This study investigated the mechanism of prolonged relaxation to ATP in the rat isolated perfused mesenteric arterial bed. In methoxamine pre-constricted preparations, ATP elicited dose-dependent, endothelium-dependent, rapid relaxation at 5 pmol – 0.05 μmol (Rmax 76±5.6%, pD2 9.2±0.2), and contraction, followed by prolonged endothelium-independent vasorelaxation at 0.05, 0.5 and 5 μmol (56±3.0, 87±2.9 and 85±4.6%). Suramin (100 μM), attenuated rapid (pD2 7.8±0.1) and prolonged relaxation to ATP. The selective P2 receptor antagonist PPADS (10 μM) reduced prolonged, but not rapid relaxation. Neither phase of relaxation was affected by 8-sulphophenyltheophylline (1 μM) or indomethacin (10 μM). α,β-methylene ATP (α,β-meATP; 10 μM) attenuated prolonged relaxation to ATP (relaxations at 0.05 and 0.5 μmol were 25±8.3 and 48±9.0%, respectively). α,β-meATP blocked contractions and revealed rapid relaxation to ATP at 0.05 – 5 μmol. Capsaicin pre-treatment did not affect either phase of vasorelaxation to ATP. α,β-meATP (10 μM) had no effect on vasorelaxation mediated by electrical stimulation of capsaicin-sensitive sensory nerves. High K+ (25 mM) attenuated prolonged relaxation to ATP (21±2.6 and 64±5.8%, at 0.05 and 0.5 μmol, respectively), but had no effect on rapid relaxation. Ouabain (1 mM), an inhibitor of Na+/K+-ATPase, and glibenclamide (10 μM), an inhibitor of KATP channels, also attenuated prolonged relaxation to ATP. Charybdotoxin (100 nM), a selective inhibitor of KCa channels, and tetraethylammonium (10 mM) had no effect on rapid or prolonged relaxations. These results show that the prolonged phase of vasorelaxation to ATP in the rat isolated mesenteric arterial bed, which may be mediated by P2Y receptors, is endothelium-independent, involves activation of Na+/K+-ATPase and KATP channels, and is inhibited by α,β-meATP. Neither prolonged nor rapid vasorelaxation to ATP involves capsaicin-sensitive sensory nerves, adenosine P1 receptors, prostanoids or KCa channels. Keywords: P2 purine receptors, ATP, α,β-methylene ATP, primary afferent neurotransmission, rat mesenteric arterial bed, capsaicin Introduction P2 receptors for purine and pyrimidine nucleotides are widely distributed in the cardiovascular system and play an important role in the regulation of vascular tone (Olsson & Pearson, 1990; Boarder & Hourani, 1998; Ralevic & Burnstock, 1998). There are two main classes of P2 receptors; P2X receptors, which are ligand-gated cation channels, and P2Y receptors, which couple to G proteins (Abbracchio & Burnstock, 1994; Fredholm et al., 1994; Ralevic & Burnstock, 1998). In most blood vessels P2X receptors are present on the smooth muscle and mediate a rapidly desensitizing vasocontractile response. P2X1 is the principal isoform of P2X receptor present in vascular smooth muscle (Collo et al., 1996), but there is also evidence for the expression of mRNA for P2X2 and P2X4 (Nori et al., 1997). P2Y1 and P2Y2 receptors are present on the endothelium and mediate vasorelaxation, and P2Y2, P2Y4 and P2Y6 receptors are expressed on the vascular smooth muscle and mediate vasoconstriction (von Kugelegen & Starke, 1990; Erlinge et al., 1998; Ralevic & Burnstock, 1998). The subtype identity of the vasorelaxant P2Y-like receptor expressed on some vascular smooth muscle (see Ralevic & Burnstock, 1998) has not been determined. In addition, there is evidence for P2X receptor expression on primary afferent nerves. Primary afferent nerves are widely distributed in the cardiovascular system and have a role in relaying information concerning the periphery to the central nervous system. Their peripheral terminals can also be activated directly by a variety of physical and chemical stimuli, which evoke release of sensory neurotransmitter and elicit a motor response (Maggi & Meli, 1988). It is known that P2X receptors (principally P2X2 and P2X3 isoforms) are expressed in dorsal root ganglia neurones (Guo et al., 1999; Ueno et al., 1999) and that ATP can excite primary afferents, via P2X receptors, indicating a role in nociception (Burnstock & Wood, 1996; Cook et al., 1997). Nociceptive effects mediated by P2X receptors on capsaicin-sensitive primary afferent neurones have been observed in a number of preparations including rat hindpaw (Bland-Ward & Humphrey, 1997) and knee joint (Dowd et al., 1998). Activation of P2X receptors expressed on primary afferent nerves of rat mesenteric arteries mediates an increase in neuronal activity (Kirkup et al., 1999). ATP, at high doses, has been observed to evoke biphasic vasorelaxation in the rat isolated mesenteric arterial bed, comprising an initial rapid relaxation, followed by a second slow and prolonged response (Stanford & Mitchell, 1998). Rapid relaxation to ATP in this preparation is mediated by activation of P2Y receptors on the endothelium (Ralevic & Burnstock, 1988; 1996) but the mechanism underlying prolonged vasorelaxation to ATP is unclear. Interestingly, the profile of the prolonged phase of ATP vasorelaxation is similar to that of calcitonin gene-related peptide (CGRP), the principal sensory motor neurotransmitter in many blood vessels including rat mesenteric arteries (Kawasaki et al., 1988). This raises the possibility that ATP activation of P2X receptors on primary afferent nerves can mediate an efferent function involving CGRP release and prolonged vasorelaxation. The present study investigated the mechanism of prolonged vasorelaxation to ATP in the rat isolated mesenteric arterial bed. A possible role of capsaicin-sensitive primary afferent nerves was investigated in mesenteric arterial beds pre-treated with capsaicin in order to cause selective desensitization and/or depletion of sensory neurotransmitter (Ralevic et al., 2000). In addition, the selective P2X receptor agonist and desensitizing agent, α,β-methylene ATP (α,β-meATP) was used in order to test the possible involvement of neuronal and/or smooth muscle P2X receptors in mediation/modulation of vasorelaxation to ATP, and in modulation of neurogenic vasorelaxation to electrical stimulation of sensory nerves. Finally, the possible involvement of adenosine P1 receptors, prostanoids, K+ channels and Na+/K+-ATPase in vasorelaxation to ATP was investigated. A preliminary account of some of these findings has been reported to the British Pharmacological Society (Ralevic, 2000).