The in vitro regulation of growth hormone secretion by orexins

Orexins, orexigenic neuropeptides, have recently been discovered in lateral hypothalamus and play an important role in the regulation of pituitary hormone secretion. Two subtypes of orexin receptors (orexin-1 and orexin-2) have been demonstrated in pituitaries. In this experiment, the effects of orexins on voltage-gated Ca2+ currents and the GH release in primary cultured ovine somatotropes were examined. Voltage-gated Ca2+ currents were isolated in ovine somatotropes as L, T, and N currents using whole-cell patch-clamp techniques and specific Ca2+ channel blocker and toxin. Application of orexin-A or orexin-B (100 nM) significantly, dose-dependently, and reversibly increased only nifedipine-sensitive L-type Ca2+ current. Inhibitors of PKC (calphostin C, PKC inhibitory peptide) but not inhibitors of PKA (H89, PKA inhibitory peptide) cancelled the increase in the L current by orexins. Co-administration of orexin-A and GHRH (10 nM) showed an additive effect on the L current. Specific intracellular Ca2+-store-depleting reagent, thapsigargin (1 microM), did not affect the orexin-induced increase in the L current. Orexin-B alone slightly increased GH release and co-administration of orexin-A and GHRH synergistically stimulated GH secretion in vitro. It is therefore suggested that orexins may play an important role in regulating GHRH-stimulated GH secretion through an increase in the L-type Ca2+ current and the PKC-mediated signaling pathways in ovine somatotropes.     

Involvement of tetrodotoxin-resistant Na+ current and protein kinase C in the action of growth hormone (GH)-releasing hormone on primary cultured somatotropes from GH-green fluorescent protein transgenic mice

GHRH depolarizes the membrane of somatotropes, leading to an increase in intracellular free Ca2+ concentration and GH secretion. Na+ channels mediate the rapid depolarization during the initial phase of the action potential, and this regulates Ca2+ influx and GH secretion. GHRH increases a tetrodotoxin-sensitive somatotrope Na+ current that is mediated by cAMP. TTX-resistant (TTX-R) Na+ channels are abundant in sensory neurons and cardiac myocytes, but their occurrence and/or function in somatotropes has not been investigated. Here we demonstrate expression of TTX-R Na+ channels and a TTX-R Na+ current, using patch-clamp method, in green fluorescent protein-GH transgenic mouse somatotropes. GHRH (100 nm) increased the TTX-R Na+ current in a reversible manner. The GHRH-induced increase in TTX-R Na+ current was not affected by the cAMP antagonist Rp-cAMP or protein kinase A inhibitors KT5720 or H89. The TTX-R current was increased by 8-bromoadenosine-cAMP (cAMP analog), forskolin (adenylyl-cyclase activator), and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), but the additional, GHRH-induced increase in TTX-R Na+ currents was not affected. U-73122 (phospholipase C inhibitor) and protein kinase C (PKC) inhibitors, G?-6983 and chelerythrine, blocked the effect of GHRH. PKC activators, phorbol dibutyrate and phorbol myristate acetate, increased the TTX-R Na+ current, but GHRH had no further effect on the current. Na+-free extracellular medium significantly reduced GHRH-stimulated GH secretion. We conclude that GHRH-induced increase in the TTX-R Na+ current in mouse somatotropes is mediated by the PKC system. An increase in the TTX-R Na+ current may contribute to the GHRH-induced exocytosis of GH granules from mouse somatotropes.     

Orexin-B augments voltage-gated L-type Ca(2+) current via protein kinase C-mediated signalling pathway in ovine somatotropes

Orexins, orexigenic neuropeptides, are secreted from lateral hypothalamus and orexin receptors are expressed in the pituitary. Since growth hormone (GH) secreted from pituitary is integrally linked to energy homeostasis and metabolism, we studied the effect of orexin-B on voltage-gated Ca(2+) currents and the related signalling mechanisms in primary cultured ovine somatotropes using whole-cell patch-clamp techniques. With a bath solution containing TEA-Cl (40 mM) and Tetrodotoxin (TTX) (1 microM), three subtypes of Ca(2+) currents, namely the long-lasting (L), transient (T), and N currents, were isolated using different holding potentials (-80 and -30 mV) in combination with specific Ca(2+) channel blockers (nifedipine and omega-conotoxin). About 75% of the total current amplitude was contributed by the L current, whereas the N and T currents accounted for the rest. Orexin-B (1-100 nM) dose-dependently and reversibly increased only the L current up to approximately 125% of the control value within 4-5 min. Neither a specific protein kinase A (PKA) blocker (H89, 1 microM) nor an inhibitory peptide (PKI, 10 microM) had any effect on the increase in L current by orexin-B. The orexin-B-induced increase in the L current was abolished by concurrent treatment with calphostin C (Cal-C, 100 nM), protein kinase C (PKC) inhibitory peptide (PKC(19-36), 1 microM), or by pretreatment with phorbol-12,13-dibutyrate (PDBu) (0.5 microM) for 16 h (a downregulator of PKC). Orexin-B also increased in vitro GH secretion in a dose-dependent manner. We conclude that orexin-B increases the L-type Ca(2+) current and GH secretion through orexin receptors and PKC-mediated signalling pathways in ovine somatotropes.     

Direct modification of somatotrope function by long-term leptin treatment of primary cultured ovine pituitary cells.

Leptin is produced primarily in adipocytes and regulates body energy balance. A close link between leptin and pituitary hormones, including GH, has been reported. The mechanisms employed by leptin to influence somatotropes are not clear, however. Here we report a direct action of recombinant ovine leptin on primary cultured ovine somatotropes by analyzing the levels of mRNA encoding for GH or the receptors for GHRH (GHRH-R) and GH-releasing peptides (GHRP). Treatment of ovine somatotropes with leptin (10(-7)-10(-9) M) for 1-3 d reduced the mRNA levels encoding GH and GHRH-R, but increased GHRP receptor mRNA levels in a time- and dose-dependent manner. Three-day treatment of cells with leptin decreased the GH response to GHRH stimulation, but the GH response to GHRP-2 stimulation was increased. The combined effect of GHRH and GHRP-2 on GH secretion was not altered by treatment of the cells with leptin. These results demonstrated a direct action of leptin on ovine pituitary cells, leading to a reduced sensitivity of somatotropes to GHRH. It is also suggested that GHRP may be useful to correct the decrease in GHRH-induced GH secretion by leptin.     

Ghrelin reduces voltage-gated potassium currents in GH3 cells via cyclic GMP pathways

Ghrelin is an endogeneous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca²⁺ concentration ([Ca²⁺]_i), which is determined by Ca²⁺ influx and release from intracellular Ca²⁺ storage sites. Ca²⁺ influx is via voltage-gated Ca²⁺ channels, which are activated by cell depolarization. Membrane potential is mainly determined by transmembrane K⁺ channels. The present study investigates the in vitro effect of ghrelin on membrane voltage-gated K⁺ channels in the GH₃ rat somatotrope cell line. Nystatin-perforated patch clamp recording was used to record K⁺ currents under voltage-clamp conditions. In the presence of Co²⁺ (1 mM, Ca²⁺ channel blocker) and tetrodotoxin (1 μM, Na⁺ channel blocker) in the bath solution, two types of voltage-gated K⁺ currents were characterized on the basis of their biophysical kinetics and pharmacological properties. We observed that transient K⁺ current (I _A) represented a significant proportion of total K⁺ currents in some cells, whereas delayed rectifier K⁺ current (I _K) existed in all cells. The application of ghrelin (10 nM) reversibly and significantly decreased the amplitude of both I _A and I _K currents to 48% and 64% of control, respectively. Application of apamin (1 μM, SK channel blocker) or charybdotoxin (1 μM, BK channel blocker) did not alter the K⁺ current or the response to ghrelin. The ghrelin-induced reduction in K⁺ currents was not affected by PKC and PKA inhibitors. KT5823, a specific PKG inhibitor, totally abolished the K⁺ current response to ghrelin. These results suggest that ghrelininduced reduction of voltage-gated K⁺ currents in GH₃ cells is mediated through a PKG-dependent pathway. A decrease in voltage-gated K⁺ currents may increase the frequency, duration, and amplitude of action potentials and contribute to GH secretion from somatotropes.     

Intracellular signaling mechanisms mediating ghrelin-stimulated growth hormone release in somatotropes

Ghrelin is a newly discovered peptide that binds the receptor for GH secretagogues (GHS-R). The presence of both ghrelin and GHS-Rs in the hypothalamic-pituitary system, together with the ability of ghrelin to increase GH release, suggests a hypophysiotropic role for this peptide. To ascertain the intracellular mechanisms mediating the action of ghrelin in somatotropes, we evaluated ghrelin-induced GH release from pig pituitary cells both under basal conditions and after specific blockade of key steps of cAMP-, inositol phosphate-, and Ca2+-dependent signaling routes. Ghrelin stimulated GH release at concentrations ranging from 10-10 to 10-6 m. Its effects were comparable with those exerted by GHRH or the GHS L-163,255. Combined treatment with ghrelin and GHRH or L-163,255 did not cause further increases in GH release, whereas somatostatin abolished the effect of ghrelin. Blockade of phospholipase C or protein kinase C inhibited ghrelin-induced GH secretion, suggesting a requisite role for this route in ghrelin action. Unexpectedly, inhibition of either adenylate cyclase or protein kinase A also suppressed ghrelin-induced GH release. In addition, ghrelin stimulated cAMP production and also had an additive effect with GHRH on cAMP accumulation. Ghrelin also increased free intracellular Ca2+ levels in somatotropes. Moreover, ghrelin-induced GH release was entirely dependent on extracellular Ca2+ influx through L-type voltage-sensitive channels. These results indicate that ghrelin exerts a direct stimulatory action on porcine GH release that is not additive with that of GHRH and requires the contribution of a multiple, complex set of interdependent intracellular signaling pathways.     

Effect of galanin on the growth hormone response to growth hormone-releasing hormone in acromegaly.

Galanin enhances growth hormone (GH)-releasing hormone (GHRH)-stimulated GH secretion in normal man. In acromegaly, circulating GH levels are increased and the GH response to GHRH may be exaggerated. Galanin has been recently shown to decrease circulating GH levels in acromegaly. The aim of our study was to investigate the effects of galanin on the GH response to GHRH in acromegalic subjects. Five acromegalic patients (three men and two women) and seven healthy adult subjects (five men and two women) were studied. GHRH-induced GH secretion was evaluated during a 40-minute intravenous (IV) infusion of saline (100 mL) or porcine galanin (12.5 micrograms/min in 100 mL saline). In normal subjects, delta GH levels after GHRH+porcine galanin administration (47 +/- 7.5 micrograms/L) were significantly higher in comparison to levels obtained with GHRH+saline (21.7 +/- 3.5 micrograms/L, P < .05). In acromegalic patients, GH responses to GHRH (delta GH, 18.8 +/- 8.6 micrograms/L) were not altered by galanin infusion (delta GH, 17.6 +/- 5 micrograms/L). Our results give the first evidence that the same dose of galanin that induces a significant enhancement of the GH response to GHRH in normal subjects has no effect on the GH response to GHRH in acromegalic patients. It can be hypothesized that galanin may interact at the pituitary level with its own receptors expressed by somatotropes independent of GHRH. Failure of galanin to enhance GH response to GHRH in acromegalic patients could be due to a change in function of the galanin receptor on GH-secreting adenomatous cells.     

Somatostatin inhibition of growth hormone secretion by somatotropes from male, female, and androgen receptor-deficient rats: evidence for differing sensitivities

To investigate the role of somatostatin (SRIF) in regulating sexually dimorphic GH secretion, we used a reverse hemolytic plaque assay and acutely dispersed somatotropes from age-matched normal male, normal female, and androgen receptor-deficient, testicular feminized (Tfm) rats. Hemolytic plaques were developed after a 90-min incubation in the presence of GH antiserum, 10 nM GH-releasing hormone (GHRH), and the following concentrations of SRIF: 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 nM. Additional studies were performed with 0 or 100 nM SRIF in the absence of GHRH. The absolute number of somatotropes (x10(6); mean +/- SEM) recovered from the pituitaries of Tfm rats (1.73 +/- 0.18) was significantly greater than that from the males (1.11 +/- 0.13; P = 0.01); the number from female rats (1.30 +/- 0.15) was not different from that of either male or Tfm animals. GHRH-stimulated GH secretion, as estimated by the mean GH plaque area (micron2 x 10(4); mean +/- SEM) in the absence of SRIF, was greater for somatotropes from male rats (3.36 +/- 0.41) than that for either Tfm (2.27 +/- 0.32; P = 0.02) or female (1.78 +/- 0.24; P = 0.001) rats; values for the latter two groups did not differ. However, mean GH plaque areas for each group during maximal SRIF inhibition in either the presence or absence of GHRH were indistinguishable from each other and from mean plaque areas obtained under basal conditions. As demonstrated by a lesser EC50 value (0.04 +/- 0.02 nM; mean +/- SEM), somatotropes from female rats were more sensitive to the inhibitory effect of SRIF than were those from either male (EC50 = 1.82 +/- 0.45; P = 0.0001) or Tfm (EC50 = 0.74 +/- 0.22, P = 0.0001) rats; values for the latter two groups were indistinguishable. These observed differences suggest that gender and/or the gonadal hormone environment may be important determinants of the inhibitory effects of SRIF on GH secretion by the somatotrope. While these gender-associated differences may represent effects of the gonadal hormones directly on the somatotrope, they could reflect modulation of the secretion of hypothalamic SRIF and/or GHRH by the prevailing gonadal hormone environment. Such gender-related differences may contribute to the overall sex-dependent patterns of GH secretion in the intact animal.     

Somatostatin increases voltage-gated K+ currents in GH3 cells through activation of multiple somatostatin receptors

The secretion of GH by somatotropes is inhibited by somatostatin (SRIF) through five specific membrane receptors (SSTRs). SRIF increases both transient outward (IA) and delayed rectifying (IK) K+ currents. We aim to clarify the subtype(s) of SSTRs involved in K+ current enhancement in GH3 somatotrope cells using specific SSTR subtype agonists. Expression of all five SSTRs was confirmed in GH3 cells by RT-PCR. Nystatin-perforated patch clamp was used to record voltage-gated K+ currents. We first established the presence of IA and IK type K+ currents in GH3 cells using different holding potentials (-40 or -70 mV) and specific blockers (4-aminopirimidine and tetraethylammonium chloride). SRIF (200 nM) increased the amplitude of both IA and IK in a fully reversible manner. Various concentrations of each specific SRTR agonist were tested on K+ currents to find the maximal effective concentration. Activation of SSTR2 and SSTR4 by their respective agonists, L-779,976 and L-803,087 (10 nM), increased K+ current amplitude without preference to IA or IK, and abolished any further increase by SRIF. Activation of SSTR1 and SSTR5 by their respective agonists, L-797,591 or L-817,818 (10 nM), increased K+ current amplitude, but SRIF evoked a further increase. The SSTR3 agonist L-797,778 (10 nM) did not affect the K+ currents or the response to SRIF. These results indicate that SSTR1, -2, -4, and -5 may all be involved in the enhancement of K+ currents by SRIF but that only the activation of SSTR2 or -4 results in the full activation of K+ current caused by SRIF.     

Ghrelin main action on the regulation of growth hormone release is exerted at hypothalamic level

Ghrelin, a recently isolated hormone, seems to participate in the physiological regulation of GH secretion. Exogenously administered ghrelin stimulates GH discharge in all species so far tested including man, but whether this action is exerted at pituitary or alternatively at hypothalamic level is not known at present. To understand the point of ghrelin action a group of patients with organic lesion mainly in the hypothalamic area and matched controls were studied. Patients showed a severe GH deficiency after hypothalamic stimulation (ITT), but partial response after GHRH administration. Cases and controls were tested on three separate days by either ghrelin; GHRH; and ghrelin plus GHRH; always at 1 micro g/Kg iv. The mean GH peak after stimulation in the patients were: 0.4 +/- 0.1 micro g/L by ITT; 3.1 +/- 0.5 micro g/L after GHRH; 2.0 +/- 0.8 micro g/L after ghrelin; and 9.6 +/- 2.9 micro g/L after the combination of GHRH plus ghrelin. In the controls GHRH induced a GH peak of 21.2 +/- 7.5 micro g/L, and 75.1 +/- 16.0 micro g/L after ghrelin with a peak after GHRH + ghrelin of 103.5 +/- 26.4 micro g/L. These data indicate that when hypothalamic structures are not operative ghrelin, either alone or in combination with GHRH, is not able to significantly release GH. In addition to postulating a hypothalamic point of action for the ghrelin-induced GH secretion, these results suggests that ghrelin will not have significant clinical utility in patients with GH deficiency due to organic lesion.     

Somatostatin actions on a protein kinase C-dependent growth hormone secretagogue cascade

In mammals, the ability of somatostatin (SS) to block growth hormone (GH) secretion is due, in part, to the inhibition of two key intracellular mediators, cAMP and Ca2+. We examined whether or not inhibition of Ca2+ signaling was mediating SS-induced inhibition basal, as well as gonadotropin-releasing hormone (GnRH; a protein kinase C (PKC)-dependent growth hormone secretagogue)-stimulated growth hormone (GH) release. Although SS reduced basal GH release from populations of pituitary cells, parallel reductions in [Ca2+]i were not observed within single, identified somatotropes. Similarly, application of GnRH and the PKC activator DiC8 elicited increases in [Ca2+]i and GH release, but abolition of the Ca2+ responses did not accompany SS inhibition of the GH responses. Surprisingly, while DiC8 potentiated SS inhibition of GH release, SS paradoxically increased DiC8-stimulated increases in [Ca2+]i. These data establish that abolition of Ca2+ signals is not a primary mechanism through which SS lowers basal, or inhibits GnRH-stimulated hormone release.     

GH-releasing peptide-6 overcomes refractoriness of somatotropes to GHRH after feeding.

After a meal, somatotropes are temporarily refractory to growth hormone-releasing hormone (GHRH), the principal hormone that stimulates secretion of growth hormone (GH). Refractoriness is particularly evident when free access to feed is restricted to a 2-h period each day. GH-releasing peptide-6 (GHRP-6), a synthetic peptide, also stimulates secretion of GH from somatotropes. Because GHRH and GHRP-6 act via different receptors, we hypothesized that GHRP-6 would increase GHRH-induced secretion of GH after feeding. Initially, we determined that intravenous injection of GHRP-6 at 1, 3 and 10 microg/kg body weight (BW) stimulated secretion of GH in a dose-dependent manner. Next, we determined that GHRP-6- and GHRH-induced secretion of GH was lower 1 h after feeding (22.5 and 20 ng/ml respectively) than 1 h before feeding (53.5 and 64.5 ng/ml respectively; pooleds.e.m.=8.5). However, a combination of GHRP-6 at 3 microg/kg BW and GHRH at 0.2 microg/kg BW synergistically induced an equal and massive release of GH before and after feeding that was fivefold greater than GHRH-induced release of GH after feeding. Furthermore, the combination of GHRP-6 and GHRH synergistically increased release of GH from somatotropes cultured in vitro. However, it was not clear if GHRP-6 acted only on somatotropes or also acted at the hypothalamus. Therefore, we wanted to determine if GHRP-6 stimulated secretion of GHRH or inhibited secretion of somatostatin, or both. GHRP-6 stimulated secretion of GHRH from bovine hypothalamic slices, but did not alter secretion of somatostatin. We conclude that GHRP-6 acts at the hypothalamus to stimulate secretion of GHRH, and at somatotropes to restore and enhance the responsiveness of somatotropes to GHRH.     

Re-assessment of growth hormone secretion in young adult patients with childhood-onset growth hormone deficiency

OBJECTIVE: Patients with childhood-onset GH deficiency (coGHD) need retesting in late adolescence or young adulthood to verify whether they need to continue GH treatment. For this purpose the Growth Hormone Research Society (GRS) recommends the insulin tolerance test (ITT), or as an alternative the arginine + growth hormone releasing hormone test (ARG + GHRH test) as a diagnostic tool in adolescents and adults. However, there are no standardized cut-off levels based on normal GH secretion for determining GHD vs. GH sufficiency in young adults for the ITT, the ARG + GHRH test or the pyridostigmine + GHRH (PD + GHRH) test, a further new GH stimulation test. PATIENTS AND MEASUREMENTS: We studied 43 patients (28 with organic coGHD, 15 with idiopathic coGHD; 30 males, 13 females; aged 20.4 years, range 16.2-25.4; body mass index 23.5, range 16.3-35.8) using the ARG [0.5 g/kg intravenously (i.v.)] + GHRH (1 micro g/kg i.v.) test, the PD (120 mg orally) + GHRH (1 micro g/kg i.v.) test and the ITT (0.1 IU/kg i.v.) and compared these data with the results of 40 healthy age- and weight-matched volunteers. RESULTS: The GH response in patients was significantly lower than in healthy controls: ARG + GHRH test, 0.8 micro g/l (interquartile range 0.3-2.6) vs. 51.8 micro g/l (32.6-71.2) in controls (P < 0.0001); PD + GHRH test, 0.9 micro g/l (0.3-1.9) vs. 40.4 micro g/l (27.1-54.4) in controls (P < 0.0001); ITT, 0.1 micro g/l (0.0-0.8) vs. 20.3 micro g/l (14.7-31.7) in controls (P < 0.0001). In the ARG + GHRH test we found a diagnostic sensitivity of 100% and a specificity of 97.5% for a cut-off range from 15.1 to 20.3 micro g/l, in the PD + GHRH test a sensitivity of 100% and a specificity of 97% (cut-off range 9.1-13.1 micro g/l) and in the ITT a sensitivity and specificity of 100% each within a cut-off range from 2.7 to 8.8 micro g/l. CONCLUSION: There were no marked differences in sensitivity and specificity in young adults among ARG + GHRH test, PD + GHRH test and the ITT in assessing GH secretion. Because of the lack of side-effects, the ARG + GHRH test is the recommended method for re-evaluation of coGHD in young adults when pituitary GHD is suspected. Furthermore, in adult patient groups where organic pituitary coGHD is common, the ITT may be completely replaced by the ARG + GHRH test. Because of the predominance of hypothalamic GHD in childhood, the ITT is commonly performed for the re-evaluation of patients with childhood-onset GHD because of its mechanism of GH stimulation. The present results confirm the high discriminatory capability of the ITT in young adults.     

Effects of hypophysiotropic factors on growth hormone and prolactin secretion from somatotroph adenomas in culture

In an attempt to characterize GH and PRL secretion in acromegaly, the effects of various stimuli on GH and PRL release by cultured pituitary adenoma cells derived from acromegalic patients were studied. In addition, the PRL responses of somatotroph adenoma cells were compared to those of prolactinoma cells. GH-releasing hormone-(1-44) (GHRH) consistently stimulated GH secretion in all 14 somatotroph adenomas studied in a dose-dependent manner. The sensitivity as well as the magnitude of the GH responses to GHRH were highly variable in individual tissues. Somatotroph adenomas that did not respond to dopamine were more sensitive and had greater GH responses to GHRH. In 8 of 9 somatotroph adenomas that concomitantly secreted PRL, the addition of GHRH likewise increased PRL release. Omission of extracellular Ca2+ blocked the stimulatory effect of GHRH on GH and PRL secretion. When cells were coincubated with 0.1 nM somatostatin, GH and PRL secretion induced by 10 nM GHRH were completely blocked in most adenomas. Similarly, coincubation of dopamine resulted in inhibition of GHRH-induced hormone secretion in some adenomas. Addition of TRH to the incubation medium, on the other hand, significantly stimulated GH secretion in 8 of 14 adenomas, while TRH stimulated PRL release in all of the adenomas. Vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH) produced an increase in GH and PRL secretion in other adenomas. In prolactinoma cells, somatostatin and dopamine unequivocally suppressed PRL secretion; however, other stimuli including GHRH, VIP, and CRF were ineffective. TRH induced a significant increase in PRL secretion in only one prolactinoma. These results suggest that responsiveness to GHRH and somatostatin is preserved in somatotroph adenomas; the responsiveness to GHRH is inversely correlated to that to dopamine; and PRL cells associated with somatotroph adenomas possess characteristics similar to those of GH cells. Further, the GH stimulatory actions of TRH and VIP are different.     

Ghrelin reduces voltage-gated calcium currents in GH₃ cells via cyclic GMP pathways

Ghrelin is an endogenous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca2+ concentration ([Ca2+]i), which is determined by Ca2+ influx and release from intracellular Ca2+ storage sites. Ca2+ influx is via voltage-gated Ca2+ channels, which are activated by cell depolarization. The mechanism underlying the effect of ghrelin on voltage-gated Ca2+ channels is still not clear. In this report, using whole cell patch-clamp recordings, we assessed the acute action of ghrelin on voltage-activated Ca2+ currents in GH3 rat somatotrope cell line. Ca2+ currents were divided into three types (T, N, and L) through two different holding potentials (-80 and -40 mV) and specific L-type channel blocker (nifedipine, NFD). We demonstrated that ghrelin significantly and reversibly decreases all three types of Ca2+ currents in GH3 cells through GHS receptors on the cell membrane and down-stream signaling systems. With different signal pathway inhibitors, we observed that ghrelin-induced reduction in voltage-gated Ca2+ currents in GH3 cells was mediated by a protein kinase G-dependent pathways. As ghrelin also stimulates Ca2+ release and prolongs the membrane depolarization, this reduction in voltage-gated Ca2+ currents may not be translated into a reduction in [Ca2+]i, or a decrease in GH secretion.     

Growth hormone secretagogue (GHS) analogue, hexarelin stimulates GH from peripheral lymphocytes.

The role of growth hormone releasing hormone (GHRH) and growth hormone releasing peptide-6 (GHRP-6) analogue hexarelin was investigated in the regulation of GH production from lymphocytes. Porcine and bovine blood mononuclear cells were separated using density gradient centrifugation method by layering the whole blood or buffy coat cells on lymphodex. Cells were incubated for 3 or 5 days with or without phytohemagglutinin (PHA-M), GHRH, GHRP-6 analogue hexarelin, somatostatin or GHRH + hexarelin. Growth hormone was fractionated from supernatants by gel chromatography and further concentrated by lyophilization at - 20 degrees C. A nearly two fold increase in basal secretion of GH (porcine: 3.5 +/- 0.1 ng/ml, bovine: 3.2 +/- 0.2 ng/ml) was achieved by GHRH and hexarelin at concentrations of 0.1, 1.0, 10 and 100 nM in both porcine and bovine cells. Lymphocytic GH release was also stimulated in response to PHA-M (10 micro g/well). Neither a dose dependent nor a synergistic nor an additive effect was apparent on GH secretion from lymphocytes. GHRH stimulated lymphocytic GH secretion, whereas, somatostatin had no effect. This study reports for the first time that hexarelin stimulates the secretion of GH from peripheral lymphocytes.     

Growth hormone (GH)-releasing factor differentially activates cyclic adenosine 3',5'-monophosphate- and inositol phosphate-dependent pathways to stimulate GH release in two porcine somatotrope subpopulations

Somatotropes comprise two morphologically and functionally distinct subpopulations of low (LD) and high (HD) density cells. We recently reported that GRF induces different patterns of increase in the cytosolic free Ca2+ concentration in single porcine LD and HD somatotropes, which for LD cells required not only Ca2+ influx but also intracellular Ca2+ mobilization. This suggested that GRF may activate multiple signaling pathways in pig LD and HD somatotropes to stimulate GH secretion. To address this question, we first assessed the direct GRF effect on second messenger activation in cultures of LD and HD cells by measuring cAMP levels and [3H]myo-inositol incorporation. Secondly, to determine the relative importance of cAMP- and inositol phosphate (IP)-dependent pathways, and of intra- and extracellular Ca2+, GRF-induced GH release from cultured LD and HD somatotropes was measured in the presence of specific blockers. GRF increased cAMP levels in both subpopulations, whereas it only augmented IP turnover in LD cells. Accordingly, adenylate cyclase inhibition by MDL-12,330A abolished GRF-stimulated GH release in both subpopulations, whereas phospholipase C inhibition by U-73122 only reduced this effect partially in LD cells. Likewise, blockade of Ca2+ influx with Cl2Co reduced GRF-stimulated GH secretion in both LD and HD somatotropes, whereas depletion of thapsigargin-sensitive intracellular Ca2+ stores only decreased the secretory response to GRF in LD cells. These results demonstrate that GRF specifically and differentially activates multiple signaling pathways in two somatotrope subpopulations to stimulate GH release. Thus, although the prevailing signaling cascade employed by GRF in both subpopulations is adenylate cyclase/cAMP/extracellular Ca2+, the peptide also requires activation of the phospholipase C/IP/intracellular Ca2+ pathway to exert its full effect in porcine LD somatotropes.