Cystic fibrosis (CF) is one of the most common lethal genetic diseases in white patients, affecting nearly 30,000 individuals in the United States. Approximately 4% of white individuals carry one mutation of the CF transmembrane conductance regulator (CFTR) gene. The pathological consequences of this mutation can impinge function of the reproductive, digestive, and respiratory systems, as well as temperature regulation and fluid balance. Although CF is a multisystem disease, lung disease results in 85% of CF mortality, according to the CF Foundation Patient Registry. As an autosomal recessive disease, an individual with CF inherits a CFTR mutation on both their paternal and maternal alleles. Thus, the defective CFTR protein that is produced results in improper ion transport because of production of a nonfunctional or mislocalized CFTR chloride (Cl−) channel on the apical membrane of epithelial cells. In the lung, this mutated CFTR leads to decreased Cl− secretion and hyperabsorption of sodium (Na+) through epithelial Na+ channels (ENaC) (23). Consequently, in the CF lung, airway surface liquid (ASL) depth and mucociliary clearance are reduced, leading to airway obstruction, infection, and inflammation. Additionally, at the sweat gland, CFTR dysfunction leads to both high rates of sweat secretion and excessive loss of both Na+ and Cl−. In contrast to the lungs, hypoabsorption of Na+ by ENaC in sweat gland ducts is linked to the lack of CFTR in plasma membranes. This altered sweat gland function can lead to dehydration, hyponatremia, and hypochloremia in patients with CF especially during exercise in the heat (4,21,22).
Exercise can provide improvements to quality of life in CF patients, with benefits including increased exercise tolerance (20), increased respiratory muscle endurance (20), reduced residual volume (3), increased sputum expectoration (25), and reduced rate of decline in pulmonary function (27). It also has been demonstrated that high levels of aerobic fitness in CF patients are associated with a significantly lower risk of death (19). In addition, exercise training is known to improve fluid balance and retention of serum electrolytes in healthy individuals, likely through the plasma volume expansion that is characteristic of exercise and possibly through a direct effect on ion regulation in the sweat glands themselves. Therefore, exercise training has the potential to improve the fluid and salt loss due to sweat gland dysfunction in CF patients by reducing thermal strain and the incidence of dehydration, possibly through direct interaction of CFTR-related mechanisms. In this review, we will first present the previous work that has led to the belief that exercise is a vital component of the therapy regimen prescribed to CF patients because of the multitudinous systemic benefits. We will then present the emerging data and our recent research supporting the mechanisms by which exercise is medicine for CF patients at a cellular level by promoting improvements in ion balance by acting directly on sweat glands and airway epithelia to improve ion dysregulation and thereby enhance pulmonary function, quality of life, and life expectancy.
EXERCISE IN CF
The ideal therapy in CF lung disease is one that demonstrates an attenuation of the characteristic 2%-3% annual decline in pulmonary function and forced expiratory volume in 1 s (FEV1) and facilitates an improvement in salt transport. Previous research demonstrated that CF patients with a maximal oxygen consumption (V˙O2peak) greater than 82% of their predicted value had an 83% 8-yr survival rate compared with only a 28% 8-yr survival rate for patients with a V˙O2peak percent predicted less than 58%. Additionally, this study found that aerobic fitness was an independent predictor of 8-yr survival, whereas FEV1, the gold standard for the assessment of disease progression and life expectancy, was not (19). Exercise tolerance, aerobic capacity, and respiratory muscle endurance all were significantly increased in a study of subjects completing a 3-month supervised running program. Unlike subjects in the control group, subjects who completed the running program demonstrated no significant decline in pulmonary function (20). These findings were confirmed in another study, which demonstrated similar improvement in V˙O2peak, work capacity, forced vital capacity (FVC), amount of time spent in vigorous physical activity, and perceived health using a 6-month partially supervised conditioning program (15). Although less effective when compared with chest physiotherapy, exercise tended to increase mean daily sputum expectoration weight 24 ± 25 at baseline versus 37 ± 47 g post-2-month training program (25). These advantageous systemic changes in response to exercise demonstrate a better quality of life and suggest increased life expectancy for CF patients who exercise, but there is limited mechanistic data on the effects of exercise on providing improvements to ion regulation at the cellular level in vivo in CF. We hypothesize that exercise can be beneficial to patients with CF at a cellular level by both increasing Cl− secretion and inhibiting Na+ hyperabsorption across airway and alveolar epithelial cells, thereby ameliorating ion dysregulation in the CF lung, and also by improving thermoregulation and fluid balance through plasma volume expansion and/or a direct effect on the sweat glands.
Ion Regulation in the Sweat Glands
Along the reabsorptive duct of the sweat gland, CFTR is essential for Cl− absorption (30). Indirectly, malfunctioning CFTR and the lack of Cl− absorption also lead to a limitation in Na+ absorption via ENaC in sweat gland ducts (24). This ion dysregulation in the sweat glands of CF patients results in abnormally high Na+ and Cl− concentration in secreted sweat, and since this was discovered by Di Sant‘Agnese et al. (13), it has become a diagnostic characteristic of CF. Exercise training activates pathways to mediate alterations in the ion composition of sweat in healthy subjects. Normal sweat secretion can be controlled by both β-adrenergic (ADBR2) receptors and cholinergic mechanisms. However, sweat glands in CF patients lack any ADBR2-stimulated sweat production because ADBR2 sweat secretion is mediated through CFTR (26). However, cholinergic sweat secretion is dependent on both calcium-dependent potassium channels and CFTR (26). The significantly elevated sweat production and sweat salt content during exercise in patients with CF is primarily related to the cholinergic activation of eccrine sweat glands. An interesting finding from studies of ADBR2-mediated sweat rate demonstrated that ADBR2-mediated sweating in healthy CF carriers is essentially half that of healthy controls. These researchers concluded that CF carriers generally have an ADBR2-stimulated sweat secretion that is significantly reduced compared with their healthy counterparts (5). If a functional CFTR is necessary for normal Cl− absorption, these findings suggest that even CF carriers may have altered, or reduced, CFTR expression in the sweat gland.
Thermoregulation and Fluid Balance
Previous work has demonstrated heat intolerance in children and adults with CF (13,22). Orenstein et al. (21) reported high sweat rates and substantially higher concentrations of both Na+ and Cl− in sweat of patients with CF. Thus, the most common underlying factors associated with this heat intolerance with CF are dehydration, hypochloremia, and/or hyponatremia (4). This suggests that during exposure to high environmental heat, patients with CF may endure significantly elevated thermal and possibly cardiovascular strain. Exploiting the known training adaptations, including plasma volume expansion and serum electrolyte retention, individuals with CF may be able to take advantage of a protective effect of chronic exercise.
Plasma volume expansion is a clear adaptation of exercise training and heat acclimation in humans with normal sweat and ion production. This plasma volume expansion may be an important mechanism for the adaptations to heat acclimation, including sweat dilution and a reduction in sweat Na+ and Cl− concentration, which would be a particularly important adaptation for patients with CF (1,16). The question of whether plasma volume expansion occurs in CF remains unanswered. Recent work demonstrating that baseline plasma vasopressin levels were significantly elevated in non-CF "salty sweaters" and tended to be higher in CF patients when compared with healthy controls and finding a positive relationship between sweat Na+ concentration and plasma vasopressin concentration support the hypothesis that plasma volume expansion may indeed be a natural adaptation to salty sweat (8). More dilute sweat with heat acclimation acts to retain serum Na+ and Cl− during subsequent heat exposure (6,22). In addition to sweat dilution, exercise training and acclimation to heat reduces the core temperature threshold for reflex vasodilation and sweating, thereby maximizing evaporative heat loss and decreasing thermal strain (18). In addition, acute plasma volume expansion reduces heart rate and increases stroke volume during heat stress, thereby decreasing cardiovascular strain for any given rise in core body temperature (29,35). In general, the physiological adaptations from exercise training and acclimation to heat act to decrease cardiovascular and thermal strain during subsequent heat exposure.
To our knowledge, only one study has examined physiological adaptations to heat acclimation, per se, in CF patients. Orenstein et al. (22) reported that subjects with CF tolerated repeated heat exposure (8 d of combined exercise and heat) remarkably well, and core temperature during exercise in the heat decreased after acclimation in subjects with CF. However, combined exercise and heat acclimation failed to decrease sweat Na+ and Cl− concentrations in patients with CF. As a consequence, serum Na+ and Cl− concentrations remained depressed in CF. Unfortunately, this study did not investigate potential adaptations in skin blood flow regulation or sudomotor control of sweating, which may have contributed to the enhanced heat tolerance after exercise and heat acclimation in these CF patients. Skin blood flow responses to local and passive whole body heating, and muscle blood flow response during exercise, seem normal in CF (28,34). Thus, improvements in reflex cutaneous vasodilation after exercise training are likely in patients with CF. The mechanisms responsible for the normal enhanced absorption of salt in the sweat gland adaptation after exercise training are not well understood. Because Orenstein et al. did not observe more dilute sweat (enhanced absorption) in CF after combined exercise and heat acclimation, the mechanism may be related to the function of CFTR and ENaC in the sweat gland.
The high sweat rate and high concentrations of Na+ and Cl− in the sweat may significantly reduce serum osmolality. The low osmolality in body fluids may deprive individuals with CF of a thirst stimulus, further exacerbating dehydration during combined exercise and heat stress. An important consideration when using exercise as medicine in patients with CF is fluid and electrolyte replacement (4). Unfortunately, replacement strategies specifically have not been identified in CF. Recent work has demonstrated that the serum salt and osmolality increased less in response to dehydration but that there was a greater relative loss of plasma volume in CF patients. There was no difference in thirst perception, but CF subjects drank approximately 30% less, leading the researchers to hypothesize that there may be negative feedback triggering slowed voluntary drinking and that thirst-guided fluid replacement, rather than forced drinking, may be most appropriate to effectively maintain body weight and decrease recovery heart rate after exercise (7).
Ion Regulation in the Lung
Epithelial cells of the bronchial tree and alveoli are covered in a surface liquid composed of mucus, ions, inflammatory proteins, and water. Airway surface liquid is a fundamental component of the pulmonary defense and one that has been shown to influence overall pulmonary function, where homeostasis is achieved by keeping the lungs moist but not overly wet. Maintaining ASL depth, normally approximately 7 μm, allows for effective ciliary beating to facilitate effective mucus clearance and prevent mucus plugging that can limit gas transfer. Maintaining the ideal hydration of the airway lumen and alveoli is achieved by the active transport of salt prompting the subsequent osmotic water flux. The absorptive pathway for removal of excess fluid is mediated through ENaC, which moves Na+ down its concentration gradient into the cell where it is then pumped out on the basolateral side, by Na+/K+-ATPase, maintaining this inward electrochemical gradient. To maintain electroneutrality, negatively charged Cl− will move paracellularly through the "loose" tight junctions to balance the positively charged Na+ influx. In the alternative, Cl− can be secreted from the airway epithelia primarily through CFTR and also, to a lesser extent, by calcium-dependent chloride channels (CaCC), with Na+ balancing this anion efflux through paracellular transport allowing for an increase in ASL depth. The outward concentration gradient for this efflux of Cl− is facilitated through the activity of the basolateral Na+/K+/2Cl− cotransporter. It is the regulation of these channels to control the net ion transport and salt load that maintains homeostasis of ASL depth (11).
During exercise, two pathways for regulating ion composition of ASL are initiated, both of which could have favorable effects on ion regulation in CF. This has led us to our emerging hypothesis that exercise is beneficial, beyond the systemic improvements mentioned previously, at a cellular level by helping to keep the lungs moist through amelioration of ion dysregulation that is a hallmark of this disease. Exercise results in endogenous stimulation of ADBR2 by elevated epinephrine (Epi) and stimulation of purinergic (P2Y2) receptors by adenosine triphosphate (ATP) and adenosine (ADO) (11). Moderate exercise, greater than 50% maximal oxygen consumption, markedly increases catecholamine (norepinephrine (NE) and Epi) levels. As a G-protein coupled receptor, binding of Epi to ADBR2s causes release of the Gsα subunit, which then activates adenylate cyclase, allowing for conversion of ATP to cyclic-ADO monophosphate (cAMP). cAMP can then mediate the activation of protein kinase A (PKA). Phosphorylation of the regulatory domain of CFTR by PKA causes channel activation, resulting in Cl− efflux to the apical side of airway epithelial cells. β-agonist stimulation has demonstrated an increase in Cl− secretion toward the lumen across the canine trachea, in vitro (17). When both ENaC and CFTR are expressed, cAMP agonists decrease ENaC activity because of the inhibitory action of CFTR on ENaC; however, when CFTR is absent, which occurs with the ΔF508 mutation, which is present in 90% of CF patients, cAMP increases Na+ reabsorption, highlighting the important inhibitory role of CFTR in restricting ENaC activity (11). In the second pathway, ATP is released from airway epithelia in response to mechanical stress that occurs with the increased ventilation of exercise. Both ATP and its metabolite ADO can interact with P2Y2 receptors on the apical membrane causing a depletion in phosphatidylinositol 4,5-bisphosphate (PIP2). Because PIP2 is required for protein kinase C-mediated ENaC activation, P2Y2 receptor activation thereby inhibits ENaC. Purinergic receptor activation also can stimulate CaCC activity through the concurrent inositol trisphosphate-mediated release of Ca+2 from endoplasmic reticulum calcium stores (11). Research has shown that nucleotide application to the airway lumen mediates ASL secretion in both healthy and CF airway epithelia (9). Additionally, the ADO-2b (A2b) receptor also is present on the apical membrane of the airway epithelia and increases PKA to activate CFTR (9) (Fig. 1). Through these pathways, it is clear that exercise could act to ameliorate ion dysregulation in CF and provide benefits to CF patients.
Recent work in CF airway submucosal gland serous cells has demonstrated that salmeterol, a long-acting β-agonist, restores normal secretory function by stabilizing CFTR and allowing for increased Cl− secretion (12). Also, there has been recent in vivo research demonstrating that exercise inhibits ENaC function in both healthy and CF subjects. At rest, nasal potential difference (NPD) was more negative in CF because of the lack of Cl− efflux and hyperabsorption of Na+ (negative inward current). With exercise, NPD became less negative in CF subjects so that, at the end of exercise, there was no difference in NPD between healthy and CF subjects (2,14). The effect of amiloride on NPD also was reduced during exercise, suggesting that exercise partially blocked ENaC conductance. These findings suggest that exercise-mediated inhibition of ENaC and possible CaCC activation could help to increase water content in the mucus, providing greater ease for expectoration (14). Because of the lack of functional CFTR in CF, ADBR2-mediated stimulation has minimal effect on Cl− secretion; however, P2Y2 agonists ATP/ADO also are increased during exercise, which can mediate inhibition of ENaC Na+ absorption and cause CaCC-mediated Cl− efflux through a CFTR-independent pathway (2).
Ameliorating Ion Dysregulation in the CF Lung
Quantitative analysis of ASL composition is extremely difficult because of the layer's thinness and limited accessibility. Exhaled air, predominantly composed of water vapor, also contains small droplets of fluid that are produced by the shear force of turbulent flow upon exhalation across the ASL. Therefore, the composition of one's exhaled breath is believed to be a surrogate, although diluted, marker of the composition of the ASL from any location in the lung, including the alveoli. The composition of the exhaled breath condensate (EBC) then can be analyzed for solutes, including ions (10). Our laboratory has been using ion composition of EBC to understand composition of ASL noninvasively and to study how different stimuli can alter this composition in both healthy subjects (n = 26) and mild-to-moderate patients with CF (n = 10, FEV1 = 70 ± 24% pred, forced vital capacity (FVC) = 85 ± 20% pred) First, we have demonstrated that the administration of the β-agonist albuterol alters EBC ion composition, significantly increasing net exhaled Cl− from the baseline in healthy subjects and showing a trend of reducing exhaled Na+ in both healthy and CF subjects (Fig. 2). Net exhaled Cl− was calculated as gross exhaled Cl− concentration plus the absolute value of the percent change in exhaled Na+ from the baseline multiplied by the gross exhaled Cl− concentration, which we have used to account for Cl− influx that would follow Na+ absorption to maintain electroneutrality. These results demonstrate that EBC composition is regulated because it can be altered by the administration of a β-agonist.
Second, we have shown that there is a negative relationship between exhaled Na+ and gas diffusion at the individual functional alveolar-capillary unit (alveolar-capillary membrane conductance/pulmonary capillary blood volume). Where increased exhaled Na+ could be indicating an increase in lung water, which consequently could limit gas transfer (31). This correlation between exhaled Na+ and gas transfer suggests that a portion of EBC droplet formation is occurring in the respiratory zone where gas diffusion takes place. Third, both healthy and CF subjects with a more active ENaC genotype have lower exhaled Na+ levels (32). When we performed maximal exercise tests with measurements of lung diffusion, we found that membrane conductance was significantly lower in the CF patients at rest, and the difference was augmented with exercise. However, exercise did mediate an improvement in lung function and lung diffusion, demonstrating an increase of 37% in forced expiratory flow at 25%-75% of FVC (FEF25-75) and 29% in membrane conductance in these CF patients (33). These data demonstrate that exercise can cause systemic benefits by improving gas transfer and mediating bronchodilation to a level that is similar to what can be achieved with the administration of a β-agonist. Essentially, these improvements in membrane conductance could be due to exercise-mediated cellular improvements in ion regulation to help moisten the lung and ameliorate some of the gas diffusion limitations present at rest. The therapeutic benefits of exercise, we hypothesize, stem from the idea that exercise could act similar to an exogenous agonist (drug therapy); with exercise, there is not only the ability to stimulate adrenergic-mediated regulation of ions and lung fluid, but exercise also facilitates the activation of purinergic mechanisms for maintaining lung fluid homeostasis. It is this addition of the purinergic stimulation of CFTR-independent Cl− secretion through CaCC and ENaC inhibition that may be the most beneficial for ameliorating ion dysregulation in the CF lung.
Despite evidence that exercise improves the quality of life in patients with CF, there is little mechanistic data available regarding the influence of exercise on ASL ion regulation (i.e., evidence for what is occurring on a cellular level) and mechanisms of thermoregulatory adaptations in this population. Because of endogenous adrenergic and purinergic ion channel regulation in the airway epithelial cells, it is likely that exercise could attenuate CFTR dysfunction and ENaC hyperactivity and additionally activate CaCC, ameliorating ion dysregulation in CF. Evidence of this exercise-induced ion regulation in the lung could provide incentive for CF subjects to implement and maintain the prescribed exercise as medicine through childhood and adult life to prevent the progression of pulmonary disease.
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