The use of antagonists to characterize the receptors mediating depolarization of the rat isolated vagus nerve by α,β‐methylene adenosine 5′‐triphosphate

Abstract

We have previously found that the P2x‐purinoceptor agonist, α,β‐methylene adenosine 5′‐triphosphate (α,β‐methylene ATP), depolarizes the rat cervical vagus nerve, measured with a ‘grease‐gap’ extracellular recording technique. This effect was attenuated by the P2 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 P2 purinoceptor antagonists, cibacron blue and pyridoxal‐phosphate‐6‐azophenyl‐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. Suramin (1 × 10−5–1 × 10−4 m) produced reversible, concentration‐related rightward displacements of the concentration‐effect curve to α,β‐methylene ATP. Schild analysis of this antagonism yielded a pA2 value of 5.90 with a slope value of 0.47. Cibacron blue (3 × 10−5–1 × 10−4 m) also antagonized depolarizations induced by α,β‐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 × 10−5 m and 3 × 10−5 m) produced enhancement of the maximal response to α,β‐methylene ATP. At the highest concentration tested (1 × 10−4 m) the concentration‐effect curve to α,β‐methylene ATP was shifted to the right in a parallel manner, yielding a pKB estimate of 4.96. Iso‐PPADS (1 × 10−6–1 × 10−5 m) produced a concentration‐related depression in the maxima of the concentration‐effect curves to α,β‐methylene ATP. Analysis of these data by a double reciprocal plot yielded a pKB 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. In the presence of suramin (1 × 10−4 m), cibacron blue (1 × 10−4 m) produced no further rightward displacement of the α,β‐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 α,β‐methylene ATP activate a receptor population which is resistant to blockade by either antagonist. The antagonistic effect of iso‐PPADS (1 × 10−5 m) was partially attenuated by suramin (1 × 10−4 m). It is possible that this interaction reflects a slow dissociation of iso‐PPADS from the receptor with which suramin and α,β‐methylene ATP interact. Suramin, cibacron blue or iso‐PPADS had no marked effect on depolarization produced by 5‐hydroxytryptamine (5‐HT, 1 × 10−7–3 × 10−5 m), indicating their specificity in antagonizing responses to α,β‐methylene ATP. Responses to α,β‐methylene ATP were not antagonized by 8‐para‐sulphophenyltheophylline (3 × 10−5 m), ondansetron (1 × 10−7 m), bicuculline (1 × 10−5 m), phentolamine (1 × 10−6 m) or hexamethonium (1 × 10−4 m), which are antagonists at P1‐purinoceptors, 5‐HT3 receptors, GABAA receptors, α‐adrenoceptors and nicotinic cholinoceptors, respectively, thereby excluding the involvement of these receptors. Indomethacin (3 × 10−6 m) had no effect on responses to α,β‐methylene ATP. The results obtained with three purinoceptor antagonists confirm and extend our original supposition that α,β‐methylene ATP‐induced depolarization of the rat vagus nerve is mediated predominantly via P2 purinoceptors, thought to be of the P2x 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. 1994 British Pharmacological Society