Pharmacological and molecular characterization of P2X receptors in rat pelvic ganglion neurons

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Abstract
1. The presence and characteristics of P2X receptors on neurons of the rat major pelvic ganglia (MPG) have been studied using whole cell voltage-clamp, in situ hybridization and immunohistochemistry. 2. Rapid application of ATP (100 microM) to isolated rat MPG neurons induced moderately large inward currents (0.33-5.3 nA) in 39% of cells (108/277). The response to ATP occurred very rapidly, with an increase in membrane conductance, and desensitized slowly. 3. The concentration-response curve for ATP yielded an EC50 of 58.9 microM. The agonist profile was ATP> or =2MeSATP=ATPgammaS>BzATP, while alpha,beta-MeATP, beta,gamma-MeATP, UTP and ADP were all inactive at concentrations up to 100 microM. 4. The response to ATP was antagonized by suramin (pA2=5.6), reactive blue-2 (IC50=0.7 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 5. Lowering the pH from 7.4 to 6.8 produced a marked potentiation (to 339% of control) of the responses to ATP (30 microM), while raising the pH to 8.0 attenuated the responses (to 20% of control). The EC50s for ATP were 28.8, 58.9 and 264 microM at pH 6.8, 7.4 and 8.0, respectively. 6. Co-application of ATP with Zn2+ produced a marked enhancement of the responses to ATP, with an EC50 of 9.55 microM. In the presence of Zn2+ (30 microM), the EC50 for ATP was decreased to 4.57 microM. 7. In situ hybridization revealed that the P2X receptor transcripts levels in rat MPG neurons are P2X2>P2X4>P2X1, P2X3, P2X5 and P2X6. The immunohistochemical staining revealed a small number of neurons with strong P2X2 immunoreactivity. 8. In conclusion, our results indicate that there are P2X receptors present on MPG neurons. The pharmacological characteristics of these receptors, the in situ hybridization and immunohistochemical evidence are consistent with them being of the P2X2 subtype, or heteromultimers. with P2X2 being the dominant component.