Role of protein kinase C in the regulation of ATP-triggered intracellular Ca2+ oscillations in chicken granulosa cells

Morley P. Chakravarthy BR, Mealing GAR, Tsang BK, Whitfield JF. Role of protein kinase C in the regulation of ATP-triggered intracellular Ca2+ oscillations in chicken granulosa cells. Eur J Endocrinol 1996;134:743–50. ISSN 0804–4643 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 obtained from the two largest preovulatory follicles (F1 and F2) of hens and [Ca2+]i was measured in cells loaded with the Ca2+-responsive fluorescent dye fura-2. Adenosine triphosphate (100 μmol/l) triggered an immediate, large [Ca2+]i spike that was followed by oscillations that returned to the resting level between spikes. The ATP (100 μmol/l) also stimulated a 1.70 ± 0.1-fold increase in membrane-associated PKC activity over control levels. The frequency of the ATP-triggered [Ca2+]i oscillations was reduced in a concentration-dependent (1–10 nmol/l) manner by treating the cells for 2 min with a PKC activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA). A higher TPA concentration (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. Interestingly, 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-methylpiperazine dihydrochloride (H7; 100 μmol/l), either before or during the ATP (100μmol/l)-triggered [Ca2+]i response, also completely 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 oscillations were prevented by either full activation or inhibition of PKC activity. Paul Morley, Institute for Biological Sciences, National Research Council of Canada, Building M54, Montreal Road, Ottawa, Ontario, K1A OR6, Canada