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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.

The synthesis and characterization of the full family of 11 pyrazoles were performed by means of UV–Vis, FTIR, 1H NMR, 13C NMR, two-dimensional NMR experiments and DFT simulations. As pyrazoles are known for showing diverse biological actions, they were also tested in the NCI-60 cancer cell line panel, showing moderate to good activity against different cell lines. Furthermore, the anti-proinflammatory activity test of a set of pyrazoles of the form (E)-4-((4-bromophenyl)diazenyl)-3,5-dimethyl-1-R-phenyl-1H-pyrazole was performed, this is based on the study of the blockage of the increase in intracellular [Ca2+] observed in response to platelet-activating factor (PAF) treatment of four pyrazoles (i.e. 6, 8, 9 and 10), which successfully displayed [Ca2+] channel inhibition. Therefore, the obtained intracellular [Ca2+] signal results indicate that the pyrazole family characterized in this study, in particular compounds 6 and 10, are potent blockers of the PAF-initiated Ca2+ signaling that mediates the hyperpermeability typically observed during the development of inflammation., FONDECYT 1201880, 1171118, 1180565. Millennium Science Initiative of the Ministry of Economy, Development and Tourism, Chile, grant Nuclei on Catalytic Processes towards Sustainable Chemistry (CSC). ANID/FONDAP/15110019. Universidad del Atlántico (primera convocatoria interna, que otorga apoyo económico para el desarrollo de trabajos de grado en investigación formativa – nivel pregrado y postgrado).

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....

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.

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Control of blood flow distribution and tissue homeostasis depend on the tight regulation of and coordination between the microvascular network and circulating blood cells. Channels formed by connexins or pannexins that connect the intra- and extracellular compartments allow the release of paracrine signals, such as ATP and prostaglandins, and thus play a central role in achieving fine regulation and coordination of vascular function. This review focuses on vascular connexin hemichannels and pannexin channels. We review their expression pattern within the arterial and venous system with a special emphasis on how post-translational modifications by phosphorylation and S-nitrosylation of these channels modulate their function and contribute to vascular homeostasis. Furthermore, we highlight the contribution of these channels in smooth muscle cells and endothelial cells in the regulation of vasomotor tone as well as how these channels in endothelial cells regulate inflammatory responses such as during ischemic and hypoxic conditions. In addition, this review will touch on recent evidence implicating a role for these proteins in regulating red blood cell and platelet function.

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.

Connexins (Cxs) and pannexins (Panxs) are ubiquitous membrane channel forming proteins that are critically involved in many aspects of vascular physiology and pathology. The permeation of ions and small metabolites through Panx channels, Cx hemichannels and gap junction channels confers a crucial role to these proteins in intercellular communication and in maintaining tissue\ud homeostasis. This review provides an overview of current knowledge with respect to the pathophysiological role of these channels in large arteries, the microcirculation, veins, the lymphatic system and platelet function. The essential nature of these membrane proteins in vascular homeostasis is further emphasized by the pathologies that are linked to mutations and polymorphisms in Cx and\ud Panx genes.

In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent Na+ (Nav) and Ca2+ channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The Nav channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The Nav channel activator veratridine induced an endothelium-dependent dilation. The Nav channel isoforms Nav1.2, Nav1.6, and Nav1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of Nav channels in the conducted response. BAPTA buffering of endothelial cell Ca2+ delayed and reduced the conducted dilation, which was almost eliminated by Ni2+, amiloride, or deletion of α1H T-type Ca2+ (Cav3.2) channels. Blockade of endothelial nitric oxide synthase or Ca2+-activated K+ channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of Nav and Cav3.2 channels. The resultant Ca2+ influx activates endothelial nitric oxide synthase and Ca2+-activated K+ channels, triggering vasodilation.

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.

The proteins that form the gap junctions (connexins) are widely expressed in organs that are central to the development of hypertension: endocrine organs, kidney, brain, heart, and vasculature (Figure 1). Surprisingly, there is little information on the modification of connexins in hypertension in any of these organs except the vasculature, the subject of our review, but it would be hoped that this lack of information might spur research on these organs. Figure 1. Possible roles for the connexins in the control of blood pressure. A, Electron micrograph of a longitudinal view of an arteriole. Vascular connexins can play multiple roles in regulating function and vasomotor tone. Cell–cell communication, both radial and longitudinal, links the cells of the arteriolar wall. B, Most every organ involved in the control of blood pressure abundantly expresses ≥1 of the connexins. Essentially no experimental evidence exists regarding a possible link between connexins and hypertension in nonvascular tissues. Augmented vasomotor tone typically plays a key role in the development of hypertension, and tone depends on cell–cell communication established by paracrine molecules and, in addition, gap junctions. Paracrine-based linkages between cells of the vasculature are well known, and the possible roles of such linkages in the genesis of hypertension have been extensively explored. Much less is known of the roles of gap junctional communication in establishing vasomotor tone and of the effects of modification of gap junctions on hypertension. The gap junctions are formed by joining 2 hexameric assemblies of connexin protein monomers, that is, hemichannels (1 in Figure 2A). In the vasculature, assays of message and protein show that the hemichannels can be assembled from combinations of 4 connexins, named according to their apparent molecular weight: Cx37, Cx40, Cx43, and Cx45. Two hemichannels are linked to form a gap junction, which can provide both homocellular …

Endothelial cells control vascular tone by releasing vasoactive substances in response to physical and chemical signals. Nitric oxide (NO) has emerged as the most notable endothelium-derived vasodilator. Elegant pharmacological studies, in isolated or in situ arteries and arterioles, have documented the pivotal role of NO in the endothelium-dependent vasodilatation induced by several humoral agents and shear stress (Ishii et al. 1990; Koller & Kaley, 1991; Kuo et al. 1991; Moncada et al. 1991). On the other hand, the mechanisms triggering NO release by the activation of endothelial NO synthase (eNOS) have been studied mainly in cultured endothelial cells derived from large vessels, or in transfected non-endothelial cells (Michel et al. 1993, 1997a; Venema et al. 1996; Garcia-Cardena et al. 1997; Feron et al. 1998). While a few recent reports address this subject in vitro in endothelial cells of microvascular origin (Thuringer et al. 2000; Kawanaka et al. 2002), much less is known about the mechanisms of eNOS activation in vivo. In cultured cells, eNOS is targeted primarily to and anchored to the plasma membrane by dual acylation with myristate and palmitate (Busconi & Michel, 1993; Robinson & Michel, 1995; Sowa et al. 1999). In the membrane, the enzyme is found mainly in an inhibitory association with caveolin-1 (Cav-1), a structural protein of caveolae (Feron et al. 1996; Garcia-Cardena et al. 1997; Ju et al. 1997). Furthermore, it has been proposed that following a rise in intracellular calcium, eNOS dissociates from Cav-1, allowing activation of the enzyme. This hypothesis is supported by reports that an increase in intracellular calcium is associated with a rise in NO production (Blatter et al. 1995; Kanai et al. 1995), as the interaction of eNOS with calcium- calmodulin dissociates the eNOS-Cav-1 complex (Garcia-Cardena et al. 1997; Michel et al. 1997a,b; Feron et al. 1998). A role for tonic Cav-1 inhibition of eNOS activity in vivo is supported by the recent observation that the aortic rings of Cav-1-null mice present enhanced NOS-dependent relaxation in response to ACh (Drab et al. 2001; Razani et al. 2001). In addition, eNOS translocation from the membrane to the cytosol and/or perinuclear compartments has been observed following stimulation with calcium-mobilising agonists (Michel et al. 1993; Prabhakar et al. 1998; Goetz et al. 1999), possibly via enzyme de-palmitoylation (Robinson et al. 1995; Yeh et al. 1999). Translocation was presumed to be an inactivation mechanism of eNOS because it was a relatively slow process compared to the rapid transient NO signal reported in aortic endothelial cells (Malinski & Taha, 1992). This interpretation is questionable because the time course of NO production was not analysed together with eNOS translocation. To what extent the mechanisms determined separately in cultured cells occur and are relevant in vivo has not been thoroughly investigated. It is conceivable that the dynamic environment in the intact circulation resulting in tonic eNOS activation may influence the kinetics and functional relevance of processes determined in tissue culture. To resolve whether eNOS translocation corresponds to activation or inactivation of the enzyme, this process should be analysed along with the time course of NO production. Furthermore, the functional relevance of such an event should be assessed in intact preparations or in vivo. Therefore, in the work presented here, we tested the hypothesis that agonist-induced NO production and NO-dependent vasodilatation is associated with eNOS translocation from the cell membrane in microvascular endothelium in vivo. We decided to investigate this hypothesis in the hamster cheek pouch because this experimental model allows the direct visualisation and quantification of blood flow in the microcirculatory bed with minimal surgical trauma, as well as detection of NO production and eNOS subcellular distribution in a single preparation (Duran et al. 2000; Figueroa et al. 2001a). We chose ACh as the test agonist because it is a recognised calcium-mobilising endothelium-dependent vasodilator. We compared eNOS fractional distribution at rest and at several times during ACh-induced vasodilatation. We report data on the correspondence between the time course of the rise in blood flow and microvessel diameter with NO production and the reduction of microsomal eNOS content. Our data support the hypothesis that translocation from the membrane represents eNOS activation in vivo.

Prostate cancer (CaP) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Androgen deprivation therapy (ADT) is the standard treatment for advanced or metastatic CaP. However, during ADT, CaP progresses from an androgen-sensitive (AS-CaP) to a more aggressive, and eventually lethal, castration-resistant (CRPC) phenotype. There is evidence to suggest that the prostate tumor mass is under tight control of endothelial microvasculature due to an increase in angiogenesis by tumor cells. Nevertheless, now there is evidence to support that this influence is not one-directional and that the endothelial cells secrete a large number of active substances (angiocrine factors), which may directly or indirectly influence tumor growth and progression. However, the direct impacts of the endothelium on prostate tumor progression or the molecular mechanisms that are involved in this communication remain unclear. Here we investigated the potential influence of endothelium-derived paracrine factors on prostate cancer biology and the role of connexins in these interactions, since connexins play a major role in cell-cell communication and form a bidirectional signaling pathway to assemble gap junctions and alter cell behaviors. We measured the effect of conditioned media (CM) obtained from a primary culture of human endothelial cells isolated from umbilical vein (HUVEC) on viability, proliferation, migration and invasion of CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) and in the metastatic potential by in vivo assays using co-injection of CaP cell with HUVEC or injection of CaP cells pre-incubated with CM from HUVEC in a zebrafish embryo model. Finally, we studied the expression and the role of connexins on this stimulation using pharmacological (GJIC inhibitors) approaches. All together, our results showed that CM from endothelial cell induces an increases in the viability and proliferation in all CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) but only increases migration of the CRPC cell lines (LNCaP-C4-2 and PC3). We also observed in our in vivo model that endothelial cells either through cell-cell interaction or by paracrine communication increases the metastatic ability of the CaP cells. Moreover, the increase in viability and migration of CaP cells observed with CM from endothelial cells was blocked using inhibitors of gap junctions. Real-time PCR analyses detected an up-regulation of Cx43 mRNA after exposition to CM from endothelial cells. Our data suggest that angiocrine communication between endothelial cells and CaP cells increases proliferation and migration of more aggressive CaP cells which could be important for the acquisition of the aggressive phenotype of the disease, and this interaction could be mediated by Cx43. Delineation of such critical players may culminate in identifying therapeutic targets or biomarkers to counteract CaP, especially advanced CaP. Citation Format: Verónica Torres-Estay, Patricia Fuenzalida, Catalina Ascencio, Carla Cembrano, Daniela Carreño, Néstor Corro, Viviana Montecinos, Gareth Owen, Xavier Figueroa, Julio Amigo, Juan Carlos Saéz, Alejandro Godoy. Paracrine effect of the endothelium on prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2017-2969

Pannexin-1 (Panx1) is expressed in smooth muscle and endothelial cells of resistance arteries. In smooth muscle, Panx1-formed channels mediate the α1-adrenergic receptor-initiated constriction, but...

1. To assess the involvement of endothelial alpha(2)-adrenoceptors in the clonidine-induced vasodilatation, the mesenteric artery of Sprague Dawley rats was cannulated and perfused with Tyrode solution (2 ml min(-1)). We measured perfusion pressure, nitric oxide (NO) in the perfusate using chemiluminescence, and tissue cyclic GMP by RIA. 2. In phenylephrine-precontracted mesenteries, clonidine elicited concentration-dependent vasodilatations associated to a rise in luminal NO. One hundred nM rauwolscine or 100 microM L(omega)-nitro-L-arginine antagonized the clonidine-induced vasodilatation. Guanabenz, guanfacine, and oxymetazoline mimicked the clonidine-induced vasorelaxation. 3. In non-contracted mesenteries, 100 nM clonidine elicited a maximal rise of NO (123+/-13 pmol); associated to a peak in tissue cyclic GMP. Endothelium removal, L(omega)-nitro-L-arginine, or rauwolscine ablated the rise in NO. One hundred nM aminoclonidine, guanfacine, guanabenz, UK14,304 and oxymetazoline mimicked the clonidine-induced surge of NO. Ten microM ODQ obliterated the clonidine-induced vasorelaxation and the associated tissue cyclic GMP accumulation; 10 - 100 nM sildenafil increased tissue cyclic GMP accumulation without altering the clonidine-induced NO release. 4. alpha(2)-Adrenergic blockers antagonized the clonidine-induced rise in NO. Consistent with a preferential alpha(2D)-adrenoceptor activation, the K(B)s for yohimbine, rauwolscine, phentolamine, WB-4101, and prazosin were: 6.8, 24, 19, 165, and 1489 nM, respectively. 5. Rat pretreatment with 100 mg kg(-1) 6-hydroxydopamine reduced 95% tissue noradrenaline and 60% neuropeptide Y. In these preparations, 100 nM clonidine elicited a rise of 91.9+/-15.5 pmol NO. Perfusion with 1 microM guanethidine or 1 microM guanethidine plus 1 microM atropine did not modify the NO surge evoked by 100 nM clonidine. 6. Clonidine and congeners activate endothelial alpha(2D)-adrenoceptors coupled to the L-arginine pathway, suggesting that the antihypertensive action of clonidine involves an endothelial vasorelaxation mediated by NO release, in addition to presynaptic mechanisms.

Abstract —We have shown previously that the kininogen-derived peptides bradykinin, prokinins, and PU-D1, given intravenously or into the duodenal lumen, block the atrial natriuretic peptide (ANP)-induced diuretic-natriuretic effect in fasting, anesthetized rats infused with isotonic glucose. HOE-140, an inhibitor of bradykinin B2 receptors, completely suppresses this ANP blockade. When intravenous glucose infusion is omitted, the above-described inhibition of ANP does not take place. Therefore, to clarify the role of glucose and/or feeding in this phenomenon, we used fasted, anesthetized rats to test how the ANP excretory response was affected by (1) short-term feeding before anesthesia, (2) 1 mL of isotonic glucose introduced into the stomach, and (3) the interaction of HOE-140 with these treatments. In addition, we tested the effects of 1 mL of intragastric glucose administration and HOE-140 on urinary excretion in awake rats. In anesthetized rats, both glucose administration and feeding significantly inhibited the diuretic-natriuretic effect of ANP for up to 90 minutes. Similarly, intragastric glucose delayed spontaneous sodium and water excretion for 90 minutes in awake rats. In all 3 cases, pretreatment with HOE-140 (2.5 μg IV) fully prevented the inhibition of ANP excretory action, ruling out osmotic effects as the cause of reduced diuresis. These results indicate that the presence of glucose in the digestive tract triggers an inhibitory effect on ANP renal actions that requires activation of kinin B2 receptors, providing strong support to our hypothesis that during the early prandial period, gastrointestinal signals elicit a transient blockade of renal excretion with a mechanism involving the kallikrein-kinin 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.

NO is generated within cells and frequently must be transferred to responsive neighboring cells, as occurs in the endothelium-dependent relaxation of smooth muscle cells observed in blood vessels. It is thought that NO diffuses freely across cell membranes, but it may also permeate through low resistant membrane pathways. Here, we describe the participation of connexin (Cx)-formed channels in the NO transport across cell membranes and between endothelial and smooth muscle cells. We used a water-soluble NO donor of high molecular weight (S-nitrosylated albumin, BSA-NO) that does not permeate through cell membranes or Cx-based channels and the NO-sensitive dye 4,5-diaminofluorescein diacetate to detect changes of intracellular NO concentration. We found that NO generated in the extracellular space was not detected intracellularly in Cx-deficient HeLa cells, suggesting that cell membrane represents a significant diffusion barrier for NO transfer. However, Cx-based channels provide efficient pathways for NO signaling because NO opened and permeated hemichannels expressed in HeLa cells transfected with Cx43, Cx40, or Cx37. In contrast, NO closed hemichannels of HeLa-Cx32 cells, which otherwise are permeable to NO if are opened by a divalent cation-free extracellular solution. Consistent with this, blockade of Cx-based channels abolished the myoendothelial NO transfer and associated NO-dependent vasodilation induced by acethylcholine. These results indicate that Cx-based channels play a key role in the NO-dependent tonic control of vascular function and may direct the NO signal to specific targets, which provides a novel mechanistic basis for the critical role of Cxs in cell-cell communication in the vessel wall. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

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.

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 Ca2+. 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 Ca2+, 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 Ca2+-dependent eNOS translocation from membrane to intracellular compartments and sustained Ca2+-independent PI3K-Akt-mediated phosphorylation.

The blood vascular system consists in a complex network of vessels that is mainly intended to provide oxygen and nutrients to all individual cells of peripheral tissues and help to dispose metabolic wastes. Several distinct functional compartments can be distinguished in the vascular network: arteries, arterioles, capillaries, venules and veins. Conduit arteries (diameter, 1 to several millimeters) carry blood away from the heart through a divergent arborescence that reaches and penetrates into the tissues via the feed arteries (diameter, 100 to 500 μm) (Davis et al., 1986; Segal, 2000, 2005). These muscular vessels give rise to the arterioles (diameter, < l00 μm), which control and coordinate the blood flow distribution in such a way that each capillary is correctly supplied at the proper pressure (Mulvany, 1990; Segal, 2005). This part of the vascular network composed of arterioles, capillaries and venules is embedded within the organ irrigated and is called microcirculation (Davis et al., 1986; Segal, 2005; Lockhart et al., 2009). Finally, veins carry blood back to the heart through a convergent arborescence.

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.

Epinephrine plays a key role in the control of vasomotor tone; however, the participation of the NO/cGMP pathway in response to β-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 (EC50 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 ( Nω-nitro-l-arginine). Blockade of β1- and β2-adrenoceptors with 1 μM propranolol or β3-adrenoceptor with 10 nM SR 59230A displaced rightward the concentration-NO production curve evoked by epinephrine. Selective stimulation of β1-, β2-, or β3-adrenoceptors also resulted in NO and cGMP production. Propranolol (1 μM) inhibited the rise in NO induced by isoproterenol or the β2-adrenoceptor agonists salbutamol, terbutaline, or fenoterol. Likewise, 10 nM SR 59230A reduced the effects of the β3-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 β1-, β2-, and β3-adrenoceptors are coupled to the NO/cGMP pathway, highlighting the role of the endothelium in the vasomotor action elicited by epinephrine and related β-adrenoceptor agonists.

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. Antioxid. Redox Signal. 11, 251–266.

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 μm 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.