PPADS, a P2X receptor antagonist, as a novel inhibitor of the reverse mode of the Na+/Ca2+ exchanger in guinea pig airway smooth muscle

The Na+/Ca2+exchanger (NCX) principal function is taking 1 Ca2+ out of the cytoplasm and introducing 3 Na+. The increase of cytoplasmic Na+ concentration induces the NCX reverse mode (NCXREV), favoring Ca2+ influx. NCXREV can be inhibited by: KB-R7943 a non-specific compound that blocks voltage-dependent and store-operated Ca2+ channels; SEA0400 that appears to be selective for NCXREV, but difficult to obtain and SN-6, which efficacy has been shown only in cardiomyocytes. We found that PPADS, a P2X receptor antagonist, acts as a NCXREV inhibitor in guinea pig tracheal myocytes. In these cells, we characterized the NCXREV by substituting NaCl and NaHCO3 with LiCl, resulting in the increase of the intracellular Ca2+ concentration ([Ca2+]i) using fura 2-AM. We analyzed 5 consecutive responses of the NCXREV every 10 min, finding no differences among them. To evaluate the effect of different NCXREV blockers, concentration response curves to KB-R7943 (1, 3.2 and 10 μM), and SN-6 (3.2, 10 and 30 μM) were constructed, whereas PPADS effect was characterized as time- and concentration-dependent (1, 3.2, 10 and 30 μM). PPADS had similar potency and efficacy as KB-R7943, whereas SN-6 was the least effective. Furthermore, KCl-induced contraction, sensitive to D600 and nifedipine, was blocked by KB-R7943, but not by PPADS. KCl-induced [Ca2+]i increment in myocytes was also significantly decreased by KBR-7943 (10 μM). Our results demonstrate that PPADS can be used as a reliable pharmacological tool to inhibit NCXREV, with the advantage that it is more specific than KB-R7943 because it does not affect L-type Ca2+ channels.