Receptor-operated Ca2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line

Prostate smooth muscle cells predominantly express alpha 1-adrenoceptors (of alpha 1-AR). alpha 1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca2+ entry pathways associated with the activation of alpha 1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PSI (a prostate smooth muscle cell line) which express endogenous alpha 1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca2+ influx which depended on the extracellular Ca2+ and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca2+ influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca2+-permeable channels mediated both alpha-agonist- and OAG-evoked Ca2+ influx. Following the sarcoplasmic reticulum (SR) Ca2+ store depletion by thapsigargin (Tg), a SERA inhibitor, OAG and PHE were both still able to activate Ca2+ influx. However, OAG failed to enhance Ca2+ influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-incluced Ca2+ influx required TRPC3 and TRPC6, whereas the Tg-activated ("capacitative") Ca2+ entry involved only TRPC3 encoded protein. It may be thus concluded that PSI cells express TRPC3 and TRPC6 proteins which function as receptor- and store-operated Ca2+ entry pathways.