Purinergic activation of Ca ²⁺-permeable TRPV4 channels is essential for mechano-sensitivity in the aldosterone-sensitive distal nephron

Mechanical forces are known to induce increases of [Ca ²⁺] _i in the aldosterone-sensitive distal nephron (ASDN) cells to regulate epithelial transport. At the same time, mechanical stress stimulates ATP release from ASDN cells. In this study, we combined ratiometric Fura-2 based monitoring of [Ca ²⁺] _i in freshly isolated split-opened ASDN with targeted deletion of P2Y2 and TRPV4 in mice to probe a role for purinergic signaling in mediating mechano-sensitive responses in ASDN cells. ATP application causes a reproducible transient Ca ²⁺ peak followed by a sustained plateau. Individual cells of the cortical collecting duct (CCD) and the connecting tubule (CNT) respond to purinergic stimulation with comparative elevations of [Ca ²⁺] _i. Furthermore, ATP-induced Ca ²⁺-responses are nearly identical in both principal (AQP2-positive) and intercalated (AQP2-negative) cells as was confirmed using immunohistochemistry in split-opened ASDN. UTP application produces elevations of [Ca ²⁺] _i similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca ²⁺] _i response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca ²⁺ responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca ²⁺ appeared to contribute to the generation of ATP-induced Ca ²⁺ response in ASDN cells. Importantly, flow- and hypotonic-induced Ca ²⁺ elevations are markedly blunted in P2Y2 -/- mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca ²⁺] _i elevation during purinergic stimulation. Consistent with this, ATP-induced Ca ²⁺ plateau are dramatically attenuated in TRV4 -/- mice. Inhibition of TRPC channels with 10 μM BTP2 also decreased ATP-induced Ca ²⁺ plateau whilst to a lower degree than that observed with TRPV4 inhibition/genetic deletion. We conclude that stimulation of purinergic signaling by mechanical stimuli leads to activation of TRPV4 and, to a lesser extent, TRPCs channels, and this is an important component of mechano-sensitive response of the ASDN.