ATP increases [Ca²⁺]iand activates a Ca²⁺‐dependent Cl⁻current in rat ventricular fibroblasts

New Findings What is the central question of this study? Although electrophysiological and biophysical characteristics of heart fibroblasts have been studied in detail, their responses to prominent paracrine agents in the myocardium have not been addressed adequately. Our experiments characterize changes in cellular electrophysiology and intracellular calcium in response to ATP. What is the main finding and its importance? In rat ventricular fibroblasts maintained in cell culture, we find that ATP activates a specific subset of Ca 2+ ‐activated Cl − channels as a consequence of binding to P2Y purinoceptors and then activating phospholipase C. This response is not dependent on [Ca 2+ ] o but requires an increase in [Ca 2+ ] i and is modulated by the type of nucleotide that is the purinergic agonist. Abstract 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 [Ca 2+ ] i . Voltage‐clamp studies (amphotericin‐perforated patch) showed that ATP (1–100 μ 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 μ m ), blocked the responses to 100 μ 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 μ m each), significantly inhibited the Cl − current produced by 100 μ m ATP. ATP was able to activate this Cl − current when [Ca 2+ ] o was removed, but not when [Ca 2+ ] i was buffered with BAPTA‐AM. In the presence of the phospholipase C inhibitor U73122, this Cl − current could not be activated. PCR analysis revealed strong signals for a number of P2Y purinoceptors and for the Ca 2+ ‐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 [Ca 2+ ] i , followed by activation of a Ca 2+ ‐dependent Cl − current in rat ventricular fibroblasts.