Figure 3. The mechanism of androgen-induced Ca²⁺ release. (A) The effect of cyproterone acetate (CPA), an antagonist of intracellular androgen receptor, and cycloheximide, a protein synthesis inhibitor, on the response of LNCaP cells to 1 µM 5-dihydrotestosterone (DHT). Cells were incubated for 3 hours with 10 µM cycloheximide and then stimulated with DHT (; 1 µM). Cycloheximide did not affect the Ca²⁺ increases induced by the hormone. Cells were incubated for 20 minutes with 1 µM CPA and then stimulated with DHT (). The use of CPA did not modify the Ca²⁺ increases produced by the hormone. The black bar indicates the time of addition of DHT. (B) Testosterone-induced rise in the intracellular concentration of Ca²⁺ ([Ca²⁺]i) of LNCaP cells. 10 nM testosterone () elicited an immediate [Ca²⁺]i increase in LNCaP cells. 10 nM T-BSA also increased [Ca²⁺]i (), while BSA alone () was ineffective. (C) Effects of genistein (a tyrosine kinase inhibitor), GDPßS, and PTX on testosterone-induced [Ca²⁺]i increases. Cells were incubated with 100 ng/mL PTX for 24 hours and then stimulated with DHT (•; 1 µM). PTX blocked the Ca²⁺ increases induced by the hormone. Cells were permeabilized with saponin and stimulated with DHT (). It is noteworthy that under these conditions the cells did not lose the capacity to respond to the hormone. Nevertheless, permeabilization in the presence of GDPßS (500 nM) blocked the DHT-induced Ca²⁺ increases (). Cells were incubated for 20 minutes with 100 µM genistein and then stimulated with DHT (). The use of genistein did not modify the Ca²⁺ increases produced by the hormone. The black bar indicates the time of addition of DHT.