Differences in localization of P2X7 during epithelial wound healing in pre-type II diabetic models

Corneal injury, accompanied by improper wound repair, is the 4th highest cause of preventable blindness according to the World Health Organization. The cornea, which is the most densely innervated structure in the human body, serves to protect the delicate internal environment of the eye from damage. The integrity of this intricate nerve structure is critical in our ability to sense even the slightest insult to the corneal surface, with reduced sensitivity leading to increased susceptibility to trauma. In diabetes, decreased corneal sensitivity secondary to diabetic peripheral neuropathy can lead to increased corneal abrasion, ulceration, and even blindness. The P2X7 purinoreceptor is an ion channel that is expressed by the epithelium along with the intra-epithelial nerves and stromal nerves. The goal of our study was to use a type 2 diabetic mouse model (DIO) to characterize the changes in P2X7 localization and potentially correlate our results with changes in trafficking and sensory nerve loss. We hypothesized that the P2X7 receptor acts to sense changes at the leading edge and this fine tuned regulation is altered during the diabetic disease state. Further understanding of the corneal changes that occur in diabetes can help us better monitor progression of diabetic complications as well as develop new therapeutics for the treatment of diabetic corneal dysfunction

Deficiency of the purinergic receptor 2X7 attenuates nonalcoholic steatohepatitis induced by high-fat diet. possible role of the NLRP3 Inflammasome

Molecular mechanisms driving transition from simple steatosis to nonalcoholic steatohepatitis (NASH), a critical step in the progression of nonalcoholic fatty liver disease (NAFLD) to cirrhosis, are poorly defined. This study aimed at investigating the role of the purinergic receptor 2X7 (PR2X7), through the NLRP3 inflammasome, in the development of NASH. To this end, mice knockout for the Pr2x7 gene (Pr2x7 −/−) and coeval wild-type (WT) mice were fed a high-fat diet (HFD) or normal-fat diet for 16 weeks. NAFLD grade and stage were lower in Pr2x7 −/− than WT mice, and only 1/7 Pr2x7 −/− animals showed evidence of NASH, as compared with 4/7 WT mice. Molecular markers of inflammation, oxidative stress, and fibrosis were markedly increased in WT-HFD mice, whereas no or significantly reduced increments were detected in Pr2x7 −/− animals, which showed also decreased modulation of genes of lipid metabolism. Deletion of Pr2x7 gene was associated with blunted or abolished activation of NLRP3 inflammasome and expression of its components, which were induced in liver sinusoidal endothelial cells challenged with appropriate stimuli. These data show that Pr2x7 gene deletion protects mice from HFD-induced NASH, possibly through blunted activation of NLRP3 inflammasome, suggesting that PR2X7 and NLRP3 may represent novel therapeutic targets

Activation of Extracellular-signal Regulated Kinase (ERK1/2) by Fluid Shear is Ca\u3csup\u3e2+\u3c/sup\u3e- and ATP-dependent in MC3T3-E1 Osteoblasts

To determine the role of Ca2+ signaling in activation of the Mitogen-Activated Protein Kinase (MAPK) pathway, we subjected MC3T3-E1 pre-osteoblastic cells to inhibitors of Ca2+ signaling during application of fluid shear stress (FSS). FSS only activated ERK1/2, rapidly inducing phosphorylation within 5 min of the onset of shear. Phosphorylation of ERK1/2 (pERK1/2) was significantly reduced when Cai2+ was chelated with BAPTA or when Ca2+ was removed from the flow media. Inhibition of both the L-type voltage-sensitive Ca2+ channel and the mechanosensitive cation-selective channel blocked FSS-induced pERK1/2. Inhibition of phospholipase C with U73122 significantly reduced pERK1/2. This inhibition did not result from blockage of intracellular Ca2+ release, but a loss of PKC activation. Recent data suggests a role of ATP release and purinergic receptor activation in mechanotransduction. Apyrase-mediated hydrolysis of extracellular ATP completely blocked FSS-induced phosphorylation of ERK1/2, while the addition of exogenous ATP to static cells mimicked the effects of FSS on pERK1/2. Two P2 receptors, P2Y2 and P2X7, have been associated with the anabolic responses of bone to mechanical loading. Using both iRNA techniques and primary osteoblasts isolated from P2X7 knockout mice, we found that the P2X7, but not the P2Y2, purinergic receptor was involved in ERK1/2 activation under FSS. These data suggest that FSS-induced ERK1/2 phosphorylation requires Ca2+-dependent ATP release, however both increased Cai2+ and PKC activation are needed for complete activation. Further, this ATP-dependent ERK1/2 phosphorylation is mediated through P2X7, but not P2Y2, purinergic receptors

Pharmacological characterization of P2X1 and P2X3 purinergic receptors in bovine chondrocytes

SummaryObjectiveThe aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X1 and P2X3 purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE2) release.MethodsSaturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X1 and P2X3 purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X1 and P2X3 purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE2 release) the potency of purinergic agonists and antagonists was evaluated.ResultsBovine chondrocytes expressed P2X1 and P2X3 purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5′-triphosphate (ATP) and α,β-methyleneATP were able to increase NO and PGE2 release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists.ConclusionsThese data demonstrate for the first time the presence of functional P2X1 and P2X3 purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE2 release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

<p>Abstract</p> <p>Background</p> <p>Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation.</p> <p>Results</p> <p>Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells.</p> <p>Conclusion</p> <p>Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.</p

Phylogenomic analysis of metabotrophic P2Y receptor family and its expression in zebrafish, Danio rerio

G-protein-coupled receptors (GPCRs) constitute the largest and most divergent class of cell surface proteins. GPCRs can be activated by extracellular signals as diverse as light, peptides, proteins, lipids, odorants, tastants, nucleotides and nucleosides. Currently, all the known chemosensory receptor genes, such as odorant, taste and pheromone receptors belong to the GPCR family, with many of them being class A or class-A related genes. Metabotropic P2Y receptors belong to class A GPCRs and are activated by extracellular nucleotides. Nucleotides are released to the environment when the organism is injured and therefore serve as one of the food stimuli. As such, it would be interesting to see if P2Y receptors play any role in olfaction in zebrafish. To date, eight functional human P2Y receptors and 25 related orphan receptors have been found. I performed extensive data mining in drosophila, ascidian, jawless, cartilaginous and bony fish, frog and human genomes to delineate the P2Y family and investigate its evolutionary origin. The P2Y family originates early in the vertebrate lineage, reflected by the presence of lamprey and the absence of ascidian orthologues. Consistent with these findings, no P2Y receptor is found in invertebrates. In total, 38 subfamilies can be distinguished within the P2Y family, at least two third of which are already present in the shark genome. Two subfamilies, p2yl-3 and p2yl-4, are lost in the human lineage and only GPR 87 subfamily is lost in all teleost species. Zebrafish has 68 P2Y receptor genes, the most of any fish species, and almost double as many than mammals. The teleost P2Y genes are widely distributed in the genome as small cluster and singletons. The vast majority of P2Y genes are intronless while the remaining genes contain up to five introns. In the teleost lineage, the genomic arrangement of P2Y genes is preserved to a large extent, and some synteny is found even with the elephant shark and human genome, possibly reflecting the functional importance of these genes. Selective pressure on teleost P2Y genes generally is high, as evidenced by a preponderance of negative selection. However, a few genes exhibit positive selection at individual sites. In early development, P2Y genes are expressed in many tissues and organs, notably the central and peripheral nervous system, pharyngeal arches, otic vesicle and kidney, suggesting an important role in the development of many tissues. However, no expression is detected in larval olfactory epithelium. In contrast, olfactory epithelium of adult fish does express several P2Y genes as shown by RT-PCR. A possible explanation would be a late onset of expression in the olfactory epithelium. In situ hybridisation of adult olfactory epithelium established an ubiquitous distribution, both in the sensory and non-sensory region, which seems to argue against a role of P2Y genes in nucleotide odor detection. However, further studies will be necessary to give a definitive answer to that question

Characterization, localization and solubilization of P2x-purinoceptors

A radioligand binding assay was established to study the biochemical characteristics of P2x-purinoceptors. α,β-Methylene ATP was tritiated, and it was found that the binding of [3H]α,β-methylene ATP ([3H]α,β-MeATP) to rat bladder membrane preparations was rapid, fully reversible, and saturable. Competitive displacement showed that the potency order of the unlabelled purinergic ligands in displacing [3H]α,β-MeATP binding was: α,β-methylene ATP > β,Y-methylene ATP > suramin > ATP > 2-methylthio ATP > ADP >> adenosine, which indicates the binding sites are, or are related, to P2X-purinoceptors. Saturation assays revealed high- and low-affinity binding state, with Kd values of approximally 6 and 80 nM respectively. The optimal pH value of the binding was around 7, and the specific binding sites could be completely denatured by heat. The binding was greatly influenced by Na+, Ca2+, and Mg2+ ions in the media. Autoradiographic localization showed that the [3H]α,β-MeATP binding sites were distributed only over the smooth muscle cells of the rat urinary bladder, vas deferens, and the rabbit ear artery, which indicates that the binding is tissue-specific. The basic criteria for the recognition of a ligand binding site as a receptor or a subunit were fulfilled. Further competitive displacement experiments with other nucleotides, base and polyphosphate suggest that the critical structure for the interaction of the ligand with P2X- purinoceptor is the polytriphosphate moiety. A comparative study carried out on the urinary bladder and urethra of rat, guinea-pig and rabbit showed that rat bladder contained the highest density of [3H]α,β-MeATP binding sites, followed by rabbit and guinea-pig. Semi-quantitation of the autoradiograms revealed a similar order of binding site densities. The urethra of the rat and guinea-pig, a tissue where the P2X-purinoceptor-mediated responses have been shown to be absent, lacked specific labelling. In human urinary bladder, P2X-purinoceptor-mediated neurogenic contractile responses were reported to be absent or to exist only in some individuals. Both radioligand binding assay and autoradiographic localization showed that specific binding sites of [3H]α,β-MeATP were observed only in about 38 - 42% of the bladder detrusor of old male subjects, and the density was much lower than those in rat, guinea-pig and rabbit bladder. In many blood vessels extracellular ATP has been reported to elicit contractions of smooth muscle. Radioligand binding assays showed that rabbit ear artery contained a high density of high-affinity [3H]α,β-MeATP binding sites. Autoradiographic localization demonstrated specific binding sites of [3H]α,β-MeATP in many blood vessels from rat, guinea-pig and rabbit. Generally, medium- and small-sized muscular arteries, such as rat saphenous, rat tail, and rabbit ear arteries, contained higher densities of binding sites than large elastic arteries like aorta. In some large arteries such as rabbit carotid, renal and hepatic arteries, the binding sites were denser over the outer region of the vascular wall than the inner region. Only sparse specific binding was observed in all veins other than the portal vein from all the three species. In the portal veins, the densities over the circular and longitudinal muscles were different. The high-affinity component of the specific [3H]α,β-MeATP binding sites were successfully solubilized from rat vas deferens with 2% digitonin. The solubilized [3H]α,β-MeATP binding sites still possessed the characteristics of the membrane-bound binding sites, i.e., rapid association and dissociation, reversibility, and saturability. The potency order of the unlabelled ligands in displacing the [3H]α,β-MeATP binding was similar to that obtained from membrane binding experiments. Sucrose density gradient ultracentrifugation showed that the sedimentation coefficient of receptor-detergent complex was 12.1 S. Upon UV irradiation [3H]α,β-MeATP was cross-linked to a molecule of 62,000 daltons in rat vas deferens membrane, which might be the functional receptor or the binding subunit

New Aspects in the Differential Diagnosis and Therapy of Bladder Pain Syndrome/Interstitial Cystitis

Diagnosis of bladder pain syndrome/interstitial cystitis (BPS/IC) is presently based on mainly clinical symptoms. BPS/IC can be considered as a worst-case scenario of bladder overactivity of unknown origin, including bladder pain. Usually, patients are partially or completely resistant to anticholinergic therapy, and therapeutical options are especially restricted in case of BPS/IC. Therefore, early detection of patients prone to develop BPS/IC symptoms is essential for successful therapy. We propose extended diagnostics including molecular markers. Differential diagnosis should be based on three diagnostical “columns”: (i) clinical diagnostics, (ii) histopathology, and (iii) molecular diagnostics. Analysis of molecular alterations of receptor expression in detrusor smooth muscle cells and urothelial integrity is necessary to develop patient-tailored therapeutical concepts. Although more research is needed to elucidate the pathomechanisms involved, extended BPS/IC diagnostics could already be integrated into routine patient care, allowing evidence-based pharmacotherapy of patients with idiopathic bladder overactivity and BPS/IC

The role of the P2X7 receptor in injury-induced calcium dynamics and cell migration in the corneal epithelium

Wound healing in the corneal epithelium is an essential process to maintain corneal clarity and organism health. The earliest events of cellular injury response include the release of nucleotides and the activation of P2 purinergic receptors. While the purinergic receptor P2X7 has been shown to promote cell migration, its role in corneal epithelial wound healing is still poorly understood. The goal of this work is to better understand the role of P2X7 in the injury response. We analyzed P2X7 expression after epithelial injury in rat corneal organ cultures and found that the receptor localizes to the leading edge of the corneal epithelium. However, overall mRNA and protein expression of P2X7 decreased after injury. Inhibition of P2X7 activation significantly delayed wound closure and prevented the leading edge-localization after injury. We found that P2X7 inhibition altered the wound-induced calcium wave in epithelial cells and altered the number and distribution of focal adhesions in the migrating cells. Live cell imaging of epithelial cells showed that P2X7 inhibition led to altered actin rearrangement, with thick actin bundles in the treated cells. In order to determine the importance of P2X7 in epithelial differentiation and stratified cell migration, we developed a stratified culture model. The cells in the stratified model expressed proliferative and differentiation markers similar to organ cultured corneas, as well as similar P2X7 expression and localization after injury. Together, these results show the importance of P2X7 in the overall purinergic response to injury, and provide tools to study P2X7 in stratified corneal cell migration. To determine if P2X7 may contribute to pathologic delayed wound healing in diseases such as type 2 diabetes, we analyzed P2X7 expression in diabetic human corneas and diabetic model rodent corneas. We showed that P2X7 expression is significantly elevated in unwounded diabetic corneas, and that wound healing is delayed in the rodent model. These data show that elevated P2X7 expression may contribute to the delayed healing in disease and may be a possible therapeutic target