Involvement of protein kinase-C, calpains, and calpastatin in prostaglandin F2 alpha-induced oxytocin secretion from the bovine corpus luteum

Abstract

An experiment was conducted to determine whether prostaglandin F2 alpha (PGF2 alpha)-induced secretion of oxytocin (OT) by the bovine corpus luteum (CL) was associated with changes in the activities of protein kinase-C (PKC), calpains, and calpastatin. On day 8 of the estrous cycle (estrus = day 0), beef heifers were restrained and given a 500-micrograms iv injection of cloprostenol, a PGF2 alpha analog. Corpora lutea were surgically removed from beef heifers 0, 2, 7.5, or 30 min (n = 4 animals/time period) after cloprostenol injection. Blood samples were collected before injection and at frequent intervals after injection. Distribution of PKC activity in cytosol and membrane fractions and activities of microcalpain, millicalpain, and calpastatin were determined for all CL. OT was measured in plasma and tissue by RIA. Relative to mean plasma levels of OT at time zero (85 +/- 7 pg/ml), peak plasma levels occurred between 1.5-10 min (270 +/- 36 pg/ml) for all animals. The mean luteal concentration of OT was greater at 0, 2, and 7.5 min (145 +/- 27, 232 +/- 82 and 269 +/- 115 ng/g, respectively) than at 30 min (93 +/- 33), but differences in tissue OT over time were not significant (P > 0.05). PKC activities (percentage over nonactivated control values) in the membrane or cytosolic fractions did not differ significantly among the times of CL removal; however, membrane PKC activity was positively correlated with the plasma OT level at the time of CL removal (r = 0.82; P < 0.0025). Luteal millicalpain activity was approximately twice that of microcalpain at each time point (P < 0.001), although the activities of the individual calpains over time after PGF2 alpha injection did not change. Calpastatin activity was significantly higher at 30 min (515 +/- 28 U/g tissue) than at 0, 2, or 7.5 min (373 +/- 26, 423 +/- 26, and 426 +/- 24 U/g tissue, respectively). PKC activity in the membrane appears to be positively correlated with OT secretion from the bovine CL, and increased calpastatin activity after PGF2 alpha injection may inhibit calpains present in the CL, thereby maintaining an active pool of PKC.