Journal of the Autonomic Nervous System
Comparison of P2X receptors in rat mesenteric, basilar and septal (coronary) arteries
Introduction
P2X receptors for ATP are ligand gated cation channels that are expressed by a variety of excitable tissues including neurons and smooth muscle cells (Burnstock, 1997). In the vascular system they are present on a range of arteries including those in the cerebral, coronary and peripheral circulation (Byrne and Large, 1986, Ramme et al., 1987, Evans and Surprenant, 1992, Corr and Burnstock, 1994). In arteries P2X receptor activation by purinergic agonists or ATP released from sympathetic nerves results in membrane depolarisation and vasoconstriction. P2X receptors are calcium permeable and ∼5–10% of the current carried by P2X receptors in smooth muscle is due to calcium influx (Benham, 1989, Schneider et al., 1991). P2X receptors can therefore provide a direct route for calcium entry and contraction as well as leading to depolarisation of the muscle and opening of L-type calcium channels.
Seven isoforms of the P2X receptor have been detected at the molecular level (P2X1–7) and these constitute a distinct family of ligand gated cation channels with two transmembrane domains, intracellular amino and carboxy termini and a large extracellular loop associated with ligand binding (Burnstock, 1997). P2X1 receptors were originally isolated from vas deferens smooth muscle and they have a widespread distribution in arteries neurons and some blood cells (Valera et al., 1994, Collo et al., 1996, Vulchanova et al., 1996, Nori et al., 1998, Sun et al., 1998). The properties of native smooth muscle P2X receptors, in particular the desensitising nature of the response and the sensitivity to the agonists α,β-methylene ATP (αβmeATP)and l-β,γ-methylene ATP (l-βγmeATP) and the antagonist 2′,3′-O-(2,4,6-trinitropheyl) ATP (TNP-ATP) suggest that the smooth muscle response is dominated by a P2X1-like receptor phenotype (Evans and Surprenant, 1996, Lewis et al., 1998). This is further supported by recent studies on a P2X1 receptor-deficient mouse which have shown that the P2X1 receptor is essential for the production of a functional vas deferens smooth muscle P2X receptor (Mulryan et al., 2000). The distribution of P2X receptors in different arteries however may be heterogeneous; for example, RNA for P2X1 was not detected in large mesenteric arteries and limited detection of P2X1 receptor subunits in arteries associated with brain sections has been reported (Collo et al., 1996). In vascular smooth muscle in addition to P2X1 receptors other P2X receptor isoforms have been detected at the mRNA level using in situ hybridisation techniques, e.g., P2X2 and P2X4 receptors (Nori et al., 1998).
P2X receptors form as multimeric assemblies of channel subunits. The exact stoichiometry of the receptor remains unclear although studies have suggested the channel may be comprised of at least three subunits (Kim et al., 1997, Nicke et al., 1998, Stoop et al., 1999). These multimers can either be homomeric channels or different subunits may come together to form heteromeric channels (Torres et al., 1999), often with composite phenotypes, e.g., P2X2/3, P2X2/4 or P2X1/5 receptors. Thus arteries may show a variety of P2X receptor phenotypes dependent on the expression of P2X receptor subunits and any heteropolymerisation within a given vessel.
In this study we have compared the properties of rat mesenteric (peripheral), basilar (cerebral) and septal (coronary) artery P2X receptors. In particular we have determined: (1) their sensitivity to the stable ATP analogue αβmeATP, (2) the properties of P2X receptor-mediated currents, (3) the immunohistochemical localisation of P2X receptor subunits and (4) the sensitivity of P2X receptor-mediated contractions to the L-type calcium channel antagonist nifedipine.
Section snippets
Methods
Male Wistar rats (250–300 g) were killed by cervical dislocation or CO2 followed by femoral exsanguination. Mesenteric (second order), basilar and septal arteries were dissected. For contraction experiments artery rings were mounted in a Mulvany myograph and perfused with physiological saline and vasoconstrictions to applied drugs measured as described previously (Lewis et al., 2000). The mean diameters of vessels were 305.2±6.6, 245.4±10.2 and 212.6±11 μm for mesenteric, basilar and septal
Sensitivity of mesenteric, basilar and septal arteries to αβmeATP
The characterisation of P2X receptors in organ bath experiments can be complicated by the breakdown of some purinergic agonists by ecto-nucleotideases. We have therefore used the metabolically stable ATP analogue αβmeATP to compare P2X receptor-mediated constrictions in different arteries. αβmeATP-evoked concentration dependent constrictions of mesenteric, basilar and septal arteries (Fig. 1, Fig. 2) (peak response to 100 μM αβmeATP, 3.1±0.5, 2.1±0.3 and 0.8±0.1 mN, respectively). At low
Discussion
In this study we have compared the properties of P2X receptor-mediated vasoconstrictions in rat mesenteric, basilar and septal arteries to determine whether artery P2X receptors have a common phenotype. In addition we have used P2X receptor isoform specific antibodies to document the receptor distribution in these vessels. This work has highlighted a number of similarities and differences in P2X receptor-mediated responses in these vessels and indicates that the properties of P2X
Acknowledgements
This work was supported by the Wellcome Trust.
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2013, Pharmacological ReportsCitation Excerpt :In the cardiovascular system, P2X receptors are expressed predominantly on smooth muscle cells [25, 52]. In rat mesenteric arteries, the predominant P2X receptor is homomeric P2X1 [30]. Cation channels formed by P2X subunits have similar permeability for Na+ and K+, and much greater permeability for Ca2+ [12, 25], e.g., relative Ca2+ over Na+ permeability (PCa/PNa) of 4.8 and 4.2 was reported for P2X1 and P2X4 receptors, respectively [11, 34].
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2003, Kidney InternationalCitation Excerpt :NA acted on α1-adrenoceptors to produce sustained contractions, while ATP, and its stable analog α,β-meATP, act on P2X1 receptors to produce a faster transient contraction. In common with other blood vessels such as the rat mesenteric, pulmonary, basilar, and coronary arteries where P2X1 receptors have been visualized immunohistochemically on vascular smooth muscle cells [33-35], the presence of P2X1 receptors on the vascular smooth muscle of the rat renal artery was demonstrated by immunostaining. P2X2 receptor immunostaining was also shown to be present in the rat renal artery.
Introduction: ATP and Its Metabolites as Potent Extracellular Agents
2003, Current Topics in MembranesCitation Excerpt :Early studies of the distribution of P2X receptor subtypes based on Northern blot and in situ hybridization studies (Collo et al., 1996) have been extended substantially, after antibodies to these receptors became available, by immunohistochemical localization at both light microscopic (Vulchanova et al., 1996; Bradbury et al., 1998; Chan et al., 1998; Xiang et al., 1998a,b, 1999; Bo et al., 1999; Gröschel-Stewart et al., 1999a,b; Bardini et al., 2000; Brouns et al., 2000; Lee et al., 2000a,b) and electron microscopic levels (Llewellyn-Smith and Burnstock, 1998; Loesch and Burnstock, 1998, 2000; Loesch et al., 1999). For example, while it was originally thought that smooth muscle contained only P2X1 receptors, there is now evidence of the presence of P2X2, P2X4, and probably P2X5 receptors as both homomultimers and heteromultimers (Nori et al., 1998; Hansen et al., 1999; Lewis and Evans, 2000). P2X1 receptors, which, in earlier studies, were not considered to be present in the brain, now have been found at postjunctional sites in synapses in the cerebellum (Loesch and Burnstock, 1998).