Extracellular nucleosides and nucleotides, such as ATP and adenosine, act on endothelial or smooth muscle cells to modulate vascular tone. Classical effects of ATP are vasoconstriction or vasodilatation, by P2X receptor-mediated depolarisation of smooth muscle cells or by P2Y receptors on endothelial cells, respectively. Adenosine acting at A_2A or A_2B receptors on smooth muscle acts to mediate vasorelaxation. We have previously reported pharmacological evidence for the presence of vasorelaxatory A_2A and P2Y₁ receptors on the smooth muscle of the porcine coronary artery (Alexander et al., 2002; Barbadillo-Muñoz et al., 2001). In the present study, we have, therefore, investigated the expression of these receptors at the molecular level.

RNA extracted from endothelium-denuded porcine coronary artery (PCA) and vein (PCV) segments was reverse transcribed and subjected to multiplex PCR with pig specific primers for hypoxanthine phosphoribosyltransferase (hprt), P2Y₁ (p2y1r) and A_2A (a2ar) receptors. PCR products, separated through 3% agarose, were quantitated by densitometric analysis. p2y1r and a2ar PCR products were expressed relative to the hprt gene product. Western blotting, employing an antibody raised against the third intracellular loop of the human P2Y₁ receptor, was used to examine the expression of p2y1r protein. Data presented are means ± S.E.M. or means ± range for 4 or 2 donor animals, respectively, and were assessed for statistical significance by two-way ANOVA.

Multiplex PCR of PCA and PCV cDNA demonstrated the presence of both p2y1r and a2ar gene products in these tissues. Densitometric analysis revealed that, whilst the levels of p2y1r mRNA did not vary significantly between arterial and vein samples (PCA 36 ± 7 %, n = 4; PCV 58 ± 3 %, n = 2), the levels of a2ar mRNA expressed in the PCV were significantly lower (p<0.01) than those observed in the PCA (PCA 251 ± 50 %, n = 4; PCV 7 ± 0.2 %, n = 2).

Western blotting demonstrated the presence of multiple bands of P2Y1-like immunoreactivity in both the artery and vein. Using HEK293 cells as a positive control revealed the presence of three major protein bands. These were observed at molecular weights of approximately 55, 115 (doublet) and 200 kDa, consistent with observations by other groups using antibodies raised against the same region of the receptor (Wang et al., 2002). The largest band could be observed in all samples from either coronary arteries or veins. The 55 and 115 kDa bands could also be visualised in all artery samples.

In summary, therefore, we have demonstrated a differential expression of a2ar mRNA in arterial and venous vessels from the same vascular bed. In contrast, p2y1r gene expression appears consistent between arterial and venous tissue samples. Whether these differences are replicated in functional studies will be the subject of future investigations.

Alexander, S.P.H. et al. (2002). Br. J. Pharmacol., 137S, 12P.
Barbadillo-Muñoz, M. et al. (2001). Br. J. Pharmacol. 133S, 96P.
Wang, L. et al. (2002). J. Cardiovasc. Pharmacol. 40, 841-853.

We thank the British Heart Foundation for financial support.