Pannexin channels do not influence endothelial function in porcine coronary arteries

Abstract

Pannexins are a newly discovered family of proteins with sequence homology to innexins (Billaud et al., 2011). The pannexin family consists of Panx1, Panx2 and Panx3. These proteins form channels in cell membranes to allow ATP to pass through to act as an intercellular messenger. It has recently been suggested that Panx1 plays an important role in endothelium‐dependent regulation of arterial tone, by facilitating endothelium‐dependent hyperpolarizing (EDH) responses (Gaynullina et al., 2015). This study aimed to determine the influence of Panx1 on endothelium‐dependent vasorelaxation induced by bradykinin (BK) in porcine coronary arteries (PCA).PCAs were dissected from pig hearts obtained from a local abattoir. Segments of PCA were prepared for isometric tension recording in oxygenated Krebs solution warmed to 37°C. After assessing tissue contractility with KCl (60 mM), tone was raised to 50% of the KCl response using U46619. Thereafter, responses to the endothelium‐dependent vasorelaxant, BK, were assessed in the absence and presence of the pannexin inhibitors, carbenoxolone (100 μM), mefloquine (20 μM) or probenecid (1 mM). Similar experiments were carried out after treating the PCA with the nitric oxide synthase inhibitor L‐NAME (100 μM) and cyclooxygenase inhibitor, indomethacin (10 μM). Vasorelaxant responses were expressed as a percentage of U46619‐induced contraction. Data were analysed by two‐way ANOVA followed by a Bonferroni post‐hoc test for comparison of multiple groups. P<0.05 was considered significant.Under control conditions, BK produced a concentration‐dependent vasorelaxation (‐log EC50 7.79 ± 0.49; Rmax = 82 ± 23%, n = 4). The response to BK was increased in the presence of carbenoxolone (‐log EC50 = 8.31 ± 0.11; Rmax = 102 ± 5%, n = 4), unaffected by probenecid (‐log EC50 = 8.34 ± 0.30; Rmax = 69 ± 9%, n = 4) but reduced by mefloquine (‐log EC50 = 7.05 ± 0.30; Rmax = 63 ± 22%, n = 4). A much greater concentration of U46619 was required to raise tone in the presence of mefloquine (raised from approximately nanomolar to micromolar concentrations). BK caused a concentration‐dependent relaxation in preparations exposed to L‐NAME and indomethacin, indicating the presence of an EDH response (‐log EC50 = 7.58 ± 0.27; Rmax = 53 ± 9%, n = 4). This EDH response was not affected by either carbenoxolone (‐log EC50 = 7.12 ± 1.63; Rmax = 41 ± 29%, n = 4) or probenecid (‐log EC50 = 7.46 ± 0.70; Rmax = 64 ± 22%, n = 4) but reduced by mefloquine (‐log EC50 = 6.87 ± 2.10; Rmax = 29 ± 15%, n = 4).The data show that BK induces vasorelaxation in the PCA, in part, by causing an EDH response and nitric oxide response. The use of three different pannexin inhibitors produced conflicting effects. Only mefloquine inhibited the response to BK, while probenecid had no effect and carbenoxolone enhanced BK effects. Thus we cannot conclude that pannexins are involved in mediating endothelium‐dependent responses in the PCA.Support or Funding InformationRoyal Embassy of Saudi ArabiaCultural Bureau in London630 Chiswick High Road London W4 5RY United Kingdom