Endogenous Acetylcholine Increases Alveolar Epithelial Fluid Transport via Activation of Alveolar Epithelial Na,K-ATPase in Mice
Introduction
The ability of the lung to remove edema fluid from the alveolar space is critically important for keeping the alveolar space relatively fluid-free for adequate gas exchange in cases of alveolar flooding during conditions such as lung lavage, drowning, newborn lung, congestive heart failure, and acute respiratory distress syndrome. Inhibition and stimulation of alveolar fluid clearance (AFC) have been extensively studied in the normal and pathological lung of many species, and both endogenous and exogenous catecholamines have been reported to stimulate AFC in newborn and adult animals via β-receptor-mediated activation of the epithelial sodium channel and Na,K-ATPase.
Clinical studies have found a rapid rate of AFC after lung lavage, with normal ranges of plasma and alveolar fluid epinephrine levels. Studies involving animals with bilateral adrenalectomy have indicated that mouse AFC can occur rapidly and independently of endogenous catecholamines during the first fifteen minutes. While these studies suggest catecholamine-independent pathways of AFC, the specific mechanisms regulating this process remain incompletely understood. Parasympathetic nerves innervate the lung, and cholinergic agonists have been reported to increase AFC. However, previous studies did not explore the role of parasympathetic nerves in the removal of alveolar fluid. The first aim of this study was to explore the role of these nerves in AFC in mice following vagotomy and vagus nerve stimulation.
There is convincing evidence that removal of edema fluid from the air spaces relies on a transepithelial sodium concentration gradient established by the basolateral sodium-potassium ATPase. Na,K-ATPase is a ubiquitous heterodimeric transmembrane ion transporter that maintains Na+ and K+ gradients across cell membranes. A decrease in the number of Na,K-ATPase molecules at the plasma membrane results in inhibition of Na+ transport and thus decreased AFC, while overexpression or upregulation of Na,K-ATPase function in alveolar epithelial cells may improve AFC. Therefore, regulation of Na,K-ATPase represents an important mechanism for modulating alveolar epithelial function. Cholinergic agonists can activate epithelial sodium channels in primary cultures of rat alveolar type II cells. However, the effect of acetylcholine, the main neurotransmitter of parasympathetic nerves, on Na,K-ATPase in alveolar epithelial cells had not been studied. Thus, the second aim of this study was to determine the effects of acetylcholine on the activity of Na,K-ATPase in cultured alveolar epithelial cells.
Materials and Methods
Chemicals
Acetylcholine chloride, albumin bovine V, and Evans blue dye were obtained from standard suppliers. The Na,K-ATPase α1 antibody was purchased from a specified manufacturer. The Na,K-ATPase activity assay kit was also obtained from a recognized bioengineering institute.
Animals
Specific pathogen-free adult male Balb/c mice weighing 28 to 31 grams (eight weeks old) and male Kunming mice weighing 28 to 31 grams (five weeks old) were obtained according to institutional protocols, and all animal procedures were approved by the institutional animal research committee. Mice had access to food and water ad libitum. For experiments, mice were anesthetized with an intraperitoneal injection of chloral hydrate solution. Balb/c mice were divided into groups for vagus nerve interventions: sham-operated controls (left cervical vagus nerve dissected but not transected), left cervical vagus nerve transection, left cervical vagus nerve transection and central end stimulation, and left cervical vagus nerve transection and peripheral end stimulation. Kunming mice were divided into groups for tracheal instillation of various concentrations of acetylcholine chloride (with or without atropine) for AFC measurements.
Vagotomy and Vagus Nerve Stimulation
Mice were anesthetized and a longitudinal incision was made above the trachea. Structures were exposed and under a dissecting microscope, the vagus nerve was carefully separated from the carotid artery then transected in the vagotomy group or left intact in the sham group. For stimulation, the peripheral and central ends of the left cervical vagus nerve were separately placed on a stimulation device and stimulated with a constant voltage pulse before and during airway fluid infusion.
Measurement of Alveolar Fluid Clearance (AFC) in Mice
After dissection, mice were positioned at a head-up angle. A catheter was inserted in the airway via tracheotomy for oxygen inhalation. A warmed isotonic saline solution containing albumin and Evans blue dye was instilled into the lung at timed intervals. After the procedure, mice were exsanguinated, and an alveolar fluid sample was collected. AFC was calculated from measurements of instilled and final fluid volumes and dye concentration.
Measurement of AFC With and Without Acetylcholine Chloride
To evaluate acetylcholine’s effect on lung fluid clearance, acetylcholine or acetylcholine plus atropine was added to the instillate with Evans blue labeled albumin. AFC was measured using established protocols, collecting samples after a set interval for analysis.
Culture of A549 Cells and Acetylcholine Stimulation
A549 cells, a human lung adenocarcinoma-derived line exhibiting alveolar epithelial properties, were cultured in appropriate medium. Cells were stimulated with acetylcholine or acetylcholine plus atropine, or exposed to vehicle alone, before assays or analysis.
Na,K-ATPase Activity Assay
After washing and harvesting, Na,K-ATPase activity in A549 cells was measured using a commercial kit.
Western Blot Analysis
For protein separation, cells were disrupted in homogenization buffer with protease inhibitors. Membrane fractions were isolated by differential centrifugation and stored for further processing.
Immunohistochemistry
A549 cells were grown on coverslips, stimulated with acetylcholine or control, then fixed and permeabilized. After incubation with primary antibody (Na,K-ATPase α1) and secondary antibody, cells were mounted and imaged using fluorescence microscopy.
Statistical Analysis
Data were expressed as mean ± standard deviation and analyzed with appropriate statistical methods. A p-value of less than 0.05 was considered statistically significant.
Results
AFC Determination in Mice Following Vagus Nerve Intervention
To evaluate the effect of the vagus nerve on AFC, mice underwent vagotomy and vagus nerve stimulation. After fluid instillation in the alveolar space, samples were taken for AFC assessment. Mice in the control group showed high AFC levels. Following left cervical vagus nerve transection (with the right nerve intact), there was a significant decrease in AFC compared to the control group. However, peripheral end stimulation of the left cervical vagus nerve inhibited the reduction of AFC caused by transection, restoring AFC to levels closer to control values. Stimulation of the central end of the left cervical vagus nerve did not produce the same effect and resulted in a further reduction of AFC.
Effect of Acetylcholine Chloride on AFC
Since left vagotomy reduced AFC, and stimulation of the peripheral end (but not central end) mitigated this effect, it was hypothesized that acetylcholine release may play a role. Tracheal instillation of acetylcholine chloride in different concentrations led to a dose-dependent increase in AFC compared to control mice, and atropine partially blocked the increase in AFC due to acetylcholine. These results were consistent with findings from earlier studies involving other cholinergic agonists.
Effect of Acetylcholine Chloride on Alveolar Epithelial Na,K-ATPase Activity
Acetylcholine chloride significantly increased Na,K-ATPase activity in A549 cells at short time points, an effect that was reversed by atropine. The activity of Na,K-ATPase decreased at later time points compared to earlier measurements.
Effect of Acetylcholine Chloride on Na,K-ATPase α1 Protein Abundance in the Cell Basolateral Membrane
Acetylcholine chloride increased the Na,K-ATPase protein level in the basolateral membrane of A549 cells, an effect that was inhibited by atropine. Acetylcholine chloride also increased the abundance of the Na,K-ATPase α1 subunit in the cell basolateral membrane, as shown by immunofluorescence microscopy, consistent with Western blot results.
Discussion
The objective of this study was to investigate the function of endogenous acetylcholine on AFC after infusion-induced lung edema. Maintaining the alveolar space free from excess fluid is crucial for gas exchange and for clinical procedures such as bronchoalveolar lavage. Previous work showed that AFC can be catecholamine-independent, prompting the exploration of parasympathetic roles. In this study, left vagotomy reduced AFC in mice, while stimulation of the peripheral end of the vagus nerve reversed this effect, indicating that the vagus nerve, through its efferent branches, promotes AFC. The increase in AFC following peripheral vagus nerve stimulation was attributed to the release of acetylcholine.
On the molecular level, AFC depends on sodium transport via apical sodium channels and basolateral Na,K-ATPase. Regulation of Na,K-ATPase is crucial for epithelial fluid clearance. Acetylcholine increased Na,K-ATPase activity and abundance of its α1 subunit at the basolateral membrane of alveolar epithelial cells, effects that could be inhibited by atropine. These observations suggest that endogenous acetylcholine enhances AFC via activating Na,K-ATPase and recruiting it from intracellular pools to the plasma membrane. Acetylcholine may also target apical sodium channels, as previously shown with other muscarinic agonists.
The experimental conditions were optimized to balance animal tolerance and sample retrieval for AFC measurement. The methods used, including dye dilution, are standard in the field although there are potential limitations, such as the possible stimulation of mucus secretion by acetylcholine, which could influence sample composition.
The results indicate that the vagus nerve can increase alveolar epithelial fluid transport via acetylcholine release. This finding contrasts with pathophysiological states, like organophosphorus poisoning, where cholinergic overactivity can cause pulmonary edema due to excessive secretion overwhelming alveolar absorptive capacity. In physiological conditions, acetylcholine appears to promote reabsorption, possibly as a compensatory mechanism. Similar dual actions have been observed with adrenergic modulation of alveolar fluid clearance.
Stimulation of the central end of the vagus nerve led to further reduction in AFC. This could be due to afferent pathway activation modifying respiratory rhythm or autonomic balance, potentially affecting fluid reabsorption.
In summary, the study demonstrates that the vagus nerve increases alveolar epithelial fluid transport by releasing endogenous acetylcholine in a mouse model of infusion-induced pulmonary edema. Acetylcholine increases Na,K-ATPase activity and its abundance at the cell membrane in alveolar epithelium, effects that are sensitive to muscarinic antagonism. Endogenous acetylcholine likely enhances AFC through activation of muscarinic receptors in alveolar epithelium and subsequent Na,K-ATPase regulation.