Orexin-A Augments Voltage-Gated Ca2+ Currents and Synergistically Increases Growth Hormone (GH) Secretion with GH-Releasing Hormone in Primary Cultured Ovine Somatotropes

Abstract

Orexins are recently discovered neuropeptides that play an important role in the regulation of hormone secretion, and their receptors have been recently demonstrated in the pituitary. The effects of orexin-A on voltage-gated Ca2+ currents and GH release in primary cultured ovine somatotropes were examined. The expression of orexin-1 receptor was demonstrated by RT-PCR in ovine somatotropes, from which Ca2+ currents were also isolated as L, T, and N currents. Application of orexin-A (100 nM) significantly and reversibly increased only the L current, and coadministration of orexin-A and GHRH (10 nM) showed an additive effect on this current, but no effect of orexin-A was observed on either T or N current. Furthermore, the orexin-A-induced increase in the L current was completely abolished by the inhibition of protein kinase C (PKC) activity using calphostin C (100 nM), phorbal 12,13-dibutyrate pretreatment (0.5 micro M) for 16 h or specific PKC inhibitory peptide PKC(19-36) (1 mM). However, the increase in L current by orexin-A was sustained when cells were preincubated with a specific protein kinase A blocker H89 (1 micro M) or a specific intracellular Ca2+ store depleting reagent thapsigargin (1 micro M). Finally, orexin-A alone did not significantly increase GH release, but coadministration of orexin-A and GHRH showed a synergistic effect on GH secretion in vitro. Our results therefore suggest that orexin-A may play an important role in regulating GHRH-stimulated GH secretion through the enhancement of the L-type Ca2+ current and the PKC-mediated signaling pathway in ovine somatotropes.

Orexin A suppresses in vivo GH secretion

“…The fact that OX-A failed to modify in vitro GH secretion suggests that its inhibitory effect was taking place at the hypothalamic level. This is in contrast to previous data obtained in porcine somatotrophs where it was found that OX increased in vitro GH responses to GHRH (16). It is likely that these discrepancies reflect interspecies differences.…”

“…Finally, we also assessed the effect of OX-A on in vitro GH secretion, since the presence of OX receptors in the human anterior pituitary has been recently reported (10), as well as the activation of voltage-gated Ca 2þ currents in porcine somatotrophs (16).…”

The Orexin OX1Receptor Regulates Ca2+Entry via Diacylglycerol-Activated Channels in Differentiated Neuroblastoma Cells

“…van den found that the same inhibitor of PKC that we used totally blocked the [Ca 2ϩ ] i increase. In several other studies, PKC has been suggested to be a crucial intermediate effector in orexin action, often with downstream effects linked to specific ion channels (Uramura et al, 2001;Xu et al, 2002;Kohlmeier et al, 2004) or less well defined ion conductances (Yang et al, 2003). PKC has long been known to modulate VGCCs (Yang and Tsien, 1993) and K ϩ conductances (Henry et al, 1996).…”

Selected hormonal and neurotransmitter mechanisms regulating feed intake in sheep

“…In addition to reproduction, ORX neurons project to the PeVN adjacent to somatostatin neurons , suggesting a possible role in regulating GH. Indeed, ORX A and B receptors are expressed in somatotropes and both molecules activate calcium channels in the pituitary via protein kinase C mechanisms (Xu et al, 2002 and. ORX B will also release GH from isolated pituitary cells.…”