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

Abstract

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 (RATP) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure RATP 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 RATP rapidly to a peak value of 1.89 ± 0.11 pmol min−1 (RATPpeak) followed by a plateau phase reaching 0.51 ± 0.07 pmol min−1 (RATPplat). Both RATPpeak and RATPplat values increased with the degree of dilution. The magnitude of RATPplat 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 RATP value, induced in the absence of cell swelling, is comparable to RATPplat. 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 RATPpeak. 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.

Purinergic signalling in the kidney in health and disease

“…Activation of P2X receptors increased both sodium and water excretion [168]. Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured A6 cells [106,172].…”

Aldosterone acts via an ATP autocrine/paracrine system: The Edelman ATP hypothesis revisited

“…Monolayers on the membrane filters were gently superfused with L-15 medium and equilibrated for 60 min in an Endohm 24 chamber, maintaining the apical and basolateral solution volumes at 2 ml. We quantified ATP release by using an ATP bioluminescent assay kit (Sigma) following a modified method previously reported (8). Briefly, a 500-l sample was taken from the basolateral compartment and added in a mixture of 100 l of luciferin͞luciferase assay reagent and 500 l of L-15 medium in a Petri dish.…”

“…In recent work, Jans et al (8) have shown that hypotonic treatment of renal epithelia induces a transient increase in intracellular Ca 2ϩ through a mechanism that is sensitive to the purinergic P2 receptor antagonist suramin applied to the basolateral border only. They demonstrated that ATP was released from the cell and then acted via an autocrine͞paracrine system to produce the suramin-sensitive rise of intracellular Ca 2ϩ .…”

ENaC, renal sodium excretion and extracellular ATP

“…However, this system remains noteworthy because it shows a robust response to changes in tonicity at the basolateral membrane. Here, hypo-osmotic solutions (140 mOsmol; Na + , 70 mM) evoke a pronounced biphasic pattern of ATP release [48]. The first phase was transient and occurred with cell swelling and was replaced by a later tonic phase of sustained ATP release.…”

“…In turn, ATP stimulates suramin-sensitive P2Rs to increase [Ca 2+ ] int and evoke further ATP release. It was proposed that an ATP plateau was reached only when the rate of ATP release was balanced by the rate of ATP consumption by ectoATPases [48].…”