Effect of galanin on the growth hormone response to growth hormone-releasing hormone in acromegaly.

Galanin enhances growth hormone (GH)-releasing hormone (GHRH)-stimulated GH secretion in normal man. In acromegaly, circulating GH levels are increased and the GH response to GHRH may be exaggerated. Galanin has been recently shown to decrease circulating GH levels in acromegaly. The aim of our study was to investigate the effects of galanin on the GH response to GHRH in acromegalic subjects. Five acromegalic patients (three men and two women) and seven healthy adult subjects (five men and two women) were studied. GHRH-induced GH secretion was evaluated during a 40-minute intravenous (IV) infusion of saline (100 mL) or porcine galanin (12.5 micrograms/min in 100 mL saline). In normal subjects, delta GH levels after GHRH+porcine galanin administration (47 +/- 7.5 micrograms/L) were significantly higher in comparison to levels obtained with GHRH+saline (21.7 +/- 3.5 micrograms/L, P < .05). In acromegalic patients, GH responses to GHRH (delta GH, 18.8 +/- 8.6 micrograms/L) were not altered by galanin infusion (delta GH, 17.6 +/- 5 micrograms/L). Our results give the first evidence that the same dose of galanin that induces a significant enhancement of the GH response to GHRH in normal subjects has no effect on the GH response to GHRH in acromegalic patients. It can be hypothesized that galanin may interact at the pituitary level with its own receptors expressed by somatotropes independent of GHRH. Failure of galanin to enhance GH response to GHRH in acromegalic patients could be due to a change in function of the galanin receptor on GH-secreting adenomatous cells.     

Decreased galanin mRNA levels in growth hormone-releasing hormone neurons after perinatally induced growth retardation

Intrauterine growth retardation (IUGR) is associated with persistent postnatal growth retardation accompanied by dysfunction of the hypothalamic components of the growth hormone (GH) axis. At the adult stage, this is reflected by increased somatostatin (SS) and decreased neuropeptide Y (NPY) mRNA levels, whereas the GH-releasing hormone (GHRH) mRNA levels are normal and the output of GH remains unchanged. To extend our insight into the hypothalamic control of GH secretion in growth retarded rats, we determined galanin (GAL) mRNA levels at the adult stage of perinatally malnourished (i.e. IUGR and early postnatally food restricted) rats. Analyses included comparison of GAL mRNA levels in GHRH neurons in perinatally malnourished adult rats using a semi-quantitative double labeling in situ hybridization technique. We report that IUGR is accompanied by a 60% decrease in GAL mRNA levels in all GHRH neurons in the male IUGR group whereas a tendency towards a decrease was observed in the male early postnatally food restricted (FR) group. These effects became more pronounced when the analysis was restricted to GHRH neurons coexpressing GAL mRNA i.e. decreased GAL mRNA levels were seen in both male and female IUGR rats and in FR males. These data show that GAL mRNA levels in GHRH neurons are persistently decreased after perinatal malnutrition. Taking these results together with our previous data on SS, NPY and GHRH mRNA levels, we can conclude that IUGR leads to a reprogramming of the hypothalamic regulation of GH secretion.     

The effect of galanin on growth hormone-releasing factor and somatostatin release from median eminence fragments in vitro.

Galanin has been reported to stimulate secretion of GH in humans and rats. Thus, to investigate whether the effect of galanin on GH release is the result of either a stimulation of GH-releasing factor (GRF) and/or an inhibition of somatostatin (SRIF) release, we have evaluated the action of galanin on the release of SRIF and GRF from median eminence (ME) fragments in vitro. The MEs from adult male rats were incubated in Krebs-Ringer bicarbonate-glucose buffer, pH 7.4, at 37 degrees C, in an atmosphere of 95% O2, 5% CO2 with constant shaking for 30 min. Medium was discarded and replaced by medium containing various concentrations of galanin (10(-10)-10(-7) M). Galanin stimulated SRIF and GRF release in a dose-related manner. This effect was significant at concentrations varying from 10(-8) to 10(-7) M. To determine the mechanism by which galanin stimulated SRIF and GRF release, MEs were incubated with pimozide (dopaminergic blocker), phentolamine (alpha-adrenergic blocker) or naloxone (opioid blocker), at concentrations of 10(-6) M, and the effect of galanin was then evaluated. Phentolamine and naloxone did not alter the stimulatory effect of galanin, but when galanin was tested with pimozide, the galanin-induced release of SRIF and GRF was blocked. To determine whether the effect of galanin is mediated through D-1 and/or D-2 dopamine receptors, selective antagonists of D-1 (SCH 23390) and D-2 receptors (domperidone) were used (10(-7) M) in the presence of galanin (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

CD36 mediates the cardiovascular action of growth hormone-releasing peptides in the heart

Growth hormone-releasing peptides (GHRPs) are known as potent growth hormone secretagogues whose actions are mediated by the ghrelin receptor, a G protein-coupled receptor cloned from pituitary libraries. Hexarelin, a hexapeptide of the GHRP family, has reported cardiovascular activity. To identify the molecular target mediating this activity, rat cardiac membranes were labeled with a radioactive photoactivatable derivative of hexarelin and purified using lectin affinity chromatography and preparative gel electrophoresis. A binding protein of M(r) 84 000 was identified. The N-terminal sequence determination of the deglycosylated protein was identical to rat CD36, a multifunctional glycoprotein, which was expressed in cardiomyocytes and microvascular endothelial cells. Activation of CD36 in perfused hearts by hexarelin was shown to elicit an increase in coronary perfusion pressure in a dose-dependent manner. This effect was lacking in hearts from CD36-null mice and hearts from spontaneous hypertensive rats genetically deficient in CD36. The coronary vasoconstrictive response correlated with expression of CD36 as assessed by immunoblotting and covalent binding with hexarelin. These data suggest that CD36 may mediate the coronary vasospasm seen in hypercholesterolemia and atherosclerosis.     

Immunohistochemical identification of galanin and growth hormone-releasing factor-containing neurons projecting to the median eminence of the rat.

Through the combined demonstration of the retrograde transport of True blue and the immunohistochemical staining of galanin (GAL), the GAL neurons that project to the median eminence were identified. Moreover, the distribution of GAL and growth hormone-releasing factor (GRF) was analyzed in the arcuate nucleus (ARC) with an elution-restaining procedure. Following the injection of True blue into the median eminence, GAL-positive cells labeled with True blue were found mainly in the ARC. In addition, a few GAL neurons in the periventricular, the paraventricular and the supraoptic nuclei were labeled with True blue. The elution-restaining results revealed that many retrogradely labeled GAL neurons in the ARC contained GRF. These findings suggest that a subpopulation of GAL neurons in the ARC sending axons to the median eminence produces, stores and releases a GRF-like peptide.     

The role of growth hormone-releasing factor and somatostatin on somatic growth in rats

The role of GH-releasing factor (GRF) in regulating somatic growth was studied in rats by passively immunizing animals with antisera raised against rat GRF. GRF antiserum (GRF-ab) treatment significantly inhibited the normal increase in body weight observed in both young male and female rats. Similar studies conducted in neonatal rats (days 1-23 of age) also demonstrated that GRF is critical in regulating neonatal growth. The biological half-life of the GRF-ab was determined to be between 2.5 and 3.5 days in these animals. The effects of GRF-ab and somatostatin antiserum (SRIF-ab) treatment on serum GH concentrations were also studied in neonatal rats. Between 1 and 20 days of age, GRF-ab treatment significantly decreased serum GH concentrations compared to those in control animals. From days 1-10 of age, SRIF-ab treatment had no effect on GH concentrations, whereas on days 15 and 20 of age, SRIF-ab treatment significantly increased GH concentrations. The results demonstrate that hypothalamic GRF is critical in regulating serum GH concentrations and somatic growth in neonatal and young animals. They also suggest that the role of SRIF in regulating GH concentrations is not established until sometime after 10 days of age.     

Cholinergic modulation of growth hormone secretion induced by galanin in rats.

The effects of cholinergic modulation by hexamethonium (HEX), tetraethylammonium (TEA), pirenzepine (PIR) and neostigmine on growth hormone (GH) secretion induced by galanin (GAL) were investigated in awake, freely moving male rats. Intracerebroventricular (i.c.v.) injection of GAL (0.6 nmol) caused a marked increase in plasma GH levels. Pretreatment with HEX (3 or 15 mg/kg BW), TEA (15 mg/kg BW) or PIR (0.5 mg/kg BW) significantly reduced the GAL-induced GH secretion. Pretreatment of animals with neostigmine (0.05 mg/kg BW) or with a specific antisomatostatin antiserum (10 microliters of 1:10 diluted with saline, i.c.v.) reversed the inhibition of GAL-induced GH secretion by HEX. These findings suggest that both nicotinic and muscarinic cholinergic mechanisms interact with the galanin-induced GH secretion by modulating hypothalamic somatostatin release.     

Effect of galanin on growth hormone-releasing hormone-stimulated growth hormone secretion in adult patients with nonendocrine diseases on long-term daily glucocorticoid treatment.

Glucocorticoids are thought to inhibit growth hormone (GH) secretion through an enhancement of endogenous somatostatin tone. The aim of our study was to evaluate the effect of galanin, a neuropeptide that stimulates GH secretion, on GH-releasing hormone (GHRH)-induced GH secretion in adult patients with nonendocrine diseases who were under daily immunosuppressive glucocorticoid therapy. Six normal subjects (four men, two women) and seven steroid-treated subjects (three men, four women) were studied. GHRH-induced GH secretion was evaluated during a 40-minute intravenous (i.v.) infusion of saline or porcine galanin (12.5 micrograms/min). During saline infusion, steroid-treated patients showed a blunted GH response to GHRH (GH peak, 8.1 +/- 2.8 micrograms/L), as compared with normal subjects (GH peak, 23.8 +/- 3.9 micrograms/L). During galanin infusion, the GH response to GHRH was significantly enhanced (GH peak, 46.6 +/- 9.4 micrograms/L, P less than .05), as compared with saline infusion in normal subjects. In contrast, galanin infusion did not enhance the GH response to GHRH (GH peak, 16.6 +/- 6.5 micrograms/L), as compared with saline infusion in steroid-treated patients. The area under the GH-response curves was also significantly (P less than .05) lower in steroid-treated subjects, as compared with normal subjects. Thus, galanin failed to normalize or enhance the GH response to GHRH in patients treated long-term with glucocorticoids. It can be hypothesized that galanin does not elicit GH secretion by decreasing hypothalamic somatostatin tone.     

The effect of cholinergic blockade on the growth hormone response to galanin in humans

The effect of cholinergic blockade with pirenzepine or atropine on growth hormone (GH) release after galanin administration was investigated in five normal male subjects. The mean peak GH response to an infusion of galanin (40 pmol/kg/min for 40 minutes) was significantly reduced from 17.2 mU/L to 2.9 mU/L (P less than .001) with prior administration of pirenzepine (30 mg IV). When galanin was infused at a higher dose (80 pmol/kg/min), this suppression of release by pirenzepine was partially overcome, with GH rising to a mean peak response of 8.0 mU/L (P less than .05). Repeated administration of atropine (two bolus doses of 0.6 mg IV) also failed to abolish the GH response to this higher dose of galanin in two subjects. It has been proposed that cholinergic pathways control GH release via somatostatin, and this study suggests that galanin may also act by modulating hypothalamic somatostatinergic tone either directly or by facilitating cholinergic transmission.     

Diminished pituitary responsiveness to growth hormone-releasing factor in aging male rats

The pattern of GH secretion undergoes substantial changes in the aging rat, resulting in decreased daily secretion of GH. In this study, the pituitary responsiveness to GH-releasing factor (GRF) was examined in young (2- to 5-month old) and aging (14- to 18-month old) male rats. In vivo studies were performed under sodium pentobarbital anesthesia. After injection of 250 ng GRF/100 g BW, young rats experienced more GH secretion [peak level, 544.5 +/- 209.5 (+/- SEM) ng/ml] than did 18-month-old rats (89.3 +/- 13.7 ng/ml). To investigate the locus of this insensitivity to GRF, anterior pituitary cells from young and aging rats were dispersed and placed in primary culture. While basal GH secretion from the cultured pituitary cells was similar in the two groups (49.7 +/- 3.5 vs. 47.8 +/- 2.7 ng/ml X 4 h for the 2- and 18-month old rats, respectively), the GH-releasing ability of GRF was partially but significantly impaired in cells derived from both 14- and 18-month old rats; 100 nM GRF stimulated the release of 96.7 +/- 1.8 ng/ml X 4 h in the 18-month old rats as opposed to 115.0 +/- 6.0 (P less than 0.05) ng/ml X 4 h in the 2-month-old rats. Since GRF stimulates GH release through the activation of adenylate cyclase, intracellular cAMP levels were measured in the cultured pituitary cells. GRF stimulated 65% less intracellular cAMP accumulation in the 18-month-old rats. In 14-month-old rats, the ability of forskolin and (Bu)2 cAMP to release GH was impaired, while phorbol ester-elicited GH secretion was unchanged. In conclusion, the GH response to GRF is blunted in aging rats. While much of the insensitivity to GRF may be mediated by the increased somatostatin tone reported in aging rats, a diminished pituitary cAMP response to GRF may also be an important etiological factor in the hyposomatotropinemia of the aging male rat.     

Withdrawal of endogenous somatostatin induces secretion of growth hormone-releasing factor in rats

Secretion of GH occurs in episodic bursts under the dual control of two hypothalamic peptides, GH-releasing factor (GRF) and GH-inhibiting factor (somatostatin, SRIF). Recent studies in rats suggest that episodic GH secretion is generated by the periodic release of GRF, which is associated with the simultaneous withdrawal of SRIF secretion. To test the possibility that GRF discharge is functionally coupled with the withdrawal of SRIF, we investigated whether acute withdrawal of SRIF can induce GRF release by the rat hypothalamus using highly specific antisera against SRIF and rat GRF. In conscious unrestrained rats, i.v. administration of SRIF antiserum at the period of the GH trough induced a rapid onset of the GH secretory surge with a peak value of 309 +/- 67 micrograms/l (mean +/- S.E.M.) 30 min after injection. Pretreatment with antiserum to rat GRF resulted in a approximately 83% suppression of the GH surge induced by SRIF antiserum without affecting the trough GH values. GRF antiserum also significantly inhibited the spontaneous GH surge. In urethane-anaesthetized rats, as in conscious rats, an acute phasic GH release was caused by SRIF antiserum despite the interference of anaesthesia with spontaneous GH secretion. A further surge-like GH secretion was not restored during the next several hours, however, with the GH secretory profile being characterized by a tonic increase in the baseline levels of GH. In the anaesthetized rat antiserum to rat GRF, having no effect on basal GH levels, similarly inhibited by approximately 66% the acute GH surge induced by SRIF antiserum and decreased by about 30% the later sustained rise in GH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations of pituitary growth hormone-releasing factor binding sites in aging rats.

This study was designed to determine whether growth hormone (GH)-releasing factor (GRF) binding sites are altered in parallel to the diminution of GH secretion that occurs in aging. Using [125I-Tyr10] hGRF (1-44)NH2 as radioligand, we performed cold saturation studies in 2, 8, 14 and 18-month-old Sprague-Dawley male rat pituitary homogenates. In young rats (2 months), analysis by Ligand revealed the presence of two distinct classes of binding sites (KDs = 0.86 +/- 0.15 and 400 +/- 210 nM; BMAXS = 269 +/- 47 fmol and 42 +/- 19 pmol/mg protein, respectively). In 8 and 14-month old rats, there was a concomitant decrease in capacity of the high affinity class of sites (P less than 0.01) and increase in capacity of the low affinity class of sites (P less than 0.05). In parallel, a transient increase in affinity of the high affinity class of sites was observed in 14-month-old rats (P less than 0.05). In old rats (18 months), the data were no longer statistically analyzed with a two site-model, indicating a severe blunting of the high affinity sites. As the GRF-induced GH secretion is still not diminished at 8 month of age in these animals, our results indicate: 1) that alterations of GRF binding sites precede the GH impairment, thus probably participate in the initiating of this phenomenon and 2) that the biological actions of GRF on GH secretion are likely mediated by the high affinity class of sites.     

Pituitary growth hormone response in rats during a 24-hour infusion of growth hormone-releasing factor

The pituitary GH response during a 24-h iv infusion of GH-releasing factor (hpGRF-44; 15 micrograms/h) and to a subsequent bolus injection of hpGRF-44 (2 micrograms) was studied in conscious, freely moving male rats pretreated with antiserum against somatostatin. Within 2 h of the initiation of the hpGRF-44 infusion, plasma GH concentrations rose from 169 +/- 16 to 2465 +/- 307 (+/- SE) ng/ml. By 6 h, plasma GH concentrations began to fall. They decreased slowly and reached a nadir of 490 +/- 107 ng/ml by 12 h. Rats infused for 24 h with hpGRF-44 failed to respond to a 2-micrograms bolus injection (iv) of hpGRF-44, whereas rats infused for 24 h with saline responded with a normal increase in plasma GH. The pituitary GH content of rats treated with saline was significantly greater than that of rats treated with hpGRF-44. These results demonstrate that the capacity of the pituitary to respond to GRF can be exhausted after the chronic administration of hpGRF-44 and that this lack of response appears to be due, in part, to a depletion of pituitary stores of GH.     

Disappearance half-life times of exogenous and growth hormone-releasing factor-stimulated endogenous growth hormone in normal rats

This study was performed to determine the disappearance half-life times of endogenous and exogenous rat GH in conscious normal rats and to compare these with the decay characteristics of GH at the end of spontaneous normal bursts. The endogenous half-life was determined in five rats by giving an i.v. injection of rat GH-releasing factor followed after 10 min by an i.v. injection of long-acting somatostatin analogue (octreotide) and taking blood samples for 85 min. The half-lives (mean +/- S.E.M.) were 3.4 +/- 0.4 min and 13.2 +/- 1.1 min for the first and second exponential respectively as determined by bi-exponential analysis. The exogenous GH half-life was determined in ten rats by giving i.v. octreotide followed after 10 min by i.v. rat GH and sampling for 85 min. The half-lives of exogenous GH were 3.3 +/- 0.2 min and 17.5 +/- 1.4 min by bi-exponential analysis and there was no significant difference between the half-lives of endogenous and exogenous GH. The half-life of the decline of GH levels at the end of spontaneous bursts in nine rats was 14.4 +/- 0.9 min, not different from the half-life of endogenous GH, the secretion of which was terminated by octreotide. This suggests that the end of spontaneous GH bursts is marked by sudden cessation of GH release and may provide an indication of the rapidity of change in the levels of the underlying hypothalamic hormones which control GH release.     

Involvement of growth hormone-releasing factor in growth hormone secretion induced by gamma-aminobutyric acid in conscious rats

Intracerebroventricular (icv) injection of gamma-aminobutyric acid (GABA) (10 mumol/rat) resulted in an increase in plasma GH in conscious freely moving rats pretreated with normal rabbit serum (0.5 ml/rat, iv). Rabbit antiserum specific for rat GH-releasing factor (GRF) (0.5 ml/rat, iv) abolished GH release induced by GABA in these animals. Rabbit anti-rat GRF serum also blunted GH release induced by a Met5-enkephalin analog, FK33-824 (10 micrograms/100 g BW, iv) in conscious rats. Considering our previous findings that rat GH release induced by FK33-824 was blunted by GABA antagonists (Endocrinology 103:1033, 1981), these results suggest that GH secretion induced by opioid peptides via GABAergic mechanisms is mediated, at least in part, by hypothalamic GRF in the rat.     

Ovarian role in the modulation of pituitary responsiveness to growth hormone-releasing hormone in rats.

The influence of ovarian function on the pituitary responsiveness to growth hormone-releasing hormone (GHRH) was studied by measuring GH levels in serum before and after the injection of GHRH 1-29 NH2 (5 micrograms/kg i.v.) in female Wistar rats under sodium pentobarbital anesthesia (50 mg/kg i.p.). In the first experiment, GHRH was administered to 90-day-old females in different phases of the estrous cycle (in the morning and afternoon of proestrus and in the morning of estrus, diestrus 1 and diestrus 2). In the second experiment, females were ovariectomized or sham-operated on day 23 and analyzed on days 30, 45, 60 and 90. Pituitary growth hormone (GH) content in females ovariectomized or sham-operated on day 23 was also analyzed at different ages. In the third experiment, females ovariectomized or sham-operated on day 83 were tested on day 90. Results showed that (a) pituitary responsiveness to GHRH changes throughout the estrous cycle is maximal in diestrus and minimal in proestrus; (b) GHRH stimulated GH secretion in intact and ovariectomized females on days 45, 60 and 90, but not on day 30; (c) the age increase of GH responsiveness to GHRH administration was slightly reduced in animals ovariectomized on day 23; (e) the age increase of pituitary GH content was similar in control and ovariectomized females; and (d) ovariectomy on day 83 reduced pituitary responsiveness to GHRH more effectively than ovariectomy on day 23.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of atenolol on the growth hormone response to growth hormone-releasing hormone in obese children.

We have evaluated the effect of acute administration of atenolol, a selective beta-adrenergic antagonist, on the GH response to GHRH in nine obese children and in eight age-matched controls. The GH response to GHRH (1-29, 1 microgram/kg iv), evaluated both as the GH peak and as integrated area under the curve, was significantly lower in the obese children than in the controls. Pretreatment with atenolol (50 or 100 mg orally in subjects with body weight less than or greater than 40 kg, respectively, administered 120 min before the GHRH injection) significantly increased the GH response to GHRH in the obese subjects, such that their mean peak GH levels and mean integrated area under the curve after atenolol plus GHRH were similar to those of the control children after GHRH. Also in control children, atenolol caused a significant augmentation of the GH response to GHRH. Mean peak GH levels and mean integrated area under the curve after atenolol plus GHRH were significantly higher in the controls than in the obese children given the same treatment. These data show that inhibition of central beta-adrenergic receptors counteracts the blunted GH response to GHRH present in the obese children. In view of the alleged mechanism of action of beta-adrenergic blockade (inhibition of endogenous SRIH release), our data suggest that the somatostatinergic system is intact in obesity, and that the suppressed GH secretion is due to other causes.     

Post-somatostatin rebound secretion of growth hormone is dependent on growth hormone-releasing factor in unrestrained female rats

To examine the characteristics of GH secretion following the termination of the infusion of somatostatin, unrestrained adult female Wistar rats were subjected to repeated infusions of somatostatin separated by 30-min control periods. When somatostatin was infused for 150 min at a dose of 3, 30 or 300 micrograms/kg body wt per h, the magnitude of the rebound GH secretion increased in a dose-dependent manner. The infusion of somatostatin at a dose of 300 micrograms/kg body wt per h for 60, 150 or 240 min progressively augmented the size of the rebound GH secretion. When an antiserum to rat GH-releasing factor (GRF) was injected i.v. 10 min before the end of the infusion, the peak amplitude of the rebound GH secretion (300 micrograms/kg body wt, 150 min) was reduced to less than 20% of that of control rats. The rebound GH secretion (300 micrograms/kg body wt per h, 150 min) was augmented by a bolus injection of human GRF (1 microgram/kg body wt). The combined effect of the end of infusion of somatostatin and a bolus injection of GRF on the amount of GH secreted was additive. The plasma GH response to GRF was completely inhibited when human GRF (3 micrograms/kg body wt per h) and somatostatin (300 micrograms/kg body wt per h) were infused simultaneously for 150 min. The magnitude of the rebound GH secretion following the termination of the co-administration was larger than that following the somatostatin infusion alone, but this rebound was not enhanced by a bolus injection of human GRF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effect of galanin on basal and pentagastrin-stimulated gastric acid secretion in rats.

The dose-dependent effects of galanin on basal and pentagastrin-stimulated gastric acid secretion in pentobarbital-anesthetized rats were examined. Intravenous infusion of galanin at doses of 0.31, 0.62, 1.25, 1.87, 3.11, and 6.22 nmol/kg-1/h-1 into pentagastrin-stimulated rats produced a diminution in gastric acid secretion which was maximal (54.7%) at the level of the 1.87 nmol/kg-1/h-1 dose. Furthermore, the effect was biphasic, since both lower and higher doses of peptide were less effective. At the optimum concentration of 1.87 nmol/kg-1/h-1 galanin also inhibited basal gastric acid secretion. We conclude that endogenous galanin might be involved in the physiologic regulation of gastric acid secretion.