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Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6)

Danny Jans*, S. P. Srinivas†, Etienne Waelkens‡, Andrei Segal*, Els Larivière*, Jeannine Simaels* and Willy Van Driessche*

In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin-luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing-type chamber. We developed a LL pulse protocol to determine the rate of ATP release (R_ATP) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure R_ATP as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 µl of A6 cells released ATP at a rate of 66 ± 8 fmol min^-1. A sudden reduction of the basolateral osmolality from 260 to 140 mosmol (kg H₂O)^-1 elevated R_ATP rapidly to a peak value of 1.89 ± 0.11 pmol min^-1 (R_ATP^peak) followed by a plateau phase reaching 0.51 ± 0.07 pmol min^-1 (R_ATP^plat). Both R_ATP^peak and R_ATP^plat values increased with the degree of dilution. The magnitude of R_ATP^plat remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 ± 0.08 pmol min^-1 was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H₂O)^-1. This R_ATP value, induced in the absence of cell swelling, is comparable to R_ATP^plat. Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose-response curves for peak [Ca²⁺]_i increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, was linearly correlated with R_ATP^peak. The link between the ATP release and the fast [Ca²⁺]_i transient was also demonstrated by the depression of both phenomena by Cl^- removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin-sensitive rise of [Ca²⁺]_i during the hyposmotic shock.

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