Influence of P2X receptors on renal medullary circulation is not altered by angiotensin II pretreatment.

BACKGROUND: Purine P2X and P2Y receptors (P2-R) are involved in control of renal circulation, especially in the medulla, wherein they appear to interact with angiotensin II (Ang II). Our experimental approach enabled examination of interaction with Ang II per se, in the absence of blood pressure elevation. In this whole-kidney functional study we focused on the influence of P2X1-R on perfusion of the renal medulla. METHODS: Acute experiments were conducted with normal rats, untreated or subjected to two weeks' infusion of Ang II (osmotic minipumps). Urethane was used for anesthesia because in Ang II-treated rats it normalized elevated blood pressure. MRS2159, a P2X1-R inhibitor, was infused intravenously or directly into the medulla. Renal blood flow (RBF, Transonic renal artery probe), perfusion of the outer and inner medulla (OM-BF, IM-BF; laser-Doppler fluxes), and sodium and water excretion and urine osmolality (Uosm) were measured. RESULTS: In untreated rats intravenous MRS2159 unexpectedly decreased RBF by 12±4% (p<0.02) and IM-BF by 7±2% (p<0.05). In Ang II-pretreated rats the inhibitor tended to increase RBF while OM-BF and IM-BF increased 14±5% and 12±2%, respectively (p<0.05 for both). Renal excretion was not affected, with or without Ang II treatment, while Uosm increased by about 150mosmol/kg H2O (p<0.05). Intramedullary MRS2159 increased IM-BF only, by 21±5% in untreated and 16±3% in Ang II-treated rats (p<0.04 for both). CONCLUSIONS: Tonic activity of P2X1 receptors participates in control of renal medullary perfusion and of the tubular processes involved in urine concentration, neither effect is modified by Ang II pretreatment.

Presynaptic P2X1-3 and α3-containing nicotinic receptors assemble into functionally interacting ion channels in the rat hippocampus.

Previous studies documented a cross-talk between purinergic P2X (P2XR) and nicotinic acetylcholine receptors (nAChR) in heterologous expression systems and peripheral preparations. We now investigated if this occurred in native brain preparations and probed its physiological function. We found that P2XR and nAChR were enriched in hippocampal terminals, where both P2X1-3R and α3, but not α4, nAChR subunits were located in the active zone and in dopamine-ß-hydroxylase-positive hippocampal terminals. Notably, P2XR ligands displaced nAChR binding and nAChR ligands displaced P2XR binding to hippocampal synaptosomes. In addition, a negative P2XR/nAChR cross-talk was observed in the control of the evoked release of noradrenaline from rat hippocampal synaptosomes, characterized by a less-than-additive facilitatory effect upon co-activation of both receptors. This activity-dependent cross-inhibition was confirmed in Xenopus oocytes transfected with P2X1-3Rs and α3ß2 (but not α4ß2) nAChR. Besides, P2X2 co-immunoprecipitated α3ß2 (but not α4ß2) nAChR, both in HEK cells and rat hippocampal membranes indicating that this functional interaction is supported by a physical association between P2XR and nAChR. Moreover, eliminating extracellular ATP with apyrase in hippocampal slices promoted the inhibitory effect of the nAChR antagonist tubocurarine on noradrenaline release induced by high- but not low-frequency stimulation. Overall, these results provide integrated biochemical, pharmacological and functional evidence showing that P2X1-3R and α3ß2 nAChR are physically and functionally interconnected at the presynaptic level to control excessive noradrenergic terminal activation upon intense synaptic firing in the hippocampus.

Propensity of red blood cells to undergo P2X7 receptor-mediated phosphatidylserine exposure does not alter during in vivo or ex vivo aging.

BACKGROUND: Phosphatidylserine (PS) exposure facilitates the removal of red blood cells (RBCs) from the circulation, potentially contributing to the loss of stored RBCs after transfusion, as well as senescent RBCs. Activation of the P2X7 receptor by extracellular adenosine 5'-triphosphate (ATP) can induce PS exposure on freshly isolated human RBCs, but whether this process occurs in stored RBCs or changes during RBC aging is unknown. STUDY DESIGN AND METHODS: RBCs were processed and stored according to Australian blood banking guidelines. PS exposure was determined by annexin V binding and flow cytometry. Efficacy of P2X antagonists was assessed by flow cytometric measurements of ATP-induced ethidium+ uptake in RPMI 8226 cells. Osmotic fragility was assessed by lysis in hypotonic saline. RBCs were fractionated by discontinuous density centrifugation. RESULTS: ATP (1 mmol/L) induced PS exposure on RBCs stored for less than 1 week. This process was near-completely inhibited by the P2X7 antagonists A438079 and AZ10606120 and the P2X1/P2X7 antagonist MRS2159 but not the P2X1 antagonist NF499. ATP-induced PS exposure on RBCs was not dependent on K+, Na+, or Cl- fluxes. ATP did not alter the osmotic fragility of stored RBCs. ATP-induced PS exposure was similar between RBCs of different densities. ATP-induced PS exposure was also similar between RBCs stored for less than 1 week or for 6 weeks. CONCLUSION: The propensity of RBCs to undergo P2X7-mediated PS exposure does not alter during in vivo and ex vivo aging. Thus, P2X7 activation is unlikely to be involved in the removal of senescent RBCs or stored RBCs after transfusion.

Extracellular ATP induces intracellular alpha-synuclein accumulation via P2X1 receptor-mediated lysosomal dysfunction.

The pathologic hallmark of Parkinson's disease (PD) is the accumulation of alpha-synuclein (αsyn) in susceptible neurons in the form of Lewy bodies and Lewy neurites. The etiology of PD remains unclear. Because brain injury has been suggested to facilitate αsyn aggregation, we investigated whether cellular breakdown products from damaged cells can act on neighboring healthy cells and cause intracellular αsyn accumulation and/or aggregation. Using 2 neuronal cell models, we found that extracellular adenosine triphosphate (ATP) induced a significant increase in intracellular αsyn levels between 24 and 48 hours after treatment. Further investigation revealed that the observed αsyn accumulation is a result of lysosome dysfunction caused by extracellular ATP-induced elevation of lysosomal pH. Interestingly, P2X1 receptor appears to mediate the cells' response to extracellular ATP. Although Ca(2+) influx via P2X1 receptor is necessary for αsyn accumulation, Ca(2+) influx per se is not sufficient for increased αsyn accumulation. These findings provide new insight into our knowledge of the role of P2X receptors in PD pathogenesis and may be helpful in identifying new therapeutic targets for PD.

Development of a P2X1-purinoceptor mediated contractile response in the aged mouse prostate gland through slowing down of ATP breakdown.

AIMS: An age-related increase in prostatic smooth muscle tone is partly responsible for the lower urinary tract symptoms associated with benign prostatic hyperplasia (BPH). Changes in the effectors of prostatic smooth muscle contraction with age may play a role in the development of these symptoms. Using a mouse model of prostate contractility, this study investigated the effect of age on the different components of contractility in the prostate gland. METHODS: The isometric force developed in response to electrical field stimulation or exogenously applied agonists by mouse prostates mounted in organ baths, was evaluated to determine the effect of age on contractile mechanisms. Changes with age in the rate of ATP breakdown and levels of the P2rx1 gene and P2X1-purinoceptor expression in mouse prostate were measured by a modified luciferin-luciferase assay, RT-PCR and western blot, respectively. RESULTS: Nerve mediated contractile responses containing a component elicited by P2X1-purinoceptors were observed in prostates taken from aged mice, but not in prostates taken from young adult mice. Furthermore, the potency of the endogenous purinoceptor agonist ATP was 50-fold greater in aged mice, whereas the potency of its stable analogue α,ß-metATP was unchanged. An age-related decrease in ATP metabolism was also observed. CONCLUSIONS: With age, a purinergic contractile response to nerve stimulation develops in the mouse prostate gland due to a decrease in the rate of ATP breakdown. This may contribute to the increase in muscular tone observed in BPH and suggests that P2X1-purinoceptors are an additional target for the treatment of BPH.

Kinetics of conformational changes revealed by voltage-clamp fluorometry give insight to desensitization at ATP-gated human P2X1 receptors.

ATP acts as an extracellular signaling molecule at cell-surface P2X receptors, mediating a variety of important physiologic and pathophysiologic roles. Homomeric P2X1 receptors open on binding ATP and then transition to an ATP-bound closed, desensitized state that requires an agonist-free washout period to recover. Voltage-clamp fluorometry was used to record ion channel activity and conformational changes simultaneously at defined positions in the extracellular loop of the human P2X1 receptor during not only agonist binding and desensitization but also during recovery. ATP evoked distinct conformational changes adjacent to the agonist binding pocket in response to channel activation and desensitization. The speed of recovery of the conformational change on agonist washout was state-dependent, with a faster time constant from the open (5 seconds) compared with the desensitized (75 seconds) form of the channel. The ability of ATP to evoke channel activity on washout after desensitization was not dependent on the degree of conformational rearrangement in the extracellular loop, and desensitization was faster from the partially recovered state. An intracellular mutation in the carboxyl terminus that slowed recovery of P2X1 receptor currents (7-fold less recovery at 30 seconds) had no effect on the time course of the extracellular conformational rearrangements. This study highlights that the intracellular portion of the receptor can regulate recovery and shows for the first time that this is by a mechanism independent of changes in the extracellular domain, suggesting the existence of a distinct desensitization gate in this novel class of ligand gated ion channels.

[Purine receptors and associated signaling systems in regulation of platelet function].

Purine receptors of platelets play key role in hemostasis and thrombogenesis and they are used as targets for antiplatelet therapy. Platelets express three subtypes of purine receptors--P2X1, P2Y1 and P2Y12. The expression, ligands and sensitivity of purine receptors of platelets and their role in thrombogenesis are discussed in this article. Authors debate information about basic molecular mechanisms, links with intracellular signaling and biological effects of purine receptors activation. In conclusion, understanding of signaling mechanisms of purine receptors is a basis for further pharmacological strategy and effective antiplatelet therapy development.

P2X1 receptors localized in lipid rafts mediate ATP motor responses in the human vas deferens longitudinal muscles.

To assess the role of the P2X1 receptors (P2X1R) in the longitudinal and circular layers of the human vas deferens, ex vivo-isolated strips or rings were prepared from tissue biopsies to record isometric contractions. To ascertain its membrane distribution, tissue extracts were analyzed by immunoblotting following sucrose gradient ultracentrifugation. ATP, alpha,beta-methylene ATP, or electrical field stimulation elicited robust contractions of the longitudinal layer but not of the circular layer which demonstrated inconsistent responses. Alpha,beta-methylene ATP generated stronger and more robust contractions than ATP. In parallel, prostatic segments of the rat vas deferens were examined. The motor responses in both species were not sustained but decayed within the first minute, showing desensitization to additional applications. Cross-desensitization was established between alpha,beta-methylene ATP or ATP-evoked contractions and electrical field stimulation-induced contractions. Full recovery of the desensitized motor responses required more than 30 min and showed a similar pattern in human and rat tissues. Immunoblot analysis of the human vas deferens extracts revealed a P2X1R oligomer of approximately 200 kDa under nonreducing conditions, whereas dithiothreitol-treated extracts showed a single band of approximately 70 kDa. The P2X1R was identified in ultracentrifugation fractions containing 15%-29% sucrose; the receptor localized in the same fractions as flotillin-1, indicating that it regionalized into smooth muscle lipid rafts. In conclusion, ATP plays a key role in human vas deferens contractile responses of the longitudinal smooth muscle layer, an effect mediated through P2X1Rs.

Extracellular UDP enhances P2X-mediated bladder smooth muscle contractility via P2Y(6) activation of the phospholipase C/inositol trisphosphate pathway.

Bladder dysfunction characterized by abnormal bladder smooth muscle (BSM) contractions is pivotal to the disease process in overactive bladder, urge incontinence, and spinal cord injury. Purinergic signaling comprises one key pathway in modulating BSM contractility, but molecular mechanisms remain unclear. Here we demonstrate, using myography, that activation of P2Y6 by either UDP or a specific agonist (MRS 2693) induced a sustained increase in BSM tone (up to 2 mN) in a concentration-dependent manner. Notably, activation of P2Y6 enhanced ATP-mediated BSM contractile force by up to 45%, indicating synergistic interactions between P2X and P2Y signaling. P2Y6-activated responses were abolished by phospholipase C (PLC) and inositol trisphosphate (IP3) receptor antagonists U73122 and xestospongin C, demonstrating involvement of the PLC/IP3 signal pathway. Mice null for Entpd1, an ectonucleotidase on BSM, demonstrated increased force generation on P2Y6 activation (150%). Thus, in vivo perturbations to purinergic signaling resulted in altered P2Y6 activity and bladder contractility. We conclude that UDP, acting on P2Y6, regulates BSM tone and in doing so selectively maximizes P2X1-mediated contraction forces. This novel neurotransmitter pathway may play an important role in urinary voiding disorders characterized by abnormal bladder motility.

P2X1 and P2X2 receptors in the central nervous system as possible drug targets.

P2X receptors are homo- or heterotrimeric ATP-gated cation channels that assemble from seven subunits, P2X1-P2X7. To our knowledge, no drug that acts on the P2X1 or P2X2 receptors in the CNS or elsewhere in the body has been approved, nor is there such a drug currently in clinical trials. Only a few non-drug-like antagonists such as the suramin derivatives NF449 and NF770 and the anthraquinone derivative PSB-1011 are available as pharmacological tools to block the P2X1 and P2X2 receptors, respectively. The focus of this review is twofold. First, we review the current knowledge of the role of the P2X1 and P2X2 receptors in normal and pathological CNS functions as inferred from experiments with wild-type, P2X1 knockout and P2X2 knockout mice. From the available data we conclude that the P2X1 and P2X2 receptors may have therapeutic potential as targets for neuroprotective drugs. Second, we review the impact of the recent resolution of the crystal structure of the zebrafish P2X4 receptor in the apo closed state and the ATP-bound open state. The P2X4 crystal structure opens the exciting possibility to generate P2X homology models for a rational drug design. In silico docking experiments with a homology-modeled rat P2X2 receptor revealed an almost perfect coordination of the nanomolar potent P2X2 antagonist NF770 through strong polar interactions between the acidic groups of NF770 and the mostly basic groups of the ATP-binding pocket. Such structural information might be helpful in designing drug-like compounds that function as selective P2X receptor antagonists without the pharmacokinetic limitations of the currently available antagonists.

Spatiotemporal and anatomical analyses of P2X receptor-mediated neuronal and glial processing of sensory signals in the rat dorsal horn.

Extracellularly released adenosine triphosphate (ATP) modulates sensory signaling in the spinal cord. We analyzed the spatiotemporal profiles of P2X receptor-mediated neuronal and glial processing of sensory signals and the distribution of P2X receptor subunits in the rat dorsal horn. Voltage imaging of spinal cord slices revealed that extracellularly applied ATP (5-500 µM), which was degraded to adenosine and acting on P1 receptors, inhibited depolarizing signals and that it also enhanced long-lasting slow depolarization, which was potentiated after ATP was washed out. This post-ATP rebound potentiation was mediated by P2X receptors and was more prominent in the deep than in the superficial layer. Patch clamp recording of neurons in the superficial layer revealed long-lasting enhancement of depolarization by ATP through P2X receptors during the slow repolarization phase at a single neuron level. This depolarization pattern was different from that in voltage imaging, which reflects both neuronal and glial activities. By immunohistochemistry, P2X(1) and P2X(3) subunits were detected in neuropils in the superficial layer. The P2X(5) subunit was found in neuronal somata. The P2X(6) subunit was widely expressed in neuropils in the whole gray matter except for the dorsal superficial layer. Astrocytes expressed the P2X(7) subunit. These findings indicate that extracellular ATP is degraded into adenosine and prevents overexcitation of the sensory system, and that ATP acts on pre- and partly on postsynaptic neuronal P2X receptors and enhances synaptic transmission, predominantly in the deep layer. Astrocytes are involved in sensitization of sensory network activity more importantly in the superficial than in the deep layer.

Hypertonic stress regulates T cell function via pannexin-1 hemichannels and P2X receptors.

Hypertonic saline (HS) resuscitation increases T cell function and inhibits posttraumatic T cell anergy, which can reduce immunosuppression and sepsis in trauma patients. We have previously shown that HS induces the release of cellular ATP and enhances T cell function. However, the mechanism by which HS induces ATP release and the subsequent regulation of T cell function by ATP remain poorly understood. In the present study, we show that inhibition of the gap junction hemichannel pannexin-1 (Panx1) blocks ATP release in response to HS, and HS exposure triggers significant changes in the expression of all P2X-type ATP receptors in Jurkat T cells. Blocking or silencing of Panx1 or of P2X1, P2X4, or P2X7 receptors blunts HS-induced p38 MAPK activation and the stimulatory effects of HS on TCR/CD28-induced IL-2 gene transcription. Moreover, treatment with HS or agonists of P2X receptors overcomes T cell suppression induced by the anti-inflammatory cytokine IL-10. These findings indicate that Panx1 hemichannels facilitate ATP release in response to hypertonic stress and that P2X1, P2X4, and P2X7 receptor activation enhances T cell function. We conclude that HS and P2 receptor agonists promote T cell function and thus, could be used to improve T cell function in trauma patients.

Pentosan polysulfate treatment preserves renal autoregulation in ANG II-infused hypertensive rats via normalization of P2X1 receptor activation.

Inflammatory factors are elevated in animal and human subjects with hypertension and renal injury. We hypothesized that inflammation contributes to hypertension-induced renal injury by impairing autoregulation and microvascular reactivity to P2X(1) receptor activation. Studies were conducted in vitro using the blood-perfused juxtamedullary nephron preparation. Rats receiving ANG II (60 ng/min) infusion were treated with the anti-inflammatory agent pentosan polysulfate (PPS) for 14 days. The magnitude and progression of hypertension were similar in ANG II and ANG II+PPS-treated rats (169 ± 5 vs. 172 ± 2 mmHg). Afferent arterioles from control rats exhibited normal autoregulatory behavior with diameter decreasing from 18.4 ± 1.6 to 11.4 ± 1.7 µm when perfusion pressure was increased from 70 to 160 mmHg. In contrast, pressure-mediated vasoconstriction was markedly attenuated in ANG II-treated rats, and diameter remained essentially unchanged over the range of perfusion pressures. However, ANG II-treated rats receiving PPS exhibited normal autoregulatory behavior compared with ANG II alone rats. Arteriolar reactivity to ATP and ß,γ-methylene ATP was significantly reduced in ANG II hypertensive rats compared with controls. Interestingly, PPS treatment preserved normal reactivity to P2 and P2X(1) receptor agonists despite the persistent hypertension. The maximal vasoconstriction was 79 ± 3 and 81 ± 2% of the control diameter for ATP and ß,γ-methylene ATP, respectively, similar to responses in control rats. PPS treatment significantly reduced α-smooth muscle actin staining in afferent arterioles and plasma transforming growth factor-ß1 concentration in ANG II-treated rats. In conclusion, PPS normalizes autoregulation without altering ANG II-induced hypertension, suggesting that inflammatory processes reduce P2X(1) receptor reactivity and thereby impair autoregulatory behavior in ANG II hypertensive rats.