Panx1 Expression Research Articles

Promises and pitfalls of a Pannexin1 transgenic mouse line

Gene targeting strategies have become a powerful technology for elucidating mammalian gene function. The recently generated knockout (KO)-first strategy produces a KO at the RNA processing level and also allows for the generation of conditional KO alleles by combining FLP/FRT and Cre/loxP systems, thereby providing high flexibility in gene manipulation. However, this multipurpose KO-first cassette might produce hypomorphic rather than complete KOs if the RNA processing module is bypassed. Moreover, the generation of a conditional phenotype is also dependent on specific activity of Cre recombinase. Here, we report the use of an efficient molecular biological approach to test pannexin1 (Panx1) mRNA expression in global and conditional Panx1 KO mice derived from the KO-first mouse line, Panx1tm1a(KOMP)Wtsi. Using qRT-PCR, we demonstrate that tissues from wild-type (WT) mice show a range of Panx1 mRNA expression levels, with highest expression in trigeminal ganglia, bladder and spleen. Unexpectedly, we found that in mice homozygous for the KO-first allele, Panx1 mRNA expression is not abolished but reduced by 70% compared to that of WT tissues. Thus, Panx1 KO-first mice present a hypomorphic phenotype. Crosses of Panx1 KO-first with FLP deleter mice generated Panx1f/f mice. Further crosses of the latter mice with mGFAP-Cre or NFH-Cre mice were used to generate astrocyte- and neuron-specific Panx1 deletions, respectively. A high incidence of ectopic Cre expression was found in offspring of both types of conditional Panx1 KO mice. Our study demonstrates that Panx1 expression levels in the global and conditional Panx1 KO mice derived from KO-first mouse lines must be carefully characterized to ensure modulation of Panx1 gene expression. The precise quantitation of Panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of Panx1 under physiological and pathological conditions.

A Comparative Antibody Analysis of Pannexin1 Expression in Four Rat Brain Regions Reveals Varying Subcellular Localizations

Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus, and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on-line viewing, and download.

Panx1 regulates neural stem and progenitor cell behaviours associated with cytoskeletal dynamics and interacts with multiple cytoskeletal elements

BackgroundPannexins (Panxs) are relatively newly discovered large-pore ion and metabolite permeable channels. Although no proteomics-based interactome has yet been published, Panx1 has been demonstrated to interact with actin in an ectopic expression system. This interaction affects both Panx1 plasma membrane stability as well as cytoskeletal remodelling. The current study builds on our recent discovery of Panx1 expression in ventricular zone (VZ) neural stem and progenitor cells (NSC/NPCs), and on the demonstrated interaction of Panx1 with the cytoskeleton.FindingsHere we demonstrate that Panx1 also plays roles in two additional cell behaviours associated with neurogenesis, including cell migration and neurite extension. Furthermore, we confirm an endogenous interaction between actin and Panx1, and identify a new interaction with actin-related protein 3, an actin cytoskeleton-modulating protein.ConclusionsThis study further establishes the importance of Panx1 in the cell biology of NSC/NPCs and strengthens and expands our knowledge of Panx1 interactions with the cytoskeleton.

Pannexin1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated free fatty acids in liver cells

Hepatocyte lipoapoptosis induced by saturated free fatty acids (FFA) contributes to hepatic inflammation in lipotoxic liver injury, and the cellular mechanisms involved have not been defined. Recent studies have shown that apoptosis in nonhepatic cells stimulates ATP release via activation of pannexin1 (panx1), and extracellular ATP functions as a proinflammatory signal for recruitment and activation of the inflammatory cells. However, it is not known whether lipoapoptosis stimulates ATP release in liver cells. We found that lipoapoptosis induced by saturated FFA stimulated ATP release in liver cells that increased extracellular ATP concentration by more than fivefold above the values observed in healthy cells. This sustained pathophysiological ATP release was not dependent on caspase-3/7 activation. Inhibition of c-Jun NH(2)-terminal kinase (JNK), a key mediator of lipoapoptosis, with SP600125 blocked pathophysiological ATP release in a dose-dependent manner. RT-PCR analysis indicated that panx1 is expressed in hepatocytes and multiple liver cell lines. Notably, inhibition of panx1 expression with short hairpin (sh)RNA inhibited in part pathophysiological ATP release. Moreover, lipoapoptosis stimulated uptake of a membrane impermeable dye YoPro-1 (indicative of panx1 activation), which was inhibited by panx1 shRNA, probenecid, and mefloquine. These results suggest that panx1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated FFA. Thus panx1 may play an important role in hepatic inflammation by mediating an increase in extracellular ATP concentration in lipotoxic liver injury.

Intrarenal localization of the plasma membrane ATP channel pannexin1

In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1(-/-) mice. Urinary ATP levels were reduced by 30% in Panx1(-/-) compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions.

Patterns of heterogeneous expression of pannexin 1 and pannexin 2 transcripts in the olfactory epithelium and olfactory bulb

Pannexins form membrane channels that release biological signals to communicate with neighboring cells. Here, we report expression patterns of pannexin 1 (Panx1) and pannexin 2 (Panx2) in the olfactory epithelium and olfactory bulb of adult mice. In situ hybridization revealed that mRNAs for Panx1 and Panx2 were both expressed in the olfactory epithelium and olfactory bulb. Expression of Panx1 and Panx2 was mainly found in cell bodies below the sustentacular cell layer in the olfactory epithelium, indicating that Panx1 and Panx2 are expressed in mature and immature olfactory neurons, and basal cells. Expression of Panx2 was observed in sustentacular cells in a few locations of the olfactory epithelium. In the olfactory bulb, Panx1 and Panx2 were expressed in spatial patterns. Many mitral cells, tufted cells, periglomerular cells and granule cells were Panx1 and Panx2 positive. Mitral cells located at the dorsal and lateral portions of the olfactory bulb showed weak Panx1 expression compared with those in the medial side. However, the opposite was true for the distribution of Panx2 positive mitral cells. There were more Panx2 mRNA positive mitral cells and granule cells compared to those expressing Panx1. Our findings on pannexin expression in the olfactory system of adult mice raise the novel possibility that pannexins play a role in information processing in the olfactory system. Demonstration of expression patterns of pannexins in the olfactory system provides an anatomical basis for future functional studies.

Loss of Pannexin 1 Attenuates Melanoma Progression by Reversion to a Melanocytic Phenotype

Pannexin 1 (Panx1) is a channel-forming glycoprotein expressed in different cell types of mammalian skin. We examined the role of Panx1 in melanoma tumorigenesis and metastasis since qPCR and Western blots revealed that mouse melanocytes exhibited low levels of Panx1 while increased Panx1 expression was correlated with tumor cell aggressiveness in the isogenic melanoma cell lines (B16-F0, -F10, and -BL6). Panx1 shRNA knockdown (Panx1-KD) generated stable BL6 cell lines, with reduced dye uptake, that showed a marked increase in melanocyte-like cell characteristics including higher melanin production, decreased cell migration and enhanced formation of cellular projections. Western blotting and proteomic analyses using 2D-gel/mass spectroscopy identified vimentin and β-catenin as two of the markers of malignant melanoma that were down-regulated in Panx1-KD cells. Xenograft Panx1-KD cells grown within the chorioallantoic membrane of avian embryos developed tumors that were significantly smaller than controls. Mouse-Alu qPCR of the excised avian embryonic organs revealed that tumor metastasis to the liver was significantly reduced upon Panx1 knockdown. These data suggest that while Panx1 is present in skin melanocytes it is up-regulated during melanoma tumor progression, and tumorigenesis can be inhibited by the knockdown of Panx1 raising the possibility that Panx1 may be a viable target for the treatment of melanoma.

Probenecid Protects Vascular Contractile Of Thoracic Aorta In Rats Under Hypotonic Condition By Blocking Pannexin‐1

Probenecid is a drug proved to act on pannexin‐1(Panx1). This study investigated the involvement of probenecid in the vascular contractile, especially when Panx1 opens under hypotonicity. We first showed the expression of Panx1 on the aortic smooth muscle cell in rats via reverse transcription polymerase chain reaction (RT‐PCR) and immunohistochemistry (Fig. 1). Then the effect of hypotonic condition (201mOsm/L) on vascular contractile of male Sprague‐Dawley rat thoracic aorta with endothelia denuded was measured, with or without adding probenecid. Finally, after incubated in the hypotonic solution with or without probenecid, the efflux adenosine triphosphate (ATP) of smooth muscle was measured. We found that under hypotonic condition, vascular contractile was greatly impaired, while probenecid markedly protected contractility of vascular smooth muscle in hypotonic stress (Fig. 2, * p<0.05, ** p<0.01). And the protection is positively related to concentration of probenecid (Fig. 3, ** p<0.01). Measurement of ATP release showed great increase when aortic rings incubating in hypotonic solution for 15 minutes (Fig. 4, * p<0.05), which was eliminated if probenecid was added in the solution with concentration of 1mM. These results indicated that probenecid protected the vascular contractile, probably via inhibiting Panx‐1 and blocking the efflux of intracellular ATP. This work was supported by grants from the Department of Science and Technology of Zhejiang Province, China (2011C23105).

Pannexin 1 knockdown in metastatic melanoma cells induces cell differentiation into a melanocytic phenotype decreasing tumor size and metastasis in vivo

Pannexin 1 (Panx1) is a channel‐forming glycoprotein expressed in different cell types of mammalian skin. We studied the role of Panx1 in melanoma tumorigenesis and metastasis since the mouse melanocyte line CRL‐2770 showed low levels of Panx1 expression, while Panx1 in isogenic melanoma cell lines (B16‐F0, ‐F10 and ‐BL6) was increasingly up‐regulated according to qPCR and Western blot assays. Panx1 shRNA knockdown generated stable BL6 cell lines (Panx1‐KD) that showed a marked increase in melanocyte‐like cell characteristics, including higher melanin production, decreased cell migration and enhanced formation of cellular projections, while compromising Panx1 dye uptake channel function. Western blotting and proteomic analyses using 2D‐SDS‐PAGE gels/mass spectroscopy identified vimentin and beta‐catenin as some of the markers of malignant melanoma that were down‐regulated in Panx1‐KD cells. In vivo, Panx1‐KD cells were implanted on chorioallantoic membranes of day 10 avian embryos. After seven days, the excised Panx1‐KD tumors were significantly smaller than controls and metastasis to the liver was significantly reduced as quantified by mouse‐Alu qPCR of the excised embryonic organs. These data suggests that Panx1 may play a role in skin melanocytes that is potentially dysregulated upon malignant transformation in metastatic melanomas. Supported by the Canadian Institutes of Health Research.

Activation of neuronal P2X7 receptor–pannexin-1 mediates death of enteric neurons during colitis

Inflammatory bowel diseases (IBD) are chronic relapsing and remitting conditions associated with long-term gut dysfunction resulting from alterations to the enteric nervous system and a loss of enteric neurons1,2. The mechanisms underlying inflammation-induced enteric neuron death are unknown. Here we report using in vivo models of experimental colitis that inflammation causes enteric neuron death by activating a neuronal signaling complex comprised of P2X7 receptors (P2X7Rs), pannexin–1 (Panx1) channels, Asc and caspases. Inhibiting P2X7Rs, Panx1, Asc or caspase activity prevents inflammation-induced neuron cell death. Preservation of enteric neurons by inhibiting Panx1 in vivo prevented the onset of inflammation-induced colonic motor dysfunction. Panx1 expression is reduced in Crohn’s disease but not ulcerative colitis. We conclude that activation of neuronal Panx1 underlies neuron death and subsequent development of the abnormal gut motility in IBD. Targeting Panx1 represents a novel neuroprotective strategy to ameliorate the progression of IBD–associated dysmotility.

Thrombin-induced ATP release from human umbilical vein endothelial cells

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.

Channel activity of recombinant pannexin 1

Mammalian taste cells of the type II release ATP, an afferent neurotransmitter, by employing unselective ATP-permeable ion channels. The molecular identity of these channels is not known with confidence, although evidence implicates certain channel proteins from the connexin and pannexin families as most likely candidates. Here we carried out the comparative analysis of biophysical features and pharmacological profiles of unselective channels operative in type II cells and recombinant pannexin 1 (Panx1), which was cloned from the taste tissue and heterologously expressed in eukaryotic cells of several lines, including HEK-293, CHO, and neuroblastoma SK-N-SH. Integral currents mediated by Panx1 hemichannels were recorded to elucidate their kinetics characteristics, such as activation and deactivation, voltage dependence, and sensitivity to a variety of blockers, including carbenoxolone, DIDS, and NPPB. It was shown that the heterologous expression of Panx1 in cells of each type induced specific conductance, which exhibited outward rectification and was effectively blockable with carbenoxolone and anionic channel blockers DIDS and NPPB. Panx1 activity was studied at the single channel level as well. As was found, transfection of HEK-293 cells with the plasmid harboring cDNA encoding Panx1 gave rise to single channel current-like events in excised patches that were inhibited by 20 μM carbenoxolone, the relatively specific blocker of Panx1. These carbenoxolone-sensitive channels were peculiar in that single-channel current versus membrane voltage was not linear but exhibited outward rectification. In addition, the open-channel probability strongly increased with membrane voltage. Taken together, the data obtained here and earlier demonstrate clearly that by their biophysical and pharmacological features, ATP-permeable channels operative in type II cells are rather distinct from recombinant Panx1 hemichannels, thus arguing against Panx1 as the main conduit of ATP release in taste cells.

Human Platelets Express Functional Pannexin-1

Abstract 1132The protein family of pannexins (Panx) contains three members (Panx1, Panx2 and Panx3) that are highly evolutionarily conserved from worms to mammals, and that form hemichannels involved in the transport of ions like Ca2+ and small molecules such as ATP into and out of cells. Panx1 is ubiquitously expressed in human tissues, and has been shown to mediate ATP release in erythrocytes, taste buds, airway epithelia and astrocytes. In platelets, ATP serves as an important paracrine and autocrine signaling molecule, binding to P2X1 purinergic receptors on the platelet surface, regulating shape change, and amplifying platelet responses to agonists such as collagen. Based on the role of Panx and the significance of ATP release in platelets function, we investigate the expression and function of Panx1 in human platelets.Because the role of Panx1 in platelet function has not been studied previously, we sought to investigate the expression and function of Panx1 in human platelets. Materials and methods.Washed human platelets, isolated from ACD anticoagulated whole blood, were resuspended in Tyrode’s saline solution-HEPES to a final concentration of 2.5 × 108/mL, stimulated with collagen and collagen related peptide (CRP), and their activation and secretion measured by whole-blood lumi-aggregometry in the presence or absence of Panx1-specific channel blockers: carbenoxolone (Cbnx) 100 μM; probenecid (Pbn) 100 μM and mefloquine (Mfq) 10 μM. Expression of Panx1 was determined by western blot, flow cytometry and fluorescence microscopy using a specific rabbit polyclonal anti-Panx 1 antibody. Panx hemichannel function was assessed by sulforhodamine B dye uptake taken up by ADP or collagen-stimulated platelet in the presence or absence of known Panx1 channel blockers. Results.Western blots of platelet lysates with rabbit anti-Panx1 antibody revealed a 47 kDa band corresponding to the known molecular weight of the Panx1 monomer. Flow cytometry performed on permeabilized platelets showed a significant shift in fluorescence intensity in platelets incubated with the anti-Panx1 antibody. Strong, specific staining was also observed by fluorescent microscopy of permeabilized platelets incubated with the anti-Panx1 antibody. Both platelet shape change and ATP release induced by CRP (0.5μg/ml) or collagen (1.0 μg/ml) were inhibited more than 50 % by Cbnx and Pbn; however, the most significant effect was obtained with platelets exposed to mefloquine 10 μM (see table):Collagen 1.0 μg/mlpCRP 0.5 μg/mlpDrugVehicleDrugVehicleShape change (mm)2.3±0.97.5±1.40.0283.0±0.56.5±0.30.012ATP release (%)5.7±3.118.5±4.60.036.2±3.333.7±2.40.011Platelet stimulation with Collagen or ADP induced an increase in sulforhodamine B uptake which could be significantly inhibited by preincubation with the Panx1 channel blocker probenecid. Conclusions.Our results demonstrated that human platelets express Panx1 Taken together, these data demonstrate that Panx1 is expressed by human platelets, evidenced by the presence of the protein by western blot, flow cytometry and immunofluorescence microscopy. From a functional standpoint, the significant effect of carbenoxolone, probenecid and especially mefloquine on collagen and CRP-induced shape change and ATP release, suggest that in human platelets Panx1 is involved in ATP release. In other cell types Panx1 channels can be opened by activation of P2 receptors; however, further studies are required to elucidate a possible association and functional interaction of Panx1 with P2X1 in platelets. Since purinergic signaling is a fundamental mechanism in platelet activation, inhibition of Panx1 hemichannels could have therapeutic potential when dampening platelet activation is desired. Disclosures:No relevant conflicts of interest to declare.

The P2X7 Receptor and Pannexin-1 Are Both Required for the Promotion of Multinucleated Macrophages by the Inflammatory Cytokine GM-CSF

The P2X(7) receptor (P2X(7)R), an ATP-gated ion channel, has been implicated in the process of cell-to-cell fusion into multinucleated macrophages (MA), but its contribution to MA fusion driven by physiological/pathological stimuli is not clearly established. Based on several lines of evidence, we demonstrate that P2X(7)R is critical for the induction of multinucleated MA by the inflammatory cytokine GM-CSF: 1) pharmacological inhibition of P2X(7)R with oxidized ATP (oATP), KN-62, and the selective antagonist A740003 abrogated GM-CSF action on rat alveolar MA and murine peritoneal MA; 2) a murine J774 P2X(7) low MA clone, selected for defective P2X(7)R function, was unresponsive; 3) MA from mice lacking P2X(7)R failed to respond to GM-CSF, in contrast to wild-type. GM-CSF also stimulated ATP-induced membrane permeabilization in J774 P2X(7) high MA and rat alveolar MA, an effect absent in the P2X(7) low MA clone and inhibited by the P2X(7) blockers oATP and KN-62. Notably, the stimulatory effects of GM-CSF on pore formation and MA fusion were both inhibited by blocking functional Pannexin-1 (Panx-1), and GM-CSF failed to stimulate MA fusion in cells from Panx-1 knockout mice. We provide further evidence that extracellular ATP release from peritoneal MA is dependent on P2X(7) but not on Panx-1 expression and that its metabolism to adenosine mediates P2X(7)-dependent MA fusion. These data demonstrate that both P2X(7) and Panx-1 are required for GM-CSF promotion of MA fusion but likely act independently through different signaling pathway(s).

Characterization of novel Pannexin 1 isoforms from rat pituitary cells and their association with ATP-gated P2X channels

Our previous studies have showed that Pannexin 1 (Panx1), a member of a recently discovered family of gap junction proteins, is expressed in the pituitary gland. Here we investigated the presence and expression pattern of Panx1 isoforms in pituitary cells, their roles in ATP release, and their association with purinergic P2X receptor subtypes that are native to pituitary cells. In addition to the full-size Panx1, termed Panx1a, pituitary cells also express two novel shorter isoforms, termed Panx1c and Panx1d, which formation reflects the existence of alternative splicing sites in exons 2 and 4, respectively. Panx1c is lacking the Phe108-Gln180 sequence and P2X1d is missing the Val307-Cys426 C-terminal end sequence. Confocal microscopy and biotin labeling revealed that Panx1a is expressed in the plasma membrane, whereas Panx1c and Panx1d show the cytoplasmic localization when expressed as homomeric proteins. The three Panx1 isoforms and Panx2 form homomeric and heteromeric complexes in any combination. These splice forms can also physically associate with ATP-gated P2X2, P2X3, P2X4, and P2X7 receptor channels. The Panx1a-mediated ATP release in AtT-20 immortalized pituitary cells is attenuated when co-expressed with Panx1c or Panx1d. These results suggest that Panx1c and Panx1d may serve as dominant-negative effectors to modulate the functions of Panx1a through formation of heteromeric channels. The complex patterns of Panx1 expression and association could also define the P2X-dependent roles of these channels in cell types co-expressing both proteins.

Luminal ATP release is enhanced in multidrug resistant protein Mdr1a knockout mouse duodenum

Extracellular ATP release is an essential component of the luminal purinergic system, which along with P2Y receptors and alkaline phosphatase (AP) helps regulate duodenal bicarbonate secretion (DBS). Since ATP release is reduced by CFTR inhibition or genetic deletion of murine intestinal AP (with downregulation of the multidrug resistant proteins Mdr1a and Mrp2), we hypothesized that Mdr1a knockout (KO) decreases ATP release. We measured DBS and ATP output in a perfused duodenal loop in male wild type (WT) or MDR1a KO mice, or in rats. The duodenal loop was perfused with the competitive AP substrate glycerol phosphate (GP) with or without the P2 receptor antagonist/ENTPDase inhibitor suramin, the MRP inhibitor MK571 or pannexin (Panx) inhibitor. AP inhibition with GP increased DBS and non-lytic ATP output in WT mouse and rat duodena. Co-perfusion of suramin inhibited GP-induced DBS, but enhanced ATP output. In KO duodenum, GP or suramin enhanced ATP output without parallel LDH release, inhibited by CFTRinh-172. Expression of Panx1 and MRP1 was selectively upregulated in KO duodenum. Luminal perfusion of MK571 or Panx inhibitor reduced GP-induced ATP release in rat duodenum. MDR1a, CFTR and Panx1 may coordinately contribute to duodenal epithelial ATP release. Enhanced ATP release may account for the induction of diarrhea followed by colitis as part of the Mdr1a KO intestinal phenotype. NIH-NIDDK R01 DK54221

Neuroinflammation Leads to Region-Dependent Alterations in Astrocyte Gap Junction Communication and Hemichannel Activity

Inflammation attenuates gap junction (GJ) communication in cultured astrocytes. Here we used a well-characterized model of experimental brain abscess as a tool to query effects of the CNS inflammatory milieu on astrocyte GJ communication and electrophysiological properties. Whole-cell patch-clamp recordings were performed on green fluorescent protein (GFP)-positive astrocytes in acute brain slices from glial fibrillary acidic protein-GFP mice at 3 or 7 d after Staphylococcus aureus infection in the striatum. Astrocyte GJ communication was significantly attenuated in regions immediately surrounding the abscess margins and progressively increased to levels typical of uninfected brain with increasing distance from the abscess proper. Conversely, astrocytes bordering the abscess demonstrated hemichannel activity as evident by enhanced ethidium bromide (EtBr) uptake that could be blocked by several pharmacological inhibitors, including the connexin 43 (Cx43) mimetic peptide Gap26, carbenoxolone, the pannexin1 (Panx1) mimetic peptide (10)Panx1, and probenecid. However, hemichannel opening was transient with astrocytic EtBr uptake observed near the abscess at day 3 but not day 7 after infection. The region-dependent pattern of hemichannel activity at day 3 directly correlated with increases in Cx43, Cx30, Panx1, and glutamate transporter expression (glial L-glutamate transporter and L-glutamate/L-aspartate transporter) along the abscess margins. Changes in astrocyte resting membrane potential and input conductance correlated with the observed changes in GJ communication and hemichannel activity. Collectively, these findings indicate that astrocyte coupling and electrical properties are most dramatically affected near the primary inflammatory site and reveal an opposing relationship between the open states of GJ channels versus hemichannels during acute infection. This relationship may extend to other CNS diseases typified with an inflammatory component.

Pannexin 1 Constitutes the Large Conductance Cation Channel of Cardiac Myocytes

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

Implications of pannexin 1 and pannexin 3 for keratinocyte differentiation

Pannexin (Panx) 1 and Panx3 are integral membrane proteins that share some sequence homology with the innexin family of invertebrate gap junctions. They are expressed in mammalian skin. Pannexins have been reported to form functional mechanosensitive single-membrane channels, but their importance in regulating cellular function is poorly understood. In this study, Panx1 and Panx3 were detected in the epidermis of 13.5 day embryonic mice. Compared with newborn mice, there was less Panx1 expression in both thin and thick murine skin, whereas Panx3 expression was unchanged. To investigate the role of pannexins in keratinocyte differentiation, we employed rat epidermal keratinocytes (REKs) that have the capacity to differentiate into organotypic epidermis, and engineered them to overexpress Panx1, Panx1-GFP or Panx3. The expression of Panx1 or Panx3 resulted in the increased ability of REKs to take up dye, suggesting that cell-surface channels were formed. Compared with monolayer REKs, endogenous Panx1 levels remained unchanged, whereas the 70 kDa immunoreactive species of Panx3 was greatly increased in the organotypic epidermis. In monolayer cultures, ectopic Panx1 and Panx1-GFP localized to the plasma membrane, whereas Panx3 displayed both intracellular and plasma-membrane profiles. Although both pannexins reduced cell proliferation, only Panx1 disrupted the architecture of the organotypic epidermis and markedly dysregulated cytokeratin 14 expression and localization. Furthermore, ectopic expression of only Panx1 reduced the vital layer thickness of the organotypic epidermis. In summary, Panx1 and Panx3 are coexpressed in the mammalian epidermis, and the regulation of Panx1 plays a key role in keratinocyte differentiation.

Pannexin 1 Contributes to ATP Release in Airway Epithelia

ATP is a paracrine regulator of critical airway epithelial cell functions, but the mechanism of its release is poorly understood. Pannexin (Panx) proteins, related to invertebrate innexins, form channels (called pannexons) that are able to release ATP from several cell types. Thus, ATP release via pannexons was examined in airway epithelial cells. Quantitative RT-PCR showed Panx1 expression in normal human airway epithelial cells during redifferentiation at the air-liquid interface (ALI), at a level comparable to that of alveolar macrophages; Panx3 was not expressed. Immunohistochemistry showed Panx1 expression at the apical pole of airway epithelia. ALI cultures exposed to hypotonic stress released ATP to an estimated maximum of 255 (+/-64) nM within 1 minute after challenge (n = 6 cultures from three different lungs) or to approximately 1.5 (+/-0.4) microM, recalculated to a normal airway surface liquid volume. Using date- and culture-matched cells (each n > or = 16 from 4 different lungs), the pannexon inhibitors carbenoxolone (10 microM) and probenecid (1 mM), but not the connexon inhibitor flufenamic acid (100 microM), inhibited ATP release by approximately 60%. The drugs affected Panx1 currents in Xenopus oocytes expressing exogenous Panx1 correspondingly. In addition, suppression of Panx1 expression using lentivirus-mediated production of shRNA in differentiated airway epithelial cells inhibited ATP release upon hypotonic stress by approximately 60% as well. These data not only show that Panx1 is expressed apically in differentiated airway epithelial cells but also that it contributes to ATP release in these cells.