title: Purinergic activation of Ca2+-permeable TRPV4 channels is essential for mechano-sensitivity in the aldosterone-sensitive distal nephron.
abstract: 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.
authors.affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America.
authors.forename: Mykola
authors.initials: M
authors.lastname: Mamenko
authors.forename: Oleg
authors.initials: O
authors.lastname: Zaika
authors.forename: Min
authors.initials: M
authors.lastname: Jin
authors.forename: Roger G
authors.initials: RG
authors.lastname: O'Neil
authors.forename: Oleh
authors.initials: O
authors.lastname: Pochynyuk
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000450:Aldosterone / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D040542:Mechanotransduction, Cellular / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D009399:Nephrons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2 / Q000235:genetics / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D058473:Receptors, Purinergic P2Y2; D050916:TRPV Cation Channels; C486576:Trpv4 protein, mouse; D000450:Aldosterone; D000255:Adenosine Triphosphate; D002118:Calcium; D014544:Uridine Triphosphate