Comparison of P2X receptors in rat mesenteric, basilar and septal (coronary) arteries

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Abstract

αβmeATP-evoked concentration-dependent, PPADS-sensitive, desensitising, P2X receptor-mediated, constrictions of mesenteric, basilar and septal artery rings with EC50 values of 1, 1 and 30 μM, respectively. In patch clamp studies on acutely dissociated artery smooth cells αβmeATP-evoked transient inward currents (τ∼100 ms) with mean current densities of ∼340, 175 and 120 pA/pF, respectively. P2X1 receptor immunoreactivity was expressed in mesenteric and basilar arteries and this receptor subunit appears to dominate the P2X receptor phenotype in these vessels. In contrast P2X1 receptor immunoreactivity was not detected in septal arteries and the αβmeATP sensitivity of constriction was not consistent with the involvement of P2X1 receptors. These results suggest that not all arteries share a common P2X receptor phenotype.

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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