Calcium mobilization in enzymically isolated single intact and skinned muscle cells of the porcine coronary artery.
The mobilization of 45Ca2+ was investigated in collagenase-treated single smooth muscle cells of the porcine coronary artery. After removal of extracellular 45Ca2+ by 10 mM-EGTA at 0 degree C, the content of exchangeable Ca2+ was estimated to be 0.42 +/- 0.02 nmol/2 X 10(5) cells at rest and 0.62 +/- 0.03 nmol/2 X 10(5) cells in 102.5 mM-external K solution. The efflux of 45Ca2+ into Ca2+-free solution, estimated from the 45Ca2+ remaining in the cells, increased temperature dependently and was reduced by oligomycin. The muscle cells at rest had a substantial amount of stored Ca2+ which was releasable by caffeine or acetylcholine. Saponin-treated (skinned) muscle cells accumulated 45Ca2+ in the presence of Mg ATP. Two mechanisms of ATP-dependent Ca2+ sequestration were observed: one exhibited a low affinity for Ca2+ but a high-capacity uptake which was sensitive to sodium azide; this was thought to be located in the mitochondria. The other had a high-affinity (1.5/microM) and low-capacity uptake (0.92 nmol/2 X 10(5) cells), which was insensitive to sodium azide, potentiated by oxalate and was thought to be mainly mediated via the sarcoplasmic reticulum (s.r.). The minimum concentration of free Ca2+ required for the ATP-dependent Ca2+ uptake in the saponin-treated cells was about 20 nM by the s.r. and 1 microM by the mitochondria. Thus, the mitochondria seem to play a minor role in regulating cytoplasmic Ca2+ during the contraction-relaxation cycle. These results indicate that enzymically isolated muscle cells are functionally intact, and may facilitate direct measurement of Ca2+ movements when attempting to estimate the physiological role of Ca2+ in vascular smooth muscles.
D000109:Acetylcholine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D010070:Oxalates / Q000494:pharmacology; D019815:Oxalic Acid; D012503:Saponins / Q000494:pharmacology; D013552:Swine; D013696:Temperature
D009840:Oligomycins; D010070:Oxalates; D012503:Saponins; D000001:Calcimycin; D002110:Caffeine; D000255:Adenosine Triphosphate; D019815:Oxalic Acid; D000109:Acetylcholine; D002118:Calcium