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

Fast nongenomic steroid actions in several cell types seem to be mediated b y 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 skelet al muscle cells. The effect of steroids on [Ca2+](i) was monitored in Fluo 3-acetoxymethyl ester-loaded myotubes by either confocal microscopy or fluo rescence 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 a ccompanied by oscillations, was evident with both hormones. A slow rise in [Ca2+](i) that reached its maximum after a 30-min exposure to aldosterone w as also observed. Calcium responses seem to be fairly specific for aldoster one 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 I P3 transient was more rapid than the fast calcium signal. Spironolactone, a n inhibitor of the intracellular aldosterone receptor, or cyproterone aceta te, an inhibitor of the testosterone receptor, had no effect on the fast [C a2+](i) signal or in the increase in IP3 mass. These signals could mean tha t there are distinct nongenomic pathways for the action of these two steroi ds in skeletal muscle cells.