The photoaffinity analog [32P]8-azidoadenosine cAMP ([32P]8-N3cAMP) was used to study available cAMPbinding sites on cAMP-dependent protein kinases in homogenates of ovine small (12-22 μm) and large (>22 fim) luteal cells. Binding of the analogs to specific proteins was detected by autoradiography after sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and was quantitated by liquid scintillation counting. In homogenates of untreated small and large cells, only the type I isoenzyme of the regulatory subunit (Ri) of protein kinase had a measurable number of available cAMPbinding sites. Small luteal cells were incubated for 2 h with increasing concentrations of ovine LH (oLH), cholera toxin, or forskolin, and media were collected for quantification of cAMP and progesterone. Cells were harvested and homogenized, and intraceilular cAMP content and photoincorporation of [32P]8- N3cAMP into Ri were measured. Treatment of small luteal cells with oLH, cholera toxin, and forskolin resulted in dose-dependent increases in cAMP in both the cells and the incubation media and in the progesterone content of the media. These increases were accompanied by a dose-dependent decrease in photoincorporation of [32P]8-N3cAMP into R1 of small cells, which was correlated (P < 0.05) with the increase in progesterone secretion. A time-dependent decrease (P < 0.05) in photoincorporation in small cells incubated with oLH (100 ng/ml), cholera toxin (1000 ng/ml), or forskolin (5 μM) preceded an increase (P < 0.05) in progesterone secretion by these cells. Large ovine luteal cells incubated with increasing concentrations of cholera toxin or forskolin demonstrated a dose-dependent decrease in photoincorporation of [32P] 8-N3cAMP into R1. The time-dependent decrease (P < 0.05) in photoincorporation in large cells incubated with cholera toxin (1000 ng/ml) or forskolin (5 nM) was not followed by enhanced progesterone secretion. These observations are consistent with a role for cAMP involvement in progesterone secretion by ovine small luteal cells and suggest a lack of cAMP involvement in progesterone synthesis by large cells.
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