Control of astrocytic Ca2+ signaling by nitric oxide-dependent S-nitrosylation of Ca2+ homeostasis modulator 1 channels.

BACKGROUND: Astrocytes Ca2+ signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca2+ signals in astrocytes. Importantly, astrocytes express the Ca2+-dependent nitric oxide (NO)-synthetizing enzymes eNOS and nNOS, which might contribute to the Ca2+ signals by triggering Ca2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca2+ signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex. RESULTS: Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptor-dependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. CONCLUSIONS: Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca2+ signaling.

Control of astrocytic Ca2+ signaling by nitric oxide-dependent S-nitrosylation of Ca2+ homeostasis modulator 1 channels

Background Astrocytes Ca2+ signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca2+ signals in astrocytes. Importantly, astrocytes express the Ca2+-dependent nitric oxide (NO)-synthe-tizing enzymes eNOS and nNOS, which might contribute to the Ca2+ signals by triggering Ca2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca2+ signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex. Results Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptordependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. Conclusions Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca2+ signaling.

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 [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.

CGRP signalling inhibits NO production through pannexin-1 channel activation in endothelial cells.

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 (O2• -) 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 O2• --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.

Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

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.-Lillo, M. A., Gaete, P. S., Puebla, M., Ardiles, N. M., Poblete, I., Becerra, A., Simon, F., Figueroa, X. F. Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

Functional role of connexins and pannexins in the interaction between vascular and nervous system.

The microvascular network of the microcirculation works in tight communication with surrounding tissues to control blood supply and exchange of solutes. In cerebral circulation, microvascular endothelial cells constitute a selective permeability barrier that controls the environment of parenchymal brain tissue, which is known as the blood-brain barrier (BBB). Connexin- and pannexin-formed channels (gap junctions and hemichannels) play a central role in the coordination of endothelial and smooth muscle cell function and connexin-mediated signaling in endothelial cells is essential in the regulation of BBB permeability. Likewise, gap junction communication between astrocyte end-feet also contributes to maintain the BBB integrity, but the participation of hemichannels in this process cannot be discarded. Sympathetic and sensory perivascular nerves are also involved in the control and coordination of vascular function through the release of vasoconstrictor or vasodilator signals and by the regulation of gap junction communication in the vessel wall. Conversely, ATP release through pannexin-1-formed channels mediates the α1-adrenergic signaling. Furthermore, here we show that capsaicin-induced CGRP release from mesenteric perivascular sensory nerves induces pannexin-1-formed channel opening, which in turn leads to reduction of pannexin-1 and endothelial nitric oxide synthase (eNOS) expression along the time. Interestingly, blockade of CGRP receptors with CGRP8-37 increased eNOS expression by ∼5-fold, suggesting that capsaicin-sensitive sensory nerves are involved in the control of key signaling proteins for vascular function. In this review, we discuss the importance of connexin-based channels in the control of BBB integrity and the functional interaction of vascular connexins and pannexins with the peripheral nervous system.

Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase.

Ca(2+)-activated K(+) channels (K(Ca)) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of K(Ca) with NO production in isolated arterial mesenteric beds of the rat. In phenylephrine-contracted mesenteries, acetylcholine (ACh)-induced vasodilation was reduced by NO synthase (NOS) inhibition with N(ω)-nitro-L-arginine (L-NA), but in the presence of tetraethylammonium, L-NA did not further affect the response. In KCl-contracted mesenteries, the relaxation elicited by 100 nM ACh or 1 µM ionomycin was abolished by L-NA, tetraethylammonium, or simultaneous blockade of small-conductance K(Ca) (SK(Ca)) channels with apamin and intermediate-conductance K(Ca) (IK(Ca)) channels with triarylmethane-34 (TRAM-34). Apamin-TRAM-34 treatment also abolished 100 nM ACh-activated NO production, which was associated with an increase in superoxide formation. Endothelial cell Ca(2+) buffering with BAPTA elicited a similar increment in superoxide. Apamin-TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOS(Thr495)). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOS(Thr495) observed during apamin and TRAM-34 application. Our results indicate that blockade of SK(Ca) and IK(Ca) activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOS(Thr495). These findings disclose a novel mechanism involved in the control of NO production.