| Objective: To explore the effects of P2X3 and P2X2/3 receptor on myocardial ischemic nociceptive signaling in nodose ganglion (NG) neurons and myocardium.Methods: (1) Behavioral test was used to observe the pain behavioral;(2) Histology of cardiac tissue, electrocardiac changes and myocardial enzyme level in the serum from control, myocardial ischemic rats and myocardial ischemic rats treated with A-317491 were characterized by hematoxylin and eosin staining (H–E stain), electrocardiogram (ECG) and automatic biochemistry analyzer to detect myocardial function changes. P2X3 mRNA and P2X3 protein in the myocardial tissue were detected by in situ hybridizationand western blotting.(3) Detections of the nodose ganglion (NG) neurons: P2X2/3 immunoreactivity and P2X2/3 protein were detected by immunohistochemistry and western blotting; P2X2/3 mRNA was detected by in situ hybridization and reverse transcriptive polymerase chain reaction (RT-PCR).Results: (1) Behavioral test reveals that myocardial ischemic rats displayed typical pain behavioral, and treated with A-317491, the myocardial ischemic animals show pain lessen behavioral; (2) Detections of myocardial changes:①H–E stain results show that the myocardial tissues from myocardial ischemic group showed severe atrophy, hypertrophy and proliferation of fiber connective tissue; the myocardial tissues showed slight atrophy and vicarious hypertrophy in myocardial ischemic rats treated with A-317491 group;②By ECG, in the myocardial ischemic injury rat models, the heart rates (264.63±32.30) were slower than that (361.38±29.08) in the control (p<0.05), and T waves became unusually tall with prominent; when A-317491 was injected intravenously into rats, the heart rates were quickened from 264.63±32.30 to 310.13±31.75 in the myocardial ischemic injury rat models, then the sign of premature beats in the myocardial ischemic injury rat model can also improved by A-317491.③The myocardial enzymes such as CK(6663.967±230.793), CK-MB(3091.553±370.284) and LDH(2469.4±147.5876) activities were significantly elevated after myocardial ischemia compared with the activities measured in the control rats, A-317491 significantly attenuated the ischemia induced elevations in serum LDH (907.0333±47.2300, p<0.05), CK (2960.567±55.906, p < 0.05) and CK-MB (1364.823±121.6325, p < 0.05) activities;④B y in situ hybridization, the signals of P2X3 mRNA in the ischemic injury hearts (147.75±5.67) appeared to be more intense than those in naive hearts (114.75±2.12, p < 0.01). A-317491 reduced the P2X3 mRNA expression to(119.86±4.82, p<0.05) in the ischemic injury hearts;⑤P2X3 expression in protein level was analyzed by western blotting. The gray value in the myocardial ischemic group (138.66±3.47) was higher than that in control group (101.38±5.62) with statistically significant difference (p<0.01); The myocardial ischemic rats treated with A-317491, the gray value (112.35±2.65) was lower than that in myocardial ischemic group (p<0.01) ; (3) The changes of P2X2/3 receptor in nodose ganglion (NG) neurons:①By the immunohistochemistry, the staining (113.77±5.58) of P2X2 receptor in the NG neurons of rats after myocardial ischemic injury appeared to be more intense than that (108.25±8.83, p<0.05) in treated with A-317491 and that (104.26±6.34, p<0.05) in naive rats, respectively; the gray value of the staining of P2X3 receptor in the NG neurons of rats, being 118.51±8.00 (myocardial ischemic rats treated with A-317491 group, p<0.05) and 121.29±8.73 (control group,p<0.05) were lower than that (134.39±9.03) in myocardial ischemic group;②By in situ hybridization (ISH), the levels of P2X2/3 mRNA expression were studied in nodose ganglia was significant difference between myocardial ischemic injury rats and naive control rats. The signals of P2X2 mRNA (153.83±9.04) in myocardial ischemic group were lower than those in control group (137.35±7.48,p<0.05). Compared with myocardial ischemic group, the data in rat treated with A-317491 group (138.07±3.57) were lower than that in myocardial ischemic rats (143.49±9.85); and the signals of P2X3 mRNA in myocardial ischemic group(143.49±9.85) were higher than those in control group (123.31±9.02, p<0.05). Compared with myocardial ischemic group, the data (127.55±4.73) in rat treated with A-317491 group were lower than that in myocardial ischemic rats (p<0.05) too.③RT-PCR revealed that the P2X2 mRNA of rat NG in myocardial ischemic group (1.407±0.09) was higher than those in myocardial ischemic rats treated with A-317491 group (1.136±0.05) and control group (1.131±0.04) (p<0.01) respectively; and the P2X3 mRNA of rat NG in myocardial ischemic group (0.847±0.05) was higher than those in control group (0.583±0.02) and myocardial ischemic rats treated with A-317491 group (0.666±0.04)(p<0.01),respectively.④By western blotting, the P2X2 protein of rat NG in myocardial ischemic group (2.13±0.09) was higher than those in control group (0.819±0.03) (p<0.01)and myocardial ischemic rats treated with A-317491 group (1.48±0.05)(p<0.05),respectively; and the P2X3 protein of rat NG in myocardial ischemic group (2.15±0.06) was higher than those in control group (0.90±0.04) (p<0.01) and myocardial ischemic rats treated with A-317491 group (0.93±0.05) (p<0.01) , respectively.Conclusions: The expression of P2X3 or P2X2/3 protein and mRNA were increased in myocardial tissues and NG neurons after myocardial ischemia, respectively. A-317491, a high affinity and selective antagonist of P2X3 and P2X2/3 receptor, could decrease the expression of P2X3 or P2X2/3 protein and mRNA in myocardial tissues and NG neurons after myocardial ischemia. These results may suggest that for the P2X3 or P2X2/3 receptors in myocardial tissue and NG neuron are involved in myocardial nociceptive transmission. |
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