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.

Opening of Cx43-formed hemichannels mediates the Ca2+ signaling associated with endothelial cell migration.

Endothelial cell migration is a key process in angiogenesis. Progress of endothelial cell migration is orchestrated by coordinated generation of Ca2+ signals through a mechanism organized in caveolar microdomains. Connexins (Cx) play a central role in coordination of endothelial cell function, directly by cell-to-cell communication via gap junction and, indirectly, by the release of autocrine/paracrine signals through Cx-formed hemichannels. However, Cx hemichannels are also permeable to Ca2+ and Cx43 can be associated with caveolin-1, a structural protein of caveolae. We proposed that endothelial cell migration relies on Cx43 hemichannel opening. Here we show a novel mechanism of Ca2+ signaling in endothelial cell migration. The Ca2+ signaling that mediates endothelial cell migration and the subsequent tubular structure formation depended on Cx43 hemichannel opening and is associated with the translocation of Cx43 with caveolae to the rear part of the cells. These findings indicate that Cx43 hemichannels play a central role in endothelial cell migration and provide new therapeutic targets for the control of deregulated angiogenesis in pathological conditions such as cancer.

Connexin 40-Mediated Regulation of Systemic Circulation and Arterial Blood Pressure.

Vascular system is a complex network in which different cell types and vascular segments must work in concert to regulate blood flow distribution and arterial blood pressure. Although paracrine/autocrine signaling is involved in the regulation of vasomotor tone, direct intercellular communication via gap junctions plays a central role in the control and coordination of vascular function in the microvascular network. Gap junctions are made up by connexin (Cx) proteins, and among the four Cxs expressed in the cardiovascular system (Cx37, Cx40, Cx43, and Cx45), Cx40 has emerged as a critical signaling pathway in the vessel wall. This Cx is predominantly found in the endothelium, but it is involved in the development of the cardiovascular system and in the coordination of endothelial and smooth muscle cell function along the length of the vessels. In addition, Cx40 participates in the control of vasomotor tone through the transmission of electrical signals from the endothelium to the underlying smooth muscle and in the regulation of arterial blood pressure by renin-angiotensin system in afferent arterioles. In this review, we discuss the participation of Cx40-formed channels in the development of cardiovascular system, control and coordination of vascular function, and regulation of arterial blood pressure.

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.

Function of P2X4 Receptors Is Directly Modulated by a 1:1 Stoichiometric Interaction With 5-HT3A Receptors.

Interacting receptors at the neuronal plasma membrane represent an additional regulatory mode for intracellular transduction pathways. P2X4 receptor triggers fast neurotransmission responses via a transient increase in intracellular Ca2+ levels. It has been proposed that the P2X4 receptor interacts with the 5-HT3A receptor in hippocampal neurons, but their binding stoichiometry and the role of P2X4 receptor activation by ATP on this crosstalking system remains unknown. Via pull-down assays, total internal reflection fluorescence (TIRF) microscopy measurements of the receptors colocalization and expression at the plasma membrane, and atomic force microscopy (AFM) imaging, we have demonstrated that P2X4/5-HT3A receptor complexes can interact with each other in a 1:1 stoichiometric manner that is preserved after ATP binding. Also, macromolecular docking followed by 100 ns molecular dynamics (MD) simulations suggested that the interaction energy of the P2X4 receptor with 5-HT3A receptor is similar at the holo and the apo state of the P2X4 receptor, and the interacting 5-HT3A receptor decreased the ATP binding energy of P2X4 receptor. Finally, the P2X4 receptor-dependent Ca2+ mobilization is inhibited by the 5-HT3A interacting receptor. Altogether, these findings provide novel molecular insights into the allosteric regulation of P2X4/5-HT3A receptor complex in lipid bilayers of living cells via stoichiometric association, rather than accumulation or unspecific clustering of complexes.

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.

Control of the neurovascular coupling by nitric oxide-dependent regulation of astrocytic Ca(2+) signaling.

Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca(2+) signaling that propagates to the endfeet, activating the release of vasoactive factors and arteriolar dilation. The astrocyte Ca(2+) signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30) and channels formed by pannexins (Panx-1). The neuronal activity-initiated Ca(2+) waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca(2+) entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO) can activate connexin hemichannel by S-nitrosylation and the Ca(2+)-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are expressed in astrocytes. Therefore, the astrocytic Ca(2+) signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca(2+) influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca(2+) signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in this process.

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.

Coordinated endothelial nitric oxide synthase activation by translocation and phosphorylation determines flow-induced nitric oxide production in resistance vessels.

BACKGROUND/AIMS: Endothelial nitric oxide synthase (eNOS) is associated with caveolin-1 (Cav-1) in plasma membrane. We tested the hypothesis that eNOS activation by shear stress in resistance vessels depends on synchronized phosphorylation, dissociation from Cav-1 and translocation of the membrane-bound enzyme to Golgi and cytosol. METHODS: In isolated, perfused rat arterial mesenteric beds, we evaluated the effect of changes in flow rate (2-10 ml/min) on nitric oxide (NO) production, eNOS phosphorylation at serine 1177, eNOS subcellular distribution and co-immunoprecipitation with Cav-1, in the presence or absence of extracellular Ca(2+). RESULTS: Increases in flow induced a biphasic rise in NO production: a rapid transient phase (3-5-min) that peaked during the first 15 s, followed by a sustained phase, which lasted until the end of stimulation. Concomitantly, flow caused a rapid translocation of eNOS from the microsomal compartment to the cytosol and Golgi, paralleled by an increase in eNOS phosphorylation and a reduction in eNOS-Cav-1 association. Transient NO production, eNOS translocation and dissociation from Cav-1 depended on extracellular Ca(2+), while sustained NO production was abolished by the PI3K-Akt blocker wortmannin. CONCLUSIONS: In intact resistance vessels, changes in flow induce NO production by transient Ca(2+)-dependent eNOS translocation from membrane to intracellular compartments and sustained Ca(2+)-independent PI3K-Akt-mediated phosphorylation.

De novo expression of connexin hemichannels in denervated fast skeletal muscles leads to atrophy.

Denervation of skeletal muscles induces atrophy, preceded by changes in sarcolemma permeability of causes not yet completely understood. Here, we show that denervation-induced Evans blue dye uptake in vivo of fast, but not slow, myofibers was acutely inhibited by connexin (Cx) hemichannel/pannexin1 (Panx1) channel and purinergic ionotropic P2X7 receptor (P2X7R) blockers. Denervated myofibers showed up-regulation of Panx1 and de novo expression of Cx39, Cx43, and Cx45 hemichannels as well as P2X7Rs and transient receptor potential subfamily V, member 2, channels, all of which are permeable to small molecules. The sarcolemma of freshly isolated WT myofibers from denervated muscles also showed high hemichannel-mediated permeability that was slightly reduced by blockade of Panx1 channels or the lack of Panx1 expression, but was completely inhibited by Cx hemichannel or P2X7R blockers, as well as by degradation of extracellular ATP. However, inhibition of transient receptor potential subfamily V, member 2, channels had no significant effect on membrane permeability. Moreover, activation of the transcription factor NFκB and higher mRNA levels of proinflammatory cytokines (TNF-α and IL-1ß) were found in denervated WT but not Cx43/Cx45-deficient muscles. The atrophy observed after 7 d of denervation was drastically reduced in Cx43/Cx45-deficient but not Panx1-deficient muscles. Therefore, expression of Cx hemichannels and P2X7R promotes a feed-forward mechanism activated by extracellular ATP, most likely released through hemichannels, that activates the inflammasome. Consequently, Cx hemichannels are potential targets for new therapeutic agents to prevent or reduce muscle atrophy induced by denervation of diverse etiologies.

Lipopolysaccharide induces a fibrotic-like phenotype in endothelial cells.

Endothelial dysfunction is crucial in endotoxaemia-derived sepsis syndrome pathogenesis. It is well accepted that lipopolysaccharide (LPS) induces endothelial dysfunction through immune system activation. However, LPS can also directly generate actions in endothelial cells (ECs) in the absence of participation by immune cells. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge. However, the mechanism that confers endothelial resistance to LPS is not known. LPS-resistant ECs exhibit a fibroblast-like morphology, suggesting that these ECs enter a fibrotic programme in response to LPS. Thus, our aim was to investigate whether LPS is able to induce endothelial fibrosis in the absence of immune cells and explore the underlying mechanism. Using primary cultures of ECs and culturing intact blood vessels, we demonstrated that LPS is a crucial factor to induce endothelial fibrosis. We demonstrated that LPS was able and sufficient to promote endothelial fibrosis, in the absence of immune cells through an activin receptor-like kinase 5 (ALK5) activity-dependent mechanism. LPS-challenged ECs showed an up-regulation of both fibroblast-specific protein expression and extracellular matrix proteins secretion, as well as a down-regulation of endothelial markers. These results demonstrate that LPS is a crucial factor in inducing endothelial fibrosis in the absence of immune cells through an ALK5-dependent mechanism. It is noteworthy that LPS-induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxaemia-derived sepsis syndrome and other inflammatory diseases.

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.

Hemichannels in the neurovascular unit and white matter under normal and inflamed conditions.

In the normal brain, cellular types that compose the neurovascular unit, including neurons, astrocytes and endothelial cells express pannexins and connexins, which are protein subunits of two families that form plasma membrane channels. Most available evidence in mammals indicated that endogenously expressed pannexins only form hemichannels, and connexins form both gap junction channels and hemichannels. While gap junction channels connect the cytoplasm of contacting cells and coordinate electrical and metabolic activities, hemichannels communicate intra- and extracellular compartments and serve as diffusional pathways for ions and small molecules. Here, evidence supporting the functional role of hemichannels in the neurovascular unit and white matter under physiological and pathological conditions are reviewed. A sub-threshold acute pathological threatening condition (e.g., stroke and brain infection) leads to glial cell activation, which maintains an active defense and restores the normal function of the neurovascular unit. However, if the stimulus is deleterious, microglia and the endothelium become overactivated, both releasing bioactive molecules (e.g., glutamate, cytokines, prostaglandins and ATP) that increase the activity of astroglial hemichannels, reducing the astrocyte neuroprotective functions, and further reducing neuronal cell viability. Moreover, ATP is known to contribute to myelin degeneration of axons. Consequently, hemichannels might play a relevant role in the excitotoxic response of oligodendrocytes observed in ischemia and encephalomyelitis. Regulated changes in hemichannel permeability in healthy brain cells can have positive consequences in terms of paracrine/autocrine signaling, whereas persistent changes in cells affected by neurological disorders can be detrimental. Therefore, blocking hemichannels expressed by glial cells and/or neurons of the inflamed central nervous system might prevent neurovascular unit dysfunction and neurodegeneration.

NO production and eNOS phosphorylation induced by epinephrine through the activation of beta-adrenoceptors.

Epinephrine plays a key role in the control of vasomotor tone; however, the participation of the NO/cGMP pathway in response to beta-adrenoceptor activation remains controversial. To evaluate the involvement of the endothelium in the vascular response to epinephrine, we assessed NO production, endothelial NO synthase phosphorylation, and tissue accumulation of cGMP in the perfused arterial mesenteric bed of rat. Epinephrine elicited a concentration-dependent increase in NO (EC(50) of 45.7 pM), which was coupled to cGMP tissue accumulation. Both NO and cGMP production were blocked by either endothelium removal (saponin) or NO synthase inhibition (N(omega)-nitro-L-arginine). Blockade of beta(1)- and beta(2)-adrenoceptors with 1 microM propranolol or beta(3)-adrenoceptor with 10 nM SR 59230A displaced rightward the concentration-NO production curve evoked by epinephrine. Selective stimulation of beta(1)-, beta(2)-, or beta(3)-adrenoceptors also resulted in NO and cGMP production. Propranolol (1 microM) inhibited the rise in NO induced by isoproterenol or the beta(2)-adrenoceptor agonists salbutamol, terbutaline, or fenoterol. Likewise, 10 nM SR 59230A reduced the effects of the beta(3)-adrenoceptor agonists BRL 37344, CGP 12177, SR 595611A, or pindolol. The NO production induced by epinephrine and BRL 37344 was associated with the activation of the phosphatidylinositol 3-kinase/Akt pathway and phosphorylation of eNOS in serine 1177. In addition, in anaesthetized rats, bolus administration of isoproterenol, salbutamol, or BRL 37344 produced NO-dependent reductions in systolic blood pressure. These findings indicate that beta(1)-, beta(2)-, and beta(3)-adrenoceptors are coupled to the NO/cGMP pathway, highlighting the role of the endothelium in the vasomotor action elicited by epinephrine and related beta-adrenoceptor agonists.

Gap junctions in the control of vascular function.

Direct intercellular communication via gap junctions is critical in the control and coordination of vascular function. In the cardiovascular system, gap junctions are made up of one or more of four connexin proteins: Cx37, Cx40, Cx43, and Cx45. The expression of more than one gap-junction protein in the vasculature is not redundant. Rather, vascular connexins work in concert, first during the development of the cardiovascular system, and then in integrating smooth muscle and endothelial cell function, and in coordinating cell function along the length of the vessel wall. In addition, connexin-based channels have emerged as an important signaling pathway in the astrocyte-mediated neurovascular coupling. Direct electrical communication between endothelial cells and vascular smooth muscle cells via gap junctions is thought to play a relevant role in the control of vasomotor tone, providing the signaling pathway known as endothelium-derived hyperpolarizing factor (EDHF). Consistent with the importance of gap junctions in the regulation of vasomotor tone and arterial blood pressure, the expression of connexins is altered in diseases associated with vascular complications. In this review, we discuss the participation of connexin-based channels in the control of vascular function in physiologic and pathologic conditions, with a special emphasis on hypertension and diabetes.

Dissection of two Cx37-independent conducted vasodilator mechanisms by deletion of Cx40: electrotonic versus regenerative conduction.

Conduction of changes in diameter plays an important role in the coordination of peripheral vascular resistance and, thereby, in the control of arterial blood pressure. It is thought that conduction of vasomotor signals relies on the electrotonic spread of changes in membrane potential from a site of stimulation through gap junctions connecting the cells of the vessel wall. To explore this idea, we stimulated a short segment of mouse cremasteric arterioles with an application, via micropipette, of ACh, an endothelium-dependent vasodilator, or pinacidil, an ATP-sensitive K+ channel opener. Vasodilations were evaluated at the stimulation site (local) and at 500, 1,000, and 2,000 microm upstream. The vasodilator response evoked by direct arteriolar hyperpolarization induced by pinacidil decayed rapidly with distance, as expected for the passive spread of an electrical signal. Deletion of the gap junction proteins connexin37 or connexin40 did not alter the conduction of pinacidil-induced vasodilation. In contrast to pinacidil, the vasodilator response activated by ACh spread along the entire vessel without decrement. Although the ACh-induced conducted vasodilation was similar in wild-type and connexin37 knockout mice, deletion of connexin40 converted the nondecremental conducted response activated by ACh into one similar to that of pinacidil, with a decline in magnitude along the vessel length. These results suggest that ACh activates a mechanism of regenerative conduction of vasodilator responses. Connexin40 is essential for the ACh-activated regenerative vasodilator mechanism. However, neither connexin40 nor connexin37 is indispensable for the electrotonic spread of hyperpolarizing signals.