Ghrelin-induced growth hormone release from goldfish pituitary cells involves voltage-sensitive calcium channels

Ghrelin (GRL) is a stimulator of growth hormone (GH) release in many organisms, including goldfish. As a first study to examine the signalling mechanisms mediating GRL action on GH release in goldfish, we tested the hypothesis that GLR induces GH release from goldfish pituitary cells by enhancing Ca2+ entry through L-type voltage-sensitive Ca2+ channels (LVSCCs) using perifusion GH release and fura-2/AM Ca2+-imaging experiments. Goldfish (g)GRL19 at 1 nM elicited reversible and repeatable GH responses from dispersed goldfish mixed pituitary cultures. However, the lack of a dose-response relationship in sequential treatments with decreasing concentrations of gGRL19 (ranging from 10 to 0.01 nM) implicated rapid desensitization of the GH response. Sequential applications of gGRL19 (1 nM) and salmon GnRH (100 nM), a known Ca2+-dependent stimulator of GH release, increased intracellular free Ca2+ levels ([Ca2+]i) from the same identified somatotropes, suggesting co-expression of GRL and GnRH receptors on single cells. In contrast, 1 nM gGRL19 failed to elicit GH release and elevation in [Ca2+]i when the cells are incubated with nominally Ca2+-free media. When GH release and [Ca2+]i increases were already stimulated by the LVSCC agonist Bay K8644 (10 μM), addition of 1 nM gGRL19 did not further elevate these responses. Finally, the LVSCC inhibitors nifedipine (1 μM) and verapamil (1 μM) abolished 1 nM gGRL19-induced GH release responses while nifedipine eliminated gGRL19-induced [Ca2+]i increase. Taken together, the results of this study provide evidence that entry of extracellular Ca2+ through LVSCCs is a key component of the GRL signalling pathway leading to GH release in the goldfish pituitary.