The response of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A pathways in porcine theca interna cells to opioid agonist FK 33-824

Opioids were found as factors affecting porcine ovarian steroidogenesis. The mechanism of opioid action, however, on porcine theca interna cells is completely unknown. Therefore, the present study was designed to investigate the possible involvement of two intracellular pathways, phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A, in opioid signal transduction in porcine theca cells treated with mu opioid receptor agonist, FK 33-824. Incubation of the cells for 4 h with FK 33-824 at the dose 1 nM resulted in decreases in inositol phosphate accumulation as well as androstenedione (A(4)), testosterone (T), and estradiol (E(2)) secretions. Protein kinase C (PKC) inhibitors, staurosporine (1-100 nM), D-sphingosine (10-500 nM), and PKCi (100-2000 nM), both added alone and together with the opioid agonist, depressed release of the steroid hormones. PKC activator, phorbol ester (PMA, 1-100 nM), used alone was without effect on theca cell steroidogenesis, but added in combination with FK 33-824 abolished inhibitory influence of the opioid on A(4), T, and E(2) output. The steroid hormone secretion by PKC-deficient theca cells was inhibited by the opioid agonist. FK 33-824 also suppressed PKC activity reducing (3)H]PDBu specific binding to theca cells, whereas ionomycin (a positive control) increased labeled phorbol ester binding to the cells. In the next experiment, cAMP release from theca cells during 2 and 4 h incubations with FK 33-824 (1-100 nM), naloxone (10 microM; opioid receptor antagonist), and LH (100 ng/mL; a positive control) was examined. FK 33-824 at the dose 1 nM inhibited cAMP secretion during 2 h incubation, but had no effect during longer incubation. LH in a manner independent on incubation time multiplied cAMP release. Protein kinase A inhibitor, PKAi (100-2000 nM), alone and in combination with FK 33-824 (1 nM), inhibited A(4), T, and E(2) secretions by theca cells. PKA activator, 8BrcAMP (10-1000 microM), stimulated the steroid hormone release, but this stimulatory effect was diminished in the presence of FK 33-824. The results allow to suggest that opioid peptides affect porcine theca cell steroidogenesis and their acute action on the cells is connected with the inhibition of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A signal transduction systems.