Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonist, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coli, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5-770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6-8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coil P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP.

The structure-activity relationships for a variety of adenine nucleotide analogues at P 2x -and P 2Y -purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N 6 -substituents, and a uridine base instead of adenine), the ribose moiety (2′ and 3′-positions), and the triphosphate group (lower phosphates, bridging oxygen substitution, and cyclization) were prepared. Pharmacological activity at P 2Y -purinoceptors was assayed in the guinea pig taenia coli, endothelial cells of the rabbit aorta, smooth muscle of the rabbit mesenteric artery, and turkey erythrocyte membranes. Activity at P 2X -purinoceptors was assayed in the rabbit saphenous artery and the guinea-pig vas deferens and urinary bladder. Some of the analogues displayed selectivity, or even specificity, for either the P 2X -or the P 2Y -purinoceptors. Certain analogues displayed selectivity or specificity within the P 2X -or P 2Y -purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2′,3′-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P 2Y -purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P 2X -purinoceptors. The potent agonist N 6 -methyl ATP and the somewhat less potent agonist 2′-deoxy-ATP were selective for P 2Y -purinoceptors in the guinea pig taenia coli, but were inactive at P 2X -purinoceptors and the vascular P 2Y -purinoceptors. 3′-Benzylamino-3′-deoxyATP was very potent at the P 2X -purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P 2Y receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P 2X -and P 2Y -purinoceptor classes.

1 The isolated hepatic artery of the rabbit contracted to exogenously applied noradrenaline (NA). There was no significant difference in the maximal contraction or the EC50 value in vessels where the endothelium was present and in endothelium-denuded preparations. 2 Acetylcholine (ACh) induced a vasodilatation of vessels preconstricted with NA which was entirely dependent on the endothelium.3 Adenosine 5'-triphosphate (ATP), 2-methylthio ATP, adenosine and sodium nitroprusside induced concentration-dependent, sustained relaxations of vessels in which tone had been induced with NA. The relaxation responses were not reduced after removal of the endothelium. 8-Phenyltheophylline antagonized the relaxation response produced by adenosine, but not that due to ATP at lower concentrations. The maximum response to ATP was reduced in the presence of 8-phenyltheophylline. 4 a,,B-Methylene ATP produced further contraction of vessels preconstricted with NA in both endothelium-denuded preparations and in vessels where the endothelium remained intact. 5 Immunohistochemical analysis was used to show the presence of nerve fibres containing substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) in the hepatic artery. Application of SP induced a concentration-dependent relaxation which was entirely dependent on the presence of an intact endothelium. CGRP and VIP, however, elicited concentration-dependent relaxations which were independent of the endothelium. 7 It is concluded that in the rabbit hepatic artery, responses to ACh are dependent on the presence of intact endothelium. P1-, P2X-and P2,-purinoceptors, mediating relaxation to adenosine, vasoconstriction to ATP and vasodilatation to ATP respectively, are located on vascular smooth muscle. Furthermore, CGRP and VIP mediate a direct vasodilatation of smooth muscle both in the absence and the presence of the endothelium, whereas SP produces a relaxation via receptors located on the endothelium.

The NANC inhibitory innervation of the longitudinal muscle of the rabbit portal vein has been examined. Neurogenic relaxations were partially inhibited by the P2-purinoceptor antagonist, suramin. Addition of the NO-synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) also significantly reduced responses to electrical stimulation and the addition of L-arginine reversed this effect. A combination of both suramin and L-NAME abolished the neurogenic relaxation. A maximum relaxation of the vein was evoked by sodium nitroprusside which was not affected by L-NAME or suramin. Histochemical staining demonstrated the presence of NADPH-diaphorase containing nerves between the longitudinal and circular muscle coats of the media and also in the adventitia. It is concluded that both ATP and NO are inhibitory neurotransmitters in the NANC nerves of the rabbit portal vein.

Transmural electrical field stimulation produced a transient contraction of the isolated hepatic artery of the rabbit that was frequency‐dependent up to 64 Hz. A contraction was rarely evoked at a stimulation frequency of less than 8 Hz and never at 4 Hz or less. All contractions were abolished in the presence of tetrodotoxin. Neurogenic contractions were partially inhibited by prazosin. After desensitization of the P2X‐purinoceptor with α,β‐methylene ATP, contractions in response to electrical stimulation were significantly reduced at all frequencies tested (4–64 Hz). In most cases, contractions were abolished by a combination of both drugs. In vessels treated with 6‐hydroxydopamine, no nerve‐mediated contractile responses were observed. In arteries from reserpine‐treated rabbits, nerve stimulation evoked contractions that were resistant to prazosin. After desentization of the P2X‐purinoceptor with α,β‐methylene ATP, no neurogenic contractile response remained. The tissue contracted to exogenously applied noradrenaline and α,β‐methylene ATP. There was an increase in sensitivity to noradrenaline in 6‐hydroxydopamine‐treated vessels compared with control vessels, but no difference in potency to α,β‐methylene ATP was detected. The potency of noradrenaline and α,β‐methylene ATP was not significantly affected by reserpine treatment. In control tissues, fluorescence histochemistry demonstrated the presence of noradrenergic nerve fibres. Noradrenaline‐containing nerves were not observed in 6‐hydroxydopamine‐treated or reserpine‐treated vessels. It is concluded that noradrenaline and ATP are cotransmitters in the sympathetic nerves supplying the hepatic artery of the rabbit. In contrast to other vessels, the hepatic artery requires a relatively high frequency of stimulation to evoke contractions and the purinergic component is not frequency‐dependent. The significance of this finding in terms of the physiological demands of blood flow to the liver are discussed.