Inducible nitric oxide synthase gene expression in the brain during systemic inflammation.

Inducible nitric oxide synthase (iNOS) is a transcriptionally regulated enzyme that synthesizes nitric oxide from L-arginine that has a key role in the pathophysiology of systemic inflammation and sepsis. Transgenic animals with a null mutation for the iNOS gene are resistant to hypotension and death caused by Escherichia coli lipopolysaccharide (LPS). The regulation of peripheral iNOS has been well studied in sepsis, but little is known about iNOS regulation in the brain during systemic inflammation or sepsis. We know that at baseline there is no detectable iNOS gene expression in the brain, but a detailed neuroanatomical study reveals that early in the course of systemic inflammation there is a profound induction of iNOS messenger RNA in vascular, glial and neuronal structures of the rat brain, accompanied by the production of nitric oxide (NO) metabolites in brain parenchyma and cerebrospinal fluid (CSF). We propose that the spillover of nitrite into the CSF has the potential to be a diagnostic marker for systemic inflammation and sepsis. Pharmacological interventions aimed at regulating iNOS function in the brain might represent a new treatment strategy in sepsis. Brain iNOS may be relevant to the pathophysiology, diagnosis and treatment of systemic inflammation and sepsis.

Induction of mating behavior in rats by luteinizing hormone-releasing factor.

Ovariectomized female rats treated with estrogen, in dosages too low to provoke mating, displayed this behavior when given subcutaneous injections of synthetic luteinizing hormone-releasing factor (LRF) 48 hours later. Two hours after the injection of LRF, components of female sexual behavior appeared. The lordosis reflex followed mounting by the male, and darting and hopping behavior was quite prevalent. On the other hand, treatment with estrogen followed by luteinizing hormone, follicle-stimulating hormone, or thyrotropin-releasing factor did not induce copulatory behavior. The results suggest that LRF may play a role in induction of mating behavior.

Paradoxical elevation of growth hormone by intraventricular somatostatin: possible ultrashort-loop feedback.

Somatostatin, the growth hormone-inhibiting factor, when microinjected into the third ventricle of the rat brain, paradoxically induced the release of growth hormone. A pituitary site of action having been ruled out, this result supports the concept that exogenous somatostatin within the hypothalamus acts either to suppress the release of somatostatin from somatostatin-containing neurons, possibly via an ultrashort-loop feedback mechanism, or to augment release of hypothalamic growth hormone-releasing factor, thereby inducing a release of growth hormone. Injection of somatostatin into the third ventricle also decreased plasma concentrations of luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone, probably by inhibiting the release of luteinizing hormone-releasing factor and thyrotropin-releasing factor.

Arginine vasopressin as a thyrotropin-releasing hormone.

Although hypothyroidism (with concomitant increased levels of thyroid-stimulating hormone) has been associated with elevated plasma vasopressin, the role that vasopressin plays in controlling thyroid-stimulating hormone secretion from the adenohypophysis is not understood. In two in vitro pituitary cell systems, vasopressin caused a specific and dose-related release of thyroid-stimulating hormone from cells that was equal in potency to that elicited by thyrotropin-releasing hormone, the primary acknowledged regulator of thyroid-stimulating hormone release. When injected into the hypothalamus, however, vasopressin specifically inhibited the release of thyroid-stimulating hormone. Thus, vasopressin may exert differential regulatory effects on thyroid-stimulating hormone secretion in the hypothalamus and pituitary gland.

Prostaglandin involvement in hypothalamic control of gonadotropin and prolactin release.

Prostaglandin E(2) (PGE(2)) injected into the third ventricle of ovariectomized rats increased plasma luteinizing hormone dramatically and follicle stimulating hormone slightly. PGE(1) elevated prolactin; PGF(1alpha) or PGF(2alpha) had no effect. PGE(2) or PGE(1) injected directly into the anterior pituitary were ineffective. These results suggest that specific prostaglandins act at the hypothalamus to control pituitary hormone release.

The role of toll-like receptors in the immune-adrenal crosstalk.

Sepsis and septic shock remain major health concerns worldwide, and rapid activation of adrenal steroid release is a key event in the organism's first line of defense during this form of severe illness. Toll-like receptors (TLRs) are critical in the early immune response upon bacterial infection, and recent data from our lab demonstrate a novel link between the innate immune system and the adrenal stress response mediated by TLRs. Glucocorticoids and TLRs regulate each other in a bidirectional way. Bacterial toxins acting through TLRs directly activate adrenocortical steroid release. TLR-2 and TLR-4 are expressed in human and mice adrenals and TLR-2 deficiency is associated with an impaired glucocorticoid response. Furthermore, TLR-2 deficiency in mice is associated with marked cellular alterations in adrenocortical tissue. TLR-2-deficient mice have an impaired adrenal corticosterone release following inflammatory stress induced by bacterial cell wall compounds. This defect appears to be associated with a decrease in systemic and intraadrenal cytokine expression. In conclusion, TLRs play a crucial role in the immune-adrenal crosstalk. This close functional relationship needs to be considered in the treatment of inflammatory diseases requiring an intact adrenal stress response.

The nitric oxide theory of aging revisited.

Bacterial and viral products, such as bacterial lipopolysaccharide (LPS), cause inducible (i) NO synthase (NOS) synthesis, which in turn produces massive amounts of nitric oxide (NO). NO, by inactivating enzymes and leading to cell death, is toxic not only to invading viruses and bacteria, but also to host cells. Injection of LPS induces interleukin (IL)-1beta, IL-1alpha, and iNOS synthesis in the anterior pituitary and pineal glands, meninges, and choroid plexus, regions outside the blood-brain barrier. Thereafter, this induction occurs in the hypothalamic regions (such as the temperature-regulating centers), paraventricular nucleus (releasing and inhibiting hormone neurons), and the arcuate nucleus (a region containing these neurons and axons bound for the median eminence). Aging of the anterior pituitary and pineal with resultant decreased secretion of pituitary hormones and the pineal hormone melatonin, respectively, may be caused by NO. The induction of iNOS in the temperature-regulating centers by infections may cause the decreased febrile response in the aged by loss of thermosensitive neurons. NO may play a role in the progression of Alzheimer's disease and parkinsonism. LPS similarly activates cytokine and iNOS production in the cardiovascular system leading to coronary heart disease. Fat is a major source of NO stimulated by leptin. As fat stores increase, leptin and NO release increases in parallel in a circadian rhythm with maxima at night. NO could be responsible for increased coronary heart disease as obesity supervenes. Antioxidants, such as melatonin, vitamin C, and vitamin E, probably play important roles in reducing or eliminating the oxidant damage produced by NO.

Calmodulin dependence of somatostatin release stimulated by growth hormone-releasing factor.

Experiments were performed in vitro to examine the possible role of calcium and calmodulin in GRF-induced somatostatin (SRIF) release from the median eminence. Adult male rats were used as tissue donors. The median eminences were first prestimulated in 0.4 ml Krebs Ringer bicarbonate glucose buffer (pH 7.4) containing bacitracin at 37C in an atmosphere of 95% O2, 5% CO2 with constant shaking for 30 min. When calcium was omitted, this medium was used during the prestimulation and stimulation periods. After prestimulation, the medium was discarded and replaced by medium containing the different substances to be tested (GRF, EGTA, calcium channel blockers, and calmodulin inhibitors). The stimulation of SRIF release induced by 10(-10) M GRF was not inhibited by omission of extracellular calcium or when the remaining CA+2 was chelated with 10(-4) M EGTA. The calcium channel blockers, nifendipine and verapamil (10(-6) M), failed to alter the increase of SRIF release induced by rGRF. Three calmodulin inhibitors were employed to examine the possible influence of calmodulin on GRF-induced SRIF release. Trifluoperazine (10(-6) M), triflupromazine (10(-6) M) and penfluridol (10(-7) M) had an inhibitory effect on the stimulation of SRIF release induced by GRF and failed to alter resting release. Thus, GRF can evoke SRIF release independently of extraterminal Ca+2 concentration and Ca+2 influx into the nerve terminals, but the releasing process involves translocation of Ca+2 from intracellular stores. The inhibitory effect of the calmodulin inhibitors on GRF-induced SRIF release, suggests that the translocated Ca+2 must bind to calmodulin in order to release SRIF.

Effect of destruction of the dorsal anterior hypothalamus on follicle-stimulating hormone secretion in the rat.

The role of the paraventricular nucleus-dorsal anterior hypothalamus (PVN-DAHA) in the control of anterior pituitary gland secretion of FSH and LH in castrated male and female rats was examined. Bilateral radiofrequency lesions of the PVN-DAHA in chronically ovariectomized (OVX) rats lowered plasma FSH levels by 33% (P less than 0.005) compared to values in unoperated and sham-operated control rats; plasma LH concentrations were unaltered. RIA of median eminence (ME) LHRH concentrations in these animals revealed no differences among the three experimental groups. Other categories of diencephalic destruction did not result in this pattern of selectively reduced FSH release. Bilateral radiofrequency destruction of the PVN-DAHA also attenuated by 50% (P less than 0.025 to P less than 0.005) the progesterone-induced surge of FSH in estrogen-primed OVX rats. Progesterone-induced LH release was unaffected by PVN-DAHA lesions. Other lesion categories failed to show the same result. Bilateral ablation of the PVN-DAHA in male rats resulted in a selective diminution of the postcastration rise of plasma FSH beginning 48 h postcastration (P less than 0.05 to P less than 0.005) and persisting for 14 days (P less than 0.005) after orchidectomy, thus revealing the time course and permanence of this procedure on plasma FSH levels. The postcastration rise of plasma LH levels was not affected by PVN-DAHA lesions. The concentration of ME LHRH was the same among orchidectomized male rats whether they bore PVN-DAHA lesions, sham lesions, or no lesions. In summary, destruction of the PVN-DAHA was found to reduce significantly the elevation of plasma FSH, but not LH, in the OVX rat and the estrogen-progesterone-stimulated OVX rat. PVN-DAHA lesions also attenuated the postcastration rise of FSH, but not that of LH, in the male. The failure of lesions of the PVN-DAHA to alter ME LHRH concentrations in the face of decreased FSH release does not prove that LHRH release is totally unaffected by this procedure. This finding is, however, consistent with the concept that diminished FSH secretion could be the result of a deficiency of a hypothalamic releasing factor (FSH-releasing factor?) other than that of the LHRH decapeptide.

Stimulation of somatostatin release in vitro by synthetic human growth hormone-releasing factor by a nondopaminergic mechanism.

The effect of synthetic human GH-releasing factor (hGRF-40) on somatostatin (SRIF) release from the median eminence of the hypothalamus was evaluated in rats with use of an in vitro incubation system. hGRF-40 stimulated SRIF release in a dose-related manner. This effect was significant at concentrations varying from 10(-11)-10(-7) M, with a minimal effective dose of 10(-11) M. Maximal stimulation was observed at 10(-10) M. Pimozide was added in vitro at a concentration of 10(-6) M to block dopamine (DA) receptors, since DA is a known stimulator of SRIF release. Pimozide was without effect on SRIF release and did not alter the stimulatory effect of hGRF-40. To evaluate the possibility that DA and GRF may share a common pathway to stimulate SRIF release, median eminence fragments were simultaneously exposed to submaximal concentrations of both DA (6 X 10(-7) M) and hGRF-40 (10(-12) M). By themselves, each of these doses had little effect on SRIF release. When they were added together, a marked stimulation was noted, which was not, however, significantly greater than the sum of the responses to each agent alone. These results suggest that DA and GRF act by separate mechanisms to stimulate SRIF release. GRF may be physiologically involved in the regulation of SRIF release. Stimulation of SRIF release may be a mechanism by which GRF exerts a negative ultrashort-loop feedback to inhibit GH release.

Stimulation of somatostatin release from median eminence tissue incubated in vitro by taurine and related amino acids.

To determine if the putative amino acid transmitter taurine could affect the release of somatostatin (SRIF) from median eminence fragments, median eminences were incubated in vitro in the presence of various concentrations of taurine and its precursors, hypotaurine, cysteamine, cysteic acid, and L-cysteine sulfinic acid. Taurine was effective in releasing SRIF at doses of 50 microM and higher. Surprisingly, cysteic acid was the most potent stimulant of SRIF release, with a minimal effective dose of 0.1 microM. Cysteamine was effective at a dose of 50 microM, hypotaurine was effective only at 200 microM, and cysteine sulfinic acid was ineffective in stimulating SRIF release at any of the doses tested. To determine if the effect of taurine was mediated by the dopaminergic system, the dopamine receptor blocker pimozide (1 microM) was added to the medium containing either taurine or cysteic acid. Pimozide did not prevent the stimulatory effect of the amino acids. In conclusion, these investigations suggest that taurine and related amino acids may be involved in SRIF release by a nondopaminergic pathway. In view of the high concentrations of taurine in the hypothalamus it must be considered a putative transmitter releasing SRIF.

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.

Growth hormone release in conscious pinealectomized and sham-operated male rats.

Conscious, adult male, sham-operated (control), and pinealectomized (Pect) rats with chronic jugular cannulae were bled during the day or night. Daytime samples were obtained from rats housed singly in small cages, while the night samples were obtained from animals on a reversed light-dark cycle kept in a larger isolation cubicle. During the day, controls showed frequent secretory bursts of GH release, as evidenced by dramatic elevations of plasma GH with a periodicity of 2.3 h. In the Pect rats, there was a more variable GH release, with some animals showing spikes similar in amplitude to those of controls and others showing no spikes. There were significantly fewer GH values in excess of 150 ng/ml after Pect, and GH release over the total period of sampling, calculated from the area under the plasma GH curve, was significantly reduced. During the night, plasma GH spikes of controls were significantly reduced in frequency and amplitude, but these were not further significantly reduced by Pect. Under our conditions, there was a diurnal change in GH release with reduced release at night. Pinealectomy significantly reduced GH release during the day but did not significantly further reduce GH release at night.

Effect of lesions in the ventral noradrenergic tract produced by microinjection of 6-hydroxydopamine on gonadotropin release in the rat.

Noradrenergic innervation to the hypothalamus is provided principally by the ventral noradrenergic tract (VNAT) which carries the axons of noradrenergic neurons whose cell bodies lie in the brain stem. To determine the importance of the VNAT in the stimulation of LH release induced by progesterone in ovariectomized, estrogen-primed rats, 6-hydroxydopamine (6-OHDA), an agent which selectively destroys catecholaninergic neurons, was microinjected bilaterally into this tract, 2 days after priming of the ovariectomized animals with 5 mug of estradiol benzoate, sc. Following microinjection, 2 mg of progesterone was injected sc to provoke LH release. A surge of FSH and LH release occurred 6 h after progesterone in control animals and those injected with the ascorbic acid diluent into the VNAT. Injections of 6-OHDA into the tract completely blocked both the LHP AND FSH surge. Control injections of 6-OHDA into the superior colliculus, the caudate-putamen or the frontal cortex did not alter the release of FSH and LH induced by progesterone. Twenty-four hours after injection of 6-OHDA into the VNAT, there was a slight reduction in norepinephrine content in the anterior hypothalamic area and a significant reduction in the region of the arcuate nucleus and median eminence. In this experiment, 6-OHDA injections into the VNAT blocked not only the FSH and LH release induced by progesterone but also the increase in serum prolactin which was present in control animals. In normal females, injections of 6-OHDA into the VNAT blocked the proestrous discharge of LH and partially blocked that of FSH. It is concluded that acute interruption of the VNAT induced by a 6-OHDA can block not only the stimulation of FSH and LH induced by progesterone, but also the preovulatory discharge of gonadotropins. The results suggest that increased impulse traffic in the VNAT on the afternoon of proestrus may be involved in induction of the proestrous gonadotropin surge.

Evidence that growth hormone-releasing factor stimulates somatostatin release in vitro via beta-endorphin.

Previous results indicate that GH-releasing factor (GRF) induces a dose-related stimulation of somatostatin (SRIF) release from median eminence fragments incubated in vitro. In the present investigation we examined whether this action was mediated by other neurotransmitters or neuromodulators. Studies using receptor blockers for dopamine (pimozide), alpha-adrenergic receptors (phentolamine), and muscarinic cholinergic receptors (atropine) revealed that these receptor blockers, at a dose of 10(-6) M, which was capable of blocking the response to the relevant transmitter in previous studies, had no effect on basal release of SRIF in the static incubation system and failed to modify the response to GRF (10(-10) M). On the other hand, the opiate receptor blocker naloxone at a dose of 10(-6) M, although failing to alter basal release, completely blocked the response to 10(-10) M GRF. To determine the opioid peptide involved in mediating the SRIF release induced by GRF, highly specific antibodies directed against beta-endorphin were added to the in vitro incubation system. These antibodies significantly depressed basal release SRIF and completely blocked the response to 10(-10) M GRF. Incubations in the presence of normal rabbit serum or highly specific antiserum directed against alpha MSH had no effect on either basal release of SRIF or that induced by GRF. These results suggest that in this incubation system there is a beta-endorphin tone which is partially responsible for the basal release of SRIF and that the stimulation of SRIF release induced by GRF is mediated via beta-endorphin terminals, which presumably synapse on the terminals of the somatostatinergic neurons in the median eminence fragment.

Interaction of alpha-melanocyte-stimulating hormone with beta-endorphin to influence anterior pituitary hormone secretion in the female rat.

The interaction of alpha MSH and beta-endorphin on the secretion of PRL, GH, and LH was determined in the ovariectomized rat. The potent stimulatory effect on PRL release of injection of 20 ng (5.8 pmol) beta-endorphin into the third cerebral ventricle was completely blocked by 100 ng (60 pmol) alpha MSH. The same dose of alpha MSH partially blocked the effect of 150 ng (44 pmol) beta-endorphin on PRL secretion. Intraventricular injection of either 20 ng beta-endorphin or 100 ng alpha MSH had no effect by itself on plasma LH. However, coinjection of these doses of beta-endorphin and alpha MSH suppressed plasma LH levels significantly within 15 min. beta-Endorphin (150 ng) produced a significant suppression of plasma LH levels. Furthermore, coadministration of 100 ng alpha MSH with 150 ng beta-endorphin lowered plasma LH for a longer period of time than this dose of beta-endorphin alone. Low doses of beta-endorphin (20 ng) or alpha MSH (100 ng) alone or in combination did not alter plasma GH levels. A higher dose of 150 ng beta-endorphin produced a slight elevation of plasma GH. This effect was potentiated 5-fold when 150 ng beta-endorphin were injected in combination with 100 ng alpha MSH. These results indicate that alpha MSH acts as an antagonist to beta-endorphin in regard to the secretion of PRL, whereas it potentiates the effect of beta-endorphin in stimulation of GH and inhibition of LH secretion.

Alterations during the estrous cycle in the responsiveness of the pituitary to subcutaneous administration of synthetic LH-releasing hormone (LHRH).

To further evaluate the alterations in responsiveness of the pituitary to synthetic LHRH during the proestrous discharge of gonadotropins, LHRH was given SC in the hope of producing a release of FSH as well as LH, and blood samples were removed prior to and at various intervals after the injections of the neurohormone while the rats were anesthetized with tribromoethanol. The procedure of anesthesia and blood sampling produced small declines in both FSH and LH, but these only achieved significance in the case of FSH on the morning of estrus and diestrus day 2. An increase in plasma LH occurred in response to LHRH at all stages of the estrous cycle but was minimal during diestrus. The increment in plasma LH was already increased by 9 AM on proestrus and the titer remained elevated for 2 h. A further increase in response to LHRH occured at 2 PM associated with an increase in initial plasma LH. The maximum response occurred at 5 PM together with maximal initial plasma LH, and the characteristics of the response changed such that there was a much larger increase in plasma LH at 20 min and a rapid decline thereafter, so that the response became pulselike. Initial plasma LH had already declined significantly by 9 PM, and this was associated with a dimished pulselike release of LH in response to LHRH. By the morning of estrus, an even smaller pulse-like release occurred. Significant FSH release in response to LHRH occurred at all stages of the cycle as indicated by the increments in plasma FSH. The relative increase in plasma titers was much less than that for LH and responsiveness to LHRH did not increase until 5 PM on proestrus. Responsiveness had already begun to decline by 9 PM on proestrus and by the morning of estrus was similar to that obtained on diestrus. Initial plasma FSH titers were first elevated by 2 PM on proestrus and they remained elevated on the morning of estrus. The results indicate that maximal responsiveness to LHRH in terms of LH release is associated with the proestrous discharge of LH, but that the maximal responsiveness in terms of FSH release terminates long before the endogenous release of hormone ceases sometime on estrus. The increased responsiveness in terms of LH release may be related to prior estrogen secretion and to the priming action of endogenous LHRH. The continued release of FSH in the face of a return of responsiveness to LHRH to low levels indicates that release of FSH on late proestrus and early estrus is not caused by endogenous release to LHRH.

Cyclic variations in the increased responsiveness of the pituitary to luteinizing hormone-releasing hormone (LHRH) induced by LHRH.

Following a single iv injection of 50 ng of LH-releasing hormone (LHRH), a small increase in plasma LH titers was observed and this response was greater on proestrus and estrus than in other stages of the cycle. When a second injection of a similar dose of the neurohormone was given one hour later, the response to the second injection was markedly enhanced on proestrus but only slightly increased at other stages of the cycle. The response to LHRH in androgenized females was similar to that observed on proestrus, and, as on proestrus, an enhanced response to a second injection occurred. Only small increases in FSH followed these injections and there was no increase in responsiveness to the second injection in any of the groups. It is postulated that rising estrogen titers can bring about a priming action of LHRH which enhances the response of the gland to a second injection on proestrus. The priming action of LHRH may magnify the response of the pituitary to endogenous decapeptide release to further increase the release of LH on the afternoon of proestrus.

Suppression of feeding and drinking activity in rats following intraventricular injection of thyrotropin releasing hormone (TRH).

Since both TRH and somatostatin (SRIF) are localized to the ventromedial hypothalamic nucleus, a region known to be involved in control of food intake, the possibility that these peptides might alter food intake was evaluated. The peptides were dissolved in 0.9% NaCl and injected into the 3d ventricle in a volume of 2 micron1 in animals bearing 3d ventricular cannulae. Food and water had been removed from the cages the night before and the intake was measured at 1 and 6 h after injection. Control injections of 0.15M NaCl or glutathione (3 nmoles) had no effect on food or water intake. At a dose of 3 nmoles, LHRH, SRIF, and TRH suppressed water intake alh. Lowering the dose of LHRH and SRIF to 0.6 nmoles led to loss of this inhibition but the suppressive effect of TRH, which was more pronounced at the higher dose than that of the other two peptides, persisted. Lowering the dose of TRH to 0.3 nmoles led to loss of the inhibitory effect. The dose of 3 nmoles of LHRH did not suppress food intake but this dose of both SRIF and TRH had a significant suppressive effect on food intake at 1 h. There was no suppressive action of a lower dose of 0.6 nmoles of SRIF, but TRH was still effective to suppress food intake at this dose. A dose of 0.3 nmoles of TRH had no effect on food intake. It is suggested that TRH, and possibly SRIF may play a physiological role in control of food intake, perhaps by altering the neural activity within the ventromedial nucleus.

The inhibition of growth hormone release by gastrin-releasing peptide involves somatostatin release.

Injection of gastrin-releasing peptide-27 (GRP) into the third ventricle (IVT) has been shown previously to lower plasma GH levels and block the GH release induced by GRF, suggesting that GRP might act via stimulation of the release of somatostatin (SRIF) into hypophysial portal vessels. Several experiments were performed to test this hypothesis. In the first experiment rat median eminence (ME) fragments were incubated in medium containing concentrations of GRP ranging from 1 pM to 1 microM, and SRIF levels were measured after the 30-min incubation period. GRP significantly stimulated SRIF release at doses of 0.1 nM to 1 microM. Microinjection of SRIF antiserum (3 microliters) IVT prevented GRP (2 micrograms, IVT) from inhibiting the GH surge induced by GRF (1 microgram/kg, iv). A slight but significant decrease in basal plasma GH levels was observed after GRP administration even in the presence of SRIF antiserum. Finally, to rule out a GRP-GRF interaction at the pituitary level, tubes containing dispersed rat pituitary cells (2.5 x 10(5) cells/tube) were incubated for 1.5 h in medium containing various concentrations of GRF (0.4-40 nM) alone or with 0.1 microM GRP. The addition of GRP to the medium had no significant effect on the dose-dependent stimulation of GH release by GRF. The results of these studies demonstrate that GRP can directly stimulate SRIF release in vitro. They further suggest that SRIF is a component of the mechanism whereby GRP inhibits GH release in vivo. Finally, the possibility that GRP acts at the pituitary level to inhibit GH release by blocking GRF receptors on somatotrophs has been ruled out.