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.

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.

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.

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.

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.