An estimate of rapid cytoplasmic calcium buffering in a single smooth muscle cell.
Cytoplasmic calcium increments in the absence of sarco (endo) plasmic reticulum function were measured with a low-affinity fluorophore Indo-1FF in single isolated smooth muscle cells from guinea-pig urinary bladder. To evaluate the Ca(2+)-buffering properties of the myoplasm, Ca2+ influx, measured as time integral of the Ica (integral of Ica), was compared with corresponding free Ca2+ increments (delta [Ca2+]i) in the cytoplasm. The ratio between integral of ICa and delta [Ca2+]i (integral Ica/delta [Ca2+]i), reflecting the Ca2+ buffering properties of the cytosol, was in the range of 4.9-9.3 pC/microM (mean 6.2 +/- 1.2, n = 12). It remained approximately constant (6.4 +/- 1.4 pC/microM, n = 8) during recordings lasting up to 25 min, suggesting that cytoplasmic Ca2+ binding does not change markedly during cell dialysis and that the endogenous Ca2+ buffer is not significantly washed out of the cell through the patch pipette. Wash-in or wash-out of BAPTA, a mobile high-affinity Ca2+ buffer, into or from the cell markedly changed the relationship between Ca2+ influx through Ca2+ channels and delta [Ca2+]i within minutes. Changes in integral of ICa/delta [Ca2+]i during the sequence of depolarizing steps, which increased free [Ca2+]i up to 5 microM, suggested lower limits for the apparent affinity of a rapid Ca2+ buffer (16 microM) and for the total buffer concentration (530 microM). Introduction of 4 mM DPTA (Kd for Ca2+ = 81 microM) into the cell more than doubled the total cytoplasmic Ca2+ buffer capacity. These results suggest that cytoplasmic Ca2+ buffer in smooth muscle cells has a low affinity for free Ca2+. The Ca(2+)-binding ratio of the cytoplasm in most cells was estimated to be between 30 and 40. The Ca(2+)-binding ratio did not differ markedly between cells isolated from neonatal (< or = 5 days) and adult animals.
Daub
B
B
Department of Physiology, University of Cologne, Germany.
Ganitkevich VYa
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D006168:Guinea Pigs; D007211:Indoles / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D001743:Urinary Bladder / Q000033:anatomy & histology / Q000254:growth & development
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; C113828:indo-1 FF; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid