Ghrelin increases growth hormone production and functional expression of NaV1.1 and Na V1.2 channels in pituitary somatotropes.

A variety of ion channels are expressed in the plasma membrane of somatotropes within the anterior pituitary gland. Modification of these channels is linked to intracellular Ca2+ levels and therefore to hormone secretion. Previous investigations have shown that the gut-derived orexigenic peptide hormone ghrelin and synthetic GH-releasing peptides (GHRPs) stimulate release of growth hormone (GH) and increase the number of functional voltage-gated Ca2+ and Na+ channels in the membrane of clonal GC somatotropes. Here, we reveal that chronic treatment with ghrelin and its synthetic analog GHRP-6 also increases GH release from bovine pituitary somatotropes in culture, and that this action is associated with a significant increase in Na+ macroscopic current. Consistent with this, Na+ current blockade with tetrodotoxin (TTX) abolished the ghrelin- and GHRP-6-induced increase in GH release. Furthermore, semi-quantitative and real-time RT-PCR analysis revealed an upregulation in the transcript levels of GH, as well as of NaV1.1 and NaV1.2, two isoforms of TTX-sensitive Na+ channels expressed in somatotropes, after treatment with ghrelin or GHRP-6. These findings improve our knowledge on (i) the cellular mechanisms involved in the control of GH secretion, (ii) the molecular diversity of Na+ channels in pituitary somatotropes, and (iii) the regulation of GH and Na+ channel gene expression by ghrelin and GHRPs.

Artificial feeding synchronizes behavioral, hormonal, metabolic and neural parameters in mother-deprived neonatal rabbit pups.

Nursing in the rabbit is under circadian control, and pups have a daily anticipatory behavioral arousal synchronized to this unique event, but it is not known which signal is the main entraining cue. In the present study, we hypothesized that food is the main entraining signal. Using mother-deprived pups, we tested the effects of artificial feeding on the synchronization of locomotor behavior, plasma glucose, corticosterone, c-Fos (FOS) and PERIOD1 (PER1) rhythms in suprachiasmatic, supraoptic, paraventricular and tuberomammillary nuclei. At postnatal day 1, an intragastric tube was placed by gastrostomy. The next day and for the rest of the experiment, pups were fed with a milk formula through the cannula at either 02:00 h or 10:00 h [feeding time = zeitgeber time (ZT)0]. At postnatal days 5-7, pups exhibited behavioral arousal, with a significant increase in locomotor behavior 60 min before feeding. Glucose levels increased after feeding, peaking at ZT4-ZT12 and then declining. Corticosterone levels were highest around the time of feeding, and then decreased to trough concentrations at ZT12-ZT16, increasing again in anticipation of the next feeding bout. In the brain, the suprachiasmatic nucleus had a rhythm of FOS and PER1 that was not significantly affected by the feeding schedule. Conversely, the supraoptic, paraventricular and tuberomammillary nuclei had rhythms of both FOS and PER1 induced by the time of scheduled feeding. We conclude that the nursing rabbit pup is a natural model of food entrainment, as food, in this case milk formula, is a strong synchronizing signal for behavioral, hormonal, metabolic and neural parameters.

Upregulation of voltage-gated Na+ channels by long-term activation of the ghrelin-growth hormone secretagogue receptor in clonal GC somatotropes.

A central question in adenohypophyseal cell physiology concerns the role of transmembrane ionic fluxes in the initiation of the hormone secretion process. In the current report, we investigated the effects of the growth hormone (GH) secretagogues ghrelin and GH-releasing peptide-6 (GHRP-6) on the regulation of the functional expression of voltage-gated Na(+) channels using the tumoral somatotrope GC cell line as a model. Cells were cultured under control conditions or in presence of the GH secretagogues (GHS) for 96 h, and Na(+) currents (I(Na)) were characterized in whole cell patch-clamp experiments. GHS treatment significantly increased I(Na) density in a dose-dependent manner. The effects of GHRP-6 were accompanied by an augment in conductance without changes in the kinetics and the voltage dependence of the currents, suggesting an increase in the number of channels in the cell membrane. Sustained inhibition of L-type Ca(2+) channel activity decreased I(Na) density and prevented the effects of the GHS, whereas long-term exposure to an L-channel agonist increased I(Na) density and enhanced the actions of GHRP-6, indicating that Ca(2+) entry through these channels plays a role in the regulation of Na(+) channel expression. Likewise, GHRP-6 failed to enhance Na(+) channel expression in the presence of membrane-permeable inhibitors of protein kinases A and C, as well as the Ca(2+)/calmodulin-dependent kinase II. Conversely, treatment with a cAMP analog or a protein kinase C activator enhanced both basal and GHS-induced secretion of GH measured by enzyme-linked immunoassay, suggesting that GHRP-6 acting through the ghrelin receptor and different signaling pathways enhances Na(+) channel membrane expression, which favors hormone release from GC somatotropes.

Up-regulation of high voltage-activated Ca(2+) channels in GC somatotropes after long-term exposure to ghrelin and growth hormone releasing peptide-6.

Activation of the growth hormone (GH)-secretagogue receptor (GHS-R) by synthetic GH-releasing peptides (GHRP) or its endogenous ligand (ghrelin) stimulates GH release. Though much is known about the signal transduction underlying short-term regulation, there is far less information on mechanisms that produce long-term effects. In the current report, using whole-cell patch-clamp recordings, we assessed the long-term actions of such regulatory factors on voltage-activated Ca(2+) currents in GH-secreting cells derived from a rat pituitary tumour (GC cell line). After 96 h in culture, all recorded GC somatotropes exhibited two main Ca(2+) currents: a medium voltage-activated (MVA; T/R-type) and a high voltage-activated (HVA; mostly dihydropyridine-sensitive L-type) current. Interestingly, L- and non-L-type channels were differentially up-regulated by GHRP-6 and ghrelin. Chronic treatment with the GHS induced a significant selective increase on Ba(2+) current through HVA Ca(2+) channels, and caused only a modest increase of currents through MVA channels. Consistent with this, in presence of D-(Lys(3))-GHRP-6, a specific antagonist of the GHS-R, the increase in HVA Ca(2+) channel activity after chronic treatment with the GHS was abolished. The stimulatory effect on HVA current density evoked by the secretagogues was accompanied by an augment in maximal conductance with no apparent changes in the kinetics and the voltage dependence of the Ca(2+) currents, suggesting an increase in the number of functional channels in the cell membrane. Lastly, in consistency with the functional data, quantitative real-time RT-PCR revealed that the expression level of transcripts encoding for the Ca(V)1.3 pore-forming subunit of the L-type channels was significantly increased after chronic treatment of the GC cells with ghrelin.

Ghrelin and GHRP-6 enhance electrical and secretory activity in GC somatotropes.

It is well established that pituitary somatotropes fire spontaneous action potentials (SAP) which generate Ca(2+) signals of sufficient amplitude to trigger growth hormone (GH) release. It is also known that ghrelin and synthetic GH-releasing peptides (GHRPs) stimulate GH secretion, though the mechanisms involved remain unclear. In the current report, we show that the chronic (96h) treatment with ghrelin and GHRP-6 increases the firing frequency of SAP in the somatotrope GC cell line. This action is associated with a significant increase in whole-cell inward current density. In addition, long-term application of Na(+) or L-type Ca(2+) current antagonists decreases GHRP-6-induced release of GH, indicating that the ionic currents that give rise to SAP play important roles for hormone secretion in the GC cells. Together, our results suggest that ghrelin and GHPR-6 may increase whole-cell inward current density thereby enhancing SAP firing frequency and facilitating GH secretion from GC somatotropes.