THE USE OF ANTAGONISTS TO CHARACTERIZE THE RECEPTORS MEDIATING DEPOLARIZATION OF THE RAT ISOLATED VAGUS NERVE BY ALPHA,BETA-METHYLENE ADENOSINE 5'-TRIPHOSPHATE

1 We have previously found that the P-2x-purinoceptor agonist, alpha,beta-methylene adenosine 5'-triphosphate (cr,beta-methylene ATP), depolarizes the rat cervical vagus nerve, measured with a 'grease-gap' extracellular recording technique. This effect was attenuated by the P-2 purinoceptor antagonist, suramin. In the present study we have investigated in more detail the antagonism produced by suramin and have also investigated the actions of two other putative P-2 purinoceptor antagonists, cibacron blue and pyridoxal-phosphate-6-azopheny1-2', 5'-disulphonic acid (iso-PPADS). Furthermore, we have studied the interactions between suramin and cibacron blue or iso-PPADS in an attempt to determine whether these antagonists act at a common receptor site. 2 Suramin (1 x 10(-5)-1 x 10(-4) M) produced reversible, concentration-related rightward displacements of the concentration-effect curve to alpha,beta-methylene ATP. Schild analysis of this antagonism yielded a pA(2) value of 5.90 with a slope value of 0.47. 3 Cibacron blue (3 x 10(-5)-1 x 10(-4) M) also antagonized depolarizations induced by alpha,beta-methylene ATP. The antagonistic effects of cibacron blue were slow to reach equilibrium but could be readily reversed on washout. At low concentations for antagonism, cibacron blue (1 x 10(-5) M and 3 x 10(-5) M) produced enhancement of the maximal response to alpha,beta-methylene ATP. At the highest concentration tested (1 x 10(-4) M) the concentration-effect curve to alpha,beta-methylene ATP was shifted to the right in a parallel manner, yielding a pK(B) estimate of 4.96. 4 Iso-PPADS (1 x 10(-6)-1 x 10(-5) M) produced a concentration-related depression in the maxima of the concentration-effect curves to alpha,beta-methylene ATP. Analysis of these data by a double reciprocal plot yielded a pK(B) estimate of 6.02. This profile of insurmountable antagonism could not be attributed to irreversible binding of iso-PPADS to the receptor since the effect of iso-PPADS could be reversed on washing, albeit slowly. 5 In the presence of suramin (1 x 10(-4) M), cibacron blue (1 x 10(-4) M) produced no further rightward displacement of the alpha,beta-methylene ATP concentration-effect curve. The mean agonist concentration-ratios in the presence of suramin or cibacron blue alone (11.7 and 10.3, respectively) were not significantly different from the mean concentration-ratio in the presence of both antagonists (11.8). This finding suggests that high concentrations of alpha,beta-methylene ATP activate a receptor population which is resistant to blockade by either antagonist. 6 The antagonistic effect of iso-PPADS (1 x 10(-5) M) was partially attenuated by suramin (1 x 10(-4) M). It is possible that this interaction reflects a slow dissociation of iso-PPADS from the receptor with which suramin and alpha,beta-methylene ATP interact. 7 Suramin, cibacron blue or iso-PPADS had no marked effect on depolarization produced by 5-hydroxytryptamine (5-HT, 1 x 10(-7)-3 x 10(-5) M), indicating their specificity in antagonizing responses to alpha,beta-methylene ATP. 8 Responses to alpha,beta-methylene ATP were not antagonized by 8-para-sulphophenyltheophylline (3 x 10(-5) M), ondansetron (1 x 10(-7) M), bicuculline (1 x 10(-5) M), phentolamine (1 x 10(-6) M) or hexamethonium (1 x 10(-4) M), which are antagonists at P-1-purinoceptors, 5-HT3 receptors, GABA(A) receptors, alpha-adrenoceptors and nicotinic cholinoceptors, respectively, thereby excluding the involvement of these receptors. Indomethacin (3 x 10(-6) M) had no effect on responses to alpha,beta-methylene ATP. 9 The results obtained with three purinoceptor antagonists confirm and extend our original supposition that alpha,beta-methylene ATP-induced depolarization of the rat vagus nerve is mediated predominantly via P-2 purinoceptors, thought to be of the P-2x subtype. The finding that responses induced by high concentrations of agonist were resistant to blockade by suramin and cibacron blue, but could be attenuated by iso-PPADS, adds further weight to our speculation that the purinoceptor population in the rat vagus nerve is heterogeneous.