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 Ca2+-buffering properties of the myoplasm, Ca2+ influx, measured as time integral of the I-Ca (integral I-Ca), was compared with corresponding free Ca2+ increments (Delta[Ca2+](i)) in the cytoplasm. The ratio between integral I-Ca and Delta[Ca2+](i) (integral I-Ca/Delta[Ca2+](i)), reflecting the Ca2+ buffering properties of the cytosol, was in the range of 4.9-9.3 pC/mu M (mean 6.2 +/- 1.2, n = 12). It remained approximately constant (6.4 +/- 1.4 pC/mu M, 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 I-Ca/Delta[Ca2+](i) during the sequence of depolarizing steps, which increased free [Ca2+](i) up to 5 mu M, suggested lower limits for the apparent affinity of a rapid Ca2+ buffer (16 mu M) and for the total buffer concentration (530 mu M). Introduction of 4 mM DPTA (K-d for Ca2+ = 81 mu M) 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 Ca2+-binding ratio of the cytoplasm in most cells was estimated to be between 30 and 40. The Ca2+-binding ratio did not differ markedly between cells isolated from neonatal (less than or equal to 5 days) and adult animals. (C) Harcourt Publishers Ltd 2000.