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

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 [Ca²⁺]_i of endothelial cells. Changes in NO-mediated vasodilation, membrane potential, superoxide anion (O₂ ^·-) formation, and endothelial cell [Ca²⁺]_i 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 ¹⁰Panx (60 μM) 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^S1177) and Akt at serine 473 (P-Akt^S473). In addition, probenecid or ¹⁰Panx application activated a rapid, tetrodotoxin (TTX, 300 nM)-sensitive, membrane potential depolarization and [Ca²⁺]_i increase in endothelial cells. Interestingly, the endothelial cell depolarization was converted into a transient spike after removing Ca²⁺ ions from the buffer solution and in the presence of 100 μM mibefradil or 10 μM Ni²⁺. As expected, Ni²⁺ also abolished the increment in [Ca²⁺]_i. Expression of Na_v1.2, Na_v1.6, and Ca_v3.2 isoforms of voltage-dependent Na⁺ and Ca²⁺ channels was confirmed by immunocytochemistry. Furthermore, the Panx-1 channel blockade was associated with an increase in O₂ ^·- production. Treatment with 10 μM TEMPOL or 100 μM apocynin prevented the increase in O₂ ^·- formation, ACh-induced vasodilation, P-eNOS^S1177, and P-Akt^S473 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_v channels and Ca_v3.2 channels, leading to an increase in NO-mediated vasodilation through a NADPH oxidase-dependent P-eNOS^S1177, which suggests that Panx-1 may be involved in the endothelium-dependent control of arterial blood pressure.