P2X (Purinergic) receptor distributions in rat blood vessels

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Abstract

The distribution of purinergic (P2X1 and P2X2) receptors on smooth muscle cells in relation to autonomic nerve varicosities in rat blood vessels has been determined using immunofluorescence and confocal microscopy. P2X1 and P2X2 receptors were visualised using rabbit polyclonal antibodies against the extracellular domain of the receptors and varicosities visualised using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2X1 receptor clusters were observed on the smooth muscle cells of mesenteric, renal, and pulmonary arteries as well as in the aorta and in veins: a large approximately elliptical cluster 1.32±0.21 μm long and 0.96±0.10 μm in diameter; and a smaller spherical cluster with a diameter of 0.32±0.05 μm. The latter occurred throughout the media of arteries of all sizes, whereas the former were restricted to the adventitial surface of the media and to endothelial cells, except for the pulmonary artery, in which large receptor clusters were found throughout the media of the vessel. At the adventitial surface, the large clusters are in general located beneath SV2 labelled varicosities. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2X and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2X receptor clusters. P2X2 receptors were located on nerves and on endothelial cells. They were also found in low density on the smooth muscle cells in the media. These observations are discussed in relation to the mechanism of purinergic transmission to the smooth muscle cells of blood vessels.

Introduction

Transmission from sympathetic nerves to a number of different blood vessels in the rat involves the release of ATP onto purinergic receptors. These include the aorta (White et al., 1985; Kitajima et al., 1994; Garcia-Velasco et al., 1995), the pulmonary artery (Inoue and Kannan, 1988), renal blood vessels (Schwartz and Malik, 1989), mesenteric blood vessels (Angus et al., 1988; Yamamoto et al., 1992; Windscheif et al., 1994; Brock and Van Helden, 1995). Autoradiographic studies of the distribution of [3H]α,β-methylene ATP over the media of rat blood vessels indicates that the small and medium-size arteries have a high density of purinergic receptors compared with the elastic and large muscular arteries, with the media of the large muscular arteries showing a higher density of the receptors towards the adventitial surface compared with the intimal surface (Bo and Burnstock, 1993; Zhao et al., 1996). However it seems likely that most of the smooth muscle cells in the media of these vessels possess purinergic receptors, as patch-clamped cells dissociated from the media show inward currents in response to exogenous application of ATP (von der Weid et al., 1993; Evans and Kennedy, 1994).

Purinergic receptors are of two main subtypes, G-protein-coupled receptors (termed P2Y) and ATP-gated channels (termed P2X), with seven of the P2X receptors now isolated and expressed (Collo et al., 1996). Two of these (P2X1 and P2X2) are found in rat mesenteric arterioles, with immunohistochemistry localising the P2X1 receptors to smooth muscle cells and the P2X2 receptors to nerve terminals (Vulchanova et al., 1996). Each of the seven P2X receptors is encoded by a different gene, with each possessing two transmembrane domains, a large extracellular domain, and intracellular N- and C-termini (Surprenant, 1996). All of these proteins are about 36% to 48% identical (Surprenant, 1996). The P2X1 and P2X2 receptors were originally cloned from complementary DNA that encoded these receptors in rat vas deferens (Valera et al., 1994) and in rat pheochromocytoma (PC12) cells (Brake et al., 1994). In the present work, we have developed antibodies against P2X1 and P2X2 receptors and localised these with respect to single varicosities using antibodies against the ubiquitous vesicle proteoglycan SV2. This has enabled the distribution of these purinergic receptors to be determined at the level of individual sympathetic varicosities at different sites within the media of rat blood vessels.

Section snippets

Materials

Anti-mouse SV2 antibody (AbSV2) specific for the synaptic vesicle proteoglycan SV2 (Buckley and Kelly, 1985), was kindly provided by Dr Buckley (Harvard University). Cy2 and Cy3 conjugates of donkey anti-mouse and donkey anti-rabbit IgGs, adsorbed against conspecific IgGs, were from Jackson Immunoresearch (West Grove, PA). All other reagents were purchased from Sigma (St. Louis, MO).

Antibody production

The consensus sequences of the rat P2X1 (Valera et al., 1994) and P2X2 (Brake et al., 1994) cloned receptors were

Results

Tissue sections were exposed to either a combination of antibodies to the P2X1 receptor and the ubiquitous synaptic vesicle proteoglycan SV2 or to P2X2 receptors and SV2.

The distribution of P2X1 and P2X2 receptor clusters in blood vessels

Two main transmitters have been identified for the sympathetic innervation of many arteries, namely noradrenaline and ATP (Sneddon and Burnstock, 1984; Neild and Kotecha, 1986; Papanicolaou and Medgett, 1986; Abe et al., 1987). These transmitters are stored in nerves that are usually confined to the adventitial surface of the media (Bennett, 1972a). However, in vessels with a large media, above about 100 μm wide, there is often an invasion of the outer 30% of the media by nerves from the

Acknowledgements

We wish to thank the National Health and Medical Research Council of Australia, Australian Research Council, National Heart Foundation of Australia, and the Clive and Vera Ramaciotti Foundation for financial support.

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