Role of protein kinase C in the regulation of ATP-triggered intracellular Ca2+ oscillations in chicken granulosa cells.
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 Ca(2+)-responsive fluorescent dye fura-2. Adenosine triphosphate (100 mumol/l) triggered an immediate, large [Ca2+]i spike that was followed by oscillations that returned to the resting level between spikes. The ATP (100 mumols/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 mumols/l), either before or during the ATP (100 mumols/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.
Morley
P
P
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Chakravarthy
B R
BR
Mealing
G A
GA
Tsang
B K
BK
Whitfield
J F
JF
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D004789:Enzyme Activation; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology
D009281:Naphthalenes; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C; D002118:Calcium