Low intensity exercise training in patients with amyotrophic lateral sclerosis - factors influencing eligibility and compliance to the clinical trial.

BACKGROUND: To date, there are no evidence-based recommendations for physical therapy in amyotrophic lateral sclerosis (ALS). The reason is a low number of related clinical trials (CTs), restricted sample sizes and a high dropout rate. It may influence the profile of the participants, while the final results might not translate to the general ALS population. OBJECTIVE: To analyze factors affecting the ALS patients' enrollment and retention to the study, and to describe a profile of participants as compared to the eligible group. METHODS: A total of 104 ALS patients were offered participation in a CT of low-intensity exercises at home. Forty-six patients were recruited. Demographic and clinical data (El Escorial criteria, site of onset, diagnosis delay, disease duration, amyotrophic lateral sclerosis functional rating scale - revised [ALSFRS-R], Medical Research Council [MRC], hand-held dynamometry) were analyzed every 3 months. RESULTS: Male gender, younger age and a higher ALSFRS predicted enrollment, while male gender, higher ALSFRS-R and MRC predicted retention in the study. A long commute to the study site and a fast disease progression were the main reasons influencing both enrollment and retention. Despite a high dropout rate, study participants were representative for the general ALS population. CONCLUSION: The above demographic, clinical and logistic factors need to be considered when designing studies in ALS population.

Regulation of alveolar epithelial function by hypoxia.

Patients with acute respiratory distress syndrome and high-altitude pulmonary oedema build up excess lung fluid, which leads to alveolar hypoxia. In patients with acute respiratory distress syndrome and hypoxia, there is a decrease in oedema fluid clearance, due in part to the downregulation of plasma membrane sodium-potassium adenosine triphosphatase (Na,K-ATPase). In alveolar epithelial cells, acute hypoxia promotes Na,K-ATPase endocytosis from the plasma membrane to intracellular compartments, resulting in inhibition of Na,K-ATPase activity. Exposure to prolonged hypoxia leads to degradation of plasma membrane Na,K-ATPase. The downregulation of plasma membrane Na,K-ATPase reduces adenosine triphosphate demand, as part of a survival mechanism of cellular adaptation to hypoxia. Hypoxia has also been shown to disassemble and degrade the keratin intermediate filament network, a fundamental component of the cell cytoskeleton, affecting epithelial barrier function. Accordingly, better understanding of the mechanisms regulating cellular adaptation to hypoxia may lead to the development of novel therapeutic strategies for acute respiratory distress syndrome and high-altitude pulmonary oedema patients.

Peripheral airways injury in acute lung injury/acute respiratory distress syndrome.

PURPOSE OF REVIEW: Peripheral airways are less than 2 mm in diameter and comprise a relatively large cross-sectional area, which allows for slower, laminar airflow. They include both membranous bronchioles and gas exchange ducts, and have been referred to in the past as the 'quiet zone', partly because these structures were felt to contribute little to lung mechanics, and partly because they are difficult to study directly. RECENT FINDINGS: Recent studies suggest that peripheral airway dysfunction plays a significant role in acute respiratory distress syndrome, which may be exacerbated by injurious mechanical ventilation strategies. The presence of elevated airways resistance, intrinsic positive end-expiratory pressure or a lower inflection point on a pressure-volume curve of the respiratory system may indicate presence of impaired peripheral airway function. In-vitro animal and human studies have begun to elucidate the signaling mechanisms responsible for stretch and shear mediated cellular injury. SUMMARY: Understanding the pathophysiology of peripheral airway dysfunction in acute respiratory distress syndrome and mechanical ventilation continues to evolve. Greater insight into the signaling mechanisms involved in cellular injury and repair will lead to further alterations in mechanical ventilation strategies, and may lead to specific treatment options.

Bench-to-bedside review: distal airways in acute respiratory distress syndrome.

Distal airways are less than 2 mm in diameter, comprising a relatively large cross-sectional area that allows for slower, laminar airflow. The airways include both membranous bronchioles and gas exchange ducts, and have been referred to in the past as the 'quiet zone', in part because these structures were felt to contribute little to lung mechanics and in part because they were difficult to study directly. More recent data suggest that distal airway dysfunction plays a significant role in acute respiratory distress syndrome. In addition, injurious mechanical ventilation strategies may contribute to distal airway dysfunction. The presence of elevated airway resistance, intrinsic positive end-expiratory pressure or a lower inflection point on a pressure-volume curve of the respiratory system may indicate the presence of impaired distal airway function. There are no proven specific treatments for distal airway dysfunction, and protective ventilation strategies to minimize distal airway injury may be the best therapeutic approach at this time.

Fibroblast growth factor-10 upregulates Na,K-ATPase via the MAPK pathway.

We studied the effects of fibroblast growth factor (FGF-10) on alveolar epithelial cell (AEC) Na,K-ATPase regulation. Within 30 min FGF-10 increased Na,K-ATPase activity and alpha1 protein abundance by 2.5-fold at the AEC plasma membrane. Pretreatment of AEC with the mitogen-activated protein kinase (MAPK) inhibitor U0126, a Grb2-SOS inhibitor (SH3-b-p peptide), or a Ras inhibitor (farnesyl transferase inhibitor (FTI 277)), as well as N17-AEC that express a Ras dominant negative protein each prevented FGF-10-mediated Na,K-ATPase recruitment to the AEC plasma membrane. Accordingly, we provide first evidence that FGF-10 upregulates (short-term) the Na,K-ATPase activity in AEC via the Grb2-SOS/Ras/MAPK pathway.

Alveolar type 1 cells express the alpha2 Na,K-ATPase, which contributes to lung liquid clearance.

The alveolar epithelium is composed of alveolar type 1 (AT1) and alveolar type 2 (AT2) cells, which represent approximately 95% and approximately 5% of the alveolar surface area, respectively. Lung liquid clearance is driven by the osmotic gradient generated by the Na,K-ATPase. AT2 cells have been shown to express the alpha1 Na,K-ATPase. We postulated that AT1 cells, because of their larger surface area, should be important in the regulation of active Na+ transport. By immunofluorescence and electron microscopy, we determined that AT1 cells express both the alpha1 and alpha2 Na,K-ATPase isoforms. In isolated, ouabain-perfused rat lungs, the alpha2 Na,K-ATPase in AT1 cells mediated 60% of the basal lung liquid clearance. The beta-adrenergic agonist isoproterenol increased lung liquid clearance by preferentially upregulating the alpha2 Na,K-ATPase protein abundance in the plasma membrane and activity in alveolar epithelial cells (AECs). Rat AECs and human A549 cells were infected with an adenovirus containing the rat Na,K-ATPase alpha2 gene (Adalpha2), which resulted in the overexpression of the alpha2 Na,K-ATPase protein and caused a 2-fold increase in Na,K-ATPase activity. Spontaneously breathing rats were also infected with Adalpha2, which increased alpha2 protein abundance and resulted in a approximately 250% increase in lung liquid clearance. These studies provide the first evidence that alpha2 Na,K-ATPase in AT1 cells contributes to most of the active Na+ transport and lung liquid clearance, which can be further increased by stimulation of the beta-adrenergic receptor or by adenovirus-mediated overexpression of the alpha2 Na,K-ATPase.

Invited review: lung edema clearance: role of Na(+)-K(+)-ATPase.

Acute hypoxemic respiratory failure is a consequence of edema accumulation due to elevation of pulmonary capillary pressures and/or increases in permeability of the alveolocapillary barrier. It has been recognized that lung edema clearance is distinct from edema accumulation and is largely effected by active Na(+) transport out of the alveoli rather than reversal of the Starling forces, which control liquid flux from the pulmonary circulation into the alveolus. The alveolar epithelial Na(+)-K(+)-ATPase has an important role in regulating cell integrity and homeostasis. In the last 15 yr, Na(+)-K(+)-ATPase has been localized to the alveolar epithelium and its contribution to lung edema clearance has been appreciated. The importance of the alveolar epithelial Na(+)-K(+)-ATPase function is reflected in the changes in the lung's ability to clear edema when the Na(+)-K(+)-ATPase is inhibited or increased. An important focus of the ongoing research is the study of the mechanisms of Na(+)-K(+)-ATPase regulation in the alveolar epithelium during lung injury and how to accelerate lung edema clearance by modulating Na(+)-K(+)-ATPase activity.

Dopamine increases lung liquid clearance during mechanical ventilation.

Short-term mechanical ventilation with high tidal volume (HVT) causes mild to moderate lung injury and impairs active Na+ transport and lung liquid clearance in rats. Dopamine (DA) enhances active Na+ transport in normal rat lungs by increasing Na+-K+-ATPase activity in the alveolar epithelium. We examined whether DA would increase alveolar fluid reabsorption in rats ventilated with HVT for 40 min compared with those ventilated with low tidal volume (LVT) and with nonventilated rats. Similar to previous reports, HVT ventilation decreased alveolar fluid reabsorption by ~50% (P < 0.001). DA increased alveolar fluid reabsorption in nonventilated control rats (by ~60%), LVT ventilated rats (by approximately 55%), and HVT ventilated rats (by ~200%). In parallel studies, DA increased Na+-K+-ATPase activity in cultured rat alveolar epithelial type II cells (ATII). Depolymerization of cellular microtubules by colchicine inhibited the effect of DA on HVT ventilated rats as well as on Na+-K+-ATPase activity in ATII cells. Neither DA nor colchicine affected the short-term Na+-K+-ATPase alpha1- and beta1-subunit mRNA steady-state levels or total alpha1- and beta1-subunit protein abundance in ATII cells. Thus we reason that DA improved alveolar fluid reabsorption in rats ventilated with HVT by upregulating the Na+-K+-ATPase function in alveolar epithelial cells.

beta(2)-adrenergic receptor overexpression increases alveolar fluid clearance and responsiveness to endogenous catecholamines in rats.

beta-Adrenergic agonists accelerate the clearance of alveolar fluid by increasing the expression and activity of epithelial solute transport proteins such as amiloride-sensitive epithelial Na(+) channels (ENaC) and Na,K-ATPases. Here we report that adenoviral-mediated overexpression of a human beta(2)-adrenergic receptor (beta(2)AR) cDNA increases beta(2)AR mRNA, membrane-bound receptor protein expression, and receptor function (procaterol-induced cAMP production) in human lung epithelial cells (A549). Receptor overexpression was associated with increased catecholamine (procaterol)-responsive active Na(+) transport and increased abundance of Na,K-ATPases in the basolateral cell membrane. beta(2)AR gene transfer to the alveolar epithelium of normal rats improved membrane-bound beta(2)AR expression and function and increased levels of ENaC (alpha subunit) abundance and Na,K-ATPases activity in apical and basolateral cell membrane fractions isolated from the peripheral lung, respectively. Alveolar fluid clearance (AFC), an index of active Na(+) transport, in beta(2)AR overexpressing rats was up to 100% greater than sham-infected controls and rats infected with an adenovirus that expresses no cDNA. The addition of the beta(2)AR-specific agonist procaterol to beta(2)AR overexpressing lungs did not increase AFC further. AFC in beta(2)AR overexpressing lungs from adrenalectomized or propranolol-treated rats revealed clearance rates that were the same or less than normal, untreated, sham-infected controls. These experiments indicate that alveolar beta(2)AR overexpression improves beta(2)AR function and maximally upregulates beta-agonist-responsive active Na(+) transport by improving responsiveness to endogenous catecholamines. These studies suggest that upregulation of beta(2)AR function may someday prove useful for the treatment of pulmonary edema.

Alveolar fluid reabsorption is impaired by increased left atrial pressures in rats.

Cardiogenic pulmonary edema results from increased hydrostatic pressures across the pulmonary circulation. We studied active Na(+) transport and alveolar fluid reabsorption in isolated perfused rat lungs exposed to increasing levels of left atrial pressure (LAP; 0--20 cmH(2)O) for 60 min. Active Na(+) transport and fluid reabsorption did not change when LAP was increased to 5 and 10 cmH(2)O compared with that in the control group (0 cmH(2)O; 0.50 +/- 0.02 ml/h). However, alveolar fluid reabsorption decreased by approximately 50% in rat lungs in which the LAP was raised to 15 cmH(2)O (0.25 +/- 0.03 ml/h). The passive movement of small solutes ((22)Na(+) and [(3)H]mannitol) and large solutes (FITC-albumin) increased progressively in rats exposed to higher LAP. There was no significant edema in lungs with a LAP of 15 cmH(2)O when all active Na(+) transport was inhibited by hypothermia or amiloride (10(-4) M) and ouabain (5 x 10(-4) M). However, when LAP was increased to 20 cmH(2)O, there was a significant influx of fluid (-0.69 +/- 0.10 ml/h), precluding the ability to assess the rate of fluid reabsorption. In additional studies, LAP was decreased from 15 to 0 cmH(2)O in the second and third hours of the experimental protocol, which resulted in normalization of lung permeability to solutes and alveolar fluid reabsorption. These data suggest that in an increased LAP model, the changes in clearance and permeability are transient, reversible, and directly related to high pulmonary circulation pressures.

Dopamine regulates Na-K-ATPase in alveolar epithelial cells via MAPK-ERK-dependent mechanisms.

Dopamine (DA) increases lung edema clearance by regulating vectorial Na+ transport and Na-K-ATPase in the pulmonary epithelium. We studied the role of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway in the DA regulation of Na-K-ATPase in alveolar epithelial cells (AEC). Incubation of AEC with DA resulted in a rapid stimulation of ERK activity via dopaminergic type 2 receptors. Analysis of total RNA and protein showed a 1.5-fold increase in the Na-K-ATPase beta1-subunit mRNA levels and up to a fivefold increase in beta1-subunit protein abundance after DA stimulation, which was blocked by the MAPK kinase (MEK) inhibitors PD-98059 and U-0126. Also, the DA-ERK pathway stimulated the synthesis of a green fluorescent protein reporter gene driven by the beta1-subunit promoter, which indicates that DA regulates the Na-K-ATPase beta1-subunit at the transcriptional level. The DA-mediated increase in beta1-subunit mRNA protein resulted in an increase in functional Na pumps in the basolateral membranes of alveolar type II cells. These results suggest that the MAPK-ERK pathway is an important mechanism in the regulation of Na-K-ATPase by DA in the alveolar epithelium.

Na,K-ATPase gene transfer mitigates an oxidant-induced decrease of active sodium transport in rat fetal ATII cells.

We investigated whether adenovirus-mediated transfer of genes encoding for subunits of the Na,K-ATPase increases transepithelial Na(+) transport in rat fetal distal lung epithelial (FDLE) monolayers and renders them more resistant to hydrogen peroxide injury. FDLE cells, isolated from rat fetuses at a gestational age of 19 to 20 d (22 d = term), were seeded on filters and infected with replication-incompetent human type 5 adenoviruses containing complementary DNAs encoding for rat Na,K-ATPase alpha(1) or beta(1) subunits (ad alpha(1) and ad beta(1), respectively). Once confluent monolayers were formed, the filters were mounted in Ussing chambers and short circuit currents (I(SC)) were measured. Increased levels of alpha(1) or beta(1) subunit proteins after infection with ad alpha(1) and ad beta(1), respectively, were confirmed by Western blot analysis. Baseline I(SC) increased after transfection with 2 plaque-forming units (pfu) of ad beta(1) from 5.1 +/- 0.3 to 6.1 +/- 0.3 microA/cm(2) (mean +/- SEM; P < 0.05). Permeabilization of the apical membrane with amphotericin B caused a large increase in I(SC); the ouabain-sensitive component of the amphotericin B-elicited I(SC) (ouab(max)) was increased from 4.0 +/- 0.2 (n = 69) in controls to 4.8 +/- 0.2 (n = 15), 5.9 +/- 0.3 (n = 53), 6.9 +/- 0.4 (n = 25), 7.7 +/- 0.9 (n = 16) in monolayers infected with 1, 2, 11, and 22 pfu of ad beta(1), respectively; transfection with ad alpha(1) had no effect on any measured variables. Further, transfection with ad beta(1) in comparison to noninfected monolayers resulted in higher baseline and ouab(max) I(SC) after injury with 500 microM H(2)O(2). We conclude that overexpression of the beta(1) subunit of the Na,K-ATPase may help maintain normal levels of vectorial Na(+) transport across ATII cell monolayers in pathologic conditions.

Catecholamines increase lung edema clearance in rats with increased left atrial pressure.

During hydrostatic pulmonary edema, active Na(+) transport and alveolar fluid reabsorption are decreased. Dopamine (DA) and isoproterenol (ISO) have been shown to increase active Na(+) transport in rat lungs by upregulating Na(+)-K(+)-ATPase in the alveolar epithelium. We studied the effects of DA and ISO in isolated rat lungs with increased left atrial pressure (Pla = 15 cmH(2)O) compared with control rats with normal Pla (Pla = 0). Alveolar fluid reabsorption decreased from control value of 0.51 +/- 0.02 to 0.27 +/- 0.02 ml/h when Pla was increased to 15 cmH(2)O (P < 0.001). DA and ISO increased the alveolar fluid reabsorption back to control levels. Treatment with the D(1) antagonist SCH-23390 inhibited the stimulatory effects of DA (0.30 +/- 0.02 ml/h), whereas fenoldopam, a specific D(1)-receptor agonist, increased alveolar fluid reabsorption in rats exposed to Pla of 15 cmH(2)O (0.47 +/- 0.04 ml/h). Propranolol, a beta-adrenergic-receptor antagonist, blocked the stimulatory effects of ISO; however, it did not affect alveolar fluid reabsorption in control or DA-treated rats. Amiloride (a Na(+) channel blocker) and ouabain (a Na(+)-K(+)-ATPase inhibitor), either alone or together, inhibited the stimulatory effects of DA. Colchicine, which disrupts the cellular microtubular transport of ion-transporting proteins to the plasma membrane, inhibited the stimulatory effects of DA, whereas the isomer beta-lumicolchicine did not block the stimulatory effects of DA. These data suggest that DA and ISO increase alveolar fluid reabsorption in a model of increased Pla by regulating active Na(+) transport in rat alveolar epithelium. The effects of DA and ISO are mediated by the activation of dopaminergic D(1) receptors and the beta-adrenergic receptors, respectively.

beta-agonists regulate Na,K-ATPase via novel MAPK/ERK and rapamycin-sensitive pathways.

We studied whether the beta-adrenergic agonist, isoproterenol (ISO), regulates Na,K-ATPase in alveolar epithelial cells (AEC) via a mitogen-activated protein kinase (MAPK)/extracellular signaling related kinase (ERK) dependent pathway. ISO increased ERK activity in AEC by 10 min via a beta-adrenergic receptor, protein kinase A (PKA)-dependent mechanism. Activation of the MAPK pathway by ISO, resulted in increased Na,K-ATPase beta1 and alpha1 subunit protein abundance in whole cell lysates, which resulted in functional Na, K-ATPases at the basolateral membranes. ISO did not change the alpha1 or beta1 mRNA steady state levels, but rapamycin, the inhibitor of the mammalian target of rapamycin, also blocked the ISO-mediated increase in Na,K-ATPase total protein abundance, suggesting a posttranscriptional regulation. We conclude that ISO, regulates the Na,K-ATPase in AEC via PKA, ERK and rapamycin-sensitive mechanisms.

Adenovirus-mediated transfer of an Na+/K+-ATPase beta1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats.

Pulmonary edema is cleared via active Na(+) transport by alveolar epithelial Na(+)/K(+)-ATPases and Na(+) channels. Rats exposed to acute hyperoxia have a high mortality rate, decreased Na(+)/K(+)-ATPase function, and decreased alveolar fluid clearance (AFC). We hypothesized that Na(+)/K(+)-ATPase subunit gene overexpression could improve AFC in rats exposed to hyperoxia. We delivered 4 x 10(9) PFU of recombinant adenoviruses containing rat alpha(1) and beta(1) Na(+)/K(+)-ATPase subunit cDNAs (adalpha(1) and adbeta(1), respectively) to rat lungs 7 days prior to exposure to 100% O(2) for 64 hr. As compared with controls and ad alpha(1), AFC in the adbeta(1) rats was increased by >300%. Permeability for large solutes was less in the ad beta(1) than in the other hyperoxia groups. Glutathione oxidation, but not superoxide dismutase activity, was increased only in the adbeta(1) group. Survival through 14 days of hyperoxia was 100% in the adbeta(1) group but was not different from hyperoxic controls in animals given adalpha(1). Our data show that overexpression of a beta(1) Na(+)/K(+)-ATPase subunit augments AFC and improves survival in this model of acute lung injury via antioxidant-independent mechanisms. Conceivably, restoration of AFC via gene transfer of Na(+)/K(+)-ATPase subunit genes may prove useful for the treatment of acute lung injury and pulmonary edema.

Continuous enteral nutrition attenuates pulmonary edema in rats exposed to 100% oxygen.

Adult rats exposed to hyperoxia develop anorexia, weight loss, and a lung injury characterized by pulmonary edema and decreased lung liquid clearance. We hypothesized that maintenance of nutrition during hyperoxia could attenuate hyperoxia-induced pulmonary edema. To test this hypothesis, we enterally fed adult male Sprague-Dawley rats via gastrostomy tubes and exposed them to oxygen (inspired O(2) fraction >0.95) for 64 h. In contrast to controls, enterally fed hyperoxic animals did not lose weight and had smaller pleural effusions and wet-to-dry weight ratios (a measure of lung edema) that were not different from room air controls. Enterally fed rats exposed to hyperoxia had increased levels of mRNA for the Na(+)-K(+)-ATPase alpha(1)- and beta(1)-subunits and glutathione peroxidase. These findings suggest that maintenance of nutrition during an oxidative lung injury reduces lung edema, perhaps by allowing for continued expression and function of protective proteins such as the Na(+)-K(+)-ATPase.

A novel role for protein phosphatase 2A in the dopaminergic regulation of Na,K-ATPase.

Stimulation of dopaminergic type 1 (D(1)) receptors increases lung edema clearance by regulating Na,K-ATPase function in the alveolar epithelium. We studied the role of serine/threonine protein phosphatases in the Na,K-ATPase regulation by D(1) agonists in A549 cells. We found that low doses of the type 1/2A protein phosphatase inhibitor okadaic acid as well as SV40 small t antigen transiently transfected into A549 cells prevented the D(1) agonist-induced increase in Na,K-ATPase activity and translocation from intracellular pools to the plasma membrane. This was associated with a rapid and transient increase in protein phosphatase 2A activity. We conclude that D(1) stimulation regulates Na,K-ATPase activity by promoting recruitment of Na,K-ATPases from intracellular pools into the basolateral membranes of A549 cells via a type 2A protein phosphatase.

beta-adrenergic stimulation restores rat lung ability to clear edema in ventilator-associated lung injury.

Mechanical ventilation with high tidal volume (HVT) causes lung injury and decreases the lung's ability to clear edema in rats. beta-adrenergic agonists increase active Na(+) transport and lung edema clearance in normal rat lungs by stimulating apical Na(+) channels and basolateral Na,K-ATPase in alveolar epithelial cells. We studied whether beta-adrenergic agonists could restore lung edema clearance in rats ventilated with HVT (40 ml/kg, peak airway pressure of 35 cm H(2)O) for 40 min. The ability of rat lungs to clear edema decreased by approximately 50% after 40 min of HVT ventilation. Terbutaline (TERB) and isoproterenol (ISO) increased lung edema clearance in control nonventilated rats (from 0.50 +/- 0. 02 ml/h to 0.81 +/- 0.04 ml/h and 0.99 +/- 0.05 ml/h, respectively) and restored the lung's ability to clear edema in HVT ventilated rats (from 0.25 +/- 0.03 ml/h to 0.64 +/- 0.02 ml/h and 0.88 +/- 0. 09 ml/h, respectively). Disruption of cell microtubular transport system by colchicine inhibited the stimulatory effects of ISO in HVT ventilated rats, whereas beta-lumicolchicine did not affect beta-adrenergic stimulation. The Na,K-ATPase alpha(1)- and beta(1)-subunit mRNA steady state levels were not affected by incubation with ISO for 60 min in alveolar type II cells isolated from control and HVT ventilated rats. The data suggest that beta-adrenergic agonists increased alveolar fluid reabsorption in rats ventilated with HVT by promoting recruitment of ion-transporting proteins from intracellular pools to the plasma membrane of alveolar epithelial cells.