Evidence that IP3 and Ryanodine-sensitive Intra-cellular Ca2+ Stores are not Involved in Acute Hyposmotically-Induced Prolactin Release in Tilapia

Abstract

Prolactin (PRL) cells from the euryhaline tilapia, Oreochromis mossambicus, behave like osmoreceptors by responding directly to reductions in medium osmolality with increased secretion of the osmoregulatory hormone PRL. Extracellular Ca²⁺ is essential for the transduction of a hyposmotic stimulus into PRL release. In the current study, the presence and possible role of intracellular Ca²⁺ stores during hyposmotic stimulation was investigated using pharmacological approaches. Changes in intracellular Ca²⁺ concentration were measured with fura-2 in isolated PRL cells. Intracellular Ca²⁺ stores were depleted in dispersed PRL cells with thapsigargin (1 µM) or cyclopiazonic acid (CPA, 10 µM). Pre-incubation with thapsigargin prevented the rise in [Ca²⁺]_i induced by lysophosphatidic acid (LPA, 1 µM), an activator of the IP₃ signalling cascade, but did not prevent the hyposmotically-induced rise in [Ca²⁺]_i in medium with normal [Ca²⁺] (2mM). Pre-treatment with CPA produced similar results. Prolactin release from dispersed cells followed a pattern that paralleled observed changes in [Ca²⁺]_i. CPA inhibited LPA-induced prolactin release but not hyposmotically-induced release. Xestospongin C (1µM), an inhibitor of IP₃ receptors, had no effect on hyposmotically-induced PRL release. Pre-exposure to caffeine (10mM) or ryanodine (1µM) did not prevent a hyposmotically-induced rise in [Ca²⁺]_i. Taken together these results indicate the presence of IP₃ and ryanodine-sensitive Ca²⁺ stores in tilapia PRL cells. However, the rapid rise in intracellular [Ca²⁺] needed for acute PRL release in response to hyposmotic medium can occur independently of these intracellular Ca²⁺ stores.