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

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??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^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??M mibefradil or 10??M 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??M TEMPOL or 100??M 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.

SUBMITTER: Lillo MA

PROVIDER: S-EPMC7914086 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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

Lillo Mauricio A MA Gaete Pablo S PS Puebla Mariela M Burboa Pía C PC Poblete Inés I Figueroa Xavier F XF

Oxidative medicine and cellular longevity 20210220

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 ...[more]

PMID: 33688389

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Project description:Tetrahydropalmatine (THP) is an active natural alkaloid isolated from Corydalis yanhusuo W.T. Wang which has been widely used for treating pain and cardiovascular disease in traditional Chinese medicine. Previous studies suggested THP have various pharmacological effects in neural and cardio tissue while the vascular reactivity of THP was not fully established. The present study found that THP relaxed rat aorta which contracted by phenylephrine (Phe), KCl, and U46619. The vascular relaxation effect of THP was partially attenuated by PI3K inhibitor wortmannin, Akt inhibitor IV, endothelial nitric oxide synthetase (eNOS) inhibitor L-NAME, guanylate cyclase inhibitors and the mechanical removal of endothelium. Also, the eNOS substrate L-arginine reversed the inhibition effect of L-NAME on THP-induced vascular relaxation. THP also induced intracellular NO production in human umbilical vein endothelial cells. However, Pre-incubation with β-adrenergic receptor blocker propranolol, angiotensin II receptor 1 (AT1) inhibitor losartan, angiotensin II receptor 2 (AT1) inhibitor PD123319 or angiotensin converting enzyme inhibitor enalapril enhanced the vascular relaxation effect of THP. THP did not affect the angiotensin II induced vascular contraction. Cyclooxygenase-2 (COX2) inhibitor indomethacin did not affect the vascular relaxation effect of THP. Furthermore, pre-treatment THP attenuated KCl and Phe induced rat aorta contraction in standard Krebs solution. In Ca2+ free Krebs solution, THP inhibited the Ca2+ induced vascular contraction under KCl or Phe stress and reduced KCl stressed Ca2+ influx in rat vascular smooth muscle cells. THP also inhibited intracellular Ca2+ release induced vascular contraction by blocking Ryr or IP3 receptors. In addition, the voltage-dependent K+ channel (Kv) blocker 4-aminopyridine, ATP-sensitive K+ channel (KATP) blocker glibenclamide and inward rectifying K+ channel blocker BaCl2 attenuated THP induced vascular relaxation regardless of the Ca2+-activated K+ channel (KCa) blocker tetraethylammonium. Thus, we could conclude that THP relaxed rat aorta in an endothelium-dependent and independent manner. The underlying mechanism of THP relaxing rat aorta involved PI3K/Akt/eNOS/NO/cGMP signaling path-way, Ca2+ channels and K+ channels rather than COX2, β-adrenergic receptor and renin-angiotensin system (RAS). These findings indicated that THP might be a potent treatment of diseases with vascular dysfunction like hypertension.

Dataset Information

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

Publications

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

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