| Ⅰ. BACKGROUND AND OBJECTIVESAcute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a common clinical critical syndrome with acute progressive respiratory failure caused by a variety of clinical insults of internal or external lung except cardiogenic factor. The main pathological features are pulmonary edema and hyaline membrane caused by an increase in the permeability of the alveolar-capillary barrier which leads to protein-rich edema fluid in alveolar. Some with pulmonary interstitial fibrosis. The main pathophysiological changes are reduced lung volume and compliance and severe ventilation-perfusion imbalance. The clinical manifestations are respiratory distress and refractory hypoxemia. The pulmonary imaging is nonuniformity of exudative process. In spite of intensive care, the mortality still reached 30-50%.In recent years, studies have shown that there is alveolar fluid balance which incorporates both formation and removal. Alveolar fluid reabsorption depends on sodium and water transport system in alveolar typeⅠ(ATⅠ)and typeⅡ(ATⅡ) cells, which includes epithelial sodium channel (ENaC), cyclic nucleotide-gated cation (CNG) channel, aquaporin (AQP) and Na+-K+-ATPase. Na+ is absorbed from the apical surfaces of cells via ENaC and via CNG channels and transported from the basal surface of cells into the interstitial space by Na+-K+-ATPase. If the directionality of net ion transport is from the apical surface to the interstitium, an osmotic gradient would be created, which would in turn direct water transport in the same direction, either through aquaporins or by diffusion.If alveolar epithelial fluid reabsorption balances the formation of alveolar edema, then a steady state can be established that may allow time for recovery from the fundamental cause of lung injury. Clinical study by Ware et al showed that the majority of patients with increased permeability edema and acute lung injury had impaired alveolar epithelial fluid transport, which were associated with more prolonged respiratory failure and a higher mortality, in contrast, a minority of patients could remove alveolar edema fluid rapidly, and these patients had a higer survival rate.Clinical evidences showed that alveolar fluid clearance decreased significantly in the state of ALI/ARDS, which was mainly due to injured function of alveolar fluid transport, but the detailed mechanism should be clarified. Apoptosis and necrosis occured in alveolar cells can destroy part of sodium and water transport system, What about transport system in the cells without apoptosis and necrosis? Our previous study found that the function of sodium transport decreased significantly in ATⅡcell at the state of ARDS. Recently, Johnson et al and Borok et al found that ENaC and Na+-K+-ATPase were also in the ATⅠcell and capacity of sodium transport in ATⅠcell was 2.5～3 times that of ATⅡcell. Based on the above facts, we can speculat that the destroy of sodium and water transport system and decrease of the function are important cause of alveolar edema. In our previous study, we observed that extravascular lung water increased significantly in rats with oleic acid-induced ARDS and decreased significantly by stimulation of terbutaline (cAMP agonist). Whole cell current in ATⅡcell of rat with ARDS decreased significantly compared with the normal rat, but still could be observed, and most of the the current was inhibited by amiloride which is inhibitor of ENaC. The whole-cell current increased significantly by stimulation of terbutaline, which was stronger than that of normal group and equaled that of normal group stimulated by terbutaline.Lucky Jain et al found that when ATⅡcells were grown on glass plates submerged in media that lacked steroids, the predominant channel was a 21-pS nonselective cation channel (NSC) with a Na+-to-K+ selectivity of 1; however, when grown on permeable supports in the presence of steroids and air interface, the predominant channel was a low-conductance (6.6±3.4 pS), highly Na+-selective channel (HSC) with a PNa/PK>80 that was inhibited by submicromolar concentrations of amiloride. Xi-Juan Chen et al found that the mechanism of the two types of channels stimulated by terbutaline was also different:NSC open probability (Po) increased when exposed to terbutaline, without any change in NSC number. In contrast, terbutaline increased HSC number with no significant change in HSC Po. Study of Johnson et al confirmed the existence of these two channels in freshly isolated ATⅡ. Therefore, terbutaline may not only increase the number of HSC, but also increase the NSC open probability to increase ENaC currents in the state of ALI/ ARDS.However, Norlin et al found alveolar fluid clearance is also affected by the amiloride-insensitive CNG channel.1-cis-diltiazem (inhibitor of CNG channel) had no effect on AFC of rats in normal group, which suggest that CNG channel does not seem to participate in AFC; but 1-cis-diltiazem inhibited AFC by 50% in terbutaline group, the same as amiloride. Kemp et al studied the channel in ATⅡcell of rat further. Studies showed that the channel was regulated by cGMP, amiloride-insensitive, nonselective cation channel (Na+/K+ permeability ratio of 0.73±0.02). Therefore, Terbutaline increases AFC may also by increasing Na+ current through CNG channel.It is more complicated in the state of ALI/ARDS. Our previous studies found that the cAMP level in the ATⅡcell was significantly decreased whereas the cGMP level and extravascular lung water (EVLW) content increased in the rat with oleic acid-induced ARDS. Terbutaline treatment increased the cAMP level but produced no significant effect on the cGMP level in the ATⅡcell of the rat with ARDS. In terbutaline-treated rat with ARDS, the EVLW content was lower than that of non-treated rat. Does this mean that (1) In the state of ALI/ARDS, Na+ current through CNG channel was increased and did not further increase by the stimulation of terbutaline. Terbutaline decreased extravascular lung water in the state of ARDS, which was not by enhancing the function of CNG channel? Or (2) in the state of ALI/ARDS, the number of CNG channel on the membrane reduced by endocytosis, even though cGMP level increased, it could not affect the current of CNG channel significantly. Terbutaline increased current of CNG channel by increasing the number of CNG channel inserted membrane.This study intends to use patch-clamp technique to reveal the mechanisms about Na+ transport of ENaC channel and CNG channel in the state of ALI/ARDS and improves knowledge about the pathophysiology of ALI/ARDS.Ⅱ. METHODSFor all experiments, male adult Sprague-Dawley rats (SPF) weighing 180-220g were used. A rat model of ALI/ARDS was established using the intravenous injection of oleic acid. Blood gas analysis and pathological section were used for evaluation of model. ATⅡcells were separated from lung by trypsin digestion which perfused in alveolar cavity. Viability of cells was identified by staining of trypan blue. Purity of ATⅡcells was identified by staining of alkaline phosphatase(AKP). Whole cell currents were recorded from ATⅡcells perfused with or without amiloride (inhibitor of ENaC channel) and ZnCl2 (inhibitor of CNG channel) in the whole cell recording mode of the patch-clamp technique, the effect of terbutaline on currents was also examined. Protocal of stimulation:whole cell current elicited by 500 ms voltage step to -100 mV from a holding potential of 0 mV. Pipette was filled with standard internal solution (SIS), and cell was perfused with standard external solution (SES), the primary current was recorded. After perfusion of cell with SES solution containing 10μM amiloride, the current was recorded again in 5 min. Finally, cell was perfused with SES solution containing 10μM amiloride and lmM ZnCl2, the current was recorded at last in 5 min. SPSS13.0 was used to statistical analysis.Ⅲ. RESULTS1. Model of ALI/ARDSAfter injection of oleic acid through vena femoralis, rats appeared shortness of breath in less than 2 min; most of rats appeared sigh-like breath, paradoxical motion of chest and abdomen; all of rats appeared cyanosis and there was red excretion in the endotracheal tube. PH decreased from 7.36±0.01 in the state before injury to 7.24±0.02 in the state after injury (P<0.05); PaO2 from 12.44±0.23kpa to 6.05±0.45kpa (P<0.05); PaO2/FiO2 from 445.28±8.39 to 216.61±16.16 (P<0.05); PaCO2 increased from 5.45±0.15 kpa to 7.22±0.59 kpa (P<0.05). Congested and swollen lung could be seen after opening the chest. The lower part of lung was more severe than the upper part. The edge of lung was blunt and pleural was tight and shiny. There was foam-like bloody excretion in the airway. Pathological section showed that cuff-like edema around bronchi, appearance of tissue was not exactly clear; there was red stain and red blood cells in alveolar space; pulmonary interstitium was expanded; neutrophils increased significantly; focal atelectasis and necrosis.2. Isolation and identification of ATⅡcellsThe ATⅡcells stained by AKP were blue-purple. The viability of cells in normal group was (91.00±2.10)%, the purity was (57.33±5.05)%; the viability of cells in group of oleic-acid-induced lung injury was (83.50±1.64)%, the purity was (51.00±4.34)%, both viability and purity were lower than those in normal group (P<0.05).3. Currents of ion channelsWhole-cell current in ATⅡcell was inhibited by amiloride or ZnCl2 (P<0.01), it shows there were amiloride-sensitive and Zn2+-sensitive current in ATⅡcell. Amiloride-sensitive current accounted for (39.42±12.36)%, Zn2+-sensitive current accounted for (29.70±8.14)%, The proportion of both currents was not different (P> 0.05). Both currents could be stimulated to increase obviously by terbutaline(F=26.963,P<0.001; F=40.786,P<0.001), amiloride-sensitive current accounted for (50.87±8.08)%, Zn2+-sensitive current accounted for (30.17±7.62)%, the proportion of amiloride-sensitive current is approximately 1.7 times that of Zn2+-sensitive current (P<0.05).Whole-cell current in ATⅡcell of the rat in group of oleic-acid-induced lung injury was also inhibited by amiloride or ZnCl2 (P<0.01), it showed there were amiloride-sensitive current and Zn2+-sensitive current still in ATⅡof the rat in group of oleic-acid-induced lung injury, but both currents lower than normal (F=25.997,P<0.001; F=17.870,P<0.001). Both currents could be stimulated to increase obviously and reach the levels of those in normal ATⅡcell by terbutaline. (F=26.963,P<0.001; F=40.786,P<0.001).Ⅳ. CONCLUSIONS1.Trypsin digestion which perfused in alveolar cavity can obtain ATⅡcells to a certain extent. It’s feasible to identify ATⅡcells from macrophages by staining of AKP. The stain can be observed by ordinary microscope as well as the step of stain is simple and result can be obtained in 30 minutes. It is simple and effectual compared with other methods.2. There are functional ENaC and CNG channels on freshly isolated ATⅡcells, and both are main channels for sodium transport. but the effect of ENaC was stronger than that of CNG channels. Under the state stimulated by terbutaline, sodium transport of both channels are incresed, the effect of ENaC is still the main effect.3. Under normal state, ENaC and CNG channels are main channels for sodium transport in ATⅠcells, but the effect of ENaC is stronger than that of CNG channels; Under the state stimulated by terbutaline, sodium transport of both channels are incresed in ATⅠcells, the effect of CNG channels is stronger than that of ENaC.4. Model of oleic-acid-induced lung injury is a preferable model of ALI/ARDS. Injection of oleic acid through vena femoralis is easy and precise for dose compared with injection through caudal vein, avoids unnecessary large trauma compared with injection through inferior vena cava. The dose of oleic acid is 0.1 ml/kg. It is quick to establish the model successfully. Symptoms, signs, blood gas and pathological changes of rats are accordant to that of ALI/ARDS.5. The sodium transport capacity of ENaC and CNG channels on ATⅡcells of the rats with ARDS still existed, but it can not match that on normal ATⅡcells, and can be recovered by terbutaline. |
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