Involvement of nitric oxide in growth hormone (GH)-releasing hormone-induced GH secretion in rat pituitary cells.

The involvement of nitric oxide (NO) in human GH-releasing hormone (hGHRH)-induced GH secretion was studied with freshly dissociated male rat pituitary cells. The cells were packed in a column of Bio-Gel-P2 and continuously perifused at 37 C. Hemoglobin (Hb; 10 microM), which is known to strongly bind NO, potentiated 0.01, 0.1, and 1 nM hGHRH-induced GH secretion by 73%, 52%, and 39%, respectively, without affecting the basal secretion of GH. As reported previously, 1-nM or higher concentrations of hGHRH elicit an increase in GH secretion during the application of hGHRH (on-response) and also a transient increase after the cessation of hGHRH application (off-response). It was found that Hb potentiated only the off-response in 1 nM hGHRH-induced GH secretion, and the same concentration of Hb had no effect on 10 nM hGHRH-induced GH secretion. N-Methyl-L-arginine (MeArg; 500 microM), a competitive inhibitor of NO synthase, also potentiated both the on- and off-responses of 1 nM hGHRH-induced GH secretion by 39% without affecting basal GH secretion. Since cAMP is thought to be an intracellular messenger of hGHRH action, the effects of Hb and MeArg on 1 mM (Bu)2AMP-induced GH secretion were examined. Their actions were found to be greater than those in hGHRH-induced GH secretion. Excess K+ (15 and 50 mM)-induced GH secretion, which does not involve cAMP, however, was not affected by either Hb or MeArg. In contrast, 3 mM sodium nitroprusside, which releases NO, suppressed the 1 nM hGHRH-induced off-response by 18%. The same concentration of sodium nitroprusside had no effect on excess K(+)-induced GH secretion. The effect of 8-bromo-cGMP on hGHRH-induced GH secretion was also examined, since NO is thought to exert its action through cGMP by activating guanylate cyclase in neural tissue. The application of 8-bromo-cGMP, however, did not affect 1 nM hGHRH-induced GH secretion. These observations suggest that hGHRH stimulates the synthesis of NO at least partly through cAMP, thereby partially inhibiting hGHRH-induced GH secretion.     

Endogenous testosterone enhances growth hormone (GH)-releasing factor-induced GH secretion in vitro

The influence of endogenous gonadal steroids in male and female rats on basal and growth hormone-releasing factor (GRF)-stimulated GH secretion from perifused anterior pituitaries was studied. After 75 min of perifusion with basal medium, freshly dissected pituitaries were exposed to human GRF(1-44) (10 nmol/l) for 15 min. Neonatal (day 1-2) or prepubertal (day 25) gonadectomy of male rats suppressed baseline GH release (ng/min per mg dry weight) as well as GRF-stimulated GH release by 40-70%. This effect was slightly more pronounced in neonatally gonadectomized animals. In prepubertally gonadectomized male rats, the suppression of GH release was completely reversed by testosterone replacement therapy. In female rats, prepubertal gonadectomy did not affect GH secretion from perfused pituitaries. However, treatment of ovariectomized female rats with oestradiol reduced baseline and GRF-induced GH release to levels lower than those observed in sham-operated or vehicle-treated ovariectomized animals. The data suggest that testicular androgen secretion in adult male rats increases the pituitary GH release in response to GRF in vitro, whereas ovarian oestrogen secretion is of less importance for the GRF responsiveness of female rat pituitaries.     

一氧化氮在L-精氨酸促生长激素分泌中的作用

氨基酸是促进垂体生长激素(GH)分泌的潜在刺激因子.在所有氨基酸中,左旋精氨酸(L-精氨酸)促GH分泌作用最为明显.口服或静脉滴注精氨酸均可刺激垂体GH的分泌.L-精氨酸促GH的分泌可能涉及多种机制,但一氧化氮(NO)依赖的可溶性鸟苷酸环化酶-环鸟苷酸-蛋白激酶G(sGC-cGMP-PKG)信号转导通路在此过程中起着重...     

Endogenous growth hormone (GH) secretion in male rats is synchronized to pulsatile GH infusions given at 3-hour intervals

The feedback effects of GH on its own secretion were studied in conscious male rats receiving intermittent iv infusions of human GH. Male Sprague-Dawley rats (150-180 g) were implanted with double bore iv cannula. Infusions of human GH (hGH) or buffer were given for up to 32 h, while frequent microsamples (20 microliters) of blood were withdrawn simultaneously using an automatic blood-sampling system. The endogenous GH pulses became synchronized to pulsatile hGH infusions (2.1 U/kg.infusion) given at 3-h intervals. After two or more hGH infusions episodic GH release was present in most rats, and all endogenous pulses occurred concomitantly with the hGH infusions. After 24 h of hGH treatment the endogenous pulses were still synchronized to the every 3 h hGH infusions. In addition, the pulse amplitude was lower than that in vehicle-treated animals (74 +/- 12 vs. 215 +/- 35 ng/ml; P less than 0.01). At this time a complete (1.5-h) phase shift of the 3-hourly hGH infusions markedly suppressed endogenous GH pulses in all rats. In another experiment where the same daily dose of hGH was given in iv infusions every 1.5 h instead of every 3 h, the endogenous GH pulses were irregular, infrequent, and suppressed. Infusions at 3-h intervals of a lower dose of hGH (0.42 U/kg.infusion) did not affect the timing or amplitude of endogenous GH pulses compared to those in buffer-infused animals. The endogenous GH pulses were not synchronized between animals given 0.42 U/kg hGH or buffer at 3-h intervals. It is concluded that the endogenous GH pulses in male rats became synchronized to intermittent infusions of hGH at 3-h (but not 1.5-h) intervals. The fact that there were no endogenous pulses between the 3-hourly infusions suggests that the feedback effect of a GH pulse lasts for approximately 3 h. This mechanism may be involved in the control of the GH secretory pattern in male rats.     

Involvement of cyclic guanosine 3',5'-monophosphate in nitric oxide-induced glucagon secretion from pancreatic alpha cells

It has been reported that nitric oxide (NO) is a positive modulator of glucagon release. The involvement of cyclic guanosine 3',5'-monophosphate (cGMP) in NO-induced glucagon secretion and the possible role of NO in glucagon release induced by l-arginine were investigated in mouse clonal alpha-cell line clone 6 (alpha TC6) cells, which predominantly secrete glucagon. NOC12, an NO donor, elicited an increase in glucagon release from alpha Tc6 cells in perifusion and static incubation. An inhibitor of cGMP-dependent protein kinase inhibited NOC12-induced glucagon release. NOC12 (1 mmol/L) also increased the cellular level of cGMP. In addition, a permeable cGMP agonist increased glucagon release. l-arginine (15 mmol/L) increased perifusate concentrations of glucagon and nitrite in alpha Tc6 cells, which were inhibited by N(G)-nitro-L-arginine methyl ester. NO synthase (NOS) activity was shown in alpha Tc6 cells by l-citrulline formation assay. Our present findings suggest that NO plays a stimulating role in glucagon release from the alpha cells, and that a cGMP-dependent pathway is involved in NO action. These findings also provide further evidence that l-arginine might play a stimulating role in regulating glucagon secretion, at least partly, through generation of NO in the islets.     

Ceramide enhances growth hormone (GH)-releasing hormone-stimulated cyclic adenosine 3',5'-monophosphate accumulation but inhibits GH release in rat anterior pituitary cells

In this study, the effect of ceramide on GH-releasing hormone (GHRH)-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. C2-, C6-, and C8-ceramide were found to enhance GHRH-stimulated cAMP accumulation. In contrast, their effects on GHRH-stimulated GH release were inhibitory. Treatment with a glucosylceramide synthase inhibitor produced a similar enhancing effect on cAMP accumulation and an inhibitory effect on GH release. To identify the pathway through which ceramide mediated its effect, it was found that ceramide inhibited GH release stimulated by KCl, BayK 8644, and a GH-releasing peptide, but not that stimulated by ionomycin or an activator of protein kinase C. Direct measurement of intracellular Ca2+ revealed that C2-ceramide inhibited GHRH- and KCl-mediated increases in intracellular Ca2+, suggesting that ceramide probably inhibits GH release through inhibition of the L-type Ca2+ channels. As for its mechanism on cAMP accumulation, the enhancing effect of ceramide on GHRH-stimulated cAMP accumulation was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine, suggesting that ceramide enhances the cAMP response through inhibition of its metabolism. Taken together, our results suggest that ceramide plays an important role in the regulation of GHRH-stimulated responses in somatotrophs. By reducing GH secretion while enhancing cAMP accumulation, ceramide may promote the synthesis and storage of GH in rat anterior pituitary cells.     

Extracellular renal guanosine cyclic 3'5'-monophosphate modulates nitric oxide and pressure-induced natriuresis

This study addresses the hypothesis that NO- and pressure-induced natriuresis are inhibited when guanosine cyclic 3',5'-monophosphate (cGMP) is prevented from being transported outside its renal synthesizing cells in vivo. Rats received a renal interstitial (RI) infusion of NO donor S-nitroso-N-acetylpenicillamine (SNAP) or SNAP+organic anion transporter inhibitor probenecid (PB) or SNAP+PB+cGMP. SNAP alone increased U(Na)V (P<0.05 at 1 hour and P<0.005 at 2 hours). In contrast, SNAP failed to increase U(Na)V when coinfused with PB, but cGMP coinfused with SNAP+probenecid restored the natriuretic response. SNAP alone increased RI cGMP (P<0.05) during the second experimental period. PB abolished the increase in RI cGMP in response to SNAP (P<0.01), but cGMP levels were restored by coinfusion with cGMP. PB also abolished SNAP-induced increases in fractional excretion of Na(+) (FE(Na)) and lithium (FE(Li)) (both P<0.01). PB also abolished the rise in RI cGMP and natriuresis induced by raising renal perfusion pressure (RPP) from 100 to 160 mm Hg in rats subjected to a standard pressure-natriuresis protocol and the natriuretic response was rescued by coinfusion with cGMP. RI administration of phosphodiesterase type V (PDE V) reduced both RIcGMP and U(Na)V in parallel (both P<0.01) without altering RIcAMP. The data demonstrate that export of cGMP from its renal synthesizing cells into the extracellular RI compartment is critical for the natriuretic action of NO donor SNAP or increased RPP and that RI cGMP controls basal Na(+) excretion. Extracellular cGMP modulates NO- and pressure-induced natriuresis.     

Involvement of nitric oxide in the regulation of growth hormone secretion in dogs

Nitric oxide (NO) is a highly reactive gas that has been suggested to function as a neurotransmitter in the neuroendocrine system. In this work, we have evaluated the role of NO pathways in growth hormone (GH) secretion by assessing the effect of L-arginine infusion, a precursor of NO formation, and L-NAME, a nitric oxide synthase (NOS) inhibitor. The experiments were carried out on 7 adult beagle dogs. A saline infusion was carried out on all the dogs as a control test. L-arginine (infusion i.v. 10 g in 100 ml of saline, from t = 0 to 30 min) and L-nitro-arginine-methyl ester, L-NAME (infusion of 300 microg/kg in 120 ml of saline, from t = -30 to 45 min) were administered alone and together with growth hormone-releasing hormone (GHRH) (i.v. bolus at 0 min, at a dose of 100 microg), the synthetic GH secretagogue GHRP-6 (i.v. bolus at 0 min, at a dose of 90 microg), and the 5-HT1D serotonin receptor agonist sumatriptan, SUM (s.c. injection at the dose of 3 mg). Plasma cGH was determined by RIA. Results were evaluated by one-way analysis of variance, followed by the Newman-Keuls test for multiple comparisons. L-arginine administration resulted in a slight increase in plasma cGH in comparison with saline controls. Combined administration of L-arginine and GHRH enhanced cGH release in comparison with GHRH alone. L-NAME alone did not modify baseline cGH levels, but completely suppressed the GH release induced by GHRH or GHRP-6. It also strongly reduced, but did not abolish the effect of the two peptides (GHRH plus GHRP-6) administered together. Finally, administration of the 5-HT1D agonist SUM induced a significant cGH secretion in all dogs, a response which was not modified when L-NAME was administered in combination with SUM. In conclusion, our data show that inhibition of NO blunts both GHRH or GHRP-6-induced cGH release, and are compatible with the hypothesis that it acts by decreasing hypothalamic somatostatin release.     

Immunocytochemical localization of cyclic guanosine monophosphate-dependent protein kinase in endocrine tissues

Antisera have been produced against purified soluble cyclic guanosine monophosphate (cGMP) dependent-protein kinase (ATP: protein phosphotransferase EC 2.7.1.37) isolated from bovine lung. No cross-reactivity was observed between the antisera and structurally related components of cAMP-dependent protein kinases (cAMP kinase), as judged by the immunodiffusion and immunoprecipitation techniques. Immunocytochemical specificity was determined by absorption of antisera with pure antigen. The distribution of cGMP kinase has been examined in several rat tissues, using an indirect immunofluorescence technique, and compared with the immunocytochemical distribution of cGMP. In skeletal muscle, cGMP kinase was localized primarily to A bands on the muscle fiber and along the Z line in I band regions. Densitometric determinations of immunoperoxidase staining indicated that absorbance over A band areas was greater than absorbance over the I band regions. In small intestine, cGMP kinase is distributed primarily along the villus brush border membrane. In testis, cGMP kinase is observed in several cell types adjacent to the seminiferous tubular wall, including Sertoli cells and spermatogonia, as well as in association with meiotic chromosomes of pachytene spermatocytes. In the cortex of the adrenal glands from dexamethasone-suppressed rats, chronic ACTH treatment induced an increase in cGMP kinase fluorescence in nuclei. In each of the tissues examined, a striking correlation was observed between the distribution of cGMP kinase and cGMP, supporting the hypothesis that cGMP-mediated actions occur via cGMP kinases.     

The somatostatin analog octreotide inhibits the secretion of growth hormone (GH)-releasing hormone, thyrotropin, and GH in man

The effects of the somatostatin analog octreotide on plasma GH, TSH, and immunoreactive GH-releasing hormone (IR-GHRH) were studied in 10 normal men. After morning sc administration of 50 or 100 micrograms octreotide or placebo, plasma GH, TSH and GHRH were measured frequently for 6 h. Plasma GH or IR-GHRH concentrations did not change after placebo injection, but plasma TSH levels gradually decreased, in conformity with a circadian rhythm during the morning. The mean plasma GH levels after sc injection of 50 or 100 micrograms octreotide declined, and no spontaneous GH pulses occurred for 5 h. Plasma TSH decreased rapidly after both doses of octreotide and was significantly lower than the level after placebo treatment from 90-315 min (P less than 0.05) and 60-360 min (P less than 0.05 or P less than 0.01), respectively. Plasma IR-GHRH levels also were significantly lower from 30-360 min (P less than 0.05) in the group given 100 micrograms octreotide compared with the value in the placebo group. We conclude that octreotide inhibits not only GH and TSH secretion from the pituitary, but also GHRH release from the hypothalamus and/or peripheral tissues. These findings suggest that somatostatin controls GH secretion not only by suppressing pituitary secretion of GH but also by suppressing GHRH release from the hypothalamus.     

SNAP对大鼠生长激素腺瘤GH3细胞增殖、生长激素分泌的影响及机制探讨

目的观察一氧化氮(NO)释放剂S-亚硝基-N-乙酰青霉胺(SNAP)对大鼠生长激素(GH)腺瘤GH3细胞增殖、GH分泌的影响,并探讨其机制。方法将细胞分为A、B、C、D组,A组常规培养,B、C、D组分别加ghrelin、SNAP、SNAP+ghrelin培养。分别于培养第0、1、2、3、4、5、6天,用MTT法检测细胞吸光度值观察细胞增殖情况;培养2 h后,用ELISA法检测GH3细胞培养液中的GH,Western blot法检测GH3细胞中的磷酸化细胞外信号调节激酶(p-ERK)。结果 A组培养第2、3、4、5、6天时细胞吸光度值分别为0.381±0.006、1.664±0.005、3.411±0.005、5.221±0.005、10.055±0.005,B组分别为1.062±0.005、3.115±0.005、4.512±0.005、6.656±0.005、11.060±0.005,C组分别为0.161±0.006、0.413±0.005、0.931±0.005、1.861±0.005、5.612±0.005,D组分别为0.219±0.004、0.865±0.005、1.712±0.005、3.059±0.005、6.561±0.005;B组与A组、C组与A组、D组与B组比较,P均<0.01。培养2 h时,A、B、C、D组GH3细胞培养液的吸光度值分别为0.080±0.002、0.165±0.011、0.041±0.003、0.106±0.005,pERK表达量分别为0.301 8±0.004 5、0.682 5±0.003 8、0.192 3±0.008 6、0.298 5±0.006 7,B组与A组、C组与A组、D组与B组比较,P均<0.01。结论 SNAP可抑制ghrelin诱导的GH3细胞增殖及GH分泌,可能与其阻断ghrelin激活的ERK信号通路有关。     

Leptin regulates GH gene expression and secretion and nitric oxide production in pig pituitary cells.

The aim of this study was to investigate the direct effect of leptin on GH gene expression and secretion and the role of nitric oxide as a possible mediator in pig anterior pituitary cells. Pituitary cells from adult sows were treated for 4 or 24 h with rhleptin (from 0.1 nM to 1 microM) alone or in association with GHRH (10 nM) or hexarelin (10 nM). At the end of incubation, medium was collected for GH and nitric oxide determination by ELISA and Griess test, respectively. Total RNA was collected from cells, and GH gene expression was measured by RT-PCR. Leptin significantly (P < 0.001) stimulated GH secretion in both incubation periods. The maximum response was induced by 10 nM leptin; furthermore, a significant interaction (P < 0.002) between leptin and GHRH (P < 0.03) and between leptin and hexarelin was observed when the molecules were used in association. GH gene expression was significantly increased (at least P < 0.05) by hexarelin, GHRH, and leptin (1000 and 100 nM) after 24 h of treatment. Leptin (10 nM and 1 microM) significantly (P < 0.05) increased nitric oxide production, whereas S-nitroso-N-acetyl-penicillamine (from 0.01-1000 nM) significantly (P < 0.05) stimulated GH secretion. These data demonstrate that leptin directly influences GH regulation at the pituitary level, and nitric oxide may be involved in this function.     

Short term continuous intravenous infusion of growth hormone (GH) inhibits GH-releasing hormone-induced GH secretion: a time-dependent effect

We previously reported that GH secretion evoked by GHRH is inhibited after 5 days of treatment with im GH. This impaired pituitary response was associated with a significant increase in the serum concentration of insulin-like growth factor I (IGF-I). To dissociate the possible effects of circulating IGF-I from other effects of GH on the pituitary response to GHRH, we carried out the following study in eight normal men. A bolus injection of GHRH (1 microgram/kg, iv) was administered 2 h after the start of a 4-h continuous iv infusion of GH (180-micrograms bolus dose, then 3 micrograms/min in 150 mmol/L NaCl) or placebo (150 mmol/L NaCl). In addition, a similar injection of GHRH was given 4 h after the start of a 6-h continuous iv GH infusion (180-micrograms bolus dose, then 3 micrograms/min). During the GH infusions, plasma GH levels reached steady state concentrations in the 9-13 micrograms/L range. The mean GHRH-induced GH response was not significantly blunted during the 4-h infusions of GH [724 +/- 163 (+/- SE) vs. 1184 +/- 373 micrograms.min/L during placebo; P = 0.29], but was significantly inhibited during the 6-h GH infusions (226 +/- 105 micrograms.min/L; P = 0.04 vs. control). Serum IGF-I or plasma glucose did not change significantly throughout the GH infusions. During the 6-h GH infusions, plasma FFA increased to levels significantly above basal values during the last 3 h of the 6-h infusion. These results indicate that short term GH infusion inhibits the plasma GH response to GHRH in a time-dependent manner. The inhibition is not due to changes in circulating IGF-I and glucose concentrations. Fluctuations in hypothalamic somatostatin secretion, changes in lipid or other GH-dependent metabolites, paracrine effects of IGF-I, or a direct effect of GH itself may cause the impaired pituitary responsiveness during short term iv GH infusion.     

Evidence that nitric oxide is involved in the regulation of growth hormone secretion in goldfish

Whether nitric oxide (NO) plays a role in regulation of growth hormone (GH) secretion from somatotropes in the pituitary of the goldfish Carassius auratus was investigated. Immunocytochemistry with two antibodies against mammalian NO synthase (NOS) revealed the presence of a NOS-like enzyme in primary cultures of dispersed goldfish pituitary cells, including morphologically identified somatotropes. NO donors S-nitroso-N-acetylpenicillamine and sodium nitroprusside (SNP), as well as a cyclic guanosine monophosphate analogue (dibutyryl guanosine 3':5'-cyclic monophosphate), all significantly increased GH secretion from dispersed goldfish pituitary cells in static culture. Somatostatin abolished the response to SNP, and NOS inhibitors aminoguanadine hemisulfate (AGH) and N-(3-aminomethyl)benzylacetamidine, dihydrochloride (1400W) decreased the GH release response to known neuroendocrine factors stimulatory to GH release (gonadotropin-releasing hormone and a dopamine D1 agonist). AGH and 1400W did not alter basal GH secretion. These data suggest that NO plays a role in mediating the GH response to endogenous neuroendocrine factors in goldfish.     

Somatostatin inhibits basal and vasoactive intestinal peptide-stimulated hormone release by different mechanisms in GH pituitary cells

Somatostatin (SRIF) inhibits both basal and vasoactive intestinal peptide (VIP)-stimulated hormone secretion by the GH4C1 clonal strain of rat pituitary tumor cells. We have previously shown that SRIF inhibits cAMP accumulation stimulated by VIP but does not alter basal cAMP levels in this cell line. To determine the importance of changes in cAMP accumulation in the mechanism of SRIF action, we have compared the effect of SRIF on hormone release stimulated by VIP and two other secretagogues which increase effective intracellular cAMP concentrations: forskolin and 8-Bromo-cAMP (8-Br-cAMP). VIP stimulated GH and PRL secretion to the same maximal extent (220% of control) with similar ED50 values (0.37 +/- 0.03 and 0.43 +/- 0.08 nM, mean +/- SE, respectively). SRIF (100 nM) reduced maximal VIP-stimulation of both GH and PRL release from 220 to 140% of control; however, it did not significantly change the ED50 values for VIP. The effect of SRIF on VIP-stimulated hormone release parallels its action on VIP-stimulated cAMP accumulation. Furthermore, the concentrations of SRIF required to produce half-maximal inhibition of VIP-stimulated GH and PRL release (0.8 +/- 0.2 nM and 0.7 +/- 0.1 nM, respectively) were similar to its potency to inhibit VIP-stimulated cAMP accumulation (1.2 +/- 0.1 nM). These data indicate that changes in cAMP levels mediate inhibition of VIP-stimulated hormone secretion by SRIF. Forskolin increased cAMP accumulation with an ED50 value of 2.4 +/- 0.5 microM. A maximal concentration of forskolin (100 microM) stimulated cAMP accumulation to a greater extent than 100 nM VIP (34 +/- 4-fold vs. 9 +/- 1-fold). Together, forskolin (100 microM) and VIP (100 nM) stimulated cAMP accumulation by more than 50-fold. However, PRL secretion in response to maximal concentrations of VIP or forskolin individually or together were the same (approximately 200% of control). These results support the conclusion that both compounds stimulate PRL secretion by a cAMP-mediated mechanism which can be fully activated by either one alone.(ABSTRACT TRUNCATED AT 400 WORDS)