Cystic fibrosis (CF) is a long-term disease that affects the respiratory system, the immune system, the digestive system, and the male reproductive system (66). According to the Cystic Fibrosis Foundation (19), the median life span of CF patients is mid-30s. CF is caused by an autosomal recessive mutation in the gene coding for the CF transmembrane conductance regulator (CFTR) protein. CFTR proteins are localized on the apical surface of the airway epithelial cell. After conformational ATP-driven changes, the CFTR gate opens and allows Cl− molecules to pass down an electrochemical gradient (24). As a result of Cl− secretion, Na+ balances flux paracellularly owing to the negative charge; H2O follows the net ion movement stoichiometrically to maintain osmolarity, and this allows the airways to be hydrated. Dysfunctional CFTR results in viscous mucus that impairs cilia beating and compromises mucus clearance. Stagnant mucus becomes a breeding ground for bacteria and infection, which is often followed by chronic inflammation, emphysema, and pulmonary disease (65).
The lumens of the bronchial passageway are covered by the airway surface liquid (ASL), which is composed of two layers: a superficial mucus layer containing secreted mucins and trapped particles and a periciliary layer that separates the mucus from the respiratory epithelia (37). The difference in consistency between the ASL layers is the result of mucin properties, which give the mucus layer viscoelastic and gel-forming characteristics, whereas the lack of mucin in the periciliary layer establishes a low viscosity for optimal for cilia beating. Maintenance of the periciliary layer is therefore crucial not only to provide cilia with a medium to beat to propel mucus to the large airways where it can be expectorated or swallowed but also to serve as a protective barrier against particle interaction with the epithelial membrane (66).
ASL volume is maintained by epithelial cells via the regulation of ionic movement across the apical and basolateral membranes through the interrelated actions of ionic pumps, channels, and active transport proteins (16). Airway epithelia switch between secretory and absorptive functions to regulate ASL hydration (77): ASL volume is reduced via Na+ absorption through the epithelial sodium channel (ENaC) and increased by Cl− secretion through CFTR and calcium-activated chloride channels. CFTR plays a dual role in determining ASL volume by secreting Cl− and inhibiting ENaC to reduce Na+ absorption (77).
Because airway epithelial cells have a low transepithelial resistance and are highly water permeable, large osmotic gradients cannot be maintained across airway epithelia (49). It is the interaction between ENaC sodium absorption and CFTR and calcium-activated chloride channel Cl− secretion that dictates the driving force for fluid reabsorption/excretion on the apical membrane (35). The basolateral Na+/K+-ATPase protein pumps out three Na+ for every two K+ absorbed and thereby generates the electrochemical gradient needed for passive sodium absorption through ENaC (42). As ENaC removes Na+ from the lumen, a negative electrical potential is developed, reducing the driving force for Cl− secretion (62). In normal epithelial cells, adenosine in the lumen binds adenoreceptors and causes an increase in the second messenger cyclic adenosine monophosphate (cAMP). Intracellular cAMP then reciprocally activates CFTR and inhibits ENaC. Because the Cl− chemical gradient is approximately fourfold higher in the ASL, epithelial cells rely on this reciprocal regulation to facilitate the transport of Cl− across the epithelial membrane (59). By inhibiting ENaC, the epithelial cell is able to hyperpolarize the apical membrane, thereby creating the electrical driving force for Cl− secretion and ASL hydration (10).
Impaired ion regulation is central to the pathophysiology of CF because it leads to dehydrated airways (7). ASL loss has been implicated as the major underlying cause in the pathogenesis of CF. cAMP-dependent transport of Cl− via CFTR across the epithelia membrane is lost in CF epithelia. Further, Na+ absorption is increased because dysfunctional CFTR cannot inhibit ENaC activity. As a consequence, Cl− and H2O enter via a paracellular route (7). CFTR dysfunction manifests in an increased negative transepithelial potential difference (TPD) owing to accelerated ENaC Na+ absorption (31). The net result is a hypopolarized epithelial membrane and ASL dehydration (42).
For cilia to beat an ASL, a periciliary layer of at least 7 μm must surround the cilia (16). Reduced ASL volume in CF, however, causes the collapse of cilia owing to the weight of the mucus layer. As a consequence of this failure in mucus clearance machinery, adhesion and expansion of dehydrated mucus form plaques on the epithelial surface, which can result in small airway occlusion. The resident mucus plaques provide the media for bacteria to colonize in and form biofilms. Consequently, the concentrations of macrophages and neutrophils increase and a persistent, yet ineffective inflammatory response is initiated, resulting in obstruction, inflammation, infection, and irreversible bronchiectasis (70). In addition, retained secretions contain high concentrations of inflammatory cytokines and proteases, which increase the risk of bronchospasm and further the development of progressive parenchyma degradation (45,80).
Exercise and physical activity have been recommended as an important part of CF therapy (80,87). The benefits of exercise and physical activity in CF have been documented and include enhanced cardiovascular function (80), increased muscular strength and coordination (61), improved quality of life (24), and increased mucus clearance (50). Regular exercise may also improve survival in CF patients. Nixon et al. (57) reported that aerobic fitness, as measured by peak oxygen consumption (V˙O2peak), was significantly correlated with a lower risk of dying. Exercise capacity as measured by V˙O2peak has also been used as a prognostic tool in predicting survival (53). More recently, Pianosi et al. (64) performed annual pulmonary and peak exercise tests on children 5–17 yr for 5 yr, followed by a 7- to 8-yr subsequent survival monitoring. Rate of decline in and final V˙O2peak were found to be significant predictors of mortality: mortality increased dramatically in patients with V˙O2peak less than 32 mL·min−1·kg−1 in contrast to a zero 8-yr mortality rate in patients with V˙O2peak in excess of 45 mL·min−1·kg−1. Therefore, by improving exercise capacity, consistent aerobic exercise in patients with CF may play a role in the disease-modifying factors that delay the progression of pulmonary pathogenesis.
EXERCISE DECREASES THE TRANSEPITHELIAL POTENTIAL
CF patients have a greater (more negative) resting TPD than normal individuals as a result of accelerated Na+ absorption and decreased Cl− secretion (16). Aerobic exercise has been shown to decrease the TPD in CF patients (2), possibly by directly affecting the activity of ionic channels in the respiratory epithelium. Hebestreit et al. (30) investigated the affect of exercise on normal and CF nasal epithelial ion channel activity in vivo. TPD was measured while either applying a Na+ conductance inhibitor (amiloride) or a cAMP-dependent Cl− channel stimulator (isoprenaline) to determine ENaC and CFTR activity, respectively. Ten minutes of cycling at approximately 80% maximal HR resulted in significant reductions (becoming more positive) in nasal TPD in CF patients but did not change TPD in normal controls. Furthermore, there was no longer a difference in nasal TPD between CF and healthy controls after the cessation of exercise. The inhibitor effect of amiloride to mediate a reduction in TPD was significantly less during exercise when compared with rest in CF patients but not in healthy controls. Thus, the change in TPD in CF subjects with exercise was likely induced by ENaC inhibition (30). This suggests that the therapeutic effects of exercise are partially due to ENaC inhibition and should result in more hydrated airways.
During exercise, respiratory rate and depth increase (exercise-induced hyperpnea) in relation to exercise intensity. The expansion of the airways and flow of air associated with increased ventilation imparts mechanical stressors on the epithelia, which result in changes in epithelial ionic channel conductance (16). Activation or inhibition of mechanosensitive ionic channels by mechanical stress is likely due to tension changes in the cytoskeleton, changes in the curvature of the lipid bilayer local to the ion channel, and/or signaling events including protein phosphorylation/dephosphorylation or lipids metabolism (10). Although exercise has a positive effect on the CF patient by reducing TPD, the cellular mechanisms whereby exercise affects ion regulation in the CF lung require further investigation. Wheatley et al. (85) hypothesized that exercise improves ion regulation at the cellular level in CF airway epithelia via the purinergic and adrenergic activated pathways. The purpose of this review was to add to this hypothesis by describing how the AMP-activated protein kinase (AMPK), atrial natriuretic peptide (ANP), and arginine-vasopressin (AVP) pathways may also facilitate exercise-induced improvements in ion regulation and airway surface hydration to promote further research regarding the use of exercise as a treatment in CF.
Changes in ASL ion concentrations and ionic channel conductances induced by mechanical stress have been suggested to be due to nucleotide release by epithelial cells into the ASL (32). A review by Wheatley et al. (85) suggested that exercise-induced paracrine ATP secretion by airway epithelial cells stimulates Cl− secretion and inhibits Na+ absorption. Airway epithelial cells release ATP into the ASL after exposure to mechanical deformation (32), fluid shear stress, compression or stretch (16,58,61), and osmotic shock (39). The release of ATP from normal and CF epithelial cells has been shown to differ neither in rate of release nor in concentration in the ASL in vivo (22). In vitro CF epithelial cultures have been shown to increase ASL height to normal levels (7 μm) after exposure to oscillatory shear stress (22). ATP in the ASL binds to apical purinergic receptors P2Y2 and P2X. Activation of P2Y2 receptors mobilize cytosolic Ca2+ from intracellular stores by coupling with G proteins and forming inositol triphosphate via phosphatidylinositol 4,5-biphosphate (PIP2) hydrolysis (40). P2X activation increases Ca2+ flux via the opening of ATP-dependent Ca2+ channels (91). Therefore, purinergic activation stimulates Ca-activated chloride channels via elevated intracellular Ca2+ and reciprocally inhibits ENaC Na+ absorption via PIP2 hydrolysis to increase ASL (41,42,60). Cilia beat frequency is regulated by both P2Y and P2X receptors (88). Cilia beat frequency has been shown to increase during oscillatory, but not static mechanical stress, by 50% in cultured epithelial cells (16). Because ATP is the most potent Ca2+-dependent stimulator of cilia beat frequency (90), it is likely that mechanical stress increases cilia beat frequency via purinoceptor activation. Thus, airway epithelial cells react in a coordinated manner to regulate ASL hydration and mucus clearance using cell-to-cell Ca2+-mediated communication via paracrine and autocrine ATP release (32).
Shear stress during resting tidal breathing has been estimated at approximately 0.4–2 dyn·cm−2 and can reach up to 1700 dyn·cm−2 during coughing. Compressive stress during resting tidal breathing is approximately 8.5 cm H2O, and this value regularly reaches 20 cm H2O in forced expirations during exercise (43). Button et al. (11) demonstrated that oscillations between 0 and 20 cm H2O generated the highest spikes in ATP without damaging cells or integrity of cellular tight junction. Button and Boucher (10) hypothesized that the process involved in sensing and transducing stress can adapt to the presence of sustained stress, such as during in vitro models of bronchoconstriction where ATP release is low. Thus, the rate in change of stress, not the presence of stress, triggers the release of ATP (11). Therefore, via increased oscillations in mechanical stress via exercise-induced hyperpnea, exercise may function as a CF physiotherapy that facilitates airway hydration via purinergic mediated improvements in ion regulation (85).
This is a ubiquitous kinase that acts as an intracellular energy sensor. Conditions of metabolic stress, such as increased AMP/ATP ratios or hypoxia activate AMPK via phosphorylation. Activated AMPK downregulates of pathways that consume ATP, upregulates of those that synthesize ATP, and alters transcriptional regulation (8). Although the effects of exercise on AMPK activation in the lung have yet to be investigated, aerobic exercise may activate AMPK in airway epithelial via increased stretch. Budinger et al. (9) investigated the effects of stretch on AMPK activation in mice airway epithelial cells in vivo. AMPK activation increased significantly after 15 min of noninjurious mechanically elevated ventilation. Budinger et al. (9) also investigated the effects of 10 min of 10% linear elongated cyclical stretch on AMPK activation in alveolar epithelial cells in vitro to determine possible pathways. Cyclic stretch increased AMPK activation nearly 50%. This response was not affected by extracellular signal–regulated kinases 1 and 2 cascade inhibition but was attenuated when dystroglycan was inhibited, suggesting that stretch activates AMPK independent of the extracellular signal–regulated kinase–mitogen-activated protein kinase cascade. Dystroglycan is a scaffolding protein that conducts mechanical deformation from laminin proteins in the extracellular matrix to intracellular signals (17,72) and has been identified in both human alveolar and bronchial epithelial cells (86). Takawira et al. (74) reported that cyclical mechanical stretch directly activates AMPK via a laminin–dystroglycan–plectin scaffolding molecule in lung epithelial cells. This suggests that exercise-induced hyperpnea may activate AMPK in the airway epithelial via increased oscillations in mechanical stress.
AMPK has been identified as a regulator of ion conductance in renal and airway epithelial cells (28). AMPK activation may improve CF lung disease by reducing airway fluid absorption via improved ion regulation (Fig. 1). Administration of AMPK agonist5-aminoimidazole-4-carboxamide-1-β-D-riboside (AICAR) to human bronchial epithelial cells was reported to maintain ASL for 120 min after administration in CF, but not non-CF control cells (55). In normal bronchial epithelial cells, AICAR administration has also been shown to directly inhibit basolateral Na+/K+-ATPase for 8 h and apical ENaC by 50% for up to 24 h (1,89). AMPK phosphorylation due to hypoxia exposure directly inhibits the Na+/K+-ATPase via protein kinase C activation (25). AMPK also directly inhibits the basolateral Ca2+-activated K+ channel (36), which, in turn, may decrease basolateral Na+ pumping and apical Na+ absorption by reducing the chemical and electrical gradients, respectively (18). Administration of AICAR directly affects Na+ absorption via two mechanisms. First, AMPK inhibits PIP2 binding, decreasing the open-state probability and thereby reducing ENaC activity. Second, AMPK activation in the presence of diminished PIP2 reduces the quantity of ENaC on the apical membrane (44). Bhalla et al. (4) reported that the combination of AICAR and nucleotide treatment increased ENaC endocytosis and degradation via Nedd4-2–dependent ENaC retrieval. This suggests that exercise may reduce TPD in CF by simultaneously activating AMPK and stimulating ATP release, thus resulting in airway hydration.
Airway epithelial cells from CF patients have been shown to have a greater abundance of total AMPK than those derived from normal patients (29). The benefits of promoting AMPK activation via exercise-induced stretch in CF patients may go beyond improvements in hydration. In CF bronchial epithelial cells, AMPK activation inhibits nuclear factor κB, decreases IκB kinase pathways, and has been shown to decrease inflammation in vivo via reduced cytokines (TNF-α, IL-6, IL-8, and GM-CSF) (29,55). Exposure of bronchial cells to severe stretch and hypoxia has been shown to increase reactive oxygen species (ROS) by 25% and has been implicated as a promoter of lung injury (15). The activation of AMPK, however, has been shown to reduce ROS in several epithelial cell species including liver, endothelial, and cardiac (33,34,79). In addition, because increased ventilation both activates AMPK and lowers intracellular PIP2 via auto/paracrine purinergic activation (85), long-term exercise may maintain a reduced TPD via Nedd4-2–mediated ENaC degradation (4). Consistent aerobic exercise may therefore be therapeutic in CF not only by promoting long-term airway hydration but also by attenuating the development of emphysema via reduced airway inflammation and oxidative stress.
The onset of exercise results in an intensity-dependent neuroendocrine response (27). The sympathetic nervous system innervates the myocardium, adrenals, and splanchnic vasculature. The hypothalamic–pituitary–adrenal axis is also activated. Corticosteroids and cathecolamines are released from the adrenals, whereas various peptide hormones are secreted by the pituitary. In addition, a volume overload in the right atrium stimulates the release of ANP by the myocardium (26), which results in increased renal Na+ and H2O excretion (65). Strenuous or prolonged exercise has been shown to significantly elevate plasma ANP. Mandroukas et al. (46) demonstrated that plasma ANP levels increased after 20 min of cycling at 75% maximal HR in a transient manner. Others have shown ANP to significantly rise at exercise intensities as low as 30% V˙O2peak in untrained subjects (47). Hebestreit et al. (30) suggested that ANP perfusion through the pulmonic circuit may decrease luminal Na+ absorption in the CF airway, thereby reducing TPD.
The in vivo effects of ANP on airway epithelia are currently in need of investigation; however, researchers have examined the effects of ANP perfusion on alveolar and tracheal epithelial cells in vitro. Saldías et al. (69) investigated the effects of increased left atrial pressure on alveolar fluid reabsorption in an isolated, perfused rat lung model. Left atrial pressure at 15 cm H2O was shown to inhibit alveolar Na+ transport. The authors suggested that mechanotransduction as a result of higher pressures may have reduced basolateral Na+/K+-ATPase activity. Fukuda et al. (23) suggested that elevated intravascular hydrostatic pressures during exercise may result in an accumulation of interstitial fluid, causing the formation of a barrier, preventing fluid reabsorption, reducing the concentration gradient of luminal to intracellular Na+, and thereby reducing ENaC activity.
Natriuretic receptors have been identified in the epithelia of the bronchioles and Type I/II alveolar cells (20). ANP signals epithelial cells via basolateral natriuretic peptide receptor A, causing changes in G proteins, and an increase in intracellular cyclic guanosine 3′,5′-monophosphate (56). Activation of the natriuretic peptide receptor inhibits apical sodium absorption in the kidneys and intestines (68) and inhibits ENaC in Type II alveolar cells (20). Olivera et al. (59) reported that when pulmonic circulation pressure was kept constant, the perfusion of ANP increased epithelial permeability and reduced Na+ absorption in isolated rat lung. Fluid clearance and active Na+ transport decreased by 38% and 32%, respectively, after the perfusion of ANP.
Sympathetic innervations to the adrenals during exercise results in the secretion epinephrine. By binding β-adrenergic receptors and generating cAMP, epinephrine stimulates Na+ absorption in alveolar Type I and II cells (48) but inhibits Na+ absorption in bronchial and tracheal epithelial cells via CFTR-activated ENaC inhibition (54). In CF, however, cAMP generation activates ENaC and induces water absorption. ANP perfusion may ameliorate β-adrenergic–induced water absorption in CF. Campbell et al. (12) reported a lack of fluid absorption after β-adrenergic agonist administration during increased left atrial pressure in anesthetized, ventilated sheep. The administration ANP and a β-adrenergic agonist together was shown to inhibit the basolateral Na+/K+-ATPase, thereby decreasing the chemical gradient, reducing the driving force for Na+ absorption, and ameliorating ASL dehydration. Exercise may therefore benefit CF patients by stimulating the release of endogenous hormones that positively affect airway ion regulation and lung hydration.
Plasma AVP concentrations increase during exercise in relation to intensity and duration as a response to increased plasma osmolality, plasma concentrations of angiotensin II, peripheral nerve stimulation, and/or reductions in plasma volume (83). Although systemic AVP is a product of posterior pituitary secretion, bronchial epithelial cells also release AVP in a dose dependent response to bradykinin and platelet activating factor (75). Plasma bradykinin is produced when activated kallikrein cleaves it from high-molecular-weight kininogen. Progressive exercise has been shown to activate plasma kallikrein and increase bradykinin secretion as exercise increases in relation to H+ and lactate (38,73,82). Exercise therefore not only increases plasma AVP but may also induce the release of AVP from airway epithelial cells.
Four different AVP receptors (V1a, V1b, V2, and VACM-1) have been identified on the epithelia of the bronchi and trachea. AVP receptors are coupled with G proteins, and when activated, V1 receptors lead to an increase in intracellular Ca2+ via protein lipase C, whereas V2 increase intracellular cAMP via G proteins and thus activates protein kinase A `(PKA) (13). The effect of AVP on airway epithelial ion conductance is biphasic in profile: Cl− rapidly peaks approximately 15 min after application then plateaus for 40 min. AVP has a high affinity for V1b receptors, and this binding has been linked with peak Cl− secretion, possibly via Ca-activated chloride channel activation (7,75). AVP-binding V2 receptor may be responsible for the Cl− secretion plateau. V2 activation at the basolateral domain stimulates the basolateral Na+/K+/2Cl− exchanger causing a rise in intracellular Na+ and Cl−, increasing the driving force for Cl− secretion via an increased chemical gradient, thus reducing TPD (48). On the other hand, V2 binding may also stimulate CFTR, ENaC, and Na+/K+-ATPase via PKA (63), thereby increasing active Na+ transport out of the cell and reducing the chemical gradient. In CF, Na+ absorption would increase because of the inability of dysfunctional CFTR to inhibit ENaC (71), therefore promoting an increase in TPD. Although Phillips and Yeates (63) reported a decrease in TPD after AVP administration in normal airway epithelial cells, more research is needed to evaluate the role of AVP in the reduction of TPD during exercise in CF patients reported by Hebestreit et al. (30).
By altering ion regulation, AVP may reduce luminal water absorption in airway epithelia (Fig. 2). Phillips and Yeates (63) reported a 17% net increase in the basolateral-to-luminal water flux in response to the luminal and basolateral domain AVP administration in epithelial tracheal cells. In addition, AVP administration resulted in a TPD reduction. The researchers attributed the decrease in TPD and net water absorption to basolateral NA+/K+/2Cl+ transporter stimulation and the opening of apical Cl− channels. In addition, Phillips and Yeates (63) suggested that the movement of aquaporins to the epithelial membrane may have also been partially responsible for the increase in basolateral to luminal water flux recorded. Although aquaporin 3 has been identified in airway epithelia and has been shown to shift from basolateral to apical membranes in secretory bronchial epithelial cells (52), information regarding the direct effects of AVP on airway aquaporin transport is scarce. In renal epithelia, aquaporin 3 does not respond to AVP (21); thus, the role of aquaporin migration to the epithelial membrane in facilitating water secretion may have been a result of luminal hyperosmolarity and not a direct function of AVP.
Exercise-induced AVP release may improve mucociliary clearance by increasing cilia beat frequency. Tamaoki et al. (76) investigated the affect of AVP on rabbit trachea epithelia. Cilia beat frequency, intracellular cAMP, intracellular Ca2+, and Ca2+ influx were measured to determine the mechanisms of action. Administration of luminal AVP significantly increased cilia beat frequency 22% after 3 min, and the response remained significantly elevated for 20 min after administration. Antagonism of receptor V1b, but not V1a, attenuated the response, and while intracellular Ca2+ was elevated, no changes in cAMP or Ca2+ influx were reported (76). The researchers concluded that AVP most likely increases cilia beat frequency via V1b-stimulated Ca2+ release. By inducing AVP secretion from both the posterior pituitary and airway epithelia, exercise may be able to positively affect CF patients by promoting airway hydration and sputum output (14).
In the development of CF, the upper lobes contract infection and inflammation and incur a greater degree of degradation compared with the lower lobes (70). As a result of gravity, the upper lungs are ventilated approximately 50% less during tidal breathing (51). The reduction in airflow and subsequent lack of perfusion and mechanical may reduce ASL height and cilia beat frequency, leading to the development of mucus plaques and contributing to the faster rate of disease in the upper versus lower lobes of the lung (78). Progressive aerobic exercise, however, results in an increased recruitment in lung ventilation in an intensity-dependent manner (84). Increased lung ventilation has been suggested to be responsible for the preserved mucociliary clearance before chronic airway obstruction in young, active CF children (11). Through ventilation-induced ATP secretion and AMPK activation and increased perfusion of ANF and AVP, exercise may stimulate airway hydration and improve cilia beating in the upper lungs of CF patients, thereby prolonging or preventing the development mucus plaques, reducing inflammation and infection, and thus delaying the progression in parenchyma degradation. Progressive aerobic exercise, therefore, is a valuable, inexpensive tool that may be used to delay and treat the pathogenesis of CF lung disease.
More research, however, is needed to evaluate the epithelial cellular response to long-term cyclical stretch using intensities and frequencies similar to those experienced during exercise on airway hydration and cilia beat frequency. In addition, more research is needed to investigate the effects of varying exercise intensities and durations on airway inflammatory cytokine secretion in CF patients. Results from these suggested investigations could provide beneficial information in the development of exercise prescriptions that delay the progression of CF lung disease.
No direct funding was received.
Neither author is affiliated with companies or manufacturers who will benefit from the results of the present study.
The results of the study do not constitute endorsement by the American College of Sports Medicine.
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