Browsing by Author "Poblete, Ines"

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    CGRP signalling inhibits NO production through pannexin-1 channel activation in endothelial cells
    (2019) Gaete, Pablo S.; Lillo, Mauricio A.; Puebla, Mariela; Poblete, Ines; Figueroa, Xavier F.
    Blood flow distribution relies on precise coordinated control of vasomotor tone of resistance arteries by complex signalling interactions between perivascular nerves and endothelial cells. Sympathetic nerves are vasoconstrictors, whereas endothelium-dependent NO production provides a vasodilator component. In addition, resistance vessels are also innervated by sensory nerves, which are activated during inflammation and cause vasodilation by the release of calcitonin gene-related peptide (CGRP). Inflammation leads to superoxide anion (O-2(center dot-)) formation and endothelial dysfunction, but the involvement of CGRP in this process has not been evaluated. Here we show a novel mechanistic relation between perivascular sensory nerve-derived CGRP and the development of endothelial dysfunction. CGRP receptor stimulation leads to pannexin-1-formed channel opening and the subsequent O-2(center dot-)-dependent connexin-based hemichannel activation in endothelial cells. The prolonged opening of these channels results in a progressive inhibition of NO production. These findings provide new therapeutic targets for the treatment of the inflammation-initiated endothelial dysfunction.
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    Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries
    (2018) Lillo, Mauricio A.; Gaete, Pablo S.; Puebla, Mariela; Ardiles, Nicolas M.; Poblete, Ines; Becerra, Alvaro; Simon, Felipe; Figueroa, Xavier
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    Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries
    (2018) Lillo, Mauricio A.; Gaete, Pablo S.; Puebla, Mariela; Ardiles, Nicolas M.; Poblete, Ines; Becerra, Alvaro; Simon, Felipe; Figueroa, Xavier F.
    Na+-Ca2+ exchanger (NCX) contributes to control the intracellular free Ca2+ concentration ([Ca2+](i)), but the functional activation of NCX reverse mode (NCXrm) in endothelial cells is controversial. We evaluated the participation of NCXrm-mediated Ca2+ uptake in the endothelium-dependent vasodilation of rat isolated mesenteric arterial beds. In phenylephrine-contracted mesenteries, the acetylcholine (ACh)-induced vasodilation was abolished by treatment with the NCXrm blockers SEA0400, KB-R7943, or SN-6. Consistent with that, the ACh-induced hyperpolarization observed in primary cultures of mesenteric endothelial cells and in smooth muscle of isolated mesenteric resistance arteries was attenuated by KB-R7943 and SEA0400, respectively. In addition, both blockers abolished the NO production activated by ACh in intact mesenteric arteries. In contrast, the inhibition of NCXrm did not affect the vasodilator responses induced by the Ca2+ ionophore, ionomycin, and the NO donor, S-nitroso-N-acetylpenicillamine. Furthermore, SEA0400, KB-R7943, and a small interference RNA directed against NCX1 blunted the increase in [Ca2+](i) induced by ACh or ATP in cultured endothelial cells. The analysis by proximity ligation assay showed that the NO-synthesizing enzyme, eNOS, and NCX1 were associated in endothelial cell caveolae of intact mesenteric resistance arteries. These results indicate that the activation of NCXrm has a central role in Ca2+-mediated vasodilation initiated by ACh in endothelial cells of resistance arteries.
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    Novel Pannexin-1-Coupled Signaling Cascade Involved in the Control of Endothelial Cell Function and NO-Dependent Relaxation
    (2021) Lillo, Mauricio A.; Gaete, Pablo S.; Puebla, Mariela; Burboa, Pia C.; Poblete, Ines; Figueroa, Xavier F.
    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 (O-2(-)) 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 (10)Panx (60 mu 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 (10)Panx 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 mu M mibefradil or 10 mu M Ni2+. As expected, Ni2+ also abolished the increment in [Ca2+](i). Expression of Na(v)1.2, Na(v)1.6, and Ca(v)3.2 isoforms of voltage-dependent Na+ and Ca2+ channels was confirmed by immunocytochemistry. Furthermore, the Panx-1 channel blockade was associated with an increase in O-2(-) production. Treatment with 10 mu M TEMPOL or 100 mu M apocynin prevented the increase in O-2(-) 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(v)3.2 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.