Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage.

BACKGROUND: Alcohol, a widely abused drug, significantly diminishes life quality, causing chronic diseases and psychiatric issues, with severe health, societal, and economic repercussions. Previously, we demonstrated that non-voluntary alcohol consumption increases the opening of Cx43 hemichannels and Panx1 channels in astrocytes from adolescent rats. However, whether ethanol directly affects astroglial hemichannels and, if so, how this impacts the function and survival of astrocytes remains to be elucidated. RESULTS: Clinically relevant concentrations of ethanol boost the opening of Cx43 hemichannels and Panx1 channels in mouse cortical astrocytes, resulting in the release of ATP and glutamate. The activation of these large-pore channels is dependent on Toll-like receptor 4, P2X7 receptors, IL-1ß and TNF-α signaling, p38 mitogen-activated protein kinase, and inducible nitric oxide (NO) synthase. Notably, the ethanol-induced opening of Cx43 hemichannels and Panx1 channels leads to alterations in cytokine secretion, NO production, gliotransmitter release, and astrocyte reactivity, ultimately impacting survival. CONCLUSION: Our study reveals a new mechanism by which ethanol impairs astrocyte function, involving the sequential stimulation of inflammatory pathways that further increase the opening of Cx43 hemichannels and Panx1 channels. We hypothesize that targeting astroglial hemichannels could be a promising pharmacological approach to preserve astrocyte function and synaptic plasticity during the progression of various alcohol use disorders.

Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala.

Astrocytes release gliotransmitters via connexin 43 (Cx43) hemichannels into neighboring synapses, which can modulate synaptic activity and are necessary for fear memory consolidation. However, the gliotransmitters released, and their mechanisms of action remain elusive. Here, we report that fear conditioning training elevated Cx43 hemichannel activity in astrocytes from the basolateral amygdala (BLA). The selective blockade of Cx43 hemichannels by microinfusion of TAT-Cx43L2 peptide into the BLA induced memory deficits 1 and 24 h after training, without affecting learning. The memory impairments were prevented by the co-injection of glutamate and D-serine, but not by the injection of either alone, suggesting a role for NMDA receptors (NMDAR). The incubation with TAT-Cx43L2 decreased NMDAR-mediated currents in BLA slices, effect that was also prevented by the addition of glutamate and D-serine. NMDARs in primary neuronal cultures were unaffected by TAT-Cx43L2, ruling out direct effects of the peptide on NMDARs. Finally, we show that D-serine permeates through purified Cx43 hemichannels reconstituted in liposomes. We propose that the release of glutamate and D-serine from astrocytes through Cx43 hemichannels is necessary for the activation of post-synaptic NMDARs during training, to allow for the formation of short-term and subsequent long-term memory, but not for learning per se.

SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels. RESULTS: We found that spike S1 time and dose-dependently increased the activity of Cx43 hemichannels in HeLa-Cx43 cells, as measured by dye uptake experiments. These responses were potentiated when the angiotensin-converting enzyme 2 (ACE2) was expressed in HeLa-Cx43 cells. Patch clamp experiments revealed that spike S1 increased unitary current events with conductances compatible with Cx43 hemichannels. In addition, Cx43 hemichannel opening evoked by spike S1 triggered the release of ATP and increased the [Ca2+]i dynamics elicited by ATP. CONCLUSIONS: We hypothesize that Cx43 hemichannels could represent potential pharmacological targets for developing therapies to counteract SARS-CoV-2 infection and their long-term consequences.

TNF-α Plus IL-1ß Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway.

Connexin 43 (Cx43) is expressed in kidney tissue where it forms hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analysis of ethidium uptake and thiobarbituric acid reactive species revealed that treatment with TNF-α plus IL-1ß increases Cx43 hemichannel activity and oxidative stress in MES-13 cells (a cell line derived from mesangial cells), and in primary mesangial cells. The latter was also accompanied by a reduction in gap junctional communication, whereas Western blotting assays showed a progressive increase in phosphorylated MYPT (a target of RhoA/ROCK) and Cx43 upon TNF-α/IL-1ß treatment. Additionally, inhibition of RhoA/ROCK strongly antagonized the TNF-α/IL-1ß-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell-cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.

Hypertensive Nephropathy: Unveiling the Possible Involvement of Hemichannels and Pannexons.

Hypertension is one of the most common risk factors for developing chronic cardiovascular diseases, including hypertensive nephropathy. Within the glomerulus, hypertension causes damage and activation of mesangial cells (MCs), eliciting the production of large amounts of vasoactive and proinflammatory agents. Accordingly, the activation of AT1 receptors by the vasoactive molecule angiotensin II (AngII) contributes to the pathogenesis of renal damage, which is mediated mostly by the dysfunction of intracellular Ca2+ ([Ca2+]i) signaling. Similarly, inflammation entails complex processes, where [Ca2+]i also play crucial roles. Deregulation of this second messenger increases cell damage and promotes fibrosis, reduces renal blood flow, and impairs the glomerular filtration barrier. In vertebrates, [Ca2+]i signaling depends, in part, on the activity of two families of large-pore channels: hemichannels and pannexons. Interestingly, the opening of these channels depends on [Ca2+]i signaling. In this review, we propose that the opening of channels formed by connexins and/or pannexins mediated by AngII induces the ATP release to the extracellular media, with the subsequent activation of purinergic receptors. This process could elicit Ca2+ overload and constitute a feed-forward mechanism, leading to kidney damage.

Astroglial Hemichannels and Pannexons: The Hidden Link between Maternal Inflammation and Neurological Disorders.

Maternal inflammation during pregnancy causes later-in-life alterations of the offspring's brain structure and function. These abnormalities increase the risk of developing several psychiatric and neurological disorders, including schizophrenia, intellectual disability, bipolar disorder, autism spectrum disorder, microcephaly, and cerebral palsy. Here, we discuss how astrocytes might contribute to postnatal brain dysfunction following maternal inflammation, focusing on the signaling mediated by two families of plasma membrane channels: hemi-channels and pannexons. [Ca2+]i imbalance linked to the opening of astrocytic hemichannels and pannexons could disturb essential functions that sustain astrocytic survival and astrocyte-to-neuron support, including energy and redox homeostasis, uptake of K+ and glutamate, and the delivery of neurotrophic factors and energy-rich metabolites. Both phenomena could make neurons more susceptible to the harmful effect of prenatal inflammation and the experience of a second immune challenge during adulthood. On the other hand, maternal inflammation could cause excitotoxicity by producing the release of high amounts of gliotransmitters via astrocytic hemichannels/pannexons, eliciting further neuronal damage. Understanding how hemichannels and pannexons participate in maternal inflammation-induced brain abnormalities could be critical for developing pharmacological therapies against neurological disorders observed in the offspring.

HIV gp120 Protein Increases the Function of Connexin 43 Hemichannels and Pannexin-1 Channels in Astrocytes: Repercussions on Astroglial Function.

At least half of human immunodeficiency virus (HIV)-infected individuals suffer from a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND). The molecular mechanisms that amplify damage within the brain of HIV-infected individuals are unknown. Recently, we described that HIV augments the opening of connexin-43 (Cx43) hemichannels in cultured human astrocytes, which result in the collapse of neuronal processes. Whether HIV soluble viral proteins such as gp120, can regulate hemichannel opening in astrocytes is still ignored. These channels communicate the cytosol with the extracellular space during pathological conditions. We found that gp120 enhances the function of both Cx43 hemichannels and pannexin-1 channels in mouse cortical astrocytes. These effects depended on the activation of IL-1ß/TNF-α, p38 MAP kinase, iNOS, cytoplasmic Ca2+ and purinergic signaling. The gp120-induced channel opening resulted in alterations in Ca2+ dynamics, nitric oxide production and ATP release. Although the channel opening evoked by gp120 in astrocytes was reproduced in ex vivo brain preparations, these responses were heterogeneous depending on the CA1 region analyzed. We speculate that soluble gp120-induced activation of astroglial Cx43 hemichannels and pannexin-1 channels could be crucial for the pathogenesis of HAND.

Role of a RhoA/ROCK-Dependent Pathway on Renal Connexin43 Regulation in the Angiotensin II-Induced Renal Damage.

In various models of chronic kidney disease, the amount and localization of Cx43 in the nephron is known to increase, but the intracellular pathways that regulate these changes have not been identified. Therefore, we proposed that: "In the model of renal damage induced by infusion of angiotensin II (AngII), a RhoA/ROCK-dependent pathway, is activated and regulates the abundance of renal Cx43". In rats, we evaluated: 1) the time-point where the renal damage induced by AngII is no longer reversible; and 2) the involvement of a RhoA/ROCK-dependent pathway and its relationship with the amount of Cx43 in this irreversible stage. Systolic blood pressure (SBP) and renal function (urinary protein/urinary creatinine: Uprot/UCrea) were evaluated as systemic and organ outcomes, respectively. In kidney tissue, we also evaluated: 1) oxidative stress (amount of thiobarbituric acid reactive species), 2) inflammation (immunoperoxidase detection of the inflammatory markers ED-1 and IL-1ß), 3) fibrosis (immune detection of type III collagen; Col III) and 4) activity of RhoA/ROCK (amount of phosphorylated MYPT1; p-MYPT1). The ratio Uprot/UCrea, SBP, oxidative stress, inflammation, amount of Cx43 and p-MYPT1 remained high 2 weeks after suspending AngII treatment in rats treated for 4 weeks with AngII. These responses were not observed in rats treated with AngII for less than 4 weeks, in which all measurements returned spontaneously close to the control values after suspending AngII treatment. Rats treated with AngII for 6 weeks and co-treated for the last 4 weeks with Fasudil, an inhibitor of ROCK, showed high SBP but did not present renal damage or increased amount of renal Cx43. Therefore, renal damage induced by AngII correlates with the activation of RhoA/ROCK and the increase in Cx43 amounts and can be prevented by inhibitors of this pathway.

Boldine Improves Kidney Damage in the Goldblatt 2K1C Model Avoiding the Increase in TGF-ß.

Boldine, a major aporphine alkaloid found in the Chilean boldo tree, is a potent antioxidant. Oxidative stress plays a detrimental role in the pathogenesis of kidney damage in renovascular hypertension (RVH). The activation of the renin-angiotensin system (RAS) is crucial to the development and progression of hypertensive renal damage and TGF-β is closely associated with the activation of RAS. In the present study, we assessed the effect of boldine on the progression of kidney disease using the 2K1C hypertension model and identifying mediators in the RAS, such as TGF-β, that could be modulated by this alkaloid. Toward this hypothesis, rats (n = 5/group) were treated with boldine (50 mg/kg/day, gavage) for six weeks after 2K1C surgery (pressure ≥ 180 mmHg). Kidney function was evaluated by measuring of proteinuria/creatininuria ratio (U prot/U Crea), oxidative stress (OS) by measuring thiobarbituric acid reactive substances (TBARS). The evolution of systolic blood pressure (SBP) was followed weekly. Alpha-smooth muscle actin (α-SMA) and Col III were used as markers of kidney damage; ED-1 and osteopontin (OPN) were used as markers of inflammation. We also explored the effect in RAS mediators, such as ACE-1 and TGF-β. Boldine treatment reduced the UProt/UCrea ratio, plasma TBARS, and slightly reduced SBP in 2K1C hypertensive rats, producing no effect in control animals. In 2K1C rats treated with boldine the levels of α-SMA, Col III, ED-1, and OPN were lower when compared to 2K1C rats. Boldine prevented the increase in ACE-1 and TGF-β in 2K1C rats, suggesting that boldine reduces kidney damage. These results suggest that boldine could potentially be used as a nutraceutic.

Angiotensin II-Induced Mesangial Cell Damaged Is Preceded by Cell Membrane Permeabilization Due to Upregulation of Non-Selective Channels.

Connexin43 (Cx43), pannexin1 (Panx1) and P2X7 receptor (P2X7R) are expressed in kidneys and are known to constitute a feedforward mechanism leading to inflammation in other tissues. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remain unknown. In the present work, we found that MES-13 cells, from a cell line derived from mesangial cells, stimulated with angiotensin II (AngII) developed oxidative stress (OS, thiobarbituric acid reactive species (TBARS) and generated pro-inflammatory cytokines (ELISA; IL-1ß and TNF-α). The membrane permeability increased progressively several hours before the latter outcome, which was a response prevented by Losartan, indicating the involvement of AT1 receptors. Western blot analysis showed that the amount of phosphorylated MYPT (a substrate of RhoA/ROCK) and Cx43 increased progressively and in parallel in cells treated with AngII, a response followed by an increase in the amount in Panx1 and P2X7R. Greater membrane permeability was partially explained by opening of Cx43 hemichannels (Cx43 HCs) and Panx1 channels (Panx1 Chs), as well as P2X7Rs activation by extracellular ATP, which was presumably released via Cx HCs and Panx1 Chs. Additionally, inhibition of RhoA/ROCK blocked the progressive increase in membrane permeability, and the remaining response was explained by the other non-selective channels. The rise of activity in the RhoA/ROCK-dependent pathway, as well as in Cx HCs, P2X7R, and to a minor extent in Panx1 Chs led to higher amounts of TBARS and pro-inflammatory cytokines. We propose that AngII-induced mesangial cell damage could be effectively inhibited by concomitantly inhibiting the RhoA/ROCK-dependent pathway and one or more non-selective channel(s) activated through this pathway.

Activation of Pannexin-1 channels causes cell dysfunction and damage in mesangial cells derived from angiotensin II-exposed mice.

Chronic kidney disease (CKD) is a prevalent health concern associated with various pathological conditions, including hypertensive nephropathy. Mesangial cells are crucial in maintaining glomerular function, yet their involvement in CKD pathogenesis remains poorly understood. Recent evidence indicates that overactivation of Pannexin-1 (Panx1) channels could contribute to the pathogenesis and progression of various diseases. Although Panx1 is expressed in the kidney, its contribution to the dysfunction of renal cells during pathological conditions remains to be elucidated. This study aimed to investigate the impact of Panx1 channels on mesangial cell function in the context of hypertensive nephropathy. Using an Ang II-infused mouse model and primary mesangial cell cultures, we demonstrated that in vivo exposure to Ang II sensitizes cultured mesangial cells to show increased alterations when they are subjected to subsequent in vitro exposure to Ang II. Particularly, mesangial cell cultures treated with Ang II showed elevated activity of Panx1 channels and increased release of ATP. The latter was associated with enhanced basal intracellular Ca2+ ([Ca2+]i) and increased ATP-mediated [Ca2+]i responses. These effects were accompanied by increased lipid peroxidation and reduced cell viability. Crucially, all the adverse impacts evoked by Ang II were prevented by the blockade of Panx1 channels, underscoring their critical role in mediating cellular dysfunction in mesangial cells. By elucidating the mechanisms by which Ang II negatively impacts mesangial cell function, this study provides valuable insights into the pathogenesis of renal damage in hypertensive nephropathy.

Acute activation of hemichannels by ethanol leads to Ca2+-dependent gliotransmitter release in astrocytes.

Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms. The uncontrolled activity of these channels could favor the collapse of ionic gradients and osmotic balance, the release of toxic levels of ATP or glutamate, cell swelling and plasma membrane breakdown and intracellular Ca2+ overload. Here, we evaluated whether acute ethanol exposure affects the activity of astrocyte hemichannels and the possible repercussions of this phenomenon on cytoplasmatic Ca2+ signaling and gliotransmitter release. Acute ethanol exposure triggered the rapid activation of connexin43 and pannexin1 hemichannels in astrocytes, as measured by time-lapse recordings of ethidium uptake. This heightened activity derived from a rapid rise in [Ca2+]i linked to extracellular Ca2+ influx and IP3-evoked Ca2+ release from intracellular Ca2+ stores. Relevantly, the acute ethanol-induced activation of hemichannels contributed to a persistent secondary increase in [Ca2+]i. The [Ca2+]i-dependent activation of hemichannels elicited by ethanol caused the increased release of ATP and glutamate in astroglial cultures and brain slices. Our findings offer fresh perspectives on the potential mechanisms behind acute alcohol-induced brain abnormalities and propose targeting connexin43 and pannexin1 hemichannels in astrocytes as a promising avenue to prevent deleterious consequences of alcohol consumption.

Activation of Melanocortin-4 Receptor by a Synthetic Agonist Inhibits Ethanolinduced Neuroinflammation in Rats.

BACKGROUND: High ethanol intake induces a neuroinflammatory response resulting in the subsequent maintenance of chronic alcohol consumption. The melanocortin system plays a pivotal role in the modulation of alcohol consumption. Interestingly, it has been shown that the activation of melanocortin-4 receptor (MC4R) in the brain decreases the neuroinflammatory response in models of brain damage other than alcohol consumption, such as LPS-induced neuroinflammation, cerebral ischemia, glutamate excitotoxicity, and spinal cord injury. OBJECTIVES: In this work, we aimed to study whether MC4R activation by a synthetic MC4R-agonist peptide prevents ethanol-induced neuroinflammation, and if alcohol consumption produces changes in MC4R expression in the hippocampus and hypothalamus. METHODS: Ethanol-preferring Sprague Dawley rats were selected offering access to 20% ethanol on alternate days for 4 weeks (intermittent access protocol). After this time, animals were i.p. administered an MC4R agonist peptide in the last 2 days of the protocol. Then, the expression of the proinflammatory cytokines interleukin 6 (IL-6), interleukin 1-beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α) were measured in the hippocampus, hypothalamus and prefrontal cortex. It was also evaluated if ethanol intake produces alterations in the expression of MC4R in the hippocampus and the hypothalamus. RESULTS: Alcohol consumption increased the expression of MC4R in the hippocampus and the hypothalamus. The administration of the MC4R agonist reduced IL-6, IL-1ß and TNF-α levels in hippocampus, hypothalamus and prefrontal cortex, to those observed in control rats that did not drink alcohol. CONCLUSION: High ethanol consumption produces an increase in the expression of MC4R in the hippocampus and hypothalamus. The administration of a synthetic MC4R-agonist peptide prevents neuroinflammation induced by alcohol consumption in the hippocampus, hypothalamus, and prefrontal cortex. These results could explain the effect of α-MSH and other synthetic MC4R agonists in decreasing alcohol intake through the reduction of the ethanol-induced inflammatory response in the brain.

The Neuroglial Dialog Between Cannabinoids and Hemichannels.

The formation of gap junctions was initially thought to be the central role of connexins, however, recent evidence had brought to light the high relevance of unopposed hemichannels as an independent mechanism for the selective release of biomolecules during physiological and pathological conditions. In the healthy brain, the physiological opening of astrocyte hemichannels modulates basal excitatory synaptic transmission. At the other end, the release of potentially neurotoxic compounds through astroglial hemichannels and pannexons has been insinuated as one of the functional alterations that negatively affect the progression of multiple brain diseases. Recent insights in this matter have suggested encannabinoids (eCBs) as molecules that could regulate the opening of these channels during diverse conditions. In this review, we discuss and hypothesize the possible interplay between the eCB system and the hemichannel/pannexon-mediated signaling in the inflamed brain and during event of synaptic plasticity. Most findings indicate that eCBs seem to counteract the activation of major neuroinflammatory pathways that lead to glia-mediated production of TNF-α and IL-1ß, both well-known triggers of astroglial hemichannel opening. In contrast to the latter, in the normal brain, eCBs apparently elicit the Ca2+-activation of astrocyte hemichannels, which could have significant consequences on eCB-dependent synaptic plasticity.

Heavy Alcohol Exposure Activates Astroglial Hemichannels and Pannexons in the Hippocampus of Adolescent Rats: Effects on Neuroinflammation and Astrocyte Arborization.

A mounting body of evidence indicates that adolescents are specially more susceptible to alcohol influence than adults. However, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity and recently, the opening of hemichannels and pannexons has been found to participate in both processes. Here, we evaluated whether adolescent rats exposed to ethanol exhibit changes in the activity of astrocyte hemichannels and pannexons in the hippocampus, as well as alterations in astrocyte arborization and cytokine levels. Adolescent rats were subjected to ethanol (3.0 g/kg) for two successive days at 48-h periods over 14 days. The opening of hemichannels and pannexons was examined in hippocampal slices by dye uptake, whereas hippocampal cytokine levels and astroglial arborization were determined by ELISA and Sholl analysis, respectively. We found that adolescent ethanol exposure increased the opening of connexin 43 (Cx43) hemichannels and pannexin-1 (Panx1) channels in astrocytes. Blockade of p38 mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS) and cyclooxygenases (COXs), as well as chelation of intracellular Ca2+, drastically reduced the ethanol-induced channel opening in astrocytes. Importantly, ethanol-induced Cx43 hemichannel and Panx1 channel activity was correlated with increased levels of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 in the hippocampus, as well as with profound alterations in astrocyte arbor complexity. Thus, we propose that uncontrolled opening of astrocyte hemichannels and pannexons may contribute not only to the glial dysfunction and neurotoxicity caused by adolescent alcohol consumption, but also to the pathogenesis of alcohol use disorders in the adulthood.

Connexin 43 Hemichannel Activity Promoted by Pro-Inflammatory Cytokines and High Glucose Alters Endothelial Cell Function.

The present work was done to elucidate whether hemichannels of a cell line derived from endothelial cells are affected by pro-inflammatory conditions (high glucose and IL-1ß/TNF-α) known to lead to vascular dysfunction. We used EAhy 926 cells treated with high glucose and IL-1ß/TNF-α. The hemichannel activity was evaluated with the dye uptake method and was abrogated with selective inhibitors or knocking down of hemichannel protein subunits with siRNA. Western blot analysis, cell surface biotinylation, and confocal microscopy were used to evaluate total and plasma membrane amounts of specific proteins and their cellular distribution, respectively. Changes in intracellular Ca2+ and nitric oxide (NO) signals were estimated by measuring FURA-2 and DAF-FM probes, respectively. High glucose concentration was found to elevate dye uptake, a response that was enhanced by IL-1ß/TNF-α. High glucose plus IL-1ß/TNF-α-induced dye uptake was abrogated by connexin 43 (Cx43) but not pannexin1 knockdown. Furthermore, Cx43 hemichannel activity was associated with enhanced ATP release and activation of p38 MAPK, inducible NO synthase, COX2, PGE2 receptor EP1, and P2X7/P2Y1 receptors. Inhibition of the above pathways prevented completely the increase in Cx43 hemichannel activity of cells treated high glucose and IL-1ß/TNF-α. Both synthetic and endogenous cannabinoids (CBs) also prevented the increment in Cx43 hemichannel opening, as well as the subsequent generation and release of ATP and NO induced by pro-inflammatory conditions. The counteracting action of CBs also was extended to other endothelial alterations evoked by IL-1ß/TNF-α and high glucose, including increased ATP-dependent Ca2+ dynamics and insulin-induced NO production. Finally, inhibition of Cx43 hemichannels also prevented the ATP release from endothelial cells treated with IL-1ß/TNF-α and high glucose. Therefore, we propose that reduction of hemichannel activity could represent a strategy against the activation of deleterious pathways that lead to endothelial dysfunction and possibly cell damage evoked by high glucose and pro-inflammatory conditions during cardiovascular diseases.