Abstract
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-eNOSS1177) and Akt at serine 473 (P-AktS473). 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 Nav1.2, Nav1.6, and Cav3.2 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-eNOSS1177, and P-AktS473 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 Nav channels and Cav3.2 channels, leading to an increase in NO-mediated vasodilation through a NADPH oxidase-dependent P-eNOSS1177, which suggests that Panx-1 may be involved in the endothelium-dependent control of arterial blood pressure.
Highlights
Control of blood flow distribution relies on coordinated changes in the diameter of resistance arteries through a complex interplay between the vasoconstrictor and vasodilator signals that determines the degree of smooth muscle constriction
A basal P-eNOSS1177 was detected in control conditions, and the larger vasodilation observed in the presence of probenecid was associated with an increment in the P-eNOSS1177 level (Figure 1(b)) and in Akt phosphorylation at serine 473 (P-AktS473, Figure 1(c)), suggesting that the endothelial Nitric oxide (NO) synthase (eNOS) phosphorylation triggered by the Panx-1 channel blockade was mediated by the activation of the PI3K/Akt signaling pathway, as previously observed in response to different stimuli such as shear stress or bradykinin [24,25,26]
In line with previous reports [19], stimulation with ACh did not alter the level of P-eNOSS1177 and PAktS473 observed in basal conditions or after inhibiting Panx-1 channels with probenecid or the Panx-1 blocking peptide 10Panx (Figures 1(b) and 1(c))
Summary
Control of blood flow distribution relies on coordinated changes in the diameter of resistance arteries through a complex interplay between the vasoconstrictor and vasodilator signals that determines the degree of smooth muscle constriction (i.e., vasomotor tone). Nitric oxide (NO) has been recognized as the major vasodilator signal generated by endothelial cells; in small resistance arteries (i.e., feed arteries and arterioles), an additional vasodilator pathway associated with the NO-independent hyperpolarization of smooth muscle cells has been described [2] As this vasodilator pathway relies on the gap junction-mediated transmission to smooth muscle cells of a hyperpolarizing current initiated in the endothelium by the opening of Ca2+-activated K+ channels (KCa) of small (SKCa) and Oxidative Medicine and Cellular Longevity intermediate (IKCa) conductance [2,3,4], this vasodilator component was termed as endothelium-derived hyperpolarization (EDH) [5]. Gap junctions play a central role in the coordination of vascular function, a functional association in endothelial cells between voltage-dependent Na+ channels (Nav) and T-type, voltage-dependent Ca2+ channels (Cav3) of the subtype Cav3.2 has been proposed to participate in this process by supporting the conduction of vasodilator signals [2, 7, 8]
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