Regulation of Ca2+ entry in rat aortic smooth muscle cells in primary culture.
We characterized Ca2+ entry in rat aortic smooth muscle cells (SMCs) maintained in primary culture by measuring uptake of 45Ca2+ or Mn2+ from a normal balanced salt solution and the extracellular Ca(2+)-induced increase in the intracellular Ca2+ concentration ([Ca2+]i) in a medium [high pH (pH 8.8)/high Mg2+ (20 mM) medium containing a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin] that inhibits removal of Ca2+ from the cytoplasm. Such measurements in the presence or absence of a dihydropyridine (DHP) calcium channel antagonist (PN200-110) or hyperpolarizing agent (valinomycin) revealed that DHP-sensitive voltage-gated Ca2+ channels (VGCCs) are activated in these SMCs under resting conditions and that DHP-sensitive Ca2+ entry occurs mostly via these VGCCs. We found that receptor stimulation by endothelin-1 in these SMCs resulted in activation of neither DHP-sensitive nor -insensitive Ca2+ entry, but rather resulted in marked suppression of the former. Utilizing the DHP-sensitive extracellular Ca(2+)-induced increase in [Ca2+]i as a monitor of activity of the DHP-sensitive VGCCs, we investigated the effects of protein kinase activators and phosphatase inhibitors on the regulation of these VGCCs. We found that the DHP-sensitive VGCCs were inhibited by endothelin-1 through the activation of protein kinase C. We also found that they were inhibited by 8Br-cGMP, okadaic acid, and calyculin A.
Ohshima
N
N
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka.
Iwamoto
T
T
Shigekawa
M
M
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism* / Q000493:pharmacokinetics; D015220:Calcium Channels / Q000502:physiology; D002132:Calcium Radioisotopes; D002478:Cells, Cultured; D004095:Dihydropyridines / Q000494:pharmacology; D016232:Endothelins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D016257:Fura-2; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008297:Male; D008345:Manganese / Q000493:pharmacokinetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D011921:Rats, Inbred WKY
D015220:Calcium Channels; D002132:Calcium Radioisotopes; D004095:Dihydropyridines; D016232:Endothelins; D008345:Manganese; C038806:1,4-dihydropyridine; D011494:Protein Kinases; D002118:Calcium; D016257:Fura-2