The fluorescent calcium indicator indo-1/AM inhibits renal proximal tubule cell volume regulation

The intracellular calcium indicator, indo-1, is a fluorescent compound related in structure and function to the calcium chelator ethylene glycol-bis(beta-aminoethyl ether) tetraacetic acid (EGTA) that binds one calcium ion per molecule. In the extracellular range of 1 to 10 microM indo-1/AM, the estimated intracellular concentration of the dye is 0.1 to 3 microM. Therefore, it is likely that intracellular calcium signals could be blunted under these experimental conditions (Cai approximately 0.1 to 0.2 microM). To evaluate the potential effects of indo-1/AM on cellular function, proximal renal tubules of the teleost Carassius auratus (goldfish) were exposed to its acetoxymethyl ester (cell permeable form) in an isotonic Ringer's solution (290 mOsm, 0.1 mM calcium) followed by exposure to a low sodium hypotonic Ringer's (110 mOsm, 0.1 mM calcium). Cellular regulatory volume decreases (RVD) were determined with videometric methods. In proximal renal tubules incubated with indo-1/AM, RVD was inhibited in a dose-dependent fashion (3 to 5 microM). No effects on RVD were observed with the impermeant salt of indo-1. Overt cellular injury was found at 10 microM indo-1/AM as evidenced by mitochondrial and cell swelling, cellular detachment from the tubular basement membrane, and different degrees of cytolysis. It is postulated by us that the inhibitory effects of indo-1/AM (3-5 microM) on RVD are due to intracellular calcium chelation followed by disruption of intracellular signalling.