Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells.
L-type Ca(2+) channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba(2+) influx in a depolarizing medium. In the absence of extracellular Ca(2+), activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba(2+) influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca(2+)-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in a Ca(2+)-free medium from 30 to approximately 80 nM. This [Ca(2+)](i) increase declined slowly, giving rise to a modest elevation of [Ca(2+)](i) that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca(2+)-free medium. However, when the Ca(2+) ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca(2+) channel activity, suggesting that a lower amplitude and prolonged release of Ca(2+) stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 microM) and ascomycin (5 microM), inhibitors of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1mM CaCl(2) to the Tg-treated cells increased [Ca(2+)](i) to approximately 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca(2+)](i) increase elicited by passive depletion of internal Ca(2+) stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca(2+)](i) via store-dependent Ca(2+) entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca(2+) channels in a myocyte model involves a balance between the differential Ca(2+) sensitivities and opposing actions of PKC and calcineurin.
Turner
Jay D
JD
Department of Pharmacology & Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103, USA.
Thomas
Andrew P
AP
Reeves
John P
JP
Hantash
Basil M
BM
D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D032448:Myoblasts, Skeletal / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000378:metabolism / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D010703:Phorbol Esters; D019284:Thapsigargin; D016572:Cyclosporine; C080287:thymeleatoxin; C058028:immunomycin; D011493:Protein Kinase C; D019703:Calcineurin; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016559:Tacrolimus