Identification of Atropine- and P2X1Receptor Antagonist-Resistant, Neurogenic Contractions of the Urinary Bladder

Abstract

Acetylcholine and ATP are excitatory cotransmitters in parasympathetic nerves. We used P2X1 receptor antagonists to further characterize the purinergic component of neurotransmission in isolated detrusor muscle of guinea pig urinary bladder. In the presence of atropine (1 microM) and prazosin (100 nM), pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (0.1-100 microM) and suramin (1-300 microM) inhibited contractions evoked by 4 Hz nerve stimulation in a concentration-dependent manner (IC50 of 6.9 and 13.4 microM, respectively). Maximum inhibition was 50-60%, which was unaffected by coadministration of the ectonucleotidase inhibitor ARL67156 (6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP) (100 microM). The remaining responses were abolished by tetrodotoxin (1 microM). PPADS and suramin also reduced contractions to exogenous ATP (300 microM) by 40-50%, but abolished those to the P2X1 agonist alpha,beta-methyleneATP (alpha,beta-meATP) (1 microM). The P2X1 antagonists reactive blue 2, NF279 (8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)] bis-1,3,5-naphthalenetrisulfonic acid), MRS2159 (pyridoxal-alpha5-phosphate-6-phenylazo-4'-carboxylic acid) (100 microM), and NF449 [4,4',4,4-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid] (3 microM) abolished contractions to alpha,beta-meATP (1 microM; n = 4-5), but only reduced contractions evoked by 4 Hz nerve stimulation by approximately 40-60% (n = 4-6) and ATP by 30-60% (n = 4-7). However, prolonged exposure to alpha,beta-meATP (50 microM) abolished contractions evoked by all three stimuli (n = 5-12). PPADS (100 microM) and suramin (300 microM) reduced the peak neurogenic contraction of the mouse urinary bladder to 30-40% of control. At the same concentrations, the P2X1 antagonists abolished the nonadrenergic, purinergic component of neurogenic contractions in the guinea pig vas deferens (n = 4-5). Thus, P2X1 receptor antagonists inhibit, but do not abolish, the noncholinergic component of neurogenic contractions of guinea pig and mouse urinary bladder, indicating a second mode of action of neuronally released ATP. This has important implications for treatment of dysfunctional urinary bladder, for which this atropine- and P2X1 antagonist-resistant site represents a novel therapeutic target.