The Mechanism Of Central Acid-sengsing Ion Channnels(ASICs) On Respiratory Regulation In Rat

Purpose Respiratory movementknown as a kind of hythmic aetivity can be regulated by ptratory canter. The central respiratory chemoreceptors (CRCs) sense the changes of extrocellular pH and the concentration of CO2, and transduct the chemical signal the electrical signal to excite the motoneuron of respiratory center. The CRCs is mainly distributed in ventrolateral medulla (VLM). Recenty, the Orexinergic neurons in the lateral hypothalamus (LH) have been noticed for its acid-sensing properties. However, the ionic mechanism of acid sensing of CRCs is unclear.1997, one of the acid sensing ion channels (ASICs) subunits which is belong to a family of proton-gating cation channel was been cloned firstly. Researches show that ASICs are involved in several physiological and pathophysiological functions such as learning and memory, nociception, sour taste, hearing and visual perception, acidosis-mediated neuronal injury. However, the function of ASICs on central regulation of respiration has not been addressed. We hypothses:1. ASICs expressed in VLM and LH, and taken part in the central regulation of breathing.2. The central mechanism of acid-sengsing ion channnels (ASICs) on respiratory regulation is that ASICs channels on the membrane of Orexin containing neurons percepted the decreace of excellular pH to excite the neurons and then respiroration. There are many fatal diseases such as central respiratory failture, central respiratory rhythm disturbance, congenital central hypoventialation syndrome (CCHS), which are due to dysfunction of respiratory center. Additionally, many pulmonary and airway diseases such as chronic obstructive pulmonary disease (COPD), obstructive sleep apnea syndromes (OSAS) also complicated with the abnormality of respiratory center. So this work is not only meanful in physiology but also in clinical medicine. Maybe ASICs is a pharmacological target for disease of respiratory system.Methods1.Experiments were performed on neonatal (1～5d) and adult (6～8w) SD rats. Expression and distribution of ASIC1-like-immunoreactivity (ASIC1-like-ir) and ASIC2a-like-immunoreactivity (ASIC2a-like-ir) in the medulla and hypothalamus, Orexin A-like-immunoreactivity (OXA-like-ir) in hypothalamu and medulla, Orexin1receptor-like-immunoreactivity (OX1R-like-ir) and the co-location of ASIC1/AISC2a with Neurofiber-H, ASIC1with ASIC2a, ASIC1with Orexin A in the medulla or hypothalamus were observed and compared by using immunohistochemistry ABC method, double immunofluorescence technique, confocal, westernblot and software of ImageMeasure.2. Adult (6-8w) SD rats were anesthetized intraperitoneally. The rats were placed on a stereotaxic frame in a prostrate position and the injection site on the brain surface was exposed. Finally, the phrenic nerve discharge (PND), respiratory rate (RR), artery blood pressure (BP) and heart rate (HR) was recorded pre-and post-aCSF with different pH microinjected into ventrolateral medulla (VLM) or lateral hypothalamus (LH) respectively. The effective dose was routinely given together with one of the test agents to the LH, which included a nonselective ASICs inhibitor, amiloride or a selective ASIC1a inhibitor, tarantula venom PcTX1.The co-microinjection was performed as follow: the inhibitors were administrated first and then the effective pH, and there is no time interval between them. The effect of post-Orexin administration into lateral ventricle (LV) was also observed.3. Adult (6-8w) SD rats were anesthetized intraperitoneally, the phrenic nerve discharge (PND), respiratory rate (RR), artery blood pressure (BP) and heart rate (HR) was recorded pre-and post-the simultaneous microinjection of the aCSF with effective pH into LH and SB408124into the nucleus tractus solitarius (NTS).4. The Orexin containing neurons of LH were lesioned by microinjection of Orexin-SAP. Two weeks after the treatment, weight of the dhult was measured and the respiratory rate (RR), artery blood pressure (BP) and heart rate (HR) was recorded. The effect of microinjection of the aCSF with effective pH on PND was also observed.Results1. The expression of ASIC1and ASIC2a positive neurons in medulla of adult and neonatal SD rats:(1) The ASIC1and ASIC2a positive neurons mainly expressed in VLM of medulla and slightly in the dorsal area of medulla (intercalated nuclus of medulla, In; external cuneate nuclus, ECu) in the adult. The location of ASIC1positive neurons in the neonatal was similar with that in the adult. But the ASIC2a positive neurons merely expressed in VLM of medulla in the neonatal. Relative optical density (ROD) and cell count of ASIC1and ASIC2a positive neurons in VLM of adult and neonatal rats was measured. ROD of ASIC1positive neurons in VLM of adult rats was higher than that of the neonatal rats. But the ROD of ASIC2a in adult rats was less than in neonatal rats (p<0.001, n=6). Cell count of ASIC1and ASIC2a positive neurons in VLM of adult rats was less than in neonatal rats (p<.001, n=6).(2) Western blot analysis of ASIC1and ASIC2a expression in the medulla of adult and neonatal SD rats showed that ASIC1/ASIC2a immunoreactivity was present in medulla. The relative levels of ASIC1and ASIC2a protein expression were less in the adult rats. Data were normalized by the mean value for control (p<0.01, p<0.001, n=6).(3) ASIC1and ASI2a summits were co-espression with Neurofilament-H in VLM of adult SD rats. ASIC1and ASI2a were also co-located with each other.2. The expression of ASIC1and ASIC2a positive neurons in hypothalamus of adult SD rats:Both of ASIC1-like-ir neurons and ASIC2a-like-ir neurons ditributed in dorsal hypoth area (DA) and in the LH. But the expression of them was different. The cell number of ASICl-like-ir neurons was larger in LH than in DA (p<0.001, n=6). Contrarily, ASIC2a-like-ir neurons were mainly in DA (p<0.001, n=6). Additionally, there were more ASIC1-like-ir neurons than ASIC2a-like-ir neurons in LH, and more ASIC2a-like-ir neurons in DA (p<0.01, p<0.001, n=6).3. Unilateral microinjection of acidosis aCSF in VLM and LH enlarged PND and ASICs antagonist inhibited the accretion effect:(1) The chemical stimulation of VLM using aCSF with different pH (pH=7.4,7.0,6.5,6.0,5.5,5.04.5,0.1μl) showed that pH6.5and pH6.0resulted in a greater increase in iPND than pH7.4(p<0.05, n=12).(2)The chemical stimulation of LH using aCSF with different pH (pH=7.4,6.5,5.5,4.5,0.1μl) showed that pH6.5resulted in a greater increase in iPND than pH7.4(p<0.001, n=6).(3) Co-microinjection into LH of the nonselective ASICs inhibitor, amiloride (10mM,0.1μl) or selective ASIC1a inhibitor, PcTX-1(10nM,0.1μl) with pH6.5almost totally blocked the iPND enhancement promoted by pH6.5(both p<.001, n=6). And singly administration of the two inhibitors did not result in alterations in PND. All the gradient of pH and agents did not elicit changes in RR, MAP and HR.4. The effect of Orexin A on the regulation of cardiorespiratory system: Expression of Orexin A-like-ir fibers and OX1R-like-ir neurons were found in the nucleus tractus solitarius (NTS). Unilateral microinjection of Orexin A at different concentrations (10,100,1000μg/ml,5μl) into LV resulted in augmentation of PND, BP and HR. The effects were dose-dependent. The acceleration of PND at all concentrations was significant (p<0.05, p<0.01, p<0.001vs N.S., n=6). The increase of RR at the three concentrations was not significant. The acceleration of HR was notable at100,1000μg/ml concentrations (p<0.01, p<0.01vs N.S., n=6). The increase of MAP was significant at100,1000μg/ml concentrations (p<0.05, p<0.01vs N.S., n=6).5. Co-expression of ASIC1and Orexin A in LH, and the synergistic effect of them in regulation of respiration:(1) Orexin A-like-ir neurons were distributed in LH and co-located with ASIC1.(2) Simultaneous microinjection of SB408124in NTS and aCSF with pH6.5in LH had no effect on PND. Simultaneous microinjection of SB408124in NTS and aCSF with pH7.4in LH didn’t decrease amplitude of PND.(3) There was significant loss of Nissl bodies and Orexin neurons in the LH of Orexin-SAP-treated rats relative to the blank-SAP-treated rats. Few residual Orexin neurons remain in Orexin-SAP-treated rats, two weeks after treatment. There was decrease in MAP and HR in LH lesion group (both p<0.01v.s. blank-SAP treated group, p<0.01, p<0.05v.s. control, respectively), but no change in RR. And microinjection of aCSF with pH6.5in LH no longer increased in LH lesioned rats.Conclusion1. ASIC1and ASIC2a subunits are expressed in the VLM and LH. There is a decrease of ASICs peptides in medulla as mature. The ROD and cell count of ASIC2a-like-ir both reduced in VLM of adult SD rats compared to the neonatal rats. Interestingly, there is a contradictory finding in ASIC1:cell count downgrade, but rod upgrade in adult VLM. Perhaps, the difference expression of ASICs means the distinct sensitivity of central nerve system to chemical stimulation. In LH, ASIC1and ASIC2a are found, especially ASIC1. The result of immunohistochemistry laid the groundwork for the research on the role of ASICs on the central regulation of respiration.2. Acidosis in VLM and LH excited breathing. The distinguished effect is elicited by stimulating LH with artificial cerebrospinal fluid with pH6.5. The effective dose is in accordance with the pHo.sof ASIC1a. And the stimulating effect of pH6.5on respiration was almost completely blocked by ASICs inhibitors (nonselective inhibitor, Amiloride or ASIC1a selective inhibitor, PcTX1), in LH. The result show ASICs take part in central regulation of respiration, and ASIC1a is highlighted.3. ASIC1co-located with orexin in LH, and lesion of Orexin neurons distressed the excitation of respiration provoked by ASICs, additionally, microinjection of Orexin A into LV increased the amplitude of respiration, which show that ASICs contribute to the excitation of respiration via neurotransmitters (Orexin A). Orexin-like-ir fiber and OXiR-like-ir neurons are located in NTS, so we blocked OX1R of NTS, and observed that the effect of acidosis in LH is inhibited. But blocked OX1R of NTS alone had no effect on respiration. This further proofed that ASICs lying in LH via orexin through medulla pathway to regulate respiration.