Targeting P2RX1 alleviates renal ischemia/reperfusion injury by preserving mitochondrial dynamics.

Renal ischemia/reperfusion injury (IRI) is the major cause of acute kidney injury. However, mechanisms underlying the sudden loss in kidney function and tissue injury remain to be fully elucidated. Here, we performed RNA sequencing to systematically compare the transcriptome differences between IR injured kidneys and sham kidneys. We observed that mitochondrial dynamics was destructed in renal IRI. Expression of mitochondrial fusion-associated genes was reduced, whereas expression of mitochondrial fission-related genes was increased in renal IRI, and these findings were further confirmed by mitochondrial morphological observations. By screening 19 purinergic receptors, we noticed that P2RX1 expression was markedly upregulated in renal IRI. RNA sequencing and mitochondrial morphological observations revealed that mitochondrial dynamics was preserved in P2RX1 genetic knockout (P2rx1-/-) mice. Neutrophil extracellular traps (NETs) were reported to be essential for tissue injury in renal IRI, but the detailed mechanism remained unclear. In the present study, we found that P2RX1 favored the formation of neutrophil extracellular traps (NETs) in IRI, and NETs was essential for the impairment of mitochondrial dynamics. Mechanistically, P2RX1-involved metabolic interaction between platelets and neutrophils supported NETs formation. Activation of P2RX1 promoted platelets ATP release, which subsequently contributed to neutrophil glycolytic metabolism and NETs generation.

Purinergic P2X1 receptor, purinergic P2X7 receptor, and angiotensin II type 1 receptor interactions in the regulation of renal afferent arterioles in angiotensin II-dependent hypertension.

In ANG II-dependent hypertension, ANG II activates ANG II type 1 receptors (AT1Rs), elevating blood pressure and increasing renal afferent arteriolar resistance (AAR). The increased arterial pressure augments interstitial ATP concentrations activating purinergic P2X receptors (P2XRs) also increasing AAR. Interestingly, P2X1R and P2X7R inhibition reduces AAR to the normal range, raising the conundrum regarding the apparent disappearance of AT1R influence. To evaluate the interactions between P2XRs and AT1Rs in mediating the increased AAR elicited by chronic ANG II infusions, experiments using the isolated blood perfused juxtamedullary nephron preparation allowed visualization of afferent arteriolar diameters (AAD). Normotensive and ANG II-infused hypertensive rats showed AAD responses to increases in renal perfusion pressure from 100 to 140 mmHg by decreasing AAD by 26 ± 10% and 19 ± 4%. Superfusion with the inhibitor P2X1Ri (NF4490; 1 µM) increased AAD. In normotensive kidneys, superfusion with ANG II (1 nM) decreased AAD by 16 ± 4% and decreased further by 19 ± 5% with an increase in renal perfusion pressure. Treatment with P2X1Ri increased AAD by 30 ± 6% to values higher than those at 100 mmHg plus ANG II. In hypertensive kidneys, the inhibitor AT1Ri (SML1394; 1 µM) increased AAD by 10 ± 7%. In contrast, treatment with P2X1Ri increased AAD by 21 ± 14%; combination with P2X1Ri plus P2X7Ri (A438079; 1 µM) increased AAD further by 25 ± 8%. The results indicate that P2X1R, P2X7R, and AT1R actions converge at receptor or postreceptor signaling pathways, but P2XR exerts a dominant influence abrogating the actions of AT1Rs on AAR in ANG II-dependent hypertension.

Identification of aurintricarboxylic acid as a potent allosteric antagonist of P2X1 and P2X3 receptors.

The homotrimeric P2X3 receptor, one of the seven members of the ATP-gated P2X receptor family, plays a crucial role in sensory neurotransmission. P2X3 receptor antagonists have been identified as promising drugs to treat chronic cough and are suggested to offer pain relief in chronic pain such as neuropathic pain. Here, we analysed whether compounds affect P2X3 receptor activity by high-throughput screening of the Spectrum Collection of 2000 approved drugs, natural products and bioactive substances. We identified aurintricarboxylic acid (ATA) as a nanomolar-potency antagonist of P2X3 receptor-mediated responses. Two-electrode voltage clamp electrophysiology-based concentration-response analysis and selectivity profiling revealed that ATA strongly inhibits the rP2X1 and rP2X3 receptors (with IC50 values of 8.6 nM and 72.9 nM, respectively) and more weakly inhibits P2X2/3, P2X2, P2X4 or P2X7 receptors (IC50 values of 0.76 µM, 22 µM, 763 µM or 118 µM, respectively). Patch-clamp analysis of mouse DRG neurons revealed that ATA inhibited native P2X3 and P2X2/3 receptors to a similar extent than rat P2X3 and P2X2/3 receptors expressed in Xenopus oocytes. In a radioligand binding assay, up to 30 µM ATA did not compete with [3H]-ATP for rP2X3 receptor binding, indicating a non-competitive mechanism of action. Molecular docking studies, site-directed mutagenesis and concentration-response analysis revealed that ATA binds to the negative allosteric site of the hP2X3 receptor. In summary, ATA as a drug-like pharmacological tool compound is a nanomolar-potency, allosteric antagonist with selectivity towards αß-methylene-ATP-sensitive P2X1 and P2X3 receptors.

A challenge finding P2X1 and P2X4 ligands.

Identifying novel small-molecule P2X1 and P2X4 ligands with sub-type specificity and high-affinity remains a pharmacological challenge. Here we use computational methods, electrophysiology and fluorescent microplate assays to screen for ligand candidates acting at these receptors. Modelling and docking identified 80 compounds for testing at P2X4 receptors, and 20 of these showed >50% inhibition in fluorescence-based assays, making them appealing for further SAR studies. Confirmation of activity by two-electrode voltage clamp, followed by their elaboration resulted in only minor improvements in potency, with the highest IC50 being 295 µM. Testing on P2X1 receptors, resulted in a series of biguanide compounds that yielded a maximum IC50 of 100 µM, but no consistent SAR could be found. Potencies of established antagonists gave expected results, although the measured potencies varied between techniques and no antagonism could be found for compounds such as paroxetine, carbamazepine, 9(10H)-acridanone, acridinol and phenoxazine-type heterocycles. This study highlights the challenge of identifying P2X4 and P2X1 ligands and suggests that a combination of complimentary approaches is needed if we are to be confident of ligand activities at these receptors.

Physiopathological implications of P2X1 and P2X7 receptors in regulation of glomerular hemodynamics in angiotensin II-induced hypertension.

Deleterious effects of purinergic P2X1 and P2X7 receptors (P2XRs) in ANG II-dependent hypertension include increased renal vascular resistance, and impaired autoregulation and pressure natriuresis. However, their specific effects on the determinants of glomerular hemodynamics remain incompletely delineated. To investigate the P2XR contributions to altered glomerular hemodynamics in hypertension, the effects of acute blockade of P2X1R, P2X7R, and P2X4R with NF449, A438079, and PSB12054, respectively, were evaluated in ANG II-infused rats (435 ng·kg-1·min-1). P2X1R or P2X7R blockade reduced afferent (6.85 ± 1.05 vs. 2.37 ± 0.20 dyn·s-1·cm-5) and efferent (2.85 ± 0.38 vs. 0.99 ± 0.07 dyn·s-1·cm-5) arteriolar resistances, leading to increases in glomerular plasma flow (75.82 ± 5.58 vs. 206.7 ± 16.38 nl/min), ultrafiltration coefficient (0.0198 ± 0.0024 vs. 0.0512 ± 0.0046 nl·min-1·mmHg-1), and single-nephron glomerular filtration rate (22.73 ± 2.02 vs. 51.56 ± 3.87 nl/min) to near normal values. Blockade of P2X4R did not elicit effects in hypertensive rats. In normotensive sham-operated rats, only the P2X1R antagonist caused an increase plasma flow and single-nephron glomerular filtration rate, whereas the P2X4R antagonist induced glomerular vasoconstriction that was consistent with evidence that P2X4R stimulation increases release of nitric oxide from endothelial cells. Mean arterial pressure remained unchanged in both hypertensive and normotensive groups. Western blot analysis showed overexpression of P2X1R, P2X7R, and P2X4R proteins in hypertensive rats. Whereas it has been generally assumed that the altered glomerular vascular resistances in ANG II hypertension are due to AT1 receptor-mediated vasoconstriction, these data indicate a predominant P2X1R and P2X7R control of glomerular hemodynamics in ANG II hypertension.

Impaired Aortic Contractility to Uridine Adenosine Tetraphosphate in Angiotensin II-Induced Hypertensive Mice: Receptor Desensitization?

OBJECTIVE: We previously showed that uridine adenosine tetraphosphate (Up4A)-mediated aortic contraction is partly mediated through purinergic P2X1 receptors (P2X1R). It has been reported that the plasma level of Up4A is elevated in hypertensive patients, implying a potential role for Up4A-P2X1R signaling in hypertension. This study investigated the vasoactive effect of Up4A in aortas isolated from angiotensin (Ang) II-infused (21 days) hypertensive mice. METHODS: Blood pressure was measured by tail cuff plethysmography. Aortas were isolated for isometric tension measurements, and protein expression was analyzed by western blot. RESULTS: Mean and systolic arterial pressures were elevated by ~50% in Ang II-infused mice. Protein levels of both AT1R and P2X1R were upregulated in Ang II-infused aortas. Surprisingly, Up4A (10-9-10-5 M)-induced concentration-dependent contraction was significantly impaired in Ang II-infused mice. Studies in control mice revealed that both P2X1R (MRS2159) and AT1R (losartan) antagonists significantly attenuated Up4A-induced aortic contraction. In addition, desensitization of AT1R by prior Ang II (100 nM) exposure had no effect on Up4A-induced aortic contraction. However, subsequent serial exposure responses to Up4A-induced aortic contraction were markedly reduced, suggesting a desensitization of purinergic receptors. This desensitization was further confirmed in control mice by prior exposure of aortas to the P2X1R desensitizer α, ß-methylene ATP (10 µM). CONCLUSION: Despite upregulation of AT1R and P2X1R in hypertension, Up4A-mediated aortic contraction was impaired in Ang II-infused mice, likely through the desensitization of P2X1R but not AT1R. This implies that vascular P2X1R activity, rather than plasma Up4A level, may determine the role of Up4A in hypertension.

Purinergic dysregulation in pulmonary hypertension.

Despite the fact that nucleotides and adenosine help regulate vascular tone through purinergic signaling pathways, little is known regarding their contributions to the pathobiology of pulmonary arterial hypertension, a condition characterized by elevated pulmonary vascular resistance and remodeling. Even less is known about the potential role that alterations in CD39 (ENTPD1), the ectonucleotidase responsible for the conversion of the nucleotides ATP and ADP to AMP, may play in pulmonary arterial hypertension. In this study we identified decreased CD39 expression on the pulmonary endothelium of patients with idiopathic pulmonary arterial hypertension. We next determined the effects of CD39 gene deletion in mice exposed to normoxia or normobaric hypoxia (10% oxygen). Compared with controls, hypoxic CD39(-/-) mice were found to have a markedly elevated ATP-to-adenosine ratio, higher pulmonary arterial pressures, more right ventricular hypertrophy, more arterial medial hypertrophy, and a pro-thrombotic phenotype. In addition, hypoxic CD39(-/-) mice exhibited a marked increase in lung P2X1 receptors. Systemic reconstitution of ATPase and ADPase enzymatic activities through continuous administration of apyrase decreased pulmonary arterial pressures in hypoxic CD39(-/-) mice to levels found in hypoxic CD39(+/+) controls. Treatment with NF279, a potent and selective P2X1 receptor antagonist, lowered pulmonary arterial pressures even further. Our study is the first to implicate decreased CD39 and resultant alterations in circulating purinergic signaling ligands and cognate receptors in the pathobiology of pulmonary arterial hypertension. Reconstitution and receptor blocking experiments suggest that phosphohydrolysis of purinergic nucleotide tri- and diphosphates, or blocking of the P2X1 receptor could serve as treatment for pulmonary arterial hypertension.

Mechanisms underlying uridine adenosine tetraphosphate-induced vascular contraction in mouse aorta: Role of thromboxane and purinergic receptors.

Uridine adenosine tetraphosphate (Up4A), a novel endothelium-derived vasoactive agent, is proposed to play a role in cardiovascular disorders and induces aortic contraction through activation of cyclooxygenases (COXs). We and others demonstrated that activation of A1 or A3 adenosine receptors (ARs) results in vascular contraction via thromboxane (TX) A2 production. However, the mechanisms of Up4A-induced vascular contraction in mouse aorta are not understood. We hypothesize that Up4A-induced aortic contraction is through COX-derived TXA2 production, which requires activation of A1 and/or A3AR. Concentration responses to Up4A were conducted in isolated aorta. The TXB2 production, a metabolite of TXA2, was also measured. Up4A (10(-9)-10(-5)M) produced a concentration-dependent contraction >70%, which was markedly attenuated by COX and COX1 but not by COX2 inhibition. Notably, Up4A-induced aortic contraction was blunted by both TX synthase inhibitor ozagrel and TXA2 receptor (TP) antagonist SQ29548. Surprisingly, A3AR deletion had no effect on Up4A-induced contraction. Moreover, A1AR deletion or antagonism as well as A1/A3AR deletion potentiated Up4A-induced aortic contraction, suggesting a vasodilator influence of A1AR. In contrast, non-selective purinergic P2 receptor antagonist PPADS significantly blunted Up4A-induced aortic contraction to a similar extent as selective P2X1R antagonist MRS2159, the latter of which was further reduced by addition of ozagrel. Endothelial denudation almost fully attenuated Up4A-induced contraction. Furthermore, Up4A (3µM) increased TXB2 formation, which was inhibited by either MRS2159 or ozagrel. In conclusion, Up4A-induced aortic contraction depends on activation of TX synthase and TP, which partially requires the activation of P2X1R but not A1 or A3AR through an endothelium-dependent mechanism.

Chronic administration of anticholinergics in rats induces a shift from muscarinic to purinergic transmission in the bladder wall.

BACKGROUND: First-line pharmacotherapy for overactive bladder consists of anticholinergics. However, patient compliance is exceptionally low, which may be due to progressive loss of effectiveness. OBJECTIVE: To decipher the involved molecular mechanisms and to evaluate the effects of chronic systemic administration of anticholinergics on bladder function and on muscarinic and purinergic receptors expression in rats. DESIGN, SETTING, AND PARTICIPANTS: Female Wistar rats were implanted with an osmotic pump that chronically administered vehicle (Vehc), 0.36 mg/kg per day oxybutynin (Oxyc), or 0.19 mg/kg per day fesoterodine (Fesoc) for 28 d. INTERVENTIONS: For cystometry experiments, a small catheter was implanted in the bladder. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Urologic phenotype was evaluated by the analysis of the micturition pattern and urodynamics. Expression of muscarinic and purinergic receptors was assessed by Western blot analysis of detrusor membrane protein. Functional responses to carbachol and adenosine triphosphate (ATP) were evaluated using muscle-strip contractility experiments. RESULTS AND LIMITATIONS: The number of voided spots was transiently decreased in Oxyc rats. In Oxyc rats, the effect of an acute high dose of oxybutynin (1mg/kg intraperitoneally [IP]) on the intermicturition interval was abolished. Expression experiments revealed a decrease of muscarinic acetylcholine receptors M2 (mAChR2) and M3 (mAChR3), whereas the purinergic receptor P2X, ligand-gated ion channel, 1 (P2X1) was enhanced in Oxyc and Fesoc rats compared to Vehc rats. In concordance with the modification of the expression pattern in Oxyc rats, the force generated by carbachol and ATP in muscle-strip contractility experiments was, respectively, lower and higher. Urodynamics revealed that the effects of systemic administration of the purinergic blocker pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (50mg/kg IP) were enhanced in Oxyc rats. As rat bladder physiology is different from that of humans, it is difficult to directly extrapolate our findings to human patients. CONCLUSIONS: Chronic administration of anticholinergics in rats induces receptor loss of efficiency and a shift from muscarinic to purinergic transmission.