Effects Of P2X Receptors Within The Nucleus Ambiguus On Hypoxian Respiration In The Rabbit

Objective:(1)The purpose of this study is to examine whether P2X receptor in the nucleus ambiguus were involved in the regulation of normoxia and hypoxia respiratory response by microinjection of P2X receptor agonist ATP and antagonist pyfidoxal-5'-phosphate-6'-azophenyl-2',4'-disulphonic acid(PPADS);(2)To observe the change of number of P2X receptor in NA before and after hypoxia using immunohistochemical methods,to investigate role of P2X receptors in acute hypoxic ventilatory response.Meanwhile,we employed immunohistochemical methods in our experiments in order to explore whether P2X receptor localize in NA neurons.Methods:1.Electrophysiological experiments:Microinjected:The experiment was performed on anesthetized,paralyzed and artificially ventilated adult rabbits.The hypoxia respiratory response was produced by ventilating the animals with 10%O₂(Nitrogen balanced).Phrenic nerve discharge was recorded as respiratory indicator.The NA was localized with extracellular recording of respiratory neurons.After microinjection of ATP into NA respectively,the changes of phrenic discharge and arterial blood pH under normoxia and hypoxia conditions were observed.To test the dose-dependency of ATP's effect on respiration when injected into raphe nuclei,a total of 4 doses(0.1,0.3,0.5,0.7μl)were injected into NA,with any two injections separated by 1 hour;Microinjection PPADS,Five minutes after the microinjection of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid(PPADS),a broad-spectrum P2X receptor antagonist,into the NA,another microinjection of ATP(0.2 mM,0.3μl)at the same site within the NA.These records were used to investigate the effects of each experimental factor.Comparisons of these records were also made among different groups to find the difference of effects caused by different experimental factors.2.Immunohistochemical experiment:The experiment was conducted on 32 healthy adult rabbits,which were divided into four groups,from Group A to Group D.Group A(n=8):microinjecting physiological saline solution;Group B(n=8): microinjecting ATP;Group C(n=8):microinjection PPADS;Group D(n=8):no microinjection.Rabbits in Group A,B,C were killed rapidly after exposed in the circumstance of acute hypoxia,Group D was killed directly.The brain stem tissue were then fixed in 10%formalin PBS solution.By using of the immunohistochemical SP method,we observed and compared the different level of P2X receptor in the nucleus ambiguus in different group.Results:1.Electrophysiological experiment:Both the amplitude and frequency of phrenic discharges were increased rapidly and reached maximum at 2 min after the onset ofhypoxia,and then tended to decline but remained higher than control as long as the hypoxia was continued.Microinjection of ATP into NA respectively had little effect with normoxic ventilation,but increased hypoxia respiratory response and increased arterial blood pH;Microinjection of ATP(0.2 mM 0.1-0.7μl)into NA caused dose-dependent increases in phrenic amplitude and respiratory frequency.Microinjection of PPADS,a broad-spectrum P2X receptor antagonist,into the NA did not cause any significant change in respiration,but it partially blocked the respiratory effects of ATP that was subsequently injected into the same sites within the NA.2.Fluorescent immunostaining:P2X receptor located in NA was observed in every group.Furthermore,the numbers of phosphortyrosine immunopositive neurons were increased after acute hypoxia.The strongest expression was found in Group A,B,C,Group D is the slightest(P＜0.01).Conclusion:The results in this part suggest that under normoxic ventilation P2X receptor located in the NA play little role in regulation of respiration,but it underly important functional hypoxia respiratory components.In addition,with immunofluorescence staining we found that P2X receptor existed in the postsynaptic membrane within rabbits' NA and increased after hypoxic challenge, which suggested there might be play especially under hypoxia condition.Therefore, we conclude that in the NA after accepting hypoxia signal,P2X receptor regulates sensitivity of hypoxia respiratory response.Significances:P2X receptors in the medullary NA obvious expression for the ATP on the regulation of respiratory rhythm provides a functional binding site for further study in the ventilatory response,the body feeling Pao₂,Paco₂ and hydrogen ion concentration changes,how will the information-respiratory center to provide a new approach.