Signal Transduction Study Of Sodium Transport In Alveolar Type â…¡ Epithelial Cells Of ARDS Rats

Objective:Acute respiratory distress syndrome(ARDS) is syndrome of acute respiratory failure,which could be resulted from various pulmonary and nonpulmonary non-cardiogenic etiological factors.Which manifests as pulmonary edema,respiratory distress and consequent severe hypoxemia.The mortality rate is 35%-58%at present,it is still one of the major causes of morbidity in patients after discharge.A large of clinical research indicated that alveolar fluid clearance early is a key to clinical treatment.Preventing and treating pulmonary edema is the main task in clinic.Endothelial injury and increased pulmonary capillary permeability resulting in pulmonary edema is a central pathology feature of ARDS.Epithelial injury is also important not only in the development but also the repair of the ARDS.How to decrease epithelial injury and improve alveolar liquid absorption are a key to preventing and treating pulmonary edema.Recently,several studies have provided that active ion transport from the air space to the lung interstitium is a primary mechanism for clearance of edema fluid.Alveolar epithelial cells,consisted of flat typeⅠcells and cuboidal typeⅡcells,are considered to be not only an important barrier to alveolar flooding but also the most likely site of fluid reabsorption after pulmonary edema,meanwhile maintain normal gas exchange.ATⅡcells(diameter 10mm),not only characterized morphologically by surfactant-containing secretory granules called lamellar bodies but also active transport sodium.A large quantity of evidences indicated there are sodium-permeable channel membrane protein on ATⅡcells.Na+ enters ATⅡcells by an apical Na+ channel and is actively pumped out of the cells by basolateral Na+-K+-ATPase.Meanwhile,accompanied with water reabsorption.The rate-limiting step in this process is the activity of the heterotrimeric apical membrane epithelial Na+ channel(ENaC).Na+ channel on the apical membrane are formed from three subunit proteins designatedα-,β-andγ-subunit.molecularbiology and electrophysiological studies have confirmed that the diversity of ENaC arises from assembling different combinations of three subunits to form channels with different biophysical properties and different menchanisms for regulation.In recent studies,active transport of sodium is regulated through different signaling molecules via different signal transduction.β-adrenergic agonists can increase lung liquid clearance via cAMP second messenger system.cAMP can increase levels of Na+ channel subunits mRNA and Na+ channel open probability.Influenza virus inhibits ENaC in ATⅡcells via a PLC- and Src-mediated activation of PKC.Interleukin-1beta decreases ENaCα-subunit expression via a p38 MAPK-dependent signaling pathway,et al.Signaling transduction study of Sodium transport is increasingly reconstructed abroad,but biophysical properties of sodium channels and regulatory mechanisms of sodium transport are unclear,especially pathological conditions such as ARDS.Remain unclear deserve further studies.The present study aims at setting up a oleic acid-induced ARDS model.We observe the levles of ENaC subunits mRNA of actutely isolated rats ATⅡcells under physiolgical and ARDS conditions.We study the signaling transduction of transalveolar fluid absorption viaβ-adrenergic agonists terbutaline-mediated.It provides a new,effective,suitable method for ARDS.Methods:Male adult Sprague-Dawley rats weighing 180-220g were used in all experiments. This study includes two parts as followed.In the first part,we observed the sodium channel subunits mRNA expressions of isolated rats ATⅡcells actutely and the changes in ARDS.34 SD rats were randomly assigned to nomral group and ARDS group.In each group,6 rats were selected to test Semiquantitation of ENaC subunits by RT-PCR,7 rats wre quantitated Extravasular lung water(EVLW) by a gravimetric measurement and observed histopathology by HE coloration.The remainders were tested the other indexs.Rat models of ARDS were established by intravenous injection of oleic acid(oleic acid 0.1ml/kg).ATⅡCells wre isolated and puried in previously described methods.And purity was identified with a tannic acid stain and electron microscope,and the changes in cellular ultrastructure were observed by electron microscope.In the second part,We studied the activation of cAMP and cGMP mediated by terbutaline in Na+ Transport of ATⅡcells.63 SD rats were were randomly divided into 3 groups:the control,ARDS group,and terbutaline treated group.ARDS were established by intravenous injection of oleic acid(oleic acid 0.1ml/kg).In terbutaline treated group,terbutaline solution(0.1mM,5ml/kg) was instilled into the trachea after making ARDS model successfully.Bloodletted and killed rats at 30 minutes after ARDS.Isolated and puried rat ATⅡCells.In each group,10 rats were measured the contents of cAMP and cGMP in the ATⅡcells by ridioimmunoassy,7 rats wre quantitated extravasular lung water(EVLW) by a gravimetric measurement and observed histopathology by HE coloration.The remainders were tested the other indexs.ATⅡCells wre isolated and puried in previously described methods.Results 1.Establishment ARDS modelAfter injection of oleic acid,experimental rats appeared obviously symptoms such as short of breath,130-140/minutes.skin of lip and legs became purple.Arterial blood gas analysis:PH(7.13±0.08),PO2(58.14±5.00) mmHg,were decreased with the nomral group contrast,there were significantly difference(P＜0.001).HE coloration shown that the alveoli and interstitial became dropsical and hemorrhagic;capillary vessel been overspread,engorgement and leukocytic infiltrate;and there are a lot of liquid like thin blood permeated into the alveoli.Smith lung injury score and EVLW were significantly higher than those in the nomral group(P=0.003).Electron microscopic observation of ATⅡcells were observed that the cells were degenerative, apoptotic even collapsed,and the osmiophilic multilamellar body of ATⅡcells had been decreased obviously or became vacuolated partly;the microvilluses of ATⅡcells disappeared.2.The expressions of ENaC mRNA in ATⅡα-ENaC mRaNA of ATⅡ:α-ENaC mRNA expressions of ATⅡin nomral group were 7.8303±1.5466;α-ENaC mRNA in ARDS group were 5.2150±1.5335,there was significantly difference between them(P=0.015).β-ENaC mRNA of ATⅡ:β-ENaC mRNA of ATⅡin nomral group were 2.1947±0.4181;β-ENaC mRNA of ATⅡin ARDS group were 1.7005±0.6343,there was no statistics difference between them(P=0.147).γ-ENaC mRNA of ATⅡ:γ-ENaC mRNA of ATⅡin nomral group were 3.0183±1.3229;γ-ENaC mRNA of ATⅡin ARDS group were 2.4862±0.6770,there were non-statistics difference between them(P=0.408).The content ofα-ENaC mRNA were the richest in the three subunits of ATⅡ.β-ENaC mRNA compared withγ-ENaC mRNA without the statistics difference(P＞0.05). 3.The effects of terbutaline on lung histopathology,EVLW and Arterial blood gas analysisIn terbutaline group,HE coloration shown that the alveoli and interstitial were dropsical and hemorrhagic,a little effusion,leukocytic infiltrate.but the degree of injury were lessened vs ARDS group.Smith lung injury score,EVLW were respectively 4.30±0.49,(0.6614±0.0729) ml/g,significantly lower than in ARDS group.but were higher than those in nomral group.Arterial blood gas analysis:PH (7.13±0.08) and Po2(61.44±4.92) mmHg compared with ARDS group without the statistics difference(P＞0.05).Pco2(47.95±3.76) mmHg were lower(P=0.010).4.The effects of terbutaline on ATⅡElectron microscopic observation of terbutaline treatment group,the cell's shape were irregularity,little cells were degenerative,apoptotic,and the vacuolated osmiophilic multilamellar body of ATⅡcells were lessener than ARDS group.5.the activation of cAMP and cGMP mediated by terbutaline in Na+ Transport of ATⅡcellsThe concentration of intracellular cAMP in ARDS group(34.826±4.626pmol/ml) were significantly lower than in nomral group(17.246±3.913pmol/ml,P＜0.001)about 50.4%.After terbutaline stimulated,The concentration of intracellular cAMP were improved to(24.448±4.799)pmol/ml,there had significantly difference.The concentration of intracellular cGMP in ARDS group(2.486±0.444 pmol/ml) were increaser than in nomral group(1.849±0.295pmol/ml,P=0.001).Compared with terbutaline group(2.457±0.406)pmol/ml,the statistic analysis indicated no significantly difference.(P=0.867).Conclusion:1.The rat ARDS model induced by oleic acid established rapidly with typical pathological change of lung tissues and higher ratio of model reproduce. 2.The express of three subunits mRNA in rat ATⅡcells ENaC are observed by RT-PCR experiments,the content ofα-subunit mRNA is the most abundant among ENaC submits.The content of ENaCβ-andγ-subunit mRNA in rat ATⅡcells were not significantly distinction.3.α-mRNA level decrease significantly under ARDS condition.It was in correspond with the degree of lung injury.Evidenced that ENaCα-subunit is a founction unit of ENaC.It was important in modulating the resolution process of pulmonary edema.4.The effective of terbutaline in signal transduction:(1)Terbutaline can improve the capacity of alveolar fliud clearance by up-regulation cAMP content,delay ARDS proceeding.(2) Terbutaline can lessen the depressant effect of cGMP to lung water absorption through inhibited the express of cGMP in ATⅡcells,assist alveolar fliud clearance.