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

Abstract

P2X 1 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) P2X 1 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 P2X 1 receptor channel function we used the cholesterol-depleting agent methyl-␤-cyclodextrin (10 mM for 1 h). This led to a redistribution of the P2X 1 receptor throughout the sucrose gradient and reduced P2X 1 receptor-mediated (␣,␤-methylene ATP, 10 M) currents in HEK293 cells by >90% and contractions of the rat tail artery by ϳ50%. However contractions evoked by potassium chloride (60 mM) were unaffected by methyl-␤-cyclodextrin and the inactive analogue ␣-cyclodextrin had no effect on P2X 1 receptor-mediated currents or contractions. P2X 1 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 P2X 1 receptor-mediated control of arteries.ATP released from nerves following tissue damage or shear stress acts at P2 receptors to regulate cardiovascular function (1). P2 receptors are divided into ligand-gated P2X receptor channels and G-protein-coupled P2Y receptors. P2X receptormediated vasoconstriction has been described in a range of arteries in the periphery (1) and in the brain (2). 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 (3, 4). The P2X receptormediated component predominates in small diameter arteries (4, 5) 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 (6). In addition P2X receptor channels in smooth muscle are permeant to calcium (ϳ10% of current flowing through the channel under physiological conditions, Refs. 7 and 8), and a substantial component of the calcium required for contraction enters by this route (4, 9). P2X receptors therefore provide a mechanism for sympathetic nerve-mediated regulation of vascular tone that is resistant to ␣-adrenoreceptor and calcium channel antagonists.Seven P2X receptor subunits have been identified (P2X 1-7 ),...

Purinergic Signaling and Blood Vessels in Health and Disease

“…P2X 1 receptor knockout mice have confirmed, in mesenteric arteries, the involvement of P2X 1 receptors in ATP-mediated vasoconstriction and transient inward currents and in sympathetic neurogenic vasoconstriction (Vial and Evans, 2002). Moreover, P2X 1 receptor clusters have been described on vascular smooth muscle in regions adjacent to sympathetic nerve varicosities (Hansen et al, 1999;Vial and Evans, 2005) and provide an explanation for why smooth muscle P2X receptors, and not P2Y receptors, are involved in ATP cotransmission.…”

Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland

“…The discrepancy between the latter results on the one hand and the results of Bannas et al (32) and our own data on the other hand might be explained by differences in cell types or in the procedures used in the preparation of rafts. More recently, Vial and Evans (34) reported that P2X 1 receptors colocalize with raft markers in arterial smooth muscle. Disruption of rafts with MCD blocked purinergicmediated currents and smooth muscle contraction.…”

Essential role of flotillin-1 palmitoylation in the intracellular localization and signaling function of IGF-1 receptor

“…To explore the regulatory role of flotillin-1 in IGF-1R signaling, independently of caveolin-1, caveolin-2 and caveolae, HEK293T cells that expressed no caveolins Torres et al, 2006;Vial and Evans, 2005) were stably transfected with shorthairpin RNA (shRNA) against flotillin-1 and tested for IGF-1-induced signaling activation (Fig. 1).…”