The Role And Molecular Mechanism Of Endogenous Acetylcholine On Removing Alveolar Fluid In Mice

There are many reasons leading to alveolar fluid accumulation, such as bronchoalveolar lavage, drowning, cardiac failure, and acute lung injury, etc. There is convincing evidence that removal of edema fluid from the air spaces, or distal air space fluid clearance, is dependent on a transepithelial sodium transporting through epithelial sodium channel, and then the sodium is transported across the basolateral membrane into the interstitium by the Na,K-ATPase, creating an osmotic gradient that leads to water reabsorption through water channels or Simple diffusion to the pulmonary interstitial. Finally the alveolar fluid is absorbed by the lymphatic or blood capillary. Pulmonary edema is closely associated with alveolar fluid clearance(AFC), improving alveolar fluid clearance can speed up removing alveoli liquid and can promote pulmonary edema fluid absorbing, which is controlled by the sodium channel on the apical membranes and Na,K-ATPase on the basolateral membrane of alveolar epithelial cells.The Na,K-ATPase is a ubiquitous heterodimeric transmembrane ion transporter, In the process of AFC. Na,K-ATPase maintains Na+ and K+ gradients across cell membranes by transporting 3 sodium ions out of the cell and 2 potassium ions into the cell at the same time. The overexpression Na,KATPase at the plasma membrane of alveolar epithelial or increase its activity can promote alveolar fluid clearance. On the contrary, a decrease in Na,KATPase or inhibit its activity results in inhibition of Na+ transport and, thus, decreased AFC. Therefore, regulation of the Na,K-ATPase represents an important and fundamental mechanism to modulate alveolar epithelial function.Cholinergic axons are widely distributed in the lung, regulating airway tension, blood supply, airway mucus secretion and breathing pattern, etc. Acetylcholine receptors(ACh Rs) have been previously described in the rat and human alveolar epithelial cells. Since 2000, A number of studies have explored the anti-inflammatory or-apoptotic effect of cholinergic nerve by vagotomy and vagus nerve stimulation in acute lung injury induced by sepsis or other reasons such as mechanical ventilation, ischemia reperfusion, oleic acid, etc. Cell-attached patch-clamp recordings indicated that cholinergic agonists dose dependence activated ENa C in primary cultures of rat AT2 cells. The M type cholinergic receptor blocker atropine can increase alveolar fluid absorption in the process of bronchoalveolar lavage. All suggest that cholinergic nerve may participate in the regulation of alveolar fluid transport. But there is no studies in exploring the role of cholinergic nerve on AFC so far. This present study will first explore the effect of acetylcholine on AFC by vagotomy and vagus nerve stimulation or administration of exogenous acetylcholine chloride, and further discusse the effect of acetylcholine chloride on Na,K-ATPase molecules at the plasma membrane. Detailed content is as follows: Part one vagus nerve intervention on alveolar fluid clearance in micePurpose: To examine the contribution of endogenous acetylcholine in the removal of alveolar fluid by vagotomy and vagus nerve stimulation.Method: Specific pathogen-free 60 adult male Balb/c mice weighing 28 to 31 g were randomly divided into 4 groups(15 for each) for vagus nerve intervention: ① control: left cervical vagus nerve was dissected but not transected, ② left cervical vagus nerve transection(LVAG), ③ left cervical vagus nerve transection and peripheral end stimulation(LVS1), ④ left cervical vagus nerve transection and central end stimulation(LVS2). Among each group 6 were selected for measurement of AFC, 6 for plasma acetylcholine, and 3 were for hematoxylin and eosin. A warmed isotonic saline solution(250 ul) containing 5% bovine serum albumin and Evans blue dye(0.15mg/ml) was then infused into the lung at a rate of 50 μL every 2 minutes once using a 1-m L syringe. After 20 minutes, the mouse was exsanguinated through the carotid artery, and an alveolar fluid sample was aspirated. AFC was calculated by measuring the concentration of Evans blue dye albumin in instilled albumin solution and final alveolar fluid. From which we could figure out the role of the vagal nerve in alveolar fluid transport. Because of the rapid metabolism of acetylcholine in plasma, we collected plasma at the end of vagus nerve stimulation and measure its evels by nanjingjiancheng kit, which indirectly reflect that the vagus nerve intervention has affected the acetylcholine levels in the plasma. To achieve a distinguished view of histopathology we prolonged the time to 30 min after instillation for collecting specimen.Result: Compared with the control group, mice in the left cervical vagal nerve transection showed lower AFC(66.88±2.64% vs 48.69±2.57%, P<0.01). Compared with the left cervical vagal nerve transection, AFC was significantly increased in mice subjected to peripheral end stimulation(48.69±2.57% vs 60.81±1.96%; P<0.01), but mice in vagus nerve central end stimulation reduced the AFC to a lower value(48.69±2.57% vs 38.49±1.45%; P<0.05). Plasma acetylcholine in control group is 4.64±0.25ug/ml. Compared with control group, plasma acetylcholine is lower in left cervical vagus nerve transection(3.59±0.27ug/ml) and the left vagus nerve central stimulation(2.98±0.18ug/ml), and is higher in peripheral end stimulation(6.01±0.54 ug/ml). There is a significantly statistical difference(P<0.01). Hematoxylin and eosin data showed mild interstitial edema in control group, more pink perfusate and significantly interstitial edema in vagus nerve transection. Compared with vagus nerve transection, interstitial edema was rarely seen in peripheral end stimulation.Conclusion:1 Left vagotomy resulted in reduced AFC in mice, suggesting that vagus nerve is involved in alveolar fluid transportation.2 Left vagus peripheral end stimulation but not central end stimulation could inhibit reduced AFC in left vagotomy.3 Left vagus peripheral end stimulation can increase plasma acetylcholine levels, which implies that enhanced AFC following peripheral end stimulation may be due to endogenous acetylcholine release. Part two The effect of acetylcholine chloride on AFC and Na,K-ATPasePurpose: To explore the effect of exogenous acetylcholine chloride on AFC and Na,K-ATPase.Method: Specific pathogen-free 75 Kunming mice were randomly divided into 5 groups( 15 for each) for tracheal instillation of several different concentrations of acetylcholine Chloride(or acetylcholine chloride plus atropine): ①control, ②10-4M Ach, ③10-5M Ach, ④10-6M Ach, ⑤10-6M Ach plus 10-6M Atropin Among each group 6 were for measureing AFC, 6 were for measuring Na,K-ATPase activity and protein expression, and 3 were for hematoxylin and eosin. A warmed isotonic saline solution(200 ul) containing 5% bovine serum albumin and Evans blue dye(0.15mg/ml) was then infused into the lung at a rate of 40 μL every 2 minutes once with a 1-m L syringe. After 20 min, an alveolar fluid sample was aspirated. AFC was calculated by measuring the concentration of Evans blue dye albumin concentrations in instilled albumin solution and final alveolar fluid. The solutions was exchanged with saline dilution containing different concentration of acetylcholine chloride. Lung tissue samples were collected for measuring of Na,K-ATP activity(nanjingjiancheng kit), membrane protein(western blot) and HE.Result: 1 Compared with control group, acetylcholine chloride dose dependent increased AFC. Ach at 10-4M was capable of stimulating AFC to a higher value(57.93±3.21% vs 42.95±2.41% P≤0.01). Atropine at 10-6M can inhibit the effect of 10-6M Ach on AFC(45.17±1.05% vs 48.99±1.42% P≤0.05). 2 The Na,K-ATPase activity was 3.69±0.19u/mgprot in control group, while 10-4-10-6M acetylcholine chloride significantly increased the Na,K-ATPase activity(P<0.05), its respective values are 10-4M Ach: 5.85±0.21 u/mgprot, 10-5M Ach: 5.14±0.22 u/mgprot, 10-6M Ach: 4.4±0.14 u/mgprot, 10-6M atropine blocked 10-6M acetylcholine chloride on the effect of Na,K-ATPase activity(3.12 ± 0.42 u/mgprot vs 4.4 ± 0.14 u/mgprot P<0.05). 3 Compared with control group, there is no significantly change in Na,K-ATPase ɑ1 protein expression in lung tissue homogenate after acetylcholine chloride stimulation.Conclusion:1 Cholinergic agonist acetylcholine chloride dose dependent increased AFC, and atropine can blocked such effect.2 Acetylcholine chloride dose dependent increased the Na,K-ATPase activity in lung tissue homogenate, and atropine abolished such effect.3 There is no change in Na,K-ATPase ɑ1 protein expression in lung tissue homogenate after acetylcholine chloride stimulation. Part three Effect of acetylcholine chloride on Alveolar Epithelial Na,K-ATPasePurpose: To explore the effect of 10-5M acetylcholine chlorid on alveolar epithelial Na,K-ATPase activity and protein expression in A549 cell.Method: A549 cells were cultivated and then were stimulated with 10-5M Ach or 10-5M Ach plus Atropin or exposed to vehicle(RPMI1640 medium) alone for 5 or 10 minutes before the assessment of Na,K-ATPase activity, or Western blotting, or fixation for Fluorescence microscopy.Result: Compare with control group, 10-5M acetylcholine Chloride significantly increased Na,K-ATPase activity at 5th minute(3.27±0.23 u/mgprot vs 5.34±0.5 u/mgprot P<0.01) and at 10 th minute(3.22±0.11 u/mgprot vs 3.91±0.22 u/mgprot P<0.05) and can be reversed by atropine. Ach stimulated Na,K-ATPase activity significantly declined at 10 minutes compared with 5minutes(5.34±0.50 u/mgprot vs 3.91±0.22 u/mgprot P<0.05). Compared with control group, 10-5M acetylcholine chloride caused an increase in abundance of the Na,K-ATPase α1 subunit in the basolateral cell membrane at 5 minutes by immunofluorescence microscopy. But we did not find a significant difference of Na,K-ATPase α1 subunit Green fluorescence in cell membrance in the two groups. Western-blot results showed that 10-5M acetylcholine chloride increased the amount of Na,K-ATPaseα1 protein in the basolateral cell membrane of A549 cells at 5 minutes and decreased by atropine.Conclusion:1 Acetylcholine chloride increased Na,K-ATPase activity and the amount of Na,K-ATPaseα1 protein in the basolateral cell membrane of A549 cells.2 Acetylcholine chlorid increased Na,K-ATPase activity and the amount of Na,K-ATPaseα1 protein can be inhibited by Cholinergic antagonist atropine, which implies that acetylcholine chloride regulate Na,K-ATPase through M receptor in alveolar epithelial cells.