Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells

Abstract

Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Caf) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Caf has been measured using the intracellular Ca-sensitive fluorescent dyes Fura 2 and Quin 2. Dye loading did not alter cell viability as assessed by measurements of ATP and ADP content or cell oxygen consumption. When basal Caf was examined over a wide range of cell dye content (from 0.04 to 180 nmol dye/mg protein) an inverse relationship was observed. At low dye content, Caf was 300-380 nM and, as dye content was increased, Caf progressively fell to 60 nM. Using low dye content cells, in which minimal alteration in Ca steady state would be expected, the role for plasma membrane Na-Ca exchange was examined using either medium sodium substitution or ouabain. While medium sodium substitution increased Caf, prolonged treatment with ouabain had no effect on Caf despite a clear increase in cell sodium content. The lack of effect of ouabain suggests that Na-Ca exchange-mediated Ca efflux plays a minimal role in the regulation of basal Caf. However, exchange-mediated Ca efflux may play a role in Caf regulation when cytosolic calcium is elevated.