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Sepsis is a life-threatening organ dysfunction resulting from a dysregulated host response to infections that is initiated by the body's innate immune system. Nearly a decade ago, we discovered that bacterial lipopolysaccharide (LPS) and serum amyloid A (SAA) upregulated Connexin 43 (Cx43) and Pannexin 1 (Panx1) hemichannels in macrophages. When overexpressed, these hemichannels contribute to sepsis pathogenesis by promoting ATP efflux, which intensifies the double-stranded RNA-activated protein kinase R (PKR)-dependent inflammasome activation, pyroptosis, and the release of pathogenic damage-associated molecular pattern (DAMP) molecules, such as HMGB1. Mimetic peptides targeting specific regions of Cx43 and Panx1 can distinctly modulate hemichannel activity in vitro , and diversely impact sepsis-induced lethality in vivo. Along with extensive supporting evidence from others, we now propose that hemichannel molecules play critical roles as endogenous regulators of innate immunity in sepsis. [ABSTRACT FROM AUTHOR]

Objective To study the correlation between expression levels of serum Spexin and Pannexin 1 protein, glucose and lipid metabolism and insulin resistance in elderly patients with diabetic cataract. Methods A total of 118 elderly diabetic cataract patients were selected as the case group, and 103 elderly diabetic non-cataract patients were selected as the control group who were treated in the same hospital during the same period. Serum levels of Spexin and Pannexin1 were detected by enzyme-linked immunosorbent assay (ELISA). Fasting blood glucose (FPG) and glycosylated hemoglobin (HbA1c) were measured. Lipid metabolism related indicators including triacylglycerol (TG), high density lipoprotein cholesterol (HDL-C), total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) were detected in two groups of patients. Insulin resistance related indexes: fasting insulin (FINS), insulin sensitivity index (ISI), insulin resistance index (HOMA-IR) and islet beta cell function index (HOMA-β) were also detected. The correlation between serum Spexin and Pannexin1 levels, glucolipid metabolism and ISI was detected by Pearson method. Receiver operating characteristic (ROC) curves were used to assess the predictive value of serum Spexin and Pannexin1 levels and their combination in the development of cataract in elderly diabetic patients. Results The level of Spexin was lower in the observation group than that in the control group, and the level of Pannexin1 was higher than that in the control group (P＜0.01). Levels of TG, TC, LDL-C, FPG, HbA1c, FINS and HOMA-IR of the observation group increased compared with the control group, while HDL-C, ISI and HOMA-β levels decreased compared with the control group (P＜0.01). Pearson correlation analysis showed that serum Spexin level was positively correlated with ISI and HOMA-β, and negatively correlated with TG, TC, LDL-C, FPG, HbA1c, FINS and HOMA-IR in elderly patients with diabetic cataract (P＜0.05). Serum Pannexin1 was negatively correlated with ISI and HOMA-β, and positively correlated with TG, TC, LDL-C, FPG, HbA1c, FINS and HOMA-IR (P＜ 0.05). There was no correlation between the above indexes and HDL-C (P＞0.05). The combination of both serum Spexin and Pannexin1 predicted the development of cataracts in elderly diabetic patients was better than serum Spexin and Pannexin1 assessed individually (Z both combination-Spexin=3.220, P=0.001, Z both combination-Pannexin1=4.838, P＜0.001). Conclusion In elderly patients with diabetic cataract, the expression of serum Spexin decreases and the expression of Pannexin1 increases, and they have a certain correlation with glucose and lipid metabolism and insulin resistance. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM), along with ischemia, is one of the top ten causes of death in the global population, according to the latest World Health Organization (WHO) data. Clinical research data have revealed a high risk of stroke and heart attacks in patients with diabetes. However, there is still a lack of understanding of the involvement of pannexin 1 (Panx1) protein in cerebral ischemia combined with DM. In the presented study, we used the Panx1 gene knockout mice in models of streptozotocin-induced diabetes and photoinduced ischemia to investigate the effect of the Panx1 on the severity of ischemic brain damage and systemic inflammation in mice with a combination of cerebral ischemia and diabetes. It has been found that under conditions of experimental diabetes, the knockout of the Panx1 gene significantly reduces the size of the ischemic lesion, stabilizes the ischemia-induced increase in the blood–brain barrier permeability, reduces the number of errors in the sensorimotor test and the level of neutrophils in the blood. It is important to note that the Panx1 knockout exhibits a protective effect only in the presence of diabetes mellitus, without significantly affecting the severity of ischemic brain injury in mice without streptozotocin-induced diabetes. Panx1 knockout also did not affect the severity of hyperglycemia in animals in this diabetes model. It can be assumed that the efficacy of the treatment for pathologies combined with diabetes mellitus can be enhanced by incorporating pannexin channel blockers into the complex therapy, representing a novel approach to addressing these serious conditions. [ABSTRACT FROM AUTHOR]

Sepsis is a life-threatening organ dysfunction resulting from a dysregulated host response to infections that is initiated by the body’s innate immune system. Nearly a decade ago, we discovered that bacterial lipopolysaccharide (LPS) and serum amyloid A (SAA) upregulated Connexin 43 (Cx43) and Pannexin 1 (Panx1) hemichannels in macrophages. When overexpressed, these hemichannels contribute to sepsis pathogenesis by promoting ATP efflux, which intensifies the double-stranded RNA-activated protein kinase R (PKR)-dependent inflammasome activation, pyroptosis, and the release of pathogenic damage-associated molecular pattern (DAMP) molecules, such as HMGB1. Mimetic peptides targeting specific regions of Cx43 and Panx1 can distinctly modulate hemichannel activity in vitro, and diversely impact sepsis-induced lethality in vivo. Along with extensive supporting evidence from others, we now propose that hemichannel molecules play critical roles as endogenous regulators of innate immunity in sepsis.

Immunotherapies for malignant melanoma seek to boost the anti‐tumoral response of CD8+ T cells, but have a limited patient response rate, in part due to limited tumoral immune cell infiltration. Genetic or pharmacological inhibition of the pannexin 1 (PANX1) channel‐forming protein is known to decrease melanoma cell tumorigenic properties in vitro and ex vivo. Here, we crossed Panx1 knockout (Panx1−/−) mice with the inducible melanoma model BrafCA, PtenloxP, Tyr::CreERT2 (BPC). We found that deleting the Panx1 gene in mice does not reduce BRAF(V600E)/Pten‐driven primary tumor formation or improve survival. However, tumors in BPC‐Panx1−/− mice exhibited a significant increase in the infiltration of CD8+ T lymphocytes, with no changes in the expression of early T‐cell activation marker CD69, lymphocyte activation gene 3 protein (LAG‐3) checkpoint receptor, or programmed cell death ligand‐1 (PD‐L1) in tumors when compared to the BPC‐Panx1+/+ genotype. Our results suggest that, although Panx1 deletion does not overturn the aggressive BRAF/Pten‐driven melanoma progression in vivo, it does increase the infiltration of effector immune T‐cell populations in the tumor microenvironment. We propose that PANX1‐targeted therapy could be explored as a strategy to increase tumor‐infiltrating lymphocytes to boost anti‐tumor immunity.

Abstract Background Duchenne muscular dystrophy (DMD) is associated with impaired muscle regeneration, progressive muscle weakness, damage, and wasting. While the cause of DMD is an X-linked loss of function mutation in the gene encoding dystrophin, the exact mechanisms that perpetuate the disease progression are unknown. Our laboratory has demonstrated that pannexin 1 (Panx1 in rodents; PANX1 in humans) is critical for the development, strength, and regeneration of male skeletal muscle. In normal skeletal muscle, Panx1 is part of a multiprotein complex with dystrophin. We and others have previously shown that Panx1 levels and channel activity are dysregulated in various mouse models of DMD. Methods We utilized myoblast cell lines derived from DMD patients to assess PANX1 expression and function. To investigate how Panx1 dysregulation contributes to DMD, we generated a dystrophic (mdx) mouse model that lacks Panx1 (Panx1 −/− /mdx). In depth characterization of this model included histological analysis, as well as locomotor, and physiological tests such as muscle force and grip strength assessments. Results Here, we demonstrate that PANX1 levels and channel function are reduced in patient-derived DMD myoblast cell lines. Panx1 −/− /mdx mice have a significantly reduced lifespan, and decreased body weight due to lean mass loss. Their tibialis anterior were more affected than their soleus muscles and displayed reduced mass, myofiber loss, increased centrally nucleated myofibers, and a lower number of muscle stem cells compared to that of Panx1 +/+ /mdx mice. These detrimental effects were associated with muscle and locomotor functional impairments. In vitro, PANX1 overexpression in patient-derived DMD myoblasts improved their differentiation and fusion. Conclusions Collectively, our findings suggest that PANX1/Panx1 dysregulation in DMD exacerbates several aspects of the disease. Moreover, our results suggest a potential therapeutic benefit to increasing PANX1 levels in dystrophic muscles.

Pannexin 1 is a protein capable of forming channels for the release of ATP from animal cells. In small arteries, it regulates the contraction of smooth muscle cells due to a functional connection with α1D-adrenoceptors. Veins differ from arteries in structure and mechanisms of regulation of contraction, but the functions of pannexin in the venous bed have been poorly studied. The purpose of this study was to investigate the involvement of pannexin 1 in regulating the rhythmic contractile activity of the longitudinal smooth muscle layer in the mouse portal vein. The contractions of longitudinal vein specimens obtained from mice with global knockout of pannexin 1 gene and from С57Bl/6J mice (wild type) were studied in isometric mode. Venous specimens from both groups of mice demonstrated spontaneous rhythmic activity but the frequency of contractions in pannexin 1 knockout mice was higher than in wild-type mice. The α1-adrenoceptor agonists phenylephrine and methoxamine stimulated venous contractions. In the knockout group, the effect of phenylephrine, which has a higher affinity for α1D-adrenoceptors, was lower, while the effect of methoxamine did not differ between groups. Evoking an increase in the frequency of vein contractions, exogenous ATP also demonstrated a less pronounced effect in pannexin 1 knockout mice compared to wild-type mice. While the change in the frequency of vein contractions induced by phenylephrine and ATP (but not methoxamine) inversely correlated with the baseline frequency of contractions in wild-type mice, such dependence was not observed in knockout mice. In the presence of apyrase, which degrades extracellular ATP, the effect of phenylephrine on venous contraction frequency became less pronounced in wild-type mice but remained unchanged in knockout mice. Thus, pannexin does not directly participate in the generation of myogenic rhythmic activity in the mouse portal vein but may regulate it. The results suggest that, in the longitudinal smooth muscle layer of the mouse portal vein, pannexin 1 channels serve as the main pathway for ATP secretion and they are functionally associated with α1D-adrenоreceptors. [ABSTRACT FROM AUTHOR]

Background: Duchenne muscular dystrophy (DMD) is associated with impaired muscle regeneration, progressive muscle weakness, damage, and wasting. While the cause of DMD is an X-linked loss of function mutation in the gene encoding dystrophin, the exact mechanisms that perpetuate the disease progression are unknown. Our laboratory has demonstrated that pannexin 1 (Panx1 in rodents; PANX1 in humans) is critical for the development, strength, and regeneration of male skeletal muscle. In normal skeletal muscle, Panx1 is part of a multiprotein complex with dystrophin. We and others have previously shown that Panx1 levels and channel activity are dysregulated in various mouse models of DMD. Methods: We utilized myoblast cell lines derived from DMD patients to assess PANX1 expression and function. To investigate how Panx1 dysregulation contributes to DMD, we generated a dystrophic (mdx) mouse model that lacks Panx1 (Panx1−/−/mdx). In depth characterization of this model included histological analysis, as well as locomotor, and physiological tests such as muscle force and grip strength assessments. Results: Here, we demonstrate that PANX1 levels and channel function are reduced in patient-derived DMD myoblast cell lines. Panx1−/−/mdx mice have a significantly reduced lifespan, and decreased body weight due to lean mass loss. Their tibialis anterior were more affected than their soleus muscles and displayed reduced mass, myofiber loss, increased centrally nucleated myofibers, and a lower number of muscle stem cells compared to that of Panx1+/+/mdx mice. These detrimental effects were associated with muscle and locomotor functional impairments. In vitro, PANX1 overexpression in patient-derived DMD myoblasts improved their differentiation and fusion. Conclusions: Collectively, our findings suggest that PANX1/Panx1 dysregulation in DMD exacerbates several aspects of the disease. Moreover, our results suggest a potential therapeutic benefit to increasing PANX1 levels in dystrophic muscles. [ABSTRACT FROM AUTHOR]

Immunotherapies for malignant melanoma seek to boost the anti‐tumoral response of CD8+ T cells, but have a limited patient response rate, in part due to limited tumoral immune cell infiltration. Genetic or pharmacological inhibition of the pannexin 1 (PANX1) channel‐forming protein is known to decrease melanoma cell tumorigenic properties in vitro and ex vivo. Here, we crossed Panx1 knockout (Panx1−/−) mice with the inducible melanoma model BrafCA, PtenloxP, Tyr::CreERT2 (BPC). We found that deleting the Panx1 gene in mice does not reduce BRAF(V600E)/Pten‐driven primary tumor formation or improve survival. However, tumors in BPC‐Panx1−/− mice exhibited a significant increase in the infiltration of CD8+ T lymphocytes, with no changes in the expression of early T‐cell activation marker CD69, lymphocyte activation gene 3 protein (LAG‐3) checkpoint receptor, or programmed cell death ligand‐1 (PD‐L1) in tumors when compared to the BPC‐Panx1+/+ genotype. Our results suggest that, although Panx1 deletion does not overturn the aggressive BRAF/Pten‐driven melanoma progression in vivo, it does increase the infiltration of effector immune T‐cell populations in the tumor microenvironment. We propose that PANX1‐targeted therapy could be explored as a strategy to increase tumor‐infiltrating lymphocytes to boost anti‐tumor immunity. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above—which were used to identify endogenous PANX1 phosphorylation at these two sites—are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Brain neurotrophin (BDNF) is synthesized by proteolysis of proneurotrophin to form mature BDNF and the prodomain, whose regulatory activity on neuromuscular transmission is just beginning to be studied. At motor synapses, the BDNF prodomain has an inhibitory effect, stimulating GIRK potassium channels via activation of p75 receptors. The aim of this work was to study the initiation and implementation of the mechanism of inhibitory action of the BDNF prodomain in mature motor synapses of the mouse diaphragm. Microelectrodes were used to record spontaneous (miniature) and multiquantal endplate potentials evoked by stimulation of motor axons (MEPPs and EPPs, respectively). Using selective antagonists, it was revealed that the inhibitory effect of the BDNF prodomain on synaptic transmission requires the participation of sortilin, but not TrkB receptors. Stimulation of GIRK induced by the BDNF prodomain requires the participation of synaptic metabotropic receptors, which ensure the action of βγ-subunits of Gi proteins on GIRK. Using selective inhibitors, it was found that M2 cholinergic receptors and P2Y13 purinoceptors negatively regulate presynaptic L-type calcium channels, but these metabotropic receptors are not functionally related to the action of the BDNF prodomain. It turned out that the inhibition of quantal release of acetylcholine in motor synapses caused by BDNF prodomain requires the activity of the adenosine A1-receptors only. In addition, when pannexin 1 was pharmacologically blocked by probenecid, the BDNF prodomain lost its inhibitory effect on neuromuscular transmission. Thus, BDNF prodomain-induced inhibition of quantal neurotransmitter release in mouse motor synapses requires the participation of sortilin and endogenous activation of adenosine A1-receptors, which requires the functioning of pannexins 1, which most likely provide an additional source of synaptic ATP to the vesicular one. [ABSTRACT FROM AUTHOR]

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Although its pathogenesis is not fully understood, connexins (Cxs) and pannexins (Panx) could be involved in the process of fibrosis. We analyzed the protein expression of Cx37, Cx40, Cx43, and Panx1 in the gastric mucosa of patients with SSc and healthy volunteers, using immunofluorescence staining. Protein levels of Cx37 were slightly increased, while the levels of Cx40 were significantly decreased in the lamina propria of the gastric mucosa of SSc patients compared to the controls. The changes were proportional to SSc severity, with the most prominent changes found in patients with severe diffuse cutaneous SSc. No differences in Cx43 or Panx1 levels were found between the analyzed groups of samples. The lack of changes in Cx43 expression, which has been previously associated with fibrosis, could be due to the weak expression of Cx43 in the gastric mucosa in general. Further studies on full-thickness gastric biopsies containing muscle layers and animal SSc models are needed to fully elucidate the role of Cxs and Panxs in SSc-associated fibrosis. [ABSTRACT FROM AUTHOR]

Pannexin 1 （Panx1）, a member of the ubiquitin family, is widely expressed in mammalian tissues. When the body is in an inflammatory state, Panx1 channel is activated and opened by high concentration of ion stimulation, caspase shearing, tyrosine phosphorylation and mechanical stretching pathway, which allows intracellular ATP to be released outside the cell and aggravates inflammatory response. Panx1 is also involved in the occurrence of pyroptosis in inflammatory response, and activates and releases a large number of interleukin-1-related inflammatory factors. Inflammatory response is the body's defense response to infection, but overexpression of Panx1 leads to uncontrolled inflammatory response. Therefore, Panx1, as a new intervention target of inflammation, has certain research value and application prospect. [ABSTRACT FROM AUTHOR]

Abstract Background Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain. Methods A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake. Results CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (10Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression. Conclusion In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses.

Snake venom l-amino acid oxidases (svLAAOs) have been recognized as promising candidates for anticancer therapeutics. However, multiple aspects of their catalytic mechanism and the overall responses of cancer cells to these redox enzymes remain ambiguous. Here, we present an analysis of the phylogenetic relationships and active site-related residues among svLAAOs and reveal that the previously proposed critical catalytic residue His 223 is highly conserved in the viperid but not the elapid svLAAO clade. To gain further insight into the action mechanism of the elapid svLAAOs, we purify and characterize the structural, biochemical, and anticancer therapeutic potentials of the Thailand elapid snake Naja kaouthia LAAO (NK-LAAO). We find that NK-LAAO, with Ser 223, exhibits high catalytic activity toward hydrophobic l-amino acid substrates. Moreover, NK-LAAO induces substantial oxidative stress-mediated cytotoxicity with the magnitude relying on both the levels of extracellular hydrogen peroxide (H2O2) and intracellular reactive oxygen species (ROS) generated during the enzymatic redox reactions, but not being influenced by the N-linked glycans on its surface. Unexpectedly, we discover a tolerant mechanism deployed by cancer cells to dampen the anticancer activities of NK-LAAO. NK-LAAO treatment amplifies interleukin (IL)-6 expression via the pannexin 1 (Panx1)-directed intracellular calcium (iCa2+) signaling pathway to confer adaptive and aggressive phenotypes on cancer cells. Accordingly, IL-6 silencing renders cancer cells vulnerable to NK-LAAO-induced oxidative stress together with abrogating NK-LAAO-stimulated metastatic acquisition. Collectively, our study urges caution when using svLAAOs in cancer treatment and identifies the Panx1/iCa2+/IL-6 axis as a therapeutic target for improving the effectiveness of svLAAOs-based anticancer therapies.

The bladder urothelium releases ATP into the lamina propria (LP) during filling, which can activate P2X receptors on afferent neurons and trigger the micturition reflex. Effective ATP concentrations are largely dependent on metabolism by membrane-bound and soluble ectonucleotidases (s-ENTDs), and the latter are released in the LP in a mechanosensitive manner. Pannexin 1 (PANX1) channel and P2X7 receptor (P2X7R) participate in urothelial ATP release and are physically and functionally coupled, hence we investigated whether they modulate s-ENTDs release. Using ultrasensitive HPLC-FLD, we evaluated the degradation of 1,N6-etheno-ATP (eATP, substrate) to eADP, eAMP, and e-adenosine (e-ADO) in extraluminal solutions that were in contact with the LP of mouse detrusor-free bladders during filling prior to substrate addition, as an indirect measure of s-ENDTS release. Deletion of Panx1 increased the distention-induced, but not the spontaneous, release of s-ENTDs, whereas activation of P2X7R by BzATP or high concentration of ATP in WT bladders increased both. In Panx1−/− bladders or WT bladders treated with the PANX1 inhibitory peptide 10Panx, however, BzATP had no effect on s-ENTDS release, suggesting that P2X7R activity depends on PANX1 channel opening. We concluded, therefore, that P2X7R and PANX1 are in complex interaction to regulate s-ENTDs release and maintain suitable ATP concentrations in the LP. Thus, while stretch-activated PANX1 hinders s-ENTDS release possibly to preserve effective ATP concentration at the end of bladder filling, P2X7R activation, presumably in cystitis, would facilitate s-ENTDs-mediated ATP degradation to counteract excessive bladder excitability. [ABSTRACT FROM AUTHOR]

Probenecid is an old uricosuric agent used in clinics to treat gout and reduce the renal excretion of antibiotics. In recent years, probenecid has gained attention due to its ability to interact with membrane proteins such as TRPV2 channels, organic anion transporters, and pannexin 1 hemichannels, which suggests new potential therapeutic utilities in medicine. Some current functions of probenecid include their use as an adjuvant to increase the bioavailability of several drugs in the Central Nervous System (CNS). Numerous studies also suggest that this drug has important neuroprotective, antiepileptic, and anti-inflammatory properties, as evidenced by their effect against neurological and neurodegenerative diseases. In these studies, the use of probenecid as a Panx1 hemichannel blocker to reduce neuroinflammation is highlighted since neuroinflammation is a major trigger for diverse CNS alterations. Although the clinical use of probenecid has declined over the years, advances in its use in preclinical research indicate that it may be useful to improve conventional therapies in the psychiatric field where the drugs used have a low bioavailability, either because of a deficient passage through the blood–brain barrier or a high efflux from the CNS or also a high urinary clearance. This review summarizes the history, pharmacological properties, and recent research uses of probenecid and discusses its future projections as a potential pharmacological strategy to intervene in neurodegeneration as an outcome of neuroinflammation. [ABSTRACT FROM AUTHOR]

Objectives: The detailed pathological mechanism of orofacial neuropathic pain remains unknown. We aimed to examine the pannexin 1 (Panx1) signaling in the trigeminal ganglion (TG) involvement in infraorbital nerve injury (IONI)‐induced orofacial neuropathic pain. Materials and Methods: Mechanical head‐withdrawal threshold (MHWT) was measured in IONI‐treated rats receiving intra‐TG Panx1 inhibitor or metabotropic glutamate receptor 5 (mGluR5) antagonist administration and MHWTs in naive rats receiving intra‐TG mGluR5 agonist administration post‐IONI. Glutamate and Panx1 in the TG were measured post‐IONI. Panx1, mGluR5, and glutamine synthetase expression in TG were immunohistochemically identified, and changes in the number of mGluR5‐P2X3‐expressed TG neurons were examined. Results: MHWT was significantly decreased post‐IONI, and this decrease was reversed by Panx1 inhibition or mGluR5 antagonism. mGluR5 agonism induced a decrease in the MHWT. IONI increased extracellular glutamate in TG. Panx1 was expressed in satellite glial cells and TG neurons, and intra‐TG mGluR5 antagonism decreased the number of mGluR5 and P2X3 positive TG neurons post‐IONI. Conclusions: IONI facilitates glutamate release via Panx1 that activates mGluR5 which was expressed in the nociceptive TG neurons innervating the orofacial region. In turn, P2X3 receptor‐expressed TG neurons are enhanced via mGluR5 signaling, resulting in orofacial neuropathic pain. [ABSTRACT FROM AUTHOR]

Approximately 60% of patients with squamous cell carcinoma (LSCC) have regional occult metastatic disease/distant metastases at the time of diagnosis, putting them at higher risk for disease progression. Therefore, biomarkers are needed for early prognostic purpose. The aim of this study was to analyze the expression pattern of connexins (Cx) 37, 40 and 45, pannexin1 (Panx1) and vimentin in LSCC and correlate with tumor grade (G) and outcome. Methods: Thirty-four patients who underwent (hemi-)laryngectomy and regional lymphadenectomy due to LSCC from 2017 to 2018 in University Hospital Split, Croatia, were studied. Samples of tumor tissue and adjacent normal mucosa embedded in paraffin blocks were stained using the immunofluorescence method and were semi-quantitatively analyzed. Results: The expression of Cx37, Cx40, and Panx1 differed between cancer and adjacent normal mucosa and between histological grades, being the highest in well-differentiated (G1) cancer and low/absent in poorly differentiated (G3) cancer (all p < 0.05). The expression of vimentin was the highest in G3 cancer. Expression of Cx45 was generally weak/absent, with no significant difference between cancer and the controls or between grades. Lower Panx1 and higher vimentin expression were found to be prognostic factors for regional metastatic disease. Lower Cx37 and 40 expressions were present in patients with disease recurrence after the three-year follow-up period. Conclusion: Cx37 and Cx40, Panx1, and vimentin have the potential to be used as prognostic biomarkers for LSCC. [ABSTRACT FROM AUTHOR]

Static cold storage (SCS) is the standard technique for organ preservation during transplantation, resulting in cold ischemic injury. Hypoxia can induce pannexin 1 (Panx1) channels to open, leading to release of adenosine triphosphate. However, it is unknown if Panx1 plays a role in SCS. In this study, livers from Panx1 -/- mice exhibited reduced adenosine triphosphate release, resulting in hepatocyte protection during preservation. The donor liver damage was decreased during SCS when Panx1 activity was blocked. Transmission electron microscopy revealed a decrease in mitochondria-associated endoplasmic reticulum membranes and improved mitochondria morphology. Mechanistically, Panx1 blockade upregulated the phosphatidylinositol 3-kinase-protein kinase B pathway and increased B cell leukemia/lymphoma 2 levels to combat apoptosis during liver preservation. The data indicate that blocking Panx1 during preservation of the donor liver can effectively improve mitochondrial function and reduce cellular stress damage thereby decreasing cold ischemia and reperfusion-related injuries in liver transplantation., Competing Interests: Declaration of competing interest The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this study, we explored the intricate relationship between Pannexin 1 (PANX1) and the Hippo signaling pathway effector, Yes-associated protein (YAP). Analysis of The Cancer Genome Atlas (TCGA) data revealed a significant positive correlation between PANX1 mRNA and core Hippo components, Yes-associated protein 1 [YAP], Transcriptional coactivator with PDZ-binding motif [TAZ], and Hippo scaffold, Ras GTPase-activating-like protein IQGAP1 [IQGAP1], in invasive cutaneous melanoma and breast carcinoma. Furthermore, we demonstrated that PANX1 expression is upregulated in invasive melanoma cell lines and is associated with increased YAP protein levels. Notably, our investigations uncovered a previously unrecognized interaction between endogenous PANX1 and the Hippo scaffold protein IQGAP1 in melanoma cells. Moreover, our findings revealed that IQGAP1 exhibits differential expression in melanoma cells and plays a regulatory role in cellular morphology. Functional studies involving PANX1 knockdown provided compelling evidence that PANX1 modulates YAP protein levels and its cotranscriptional activity in melanoma and breast carcinoma cells. Importantly, our study highlights the potential therapeutic significance of targeting PANX1. Pharmacological inhibition of PANX1 using selective FDA-approved inhibitors or PANX1 knockdown reduced YAP levels in melanoma cells. Furthermore, our Clariom™ S analysis unveiled key genes implicated in cell proliferation, such as neuroglin1 (NRG1), β-galactoside binding protein and galectin-3 (LGALS3), that are affected in PANX1-deficient cells. In summary, our investigation delves into the intricate interplay between PANX1 and YAP in the context of invasive melanoma, offering valuable insights into potential therapeutic strategies for effective treatment., (© 2025 Federation of European Biochemical Societies.)

AIM: To evaluate plasma and aqueous levels of adiponutrin and pannexin 1 in patients with and without diabetic retinopathy. METHODS: The study included three age and gender-matched groups of 20 cataract patients with no diabetes or additional disease (Group C), 20 cataract patients with diabetes and no retinopathy (Group DM+C), and 20 cataract patients with diabetic retinopathy (Group DR+C). All the patients were examined with respect to body mass index (BMI), fasting plasma glucose, hemoglobin A1c (HbA1c), and lipid profile. Phacoemulsification and intraocular lens (Phaco+IOL) implantation were performed to all patients in all the groups, and aqueous samples were taken during the operation. The plasma and aqueous adiponutrin and pannexin 1 levels were analyzed using enzyme-linked immunosorbent assays. RESULTS: A statistically significant difference was determined between the groups with respect to BMI, fasting plasma glucose, and HbA1c levels (P

BackgroundAbdominal aortic aneurysms (AAA) are characterized by vascular inflammation and remodeling that can lead to aortic rupture resulting in significant mortality. Pannexin-1 channels on endothelial cells (ECs) can modulate ATP secretion to regulate the pathogenesis of AAA formation. Our hypothesis focused on potential of spironolactone to inhibit EC-mediated ATP release for the mitigation of AAA formation.MethodsA topical elastase AAA model was used initially in C57BL/6 (wild-type; WT) male mice. Mice were administered either a vehicle control (saline) or spironolactone and analyzed on day 14. In a second chronic AAA model, mice were subjected to elastase and β-aminopropionitrile (BAPN) treatment with/without administration of spironolactone to pre-formed aneurysms starting on day 14 and analyzed on day 28. Aortic diameter was evaluated by video micrometry and aortic tissue was analyzed for cytokine expression and histology. ATP measurement and matrix metalloproteinase (MMP2) activity was evaluated in aortic tissue on days 14 or -28. In vitro studies were performed to evaluate the crosstalk between aortic ECs with macrophages or smooth muscle cells.ResultsIn the elastase AAA model, spironolactone treatment displayed a significant decrease in aortic diameter compared to elastase-treated controls on day 14. A significant increase in smooth muscle α-actin expression as well as decrease in elastic fiber disruption and immune cell (macrophages and neutrophils) infiltration was observed in mice treated with spironolactone compared to saline-treated controls. Spironolactone treatment also significantly mitigated pro-inflammatory cytokine expression, MMP2 activity and ATP content in aortic tissue compared to controls. Moreover, in the chronic AAA model, spironolactone treatment of pre-formed aneurysms significantly attenuated vascular inflammation and remodeling to attenuate the progression of AAAs compared to controls. Mechanistically, in vitro data demonstrated that spironolactone treatment attenuates extracellular ATP release from endothelial cells to mitigate macrophage activation (IL-1β and HMGB1 expression) and smooth muscle cell-dependent vascular remodeling (MMP2 activity).ConclusionThese results demonstrate that spironolactone can mitigate aortic inflammation and remodeling to attenuate AAA formation as well as decrease growth of pre-formed aneurysms via inhibition of EC-dependent ATP release. Therefore, this study implicates a therapeutic application of spironolactone in the treatment of AAAs.

Enhanced activity and overexpression of Pannexin 1 (Panx1) channels contribute to neuronal pathologies such as epilepsy and Alzheimer's disease (AD). The Panx1 channel ablation alters the hippocampus's glutamatergic neurotransmission, synaptic plasticity, and memory flexibility. Nevertheless, Panx1-knockout (Panx1-KO) mice still retain the ability to learn, suggesting that compensatory mechanisms stabilize their neuronal activity. Here, we show that the absence of Panx1 in the adult brain promotes a series of structural and functional modifications in the Panx1-KO hippocampal synapses, preserving spontaneous activity. Compared to the wild-type (WT) condition, the adult hippocampal neurons of Panx1-KO mice exhibit enhanced excitability, a more complex dendritic branching, enhanced spine maturation, and an increased proportion of multiple synaptic contacts. These modifications seem to rely on the actin–cytoskeleton dynamics as an increase in the actin polymerization and an imbalance between the Rac1 and the RhoA GTPase activities were observed in Panx1-KO brain tissues. Our findings highlight a novel interaction between Panx1 channels, actin, and Rho GTPases, which appear to be relevant for synapse stability. [ABSTRACT FROM AUTHOR]

Background: Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain.Methods: A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake.Results: CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (10Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression.Conclusion: In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses. [ABSTRACT FROM AUTHOR]

The development and regeneration of skeletal muscle are mediated by satellite cells (SCs), which ensure the efficient formation of myofibers while repopulating the niche that allows muscle repair following injuries. Pannexin 1 (Panx1) channels are expressed in SCs and their levels increase during differentiation in vitro, as well as during skeletal muscle development and regeneration in vivo. Panx1 has recently been shown to regulate muscle regeneration by promoting bleb‐based myoblast migration and fusion. While skeletal muscle is largely influenced in a sex‐specific way, the sex‐dependent roles of Panx1 in regulating skeletal muscle and SC function remain to be investigated. Here, using global Panx1 knockout (KO) mice, we demonstrate that Panx1 loss reduces muscle fiber size and strength, decreases SC number, and alters early SC differentiation and myoblast fusion in male, but not in female mice. Interestingly, while both male and female Panx1 KO mice display an increase in the number of regenerating fibers following acute injury, the newly formed fibers in male Panx1 KO mice are smaller. Overall, our results demonstrate that Panx1 plays a significant role in regulating muscle development, regeneration, and SC number and function in male mice and reveal distinct sex‐dependent functions of Panx1 in skeletal muscle. [ABSTRACT FROM AUTHOR]

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Although its pathogenesis is not fully understood, connexins (Cxs) and pannexins (Panx) could be involved in the process of fibrosis. We analyzed the protein expression of Cx37, Cx40, Cx43, and Panx1 in the gastric mucosa of patients with SSc and healthy volunteers, using immunofluorescence staining. Protein levels of Cx37 were slightly increased, while the levels of Cx40 were significantly decreased in the lamina propria of the gastric mucosa of SSc patients compared to the controls. The changes were proportional to SSc severity, with the most prominent changes found in patients with severe diffuse cutaneous SSc. No differences in Cx43 or Panx1 levels were found between the analyzed groups of samples. The lack of changes in Cx43 expression, which has been previously associated with fibrosis, could be due to the weak expression of Cx43 in the gastric mucosa in general. Further studies on full-thickness gastric biopsies containing muscle layers and animal SSc models are needed to fully elucidate the role of Cxs and Panxs in SSc-associated fibrosis.

Introduction: Regular exercise training reduces arterial blood pressure, but the underlying mechanisms are unclear. Here, we evaluated the potential involvement of pannexin 1, an ATP releasing channel, in the blood pressure–reducing effect of training. Methods: Middle-age men, 13 normotensive and 14 nonmedicated stage 1 hypertensive, completed 8 wk of intensive aerobic cycle training. Before and after training, blood pressure and changes in leg vascular conductance, induced by femoral arterial infusion of tyramine (induces endogenous noradrenaline release), acetylcholine, or sodium nitroprusside, were measured during control conditions and after acute pannexin 1 inhibition by probenecid. A skeletal muscle biopsy was obtained from the thigh, pre- and posttraining. Results: Exercise training reduced mean systolic and diastolic blood pressure by ~5 (P = 0.013) and 5 mm Hg (P < 0.001), respectively, in the hypertensive group only. The reduction in blood pressure was not related to changes in pannexin 1 function because mean arterial blood pressure and tyramine-induced vasoconstriction remain unaltered by pannexin 1 inhibition after training in both groups. After training, pannexin 1 inhibition enhanced leg vascular conductance in the normo- and hypertensive groups at baseline (41.5%, P = 0.0036, and 37.7%, P = 0.024, respectively) and in response to sodium nitroprusside infusion (275%, P = 0.038, and 188%, P = 0.038, respectively). Training did not alter the pannexin 1 protein expression in skeletal muscle. Training enhanced the vasodilator response to acetylcholine infusion and increased the expression of microvascular function-relevant proteins. Conclusions: The exercise training-induced lowering of arterial blood pressure in nonmedicated hypertensive men does not involve an altered function of pannexin 1. [ABSTRACT FROM AUTHOR]

Aims Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia that diverts blood flow from poorly ventilated to better aerated lung areas to optimize ventilation-perfusion matching. Yet, the exact sensory and signalling mechanisms by which hypoxia triggers pulmonary vasoconstriction remain incompletely understood. Recently, ATP release via pannexin 1 (Panx1) and subsequent signalling via purinergic P2Y receptors has been identified as regulator of vasoconstriction in systemic arterioles. Here, we probed for the role of Panx1-mediated ATP release in HPV and chronic hypoxic pulmonary hypertension (PH). Methods and results Pharmacological inhibition of Panx1 by probenecid, spironolactone, the Panx1 specific inhibitory peptide (10Panx1), and genetic deletion of Panx1 specifically in smooth muscle attenuated HPV in isolated perfused mouse lungs. In pulmonary artery smooth muscle cells (PASMCs), both spironolactone and 10Panx1 attenuated the increase in intracellular Ca2+ concentration ([Ca2+]i) in response to hypoxia. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Unexpectedly, ATP release in response to hypoxia was not detectable in PASMC, and inhibition of purinergic receptors or ATP degradation by ATPase failed to attenuate HPV. Rather, transient receptor potential vanilloid 4 (TRPV4) antagonism and Panx1 inhibition inhibited the hypoxia-induced [Ca2+]i increase in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP-P2Y-TRPV4 pathway. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells. Conclusion In the present study, we identify Panx1 as novel regulator of HPV. Yet, the role of Panx1 in HPV was not attributable to ATP release and downstream signalling via P2Y receptors or TRPV4 activation, but relates to a role of Panx1 as direct or indirect modulator of the PASMC Ca2+ response to hypoxia. Panx1 did not affect the development of chronic hypoxic PH. [ABSTRACT FROM AUTHOR]

Pannexin 1 (Panx 1), as a large-pore membrane channel, is highly permeable to ATP and other signaling molecules. Previous studies have demonstrated the expression of Panx 1 in the nervous system, including astrocytes, microglia, and neurons. However, the distribution and function of Panx 1 in the peripheral nervous system are not clear. Blocking the function of Panx 1 pharmacologically (carbenoxolone and probenecid) or with small interfering RNA targeting pannexins can greatly reduce hypotonicity-induced ATP release. Treatment of Schwann cells with a Ras homolog family member (Rho) GTPase inhibitor and small interfering RNA targeting Rho or cytoskeleton disrupting agents, such as nocodazole or cytochalasin D, revealed that hypotonicity-induced ATP release depended on intracellular RhoA and the cytoskeleton. These findings suggest that Panx 1 participates in ATP release in Schwann cells by regulating RhoA and the cytoskeleton arrangement. This study was approved by the Animal Ethics Committee of Nantong University, China (No. S20180806-002) on August 5, 2018.

Pannexin 1 (Panx1) is involved in the spinal central sensitization process in rats with neuropathic pain, but its interaction with well-known, pain-related, ligand-dependent receptors, such as NMDA receptors (NMDAR) and P2X7 purinoceptors (P2X7R), remains largely unexplored. Here, we studied whether NMDAR- and P2X7R-dependent nociceptive signaling in neuropathic rats require the activation of Panx1 channels to generate spinal central sensitization, as assessed by behavioral (mechanical hyperalgesia) and electrophysiological (C-reflex wind-up potentiation) indexes. Administration of either a selective NMDAR agonist i.t. (NMDA, 2 mM) or a P2X7R agonist (BzATP, 150 μM) significantly increased both the mechanical hyperalgesia and the C-reflex wind-up potentiation, effects that were rapidly reversed (minutes) by i.t. administration of a selective pannexin 1 antagonist (10panx peptide, 300 μM), with the scores even reaching values of rats without neuropathy. Accordingly, 300 μM 10panx completely prevented the effects of NMDA and BzATP administered 1 h later, on mechanical hyperalgesia and C-reflex wind-up potentiation. Confocal immunofluorescence imaging revealed coexpression of Panx1 with NeuN protein in intrinsic dorsal horn neurons of neuropathic rats. The results indicate that both NMDAR- and P2X7R-mediated increases in mechanical hyperalgesia and C-reflex wind-up potentiation require neuronal Panx1 channel activation to initiate and maintain nociceptive signaling in neuropathic rats. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the effects of altered ganglioside composition on the expression of Cx37, Cx40, Cx43, Cx45, and Panx1 in different kidney regions of St8sia1 gene knockout mice (St8sia1 KO) lacking the GD3 synthase enzyme. Experiments were performed in twelve male 6-month-old mice: four wild-type (C57BL/6-type, WT) and eight St8sia1 KO mice. After euthanasia, kidney tissue was harvested, embedded in paraffin wax, and processed for immunohistochemistry. The expression of connexins and Panx1 was determined in different regions of the kidney: cortex (CTX.), outer stripe of outer medulla (O.S.), inner stripe of outer medulla (IN.S.), and inner medulla (IN.MED.). We determined significantly lower expression of Cx37, Cx40, Cx45, and Panx1 in different parts of the kidneys of St8sia1 KO mice compared with WT. The most consistent decrease was found in the O.S. where all markers (Cx 37, 40, 45 and Panx1) were disrupted in St8si1 KO mice. In the CTX. region, we observed decrease in the expression of Cx37, Cx45, and Panx1, while reduced expression of Cx37 and Panx1 was more specific to IN.S. The results of the present study suggest that deficiency of GD3 synthase in St8sia1 KO mice leads to disruption of renal Cx expression, which is probably related to alteration of ganglioside composition. [ABSTRACT FROM AUTHOR]

Purpose: Ferroptosis is associated with oxidative stress (OS) and is caused by iron-dependent lipid-peroxidative damage, but its role in PE is unclear. The aim of this study is to determine whether pannexin 1 (Panx1) and toll-like receptor 4 (TLR4) are key regulators of ferroptosis in PE. Methods: The study included 65 patients with PE and 25 healthy pregnant women. In normal and PE placental tissues, OS and ferroptosis markers, including Fe2+, malondialdehyde (MDA), reduced glutathione (GSH) levels, heme oxygenase-1 (HO-1) and glutathione peroxidase 4 (Gpx4) activity, were estimated. Panx1 and solute carrier family 7 member 11 (SLC7A11) mRNA expression levels were relatively quantified in placental tissues using real‐time polymerase chain reaction (RT‐PCR), while serum Panx1, serum TLR4, and placental activating transcription factor 3 (ATF3) levels were measured by ELISA. Results: In placental tissues, Panx1 and TLR4 expression levels were significantly increased in patients with PE compared to controls and were positively correlated with pro-ferroptosis mediators such as placental Fe2+ and MDA levels and negatively correlated with anti-ferroptosis regulators such as placental GSH level, HO-1, and Gpx4 activity. Additionally, Panx1 and TLR4 had a positive correlation with ATF3 and a negative correlation with SLC7A11. Serum Panx1 and TLR4 levels were positively correlated with their placental tissue expression and showed good diagnostic capabilities for ferroptosis in PE. Conclusion: Therefore, Panx1 and TLR4 are suggested to induce ferroptosis in PE via SLC7A11-mediated signaling pathways, offering a novel perspective on PE pathogenesis and novel diagnostic tools for PE. [ABSTRACT FROM AUTHOR]

Pannexins are transmembrane glycoproteins that constitute channels involved in purinergic signaling through ATP release from cells in various physiological and pathological processes. In this study, the distribution of Panx1 expression in different cell populations of healthy postnatal human kidneys and during human embryonic and early fetal development was investigated by double immunohistochemistry. In addition, the glomerular and tubular expression of Panx1 was examined in patients with type 2 diabetes mellitus (DM2) and the control group, and renal Panx1 expression was correlated with serum creatinine. In the 6th week of embryonic development (DW), Panx1 expression was found in mesonephric glomeruli and mesonephric tubules. At the transition from 6th to 7th DW, Panx1 immunoreactivity was found in the mesonephric tubules and mesonephric duct, as well as in the metanephric ureteric bud and ampullae. In the 7th DW, strong Panx1 immunoreactivity was observed in the developing ureteric bud in the metanephros, whereas no Panx1 immunoreactivity was found in the metanephric cup. In the 8th DW, Panx1 expression was also found in the ureteric bud of the metanephros, the renal vesicle and comma-shaped nephron, and the epithelial cells of Bowman's capsule. Expression of Panx1 was found at an early stage in both the paramesonephric duct and the mesonephric duct and diminished toward the 8th DW. During the 6th–10th DW, colocalization of Panx1 with alpha smooth actin (aSMA) was found in developing blood vessels. In the postnatal kidney, strong Panx1 immunoreactivity was present in medullary and cortical collecting duct cells, renin-producing cells, and proximal tubules. Very weak Panx1 immunoreactivity was found in certain distal tubule cells and the thin descending limbs of the loop of Henle. Panx1 immunoreactivity was also found in nephrin-immunoreactive podocytes. Panx1 was not colocalized with aSMA immunoreactivity in the vessels of the postnatal human kidney, but it was present in the endothelium. A significant positive correlation was found between Panx1 expression in glomeruli and serum creatinine only in diabetic patients and was not found in the nondiabetic group. The spatiotemporal expression of Panx1 during the early stages of human kidney development supports its possible role in cellular differentiation, migration, and positioning in the developing human kidney. In addition, our data suggest that glomerular Panx1 expression is a potential indicator of worsening renal function in patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

Probenecid is an old uricosuric agent used in clinics to treat gout and reduce the renal excretion of antibiotics. In recent years, probenecid has gained attention due to its ability to interact with membrane proteins such as TRPV2 channels, organic anion transporters, and pannexin 1 hemichannels, which suggests new potential therapeutic utilities in medicine. Some current functions of probenecid include their use as an adjuvant to increase the bioavailability of several drugs in the Central Nervous System (CNS). Numerous studies also suggest that this drug has important neuroprotective, antiepileptic, and anti-inflammatory properties, as evidenced by their effect against neurological and neurodegenerative diseases. In these studies, the use of probenecid as a Panx1 hemichannel blocker to reduce neuroinflammation is highlighted since neuroinflammation is a major trigger for diverse CNS alterations. Although the clinical use of probenecid has declined over the years, advances in its use in preclinical research indicate that it may be useful to improve conventional therapies in the psychiatric field where the drugs used have a low bioavailability, either because of a deficient passage through the blood–brain barrier or a high efflux from the CNS or also a high urinary clearance. This review summarizes the history, pharmacological properties, and recent research uses of probenecid and discusses its future projections as a potential pharmacological strategy to intervene in neurodegeneration as an outcome of neuroinflammation.

Pannexin 1 (Panx1) is a ubiquitously expressed protein forming large conductance channels that are central to many distinct inflammation and injury responses. There is accumulating evidence showing ATP released from Panx1 channels, as well as metabolites, provide effective paracrine and autocrine signaling molecules that regulate different elements of the injury response. As channels with a broad range of permselectivity, Panx1 channels mediate the secretion and uptake of multiple solutes, ranging from calcium to bacterial derived molecules. In this review, we describe how Panx1 functions in response to different pro-inflammatory stimuli, focusing mainly on signaling coordinated by the vasculature. How Panx1 mediates ATP release by injured cells is also discussed. The ability of Panx1 to serve as a central component of many diverse physiologic responses has proven to be critically dependent on the context of expression, post-translational modification, interacting partners, and the mode of stimulation. [ABSTRACT FROM AUTHOR]

Purinergic signaling mediated by ATP and its metabolites contributes to various brain physiological processes as well as to several pathological conditions, including neurodegenerative and neurological disorders, such as epilepsy. Among the different ATP release pathways, pannexin 1 channels represent one of the major conduits being primarily activated in pathological contexts. Investigations on in vitro and in vivo models of epileptiform activity and seizures in mice and human tissues revealed pannexin 1 involvement in aberrant network activity and epilepsy, and highlighted that pannexin 1 exerts a complex role. Pannexin 1 can indeed either sustain seizures through release of ATP that can directly activate purinergic receptors, or tune down epileptic activity via ATP-derived adenosine that decreases neuronal excitability. Interestingly, in-depth analysis of the literature unveils that this dichotomy is only apparent, as it depends on the model of seizure induction and the type of evoked epileptiform activity, two factors that can differentially activate pannexin 1 channels and trigger distinct intracellular signaling cascades. Here, we review the general properties and ATP permeability of pannexin 1 channels, and discuss their impact on acute epileptiform activity and chronic epilepsy according to the regime of activity and disease state. These data pave the way for the development of new antiepileptic strategies selectively targeting pannexin 1 channels in a context-dependent manner. [ABSTRACT FROM AUTHOR]

The effects of acute blockade of pannexin 1 (with the pharmacological agents probenecid, mefloquine, and 10Panx1) and the absence of its expression due to gene knockout on the release of acetylcholine (ACh) in the neuromuscular synapses of the mouse diaphragm were compared. Pannexin 1 forms a channel that can pass ATP into the synaptic cleft, thereby affecting neuromuscular transmission. Miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in neuromuscular preparations of wild-type mice under exposure to various pannexin 1 blockers and P2X7 receptor agonist (BzATP) were recorded. The obtained results were compared with those for pannexin 1 knockout mice. The absence of expression due to the genetic knockout or blockade of pannexin 1 under the action of probenecid or 10Panx1 in wild-type mice did not affect the release of ACh; however, it caused a potentiating effect on the release of ACh from P2X7 receptors when their agonist was applied, which did not occur without the blockade of pannexin 1. In both cases, the increase in ACh release was due to an increase in the size of the readily releasable pool of synaptic vesicles. On this basis, it was concluded that the lack of expression of the pannexin 1 gene caused by its knockout and acute blockade of pannexin 1 equally affected the secretion of ACh due to a decrease in the concentration of purines in the synaptic cleft; probably, the mechanisms of these effects were similar. Pannexin 1 was involved in them as an important participant in the purinergic regulation of the motor synapse: when it was blocked, the activation conditions of presynaptic purinoreceptors significantly changed. [ABSTRACT FROM AUTHOR]

Pannexin 1 (Panx1), an ATP-efflux pathway, has been linked with inflammation in pulmonary capillaries. However, the physiological roles of endothelial Panx1 in the pulmonary vasculature are unknown. Endothelial transient receptor potential vanilloid 4 (TRPV4) channels lower pulmonary artery (PA) contractility and exogenous ATP activates endothelial TRPV4 channels. We hypothesized that endothelial Panx1–ATP–TRPV4 channel signaling promotes vasodilation and lowers pulmonary arterial pressure (PAP). Endothelial, but not smooth muscle, knockout of Panx1 increased PA contractility and raised PAP in mice. Flow/shear stress increased ATP efflux through endothelial Panx1 in PAs. Panx1-effluxed extracellular ATP signaled through purinergic P2Y2 receptor (P2Y2R) to activate protein kinase Cα (PKCα), which in turn activated endothelial TRPV4 channels. Finally, caveolin-1 provided a signaling scaffold for endothelial Panx1, P2Y2R, PKCα, and TRPV4 channels in PAs, promoting their spatial proximity and enabling signaling interactions. These results indicate that endothelial Panx1–P2Y2R–TRPV4 channel signaling, facilitated by caveolin-1, reduces PA contractility and lowers PAP in mice.

Reactive astrogliosis is a key hallmark of inflammatory responses in the pathogenesis of brain injury, including Parkinson’s disease (PD), but its role and regulatory mechanisms are not fully understood. Pannexin 1 (Panx 1) is a membrane channel that mediates substance release in many neurodegenerative diseases. However, the role of astrocyte Panx 1 in the regulation of PD-like neuroinflammation remains elusive. Here, we characterized the expression of Panx 1 in isolated primary astrocytes and a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model. The functions of Panx 1 in inflammatory cytokines expression and the viability of neuronal SH-SY5Y cells were examined in cultured cells treated with lipopolysaccharide (LPS) and 1-methyl-4-phenylpyridinium (MPP+). We found that Panx 1 expression was significantly increased under both LPS- and MPP+-treated conditions. Panx 1 downregulation suppressed LPS-induced pro-inflammatory cytokine expression but did not significantly affect MPP+-induced astrocyte apoptosis or inflammatory cytokine expression through treatment with the Panx 1 inhibitor carbenoxolone (CBX) and Panx 1 siRNA. Moreover, silencing Panx 1 in reactive astrocytes had a potentially protective effect on the viability of neuronal SH-SY5Y cells. Therefore, we propose that Panx 1 may serve as a key regulator in reactive astrocytes to intervene in the inflammatory response and maintain neuronal viability in the context of PD-like conditions.

Reactive astrogliosis is a key hallmark of inflammatory responses in the pathogenesis of brain injury, including Parkinson's disease (PD), but its role and regulatory mechanisms are not fully understood. Pannexin 1 (Panx 1) is a membrane channel that mediates substance release in many neurodegenerative diseases. However, the role of astrocyte Panx 1 in the regulation of PD-like neuroinflammation remains elusive. Here, we characterized the expression of Panx 1 in isolated primary astrocytes and a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model. The functions of Panx 1 in inflammatory cytokines expression and the viability of neuronal SH-SY5Y cells were examined in cultured cells treated with lipopolysaccharide (LPS) and 1-methyl-4-phenylpyridinium (MPP+). We found that Panx 1 expression was significantly increased under both LPS- and MPP+-treated conditions. Panx 1 downregulation suppressed LPS-induced pro-inflammatory cytokine expression but did not significantly affect MPP+-induced astrocyte apoptosis or inflammatory cytokine expression through treatment with the Panx 1 inhibitor carbenoxolone (CBX) and Panx 1 siRNA. Moreover, silencing Panx 1 in reactive astrocytes had a potentially protective effect on the viability of neuronal SH-SY5Y cells. Therefore, we propose that Panx 1 may serve as a key regulator in reactive astrocytes to intervene in the inflammatory response and maintain neuronal viability in the context of PD-like conditions. [ABSTRACT FROM AUTHOR]

In this study, we explored the intricate relationship between Pannexin 1 (PANX1) and the Hippo signaling pathway effector, Yes-associated protein (YAP). Analysis of The Cancer Genome Atlas (TCGA) data revealed a significant positive correlation between PANX1 mRNA and core Hippo components, YAP, TAZ, and Hippo scaffold, IQGAP1, in invasive cutaneous melanoma and breast carcinoma. Furthermore, we demonstrated that PANX1 expression is upregulated in invasive melanoma cell lines and is associated with increased YAP protein levels. Notably, our investigations uncovered a previously unrecognized interaction between endogenous PANX1 and the Hippo scaffold protein IQGAP1 in melanoma cells. Moreover, our findings revealed that IQGAP1 exhibits differential expression in melanoma cells and plays a regulatory role in cellular morphology. Functional studies involving PANX1 knockdown provided compelling evidence that PANX1 modulates YAP protein levels and its co-transcriptional activity in both melanoma and breast carcinoma cells. Importantly, our study showcases the potential therapeutic relevance of targeting PANX1, as pharmacological inhibition of PANX1 using selective FDA-approved inhibitors or PANX1 knockdown reduced YAP abundance in melanoma cells. Furthermore, our Clariom ™ S analysis unveiled key genes implicated in cell proliferation, such as neuroglin1 (NRG1), β-galactoside binding protein, galectin-3 (LGALS3), that are affected in PANX1-deficient cells. In summary, our investigation delves into the intricate interplay between PANX1 and YAP in the context of invasive melanoma, offering valuable insights into potential therapeutic strategies for effective treatment., Competing Interests: Conflict of Interest Statement The authors declare no conflict of interest with this manuscript.

Pannexins are transmembrane glycoproteins that constitute channels involved in purinergic signaling through ATP release from cells in various physiological and pathological processes. In this study, the distribution of Panx1 expression in different cell populations of healthy postnatal human kidneys and during human embryonic and early fetal development was investigated by double immunohistochemistry. In addition, the glomerular and tubular expression of Panx1 was examined in patients with type 2 diabetes mellitus (DM2) and the control group, and renal Panx1 expression was correlated with serum creatinine. In the 6th week of embryonic development (DW), Panx1 expression was found in mesonephric glomeruli and mesonephric tubules. At the transition from 6th to 7th DW, Panx1 immunoreactivity was found in the mesonephric tubules and mesonephric duct, as well as in the metanephric ureteric bud and ampullae. In the 7th DW, strong Panx1 immunoreactivity was observed in the developing ureteric bud in the metanephros, whereas no Panx1 immunoreactivity was found in the metanephric cup. In the 8th DW, Panx1 expression was also found in the ureteric bud of the metanephros, the renal vesicle and comma-shaped nephron, and the epithelial cells of Bowman’s capsule. Expression of Panx1 was found at an early stage in both the paramesonephric duct and the mesonephric duct and diminished toward the 8th DW. During the 6th–10th DW, colocalization of Panx1 with alpha smooth actin (aSMA) was found in developing blood vessels. In the postnatal kidney, strong Panx1 immunoreactivity was present in medullary and cortical collecting duct cells, renin-producing cells, and proximal tubules. Very weak Panx1 immunoreactivity was found in certain distal tubule cells and the thin descending limbs of the loop of Henle. Panx1 immunoreactivity was also found in nephrin-immunoreactive podocytes. Panx1 was not colocalized with aSMA immunoreactivity in the vessels of the postnatal human kidney, but it was present in the endothelium. A significant positive correlation was found between Panx1 expression in glomeruli and serum creatinine only in diabetic patients and was not found in the nondiabetic group. The spatiotemporal expression of Panx1 during the early stages of human kidney development supports its possible role in cellular differentiation, migration, and positioning in the developing human kidney. In addition, our data suggest that glomerular Panx1 expression is a potential indicator of worsening renal function in patients with type 2 diabetes.

In mouse motor synapses tetanic neuromuscular activity (30 Hz, 2 min) led to a delayed posttetanic potentiation of amplitude and duration of spontaneous miniature endplate potentials (MEPPs). Microelectrode recordings of MEPPs before and after nerve stimulation showed an increase in MEPP amplitude and time course by 30% and 15%, respectively, without changes in their frequency. Peak effect was detected 20 min after tetanic activity and progressively faded throughout the next 40 min of recording. The revealed potentiation of MEPPs was fully preserved in preparations from pannexin 1 knockout mice. It means, that myogenic ATP released via pannexin 1 channels from contracting muscle fibers is not likely to participate in the described phenomenon. But posttetanic potentiation of MEPPs was fully prevented by competitive antagonist of calcitonin gene‐related peptide (CGRP) receptors CGRP8‐37, ryanodine receptors inhibitor ryanodine and by vesicular acetylcholine transporter inhibitor vesamicol. It is suggested that the combination of intensive synaptic and contractile activity in neuromuscular junctions is required to induce Ca2+‐dependent exocytosis of endogenous CGRP. The accumulation of CGRP in the synaptic cleft and its presynaptic activity may induce posttetanic potentiation of MEPP amplitude due to CGRP‐stimulated acetylcholine loading into vesicles and subsequent increase of quantal size. [ABSTRACT FROM AUTHOR]

Synaptic loss induced by soluble oligomeric forms of the amyloid β peptide (sAβos) is one of the earliest events in Alzheimer’s disease (AD) and is thought to be the major cause of the cognitive deficits. These abnormalities rely on defects in synaptic plasticity, a series of events manifested as activity-dependent modifications in synaptic structure and function. It has been reported that pannexin 1 (Panx1), a nonselective channel implicated in cell communication and intracellular signaling, modulates the induction of excitatory synaptic plasticity under physiological contexts and contributes to neuronal death under inflammatory conditions. Here, we decided to study the involvement of Panx1 in functional and structural defects observed in excitatory synapses of the amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic (Tg) mice, an animal model of AD. We found an age-dependent increase in the Panx1 expression that correlates with increased Aβ levels in hippocampal tissue from Tg mice. Congruently, we also observed an exacerbated Panx1 activity upon basal conditions and in response to glutamate receptor activation. The acute inhibition of Panx1 activity with the drug probenecid (PBN) did not change neurodegenerative parameters such as amyloid deposition or astrogliosis, but it significantly reduced excitatory synaptic defects in the AD model by normalizing long-term potentiation (LTP) and depression and improving dendritic arborization and spine density in hippocampal neurons of the Tg mice. These results suggest a major contribution of Panx1 in the early mechanisms leading to the synaptopathy in AD. Indeed, PBN induced a reduction in the activation of p38 mitogen-activated protein kinase (MAPK), a kinase widely implicated in the early neurotoxic signaling in AD. Our data strongly suggest that an enhanced expression and activation of Panx1 channels contribute to the Aβ-induced cascades leading to synaptic dysfunction in AD.

Synaptic loss induced by soluble oligomeric forms of the amyloid β peptide (sAβos) is one of the earliest events in Alzheimer's disease (AD) and is thought to be the major cause of the cognitive deficits. These abnormalities rely on defects in synaptic plasticity, a series of events manifested as activity-dependent modifications in synaptic structure and function. It has been reported that pannexin 1 (Panx1), a nonselective channel implicated in cell communication and intracellular signaling, modulates the induction of excitatory synaptic plasticity under physiological contexts and contributes to neuronal death under inflammatory conditions. Here, we decided to study the involvement of Panx1 in functional and structural defects observed in excitatory synapses of the amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic (Tg) mice, an animal model of AD. We found an age-dependent increase in the Panx1 expression that correlates with increased Aβ levels in hippocampal tissue from Tg mice. Congruently, we also observed an exacerbated Panx1 activity upon basal conditions and in response to glutamate receptor activation. The acute inhibition of Panx1 activity with the drug probenecid (PBN) did not change neurodegenerative parameters such as amyloid deposition or astrogliosis, but it significantly reduced excitatory synaptic defects in the AD model by normalizing long-term potentiation (LTP) and depression and improving dendritic arborization and spine density in hippocampal neurons of the Tg mice. These results suggest a major contribution of Panx1 in the early mechanisms leading to the synaptopathy in AD. Indeed, PBN induced a reduction in the activation of p38 mitogen-activated protein kinase (MAPK), a kinase widely implicated in the early neurotoxic signaling in AD. Our data strongly suggest that an enhanced expression and activation of Panx1 channels contribute to the Aβ-induced cascades leading to synaptic dysfunction in AD. [ABSTRACT FROM AUTHOR]