FIGURE 3.

Effects of CAY10499 and H-89 on Bt₂cAMP-treated HSL activity, P-HSL, StAR, P-StAR, and progesterone synthesis, and PKA activity. MA-10 cells were pretreated without or with CAY10499 (10 μm, A and B; or 1–20 μm, C and D) for 45 min, and then treated in the absence or presence of Bt₂cAMP with either increasing doses (0.01–1.0 mm; A) or a fixed dose (0.5 mm; B-D) for an additional 4 (B) or 6 h (A, C, and D). Cells were then processed for determining HSL activity (A and D), PKA activity (B), and immunoblotting (C), as described under “Experimental Procedures.” [¹⁴C]Cholesteryl oleate was used as a substrate for determining HSL activity (A and D). [¹⁴C]Oleic acid released was measured and expressed in terms of HSL activity (pmol/min/μg of protein), which represent the mean ± S.E. five independent experiments (A and D). Representative immunoblots illustrate P-HSL (Ser-660), HSL, StAR, P-StAR, and CYP11A1 in different groups using 20–30 μg of total cellular protein (C). Immunoblots shown are representative of five independent experiments. Accumulation of progesterone in media was determined (±S.E., n = 5) and expressed as ng/mg of protein (A and D; right panels).