ИСТИНА

Introduction Vertebrate pannexins were discovered as homologs to invertebrate gap junction proteins (innexins; Panchin et al., 2000). Pannexin family consists of three members, Panx 1, 2 and 3, which are structurally very similar to connexins, the vertebrate gap junction proteins. Panx1 is ubiquitously expressed in vertebrate tissues and was shown to participate in numerous physiological functions, including calcium waves generation and purinergic signaling. In murine systemic arterial network Panx1 is the primary expressed isoform. Panx1 is abundant in endothelium of all arteries and capillaries, but the pattern of Panx1 expression depends on the vessel size. In contrast to larger conduit arteries where Panx1 is expressed primarily in the endothelium, in smaller resistance arteries it is also expressed in smooth muscle cells (Lohman et al., 2012). In arterial smooth muscle Panx1 was shown to mediate ATP release, thereby potentiating arterial contractile response to alpha1-adrenoceptor agonist (Billaud et al., 2011). However, the role of Panx1 in the functioning of endothelium has never been studied before. Thus, we tested the hypothesis, that Panx1 may regulate vascular tone not only by affecting smooth muscle cell contractility, but also via endothelium-dependent mechanisms. Methods Since truly selective pharmacological blockers of the Panx1 channel currently are not available, we utilized Panx1-/- mice as the ultimate test model for investigating functional significance of Panx1 activity in vascular system regulation. Wild type (WT) animals were age-matched (2-3 months old) male mice of the C57BL/6 background. The experiments were performed using the preparations of the endothelium-denuded mesenteric and endothelium-intact or –denuded saphenous arteries, representing different patterns on Panx1 expression: in relatively small mesenteric arteries Panx1 is proposed to be expressed in both smooth muscle and endothelium, while in relatively large conduit saphenous artery Panx1 is expressed only in endothelium. For force recording, 2-mm ring preparations were mounted in isometric myograph. qPCR was used for gene expression analysis. Results In order to identify the role of Panx1 in the modulation of smooth muscle cell functioning, we studied contractile reactions of mesenteric arteries. Despite previous findings, showing that Panx1 potentiates smooth muscle contraction to alpha1 – adrenoceptor agonist phenylephrine (Billaud et al., 2011), we didn’t observed any difference in the contractile responses to phenylephrine in mesenteric arteries of Panx1-/- and WT mice. However, the difference between the reactions of mesenteric arteries of Panx1-/- and WT mice appeared, when the vessels were incubated with ecto-ATPase inhibitor ARL67156. ARL67156 augmented the contractile responses to phenylephrine in mesenteric arteries of WT mice, but didn’t affect the responses of Panx1-/- mice. Importantly, ARL67156 didn’t modify the contractile responses to thromboxane A2 analogue U46619 neither in Panx1-/- nor in WT mice. The role Panx1 plays in the functioning of endothelium was tested in the saphenous arteries of Panx1-/- and WT mice. We found that the ablation of endothelium caused significant reduction of the transcripts of Panx1 and endothelial marker CD31. Thus, Panx1 is expressed predominantly in endothelial cells of murine saphenous artery. Phenylephrine-induced contractile responses of endothelium-denuded arteries did not differ in WT and Panx1-/- mice, which is in good agreement with predominantly endothelial localization of Panx1in murine saphenous arteries. However, the contractile response to phenylephrine in endothelium-intact arteries of Panx1-/- mice was stronger than of WT mice, indicating that in saphenous arteries of Panx1-/- mice the anticontractile role of endothelium is impaired. Similar observations were found for contractile responses to another alpha1 – adrenoceptor agonist methoxamine and non-receptor activation by high-K+ depolarization. Thus, Panx1 modulates anticontractile effect of the endothelium. Another strong evidence for functional role of Panx1 in the endothelium was obtained by comparing the acetylcholine-induced relaxations of endothelium-intact arteries in WT and Panx1-/- mice. We found that the response to acetylcholine was significantly weaker in Panx1-/- vs. WT saphenous arteries, indicating that Panx1 participates in the regulation of the endothelium-dependent dilatory mechanisms. Conclusions Our data show that Panx1 may modulate arterial smooth muscle contractile responses to agonist of alpha1-adrenoceptors, but these modulatory influences could be masked by the activity of ecto-ATPases. Moreover, we demonstrate for the first time that Panx1 is involved in the regulation of anticontractile effect of endothelium and endothelium-dependent relaxation of conduit arteries.