In vivo and in vitro effects of ghrelin/motilin-related peptide on growth hormone secretion in the rat

Ghrelin (Ghr), a 28 amino acid gastric peptide with an n-octanoylation on Ser 3, has recently been identified as an endogenous ligand of the growth hormone secretagogue (GHS) receptor. A cDNA was also isolated from a mouse stomach library encoding a protein named prepromotilin-related peptide (ppMTLRP) which shares sequence similarities with prepromotilin. Mouse and rat ppMTLRP sequences (rGhr) are identical and show 89% identity with human ghrelin (hGhr). By analogy with promotilin, cleavage of proMTLRP into an 18 amino acid endogenous processed peptide can be assumed on the basis of a conserved dibasic motif in position 9-10 of its sequence. In the present work, we compared the GH-releasing activity of rGhr28/MTLRP and of hGhr28/MTRLP with that of a shorter form of the peptide, hGhr18. A short peptide devoid of Ser-3 n-octanoylation hGhr18[-] was also tested. Addition of rGhr28, hGhr28 and hGhr18 stimulated GH release to the same extent from superfused pituitaries. The effect was dose dependent in a 10(-8) to 10(-6) M concentration range. In contrast, hGhr 18[-] was inactive. In freely moving animals, both rGhr28 and hGhr28 (10 microg, i.v.) stimulated GH release, whereas the same dose of hGhr18 or of hGhr18[-] was ineffective. After rGhr28, GH plasma levels increased as early as 5 min after injection and returned to basal values within 40-60 min. Expressed as percent stimulation, administration of rGhr28 was equally effective when injected during troughs or peaks of GH. Plasma concentrations of prolactin, adrenocorticotropin and leptin were not modified. Spontaneous GH secretory episodes were no longer observed within 3 h of rGhr28 treatment, but repeated administration of the secretagogue at 3- to 4-hour intervals resulted in a similar GH response. Activation of somatostatin (SRIH) release by ether stress did not blunt the GH response to rGhr28. This suggests that the secretagogue acts in part by inhibiting endogenous SRIH, as further substantiated by the ability of rGhr28 (10(-6) M), to decrease the amplitude of 25 mM K+-induced SRIH release from perifused hypothalami. In conclusion, (1) n-octanoylation of Ghrs and the shorter form hGhr18 is essential for the direct pituitary GH-releasing effect of this new family of endogenous GHSs; (2) only the longer forms are active in vivo and (3) inhibition of SRIH release appears involved in the mechanism of Ghr action.     

Prolonged inhibition of growth hormone secretion by peripheral injection of bombesin is mediated by somatostatin in the rat

Peripherally injected bombesin inhibits GH secretion in conscious, freely moving rats and in sodium pentobarbital-anesthetized rats. This inhibition of GH secretion is unusually prolonged, lasting up to 90 min after a single ip injection. The duration of inhibition of GH secretion by bombesin is greater than that observed for somatostatin (SRIF) in the same bioassay. The inhibition of GH release occurs concomitantly with stimulation of gastrin release and is independent of stimulatory effects of bombesin on plasma glucose. The structurally related mammalian gastrin-releasing peptide also inhibits GH secretion in the pentobarbital-anesthetized rat after peripheral injection. Peripherally administered bombesin blocks GH-releasing factor stimulation of GH secretion. Prior treatment of pentobarbital-anesthetized rats with SRIF-specific anti-serum blocks the inhibitory effect of bombesin on GH secretion. No effect of bombesin on GH secretion was observed in primary cultures of rat anterior pituitary cells. These data suggest that peripherally administered bombesin stimulates SRIF secretion, most probably of hypothalamic origin, which, in turn, inhibits pituitary secretion of GH. This sensitivity of the hypothalamus to a peripherally rather than centrally administered peptide has important mechanistic and therapeutic implications.     

Effects of a novel bombesin antagonist analogue on bombesin-stimulated gastric acid secretion and growth hormone release in the pentobarbital-anesthetized rat

A new and specific bombesin receptor antagonist analogue, Leu13 psi [CH2NH]Leu14-bombesin, was studied for inhibition of bombesin-stimulated gastric acid secretion in pentobarbital-anesthetized rats. The analogue potently inhibited bombesin-stimulated gastric acid secretion in a dose-dependent fashion, exhibiting an ID50 of 0.66 mumol/250 g, which corresponds to a molar ratio of bombesin to antagonist of approximately 1:12. This agrees well with antagonist to agonist potency ratios previously reported for inhibition of bombesin-stimulated amylase release from guinea pig pancreatic acinar cells and the growth of murine Swiss 3T3 cells, suggesting functional similarities between the receptor sites involved. Conversely, the analogue failed to inhibit bombesin inhibition of growth hormone release in the sodium pentobarbital-anesthetized rat model and was, in fact, a weak agonist at higher dose levels. This indicates either that this system is not particularly bombesin-specific or that bombesin receptor recognition and signaling requirements are substantially different in the gut and hypothalamus.     

Influence of streptozotocin-induced diabetes on growth hormone secretion in the rat

The effects of streptozotocin-induced diabetes on pituitary growth hormone (GH) content and release from incubated pituitaries were investigated. Male rats were made diabetic by a single injection of streptozotocin (65 mg/kg) and sacrificed by decapitation 15 days later. Pituitary GH concentration was significantly reduced in streptozotocin diabetic rats as compared to that observed in control animals. The amount of GH released from hemipituitaries was also lower in diabetic rats than in controls. Kinetic characteristics of somatostatin (SRIF) inhibition of GH release were not affected by the treatment. These results suggest that the decrease in plasma GH observed by some investigators in streptozotocin diabetic rats is probably due to a deficiency in GH storage and/or synthesis rather than a change in the responsiveness of pituitary GH cells to SRIF.     

Differential effects of arginine on growth hormone releasing hormone and insulin induced growth hormone secretion

OBJECTIVE: We wished to investigate the interaction of arginine, GHRH and insulin stress on GH secretion. DESIGN: Six healthy, non-obese volunteers underwent seven separate studes in random order. They received (1) insulin alone at 0 minutes; (2) GHRH alone at 15 minutes; (3) arginine alone at 0-30 minutes; (4) arginine at 0-30 minutes and GHRH at 15 minutes; (5) insulin at 0 minutes and arginine at 0-30 minutes; (6) insulin at 0 minutes, GHRH at 15 minutes and arginine at 0-30 minutes; (7) insulin at 0 minutes and GHRH at 15 minutes. MEASUREMENTS: GH and PRL were measured from -30 to 150 minutes at intervals of 15 minutes. RESULTS: Arginine increased GH responses to GHRH and decreased GH responses to hypoglycaemia, but this inhibitory effect of arginine was reversed by GHRH. CONCLUSIONS: The findings suggest that arginine-induced GH release is mainly mediated by a decrease in somatostatinergic tone, while GH responses to insulin stress are probably mediated by both an increase in hypothalamic GHRH release and inhibition of somatostatin.     

The effect of gastrin-releasing peptide on growth hormone secretion in the rat

Several investigators have reported gastrin-releasing peptide (GRP)-like immunostaining in several regions of the rat brain. The objective of this study was to determine the possible effects of this peptide on GH release. Porcine GRP was injected intraventricularly (third ventricular) in a volume of 2 microliters into ovariectomized female rats. A significant decrease in basal GH release, as evidenced by decreased plasma GH levels, was observed within 10 min which lasted for 90 min after the injection of 2 micrograms (0.7 nmol) GRP (P less than 0.001). In addition, all GH pulses were abolished during this time. In subsequent experiments, varying doses of GRP were administered, and human pancreatic GH-releasing factor (GRF) was injected iv at a dose of 0.1 microgram/kg 20 min later to determine the responsiveness of the pituitary. The minimal effective dose of GRP to lower plasma GH was approximately 10 ng (3.6 pmol); however, the GH-releasing action of GRF was blocked by even the lowest dose of the peptide tested (5 ng; 1.8 pmol). To determine if GRP had any direct action on the pituitary, overnight-cultured pituitary cells from ovariectomized animals were incubated for 1 h with GRP in various concentrations. There was a slight dose-dependent stimulation of GH release with concentrations of GRP ranging from 10(-9)-10(-6) M; however, the GH-releasing action was much less than that of GRF. To confirm the direct stimulatory effect of GRP on GH release, dispersed pituitary cells were perifused with medium containing 2 X 10(-6) M GRP. An immediate increase in GH release was observed in the perfusate. Since GRP has a direct stimulatory action to release GH in the pituitary, but ivt injection of the peptide inhibits GH release and blocks the response to GRF, we suggest that GRP may act on periventricular structures to release somatostatin, which reduces GH release and blocks the response to GRF.     

Evidence for a growth hormone releasing factor mediating alpha-adrenergic influence on growth hormone secretion in the rat

The effects of adrenergic receptor agonists on GH secretion were studied in adult, male rats pretreated with reserpine and somatostatin antiserum. Frequent blood samples were obtained from intra-aortic cannulae. Plasma GH was determined by radioimmunoassay. Reserpine (10 mg/kg i.p.) caused a complete suppression of the normal, pulsatile secretion of GH in all animals. Administration of somatostatin antiserum resulted in rapid elevations of plasma GH in reserpine-pretreated rats with peak levels at 30 min. GH levels then fell but remained slightly elevated for the duration of the sampling period (8 h). Apomorphine (0.5 mg/kg i.p.) had no effect on plasma GH levels, whereas clonidine (0.5 mg/kg i.p.) induced release of GH in both antiserum treated and control rats. The results indicate that the alpha-adrenergic influence on the secretion of GH is mediated not by inhibition of somatostatin release but rather by effects on the release of a GHRF.     

Chlorpromazine inhibits episodic growth hormone secretion in the rat.

Effects of chlorpromazine (CPZ) on plasma GH and prolactin levels were observed in conscious rats provided with chronic indwelling right atrial cannulae. The administration of CPZ (200 micrograms/100 g b.w. iv) suppressed episodic plasma GH burst and resulted in significant elevations of plasma prolactin levels. These were also observed in rats in which two types of hypothalamic deafferentation, i.e. anterior and complete, had been carried out. The data suggest that CPZ acts within the medial basal hypothalamus and inhibits episodic plasma GH secretion. In addition, it is inferred that catecholamines are involved in the generation of episodic plasma GH burst.     

Development of hypothalamic control of growth hormone secretion in the rat

The development of hypothalamic control of GH in the late prenatal and early postnatal periods in the rat was studied by employing a static system for the incubation of pituitaries. The basal secretion of GH into the medium after a 3-h incubation period showed a gradual increase from day 18 prenatally to day 1 postnatally. This was followed by a gradual decline in GH release on postnatal days 5 and 8. There was a sustained rise in the total pituitary GH content from prenatal day 18 to postnatal day 8. The percentage of the total GH that was released into the medium was high from fetal pituitaries and lower from neonatal pituitaries. TRH (100 ng/ml) stimulated GH secretion starting on prenatal day 21. This TRH effect persisted through day 8 postnatally. Hypothalamic extracts from fetuses and neonates stimulated the secretion of GH when coincubated with pituitaries of the same age and with adult male rat pituitaries. Similarly, adult male rat hypothalamic extract stimulated the secretion of GH from pituitaries of 1-day-old neonates. Pronase treatment of neonatal hypothalamic extract completely abolished its stimulatory effect on GH release. Incubation of 1-day postnatal pituitaries with cerebral cortical extract obtained from neonates of the same age did not alter the secretion of GH; however, cerebral cortical extract from adult males did cause a significant stimulation of GH release from the neonatal pituitaries. Somatostatin (100 ng/ml) failed to inhibit GH release by pituitaries until day 5 postnatally, but a 10-fold increase in the concentration of somatostatin significantly inhibited GH secretion from pituitaries of rats as early as day 21 prenatally. Coincubation of hypothalamic extract with the high concentration of somatostatin significantly attenuated the effect of the extract in stimulating GH release from pituitaries of 1-day-old rats. The results suggest that the high circulating levels of GH during the late prenatal and early neonatal periods are maintained by a combination of factors including the release of a hypothalamic peptidergic GH-releasing factor, the relative insensitivity of the pituitary to somatostatin, and changes in the relative size of storage vs. releasable pools of GH during development.     

In vivo effects of serotonergic agents on alpha-melanocyte-stimulating hormone secretion.

We used an in vivo pharmacological approach to investigate the potential influence of serotonin (5-HT) on peptide release from the intermediate lobe (IL) of the rat pituitary. Plasma levels of alpha-melanocyte-stimulating hormone (alpha-MSH) as well as the ultrastructural appearance of IL cells were used as indicators of IL secretory activity. Plasma beta-endorphin levels were also measured to assess the effectiveness of 5-HT-acting drugs. Intraperitoneal administration of 5-hydroxy-L-tryptophan, the synthetic precursor of 5-HT, dramatically elevated the content of 5-HT in the neurointermediate lobe but did not alter plasma titers of alpha-MSH. Treatment with the 5-HT reuptake blocker fluoxetine elevated plasma levels of beta-endorphin but not alpha-MSH. Administration of the 5-HT1/5-HT2 agonist MK-212 produced an elevation in both plasma alpha-MSH and beta-endorphin levels. Quantitative morphometry of IL cells at the ultrastructural level revealed that MK-212 treatment selectively causes depletion of electron-lucent but not electron-dense secretory granules from the cytoplasm of IL cells. Pretreatment with the selective D2 dopamine agonist apomorphine blocked MK-212-induced release of alpha-MSH but not beta-endorphin. Our results show that manipulation of 5-HT synthesis/reuptake does not affect release of alpha-MSH, but that direct activation of 5-HT receptors with the nonselective agonist MK-212 stimulates alpha-MSH release. The ability of apomorphine to block MK-212-induced release of alpha-MSH suggests a direct antagonism between dopaminergic and serotonergic regulation of alpha-MSH release.     

In vivo action of bombesin on exocrine pancreatic secretion in the rat: independent of cholecystokinin and cholinergic mediation

This study evaluates the effect of bombesin on pancreatic enzyme secretion in the rat and determines whether the stimulatory action of bombesin is mediated through the release of cholecystokinin (CCK) or via a cholinergic pathway. We performed in vivo experiments on conscious rats prepared with cannulae inserted in the pancreatic duct, in the external jugular vein, and in the duodenum. Intravenous infusion of bombesin stimulated pancreatic protein output in a dose-dependent fashion. Bombesin infused at 5 micrograms/kg/h stimulated pancreatic protein secretion from a basal of 12 +/- 5 to 42 +/- 10 mg/h. Infusion of proglumide (400 mg/kg/h) did not affect the stimulatory effect of bombesin on pancreatic protein secretion (38 +/- 5 mg/h). In contrast, infusion of proglumide abolished the pancreatic protein output elicited by intravenous infusion of CCK8 (500 ng/kg/h). This suggests that bombesin does not act through CCK to mediate exocrine pancreatic secretion. In separate studies we intravenously infused rats with atropine (100 micrograms/kg/h) prior to infusion with bombesin. Administration of atropine slightly decreased secretory volume but did not affect the action of bombesin. Combined administration of atropine and proglumide also did not affect pancreatic protein output stimulated by bombesin. Since infusion of neither proglumide nor atropine inhibited the stimulatory action of bombesin, the action of bombesin in the rat is probably direct and not through the release of CCK or via a cholinergic pathway.     

Antiserum to somatostatin-28 augments growth hormone secretion in the rat

To determine directly whether somatostatin-28 (S-28) physiologically regulates GH secretion, and what its contribution is relative to somatostatin-14 (S-14), we have compared the effects of immunoneutralization with a specific S-28 antibody with those of an anti S-14/S-28 serum on GH secretory dynamics in the rat. Plasma samples were obtained every 15 min for 7 h (1000-1700 h) from conscious, chronically cannulated rats after iv administration of 1 ml of one of the following sera: 1) rabbit anti S-28 (reacts with S-28, but not with S-14; maximum binding, 297 pmol/ml), 2) nonimmune rabbit serum, 3) sheep anti-S-14/S-28 serum (maximum binding, 9.4 nmol S-14 or S-28/ml), and 4) nonimmune sheep serum. A comparison of the mean integrated plasma GH levels during peak and trough periods showed significantly higher trough GH levels in both antibody-treated groups compared to those in the corresponding controls. In the anti-S-14/S-28-treated group, the elevation of trough period GH levels (40.5 +/- 3.5 ng/ml) represented a 3.25-fold increase (P less than 0.01) compared to the control value (12.5 +/- 1.5 ng/ml). In the anti-S-28-treated group, trough period GH levels (14 +/- 1.6 ng/ml) showed a 2.3-fold increase (P less than 0.01) compared to the control value (6.1 +/- 0.9 ng/ml). Mean peak period GH levels were 1.35-fold higher (P less than 0.05) than control values in the anti-S-14/S-28-treated group; anti-S-28 serum did not change mean peak GH levels. These data provide strong evidence that circulating S-28 (like S-14) physiologically regulates trough GH secretion and that the contribution of S-28 to GH inhibition is as important as that of S-14.     

Intrathecal injection of bombesin inhibits gastric acid secretion in the rat

Bombesin (100-500 ng) injected intrathecally (T9-10) inhibited gastric acid secretion stimulated by pentagastrin and the GABAB agonist baclofen in urethane-anesthetized rats and basal gastric acid secretion in conscious, pylorus-ligated rats. Peptide action was dose-related, occurred within 30 min, and lasted for greater than 1 h. Bombesin-induced inhibition of pentagastrin-stimulated gastric acid secretion was not altered by cervical cord transection. Intravenous infusion of the monoclonal bombesin antibody 2A11 abolished intravenous bombesin (10 micrograms/kg.h)-induced 33% inhibition of gastric response to pentagastrin but did not alter intrathecal bombesin (200 ng)-induced 38% inhibition of gastric response to pentagastrin. The inhibitory effect of bombesin (200 ng) on pentagastrin-stimulated gastric secretion was reversed by bilateral adrenalectomy or removal of celiac and mesenteric ganglia. Intrathecal injections of rat calcitonin gene-related peptide, neuromedin B, neuromedin U, and the stable substance P analogue (pGlu5, MePhe8, MeGly9)-substance P(5-11) did not alter pentagastrin-stimulated gastric acid secretion. These results demonstrate that bombesin injected into the subarachnoid space of the spinal cord inhibits vagally stimulated and pentagastrin-stimulated gastric secretion in rats. Bombesin action is peptide specific, exerted at a spinal site, and expressed through the sympathetic nervous system.     

Acute effects of ethanol on hepatic glycoprotein secretion in the rat in vivo

The in vivo effects of acute ethanol administration on the secretion of plasma glycoproteins and albumin were studied. Ethanol (6 g/kg body wt) was administered intragastrically to nonfasted rats, while control animals received isocaloric glucose. Hepatic protein and glycoprotein secretion in these animals was assessed by determining the rate of appearance of [3H]leucine- and [14C]fucose-labeled proteins into the plasma. Ethanol treatment decreased the secretion of both leucine- and fucose-labeled proteins into the plasma. This inhibition of secretion was accompanied by a corresponding increase in the hepatic retention of both leucine- and fucose-labeled immunoprecipitable secretory proteins. Pretreatment of the ethanol-treated animals with pyrazole, an inhibitor of ethanol metabolism, prevented the inhibition of hepatic protein secretion. These in vivo studies confirm our earlier in vitro findings that acute ethanol administration inhibits the secretion of plasma proteins, resulting in their hepatocellular retention.     

Further evidence that thyrotropin-releasing hormone participate in the regulation of growth hormone secretion in the rat

Effects of thyrotropin-releasing hormone (TRH) on growth hormone (GH) secretion were investigated in vivo (on intact or mediobasal hypothalamic lesioned rats tested under either anesthesia or free moving conditions) as well as in vitro (in incubation or perifusion systems of anterior pituitary tissue). The peptide induced a rapid, dose-dependent increase of plasma GH levels in free moving animals bearing an extensive lesion of the mediobasal hypothalamus including the median eminence. Under comparable conditions, TRH was ineffective in intact animals. After chloral hydrate anesthesia a GH response to TRH was recorded in both groups, but lesioned rats exhibited a better responsiveness to all doses tested. In vitro TRH increased GH release from incubated or perifused pituitaries sampled from both intact and lesioned rats in a transient and concentration-dependent manner. A similar effect was obtained with the (3 Me His2) analogue of TRH. These findings indicate that TRH can affect GH secretion at the pituitary level under specific experimental conditions and support the hypothesis that either peripheral hormones or other, still unidentified hypothalamic neurohormones may modulate this effect.