Study Of Myocardial Ischemic Nociceptive Signaling Mediated By P2X₃ Receptor In Rat Stellate Ganglion Neurons

Objective To explore the effects of P2X3 receptor on myocardial ischemic nociceptive signaling in stellate ganglion (SG) neurons.Methods (1) P2X3 immunoreactivity and P2X3 protein were detected by immunohistochemistry and western blotting; (2) P2X3 mRNA was detected by in situ hybridization and reverse transcriptive polymerase chain reaction (RT-PCR); (3)P2X3 receptor agonist-activated currents and the effects of A-317491 on P2X receptor agonist-activated currents were recorded by whole-cell patch-clamp technique.Results (1)By the immunohistochemistry, the staining of P2X3 receptor in the SG neurons of rats after myocardial ischemic injury appeared to be more intense than those treated with A-317491 and those of naive rats, being 219.87±7.59(myocardial ischemic group, n = 8), 203.43±10.67(myocardial ischemic rats treated with A-317491 group, n=7 ;p<0.05) and 198.09±24.43(control group, n=8; p<0.01) respectively;(2) By western blotting, the P2X3 protein of rat SG in myocardial ischemic group, control group and myocardial ischemic rats treated with A-317491 group were 1.48±0.27, 1.35±0.33(P<0.01) and 1.32±0.48(P<0.01) respectively; (3) By in situ hybridization, the signals of P2X3 mRNA in the SG neurons of rats after myocardial ischemic injury appeared to be more intense than those treated with A-317491 and those of naive rats, being177.21±21.99,148.52±32.12(P<0.01) and 151.91±13.91 (P<0.01) respectively;(4) RT-PCR revealed that the P2X3 mRNA of rat SG in myocardial ischemic group, control group and myocardial ischemic rats treated with A-317491 group were 0.231±0.0040. 0.120±0.004 (P<0.01)and 0.135±0.004 (P<0.01) respectively;(5) The amplitude of the currents increased with the enlarging concentration of ATP(1-1000μmol/L). It was much larger in myocardial ischemic group than those obtained in control group after application of same concentration ATP. The mean peak of currents measured in myocardial ischemic rats was 2.4 times higher than that measured in control rats when the concentration of ATP was 100μmol/L. After application ofα,β-meATP(10μmol/L ), the amplitude of the current in myocardial ischemic group was also larger than that in control group. A-317491, a high affinity and selective antagonist of P2X3 receptor, could inhibit ATP-activated currents(100μmol/L)andα,β-meATP-activated currents(10μmol/L) in the concentration range from 100 to 10000 nmol/L.The inhibition of A-317491 was stronger in myocardial ischemic group than that in control group after application of same concentration of A-317491.Conclusions The expression of P2X3 protein and P2X3 mRNA increased in SG neurons after myocardial ischemia. P2X receptor agonist activated currents of SG neurons increased after myocardial ischemia and decreased after the treatment of A-317491. A-317491, a high affinity and selective antagonist of P2X3 receptor, could decrease the expression of P2X3 protein and P2X3 mRNA increased in SG neurons after myocardial ischemia and inhibit the enhanced currents activated by P2X receptor agonists after myocardial ischemia. These results may be suggestive for the involvement of SG neuron P2X3 receptor in cardiac nociceptive transmission.