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.