The role of protein kinase C activation in the control of cortisol synthesis was studied using the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). Bovine zona fasciculata cells were incubated with various concentrations of TPA in the presence and absence of EGTA, verapamil or nitrendipine to see whether cortisol stimulation was dependent on extracellular calcium ions. When free extracellular concentration of Ca2+ was reduced to approximately 10 μmol/l the cortisol response at all concentrations of TPA was reduced by approximately 25% indicating that protein kinase C activation is only partially dependent on extracellular calcium ions. This is confirmed by the effects of the voltage-dependant calcium channel blocker verapamil, which partially inhibited the cortisol response to a maximally effective concentration of TPA (1 μmol/l). However, a second channel blocker, nitrendipine, proved to be ten times more potent than verapamil and totally inhibited the TPA response. The partial effects of EGTA and verapamil and the contrast between verapamil and nitrendipine do not exclude the possibility that intracellular calcium ions are important in protein kinase C activation and may indicate that nitrendipine has better access to an additional site of inhibitory action than verapamil. It is significant that the ionophore A23187, which facilitates Ca2+ entry independently of voltage-sensitive channels, failed to overcome the inhibitory effects of nitrendipine in TPA-stimulated cells.
In some other tissues, the effects of protein kinase C activation are mediated by the opening of Na+/H+ exchange ports. The involvement of this port in the cortisol response has been tested by incubating TAP-and ACTH-treated cells with amiloride, an inhibitor of Na+/H+ exchange. It would seem unlikely that the steroidogenic effects of TPA are mediated by altered Na+/H+ exchange because, at the high concentration of amiloride used to block cortisol synthesis (1 mmol/l), non-specific side effects may occur and also because amiloride at this concentration inhibited the cortisol response to ACTH whose mechanism of action is not solely dependent on protein kinase C activation.
Cells were also incubated with increasing concentration of TPA in the presence and absence of ACTH and angiotensin. TPA did not further stimulate ACTH-treated cells and at 0·32 μmol/l inhibited cortisol synthesis. The effects of angiotensin and TPA were additive which suggests that the steroidogenic effects of angiotensin II are largely independent of protein kinase C activation.