SRIF Release Research Articles

Evidence thatα2-Adrenergic Pathways Play a Major Role in Growth Hormone (GH) Neuroregulation:α2-Adrenergic Agonism Counteracts the Inhibitory Effect of Muscarinic Cholinergic Receptor Blockade on the GH Response to GH-Releasing Hormone, whileα2-Adrenergic Blockade Diminishes the Potentiating Effect of Increased Cholinergic Tone on such Stimulation in Normal Men*

The aim of this study was to investigate the interrelationships between alpha 2-adrenergic and cholinergic pathways in the control of hypothalamic somatostatin (SRIF) secretion in humans. In eight normal volunteers subjects we compared the pattern of GHRH-induced GH release to that elicited by similar challenge given 60 min after a pretreatment with drugs affecting alpha 2-adrenergic and muscarinic cholinergic neurotransmission. In a control study, synthetic GHRH [GRF-(1-29); 1 microgram/kg, iv] was administered 60 min after giving placebo. In other experiments, the administration of atropine (1 mg, im), or clonidine (0.300 mg, orally), or atropine plus clonidine, or pyridostigmine (120 mg, orally), or yohimbine (30 mg, orally), or pyridostigmine plus yohimbine, at 0 min was followed by GHRH administration 60 min later. The administration of both clonidine and pyridostigmine significantly (P less than 0.01) enhanced the GH responses to GHRH compared to those elicited by this challenge when given after placebo. Conversely, atropine pretreatment significantly (P less than 0.01) blocked the GH response to GHRH challenge, whereas yohimbine did not significantly affect it. When atropine and clonidine were given together, the inhibitory effect of the former was overcame and mean GHRH-elicited GH peak response was significantly (P less than 0.05) higher than that in the control study. In contrast, pretreatment with yohimbine significantly (P less than 0.05) blunted the pyridostigmine-induced enhancement of GHRH-elicited GH release. These data confirm our previous postulate suggesting that the stimulatory effect of clonidine on GH release is mainly exerted by inhibiting the hypothalamic SRIF release. Moreover, the effect of cholinergic neurons on SRIF release seems to be, at least in part, dependent on alpha 2-adrenergic pathways. Based on these data, it can be proposed that the alpha 2-adrenergic system plays a major role in the control of hypothalamic SRIF release, and hence in GH neuroregulation, whereas the muscarinic cholinergic system would participate in such regulation by modulating the functional activity of the former.

Sexual Dimorphism of Somatostatin and Growth Hormone-Releasing Factor Signaling in the Control of Pulsatile Growth Hormone Secretion in the Rat*

A striking sexual dimorphism exists in the pattern of GH secretion and rate of somatic growth; however, the mechanism(s) mediating this sex difference is unknown. To elucidate the physiological roles of the hypothalamic neuropeptides, somatostatin (SRIF) and GRF, and their interrelation, in generating the sexually dimorphic GH secretory pattern we examined: 1) GH responsiveness to exogenous GRF and 2) the effects of immunoneutralization of endogenous SRIF and GRF on GH secretory dynamics, in free-moving male and female rats. In males, the GH response to 1 microgram rat(r)GRF(1-29)NH2 iv was significantly greater at peak compared to trough times of GH secretion (925.2 +/- 250.8 vs. 95.6 +/- 27.8 ng/ml; P less than 0.02), the latter known to be due to antagonization by the cyclic increased release of endogenous SRIF. In contrast, females failed to exhibit a time-dependent difference in GH responsiveness to GRF. Passive immunization with a specific antiserum to SRIF in males resulted in significant elevation of GH nadir levels but had no effect on GH peak amplitude. In contrast, immunoneutralization of endogenous SRIF in females caused a marked augmentation of plasma GH levels at all time points; there was a significant increase in GH peak amplitude (171.3 +/- 39.9 vs. 67.5 +/- 11.3 ng/ml; P less than 0.05), GH nadir (18.3 +/- 2.7 vs. 5.8 +/- 1.1 ng/ml; P less than 0.01) and mean 6-h plasma GH level (78.7 +/- 4.1 vs. 33.1 +/- 5.8 ng/ml; P less than 0.001), compared to normal sheep serum-treated controls. These results indicate that the pattern of hypothalamic SRIF secretion in females does not follow the male-like ultradian rhythm. Passive immunization with a specific antiserum to GRF obliterated spontaneous GH pulses in both sexes. Moreover, in females, anti-GRF serum attenuated GH nadir levels (4.3 +/- 1.7 vs. 21.4 +/- 3.5 ng/ml; P less than 0.01) indicating a physiological role for GRF in maintaining the elevated basal GH level of females, in addition to its important role in generating the episodic GH pulses. Taken together, these findings provide support for the hypothesis that, in female rats, the pattern of hypothalamic SRIF secretion into hypophyseal portal blood is continuous, rather than cyclical, as in the male; whereas in the case of GRF secretion, in addition to steady-state release which occurs at a higher level in females than males, there is also episodic GRF bursting which does not follow a specific rhythm, as in the male.(ABSTRACT TRUNCATED AT 400 WORDS)

On the Actions of the Growth Hormone-Releasing Hexapeptide, GHRP*

GH-releasing peptide (His-DTrp-Ala-Trp-DPhe-Lys-NH2 or GHRP) releases GH by a unique and complementary dual site of action on the hypothalamus and pituitary. These effects are mediated via non-GH-releasing hormone (non-GHRH) and nonopiate receptors in rats. Select types of opiates are known to release GH by a hypothalamic site of action, and thus, the dermorphin heptapeptide and benzomorphan opiate agonist 2549 used in this study presumably act on the hypothalamus to release GH. Neither dermorphin nor 2549 released GH or augmented the GH responses of GHRP or GHRH in vitro by a direct pituitary action, while GHRH antiserum inhibited the GH response of both dermorphin and 2549 in vivo. Evidence indicates that these opiates and GHRP administered together synergistically release GH, demonstrating the independent action(s) of GHRP and the opiates. Present data indicate that one of the major differences in the actions of dermorphin, 2549, and GHRP is the inhibition of somatostatin (SRIF) release by the opiates but not by GHRP. Although the actions of dermorphin, 2549, and GHRP on GH release are GHRH dependent, release of endogenous GHRH does not explain how GH is released synergistically by the combination of these peptides. It is proposed that dermorphin/2549 synergistically release GH with GHRP or GHRH because these opiates inhibit SRIF release. Since the GHRP plus GHRH synergistic GH release was not explained by inhibition of SRIF or stimulation of GHRH, an alternative mechanism is proposed to explain how GHRP synergistically release GH in combination with GHRH. The complementary, rather dramatic synergistic interaction of GHRP, GHRH, and dermorphin or GHRP, GHRH, and 2549 in releasing GH again strongly supports the independent actions of these compounds.

Effect of restricted feeding on the relationship between hypophysial portal concentrations of growth hormone (GH)-releasing factor and somatostatin, and jugular concentrations of GH in ovariectomized ewes.

These studies characterized the secretion of GH-releasing factor (GRF) and somatostatin (SRIF) into the hypophysial portal circulation in ewes after long term restricted feeding. In addition, we examined the temporal relationship between the concentrations of these two hypothalamic peptides in portal blood and the concentration of GH in jugular blood. Six sheep were fed 1000 g hay/day (normal feeding) and 6 sheep were fed 400-600 g hay/day (restricted feeding). This resulted in a wt loss of 35% in restricted animals compared with 6% in control animals after 20 weeks. Fluctuations in portal levels of GRF indicated a pulsatile pattern of secretion with approximately 60% of pulses coincident with, or immediately preceding, a GH pulse. Similarly, 65% of GH pulses were associated with GRF pulses. Restricted feeding increased (P less than 0.01) mean ( +/- SEM) plasma GH levels (9.8 +/- 1.4 vs. 2.9 +/- 0.6 ng/ml) and mean GH pulse amplitude (7.9 +/- 1.8 vs. 2.8 +/- 0.3 ng/ml) but did not affect mean GH pulse frequency (6.0 +/- 1.1 vs. 5.7 +/- 1.1 pulses/8 h). The level of feeding had no effect on mean portal concentration of GRF (restricted: 5.5 +/- 0.8, normal: 6.6 +/- 1.4 pg/ml), GRF pulse amplitude (14.7 +/- 2.3 vs. 13.5 +/- 0.7 pg/ml), or GRF pulse frequency (5.3 +/- 1.1 vs. 6.7 +/- 0.9 pulses/8 h). Portal concentrations of SRIF in sheep on a restricted diet were half (P less than 0.01) those of sheep fed a normal diet (10.2 +/- 2.3 vs. 19.6 +/- 1.6 pg/ml). Pulses of SRIF were not significantly associated with changes in GH or GRF concentrations. These data indicate a functional role for hypothalamic GRF in initiating GH pulses. Furthermore, the increase in GH secretion in underfed sheep was most probably due to a decrease in the release of SRIF into hypophysial portal blood. Restricted feeding had no affect on GRF secretion, but because of the reduced exposure of the pituitary gland to SRIF, it is possible that responsiveness to GRF is enhanced.

Rat growth hormone-releasing factor stimulates cyclic GMP formation and phosphatidylinositol metabolism in the median eminence

The effects of rat growth hormone releasing factor (rGRF) on somatostatin (SRIF) secretion, cyclic nucleotide production and phosphatidylinositol metabolism were investigated in the median eminence (ME), using an in vitro system. Medium was discarded and replaced by medium containing various concentrations of rGRF or rGRF plus epinephrine (E, 6×10 −7M). rGRF had no effect on basal or E-stimulated release of cAMP. In the same experiments rGRF markedly stimulated SRIF release. These results suggested that cAMP is not involved in the stimulatory effect of GRF on SRIF release. However, GRF significantly stimulated release of both SRIF and cGMP in a dose-related manner. Maximal stimulation was observed at 10 −10M GRF (p<0.005) which also produces maximal SRIF release. 2′0-monobutyrylguanosine 3′5′ cyclic phosphate (mbcGMP, 10 −11 to 10 −10M) stimulated SRIF release from ME fragments (p<0.001 at 10 −10M) whereas the control, sodium butyrate (10 −6M), had no effect. GRF caused significant elevation of 30.6% in the concentration of labelled inositol phosphates ([ 3H]-IPs) in the ME. These data indicate that GRF stimulation of SRIF release is accompanied by increased cGMP production and phosphatidylinositol (PI) metabolism but does not alter cAMP production. Because mbcGMP can directly stimulate SRIF release, we suggest that GRF causes a receptor-mediated increase in the metabolism of phosphatidylinositol and cGMP formation. These actions therefore may be among the early metabolic events in the mechanism of GRF-stimulated SRIF release from the ME.

Roles of Somatostatin and Growth Hormone-Releasing Factor in Ether Stress Inhibition of Growth Hormone Release

In order to evaluate the release of somatostatin (SRIF) and growth hormone-releasing factor (GRF) into the pituitary gland in response to ether stress, a push-pull perifusion (PPP) technique has been used in freely moving rats. Push-pull cannulae (PPC) were implanted into the anterior pituitary (AP) glands of male rats. After a 7-day recovery period the rats were fitted with indwelling jugular catheters. The next day the animals were subjected to PPP of the AP during 1 h followed by ether stress (2 min) and another hour of perifusion. The perifusion flow was 20 microliters/min and 10-min fractions were collected and assayed for SRIF and GRF by RIA. Plasma growth hormone (GH) levels were assayed every 10 min. At the end of the experiments, the accuracy of PPC tip placements was ascertained with a dissecting microscope. Under basal conditions there were 2.9 pulses/h of SRIF with an amplitude of 12.76 +/- 0.46 pg. The output of SRIF and GRF in the 10-min period beginning with application of ether was increased 2-fold (p less than 0.005 and p less than 0.01, respectively). Interestingly, the increased release of SRIF continued for an additional 10 min, whereas GRF output decreased and was almost undetectable. The release of both GRF and SRIF had returned to basal values 20-30 min after stress. Mean plasma GH levels were significantly lowered 10 min after stress. Each of the 9 animals showed a restoration of pulsatile GH release to basal levels within 20-30 min after stress. Our findings provide compelling evidence that SRIF plays a prominent role in stress-induced inhibition of GH release in the rat by blocking the response to the transient elevation of GRF and continuing to suppress GH release for 20 min.

Thyrotrophin-releasing hormone-induced growth hormone (GH) secretion in anaesthetized chickens: inhibition by GH-releasing factor at central sites.

Intracerebroventricular (i.c.v.) administration of GH-releasing factor (GRF) (at 1 or 10 micrograms) to anaesthetized immature (6- to 8-weeks-old) or adult (greater than 24-weeks-old) domestic fowl had no effect on basal GH concentrations in peripheral plasma, but suppressed (after 20 min) the acute GH response to exogenous (i.v.) thyrotrophin-releasing hormone (TRH) (1 micrograms/kg). The i.c.v. injection of GRF also reduced the content of somatostatin (SRIF) and dopamine (DA) in the hypothalamus, while increasing the concentration of the DA metabolite 3,4-dihydroxyphenyl acetic acid (DOPAC) and the DOPAC/DA ratio. The release of SRIF from hypothalamic tissue was stimulated in vitro by 100 nmol GRF/l. The inhibitory effect of i.c.v. GRF on TRH-induced GH secretion was blocked when it was simultaneously injected i.c.v. with SRIF antiserum. These results demonstrate central effects of GRF on avian hypothalamic function and suggest an inhibitory role for this peptide in GH regulation, possibly mediated through increased SRIF secretion.

Hypothalamic Secretion of Somatostatin and Growth Hormone-Releasing Factor into the Hypophysial-Portal Circulation Is Reduced in Streptozotocin Diabetic Male Rats

Growth hormone (GH) secretory patterns are disrupted in streptozotocin (STZ) diabetic rats. Alterations in hypothalamic growth hormone-releasing factor (GRF) or hypothalamic somatostatin (SRIF) secretion, increased systemic SRIF secretion, and changes in somatotroph sensitivity to these hypothalamic factors have been advanced as potential underlying mechanisms for the observed attenuation of GH secretion after STZ treatment. Two weeks after STZ treatment, the mean circulating GH level was attenuated by 58%, plasma glucose concentration was 4-fold higher, and systemic SRIF concentration was elevated 6.8-fold with respect to vehicle (VEH)-treated rats. The hypothalamic content of neither SRIF nor GRF was significantly different between VEH and STZ groups. Total hypophysial-portal SRIF concentration, which represents the sum of both peripheral and hypothalamic SRIF contributions, was significantly elevated in the STZ versus VEH group (p less than 0.007). However, when corrected for the contribution of peripheral SRIF, the mean portal SRIF concentration in STZ rats was only 44% of the mean portal SRIF in VEH rats. A reduction in hypophysial-portal GRF concentration in STZ diabetic rats was also observed. Overall, these observations suggest that elevated peripheral SRIF levels characteristic of STZ diabetic rats play a major role in mediating the suppression of GH secretion in this model. Furthermore, the data are suggestive of an inhibitory effect of peripheral SRIF on hypothalamic SRIF release.

Functional maturation of somatostatin neurons and somatostatin receptors during development of mouse hypothalamus in vivo and in vitro

Ontogenesis of somatostatin (SRIF) neurons and receptors was studied in fetal hypothalamic cell cultures kept in serum-free medium, and compared to the in vivo developmental pattern. Initial rise in neuronal content of SRIF occurred later in vitro than in vivo. In vitro, K +-induced SRIF release was only present after synaptogenesis. SRIF binding sites were measurable as early as 1 day after birth and at an equivalent time in culture, after 6 days in vitro (DIV); their affinity was in the nanomolar range. In cultured cells, binding reached a maximum at two weeks in vitro and decreased sharply thereafter as a consequence of binding site occupancy by the endogenous ligand. Indeed, pretreatment with cysteamine decreased SRIF concentration in the neuronal cultures and twice as many binding sites as in control cultures of 21 DIV were measured. Competition kinetics using unlabelled SMS 201–995 to displace [ 125I]SRIF revealed two distinct binding sites in the neuronal preparations (IC 50 = 11 ± 3 pM and 4.5 ± 0.8 nM). In contrast, only the lower affinity site was present on glial cell preparations (1.7 ± 0.4 nM) SRIF inhibited adenylate cyclase activity in glia and neurons, and the onset of SRIF coupling to the second messenger occurred earlier in vitro than in vivo. Pertussis toxin pretreatment was equally effective in neuronal and glial cell preparations to decrease SRIF binding and to inhibit adenylate cyclase activity

Somatostatin regulation by cytokines

Inflammatory states are associated with nervous and neuroendocrine responses, which appear to be mediated through the actions of cytokines. Since endotoxin treatment in the rat is associated with declines in thyrotropin (TSH) secretion and growth hormone (GH) secretion, changes that may be explained by stimulation of hypothalamic somatostatin (SRIF), the effects of cytokines on SRIF were examined. In an in vitro model system consisting of fetal rat diencephalic cells interleukin-1 (IL-1), tumor necrosis factor (TNF) and interleukin-6 (IL-6) were found to stimulate the synthesis and release of SRIF. This effect developed slowly over 24 hours and was dose- and time-dependent. Acute release of SRIF over periods up to 1 hour was not found. The mechanism of cytokine stimulation of SRIF is not known. Since the depletion of glial cells in the cultures inhibits the effect, mediators that depend on the presence glia may be involved. The ability of cytokines to stimulate brain SRIF is likely to prove relevant to our understanding in many areas, including brain development, brain responses to injury, and neuroendocrine changes in chronic illness.

Physiologically Significant Effect of Neuropeptide Y to Suppress Growth Hormone Release by Stimulating Somatostatin Discharge*

Neuropeptide Y (NPY) is a peptide found in a variety of hypothalamic loci which is frequently colocalized with catecholamines. It is also secreted into hypophyseal portal vessels. The injection of NPY into the third ventricle (3V) lowered plasma GH levels in conscious, freely moving male rats. To determine the physiological significance of the hypothalamic inhibitory action of the peptide, highly specific antiserum directed against NPY was injected into the 3V of conscious rats. 3V injection of the antiserum evoked a significant elevation of plasma GH within 2 h on comparison to values in normal rabbit serum-injected, ovariectomized rats. The difference increased and reached a maximum at 6 h after injection. On the other hand, there was no effect of the antiserum in ovariectomized, estrogen, progesterone-blocked rats. Intraventricular injection of the anti-NPY serum also caused a significant elevation of plasma GH within 2 h in normal male rats and the increases above values in normal rat serum-injected control animals became even more significant at 3 and 4 h. To determine the mechanism by which NPY lowers GH after its intraventricular injection, its effect on the release of somatostatin (SRIF) from median eminence fragments incubated in vitro was examined. NPY stimulated SRIF release with a highly significant effect at a concentration of 10(-9) M. Borderline stimulation was observed at doses as low as 10(-11) M. The curve was bell-shaped with a declining release at 10(-8) M and 10(-7) M. The releasing action of NPY was blocked by either the alpha 1-receptor blocker, prazosin (10(-6) M), or the beta-receptor blocker, propranolol (10(-6) M), but was not affected by the alpha 2-receptor blocker, yohimbine (10(-6) M). We conclude that NPY has a physiologically significant inhibitory action within the hypothalamus to suppress GH release in ovariectomized female and intact male rats by stimulation of SRIF release by alpha 1 and beta-adrenergic receptor-mediated mechanisms.

Peripheral administration of picrotoxin and bicuculline stimulates in vivo somatostatin release from rat median eminence

The γ-Aminobutyric acid-A (GABA A) antagonist picrotoxin and bicuculline were administered to male rats to determine their effects on somatostatin (SRIF) release, measured in unanesthetized animals stereotaxically implanted with push-pull cannula in the median eminence (ME). I.p. injection (3 mg/kg) of picrotoxin ( n=5) or bicuculline ( n=6) significantly increased (35.4±10.8 vs 13.7±4.3 pg/15 min; P<0.03 and 38±3.5 vs 14±1.8 pg/15 min; P<0.001, respectively) SRIF release from the ME compared to baseline levels measured in the same animals. In contrast, with local perfusion of picrotoxin, (10 −4 to 10 −6 M) SRIF release from the ME was not affected. These data suggest a physiological endogenous inhibitory tone of GABA on SRIF release.

Epoxygenase Products of Arachidonic Acid Are Endogenous Constituents of the Hypothalamus Involved in D2Receptor-Mediated, Dopamine-Induced Release of Somatostatin

The epoxyeicosatrienoic acids (EETs) were discovered as products of a cyclooxygenase/lipoxygenase-independent, cytochrome P-450 catalyzed metabolism of arachidonic acid (AA) termed the "epoxygenase" pathway. The rat hypothalamus is able to synthesize EETs from exogenous AA, and 5,6-EET has been found to release the neuropeptide somatostatin (SRIF) from hypothalamic nerve terminals of the median eminence (ME). In the present study, hypothalami from male rats were examined for the presence of endogenous EETs, using chemical, chromatographic, and mass spectral analysis procedures. The samples were initially separated in a C18 Sepralyte column, fractionated on TLC plates, and purified by reverse phase HPLC. Thereafter, they were esterified (pentafluorobenzyl esters) and subjected to negative ion chemical ionization/gas chromatography (GC)/mass spectral (MS) analysis. The GC retention time and the MS fragmentation patterns revealed the presence of a mixture of 8,9-, 11,12- and 14,15-EETs; instability of 5,6-EET during the isolation protocol precluded its identification. Total hypothalamic EET concentration was estimated to be 120 ng/g wet tissue. The 8,9-regiosomer released SRIF from ME nerve terminals with an ED50 of 5 x 10(-12) M; Dopamine (DA) and the D2 receptor agonist PPHT, but not the D1 receptor agonist SKF-38393, induced SRIF release from the ME. This effect was blocked by clotrimazole and ketoconazole, two inhibitors of microsomal cytochrome P-450 function and AA epoxygenase in particular. In contrast, the inhibitors failed to affect the increase in SRIF release induced by 8,9-EET. These results indicate that: 1) in addition to cyclooxygenase and lipoxygenase products, epoxygenase metabolites of AA are endogenous compounds of the hypothalamus, and 2) EETs may mediate the increase in SRIF release from hypothalamic neurons induced by the interaction of DA with D2 receptors.

Inhibition of Hypothalamic Somatostatin Release by β-Adrenergic Antagonists*

A number of in vivo studies suggest that hypothalamic somatostatin (SRIF) tone is stimulated by the beta-adrenergic system. Employing dispersed adult male rat hypothalamic cells, we studied the effects of beta-adrenergic antagonists on the release of hypothalamic SRIF. Propranolol, at concentrations of 1-100 microM, had no detectable effect on basal SRIF release, but caused dose-dependent inhibition of SRIF release stimulated by ouabain. Two other beta-adrenergic antagonists, labetolol and metoprolol, also caused inhibition of ouabain-stimulated SRIF release. The alpha 2-agonist clonidine was without effect on SRIF release under basal or stimulated conditions. GH secretion from monolayers of dispersed rat anterior pituitary cells was also examined. Propranolol (1-100 microM) had no significant effect on basal GH secretion or GH secretion stimulated by rat GRF. In conclusion, 1) beta-adrenergic antagonists caused inhibition of stimulated SRIF release; 2) clonidine had no detectable effect on SRIF release; and 3) propranolol did not affect GH secretion in vitro. These findings support the hypothesis that beta-adrenergic antagonists augment GH responsivity by inhibiting hypothalamic SRIF release.

Role of Arachidonic Acid or Its Metabolites in Growth-Hormone-Releasing Factor-Induced Release of Somatostatin from the Median Eminence

The possible involvement of arachidonic acid (AA) release in growth-hormone-releasing factor (GRF)-induced somatostatin (SRIF) release from the median eminence (ME) of the hypothalamus was evaluated in adult male rats using an in vitro incubation system. The MEs were preincubated with [14C]-AA, then washed and incubated with vehicle or test agents, and the release of SRIF and [14C]-AA into the medium was measured. In the experiments designed only to determine SRIF release, the MEs were first preincubated for 30 min. The medium was then discarded and replaced with fresh buffer or test substances and incubated for 10, 20 and/or 30 min. GRF (10(-10) M) stimulated both AA and SRIF release significantly within 20 min, with maximum release occurring at 30 min. The stimulatory effect of GRF on AA release was coincident with the release of SRIF. A phospholipase A2 inhibitor (10(-6) M, quinacrine) completely abolished the stimulatory effect of GRF on both AA and SRIF release. The release of SRIF induced by GRF was also inhibited by both indomethacin (10(-6) M, a cyclooxygenase inhibitor) and metyrapone (10(-6) M, a cytochrome P-450 inhibitor). On the other hand, nordihydroguaiaretic acid (10(-6) M, a lipoxygenase inhibitor) had no effect on GRF-evoked SRIF release. The data presented here suggest that an important GRF-mediated event leading to SRIF secretion is an elevated release of AA from ME fragments in vitro. In conclusion, our data are suggestive that the stimulatory effect of GRF on SRIF release is due, in part, to the release and subsequent metabolism of AA to one or more metabolites.

Characterization of the Glucose-Dependent Release of Growth Hormone-Releasing Factor and Somatostatin from Superfused Rat Hypothalami

The glucose-dependent secretion of the neuropeptides, growth hormone-releasing factor (GRF) and somatostatin (SRIF), by hypothalamic fragments was studied in vitro using a superfusion system. After equilibration of mediobasal hypothalami in HEPES-buffered Krebs-Ringer solution containing 5.5 mM glucose, glucose levels in the superfusion medium were altered. Lowering the glucose concentration in the medium from 5.5 to 2.7 or 1.1 mM provoked a rapid increase in GRF and SRIF release in a concentration and Ca2+-dependent manner. At 1.1 mM glucose, neuropeptide secretion was elevated 3- to 4-fold. The increase of GRF and SRIF release induced by low glucose was transient since stimulated neuropeptide secretion declined to basal levels in the continued presence of low glucose. Furthermore, after reequilibration in 5.5 mM glucose, no second stimulation of neuropeptide release could be induced by reduced glucose. Intracellular glucopenia induced by addition of 2-deoxy-D-glucose (16.5 mM) to the superfusion medium containing 5.5 mM glucose, also evoked increases in GRF and SRIF release. The sensitivity of GRF and SRIF neurons to glucose was absent in the postnatal period until day 9 after birth and then gradually increased. The parallel increases of GRF and SRIF release in response to low glucose observed in the present in vitro study, together with the suppression of plasma GH levels occurring in hypoglycemia in the rat, suggest that, in this condition, the inhibition of GH release induced by elevated SRIF levels predominates whereas the increase of GRF release might serve to attenuate this effect of SRIF.

Galanin stimulates immunoreactive growth hormone-releasing factor secretion from rat hypothalamic slices perifused in vitro

The effect of galanin (GAL) on the release of GH-releasing factor (GRF) and somatostatin (SRIF) was examined in an in vitro perifusion system of rat hypothalamic slices. GAL at doses at 10 −7 and 10 −6M stimulated the release of immunoreactive GRF while it failed to affect SRIF release. Therefore, in vivo stimulation of GH release by GAL may be explained in part by the GRF-releasing effect of this peptide.

Effects of Active Immunization Against Somatostatin on Serum Growth Hormone Concentration in Growing Pigs: Influence of Fasting and Repetitive Somatocrinin Injections*

Three experiments were conducted with growing pigs actively immunized against a protein-conjugated somatostatin (SRIF) in Freund's adjuvant. In the first experiment, blood from 24-week-old pigs (seven immunized and eight control) was sampled at 20-min intervals for 6 h to evaluate basal GH concentrations. The animals were then injected iv with porcine GH-releasing factor (GRF)-(1-29)NH2 (10 micrograms/kg). Before GRF stimulation, immunized animals had higher (P less than 0.05) baseline mean GH levels (2.6 vs. 1.4 ng/ml) and area under the GH curve (AUC; 1632 vs. 779 ng/min.ml); they also had higher AUC after GRF administration (4268 vs. 1972 ng/min.ml). In a second experiment eight immunized and eight control pigs were injected iv four times at 90-min intervals with porcine GRF (10 micrograms/kg). Control pigs responding to the first injection did not respond to the second and third, and those responding to the second did not respond to the first, third, and fourth, indicating a decreased responsiveness that was longer than 3 h post-GRF response in control pigs. SRIF-immunized pigs had a more consistent GH response to the GRF injections. Overall, a reduced response was observed after the second and the fourth injections in immunized pigs, although five and six of eight animals had a GH peak response higher than 10 ng/ml during these periods. In a third experiment, effects of fasting, GRF, and SRIF immunization were studied. Immunization and fasting had their own positive effects on serum GH levels. Immunization increased baseline mean GH levels (5.0 vs. 2.2 ng/ml) and total AUC before (2318 vs. 1073 ng/min.ml) and after (1886 vs. 910 ng/min.ml) iv GRF stimulation (10 micrograms/kg) compared to controls. Fasting increased the mean baseline GH level (4.5 vs. 2.6 ng/ml), and it increased AUC before exogenous GRF stimulation (2009 vs. 1392 ng/min.ml). In conclusion, SRIF in pigs seems to be a potent GH-governing factor, since, when inhibited, baseline mean GH levels increase, and a consistent response to GRF is observed. Fasting could increase GH concentrations by different ways: decreasing SRIF release and increasing GRF release or modifying the sensitivity of the somatotrophs to both factors.

Glucopenia-Mediated Release of Somatostatin from Incubated Rat Hypothalamus: Monosaccharide Specificity and Role of Glycolytic Intermediates*

Rat hypothalamic somatostatin (SRIF) release in vitro is inversely related to medium glucose concentration and is stimulated by agents interfering with neural glucose uptake or metabolism. The present studies examined monosaccharide specificity of this effect by comparing the ability of glucose and nonmetabolized sugars (ribose, fructose, and galactose) to reverse glucopenia-mediated SRIF release. Removal of glucose from incubation medium resulted in a 7-fold stimulation of hypothalamic SRIF release (24.9 +/- 3.0 to 169.0 +/- 46.2 pg/5 min) that was promptly and fully reversed by readdition of glucose (21.5 +/- 6.0 pg/5 min). Depolarizing concentrations of potassium (40 mM) added to medium after incubation in the presence or absence of glucose produced similar 4-fold biphasic stimulation of SRIF release, suggesting that tissue viability was unimpaired by short term glucopenia. By contrast, nonmetabolized sugars (ribose, fructose, or galactose) at concentrations up to 28 mM were unable to reverse glucopenia-mediated hypothalamic SRIF release. These findings suggest that neural metabolism of glucose specifically influences hypothalamic SRIF release, an effect likely to depend on glycolysis with resulting energy production. This explanation is supported by the finding that the glycolytic intermediates dihydroxyacetone and glyceraldehyde were partly, and pyruvate completely, able to restore glucopenia-mediated SRIF release toward basal, with a maximal effect at 14 mM. Our present studies suggest that rat hypothalamic SRIF release reflects ambient glucose status, is stimulated by glucopenia, and may explain inhibition of GH secretion in the rat during hypoglycemia.

Somatostatin inhibition of VIP-induced somatostatin release, cyclic AMP accumulation and 45Ca 2+ uptake in diencephalic cells

The effect of somatostatin (SRIF) on VIP induction of SRIF secretion, cyclic AMP accumulation and 45Ca 2+ influx was investigated in cultured diencephalic cells. [D-Trp 8]SRIF suppressed VIP-stimulated SRIF release and decreased VIP-stimulated cyclic AMP accumulation in a dose-dependent manner. SRIF-14 blocked basal and VIP-stimulated 45Ca 2+ entry into cells. The data suggest that the inhibitory effect of SRIF on VIP-induced SRIF release is partly due to a decrease in Ca 2+ entry into cells.