Effects of Trypsin, Protease Inhibitors and Ethanol on Corpus Luteum Adenylyl Cyclase1

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Abstract
The effects of trypsin, 2 protease inhibitors (N-alpha-p-tosyl-L-lysine chloromethyl ketone HCl [TLCK] and L-1-tosylamide-2-phenylethyl chloromethyl ketone [TPCK]) and ethanol on rabbit and rat corpus luteum adenylyl cyclase were studied. Trypsin stimulated adenylyl cyclase activity in rabbit corpus luteum membranes. Maximal stimulation was observed at 2.5 μg/ml trypsin. Higher trypsin concentrations inhibited rabbit corpus luteum adenylyl cyclase activity. Addition of 10 μM GTP did not alter the activation of adenylyl cyclase by trypsin. In the presence of ethanol, TLCK inhibited hCG and LH stimulation of rabbit and rat corpus luteum adenylyl cyclase. Ethanol, alone, enhanced hCG and LH stimulation of adenylyl cyclase up to a concentration of 5% ethanol, while higher concentrations of ethanol inhibited hCG and LH stimulation of ovarian adenylyl cyclase. These responses in the rat ovarian adenylyl cyclase were not altered by GTP; in the rabbit corpus luteum adenylyl cyclase, TLCK in the presence of ethanol and GTP actually enhanced hCG and LH stimulation of adenylyl cyclase up to a concentration of 0.625 mM TLCK. Higher concentration of TLCK in the presence of ethanol and GTP inhibited hCG and LH stimulation of the rabbit enzyme. The combined effects of ethanol and TLCK on ovarian adenylyl cyclase do not appear to involve alteration of the apparent affinity of hCG for its receptor and are due primarily to a reduction in the maximal velocity of the reaction. It appears that TLCK may be enhancing the inhibitory action of high concentrations of ethanol on hCG and LH stimulation of ovarian adenylyl cyclase. Based on the findings of the present study, it would appear to be premature to propose a central role for proteases in hCG and LH stimulation of ovarian adenylyl cyclase.