Na(+)-K(+)-ATPase activity in alveolar epithelial cells increases with cyclic stretch

Na(+)-K(+)-ATPase pumps (Na(+) pumps) in the alveolar epithelium create a transepithelial Na(+) gradient crucial to keeping fluid from the pulmonary air space. We hypothesized that alveolar epithelial stretch stimulates Na(+) pump trafficking to the basolateral membrane (BLM) and, thereby, increases overall Na(+) pump activity. Alveolar type II cells were isolated from Sprague-Dawley rats and seeded onto elastic membranes coated with fibronectin or 5-day-conditioned extracellular matrix. After 2 days in culture, cells were uniformly stretched for 1 h in a custom-made device. Na(+) pump activity was subsequently assessed by ouabain-inhibitable uptake of (86)Rb(+), a K(+) tracer, and BLM Na(+) pump abundance was measured. In support of our hypothesis, cells increased Na(+) pump activity in a "dose-dependent" manner when stretched to 12, 25, or 37% change in surface area (DeltaSA), and cells stretched to 25% DeltaSA more than doubled Na(+) pump abundance in the BLM. Cells on 5-day matrix tolerated higher strain than cells on fibronectin before the onset of Na(+) pump upregulation. Treatment with Gd(3+), a stretch-activated channel blocker, amiloride, a Na(+) channel blocker, or both reduced but did not abolish stretch-induced effects. Sustained tonic stretch, unlike cyclic stretch, elicited no significant Na(+) pump response.     

Reduced activity of muscle Na(+)-K(+)-ATPase after prolonged running in rats.

The purpose of this study was to investigate the hypothesis that Na(+)-K(+)-ATPase activity is reduced in muscle of different fiber composition after a single session of aerobic exercise in rats. In one experiment, untrained female Sprague-Dawley rats (weight 275 +/- 21 g; means +/- SE; n = 30) were run (Run) on a treadmill at 21 m/min and 8% grade until fatigue, or to a maximum of 2 h, which served as control (Con), or performed an additional 45 min of low-intensity exercise at 10 m/min (Run+). In a second experiment, utilizing rats of similar characteristics (weight 258 +/- 18 g; n = 32), Run was followed by passive recovery (Rec). Directly after exercise, rats were anesthetized, and tissue was extracted from Soleus (Sol), red vastus lateralis (RV), white vastus lateralis (WV), and extensor digitorum longus (EDL) and frozen for later analysis. 3-O-methylfluorescein phosphatase activity (3-O-MFPase) was determined as an indicator of Na(+)-K(+)-ATPase activity, and glycogen depletion identified recruitment of each muscle during exercise. 3-O-MFPase was decreased (P < 0.05) at Run+ by an average of 12% from Con in all muscles (P < 0.05). No difference was found between Con and Run. Glycogen was lower (P < 0.05) by 65, 57, 44, and 33% (Sol, EDL, RV, and WV, respectively) at Run, and there was no further depletion during the continued low-intensity exercise period. No differences in Na(+)-K(+)-ATPase activity was observed between Con and Rec. The results of this study indicate that inactivation of Na(+)-K(+)-ATPase can be induced by aerobic exercise in a volume-dependent manner and that the inactivation that occurs is not specific to muscles of different fiber-type composition. Inactivation of Na(+)-K(+)-ATPase suggests intrinsic structural modifications by mechanisms that are unclear.     

Modeling the effect of stretch and plasma membrane tension on Na+-K+-ATPase activity in alveolar epithelial cells

While a number of whole cell mechanical models have been proposed, few, if any, have focused on the relationship among plasma membrane tension, plasma membrane unfolding, and plasma membrane expansion and relaxation via lipid insertion. The goal of this communication is to develop such a model to better understand how plasma membrane tension, which we propose stimulates Na(+)-K(+)-ATPase activity but possibly also causes cell injury, may be generated in alveolar epithelial cells during mechanical ventilation. Assuming basic relationships between plasma membrane unfolding and tension and lipid insertion as the result of tension, we have captured plasma membrane mechanical responses observed in alveolar epithelial cells: fast deformation during fast cyclic stretch, slower, time-dependent deformation via lipid insertion during tonic stretch, and cell recovery after release from stretch. The model estimates plasma membrane tension and predicts Na(+)-K(+)-ATPase activation for a specified cell deformation time course. Model parameters were fit to plasma membrane tension, whole cell capacitance, and plasma membrane area data collected from the literature for osmotically swollen and shrunken cells. Predictions of membrane tension and stretch-stimulated Na(+)-K(+)-ATPase activity were validated with measurements from previous studies. As a proof of concept, we demonstrate experimentally that tonic stretch and consequent plasma membrane recruitment can be exploited to condition cells against subsequent cyclic stretch and hence mitigate stretch-induced responses, including stretch-induced cell death and stretch-induced modulation of Na(+)-K(+)-ATPase activity. Finally, the model was exercised to evaluate plasma membrane tension and potential Na(+)-K(+)-ATPase stimulation for an assortment of traditional and novel ventilation techniques.     

Effect of hydroxyl radical on Na(+)-K(+)-ATPase activity of the brain microsomal membranes.

Sphingomyelin liposomes and brain microsomes were oxidized by exposure to hydrogen peroxide and ferrous ion. Lipid peroxidation were measured by the formation of thiobarbituric acid- reactive substances (TBAR). Hydroxyl radical was detected using the spin- trapping technique. Incubation of sphingomyelin liposomes with H2O2-Fe2+ resulted in an increase in the formation of TBAR. Na(+)-K(+)-ATPase activity was markedly inhibited and the SH group content decreased during incubation of microsomes in the presence of H2O2-Fe2+. Sodium ferulate effectively inhibited TBAR formation, protected Na(+)-K(+)-ATPase activity and prevented the oxidative modification of SH groups. Spin-trapping experiments showed that sodium ferulate effectively scavenged the hydroxyl radicals.     

Regulation of Na(+)-K(+)-2Cl- cotransporter activity in rat skeletal muscle and intestinal epithelial cells

In mammalian cells, Na(+)-K(+)-2Cl- cotransporter activity participates in regulation of ion and volume homeostasis. This makes NKCC indispensable for normal cell growth and proliferation. We recently reported the existence of two mechanisms that can regulate NKCC activity in mature skeletal muscle. In isosmotic conditions, signaling through ERK MAPK pathway is necessary, while inhibition of the cAMP-dependent protein kinase A (PKA) pathway stimulates NKCC activity during hyperosmotic challenge. Both pathways are involved in regulating cell proliferation in wide variety of cells of epithelial and non-epithelial origin, so we tested which pathway regulated NKCC activity in cultured cells. In cultured rat skeletal muscle (L6) and intestinal epithelial (IEC-6) cells, NKCC activity in the basal state comprised 30 to 50% of total potassium influx, assessed as bumetanide-sensitive 38Rb-uptake. This NKCC activity could not be abolished by inhibitors of ERK MAPK (PD98059 and U0126), PKC (GF109203X), or PI 3-K (wortmannin, LY294002). In L6 myoblasts and in IEC-6 cells, elevation of cAMP levels with isoproterenol or forskolin led to a 60% inhibition on NKCC activity. In contrast, incubation of IEC-6 cells with the PKA-inhibitor H-89 resulted in 50% increase of NKCC activity compared with the basal level. In conclusion, it appears that in cultured cells the cAMP--PKA pathway regulates NKCC activity. This resembles hyperosmotic regulation of NKCC activity.     

Dexamethasone prevents transport inhibition by hypoxia in rat lung and alveolar epithelial cells by stimulating activity and expression of Na+-K+-ATPase and epithelial Na+ channels

Hypoxia inhibits Na and lung fluid reabsorption, which contributes to the formation of pulmonary edema. We tested whether dexamethasone prevents hypoxia-induced inhibition of reabsorption by stimulation of alveolar Na transport. Fluid reabsorption, transport activity, and expression of Na transporters were measured in hypoxia-exposed rats and in primary alveolar type II (ATII) cells. Rats were treated with dexamethasone (DEX; 2 mg/kg) on 3 consecutive days and exposed to 10% O(2) on the 2nd and 3rd day of treatment to measure hypoxia effects on reabsorption of fluid instilled into lungs. ATII cells were treated with DEX (1 muM) for 3 days before exposure to hypoxia (1.5% O(2)). In normoxic rats, DEX induced a twofold increase in alveolar fluid clearance. Hypoxia decreased reabsorption (-30%) by decreasing its amiloride-sensitive component; pretreatment with DEX prevented the hypoxia-induced inhibition. DEX increased short-circuit currents (ISC) of ATII monolayers in normoxia and blunted hypoxic transport inhibition by increasing the capacity of Na(+)-K(+)-ATPase and epithelial Na(+) channels (ENaC) and amiloride-sensitive ISC. DEX slightly increased the mRNA of alpha- and gamma-ENaC in whole rat lung. In ATII cells from DEX-treated rats, mRNA of alpha(1)-Na(+)-K(+)-ATPase and alpha-ENaC increased in normoxia and hypoxia, and gamma-ENaC was increased in normoxia only. DEX stimulated the mRNA expression of alpha(1)-Na(+)-K(+)-ATPase and alpha-, beta-, and gamma-ENaC of A549 cells in normoxia and hypoxia (1.5% O(2)) when DEX treatment was begun before or during hypoxic exposure. These results indicate that DEX prevents inhibition of alveolar reabsorption by hypoxia and stimulates the expression of Na transporters even when it is applied in hypoxia.     

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.