Dual control of local blood flow by perivascular nerves and endothelial cells: changes in development, atherosclerosis and acrylamide neuropathy

This thesis examines the influence of perivascular nerves and the vascular endothelium on the local control of vascular tone with particular emphasis on how these mechanisms are affected by age and in atherosclerosis. Using the method of in vitro pharmacology, it was shown that ATP is a cotransmitter with noradrenaline (NA) in the hepatic artery of the rabbit and that the response of the vessel to the purine is mediated by ATP acting through post-junctional P2x-purinoceptors. Acetylcholine (ACh) induced a vasodilatation that was entirely dependent on the presence of an intact endothelium whereas adenosine, ATP and 2-methylthio ATP (a selective P2y-purinoceptor agonist) were shown to elicit a relaxation that was independent of the endothelium. The presence of nerve fibres containing substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were demonstrated in the hepatic artery of the rabbit. Furthermore, it was shown that CGRP and VIP mediated a vasodilatation in the absence of endothelium whereas SP produced a relaxation that was endothelium-dependent. Changes in the reactivity of hepatic and saphenous arteries from male and female rabbits (2-36 months) in response to directly acting vasoconstrictor agents (NA, a, methylene ATP (a, meATP), KCl); endothelium-dependent vasodilator agents (ACh, SP); endothelium-independent vasodilator agents (CGRP, VIP, ATP) and transmural nerve stimulation are described. Male and female Watanabe Heritable Hyperlipidaemic (WHHL) rabbits (4-12 months) were used as a model for human homozygous hypercholesterolemia. After an initial reduction in endothelium-dependent vasodilatation, there was an increase in relaxation at 12 months, when plaques were present. Contractions to sympathetic nerve stimulation was also reduced at 12 months. The effects of acrylamide, which is regarded as a model for autonomic neuropathy, on the response of the hepatic, saphenous and basilar arteries are also described

Structure activity relationships for derivatives of adenosine-5?-triphosphate as agonists at P2 purinoceptors: Heterogeneity within P2x and P2y subtypes

The structure-activity relationships for a variety of adenine nucleotide analogues at P2x- and P2Y-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N6-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 P2Y-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 P2X-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 P2X- or the P2Y-purinoceptors. Certain analogues displayed selectivity or specificity within the P2X- or P2Y-purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2′,3′-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P2Y-purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P2X-purinoceptors. The potent agonist N6-methyl ATP and the somewhat less potent agonist 2′-deoxy-ATP were selective for P2Y-purinoceptors in the guinea pig taenia coli, but were inactive at P2X-purinoceptors and the vascular P2Y-purinoceptors. 3′-Benzylamino-3′-deoxyATP was very potent at the P2X-purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P2Y receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P2X- and P2Y-purinoceptor classes

Identification of potent, selective P2Y-purinoceptor agonists: structure-activity relationships for 2-thioether derivatives of adenosine 5'-triphosphate

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 agonists, 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 coil, 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 coli 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