Modulation of Ca(2+) release through ryanodine receptors in vascular smooth muscle by protein kinase Calpha.
The role of protein kinase C (PKC) in Ca(2+) release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in both freshly isolated and cultured mouse aortic SMCs (ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca(2+)](i) transients. Application of the PKC inhibitor calphostin C caused [Ca(2+)](i) transients which were not blocked by nifedipine or by removing extracellular Ca(2+) but were abolished after inhibition of the SR Ca(2+)-ATPase with thapsigargin or after inhibition of RyRs with ryanodine. In addition, chelerythrine and GF109203X also elevated resting [Ca(2+)](i) but no further [Ca(2+)](i) increase was seen with subsequent application of caffeine. Selective inhibition of PKCalpha with safingol blocked caffeine-induced [Ca(2+)](i) transients, but the PKCepsilon inhibitory peptide V1-2 did not. In cells expressing a EGFP-tagged PKCalpha, caffeine-induced [Ca(2+)](i) transients were associated with a rapid focal translocation near the cell periphery, while application of ionomycin and DOG caused translocation to the plasma membrane. Western blot showed that caffeine increased the relative amount of PKCalpha in the particulate fraction in a time-dependent manner. Co-immunoprecipitation of RyRs and PKCalpha indicated that they interact. In conclusion, our studies suggest that PKC activation can inhibit the gating activity of RyRs in the SR of ASMCs, and this regulation is most likely mediated by the Ca(2+)-dependent PKCalpha isoform.
Peng
HongLi
H
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
Yaney
Gordon C
GC
Kirber
Michael T
MT
D000818:Animals; D015153:Blotting, Western; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047468:Immunoprecipitation; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism*
D004791:Enzyme Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; C496630:Prkca protein, mouse; D051571:Protein Kinase C-alpha; D002118:Calcium