ATP increases [Ca2+]i and activates a Ca2+-dependent Cl- current in rat ventricular fibroblasts

Effects of ATP on enzymatically isolated rat ventricular fibroblasts maintained in short-term (36-72 h) cell culture were examined. Immunocytochemical staining of these cells revealed that a fibroblast, as opposed to a myofibroblast, phenotype was predominant. ATP, ADP or uridine 5-triphosphate (UTP) all produced large increases in [Ca2+](i). Voltage-clamp studies (amphotericin-perforated patch) showed that ATP (1-100 m) activated an outwardly rectifying current, with a reversal potential very close to the Nernst potential for Cl-. In contrast, ADP was much less effective, and UTP produced no detectable current. The non-selective Cl- channel blockers niflumic acid, DIDS and NPPB (each at 100 m), blocked the responses to 100 m ATP. An agonist for P2Y purinoceptors, 2-MTATP, activated a very similar outwardly rectifying C1(-) current. The P2Y receptor antagonists, suramin and PPADS (100 m each), significantly inhibited the Cl- current produced by 100 m ATP. ATP was able to activate this Cl- current when [Ca2+](o) was removed, but not when [Ca2+](i) was buffered with BAPTA-AM. In the presence of the phospholipaseC inhibitor U73122, this Cl- current could not be activated. PCR analysis revealed strong signals for a number of P2Y purinoceptors and for the Ca2+-activated Cl- channel, TMEM16F (also denoted ANO6). In summary, these results demonstrate that activation of P2Y receptors by ATP causes a phospholipase C-dependent increase in [Ca2+](i), followed by activation of a Ca2+-dependent Cl- current in rat ventricular fibroblasts.