Catherine Vial

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.

Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors

P2X1 receptors for ATP are ligand-gated cation channels, present on many excitable cells including vas deferens smooth muscle cells. A substantial component of the contractile response of the vas deferens to sympathetic nerve stimulation, which propels sperm into the ejaculate, is mediated through P2X receptors. Here we show that male fertility is reduced by ∼90% in mice with a targeted deletion of the P2X1 receptor gene. Male mice copulate normally—reduced fertility results from a reduction of sperm in the ejaculate and not from sperm dysfunction. Female mice and heterozygote mice are unaffected. In P2X1-receptor-deficient mice, contraction of the vas deferens to sympathetic nerve stimulation is reduced by up to 60% and responses to P2X receptor agonists are abolished. These results show that P2X1 receptors are essential for normal male reproductive function and suggest that the development of selective P2X1 receptor antagonists may provide an effective non-hormonal male contraceptive pill. Also, agents that potentiate the actions of ATP at P2X1 receptors may be useful in the treatment of male infertility.

The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells

© 1997 Federation of European Biochemical Societies.

A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo.

The P2X1 receptor is a fast ATP-gated cation channel expressed in blood platelets, where its role has been difficult to assess due to its rapid desensitization and the lack of pharmacological tools. In this paper, we have used P2X1 −/− and wild-type mouse platelets, treated with apyrase to prevent desensitization, to demonstrate the function of P2X1 in the response to thrombogenic stimuli. In vitro, the collagen-induced aggregation and secretion of P2X1-deficient platelets was decreased, as was adhesion and thrombus growth on a collagen-coated surface, particularly when the wall shear rate was elevated. In vivo, the functional role of P2X1 could be demonstrated using two models of platelet-dependent thrombotic occlusion of small arteries, in which blood flow is characterized by a high shear rate. The mortality of P2X1 −/− mice in a model of systemic thromboembolism was reduced and the size of mural thrombi formed after a laser-induced vessel wall injury was decreased as compared with normal mice, whereas the time for complete thrombus removal was shortened. Overall, the P2X1 receptor appears to contribute to the formation of platelet thrombi, particularly in arteries in which shear forces are high.

Physiological role for P2X1 receptors in renal microvascular autoregulatory behavior

This study tests the hypothesis that P2X1 receptors mediate pressure-induced afferent arteriolar autoregulatory responses. Afferent arterioles from rats and P2X1 KO mice were examined using the juxtamedullary nephron technique. Arteriolar diameter was measured in response to step increases in renal perfusion pressure (RPP). Autoregulatory adjustments in diameter were measured before and during P2X receptor blockade with NF279 or A1 receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Acute papillectomy or furosemide perfusion was performed to interrupt distal tubular fluid flow past the macula densa, thus minimizing tubuloglomerular feedback‐dependent influences on afferent arteriolar function. Under control conditions, arteriolar diameter decreased by 17% and 29% at RPP of 130 and 160 mmHg, respectively. Blockade of P2X1 receptors with NF279 blocked pressuremediated vasoconstriction, reflecting an attenuated autoregulatory response. The A1 receptor blocker DPCPX did not alter autoregulatory behavior or the response to ATP. Deletion of P2X1 receptors in KO mice significantly blunted autoregulatory responses induced by an increase in RPP, and this response was not further impaired by papillectomy or furosemide. WT control mice exhibited typical RPPdependent vasoconstriction that was significantly attenuated by papillectomy. These data provide compelling new evidence indicating that tubuloglomerular feedback signals are coupled to autoregulatory preglomerular vasoconstriction through ATP-mediated activation of P2X1 receptors.

Molecular properties of P2X receptors

P2X receptors for adenosine tri-phosphate (ATP) are a distinct family of ligand-gated cation channels with two transmembrane domains, intracellular amino and carboxy termini and a large extracellular ligand binding loop. Seven genes (P2X1-7) have been cloned and the channels form as either homo or heterotrimeric channels giving rise to a wide range of phenotypes. This review aims to give an account of recent work on the molecular basis of the properties of P2X receptors. In particular, to consider emerging information on the assembly of P2X receptor subunits, channel regulation and desensitisation, targeting, the molecular basis of drug action and the functional contribution of P2X receptors to physiological processes.

P2X receptor expression in mouse urinary bladder and the requirement of P2X1 receptors for functional P2X receptor responses in the mouse urinary bladder smooth muscle

We have used subtype selective P2X receptor antibodies to determine the expression of P2X1–7 receptor subunits in the mouse urinary bladder. In addition we have compared P2X receptor mediated responses in normal and P2X1 receptor deficient mice to determine the contribution of the P2X1 receptor to the mouse bladder smooth muscle P2X receptor phenotype. P2X1 receptor immunoreactivity was restricted to smooth muscle of the bladder and arteries and was predominantly associated with the extracellular membrane. Diffuse P2X2 and P2X4 receptor immunoreactivity not associated with the extracellular membrane was detected in the smooth muscle and epithelial layers. Immunoreactivity for the P2X7 receptor was associated with the innermost epithelial layers and some diffuse staining was seen in the smooth muscle layer. P2X3, P2X5 and P2X6 receptor immunoreactivity was not detected. P2X receptor mediated inward currents and contractions were abolished in bladder smooth muscle from P2X1 receptor deficient mice. In normal bladder nerve stimulation evoked contractions with P2X and muscarinic acetylcholine (mACh) receptor mediated components. In bladder from the P2X1 receptor deficient mouse the contraction was mediated solely by mACh receptors. Contractions to carbachol were unaffected in P2X1 receptor deficient mice demonstrating that there had been no compensatory effect on mACh receptors. These results indicate that homomeric P2X1 receptors underlie the bladder smooth muscle P2X receptor phenotype and suggest that mouse bladder from P2X1 receptor deficient and normal animals may be models of human bladder function in normal and diseased states.

A study of P2X1 receptor function in murine megakaryocytes and human platelets reveals synergy with P2Y receptors

We have examined the role of ATP‐dependent P2X1 receptors in megakaryocytes (MKs) and platelets using receptor‐deficient mice and selective agonists. α,β‐meATP‐ and ATP‐ evoked ionotropic inward currents were absent in whole‐cell recordings from MKs of P2X1−/− mice, demonstrating that the P2X receptor phenotype in MKs, and by inference, platelets, is due to expression of homomeric P2X1 receptors. P2X1 receptor deficiency had no effect on MK (CD 41) numbers or size distribution, showing that it is not essential for normal MK development. P2Y receptor‐stimulated [Ca2+]i responses were unaffected in MKs from P2X1−/− mice, however the inward cation current associated with Ca2+ release was reduced by ∼50%, suggesting an interaction between the membrane conductances activated by P2X1 and P2Y receptors. Interaction between P2X1 and P2Y receptors in human platelets was also examined using [Ca2+]i recordings from cell suspensions. α,β‐meATP (10 μM) evoked a rapid transient P2X1 receptor‐mediated increase in [Ca2+]i, whereas ADP‐(10 μM) evoked P2Y receptor responses were slower, peaked at a higher level and remained elevated for longer periods. Co‐application of α,β‐meATP and ADP resulted in marked acceleration and amplification of the peak [Ca2+]i response. We conclude that ionotropic P2X1 receptors may play a priming role in the subsequent activation of metabotropic P2Y receptors during platelet stimulation.

Cloning and sequencing of a human cDNA encoding endothelial P2Y1 purinoceptor

We have cloned and sequenced a 3055-bp human placenta cDNA encoding a P2 purinoceptor. An identical receptor has also been found in human saphenous vein endothelial cells. Comparison of the deduced amino acid (aa) sequence to those of previously described P2Y1 purinoceptors revealed greater than 95% aa identity with the rat and mouse counterparts, and 83% with the chick P2Y1 purinoceptor. Northern blot experiments revealed two mRNAs of 7.5 and 4.6 kb whose expression varies according to the tissue examined.

Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in human lung mast cells.

Background and purpose:  P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs).