Disruption of Lipid Rafts Inhibits P2X1 Receptor-mediated Currents and Arterial Vasoconstriction

Abstract

P2X1 receptors for ATP are ligand-gated cation channels expressed on a range of smooth muscle preparations and blood platelets. The receptors appear to be clustered close to sympathetic nerve varicosities and mediate the underlying membrane potential changes and constriction following nerve stimulation in a range of arteries and resistance arterioles. In this study we have used discontinuous sucrose density gradients, Western blot analysis, and cholesterol measurements to show that recombinant and smooth muscle (rat tail artery, vas deferens, and bladder) P2X1 receptors are present in cholesterol-rich lipid rafts and co-localize with the lipid raft markers flotillin-1 and -2. Lipid rafts are specialized lipid membrane microdomains involved in signaling and trafficking. To determine whether lipid raft association was essential for P2X1 receptor channel function we used the cholesterol-depleting agent methyl-β-cyclodextrin (10 mm for 1 h). This led to a redistribution of the P2X1 receptor throughout the sucrose gradient and reduced P2X1 receptor-mediated (α,β-methylene ATP, 10 μm) currents in HEK293 cells by >90% and contractions of the rat tail artery by ∼50%. However contractions evoked by potassium chloride (60 mm) were unaffected by methyl-β-cyclodextrin and the inactive analogue α-cyclodextrin had no effect on P2X1 receptor-mediated currents or contractions. P2X1 receptors are subject to ongoing regulation by receptors and kinases, and the present results suggest that lipid rafts are an essential component in the maintenance of these localized signaling domains and play an important role in P2X1 receptor-mediated control of arteries. P2X1 receptors for ATP are ligand-gated cation channels expressed on a range of smooth muscle preparations and blood platelets. The receptors appear to be clustered close to sympathetic nerve varicosities and mediate the underlying membrane potential changes and constriction following nerve stimulation in a range of arteries and resistance arterioles. In this study we have used discontinuous sucrose density gradients, Western blot analysis, and cholesterol measurements to show that recombinant and smooth muscle (rat tail artery, vas deferens, and bladder) P2X1 receptors are present in cholesterol-rich lipid rafts and co-localize with the lipid raft markers flotillin-1 and -2. Lipid rafts are specialized lipid membrane microdomains involved in signaling and trafficking. To determine whether lipid raft association was essential for P2X1 receptor channel function we used the cholesterol-depleting agent methyl-β-cyclodextrin (10 mm for 1 h). This led to a redistribution of the P2X1 receptor throughout the sucrose gradient and reduced P2X1 receptor-mediated (α,β-methylene ATP, 10 μm) currents in HEK293 cells by >90% and contractions of the rat tail artery by ∼50%. However contractions evoked by potassium chloride (60 mm) were unaffected by methyl-β-cyclodextrin and the inactive analogue α-cyclodextrin had no effect on P2X1 receptor-mediated currents or contractions. P2X1 receptors are subject to ongoing regulation by receptors and kinases, and the present results suggest that lipid rafts are an essential component in the maintenance of these localized signaling domains and play an important role in P2X1 receptor-mediated control of arteries. ATP released from nerves following tissue damage or shear stress acts at P2 receptors to regulate cardiovascular function (1Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). P2 receptors are divided into ligand-gated P2X receptor channels and G-protein-coupled P2Y receptors. P2X receptor-mediated vasoconstriction has been described in a range of arteries in the periphery (1Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar) and in the brain (2Lewis C.J. Ennion S.J. Evans R.J. J. Physiol. 2000; 527: 315-324Crossref PubMed Scopus (51) Google Scholar). Sympathetic nerves co-store and co-release ATP and noradrenaline, and the relative contribution of purinergic and noradrenergic mechanisms to vasoconstriction is dependent on the size of the vessel and the parameters of stimulation (3Evans R.J. Cunnane T.C. Naunyn Schmiedeberg's Arch. Pharmacol. 1992; 345: 424-430Crossref PubMed Scopus (34) Google Scholar, 4Gitterman D.P. Evans R.J. Br. J. Pharmacol. 2001; 132: 1201-1208Crossref PubMed Scopus (66) Google Scholar). The P2X receptor-mediated component predominates in small diameter arteries (4Gitterman D.P. Evans R.J. Br. J. Pharmacol. 2001; 132: 1201-1208Crossref PubMed Scopus (66) Google Scholar, 5Ramme D. Regenold J.T. Starke K. Busse R. Illes P. Naunyn Schmiedeberg's Arch. Pharmacol. 1987; 336: 267-273Crossref PubMed Scopus (100) Google Scholar) and in submucosal resistance arterioles P2X receptor activation is solely responsible for sympathetic neurogenic vasoconstriction with noradrenaline acting through presynaptic mechanisms to regulate transmitter release (6Evans R.J. Surprenant A. Br. J. Pharmacol. 1992; 106: 242-249Crossref PubMed Scopus (157) Google Scholar). In addition P2X receptor channels in smooth muscle are permeant to calcium (∼10% of current flowing through the channel under physiological conditions, Refs. 7Benham C.D. Tsien R.W. Nature. 1987; 328: 275-278Crossref PubMed Scopus (574) Google Scholar and 8Benham C.D. J. Physiol. 1989; 419: 689-701Crossref PubMed Scopus (111) Google Scholar), and a substantial component of the calcium required for contraction enters by this route (4Gitterman D.P. Evans R.J. Br. J. Pharmacol. 2001; 132: 1201-1208Crossref PubMed Scopus (66) Google Scholar, 9Lamont C. Wier W.G. Circ. Res. 2002; 91: 454-456Crossref PubMed Scopus (48) Google Scholar). P2X receptors therefore provide a mechanism for sympathetic nerve-mediated regulation of vascular tone that is resistant to α-adrenoreceptor and calcium channel antagonists. Seven P2X receptor subunits have been identified (P2X1–7), and these can assemble as homo- and heterotrimeric receptors with a range of properties (10North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2413) Google Scholar). The characteristic features of artery smooth muscle P2X receptors; (i) sensitivity to the ATP analogues: α,β-methylene and l-β,γ-methylene ATP, and (ii) responses that desensitize during agonist application, are consistent with the expression of P2X1 receptor subunits. In arterial smooth muscle the P2X1 receptor is the dominant isoform, and P2X receptor-mediated responses are abolished in arteries from P2X1 receptor-deficient mice (11Vial C. Evans R.J. Mol. Pharmacol. 2002; 62: 1438-1445Crossref PubMed Scopus (103) Google Scholar, 12Inscho E.W. Cook A.K. Imig J.D. Vial C. Evans R.J. J. Clin. Investig. 2003; 112: 1895-1905Crossref PubMed Scopus (147) Google Scholar). Studies with these P2X1 receptor-deficient mice have established a role for these receptors in sympathetic nerve-mediated vasoconstriction (11Vial C. Evans R.J. Mol. Pharmacol. 2002; 62: 1438-1445Crossref PubMed Scopus (103) Google Scholar) and autoregulation of blood flow in the kidney (12Inscho E.W. Cook A.K. Imig J.D. Vial C. Evans R.J. J. Clin. Investig. 2003; 112: 1895-1905Crossref PubMed Scopus (147) Google Scholar, 13Inscho E.W. Cook A.K. Imig J.D. Vial C. Evans R.J. Acta. Physiol. Scand. 2004; 181: 445-453Crossref PubMed Scopus (57) Google Scholar). The level of P2X1 receptors can be regulated by congestive heart failure (14Malmsjo M. Bergdahl A. Moller S. Zhao X.-H. Sun X.-Y. Hedner T. Edvinsson L. Erlinge D. Cardiovasc. Res. 1999; 43: 219-227Crossref PubMed Scopus (20) Google Scholar), cardiomyopathy (15Berry D.A. Barden J.A. Balcar V.J. Keogh A. dos Remedios C.G. Electrophoresis. 1999; 20: 2059-2064Crossref PubMed Scopus (21) Google Scholar), and shear stress (16Wang L. Andersson M. Karlsson L. Watson M.A. Cousens D.J. Jern S. Erlinge D. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1370-1376Crossref PubMed Scopus (27) Google Scholar). In addition it has been shown recently that P2X receptors are involved in sensitizing responses following heart failure (17Li J. Sinoway A.N. Gao Z. Maile M.D. Pu M. Sinoway L.I. Circulation. 2004; 110: 3049-3054Crossref PubMed Scopus (82) Google Scholar). P2X1 receptors are also expressed on blood cells, including platelets and P2X1 receptors contribute to platelet activation (18Vial C. Rolf M.G. Mahaut-Smith M.P. Evans R.J. Br. J. Pharmacol. 2002; 135: 363-372Crossref PubMed Scopus (75) Google Scholar) and aggregation (19Toth-Zsamboki E. Oury C. Cornelissen H. De Vos R. Vermylen J. Hoylaerts M.F. J. Biol. Chem. 2003; 278: 46661-46667Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 20Oury C. Sticker E. Cornelissen H. De Vos R. Vermylen J. Hoylaerts M.F. J. Biol. Chem. 2004; 279: 26266-26273Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 21Oury C. Kuijpers M.J. Toth-Zsamboki E. Bonnefoy A. S. M.A. De Vos R. Vermylen J. Hoylaerts M.F. 2003; PubMed Scopus Google Scholar) and P2X1 receptor is P. Vial C. V. M. M. Evans R.J. C. J. 2003; PubMed Scopus Google Scholar). is that P2X1 receptors can play important in the cardiovascular and regulation of blood In arteries P2X1 receptors appear to be throughout the membrane as P2X1 receptor in M.A. Balcar V.J. Barden J.A. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). This of receptors is by on artery membrane had P2X receptor channels channel was in C.J. Evans R.J. Res. 2001; Scopus Google Scholar). also that the P2X1 receptors can be regulated by of C. Evans R.J. J. 2004; PubMed Scopus Google Scholar), that the P2X1 receptor in an signaling for the of P2X1 receptors be the in membrane lipid rafts K. D. Rev. Mol. Biol. 2000; PubMed Scopus Google Scholar). Lipid rafts are in cholesterol and that results in microdomains the membrane J. 2004; PubMed Scopus Google Scholar, Gao E. L. K. L. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). that is in lipid rafts and that a range of domains can be on in a J. 2004; PubMed Scopus Google Scholar). range of including signaling have been shown to be with rafts De M. S. A. 2003; PubMed Scopus Google Scholar) including a range of channels a K. M. Pharmacol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In this study we have shown that P2X1 receptors are in lipid rafts and that of rafts P2X1 receptor signaling in arteries. used α-cyclodextrin and methyl-β-cyclodextrin were from and kidney cells and HEK293 cells with the P2X1 receptor were in as described C. Evans R.J. J. 2004; PubMed Scopus Google Scholar). HEK293 cells were with the rat P2X1 receptor or P2X1 receptor with at and cells (10 mm for 1 at were a on a of for and to at was were by and membrane tail artery, vas deferens, and were and or in for contraction tail arteries were at in physiological mm mm 10 mm mm to with for a of and rat were a from Refs. C. Mol. Pharmacol. 2002; 62: PubMed Scopus Google Scholar and Mol. Pharmacol. 2002; PubMed Scopus Google tissue of HEK293 cells were with and into of mm and on for tail vas deferens, or from rat were in a and a reduced to the were into of mm a tissue and on for The cells and rat were to of were to sucrose by addition of an of sucrose in mm and mm and in an discontinuous sucrose gradient was on of the by of sucrose in with mm of sucrose in The gradient was at on a in a for at In HEK293 were in of or and were a tissue and for on The were to sucrose by addition of an of sucrose in and in an discontinuous sucrose gradient and sucrose in was the and as described for the of 1 were from the to the of measurements were with the cholesterol The The or tissue used for the preparations described of was Western level of was used as a To the of were for for in for tail artery, vas deferens, or and for HEK293 of the on and the membrane was with the receptor or were an and and were as described S.J. Evans R.J. 2001; PubMed Scopus Google Scholar) and with from tissue were for membrane as described and with in a were for S.J. Evans R.J. 2001; PubMed Scopus Google Scholar). The were in of on In an of of was with was and were at a potential of at as described (11Vial C. Evans R.J. Mol. Pharmacol. 2002; 62: 1438-1445Crossref PubMed Scopus (103) Google Scholar, C.J. Evans R.J. Br. J. Pharmacol. 2000; PubMed Scopus Google Scholar). The agonist 10 μm) was a at the effect of on the P2X1 receptor responses to the cells were with the at a of 10 mm for 1 at for cholesterol cells were for 1 at with cholesterol (10 μm) was with the cells for at tail artery were in a and with physiological at and to as described C.J. D.P. H. Evans R.J. Br. J. Pharmacol. 2000; PubMed Scopus Google Scholar). responses to 10 be the agonist was at The was the arteries were in the of (10 mm for 1 at In the control the was also for 1 contraction responses to and (60 the was and the arteries were with physiological are expressed as throughout and of cells, of arteries. were by and a of was P2X1 with Lipid can show a to the lipid of the with a of in cholesterol-rich lipid expression of P2X1 receptors in HEK293 cells an to the role of lipid rafts in receptor were under and on a discontinuous sucrose gradient C. Mol. Pharmacol. 2002; 62: PubMed Scopus Google Scholar). P2X1 receptor was with the lipid raft markers flotillin-1 and in and P. M.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and was from cells and of the cholesterol of the the of the P2X1 receptor in the the P2X1 receptor is a was that the the membrane in and and this for the of the P2X1 receptors in these and lipid raft To this we used to and determine on the were throughout the that the membrane the was for as no of the was at the present in the these that P2X1 receptors are localized to cholesterol lipid P2X1 in the Lipid following and of for the to of the lipid rafts and a in signaling M.P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). P2X1 receptors desensitize following agonist and for the that the is dependent on lipid rafts and the of of the P2X1 receptor with (10 μm) for 10 desensitize the it had no effect on the of the receptor in the lipid raft that of the lipid rafts is with of the P2X1 receptor Lipid P2X1 of cholesterol to of lipid The cholesterol-depleting agent (10 mm for 1 reduced by the cholesterol and a redistribution of the P2X1 receptor the gradient cholesterol the P2X1 receptor was in is membrane and cholesterol from this in for the P2X1 receptor in and that to receptors on In the ATP analogue (10 a currents through P2X1 receptors from HEK293 cells of cholesterol with or (10 mm for 1 reduced the of responses by >90% with no effect on the or of of the The reduced of is to from a in agonist at the as for the of cholesterol on channels J.D. T.C. K. C.J. L. Mol. Pharmacol. 2004; PubMed Scopus Google Scholar), as was no effect on the of the and a of ATP the was also reduced by >90% or also has no effect on the of P2X1 receptors from the as responses to were evoked at the shown C.J. Evans R.J. Br. J. Pharmacol. 2000; PubMed Scopus Google Scholar) from control cells and following or The inactive (10 mm for 1 H. A. A. H. J. K. R. E. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) had no effect on responses responses to (10 μm) were in with by cholesterol for and for and cholesterol The in current in to is to from a in the of P2X1 receptor channels at the as the of P2X1 receptors at the membrane currents through these receptors are also reduced by by and for control and was unaffected by of and cells, for control and Lipid rafts also be by acts to with membrane cholesterol P. C. L. M. A. J. 2004; PubMed Scopus (27) Google Scholar, R. PubMed Scopus Google Scholar). 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P2X1 receptor and the lipid raft were rat vas and in and is to that were in the of lipid rafts HEK293 cells and smooth muscle P2X1 receptor and lipid were present in and of HEK293 cells in and of smooth muscle This the of cholesterol and HEK293 cells and smooth are the membrane are to be by membrane P2X1 of rat tail arteries with the cholesterol agent (10 mm for 1 lipid rafts and in a redistribution of the P2X1 receptor the gradient to on HEK293 artery constriction to (10 for 10 were reduced by (10 mm for 1 the inactive analogue (10 mm for 1 had no effect and of the control for and from and and In by mm were unaffected by with or and of the control for and from and that the with the of the smooth muscle to these suggest that P2X1 receptors are with lipid rafts in arteries and that of these rafts responses through the The of the P2X1 receptor to the cholesterol-rich and the with lipid raft markers 1 and and in addition for the smooth muscle preparations and with the of P2X1 receptor responses following with lipid raft that P2X1 receptors are present in lipid rafts in HEK293 cells as as smooth vas deferens, and Lipid rafts are as and of of cholesterol-rich lipid rafts has shown that is in the rafts H. 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Pharmacol. 1992; 106: 242-249Crossref PubMed Scopus (157) Google Scholar). suggest that sympathetic nerves release ATP and noradrenaline under P2X receptors appear to be for this be the membrane of P2X and This in this study we have shown that the P2X1 receptor is localized in lipid and in it has been shown that are present in the lipid raft and contractions are by cholesterol with K. M. J. J. P. K. Arterioscler. Thromb. Vasc. Biol. 2002; PubMed Scopus Google Scholar). This provide a mechanism P2X1 receptors in lipid rafts be close to the of transmitter release be from these domains and be and of nerve stimulation to be C. E. Wier W.G. J. Physiol. 2003; PubMed Scopus Google Scholar). In the present study for the that P2X1 receptors are with lipid rafts and that of these rafts P2X1 receptor through with signaling This has for cardiovascular function P2X1 receptors have been shown to have important signaling for in the control of resistance artery vasoconstriction following sympathetic nerve stimulation (6Evans R.J. Surprenant A. Br. J. Pharmacol. 1992; 106: 242-249Crossref PubMed Scopus (157) Google Scholar, C. Evans R.J. Mol. Pharmacol. 2002; 62: 1438-1445Crossref PubMed Scopus (103) Google Scholar), autoregulation in the kidney (12Inscho E.W. Cook A.K. Imig J.D. Vial C. Evans R.J. J. Clin. Investig. 2003; 112: 1895-1905Crossref PubMed Scopus (147) Google Scholar), and platelet regulation (18Vial C. Rolf M.G. Mahaut-Smith M.P. Evans R.J. Br. J. Pharmacol. 2002; 135: 363-372Crossref PubMed Scopus (75) Google Scholar, E. Oury C. Cornelissen H. De Vos R. Vermylen J. Hoylaerts M.F. J. Biol. 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