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1. The effects of NF023, the symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid), and its parent compound suramin were investigated on vasoconstrictor responses to alpha, beta-methylene ATP in rabbit isolated saphenous artery and vasodilator responses to ATP in noradrenaline-precontracted rabbit isolated thoracic aorta. 2. In rabbit isolated saphenous artery, alpha, beta-methylene ATP-induced vasoconstrictor responses via P2X-receptors were concentration-dependently and competitively antagonised by NF023 (30-300 microM; pA2 = 5.69 +/- 0.04). Suramin (100-1000 microM) also competitively blocked vasoconstrictor responses to alpha, beta-methylene ATP, albeit with lower potency (pA2 = 4.79 +/- 0.05). In contrast, NF023 (100 microM) did not significantly affect contractile responses to noradrenaline or histamine in the saphenous artery. 3. In noradrenaline-precontracted rabbit isolated thoracic aorta preparations, ATP (3-3000 microM) concentration-dependently induced relaxations via endothelium-dependent or smooth muscle P2Y-receptor subtypes. NF023 (30-300 microM) failed to block relaxant responses to ATP at endothelium-dependent P2Y-receptors, whereas suramin (100-1000 microM) did antagonise endothelium-dependent vasodilator responses to ATP. Neither NF023 (100 microM) nor suramin (300 microM) influenced vasorelaxant responses to ATP via endothelium-independent P2Y-receptors. 4. In conclusion, this study outlines the selectivity of NF023 as an effective P2X-receptor antagonist in rabbit isolated blood vessels without affecting endothelium-dependent or endothelium-independent P2Y-receptor subtypes, adrenoceptors or histamine receptors.

8,8'-(Carbonylbis(imino-4, 1 -phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3, 5-naphthalenetrisulfonic acid) (NF279) antagonized P2X receptor-mediated contractions in rat vas deferens, evoked by alpha,beta-methylene ATP (10 microM; pIC50=5.71) without affecting responses mediated via alpha1A-adrenoceptors, adenosine A1 and A2B receptors, histamine H1, muscarinic M3 and nicotinic receptors. The low inhibitory potency of NF279 on P2Y receptors in guinea-pig taenia coli (pA2=4.10) and at ecto-nucleotidases in folliculated Xenopus laevis oocytes (IC50 > 100 microM) indicates that NF279 is a novel specific and selective P2X receptor antagonist.

At least five distinct P2-purinoceptor subtypes have been characterized to date, based on the rank order of potency of several ATP analogues: P2X, P2Y, P2U, P2T and P2Z. However, the characterization of P2-purinoceptor subtypes is hampered by an unavailability of potent, highly selective, competitive antagonists. In the search for selective P2-purinoceptor antagonists, the structure-activity relationships for a series of analogues of pyridoxal-5-phosphate and suramin at P2-purinoceptor subtypes were investigated in our laboratories. Two of these compounds were the subject of a more detailed pharmacological characterization: pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) and the symmetrical 3′-urea of 8-(benzamido)naphthalene-1,3,5-trisulfonic acid (NF023). The results demonstrate that the two parent compounds, pyridoxal-5-phosphate and suramin, do not differentiate between P2X- and P2Y-purinoceptors. In contrast, PPADS and NF023 were found to be selective antagonists of P2X-purinoceptor-mediated responses in several smooth muscle preparations. In addition, PPADS and NF023 were shown to displace competitively the binding of [3H]α,β-methylene ATP to rabbit and rat bladder membranes, respectively, which indicates that these two compounds act directly on P2X-receptors. PPADS and NF023 were ineffective at P2U-purinoceptors in rat mesenteric arterial bed. P2T-purinoceptor-mediated platelet aggregation was only affected by PPADS in concentrations higher than 100 μM. Suramin and NF023 were inhibitors of ecto-ATPase activity in the same concentration range needed for P2-purinoceptor antagonism. In contrast, PPADS was only very weakly active in inhibiting ecto-ATPase activity. AT 100 μM, PPADS and NF023 did not interact with α1-adrenoceptors, adenosine A1- and A2-, histamine H1- and muscarinic M1-, M2- and M3-receptors. In conclusion, PPADS and NF023 are specific P2-purinoceptor antagonists showing a high selectivity for the P2X-subtype. These two compounds may prove to be useful starting points in the synthesis of novel, highly potent and selective antagonists at P2-purinoceptor subtypes.