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

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Abstract
Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Ca f ) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Ca f has been measured using the intracellular Casensitive 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 Ca f 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, Ca f was 300–380 nM and, as dye content was increased, Ca f 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 Ca f , prolonged treatment with ouabain had no effect on Ca f 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 Ca f . However, exchange-mediated Ca efflux may play a role in Ca f regulation when cytosolic calcium is elevated.

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