Method of transfection affects the cAMP-mediated induction of the RII betasubunit of protein kinase A in Sertoli cells: inhibition of response by increase in intracellullar calcium

mRNA for the regulatory subunit RII beta of cAMP-dependent protein kinase i s stimulated more than 50-fold by cAMP in primary cultures of rat Sertoli c ells. We have previously shown that this induction involves regulation of t ranscriptional activation as well as mRNA stabilization. The rat RII beta g ene contains no cAMP response element (CRE), and the induction of RII beta mRNA is slow and requires on-going protein synthesis. When a construct cont aining the 5'-flanking region of the RII beta gene upstream of a CAT report er was transfected into Sertoli cells by the calcium phosphate method, low and variable responses to cAMP (three- to fivefold) were observed, whereas a 15- to 20-fold increase in reporter activity by cAMP was observed after l ipofectamine transfection. Interestingly, when a vector containing CRE elem ents upstream of a reporter gene was transfected into Sertoli cells, the re sponses to cAMP were similar regardless of the transfection method used. We have also demonstrated that increased intracellular levels of calcium by A 23187 and thapsigargin dramatically inhibit cAMP-mediated induction of RII beta mRNA, but not the mRNA for the CRE-containing RI alpha gene. Furthermo re, decreased cAMP responsiveness of endogenous RII beta mRNA (but not RI a lpha) was also observed in calcium phosphate-transfected Sertoli cells but not in lipofectamine-transfected cells. Thus, calcium-mediated reduction in cAMP response appears to be a gene-specific phenomenon.