Differential contribution of nitric oxide and cGMP to the stimulatory effects of growth hormone-releasing hormone and low-concentration somatostatin on growth hormone release from somatotrophs

There is increasing evidence that nitric oxide (NO) produced by NO synthase (NOS), and their signalling partners, guanylyl cyclase and cGMP, play a relevant role in growth hormone (GH) secretion from somatotrophs. We previously demonstrated that both GH-releasing hormone (GHRH; 10(-8) M) and low concentrations of somatostatin (10(-15) M) stimulate pig GH release in vitro, whereas a high somatostatin concentration (10(-7) M) inhibits GHRH-induced GH secretion. To ascertain the possible contribution of the NOS-NO and guanylyl cyclase-cGMP routes to these responses, cultures of pituitary cells from prepubertal female pigs were treated (30 min) with GHRH (10(-8) M) or somatostatin (10(-7) or 10(-15) M) in the absence or presence of activators or blockers of key steps of these signalling cascades, and GH release was measured. Two distinct activators of NO route, SNAP (5x10(-4) M) or L-AME (10(-3) M), similarly stimulated GH release when applied alone (with this effect being blocked by 10(-7) M somatostatin), but did not alter the stimulatory effect of GHRH or 10(-15) M somatostatin. Conversely, two NO pathway inhibitors, NAME (10(-5) M) or haemoglobin (20 microg/ml) similarly blocked GHRH- or 10(-15) M somatostatin-stimulated GH release. 8-Br-cGMP (10(-8) to 10(-4) M) strongly stimulated GH release, suggesting that cGMP may function as a subsequent step in the NO pathway in this system. Interestingly, 10(-7) M somatostatin did not inhibit the stimulatory effect of 8-Br-cGMP. Moreover, although 8-Br-cGMP did not modify the effect of GHRH, it enhanced GH release stimulated by 10(-15) M somatostatin. Accordingly, a specific guanylyl cyclase inhibitor, LY-83, 583 (10(-5) M) did not alter 10(-15) M somatostatin-induced GH release, whereas it blocked GHRH-induced GH secretion. These results demonstrate for the first time that the NOS/NO signalling pathway contributes critically to the stimulatory effects of both GHRH and low-concentration somatostatin on GH release, and that, conversely, the subsequent guanylyl cyclase/cGMP step only mediates GHRH- and not low-concentration somatostatin-induced GH secretion from somatotrophs.     

Somatostatin dramatically stimulates growth hormone release from primate somatotrophs acting at low doses via somatostatin receptor 5 and cyclic AMP

Somatostatin and cortistatin have been shown to act directly on pituitary somatotrophs to inhibit growth hormone (GH) release. However, previous results from nonprimate species indicate that these peptides can also directly stimulate GH secretion, at low concentrations. The relevance of this phenomenon in a nonhuman primate model was investigated in the present study by testing the impact of somatostatin/cortistatin on GH release in primary pituitary cell cultures from baboons. High doses (> 10(-10) m) of somatostatin/cortistatin did not alter basal GH secretion but blocked GH-releasing hormone (GHRH)- and ghrelin-induced GH release. However, at low concentrations (10(-17)-10(-13) m), somatostatin/cortistatin dramatically stimulated GH release to levels comparable to those evoked by GHRH or ghrelin. Use of somatostatin receptor (sst) specific agonists/antagonists, and signal transduction blockers indicated that sst2 and sst1 activation via intact adenylate cylcase and mitogen-activated protein kinase systems mediated the inhibitory actions of high-concentration somatostatin. By contrast, the stimulatory actions of low-dose somatostatin on GH release were mediated by sst5 signalling through adenylate cylcase/cAMP/protein kinase A and intracellular Ca(2+) pathways, and were additive with ghrelin (not GHRH). Notably, low-concentrations of somatostatin, similar to sst5-agonists, inhibited prolactin release. These results clearly demonstrate that the ultimate impact of somatostatin/cortistatin on hormone release is dose-dependent, cell type-selective and receptor-specific, where the stimulatory effects of low-concentration somatostatin/cortistatin on GH release extend to primates, thereby supporting the notion that this action is relevant in regulating GH secretion in humans.     

Role of Nitric Oxide/Cyclic GMP Pathway in the Inhibitory Effect of GABA and Dopamine on Prolactin Release

The anterior pituitary gland is a site of nitric oxide (NO) production and action, suggesting a local regulatory function. We recently reported that NO inhibits in vitro prolactin release. The aim of the present study was to establish the mechanism of action of NO on prolactin release and to determine whether NO is involved in the inhibitory effect of GABA on prolactin release. Since NO exerts its action through cGMP by activating guanylate cyclase in different tissues, we examined the effect of sodium nitroprusside (NP), a NO releaser, on intrapituitary cGMP levels. Incubation of anterior pituitary glands with 0.5 mM NP 4-fold increased intrapituitary cGMP content, but decreased intrapituitary cAMP levels. In addition, we studied the effect of NP on prolactin release in the presence of LY 83583, an inhibitor of guanylate cyclase activity and 3-lsobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase activity. 10μM LY 83583 and 0.5 mM IBMX blocked the inhibitory effect of NP on prolactin release. (10^-3 M) 8Br-cGMP, an analogue of cGMP, mimicked the effect of NP on prolactin release. On the other hand, NO seems to be involved in the inhibitory effect of GABA on prolactin release since hemoglobin, a scavenger of NO, and N^w-nitro-L-arginine methyl ester, an inhibitor of NO synthase (NOS), blocked the pituitary response to GABA. Moreover, GABA (10 ⁶ M) stimulated NOS activity by almost 50%. GABA increased intrapituitary cGMP levels and decreased cAMP. Dopamine stimulated NOS activity weakly.
These observations suggest that NO, acting through the guanylate cyclase-cGMP pathway, inhibits prolactin secretion. In addition, NO may be involved in the inhibitory effect of GABA and dopamine on prolactin release.     

Nitric oxide directly inhibits ghrelin-activated neurons of the arcuate nucleus

The hypothalamic arcuate nucleus (Arc) is a target site for signals regulating energy homeostasis. The orexigenic hormone ghrelin directly activates neurons of the medial arcuate nucleus (ArcM) in rats. Nitric oxide (NO) is a neuromodulator implicated in the control of food intake and body weight. NO is produced by nitric oxide synthase (NOS) and induces the formation of cyclic guanosine monophosphate (cGMP) via a stimulation of soluble guanylate cyclase (sGC). Both enzymes NOS and sGC have been identified in the Arc. Using extracellular recordings we characterized the effects of NO signaling on ArcM neurons and their co-sensitivity to ghrelin. The artificial NO donor sodium nitroprusside (10(-4) M) reversibly inhibited 91% of all ArcM neurons by a direct postsynaptic mechanism. 52% of ArcM neurons were excited by ghrelin. In all but one of these neurons SNP caused inhibitory responses. The SNP-induced inhibitions were mediated by cGMP since they were blocked by the specific sGC inhibitor ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, 10(-4) M). Furthermore, the membrane permeating cGMP analogue 8-Br-cGMP (10(-4) M) mimicked the inhibitory responses of SNP. In immunohistological in vitro studies SNP induced a cGMP formation, which could also be blocked by ODQ. The current studies demonstrate that NO/cGMP signaling inhibits a large population of ArcM neurons including ghrelin-excited cells. Since an activation of the latter neurons is regarded as a correlate of negative energy balance, NO may represent an anorectic neuromodulator in the Arc and/or restrain the action of signals promoting energy intake. NO signaling in the Arc is also induced following inflammation suggesting a possible role of Arc-intrinsic NO in disease-related anorexia.     

The pregnancy-induced increase in baseline circulating growth hormone in rats is not induced by ghrelin

The elevation in baseline circulating growth hormone (GH) that occurs in pregnant rats is thought to arise from increased pituitary GH secretion, but the underlying mechanism remains unclear. Distribution, Fourier and algorithmic analyses confirmed that the pregnancy-induced increase in circulating GH in 3-week pregnant rats was due to a 13-fold increase in baseline circulating GH (P < 0.01), without any significant alteration in the parameters of episodic secretion. Electron microscopy revealed that pregnancy resulted in a reduction in the proportion of mammosomatotrophs (P < 0.01) and an increase in type II lactotrophs (P < 0.05), without any significant change in the somatotroph population. However, the density of the secretory granules in somatotrophs from 3-week pregnant rats was reduced (P < 0.05), and their distribution markedly polarised; the granules being grouped nearest the vasculature. Pituitary GH content was not increased, but steady-state GH mRNA levels declined progressively during pregnancy (P < 0.05). In situ hybridisation revealed that pregnancy was accompanied by a suppression of GH-releasing hormone mRNA expression in the arcuate nuclei (P < 0.05) and enhanced somatostatin mRNA expression in the periventricular nuclei (P < 0.05), an expression pattern normally associated with increased GH feedback. Although gastric ghrelin mRNA expression was elevated by 50% in 3-week pregnant rats (P < 0.01), circulating ghrelin, GH-secretagogue receptor mRNA expression and the GH response to a bolus i.v. injection of exogenous ghrelin were all largely unaffected during pregnancy. Although trace amounts of 'pituitary' GH could be detected in the placenta with radioimmunoassay, significant GH-immunoreactivity could not be observed by immunohistochemistry, indicating that rat placenta itself does not produce 'pituitary' GH. Although not excluding the possibility that the pregnancy-associated elevation in baseline circulating GH could arise from alternative extra-pituitary sources (e.g. the ovary), our data indicate that this phenomenon is most likely to result from a direct alteration of somatotroph function.     

Differential involvement of phosphoinositide 3-kinase in gonadotrophin-releasing hormone actions in gonadotrophs and somatotrophs of goldfish, Carassius auratus

In goldfish, two endogenous gonadotrophin-releasing hormones (GnRHs) [salmon (s)GnRH and chicken (c)GnRH-II] control maturational gonadotrophin-II [lutenising hormone (LH)] and growth hormone (GH) secretion via Ca(2+)-dependent intracellular signalling pathways. We investigated the involvement of phosphoinositide 3-kinase (PI3K) in GnRH-evoked LH and GH release and associated intracellular Ca(2+) increases ([Ca(2+)](i) ) in goldfish gonadotrophs and somatotrophs. Immunoreactive PI3K p85α, the predominant regulatory subunit for class IA PI3Ks, was detected in goldfish pituitary tissue extracts and both endogenous GnRH isoforms increased phosphorylation of PI3K p85α in excised pituitary fragments. sGnRH- and cGnRH-II-elicited LH release responses from primary cultures of pituitary cells and [Ca(2+)](i) increases in identified gonadotrophs were significantly reduced in the presence of PI3K inhibitors wortmannin (100 nm) and LY294002 (10 μm). Unexpectedly, wortmannin and LY294002 inhibited GnRH-evoked GH release but only attenuated the [Ca(2+)](i) response in identified somatotrophs to cGnRH-II, and not sGnRH. On the other hand, Ca(2+) ionophore-evoked LH and GH secretion remained unaltered in the presence of the PI3K inhibitors, suggesting that general decreases in the releasable hormone pool or sensitivity to [Ca(2+)](i) changes did not underlie the ability of wortmannin and LY294002 to reduce the actions of GnRH. These results provide the first evidence for the presence and involvement of PI3K in GnRH-induced LH and GH release in any primary pituitary cell system. In gonadotrophs, the inhibitory action of PI3K on both sGnRH and cGnRH-II involves the attenuation of their evoked [Ca(2+)](i); in contrast, GnRH isoform-specific effects occur in somatotrophs.     

Spinal cord nitric oxide synthase and heme oxygenase limit morphine induced analgesia.

Spinal cord tissue contains two enzyme systems capable of producing monoxide gases which in turn are linked to the stimulation of soluble guanylate cyclase, nitric oxide synthase (NOS) which produces NO and heme oxygenase (HO) which produces CO. Reports from several laboratories link these two enzyme systems to pain of inflammatory and neuropathic etiologies. Additional studies have demonstrated that the activation of the NOS system by morphine limits the spinal analgesic action of this drug. In this study we first employed the hot plate model of pain to demonstrate that the NOS inhibitor L-NAME and the HO inhibitor Sn-P potentiate the analgesic actions of intrathecally administered morphine while having no intrinsic analgesic action at the doses used. We then determined that L-NAME loses its ability to potentiate morphine in nNOS null-mutant mice, while Sn-P no longer potentiates morphine in mice lacking a functional HO-2 gene. The intrathecal injection of the cGMP analog 8-Br cGMP caused hyperalgesia in the hot plate assay. Focusing on the possible involvement of cGMP metabolism, we documented that morphine stimulates cGMP production in a spinal cord slice model in a concentration dependent and naloxone reversible manner. Both L-NAME and Sn-P were potent inhibitors of morphine-stimulated cGMP production. Buffer containing either CO or the NO donor compound SNAP stimulated cGMP production as well. In spinal cord slices from either nNOS or HO-2 null-mutant animals morphine did not stimulate cGMP production. Taken together our data suggest that spinal monoxide generation modifies the acute analgesic actions of morphine.     

The Nitric Oxide - Cyclic GMP Pathway and Synaptic Depression in Rat Hippocampal Slices

The ability of exogenous nitric oxide (NO) to modify synaptic transmission was investigated in area CA1 of the rat hippocampal slice. The NO donors S -nitroso- N -acetylpenicillamine (SNAP) and S -nitrosoglutathione (SNOG) depressed field excitatory postsynaptic potentials evoked by low frequency stimulation of the Schaffer collateral - commissural pathway. Upon washout of the NO donors, synaptic transmission rapidly returned to control levels. A similar reversible synaptic depression was produced by SNAP when tetanic stimulation (100 Hz; 1 s) was delivered in its presence. The effect of SNAP was not mimicked by its precursor or breakdown product and was blocked by haemoglobin, indicating that the effect involved NO. Roussin's black salt, a photolabile NO donor, also depressed transiently field excitatory postsynaptic potentials following photolysis. The depression was induced rapidly following a flash of UV light (20 s duration) focused onto the slice using a confocal microscope. The depressant effect of the NO donors on synaptic transmission was mimicked by zaprinast, a specific cGMP - phosphodiesterase inhibitor. Zaprinast depressed to a similar extent both the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate and N -methyl- d -aspartate receptor-mediated components of excitatory postsynaptic currents without affecting passive membrane properties, indicating a presynaptic locus of action. SNAP, SNOG and zaprinast all elevated cGMP levels in rat hippocampal slices. Immunocytochemical staining revealed that the cGMP accumulation was mainly in a network of varicose fibres running throughout the CA1 region, consistent with a presynaptic site of action of NO. We conclude that NO, possibly through activation of guanylate cyclase, may be involved in transient presynaptic depression in the CA1 region of the hippocampus.     

Nitric oxide involvement in the anxiogenic-like effect of substance P

This study investigates whether nitric oxide (NO) is involved in the anxiogenic profile of action of substance P (SP) in mice in the elevated plus-maze (EPM). Adult Swiss mice were injected with NOS inhibitors such as L-NOARG (20 nmol/kg) i.p., L-NAME (3 nmol per site), 7-NI (0.25 nmol per site) i.c.v. or vehicle (NaCl 0.9% i.p. or PBS i.c.v.). About 30 min (i.p. pretreatment) or 5 min later (i.c.v. pretreatment), the animals received i.c.v. injections of SP (10 pmol) or phosphate buffered saline (PBS) (2 microl). Afterwards, they were observed in the EPM. SP per se reduced the time spent on open arms, an anxiogenic-like effect. This effect was reverted by different NOS inhibitors and the NO donor. NOS inhibitors had no influence on the EPM parameters but the NO-releasing compound SNAP, as well as its parent thiol NAP, increased the animals' locomotor activity. 8-Br-cGMP (20 nmol), a permeable cGMP analog, promoted an anxiogenic-like effect per se and enhanced the SP effect on the EPM. Altogether, these results suggest a putative NO role in the mediation of the anxiogenic-like effect of SP.     

Motilin: A novel growth hormone releasing agent

Motilin, a gastrointestinal peptide recently detected in the rat brain, was capable of stimulating growth hormone (GH) release from dispersed anterior pituitary cells in a dose-related fashion. In initial experiments, the minimum effective concentration was 10(-7) M and the effect was specific for just GH. Subsequent experiments demonstrated that concentrations of synthetic motilin as low as 10(-9) M could significantly stimulate GH release. Only large IV doses (100 micrograms) of motilin significantly elevated circulating GH levels in vivo. However, administration of antiserum to porcine motilin (100 microliters, IV) significantly depressed plasma GH levels, suggesting a physiologic role for median eminence and hypothalamic motilin in the control of GH secretion. Furthermore, infusion of motilin into the third ventricle of conscious rats resulted in a significant depression of GH levels, suggesting an ultrashort loop feedback action of motilin on the release of motilin itself or somatostatin. In light of motilin's only minor structural similarity to human pancreatic tumor GH-releasing factor (GRF) and the ability of passive immunoneutralization of motilin to lower GH, this 22-amino acid peptide must now be considered a physiologic GRF.     

d-Lys-GHRP-6 does not modify the endocrine response to acylated ghrelin or hexarelin in humans

Acylated ghrelin exerts numerous endocrine and non-endocrine activities via the GH Secretagogue receptor type 1a (GHS-R1a). D-Lys-GHRP-6 has been widely studied in vitro and in vivo in animal studies as GHS-R1a antagonist; its action in humans has, however, never been tested so far. Aim of our study was to verify the antagonistic action of D-Lys-GHRP-6 on the endocrine responses to acylated ghrelin and hexarelin, a peptidyl synthetic GHS, in humans. The effects of different doses of D-Lys-GHRP-6 (2.0microg/kg iv as bolus or 2.0microg/kg/h iv as infusion) on both spontaneous and acylated ghrelin- or hexarelin (1.0microg/kg iv as bolus) -stimulated GH, PRL, ACTH and cortisol levels were studied in six normal volunteers (age [mean+/-SEM]: 25.4+/-1.2yr; BMI: 22.3+/-1.0kg/m(2)). The effects of D-Lys-GHRP-6 (2.0microg/kg iv as bolus+4.0microg/kg/h iv) on the GH response to 0.25microg/kg iv as bolus acylated ghrelin was also studied. During saline, spontaneous ACTH and cortisol decrease was observed while non changes occurred in GH and PRL levels. Acylated ghrelin and hexarelin stimulated (p<0.05) GH, PRL, ACTH and cortisol secretions. D-Lys-GHRP-6 administered either as bolus or a continuous infusion did not modify both spontaneous and acylated ghrelin- or hexarelin-stimulated GH, PRL, ACTH and cortisol secretion. D-Lys-GHRP-6 did not modify even the GH response to 0.25microg/kg iv acylated ghrelin. In conclusion, D-Lys-GHRP-6 does not affect the neuroendocrine response to both ghrelin and hexarelin. These findings question D-Lys-GHRP-6 as an effective GHS-R1a antagonist for human studies.     

Regulational effect of ghrelin on growth hormone secretion from perifused rat anterior pituitary cells

Ghrelin, a novel growth hormone (GH)-releasing peptide, was recently isolated from the rat stomach as an endogenous ligand to growth hormone secretagogue receptor (GHS-R). Ghrelin specifically stimulates the release of GH from the rat anterior pituitary gland, but the regulational effect of ghrelin on GH secretion has not yet been clarified. We used a perifusion system to examine the single effect and combined effects of ghrelin with growth hormone-releasing hormone (GHRH) and somatostatin on GH secretion from rat anterior pituitary cells. The increase in GH concentration due to ghrelin stimulation showed a transitory peak that was almost the same as that previously reported for GHS, but apparently distinct from that of GHRH. Ghrelin (10(-10) M to 10(-8) M) stimulated GH secretion from the rat anterior pituitary cells in a dose-dependent manner. Serial ghrelin stimulation of the dispersed cells at 1-h intervals decreased the GH response, but the response recovered with stimulation at 3-h intervals, indicating that ghrelin strongly desensitized cells. Costimulation with ghrelin and GHRH elicited neither a synergistic nor an additive GH response from the rat pituitary cells. Furthermore, pretreatment to anterior pituitary cells with somatostatin strongly abolished ghrelin- and/or GHRH-stimulated GH secretion. In this study, we demonstrated that ghrelin caused weaker GH secretion than that caused by GHRH, and we also showed that costimulation with GHRH had no additive or synergistic effect on GH secretion, suggesting that ghrelin indirectly affects coordinated GH release from pituitary gland, as found in vivo.     

Nocturnal ghrelin, ACTH, GH and cortisol secretion after sleep deprivation in humans

Ghrelin is an endogenous ligand of the growth hormone (GH) secretagogue (GHS) receptor. It is hypothesised to play a key role in energy balance stimulating food intake and body weight. Besides GH-releasing hormone (GHRH) and somatostatin, it is thought to be a regulating factor of GH release. Ghrelin also appears to be involved in sleep regulation. We showed recently that ghrelin promotes slow-wave sleep and the nocturnal release of GH, cortisol and prolactin in humans. Similarly, promotion of non-rapid-eye-movement (NREM) sleep was reported in mice after systemic ghrelin. If ghrelin is a factor that induces and/or maintains sleep, it should be enhanced after a period of sleep deprivation (SD). To clarify this issue, nocturnal ghrelin, GH, ACTH and cortisol plasma concentrations were determined and simultaneously sleep electroencephalogram (EEG) was recorded (2300-0700 h) during sleep before and after 1 night of total SD in 8 healthy subjects. Compared to baseline, ghrelin levels increased earlier by a non-significant trend, already before the beginning of recovery sleep. Further a non-significant trend occurred, suggesting higher ghrelin secretion in the first half of the night. The ghrelin maximum was found significantly earlier after SD than at baseline. GH secretion during the first half of the night and total night after SD were elevated. ACTH and cortisol were also elevated, which was most pronounced during the second half of the night. No effects of SD on the time of the maximum were found for GH, ACTH and cortisol. The increase in ACTH after SD is a novel finding. Whereas the effects of SD on ghrelin levels were relatively weak, our findings are in line with the hypothesis that ghrelin is a sleep-promoting factor in humans. Ghrelin may be involved in sleep promotion after SD.     

Ghrelin and obestatin modulate growth hormone-releasing hormone release and synaptic inputs onto growth hormone-releasing hormone neurons

Ghrelin, a natural ligand of the growth hormone secretagogue receptor (GHS-R), is synthesized in the stomach but may also be expressed in lesser quantity in the hypothalamus where the GHS-R is located on growth hormone-releasing hormone (GHRH) neurons. Obestatin, a peptide derived from the same precursor as ghrelin, is able to antagonize the ghrelin-induced increase of growth hormone (GH) secretion in vivo but not from pituitary explants in vitro. Thus, the blockade of ghrelin-induced GH release by obestatin could be mediated at the hypothalamic level by the neuronal network that controls pituitary GH secretion. Ghrelin increased GHRH and decreased somatostatin (somatotropin-releasing inhibitory factor) release from hypothalamic explants, whereas obestatin only reduced the ghrelin-induced increase of GHRH release, thus indicating that the effect of ghrelin and obestatin is targeted to GHRH neurons. Patch-clamp recordings on mouse GHRH-enhanced green fluorescent protein neurons indicated that ghrelin and obestatin had no significant effects on glutamatergic synaptic transmission. Ghrelin decreased GABAergic synaptic transmission in 44% of the recorded neurons, an effect blocked in the presence of the GHS-R antagonist BIM28163, and stimulated the firing rate of 78% of GHRH neurons. Obestatin blocked the effects of ghrelin by acting on a receptor different from the GHS-R. These data suggest that: (i) ghrelin increases GHRH neuron excitability by increasing their action potential firing rate and decreasing the strength of GABA inhibitory inputs, thereby leading to an enhanced GHRH release; and (ii) obestatin counteracts ghrelin actions. Such interactions on GHRH neurons probably participate in the control of GH secretion.     

Ghrelin-induced growth hormone release from isolated rat anterior pituitary cells depends on intracellullar and extracellular Ca2+ sources

Ghrelin, a novel growth hormone (GH)-releasing peptide, was isolated from the rat stomach as an endogenous ligand to the growth hormone secretagogues receptor. It is known that ghrelin stimulates the release of GH from the rat anterior pituitary gland, but the intracellular signal cascade in somatotrophs has not yet been clarified. In this study, using an isolated cell perifusion system, we examined whether ghrelin- and growth hormone-releasing hormone (GHRH)-induced GH secretion from rat pituitary cells depends on intra- and extracellular Ca(2+) and voltage-gated Ca(2+) channels. For this purpose, we first measured ghrelin- or GHRH-stimulated GH concentration following treatment with reduced extracellular Ca(2+) and/or thapsigargin, an endoplasmic reticulum Ca(2+) ATPase inhibitor. Reductions in the extracellular Ca(2+) concentration to 0.25 mM and to 0 mM resulted in decreases in ghrelin-stimulated GH secretion to 81% and 39% and decreases in GHRH-induced GH secretion to 83% and 13%, respectively, compared to the levels in the case of 2.5 mM Ca(2+) concentration, suggesting that extracellular Ca(2+) is essential for both ghrelin- and GHRH-induced GH secretion. Pretreatment with thapsigargin resulted in a reduction in ghrelin-induced GH secretion to approximately 60% of the control level, but GHRH treatment had not effect on the GH secretion. Moreover, preincubation with thapsigargin and 0 mM extracellular Ca(2+) concentration resulted in significant inhibition of GHRH- and ghrelin-induced GH secretion. Subsequently, to determine whether ghrelin-stimulated GH secretion was induced through voltage-gated Ca(2+) channels, we measured the ghrelin-stimulated GH concentration following treatment with nifedipine, an L-type Ca(2+) channel inhibitor, and found that the amount of GH secretion was reduced to 44% of the control level. Furthermore, by replacement of extracellular Na(2+) in the medium with N-methyl-D(-)-glucamine, an impermeable molecule, GH secretion was reduced to 47%. In this study, we demonstrated that the GH-stimulatory effect of ghrelin, unlike that of GHRH, is achieved through both intracellular and extracellular Ca(2+) sources and that ghrelin-induced extracellular Ca(2+) influx involves an L-type voltage-gated Ca(2+) channel and Na(+) influx.     

Estimation of parameters for a mathematical model of growth hormone secretion

Here, we describe partial calibration of a parsimonious mathematical model of growth hormone (GH) secretion. From first principles, we derived a model of the effects on GH secretion from pituitary somatotrophs of stimulation by GH-releasing factor (GRF) or GH secretagogue, and of inhibition by somatostatin. We obtained a concise model by collapsing the many processes of the signal transduction cascade into a single step broadly reflecting the initial binding of GRF to its receptors. In the model, GH secretion is proportional to the rate of binding of GRF to activatable receptors. Desensitization occurs because of reduction of free receptors/available effector units, and resensitization occurs as those lost are replaced. This replacement is speeded up in the presence of somatostatin, which also inhibits GH secretion by reducing the constant of proportionality between the rate of GH secretion and the rate of GRF binding. We derived simple mathematical equations for the rate of GH secretion and cumulative secretion. Using these, we tested the model against data obtained from experiments performed in vitro, and made it quantitative using rigorous statistical approaches to optimize parameter estimates. The behaviour of the calibrated model matches experimental observations closely.     

Effect of Growth Hormone-Releasing Peptide-2 (GHRP-2) and GH-Releasing Hormone (GHRH) on the cAMP Levels and GH Release from Cultured Acromegalic Tumours

There is a difference between the sheep and rat somatotrophs in the response to growth hormone-releasing peptide-2 (GHRP-2), which raises the question of what the response may be in human somatotrophs. In the present study, cells were obtained from seven human acromegalic tumours and the effects of GHRP-2 were studied. Cells were dissociated and kept in primary culture for 1–3 weeks before experimentation. Application of GHRP-2 for 30 min induced a significant increase in GH secretion from the cultured cells from all seven tumours whereas human GH-releasing hormone (hGHRH) at a dose of 10 nM induced a significant GH release in only four of seven tumours. The intracellular levels of cAMP in all seven tumours were significantly increased by both 10 nM GHRP-2 and GHRH, but the response to GHRH was significantly higher than the response to GHRP-2. The adenylyl cyclase inhibitor, MDL 12330A, blocked the effect of GHRH and GHRP-2 on intracellular cAMP levels, whereas the Ca²⁺ channel blocker Co²⁺ (0.5 mM) did not attenuate the cAMP response. For the tumours in which GH secretion was increased by GHRH and GHRP-2, the cAMP antagonist Rp-cAMP blocked the GH response to GHRH but not to GHRP-2. When a protein kinase A (PKA) inhibitor (H₈₉) was applied, GHRH stimulated GH release was blocked, but cAMP accumulation was not affected. The response to GHRP-2 was not altered by H₈₉. Calphostin C [a protein kinase C (PKC) inhibitor] reduced the effect of GHRP-2 on the secretion of GH but did not affect the response to GHRH. Both GHRH and GHRP-2 increased the intracellular Ca²⁺ concentration in a concentration-dependent manner. We conclude that (1) GHRH increases GH secretion from human GH tumours via the cAMP pathway whereas GHRP-2 increases GH secretion mainly via the PKC pathway; (2) GHRH increases cAMP (without GH release) in a subset of tumours whereas GHRP-2 increases cAMP levels (slightly) and GH secretion in all tumours; and (3) GHRP-2 and GHRH do not act on the same receptor on human somatotrophs derived from acromegalic tumours.     

Ghrelin stimulates adrenocorticotrophic hormone (ACTH) secretion by human ACTH-secreting pituitary adenomas in vitro

Ghrelin is a brain-gut peptide with wide-ranging endocrine, metabolic, cardiovascular and neural effects. Ghrelin, like its synthetic counterparts, the growth hormone (GH) secretagogues, has been shown to markedly stimulate adrenocorticotrophic hormone (ACTH) and cortisol secretion in humans and the ACTH-releasing effect of GH secretagogues is even greater in patients with pituitary ACTH-secreting tumours. Furthermore, these tumours synthesize ghrelin itself, suggesting an intrapituitary ghrelin circuit. The aim of the present study was to evaluate the effect of ghrelin on ACTH secretion by human pituitary corticotroph tumours in vitro to test the functionality of this circuit. Nine ACTH-secreting pituitary tumours (four microadenomas, five macroadenomas) were collected during surgery and incubated with 10-100 nM human ghrelin or with 10 nM human corticotrophin-releasing hormone (CRH). Control experiments were performed in rat anterior pituitary primary cultures. ACTH secretion was assessed after 4 h and 24 h incubation by immunometric assay. After 4 h of incubation with ghrelin, medium ACTH concentrations were two- to ten-fold higher compared to ACTH concentrations in unstimulated wells. The ACTH-releasing effect of ghrelin was significantly less than the response elicited by 10 nM CRH (up to 40-fold) Similar results were obtained after 24 h of incubation and a superimposable response pattern was observed in rat anterior pituitary primary cultures. The present study demonstrates that the endogenous GH secretagogue, ghrelin, stimulates ACTH secretion directly from human tumoural corticotrophs, as well as from normal rat pituitary, and indicates that the marked ACTH release elicited by ghrelin in patients with Cushing's disease in vivo is due, at least in part, to its action on the pituitary tumour. However, the reversal of the response pattern reported in vivo, with ghrelin proving a lesser stimulant than CRH in vitro, suggests that additional, suprapituitary mechanisms are involved in the in vivo response. Moreover, these data uphold the concept of a functional intratumoural ghrelin paracrine circuit in human corticotroph adenomas.     

Role of nitric oxide on ATP-induced Ca2+ signaling in outer hair cells of the guinea pig cochlea

Recently, a negative feedback effect of nitric oxide (NO) on the adenosine 5'-triphosphate (ATP)-induced Ca2+ response has been described in cochlear inner hair cells. We here investigated the role of NO on the ATP-induced Ca2+ response in outer hair cells (OHCs) of the guinea pig cochlea using the NO-sensitive dye DAF-2 and Ca2+ -sensitive dye fura-2. Extracellular ATP induced NO production in OHCs, which was inhibited by L-NG-nitroarginine methyl ester (L-NAME), a non-specific NO synthase (NOS) inhibitor, and suramin, a P2 receptor antagonist. ATP failed to induce NO production in the Ca2+ -free solution. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, enhanced the ATP-induced increase of the intracellular Ca2+ concentrations ([Ca2+]i), while L-NAME inhibited it. SNAP accelerated ATP-induced Mn2+ quenching in fura-2 fluorescence, while L-NAME suppressed it. 8-Bromoguanosine-cGMP, a membrane permeable analog of cGMP, mimicked the effects of SNAP. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase inhibited the ATP-induced [Ca2+]i increase. Selective neuronal NOS inhibitors, namely either 7-nitro-indazole or 1-(2-trifluoromethylphenyl) imidazole, mimicked the effects of L-NAME regarding both ATP-induced Ca2+ response and NO production. Immunofluorescent staining of neuronal nitric oxide synthase (nNOS) in isolated OHCs showed the localization of nNOS in the apical region of OHCs. These results suggest that the ATP-induced Ca2+ influx via a direct action of P2X receptors may be the principal source for nNOS activity in the apical region of OHCs. Thereafter, NO can be produced while conversely enhancing the Ca2+ influx via the NO-cGMP-PKG pathway by a feedback mechanism.     

Preferential potentiation by nitric oxide of spontaneous inhibitory postsynaptic currents in rat supraoptic neurones

Magnocellular neurones in the supraoptic nucleus and paraventricular nucleus express mRNA for nitric oxide synthase (NOS) and the expression becomes more prominent when the release of vasopressin or oxytocin is stimulated. It has also been reported that NO donors inhibit the electrical activity of supraoptic nucleus neurones, but the mechanism involved in the inhibition remains unclear. In the present study, to know whether modulation of synaptic inputs into supraoptic neurones is involved in the inhibitory effect of NO, we measured spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) from rat supraoptic nucleus neurones in slice preparations identified under a microscope using the whole-cell mode of the slice-patch-clamp technique. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reversibly increased the frequency of spontaneous IPSCs mediated by GABAA receptors, without affecting the amplitude, indicating that NO potentiated IPSCs via a presynaptic mechanism. The NO scavenger, haemoglobin, suppressed the potentiation of IPSCs by SNAP. On the other hand, SNAP did not cause significant effects on EPSCs mediated by non-NMDA glutamate receptors. The membrane permeable analogue of cGMP, 8-bromo cGMP, caused a significant reduction in the frequency and amplitude of both IPSCs and EPSCs. The results suggest that NO preferentially potentiates the inhibitory synaptic inputs into supraoptic nucleus neurones by acting on GABA terminals in the supraoptic nucleus, possibly via a cGMP-independent mechanism. The potentiation may, at least in part, account for the inhibitory action of NO on the neural activity of supraoptic neurones.