Aldosterone- and testosterone-mediated intracellular calcium response in skeletal muscle cell cultures

Abstract

Fast nongenomic steroid actions in several cell types seem to be mediated by second messengers such as intracellular calcium ([Ca2+](i)) and inositol 1,4,5-trisphosphate (IP3). We have shown the presence of both slow calcium transients and IP3 receptors associated with cell nuclei in cultured skeletal muscle cells. The effect of steroids on [Ca2+](i) was monitored in Fluo 3-acetoxymethyl ester-loaded myotubes by either confocal microscopy or fluorescence microscopy, with the use of out-of-focus fluorescence elimination. The mass of IP3 was determined by radioreceptor displacement assay. [Ca2+](i) changes after either aldosterone (10-100 nM) or testosterone (50-100 nM) were observed; a relatively fast (<2 min) calcium transient, frequently accompanied by oscillations, was evident with both hormones. A slow rise in [Ca2+](i) that reached its maximum after a 30-min exposure to aldosterone was also observed. Calcium responses seem to be fairly specific for aldosterone and testosterone, because several other steroid hormones do not induce detectable changes in fluorescence, even at 100-fold higher concentrations. The mass of IP3 increased transiently to reach two- to threefold the basal level 45 s after addition of either aldosterone or testosterone, and the IP3 transient was more rapid than the fast calcium signal. Spironolactone, an inhibitor of the intracellular aldosterone receptor, or cyproterone acetate, an inhibitor of the testosterone receptor, had no effect on the fast [Ca2+](i) signal or in the increase in IP3 mass. These signals could mean that there are distinct nongenomic pathways for the action of these two steroids in skeletal muscle cells.