ABSTRACT
1. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a P2-purinoceptor antagonist, was investigated for its ability to antagonize: (1) P2X-purinoceptor-mediated contractions of the rabbit central ear artery and saphenous artery evoked by either alpha,beta-methylene ATP (alpha,beta-MeATP) or electrical field stimulation (EFS); (2) P2Y-purinoceptor-mediated relaxations of the rabbit mesenteric artery; (3) endothelium-dependent and endothelium-independent, P2Y-purinoceptor-mediated relaxations of the rabbit aorta. 2. alpha,beta-MeATP (0.1-100 microM) caused concentration-dependent contractions of the rabbit ear and saphenous arteries. The negative log[alpha,beta-MeATP] that produced a contraction equivalent to the EC25 for noradrenaline (ear artery) or histamine (saphenous artery) in the absence of PPADS was 6.60 +/- 0.18 (9) and 6.18 +/- 0.17 (9) in the ear artery and saphenous artery, respectively. These effects of exogenous alpha,beta-MeATP were concentration-dependently inhibited by PPADS (1-30 microM). In the ear artery, the negative log[alpha,beta-MeATP] producing a contractile response equivalent to the EC25 of noradrenaline, in the presence of PPADS at 1, 3 and 10 microM was 6.16 +/- 0.18 (8), 5.90 +/- 0.18 (8) and 4.72 +/- 0.36 (8), respectively (P < 0.01). In the saphenous artery, the negative log[alpha,beta-MeATP] values equivalent to the EC25 for histamine in the presence of PPADS at concentrations of 1, 3, 10 and 30 microM were 5.90 +/- 0.19 (8), 5.73 +/- 0.16 (8), 4.99 +/- 0.14 (8) and 4.51 +/- 0.13 (8), respectively (P < 0.01). 3. PPADS at a concentration of 1 microM had no effect on contractions of the ear artery evoked by EFS (4-64 Hz; 1 microM phentolamine present). At higher concentrations (3-30 MicroM) it caused concentration dependent inhibition of neurogenic contractions. In the saphenous artery, PPADS (1-30 MicroM) concentration-dependently inhibited contractions evoked by EFS at frequencies of 4, 8 and 16 Hz. Contractions evoked by EFS at frequencies of 32 and 64 Hz were significantly inhibited by PPADS only at concentrations of 10 and 30 MicroM.4. PPADS (30 MicroM) had no effect on relaxations to 2-methylthio ATP (3 nM-3 MicroM) in rabbit mesenteric artery and to ATP (1 MicroM-I mM) in rabbit aorta (with endothelium intact or removed). In addition,PPADS (30 MicroM) had no significant influence on the contractile potency of noradrenaline and histamine in rabbit ear and saphenous artery, respectively.5. In conclusion, these results support the evidence that PPADS is a selective antagonist of P2X-purinoceptor-mediated responses.
Subject(s)
Blood Vessels/drug effects , Blood Vessels/ultrastructure , Purinergic P2 Receptor Antagonists , Pyridoxal Phosphate/analogs & derivatives , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Aorta/drug effects , Aorta/physiology , Ear/blood supply , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Histamine/pharmacology , In Vitro Techniques , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiology , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle Relaxation/drug effects , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/ultrastructure , Norepinephrine/pharmacology , Pyridoxal Phosphate/pharmacology , RabbitsABSTRACT
The aim of the present study was to characterize the prejunctional modulation of evoked release of endogenous noradrenaline in rabbit vas deferens by the use of muscarinic receptor agonists and subtype-preferring antagonists. Vasa deferentia of the rabbit were stimulated electrically by trains of 120 pulses delivered at 4 Hz or trains of 30 pulses at 1 Hz. The inhibition by muscarinic agonists of the stimulation-evoked overflow of endogenous noradrenaline in the absence and presence of antagonists was used to determine affinity constants for antagonists. These values were compared with those observed at putative M1 receptors inhibiting neurogenic twitch contractions in the rabbit vas deferens and with affinity data obtained at M1(m1)-M4(m5) receptors in functional studies and binding experiments. The evoked overflow of noradrenaline from sympathetic nerves was enhanced by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and indomethacin, indicating a tonic inhibition by endogenous A1 and P2 purinoceptor agonists and prostanoids, respectively. The stimulation-evoked overflow at 4 Hz was not sensitive to inhibition by the muscarinic agonists methacholine or 4-(4-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). In contrast, at a stimulation frequency of 1 Hz the evoked noradrenaline release was decreased by muscarinic agonists (EC50): arecaidine propargyl ester (0.062 microM), 4-Cl-McN-A-343 (0.32 microM), 4-(4-fluorophenylcarbamoyloxy)-2-butynyl-N-methyl-pyrrolidinium tosylate (4-F-PyMcN+; 0.48 microM) and methacholine (0.86 microM). The affinity constants of most of the muscarinic antagonists [atropine: pKB = 9.47; (R)-trihexyphenidyl: pKB = 9.18; pirenzepine: pA2 = 7.68; methoctramine: pKB = 6.90] are consistent with estimates of these antagonists at M1(m1) receptors determined in various functional and binding studies. The high antagonistic potency of pirenzepine and (R)-trihexyphenidyl and the agonistic activity of 4-F-PyMcN+ argue for the involvement of M1, and against that of M2 and M3 receptors in the inhibition of evoked noradrenaline overflow. However, the high apparent pKB of 8.30 for himbacine is not in accordance with an M1 receptor; by contrast, it would be compatible with the presence of M2 or M4 receptors. The potencies of the tested muscarinic agonists and antagonists largely agree with those obtained for the inhibition of neurogenic twitch responses (0.05 Hz) in the rabbit vas deferens.(ABSTRACT TRUNCATED AT 400 WORDS)
