ROLE OF PROTEIN-KINASE-C IN THE REGULATION OF ATP-TRIGGERED INTRACELLULAR CA2-CELLS( OSCILLATIONS IN CHICKEN GRANULOSA)

These studies were designed to investigate the role of protein kinase C (PKC) in the regulation of ATP-triggered intracellular Ca2+ ([Ca2+]( i)) oscillations in chicken granulosa cells. Granulosa cells were obta ined from the two largest preovulatory follicles (F-1 and F-2) of hens and [Ca2+](i) was measured in cells loaded with the Ca2+-responsive f luorescent dye fura-2, Adenosine triphosphate (100 mu mol/l) triggered an immediate, large [Ca2+](i) spike that was followed by oscillations that returned to the resting level between spikes, The ATP (100 mu mo l/l) also stimulated a 1.70 +/- 0.1-fold increase in membrane-associat ed PKC activity over control levels. The frequency of the ATP-triggere d [Ca2+](i) oscillations was reduced in a concentration-dependent (1-1 0 nmol/l) manner by treating the cells for 2 min with a PKC activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA). A higher TPA concentrati on (100 nmol/l) completely prevented ATP from triggering the initial ( Ca2+](i) spike and oscillations. Adding TPA during the ATP-triggered [ Ca2+](i) oscillations immediately stopped the oscillatory activity. In terestingly, PKC inhibitors failed to amplify the ATP-triggered [Ca2+] (i) oscillations, Instead, adding the PKC inhibitors staurosporine (20 nmol/l), calphostin C (200 nmol/l) or 1-(5-isoquinolinesulfonyl)-2-me thylpiperazine dihydrochloride (H7; 100 mu mol/l), either before or du ring the ATP (100 mu mol/l)-triggered [Ca2+](i) response, also complet ely blocked the [Ca2+](i) oscillations. Therefore, ATP-triggered [Ca2](i) oscillations in chicken granulosa cells appear to be regulated by a negative feedback loop requiring PKC, because the [Ca2+](i) oscilla tions were prevented by either full activation or inhibition of PKC ac tivity.