animal plays a role in PPARalpha-induced hepatocellular proliferation and liver cell enlargement. Both these events are known to contribute to the expression of liver cancer in response to the activation of PPARalpha.     

Impact of euglycaemia and hyperglycaemia on the release of growth hormone and prolactin in type II-diabetics

The influence of different blood glucose concentrations on the arginine (30 g/30 min i.v.) and TRH (400 micrograms i.v.) induced release of growth hormone and prolactin was studied in six male type II-diabetic patients. Blood glucose concentrations were clamped at euglycaemic (4-5 mmol/l) or hyperglycaemic (12-18 mmol/l) levels by means of an automated glucose-controlled insulin infusion system. The response of growth hormone to arginine, and irregular spikes in growth hormone concentrations following TRH seen in the euglycaemic state were suppressed during hyperglycaemia. The suppression of the arginine-induced release of growth hormone by hyperglycaemia was observed both with and without concomitant administration of exogenous insulin. The rise in serum prolactin concentrations in response to arginine was unaffected by hyperglycaemia, whereas the TRH-induced release of prolactin was suppressed. Since arginine induces the release of growth hormone and prolactin via the hypothalamus, while TRH acts at the pituitary level, the glycaemic state appears to exert a modulatory effect on the secretion of growth hormone and prolactin in type II-diabetics at both locations.     

The effects of partial opiate agonists on plasma prolactin and growth hormone levels in the rat.

Opiate agonists, partial agonists, and antagonists differed in their effects on release of prolactin and growth hormone. Agonists (morphine, methadone or meperidine) elevated plasma levels of both hormones. An antagonist (naloxone) lowered levels of prolactin but not growth hormone. All partial agonists studied raised growth hormone levels; among these, levallorphan, nalorphine, and ciramadol lowered prolactin levels while pentazocine and meptazinol did not. Naloxone blocked morphine-induced release of prolactin and growth hormone. The partial agonists suppressed morphine-induced prolactin release, and several suppressed the elevated growth hormone levels as well. Data from the opiate radioreceptor assay (displacement of ³H-naloxone) in the presence and absence of sodium agrees with the above placement of agents into three classes. These results suggest that classification of opioid compounds into agonists, partial agonists and antagonists may be made by their effects on prolactin and growth hormone release.     

Effects of TRH and somatostatin on releases of prolactin and growth hormone in vitro by the pituitary of Poecilia latipinna

Summary Pituitary glands from a teleost fish were incubated in the presence of the synthetic hypophysiotropic peptides, thyrotrophin-releasing hormone and somatostatin, in two media of different osmotic pressure.The effects on prolactin and growth hormone cells were detected by electron-microscopic morphometry with the aid of an image analyser. Thyrotrophin-releasing hormone caused changes in prolactin cell ultrastructure consistent with stimulated hormone release and, in the low osmotic pressure medium, appeared to increase synthetic activity. There was no effect on growth hormone cells. After somatostatin treatment, both synthesis and release in prolactin cells appeared to be inhibited, and there was an obvious inhibition of synthesis and release in growth hormone cells. The response of both cell types to somatostatin did not appear to be dependent on the osmotic pressure of the medium.     

Altered release of growth hormone from dispersed adenohypophysial cells of streptozotocin diabetic rats. I. Effects of growth hormone releasing factor and somatostatin

As growth hormone has been implicated in the "dawn phenomenon," an early morning rise in serum glucose, we have studied the control of growth hormone release in diabetes using an acutely dispersed system of adenohypophysial cells from normal or diabetic rats (65 mg/kg streptozotocin, 8 days before sacrifice; serum glucose, 490 +/- 17 mg/dL). Growth hormone release is normally controlled by the two hypothalamic hormones, growth hormone releasing factor and somatostatin. We have found cells of the diabetic rats exhibit changes in sensitivity that result in increased growth hormone release in static incubation. In normal cells, rat growth hormone releasing factor increases growth hormone release three- to four-fold with an EC50 of 151 +/- 27 pM (n = 7). In contrast, in cells from diabetic rats, there was a significant (twofold) increase in sensitivity to growth hormone releasing factor (EC50 = 75 +/- 15 pM, n = 7) which resulted in increased growth hormone release with lower but not maximal (10 nM) growth hormone releasing factor. Basal nonstimulated release was unchanged. Somatostatin inhibition of stimulated growth hormone release was reduced (n = 7); half-maximal inhibition occurred with 0.21 +/- 0.03 nM (normal) and 0.76 +/- 0.17 nM somatostatin (diabetic). In perifusion the peak secretion rate was significantly lower for diabetic cells stimulated by a maximal dose of growth hormone releasing factor. These studies suggest somatotrophs of diabetic rats have altered sensitivity in vitro to the controlling hormones growth hormone releasing factor and somatostatin.     

Effect of peroxisome proliferators on the methylation and protein level of the c-myc protooncogene in B6C3F1 mice liver

Peroxisome proliferators in general are nongenotoxic mouse liver carcinogens for which DNA hypomethylation and altered gene expression are proposed mechanisms. Therefore, the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D), dibutyl phthalate (DBP), gemfibrozil, and Wy-14,643 were evaluated for the ability to alter the methylation and expression of the c-myc protooncogene. Male B6C3F1 mice were administered for 6 days in their diet Wy-14,643 (5-500 ppm), 2,4-D (1,680 ppm), DBP (20,000 ppm), or gemfibrozil (8,000 ppm). All four peroxisome proliferators caused hypomethylation of the c-myc gene in the liver. Wy-14,643 appeared to be the most efficacious with a threshold between 10 and 50 ppm. The level of the c-myc protein was increased by Wy-14,643, but not the other peroxisome proliferators. When female B6C3F1 mice received a two-thirds partially hepatectomy and 16 h later were administered 50 mg/kg Wy-14,643 by gavage, hypomethylation of the gene occurred 24 h later. Hypomethylation was not found in mice that received Wy-14,643 following a sham operation. Hypomethylation of the c-myc gene within 24 h of administering Wy-14,643 after a partial hepatectomy but not after a sham operation supports the hypothesis that the peroxisome proliferators prevent methylation of hemimethylated sites formed by DNA replication.     

The effects of the acute administration of buprenorphine hydrochloride on the release of anterior pituitary hormones in the rat: evidence for the involvement of multiple opiate receptors

Multiple opiate receptor agonists and antagonists have been found to produce different patterns of anterior pituitary hormone release. The present studies examined the pattern of anterior pituitary hormone release produced by buprenorphine. The effects of the kappa agonist ethylketocyclazocine on thyroid stimulating hormone release were also examined. Following buprenorphine, serum levels of corticosterone and luteinizing hormone were not changed while growth hormone release was stimulated in a dose-dependent manner. Prolactin release was stimulated after the lowest dose of buprenorphine while the highest dose induced a fall in serum prolactin. Similar biphasic effects on thyroid stimulating hormone were seen after either buprenorphine or ethylketocyclazocine. The results provide support for the role of multiple opiate receptors in opiate-induced changes in anterior pituitary hormone release.     

Inhibition of growth hormone synthesis by somatostatin in cultured pituitary of rainbow trout

Summary When the pituitary of rainbow trout (Oncorhynchus mykiss) was incubated in a serum-free medium, a high level of growth hormone release as well as an activation of growth hormone synthesis were observed, suggesting the existence of hypothalamic inhibitory factor(s) on growth hormone synthesis. Although an inhibitory effect of somatostatin on growth hormone release is well established in both mammals and teleosts, an effect on growth hormone synthesis has not been demonstrated. In this study, we examined the effect of somatostatin on growth hormone synthesis in organ-cultured trout pituitary using immunoprecipitation and Northern blot analysis. Somatostatin inhibited growth hormone release from the cultured pituitary within 10 min after addition without affecting prolactin release. Incubation of the pituitary with somatostatin also caused a significant reduction in newly-synthesized growth hormone in a dose-related manner, as assessed by incorporation of [³H]leucine into immunoprecipitable growth hormone. There were no changes in the level or molecular length of growth hormone mRNA after somatostatin treatment, as assessed by Northern slot blot and Northern gel blot analyses. Human growth hormone-releasing factor stimulated growth hormone release, although the spontaneous synthesis of growth hormone was not augmented. However, somatostatin-inhibited growth hormone synthesis was restored by growth hormone-releasing factor to the control level. The spontaneous increase in growth hormone synthesis observed in the organ-cultured trout pituitary may be caused, at least in part, by the removal of the inhibitory effect of hypothalamic somatostatin.Abbreviations GH growth hormone - GHRF GH-releasing factor - PRL prolactin - SDS sodium dodecyl sulphate - SRIF somatostatin (somatropin release-inhibiting factor)     

The effects of opiate agonists on growth hormone and prolactin release in rats

Various opioid receptor agonists, including Met5-enkephalin amide, Leu5-enkephalin amide, [D-Ala]2-Met5-enkephalin amide, [D-Ala]2-Leu5-enkephalin amide, morphine sulfate, d-methadone hydrochloride, and l-methadone hydrochloride were administered to adult male rats by subcutaneous injection. All opioid receptor agonists except Leu5-enkephalin amide significantly stimulated growth hormone and prolactin release. Naloxone and naltrexone blocked the hormone stimulatory effects of the opioids and both naloxone and naltrexone, when administered alone, significantly reduced serum growth hormone and prolactin concentrations. The dopaminergic agonist apomorphine, but not the alpha-adrenergic agonist clonidine, blocked opiate stimulation of prolactin. Morphine sulfate caused growth hormone release in rats pretreated with alpha-methyl-p-tryosine, a catecholamine synthesis inhibitor. Cholinergic agonists, physostigmine and pilocarpine, antagonized the growth hormone and prolactin release induced by morphine sulfate. The data suggest that the opiates stimulate prolactin via an interaction with catecholaminergic neurons controlling prolactin release and stimulate growth hormone via a mechanism independent of alpha-adrenergic or general catecholaminergic influence. The mechanism through which cholinergic agonists act to inhibit opiate agonist stimulation of growth hormone is presently unknown.     

Naloxone inhibits prolactin and growth hormone release induced by intracellular glucopenia in the rats

Intraventricular administration of 2-deoxy-D-glucose (2DG), which causes intracellular glucopenia in the central nervous system, increased plasma prolactin and growth hormone levels in the urethane anesthetized male rats. Naloxone, an opiate antagonist, inhibited the 2DG-induced prolactin and growth hormone release. Apomorphine, a dopaminergic agonist, also inhibited the release of these hormones induced by 2DG. These results suggest that endorphins play a role in hypoglycemia-induced prolactin and growth hormone release and that the dopaminergic mechanism may be involved in this phenomenon.     

Cycloheximide blocks insulin-like growth factor I but not somatostatin inhibition of growth hormone secretion

Growth hormone secretion is controlled by the two hypothalamic hormones, growth hormone releasing factor (GRF) and somatostatin. In addition, the insulin-like growth factors (IGF or somatomedins) which are themselves growth hormone dependent, inhibit growth hormone release in vitro, therefore acting to close the negative feedback loop. The studies reported here examine some of the differences between inhibition of growth hormone secretion by somatostatin and IGF-I in vitro. The major finding is that cycloheximide, a protein synthesis inhibitor, blocks inhibition of GRF-stimulated growth hormone release caused by IGF-I, without changing the inhibition caused by somatostatin. The experiments were done by exposing mixed rat adenohypophysial cells to secretagogues with or without cycloheximide for 24 h in a short term culture. Somatostatin (0.6 nM) totally blocked rat GRF (1 nM) stimulated growth hormone release to values 48% of control (nonstimulated values), while IGF-I (27 nM) only reduced the GRF-stimulated growth hormone release by 27 +/- 3% (N = 5). Cycloheximide (15 micrograms/mL) totally blocked the effect of IGF-I but not somatostatin. A low concentration (0.12 nM) of somatostatin, which only partly inhibited growth hormone release, was also unaffected by cycloheximide. In purified rat somatotrophs, somatostatin (0.1 nM) inhibited GRF-stimulated cAMP levels slightly and reduced growth hormone release while IGF-I (40 nM) had no effect. We suggest that IGF-I inhibits only the secretion of newly synthesized growth hormone, while somatostatin inhibits both stored and newly synthesized growth hormone pools.     

Dissociation of somatostatin effects. Peptides inhibiting the release of growth hormone but not glucagon or insulin in rats

Two analogs of somatostatin were tested for their effects on release of growth hormone, glucagon, and insulin after subcutaneous injection into rats. These peptides significantly suppressed pentobarbital-stimulated growth hormone release but showed no effect on arginine-stimulated glucagon or insulin release at dosages greater than 2 mg/kg. Somotostatin acts on all three secretions at dosages below 200 μg/kg.     

Cholecystokinin antagonist,proglumide, stimulates growth hormone release in the rat

Previous studies have revealed a stimulatory action of cholecystokinin on growth hormone release in the rat. To evaluate the physiologic significance of these effects we employed the cholecystokinin antagonist, proglumide and injected it intravenously and intraventricularly (third cerebral ventricle, 3V) to determine its actions on growth hormone. The experiments were performed in conscious, freely moving rats with indwelling cannulae in the 3V and/or external jugular vein. Intraventricular injection of 2 or 10 □g of proglumide significantly elevated plasma growth hormone concentrations in intact and castrated male rats and in ovariectomized females. Intravenous injections of 10 or 100 □g of proglumide were also effective in elevating growth hormone in a dose-related manner. Surprisingly, the response to the lower dose given intraventricularly was somewhat greater than that of the higher dose. We speculate that these stimulatory effects of proglumide given intraventricularly are due to the agonist action of proglumide at these doses since action of cholecystokinin itself is to increase plasma growth hormone following its intraventricular injection. The studies therefore do not establish a physiologically significant growth hormone-releasing action of brain cholecystokinin but provide more evidence that activation of cholecystokinin receptors in the brain can induce a stimulation of growth hormone release either by activation of the release of growth hormone-releasing hormone or by inhibition of the release of somatostatin or by a combination of these two actions.     

Effect of substance P/bombesin antagonists on the release of growth hormone by GHRP and GHRH.

The substance P(SP)/bombesin (Bn) antagonists [DArg1DTrp7,9Leu11] SP(P-7482), [DArg1-DPro2DTrp7,9Leu11]SP (P-7483), [DArg1DPhe5DTrp7,9Leu11]SP(P-7492), and the growth hormone releasing hormone (GHRH) antagonist [DArg2Ala8,9,15]GHRH(1-29)(DC21-366) were tested for their in vitro effects on the release of growth hormone (GH) in the presence of GHRH and growth hormone releasing peptide, HisDTrpAlaTrpDPheLysNH2(GHRP). P-7492, P-7483, and P-7482 decreased, dose-dependently, the release of GH by GHRP (IC50 = 0.2 microM, 0.85 microM, and 6 microM, respectively). These antagonists had only a 10-15% inhibitory effect on the stimulated GH release of GHRH even at high dosage. DC21-366 decreased the stimulated release of GH by GHRH (IC50 = 0.16 microM) but not by GHRP. Neither SP nor Bn had GH releasing or inhibitory effects in this system.     

Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methane sulfonamide) an orally active leukotriene D4 antagonist: pharmacological characterization in vitro and in vivo in the guinea pig

The following communicates the pharmacology of Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methanesulfonamide) a chemically novel and orally potent leukotriene (LT) D4 receptor antagonist. In the isolated guinea-pig trachea pretreated with indomethacin (5 microM) and L-cysteine (10 mM), Wy-48,252 antagonized TD4-induced contraction with a pKB = 7.6. Against LTC4 on tissues pretreated with IND and glutathione (10 mM), Wy-48,252 had a pKB greater than 5. Wy-48,252 (10 microM) did not antagonize pilocarpine-, histamine- or PGF2 alpha-induced tracheal contraction. Further, in the presence of indomethacin and chlorpheniramine (1 microM), Wy-48,252 dose-dependently inhibited the antigen-induced contraction of guinea-pig trachea in a manner consistent with antagonism at the LTD4 receptor and inhibition of LT synthesis. In the Konzett-Rossler model of i.v. LTD4-induced bronchoconstriction in indomethacin treated guinea pigs, intragastric Wy-48,252 (2 hr) had an ID50 of 100 micrograms/kg and a functional half-life of 5 hr. Against i.v. antigen-induced bronchoconstriction in guinea pigs treated with indomethacin and chlorpheniramine, intragastric Wy-48,252 (2 hr) had an ID50 of 0.6 mg/kg and a 5 hr half life. Intragastric Wy-48,252 also selectively blocked the cutaneous wheal reaction to intradermal LTD4 but not histamine. We conclude that Wy-48,252 is distinguished from other selective LTD4 receptor antagonists by its oral potency and should be useful in ascertaining the role of LTD4 mediated processes in asthma, allergy and animal models.     

In vivo assay of antisomatotropic activity of somatostatin and two of its analogs]

Somatostatin (Growth hormone release inhibiting hormone or somatotrophin-release inhibiting factor) and its analogues can be measured in vivo by inhibiting the pentobarbital induced growth hormone release in male rats. Experimental conditions must be carefully defined to become optimal.     

The effect of d-fenfluramine on anterior pituitary hormone release in the rat: in vivo and in vitro studies

Administration of d-fenfluramine, a serotonin-releasing drug, to male rats induced a dose-dependent increase in both serum prolactin and corticosterone concentrations. Serum growth hormone levels increased, but not significantly, at a dose of 1.25 mg/kg i.p. and decreased significantly at higher doses. When rats were pretreated with the serotonin uptake inhibitor fluoxetine (10 mg/kg i.p.) 30 min prior to injection of d-fenfluramine (5 mg/kg i.p.), the serum prolactin response to d-fenfluramine was partially inhibited, whereas the growth hormone response was not significantly modified. Fluoxetine pretreatment increased the serum corticosterone to the same level as did d-fenfluramine. d-Fenfluramine's effect on prolactin and growth hormone release was further tested in a hypothalamic-pituitary in vitro system. The addition of d-fenfluramine (5-500 ng/mL) for 30 min to rat hypothalami resulted in an enhancement of prolactin and growth hormone-releasing activities. These were expressed as the ability of the media in which the hypothalami had been incubated to stimulate prolactin and growth hormone release by cultured pituitary cells. The data suggest that the effect of d-fenfluramine on prolactin secretion is exerted through the hypothalamus and is probably mediated, at least partially, by a serotoninergic mechanism. The mechanism of d-fenfluramine's effect on corticosterone and growth hormone release needs further evaluation.     

Wy-14,643 and fenofibrate inhibit mitochondrial respiration in isolated rat cardiac mitochondria

We investigated the direct effects of two selective PPARalpha ligands, fenofibrate and Wy-14,643, on mitochondrial respiratory function using isolated rat cardiac mitochondria. Isolated left ventricular mitochondria were incubated with increasing concentrations of fenofibrate or Wy-14,643 (10, 100, and 500 microM) and mitochondrial respiration determined using: malate/glutamate (complex I), succinate (complex II) and palmitoyl-l-carnitine as oxidative substrates. Our data show that acute exposure to Wy-14,643 and fenofibrate differentially perturb cardiac mitochondrial respiration i.e., fenofibrate more potently inhibited mitochondrial respiration and bioenergetic capacity compared to Wy-14,643. Moreover, we found that both agents increased uncoupling of mitochondrial oxidative phosphorylation.