An estimate of rapid cytoplasmic calcium buffering in a single smooth muscle cell

Abstract

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 ICa(∫ICa), was compared with corresponding free Ca2+increments (Δ[Ca2+]i) in the cytoplasm. The ratio between ∫ICaand Δ[Ca2+]i(∫ICa/Δ[Ca2+]i), reflecting the Ca2+buffering properties of the cytosol, was in the range of 4.9–9.3 pC/μM (mean 6.2 ± 1.2, n = 12). It remained approximately constant (6.4 ± 1.4 pC/μM, 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 Δ[Ca2+]iwithin minutes. Changes in ∫ICa/Δ[Ca2+]iduring the sequence of depolarizing steps, which increased free [Ca2+]iup to 5 μM, suggested lower limits for the apparent affinity of a rapid Ca2+buffer (16 μM) and for the total buffer concentration (530 μM). Introduction of 4 mM DPTA (Kdfor Ca2+= 81 μM) 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 (≤ 5 days) and adult animals.