Add time:08/16/2019         Source:sciencedirect.com

Signal-response coupling was studied in an exocrine female accessory sex gland (albumen gland) of the freshwater snail Lymnaea stagnalis. Glands were incubated in vitro with Calfluxin (CaFl), a neuropeptide which stimulates the influx of Ca2+ into the mitochondria of the secretory cells. This influx, which is considered to reflect an increase of Ca2+ in the cytosol, was measured as the percentage mitochondria containing Ca deposits. Ca deposits were visualized at the ultrastructural level with the pyroantimonate precipitation technique. The origin of the Ca2+ and the mechanism by which the Ca2+ concentration in the cytosol is elevated were investigated. The results indicate that CaFl stimulates the influx of extracellular Ca2+ and mobilizes intracellular Ca2+. The increase of the percentage of mitochondria containing Ca deposits is sensitive to Ca2+ channel blockers (D600, Co2+, La3+), indicating that Ca2+ channels are involved. Li+ ions suppress the CaFl response, which suggests that the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), and thus the production of myo-inositol-1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) is involved in the Ca2+ mobilization. The protein kinase-C (PKC) stimulator 4-β-phorbol 12-β-myrastate 13-α-acetate (PMA) mimicked the response to CaFl. The PKC inhibitors trifluoperazine (TFP) and chlorpromazine (CP) markedly decreased the CaFl-stimulated influx of Ca2+ into the mitochondria. The PMA-stimulated influx of Ca2+ into the mitochondria is not dependent on extracellular Ca2+ and is not sensitive to Ca2+ channel blockers. In PMA-stimulated glands, the Na+H+ exchange blocker amiloride completely abolished the Ca2+ influx into mitochondria. In CaFl-stimulated glands the influx was partly blocked. Increasing the internal pH of the glandular cells with the Na+H+ ionophore monensin or with NH4Cl mimicked the CaFl response. It is proposed that upon stimulation with CaFl, mobilization of intracellular Ca2+ is mediated via the PKC-stimulated activation of the Na+H+ exchange, thus leading to an increase of the internal pH. The role of IP3 in the mobilization of intracellular Ca2+ is uncertain.

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