Peptide T blocks GP120/CCR5 chemokine receptor-mediated chemotaxis.

We previously reported that certain short gp120 V2 region peptides homologous to vasaoactive intestinal peptide (VIP), such as "peptide T," were potent inhibitors of gp120 binding, infectivity, and neurotoxicity. The present study shows that synthetic V2-region-derived peptides have potent intrinsic chemotaxis agonist activity for human monocytes and also act as antagonists of high-affinity (0.1 pM) gp120-mediated monocyte chemotaxis. Selectivity is shown in that peptide T is more potent at suppressing M-tropic than T-tropic gp120 chemotaxis. Peptide T was also able to suppress monocyte chemotaxis to MIP-1beta, a chemokine with selectivity for CCR5 chemokine receptors, while chemotaxis of the more promiscuous ligand RANTES was not inhibited, nor was chemotaxis mediated by SDF-1alpha. In order to determine if peptide T mediated its gp120 antagonistic effects via modulation of CCR5 receptors, RANTES chemotaxis was studied using a CCR5 receptor-transfected HOS cell line. In this case, RANTES chemotaxis was potently inhibited by V2-region-derived short peptides. Peptide T also partially suppressed (125)I-MIP1-beta binding to human monocytes, suggesting action at a subset of MIP1-beta receptors. The V2 region of gp120 thus contains a potent receptor binding domain and synthetic peptides derived from this region modulate CCR5 chemokine receptor chemotactic signaling caused by either gp120 or chemokine ligands. The results have therapeutic implications and may explain recent clinical improvements, in that HIV/gp120 actions at CCR5 receptors, such as occur in the brain or early infection, would be susceptible to peptide T inhibition.

HIV gp120 inhibits the somatotropic axis: a possible GH-releasing hormone receptor mechanism for the pathogenesis of AIDS wasting.

AIDS is often associated with growth retardation in children and wasting in adults. The dissociated envelope protein of the HIV (HIV-1), gp120, can be found in significant concentrations in the parenchyma and cerebrospinal fluid of brains in infected individuals, even in the earliest stages of HIV-1 disease. On the basis of this and the fact that we observed pentapeptide sequence homology between GH-releasing hormone (GHRH) and the V2 receptor-binding region of gp120, we initiated experiments to determine whether gp120 could affect GH secretion and growth in vivo and/or interact with anterior pituitary GHRH receptors in vitro. Although acute IV administration of gp120 in conscious rats had no effect on plasma GH levels, acute administration of gp120 (400 ng) into the brain significantly suppressed pulsatile GH release over a 6-h period compared with saline-injected controls. Furthermore, the putative gp120 antagonist, Peptide T (DAPTA), prevented the suppression of GH by gp120. In support of these in vivo findings, gp120 also significantly (P < 0.05) suppressed GHRH-stimulated GH release in static cultures of dispersed pituitary cells and from cells undergoing perifusion with the peptides. DAPTA prevented the GH suppression by gp120 in both of the pituitary cell paradigms. Furthermore, chronic administration of gp120 into the third ventricle significantly reduced body weight in juvenile rats, compared with saline-injected controls. Thus, gp120 appears to act both at the hypothalamus and pituitary to suppress GH release, and its action at these two locations is associated with a significant loss in body weight in chronically treated young animals. These findings may suggest a specific mechanism for the pathogenesis of wasting in HIV-1 patients that involves blockade of endogenous GHRH receptors by gp120.

Endotoxin-induced suppression of the somatotropic axis is mediated by interleukin-1 beta and corticotropin-releasing factor in the juvenile rat.

This study extends the neuroendocrine role of central interleukin-1 beta (IL-1 beta) during the stress of lipopolysaccharide (LPS) challenge to include inhibition of the somatotropic [GH-releasing hormone (GHRH)-somatostatin (SRIF)-GH] axis in juvenile male rats and clarifies the role of CRF in the mediation of LPS/IL-1-induced changes in GHRH and SRIF neurosecretion. The results of the in vivo component of this study demonstrated that LPS treatment (2.5 mg/kg twice daily for 5 days) caused a significant attenuation of body weight gain for 2 days (2.4 +/- 1.7% vs. 10.3 +/- 1.8% BW/day in saline controls; P < 0.05) and failure of catch-up growth thereafter even though a small transient suppression of food intake returned to normal by the second of 4 days of treatment. Associated with the first day of growth attenuation was an acute suppression of all plasma GH parameters, including GH mass (area under the curve, 1.972 +/- 0.1837 vs. 6.402 +/- 1.7 micrograms/ml.6 h for saline controls; P < 0.05), in animals receiving an acute bolus of LPS, which was blocked by prior microinjection of IL receptor antagonist protein (IRAP) into the third ventricle. In contrast, GH parameters associated with the second day of LPS-suppressed body weight gain were increased (GH mass, 9.4 +/- 2.2 vs. 3.5 +/- 0.5 micrograms/ml.4 h in saline controls; P < 0.05). These increases were reversed after another 2 days of LPS treatment. In a series of in vitro experiments using medial basal hypothalamic (MBH) explants incubated with LPS [100 ng/ml alone or with 10(-7) M IRAP or 10(-6) M CRF antagonist (CRF-ANT)], GHRH release from MBH incubated with LPS was significantly greater than that in controls (231 +/- 79% vs. 71 +/- 34% of baseline release; P < 0.05), and this stimulation was antagonized by both IRAP and CRF-ANT. SRIF release was significantly increased by incubation with LPS (163 +/- 28% vs. 97 +/- 20% of the baseline for controls; P < 0.05) and blocked (to 88 +/- 14% of the baseline) by IRAP, but not by CRF-ANT. Finally, when MBH explants were incubated with IL-1 beta (10(-9) M), there was a significant inhibition of in vitro GHRH release (37.9 +/- 6.7% vs. 74.9 +/- 16.6% for controls), which was reversed by IRAP and CRF-ANT, and a significant stimulation of SRIF release (168.7 +/- 37.5% vs. 98.0 +/- 11.6% for controls), which was reversed by IRAP, but not CRF-ANT.(ABSTRACT TRUNCATED AT 400 WORDS)

Early increase in pulsatile growth hormone release after unilateral nephrectomy in adult rats.

Removal of one kidney results, within days, in accelerated growth of the remaining kidney. However, the mechanisms that underlie this compensatory renal hypertrophic response, particularly in the early time period following nephrectomy, are not understood. In this study we tested the hypothesis that removal of one kidney leads to a change in the pulsatile release of growth hormone (GH), which facilitates compensatory renal growth. Adult Wistar rats were implanted with Silastic cannulas in jugular veins and underwent either unilateral nephrectomy (UNX) or sham operation. Plasma levels of GH were determined 24 and 48 h after sham operation or UNX. Blood samples were taken every 20 min over a 6-h period from conscious, unrestrained animals. Pulsatile GH release was markedly elevated 24 h after UNX in both the amplitude of the surges as well as in the duration of release. Peak GH levels after 24 h were three- to fourfold higher in UNX rats compared with sham controls (417 +/- 75 vs. 119 +/- 23 ng/ml, P < 0.05). However, this enhanced release of GH appeared to be of short duration and began declining by 48 h post-UNX (peak level of 227 +/- 37 ng/ml, P < 0.05 vs. both 24 h UNX and sham controls). To examine whether this rise in GH release post-UNX contributed to the compensatory renal growth, rats underwent UNX and were immediately treated with an antagonist to GH-releasing factor (GRF-AN; i.e., [N-Ac-Tyr1,D-Arg2]GRF-(1-29) amide, 200 micrograms/kg twice daily), and the effects on GH release and renal growth were determined. Administration of GRF-AN significantly suppressed the increase in GH release post-UNX and was associated with a significant attenuation in renal growth 48 h post-UNX in GRF-AN-treated rats (8.7 +/- 2.6% vs. 22.7 +/- 3.0% in UNX controls, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Tandospirone stimulates prolactin secretion in the rat by an action at serotonin-1A receptors.

Tandospirone is an azapirone drug that has high affinity for serotonin-1A (5-HT-1A) receptors and preclinical effects predictive of antidepressant and/or anxiolytic efficacy. 5-HT-1A receptor agonists, such as 8-hydroxy-2-(di-n-propyl-amino)tetralin, increase the plasma prolactin concentration in rats, probably by an action in the brain that leads to an increase in prolactin release from the pituitary gland. The purpose of this study was to examine the effect of tandospirone on plasma prolactin concentration in awake, freely moving male rats. We found that i.v. administration of tandospirone results in a rapid and dose-related increase in the plasma prolactin concentration. The plasma prolactin level peaks about 10 min after injection and returns to base-line values within 30 min after injection. The ED50 of tandospirone to increase plasma prolactin levels is approximately 0.3 mg/kg. This effect of tandospirone is blocked by pretreatment with the 5-HT antagonists metergoline and NAN-190 and shows cross-desensitization with the 5-HT-1A agonist 8-hydroxy-2-(di-n-propyl-amino)tetralin. Thus the effect of tandospirone on plasma prolactin concentration appears to be mediated by 5-HT-1A receptors. In contrast to this effect of tandospirone, 1-(2-pyrimidyl)-piperazine (1-PP), the common metabolite of tandospirone and other azapirone drugs, has no effect on plasma prolactin levels. The effect of chronic administration of tandospirone was examined by measuring the prolactin response to an acute i.v. injection of tandospirone 24 hr after the last of chronic injections of tandospirone (10 mg/kg, s.c., twice a day for 2 weeks).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of serotonin1A receptors increases release of prolactin in the rat.

The effect of serotonin1A receptor agonists on release of prolactin was examined in awake, freely-moving male rats in which a catheter in the jugular vein allowed samples of blood to be collected periodically after intravenous injection of the agonist. The serotonin1A receptor agonist, 8-hydroxy-2(di-n-propylamino) tetralin (8-OHDPAT) increased concentrations of prolactin in plasma rapidly and in a dose-related manner. Concentrations of prolactin peaked within 9 min after intravenous injection of 8-OHDPAT and returned to baseline values within 30 min. Another serotonin1A receptor agonist, 5-methylurapidil (5-MeU), produced a similar response of prolactin. The effects of these agonists on release of prolactin were completely blocked by pretreatment with the serotonin receptor antagonists, methysergide and metergoline, administered 1 or 2 hr before the agonist. These results demonstrated that serotonin1A receptors can mediate the effects of serotonin on release of prolactin in the male rat.

Effect of a growth hormone-releasing factor antagonist on compensatory renal growth, insulin-like growth factor-I (IGF-I), and IGF-I receptor gene expression after unilateral nephrectomy in immature rats.

We have recently reported that pulsatile GH secretion is elevated 24 h after unilateral nephrectomy (UNX) in adult rats. In addition, suppression of the increase in GH with an antagonist to GH-releasing factor (GRF-AN) significantly attenuated compensatory renal growth (CRG) in adult rats. The present study examined the role of GH in CRG in immature animals. Pulsatile GH release was determined 24 h post-UNX in immature (26-28 days of age) sham-operated and UNX male Wistar rats. In contrast to the adult UNX rats, no increase in GH secretion was seen in the immature UNX rats compared with that in the controls. When pulsatile GH release was suppressed with GRF-AN, there was preferential growth of the remnant kidney despite the attenuated gain in whole body weight. In addition, insulin-like growth factor-I (IGF-I) and IGF-I receptor mRNA levels were elevated 3-fold in the remnant kidneys of GRF-AN-treated rats, despite the suppression of pulsatile GH release. These findings suggest that the initial phase of CRG is GH independent in the immature rat and, further, that CRG is associated with an increase in IGF-I and IGF-I receptor gene expression that is independent of episodic GH secretion.

Partial characterization of a neurotransmitter pathway regulating the in vivo release of prolactin.

Nicotinic cholinergic, opiate and serotonergic agonists as well as dopaminergic antagonists induce the release of pituitary prolactin. The purposes of the present studies were to determine if nicotine, morphine and the serotonin1A (5-HT1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) utilize a common synaptic pathway to release prolactin and, if so, to establish the serial order of the receptors involved. We also sought to determine whether the pathway under investigation leads to the secretion of prolactin via a mechanism involving dopamine, the prolactin inhibitory factor. Male rats with indwelling jugular catheters were pretreated with saline, mecamylamine, naltrexone, methysergide or bromocriptine. In the saline-treated animals, administration of nicotine, morphine, 8-OH-DPAT and haloperidol resulted in significant increases in plasma prolactin levels. Mecamylamine pretreatment prevented the prolactin response to nicotine only. Naltrexone blocked the stimulation of prolactin release by morphine and by nicotine. Methysergide inhibited the effects of 8-OH-DPAT, morphine and nicotine but not haloperidol. Bromocriptine blocked the prolactin secretion induced by haloperidol as well as by each of the above agonists. Also, in dual-immunocytochemically stained sections, tyrosine hydroxylase-immunoreactive cells and serotonin-immunoreactive processes were detected in close anatomical proximity in the dorsomedial arcuate nucleus. These data indicate that nicotine, morphine and 8-OH-DPAT act to release prolactin via a common synaptic pathway expressing nicotinic cholinergic, opiate, and 5-HT1A receptors at synapses arranged serially in that functional order. Furthermore, the data indicate that the in vivo secretion of prolactin via this pathway may ultimately occur through the inhibition of dopamine release.

Suppression of growth hormone release restores phosphaturic response to PTH in immature rats.

Immature rats display a blunted rise in urinary phosphate but not adenosine 3',5'-cyclic monophosphate (cAMP) excretion in response to parathyroid hormone (PTH), perhaps as a consequence of the increased demand for phosphate during growth. Because a major driving force for growth is growth hormone (GH), and in view of the fact that GH has been shown to promote renal phosphate retention in the immature animal, it is possible that GH may attenuate the phosphaturic effect of PTH. The objective of this study was to determine whether suppression of pulsatile GH release, during administration of a synthetic peptide antagonist to GH-releasing factor, i.e., [N-acetyl-Tyr1-D-Arg2]-GRF-(1-29)-NH2 (GRF-AN), alters the renal response to increasing doses of PTH (1.5-15.0 micrograms.100 g-1.h-1) in the acutely thyroparathyroidectomized immature rat. Baseline fractional excretion of phosphate (FEPi), before administration of PTH, was negligible in all groups (less than 0.05%). Infusion of PTH resulted in an attenuated rise in FEPi in immature control rats compared with adult control rats (from 3.8 +/- 1.4% at lowest PTH dose to 16.7 +/- 3.1% at highest dose in immature rats vs. 21.1 +/- 3.5 to 31.9 +/- 4.4% in adult rats, P less than 0.05). In contrast, immature rats treated for 2 days with GRF-AN (100 micrograms/kg, twice daily) displayed an enhanced phosphaturic response (FEPi from 12.0 +/- 4.2 to 42.9 +/- 3.7%, P less than 0.05) compared with immature control rats, which was not different from that observed in control adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of renal phosphate reabsorption during development: implications from a new model of growth hormone deficiency.

It has been hypothesized that the high rate of renal phosphate (Pi) reabsorption in the immature animal is a consequence of the increased demand for Pi associated with the rapid rate of growth. Although growth hormone (GH) has been proposed to play a role in this process, investigations of the relationship between GH, growth and the renal Pi transport have been hampered by the lack of methods available to specifically alter circulating GH levels. This review summarizes the findings from recent studies using a newly developed peptidic antagonist to GH-releasing factor (GRF-AN) as a method of specifically inhibiting GH release. Systemic injection of GRF-AN was effective in suppressing the pulsatile release of GH, and in significantly attenuating the rate of growth, in both immature and adult rats. However, the inhibition of growth was associated with a reduction in net Pi retention only in immature rats, resulting in a doubling in the urinary excretion of Pi. GRF-AN treatment of immature rats lead to a decrease in the maximum tubular capacity to transport Pi-down to the level seen in adult rats. However, GRF-AN treatment did not alter renal Pi reabsorption in adult rats. We conclude that chronic administration of an antagonist to GRF in rats provides a new model of GH deficiency with which to study the interrelationships between growth, GH and other physiological systems. Furthermore, the findings suggest that the pulsatile release of GH, directly or indirectly, contributes to the high rate of renal Pi reabsorption in young, growing animals and may play a critical role in regulating Pi homeostasis during development.

Neuropeptide Y in the hypothalamus: effect on corticosterone and single-unit activity.

The purpose of the present study was to determine whether neuropeptide Y (NPY) acts within the hypothalamic paraventricular nucleus (PVN) or the suprachiasmatic nucleus (SCN) to alter circulating levels of corticosterone and to evaluate the effects of NPY on the single-unit response of PVN and SCN neurons using the hypothalamic slice preparation. Blood levels of corticosterone were determined in groups of rats that received microinjections of NPY or saline (Sal) into the PVN or SCN. NPY injected into the PVN 4 h after light onset resulted in corticosterone levels of 13.15 +/- 2.18 (SE) micrograms/dl within 1 h, which were significantly higher than the corticosterone levels of 4.08 +/- 1.78 micrograms/dl seen in rats receiving Sal injections. In contrast, no significant differences were observed in circulating levels of corticosterone between groups of rats 1 or 4 h after NPY or Sal microinjection into the SCN. In the hypothalamic slice, NPY was found to produce primarily inhibitory responses in both SCN and PVN neurons. Forty-nine percent of the SCN units examined were inhibited. In addition, another 20% of the neurons tested in the SCN displayed excitation followed by more sustained inhibition. In the PVN, 45% of the units examined were inhibited by NPY, however, nearly 30% of the remaining neurons were significantly excited by NPY. In summary, NPY alters the electrical activity of both SCN and PVN neurons but appears to act only within the PVN to influence circulating levels of corticosterone. These and other data indicate that NPY acts as an important neurochemical messenger within several hypothalamic sites.

Acute effects of nicotine on prolactin release in the rat: agonist and antagonist effects of a single injection of nicotine.

The effects of nicotine on prolactin release were studied in conscious, unrestrained rats in which an indwelling jugular cannula allowed multiple samplings of blood after i.v. administration of nicotine. Intravenous administration of nicotine bitartrate dihydrate increases plasma prolactin concentrations in a dose-dependent manner with an ED50 of approximately 100 micrograms/kg (200 nmol/kg) and this effect is blocked completely by pretreatment with mecamylamine, indicating that it is mediated by a nicotinic cholinergic receptor. Intracerebral ventricular injection of 1 microgram of nicotine also increases plasma prolactin levels, but i.v. injection of this same amount of nicotine has no effect, indicating that nicotine acts within the brain to release prolactin. A single i.v. injection of nicotine resulted in desensitization of the prolactin response to a subsequent injection of nicotine given 1 to 2 hr later, thus confirming a previous report by Sharp and Beyer (J. Pharmacol. Exp. Ther. 238: 486-491, 1986). The prolactin response to nicotine was restored within 24 hr after a single injection. The acute desensitization after a single injection of nicotine appears to be specific to release of prolactin by nicotine because the prolactin response to morphine was unaffected 1 hr after injection of nicotine. A single injection of nicotine appears to desensitize the prolactin response to a subsequent injection of nicotine with an ED50 of approximately 20 micrograms/kg (40 nmol/kg), indicating that nicotine is even more potent in stimulating desensitization of nicotinic cholinergic receptors than in stimulating prolactin release. These results support the concept that nicotine acts as a time-averaged antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic administration of nicotine on prolactin release in the rat: inactivation of prolactin response by repeated injections of nicotine.

The effects of chronic injections of nicotine on nicotine-induced prolactin release in the rat were measured and compared to the effects of this treatment on [3H]acetylcholine binding to nicotinic cholinergic sites in the hypothalamus. Treatment with nicotine for 10 days (s.c. injections twice daily) abolished prolactin release in response to an acute i.v. injection of nicotine given 2, 6 or 8 days after the last of the chronic injections of nicotine. At each of these time points, the binding of [3H]acetylcholine in the hypothalamus from rats treated chronically with nicotine was significantly higher than in the hypothalamus from control rats. By 14 days after the last chronic injection of nicotine, the prolactin response to an acute injection of nicotine was restored. Coinciding with the return of the nicotine-induced prolactin response, the binding of [3H]acetylcholine had returned to control values. These results are consistent with the hypothesis that nicotine inactivates nicotinic cholinergic receptors in brain by an allosteric mechanism, and that prolonged inactivation of nicotinic cholinergic receptors leads to their increased number.

Destruction of the dorsal anterior hypothalamic region suppresses pulsatile release of follicle stimulating hormone but not luteinizing hormone.

The region of the paraventricular nucleus-dorsal anterior hypothalamic area (PVN-DAHA) previously was implicated in the control of tonic FSH secretion. However, the role that this hypothalamic area plays in governing pulsatile FSH release is unknown. To examine this question, radiofrequency (RF) lesions were produced bilaterally in the PVN-DAHA of adult female rats which had been ovariectomized for 4 weeks. Control animals received sham lesions. After a recovery period of 1 week, all rats were fitted with jugular cannulae. The next day sequential blood samples were withdrawn from conscious, undisturbed rats at 10-min intervals for 3 h. Control animals displayed secretory peaks of FSH in plasma with a frequency of 4.0 +/- 0.44/3-hour period (or 1 peak/40-50 min). LH in plasma pulsed at a frequency of 5.8 +/- 0.49 peaks/3 h (or 1 peak/20-30 min). Both of these control values were in agreement with previous studies. RF lesion of the PVN-DAHA reduced FSH peak frequency to 1.2 +/- 0.37 peaks/3 h (p less than 0.001) and also significantly suppressed the mean peak height and trough values for FSH (p less than 0.001). In contrast, none of these parameters for pulsatile LH secretion was altered by the lesion. Immediately after the 3-hour sampling period, synthetic LHRH (50 ng/100 g BW) was infused intravenously into rats and blood samples withdrawn 10 and 40 min later to determine whether the responsiveness of the pituitary gland had changed as a result of the lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonist to GH-releasing factor inhibits growth and renal Pi reabsorption in immature rats.

Compared with adult rats, the immature rat has an enhanced tubular capacity for phosphate reabsorption, which presumably facilitates the growth process. Since the main driving force for growth is thought to be the pulsatile release of growth hormone, we examined the possibility that the adaptation in phosphate handling by the immature kidney is promoted by growth hormone (GH). To address this issue, we used a synthetic peptide antagonist to GH-releasing factor (GRF-AN) that we have shown blocks episodic GH secretion, and attenuates somatic growth. Immature male Wistar rats (4-5 wk of age) were catheterized with Silastic jugular cannulas and placed in metabolic cages. The rats were injected intravenously with either saline or GRF-AN (100 micrograms/kg) twice daily for 4 days. On the 4th day, they were prepared for renal clearance experiments to assess the maximum capacity for phosphate transport (TmPi). In animals treated with GRF-AN, there was an attenuated gain in body weight over 4 days of treatment (5 +/- 2 vs. 23 +/- 2% in saline controls, P less than 0.01). The suppressed growth was associated with a doubling of daily urinary phosphate excretion, and a reduction in the TmPi (3.3 +/- 0.1 vs. 4.6 +/- 0.3 mumol/ml in controls, P less than 0.01). A single injection of the antagonist to a separate group of immature rats did not alter TmPi. Thus injections of this new antagonist to GH-releasing factor over a 4-day period inhibit the pulsatile release of GH and significantly attenuate growth. The decline in growth of the immature rat was associated with a decrease in the renal capacity for phosphate reabsorption, down to levels seen in normal adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide-Y suppresses pulsatile secretion of luteinizing hormone in ovariectomized rats: possible site of action.

The hypothesis that neuropeptide-Y (NPY) suppresses pulsatile LH secretion in ovariectomized (OVX) rats was examined. Rats were bilaterally OVX and 6 weeks (Exp 1) or 2 weeks (Exp 2) later a stainless steel cannula was implanted in the third cerebral ventricle (3V). Seven to 10 days later, an intraatrial cannula was inserted. The next day, a blood sample was withdrawn, and each conscious unrestrained animal received a 3V injection of synthetic porcine NPY (5 or 0.5 micrograms/2 microliters saline) or vehicle in Exp 1. Blood samples were taken every 10 min for 2 h and centrifuged, and the plasma was analyzed for LH by RIA. In Exp 2, OVX rats received a 3V injection of NPY (5 micrograms/2 microliters) or vehicle. Blood samples were taken before and 60 min after injection. At 60 min, LHRH (10 ng/100 g BW) was injected iv, and blood was withdrawn 10, 20, 60, and 120 min later. NPY caused a dramatic dose-related reduction in the pulsatile release of LH compared to that in vehicle-treated rats. The 5.0-micrograms dose of NPY significantly reduced LH pulse frequency (P less than 0.05), pulse amplitude (P less than 0.01), and trough levels (P less than 0.01) compared to those in saline-injected controls. The lower dose of NPY (0.5 micrograms) significantly decreased the mean LH levels throughout the 2-h sampling period and slightly, though not significantly, the pulse frequency. Administration of LHRH increased plasma LH levels by 124% in control animals and by 1239% in NPY-injected rats. The results of these studies indicate that the suppressive effects of NPY on pulsatile LH release appear to be exerted through inhibition of pulsatile LHRH secretion from the hypothalamus.

Blockade of growth hormone-releasing factor (GRF) activity in the pituitary and hypothalamus of the conscious rat with a peptidic GRF antagonist.

Microinjection of synthetic GRF into the cerebroventricles or hypothalamus of the rat produces a number of neural effects, including the suppression of GH secretion, possibly representing a negative ultrashort loop autoregulation of GRF and/or stimulation of somatostatin neurosecretion. To demonstrate that such neuromodulation acts physiologically through endogenous GRF activity, the peptidic GRF antagonist (N-Ac-Tyr1,D-Arg2)GRF-(1-29)-NH2 was used to block the action of GRF on its presumed receptors in the hypothalamus. First, to establish the efficacy of the antagonist to block GRF receptors in the anterior pituitary, we injected the antagonist iv at doses of 2, 20, and 50 micrograms or saline (controls) into conscious male rats fitted with jugular cannulae. Sequential blood sampling every 15 min for 6 h between 1000-1600 h showed that 50 micrograms antagonist, iv, significantly suppressed the two periods of spontaneous release of radioimmunoassayable GH in controls in the morning and afternoon. A dose of 20 micrograms, iv, lowered mean plasma GH between 1400-1500 h (P less than 0.025), while the 2-microgram dose was without effect. The GRF antagonist was then microinjected into the third ventricle (3V) of conscious male rats at doses of 0.5 and 8.0 ng in 2 microliter sterile saline. The 8.0-ng dose of 3V antagonist elicited a 3-fold increase in the morning peak of GH (nanograms per ml): 3V antagonist, 159.0 +/- 62.0; 3V control, 51.0 +/- 21.9 (P less than 0.05). The 0.5-ng dose was without effect. Finally, we observed that pretreatment with the GRF antagonist 3V (10 ng), followed 15 min later by 10 ng rat GRF administered 3V, completely blocked the GRF-induced suppression of pulsatile GH release observed earlier. Both the systemic and central effects of the antagonist were specific to the control of GH, since PRL concentrations were unaltered. These results 1) have demonstrated the ability of a peptidic GRF antagonist to specifically suppress pulsatile GH release after its systemic administration, presumably by acting on pituitary GRF receptors, and 2) support the notion that GRF receptors are also present in the hypothalamus and are available for the physiological mediation of GRF-induced inhibition of GH release by a central mechanism.

Inhibition of pulsatile growth hormone (GH) secretion and somatic growth in immature rats with a synthetic GH-releasing factor antagonist.

We previously reported that systemic administration of the recently described GRF peptide antagonist (N-Ac-Tyr1,D-Arg2)GRF-(1-29)-NH2 to adult male rats would suppress the pulsatile release of GH. In the present study, we have sought to determine whether this same antagonist would be efficacious in immature male rats to block spontaneous GH secretion and, as a result, retard several parameters of somatic growth. Indwelling Silastic catheters were placed into the jugular veins of immature male rats (120-140 g) at 29 days of age. After a recovery period of 48 h, beginning at 1000 h, 100-400 micrograms/kg GRF antagonist or its vehicle (controls) were injected iv immediately after withdrawing an initial blood sample from conscious undisturbed animals. Subsequent samples were obtained every 20 min until 1520 h. Red blood cells were resuspended in a restorative volume of saline and reinjected after each blood sample. Results showed that both doses of antagonist prevented the two major periods of episodic GH release observed in controls. For example, mean plasma GH (+/- SEM; nanograms per ml) at 1120 h was 9.0 +/- 2.7 in antagonist-treated rats and 37.1 +/- 5.1 in controls (P less than 0.05). Mean plasma GH (+/- SEM) at 1340 h was 10.8 +/- 3.7 in antagonist-treated rats and 38.8 +/- 9.6 in controls (P less than 0.05). Injection of 400 micrograms/kg of the structurally related VIP antagonist (N-Ac-Tyr1,D-Phe2)GRF-(1-29)-NH2, iv failed to suppress spontaneous GH release. GRF antagonist (100 micrograms/kg) was next administered twice daily iv for 4 days to 31-day-old rats in metabolic cages. This treatment essentially arrested the normal rapid body weight gain, significantly suppressed increases in body and tail lengths, and reduced increases in heart and kidney weights (P less than 0.01). Food intake and fecal output were unchanged by antagonist treatment and, therefore, did not contribute to the observed effects. These results support the idea that a number of tissues and organs are stimulated by the pulsatile secretion of GH and that a peptidic GRF receptor antagonist is useful in blocking episodic GH release in immature animals. As a consequence, this specific antagonist is effective in suppressing numerous aspects of somatic growth.