08 July 2003
Research on the mechanism for growth hormone secretagogue (GHS) induction of growth hormone secretion led to the discovery of the GHS receptor (GHS-R) and later to ghrelin, an endogenous ligand for GHS-R. The ability of ghrelin to induce an increase in the intracellular Ca<sup>2+</sup> concentration – [Ca<sup>2+</sup>]<sub>i</sub> – in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone. Ghrelin increased the [Ca<sup>2+</sup>]<sub>i</sub> in a dose-dependent manner in 98% of the cells that responded to human growth hormone releasing hormone. In the presence of ( D-Lys<sup>3</sup>)-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca<sup>2+</sup>]<sub>i</sub> evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca<sup>2+</sup>]<sub>i</sub>. The stimulatory effect of ghrelin on somatotropes was greatly attentuated in low-calcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na<sup>+</sup> solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca<sup>2+</sup>]<sub>i</sub> transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The nonpeptidyl GHS, L-692,585 (L-585), induced changes in [Ca<sup>2+</sup>]<sub>i</sub> similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca<sup>2+</sup>]<sub>i</sub>. Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Simultaneous application of ghrelin and L-585 did not cause an additive increase in [Ca<sup>2+</sup>]<sub>i</sub>. Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion.