Mechanism of spironolactone-induced Ca²⁺ increase in rat testicular arteriole smooth muscle cells revealed by real-time laser scanning confocal microscopy

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We reported previously that spironolactone (SPL) induced an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in rat testicular arteriole smooth muscle cells. In the present study, we further investigated the mechanism of SPL-induced [Ca²⁺]_i dynamics in rat arteriole smooth muscles. The increase in [Ca²⁺]_i induced by SPL (300 μM) was markedly inhibited in extracellular Ca²⁺-free conditions and in the presence of diltiazem or gadolinium. In contrast, the phospholipase C inhibitor (U73122), did not affect the SPL-induced increase in [Ca²⁺]_i, similar to what was observed for 2-aminoethoxydiphenyl borate (2- APB: an inhibitor of inositol triphosphate (IP₃)dependent Ca²⁺ mobilization). Moreover, the protein kinase A (PKA) inhibitor H89 partially inhibited the SPL-induced increase in [Ca²⁺]_i, whereas the protein kinase C (PKC) inhibitor GF109203X did not. Either suramin (a non-specific G protein antagonist) or NF449 (an inhibitor of the α-subunit of the stimulatory G protein G_sα), partially blocked the SPL-induced increase in [Ca²⁺]_i. Similarily, either mifepristone, a glucocorticoid-receptor antagonist, or flutamide, a non-steroidal antiandrogen drug, partially blocked the SPL-induced increase in [Ca²⁺]_i. We suggest that the SPL-induced increase in [Ca²⁺]_i in arterioles is mediated both by Ca²⁺ influx from the extracellular fluid and by Ca²⁺ mobilization from internal Ca²⁺ stores, with the former being dominant. We thus propose that SPL interacts with both extracellular (i.e., G-proteincoupled-type) and intracellular (e.g., glucocorticoid) receptors in rat testicular arterioles, which is followed by an increase in intracellular Ca²⁺ that causes smooth-muscle contraction.