Requirement of extracellular calcium in fish pituitary gonadotropin release by gonadotropin hormone-releasing hormone

Abstract

Influence of extracellular calcium on gonadotropin hormone-releasing hormone (GnRH)-stimulated gonadotropin hormone (GtH) release from a teleostean fish (Channa punctatus) pituitary was examined in vitro by preparing enzymatically dispersed pituitary cell incubation. Effect of Ca²⁺ on GnRH-augmented GtH release was evaluated with partially purified C. punctatus GnRH (cGnRH) and synthetic mammalian GnRH (mGnRH). Cells were dispersed by 0.3% collagenase plus 0.05% trypsin in culture medium and a high yield of viable cells were obtained. Addition of cGnRH (10μg/ml) to pituitary cells in Ca²⁺-free medium resulted in a significant increase in GtH release, but the addition of Ca²⁺ (2mM) enhanced it to about four- and threefold over cGnRH and mGnRH, respectively. Increasing concentrations of Ca²⁺ (0.1-2.0 mM/well) with fixed concentrations of GnRH (10 μg/ml) or increasing doses of GnRH (2.5 to 20 μg/ml) with fixed amount of Ca²⁺ (2 mM/well) resulted in a dose dependent increase in GtH release. EDTA or EGTA (2 mM/well) completely suppressed the Ca²⁺-augmenting effect of GnRH-stimulated GtH release. Addition of lanthanum (La³⁺, 4 mM/well), a competitive inhibitor of Ca²⁺, reduced 60% of the Ca²⁺ (2 mM/well) stimulation. Verapamil, a specific Ca²⁺ channel blocker, when added in increasing concentrations (1-100 μM/well) to pituitary cell incubations containing GnRH plus Ca²⁺, inhibited GtH release in a dose dependent manner. Interestingly, GnRH-stimulated GtH release in Ca²⁺-free medium could be waived by EGTA (2 mM/well), indicating availability of extracellular calcium from tissue sources. The uptake of radioactive Ca²⁺ by pituitary cells was greatly enhanced by GnRH while the addition of verapamil (10 μM/well) not only inhibited the GnRH-stimulated uptake, but also reduced it below the control level. Based on the results, it was concluded that GnRH-stimulated GtH release from fish pituitary cells requires an influx of extracellular Ca²⁺.