Novel Pannexin-1-Coupled Signaling Cascade Involved in the Control of Endothelial Cell Function and NO-Dependent Relaxation.

Deletion of pannexin-1 (Panx-1) leads not only to a reduction in endothelium-derived hyperpolarization but also to an increase in NO-mediated vasodilation. Therefore, we evaluated the participation of Panx-1-formed channels in the control of membrane potential and [Ca] of endothelial cells. Changes in NO-mediated vasodilation, membrane potential, superoxide anion (O ) formation, and endothelial cell [Ca] were analyzed in rat isolated mesenteric arterial beds and primary cultures of mesenteric endothelial cells. Inhibition of Panx-1 channels with probenecid (1 mM) or the Panx-1 blocking peptide Panx (60 M) evoked an increase in the ACh (100 nM)-induced vasodilation of KCl-contracted mesenteries and in the phosphorylation level of endothelial NO synthase (eNOS) at serine 1177 (P-eNOS) and Akt at serine 473 (P-Akt). In addition, probenecid or Panx application activated a rapid, tetrodotoxin (TTX, 300 nM)-sensitive, membrane potential depolarization and [Ca] increase in endothelial cells. Interestingly, the endothelial cell depolarization was converted into a transient spike after removing Ca ions from the buffer solution and in the presence of 100 M mibefradil or 10 M Ni. As expected, Ni also abolished the increment in [Ca]. Expression of Na1.2, Na1.6, and Ca3.2 isoforms of voltage-dependent Na and Ca channels was confirmed by immunocytochemistry. Furthermore, the Panx-1 channel blockade was associated with an increase in O production. Treatment with 10 M TEMPOL or 100 M apocynin prevented the increase in O formation, ACh-induced vasodilation, P-eNOS, and P-Akt observed in response to Panx-1 inhibition. These findings indicate that the Panx-1 channel blockade triggers a novel complex signaling pathway initiated by the sequential activation of TTX-sensitive Na channels and Ca3.2 channels, leading to an increase in NO-mediated vasodilation through a NADPH oxidase-dependent P-eNOS, which suggests that Panx-1 may be involved in the endothelium-dependent control of arterial blood pressure.