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Calcium mobilization in enzymically isolated single intact and skinned muscle cells of the porcine coronary artery.
Author(s) -
Ueno H
Publication year - 1985
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1985.sp015698
Subject(s) - chemistry , oligomycin , extracellular , calcium , biophysics , egta , endoplasmic reticulum , ouabain , sodium azide , sodium , oxalate , biochemistry , atpase , biology , enzyme , organic chemistry
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.