Orexin-A augments voltage-gated Ca2+ currents and synergistically increases growth hormone (GH) secretion with GH-releasing hormone in primary cultured ovine somatotropes.
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.
Xu
Ruwei
R
Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia.
Wang
Qinling
Q
Yan
Ming
M
Hernandez
Maria
M
Gong
Changhong
C
Boon
Wah Chin
WC
Murata
Yoko
Y
Ueta
Yoichi
Y
Chen
Chen
C
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000494:pharmacology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004357:Drug Synergism; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D013006:Growth Hormone / Q000378:metabolism*; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D009479:Neuropeptides / Q000494:pharmacology*; D064446:Orexin Receptors; D000068797:Orexins; D010903:Pituitary Gland, Anterior / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012313:RNA / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D015398:Signal Transduction
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D012313:RNA; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium