Ghrelin-induced growth hormone release from isolated rat anterior pituitary cells depends on intracellullar and extracellular Ca2+ sources

Ghrelin, a novel growth hormone (GH)-releasing peptide, was isolated from the rat stomach as an endogenous ligand to the growth hormone secretagogues receptor. It is known that ghrelin stimulates the release of GH from the rat anterior pituitary gland, but the intracellular signal cascade in somatotrophs has not yet been clarified. In this study, using an isolated cell perifusion system, we examined whether ghrelin- and growth hormone-releasing hormone (GHRH)-induced GH secretion from rat pituitary cells depends on intra- and extracellular Ca2+ and voltage-gated Ca2+ channels. For this purpose, we first measured ghrelin- or GHRH-stimulated GH concentration following treatment with reduced extracellular Ca2+ and/or thapsigargin, an endoplasmic reticulum Ca2+ ATPase inhibitor. Reductions in the extracellular Ca2+ concentration to 0.25 mM and to 0 mM resulted in decreases in ghrelin-stimulated GH secretion to 81% and 39% and decreases in GHRH-induced GH secretion to 83% and 13%, respectively, compared to the levels in the case of 2.5 mM Ca2+ concentration, suggesting that extracellular Ca2+ is essential for both ghrelin- and GHRH-induced GH secretion. Pretreatment with thapsigargin resulted in a reduction in ghrelin-induced GH secretion to approximately 60% of the control level, but GHRH treatment had not effect on the GH secretion. Moreover, preincubation with thapsigargin and 0 mM extracellular Ca2+ concentration resulted in significant inhibition of GHRH- and ghrelin-induced GH secretion. Subsequently, to determine whether ghrelin-stimulated GH secretion was induced through voltage-gated Ca2+ channels, we measured the ghrelin-stimulated GH concentration following treatment with nifedipine, an L-type Ca2+ channel inhibitor, and found that the amount of GH secretion was reduced to 44% of the control level. Furthermore, by replacement of extracellular Na2+ in the medium with N-methyl-D(-)-glucamine, an impermeable molecule, GH secretion was reduced to 47%. In this study, we demonstrated that the GH-stimulatory effect of ghrelin, unlike that of GHRH, is achieved through both intracellular and extracellular Ca2+ sources and that ghrelin-induced extracellular Ca2+ influx involves an L-type voltage-gated Ca2+ channel and Na+ influx.