The involvement of protein kinases in signalling of opioid agonist FK 33-824 in porcine granulosa cells

It is known that acute action of μ opioid receptor agonist, FK 33-824, results in an inhibition of oestradiol (E2) secretion by porcine granulosa cells from large follicles, but the opioid mode of action is unknown. In the present study, the involvement of two signal transduction pathways, phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A, in mechanism of the opioid action was investigated. Treatment of pig granulosa cells with FK 33-824 at the dose 1 nM suppressed E2 secretion. Protein kinase C (PKC) inhibitors - staurosporine (1-100 nM), d-sphingosine (10-500 nM) and PKCi (100-2000 nM) - both alone and in combination with FK 33-824 reduced E2 release from the cells in relation to the control group. The inhibitory effect of the opioid on E2 output was also observed in PKC-deficient granulosa cells. PKC activator, PMA (10 and 100 nM) significantly attenuated the inhibitory effect of the opioid agonist. FK 33-824 also inhibited 3[H]phorbol 12,13 dibutyrate (3[H]PDBu) specific binding by granulosa cells. Adenylyl cyclase (AC) engagement in opioid signal transduction was assayed after 2-h and 4-h incubations of granulosa cells. During 2-h incubation, FK 33-824 at the dose 1 nM decreased cAMP secretion. Prolongation of the incubation up to 4 h caused disappearance of the opioid action. The addition of protein kinase A (PKC) inhibitor, PKAi (100-2000 nM), alone or together with FK 33-824, was followed by an inhibition of E2 secretion. FK 33-824 with the highest dose of PKAi (2000 nM) significantly inhibited E2 secretion by the cells in comparison to these agents tested separately. The opioid added in combination with PKA activator, 8BrcAMP (1000 μM), caused attenuation of stimulatory effect of 8BrcAMP. Collectively, these results suggest that acute action of μ opioid agonist on porcine granulosa cells leads to decrease of enzymatic activity of PKC and AC/PKA. It is not ruled out that other signal transduction pathways - not considered in this study - may also be engaged in the opioid mechanism of action in these cells.