The Na(+)-K(+)-ATPase alpha2-subunit isoform modulates contractility in the perinatal mouse diaphragm

This study uses genetically altered mice to examine the contribution of the Na⁺-K⁺-ATPase ₂ catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The ₂ protein is reduced by 38% in ₂-heterozygous and absent in ₂-knockout mice, and ₁-isoform is upregulated 1.9-fold in ₂-knockout. Resting potentials are depolarized by 0.8–4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced ₂ content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the ₂-isoform at a step after membrane excitation, which cannot be restored simply by increasing ₁ content. Na⁺/Ca²⁺ exchanger expression decreases in parallel with ₂-isoform, suggesting that Ca²⁺ extrusion is affected by the altered ₂ genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca²⁺-ATPase, phospholamban, or plasma membrane Ca²⁺-ATPase. These results demonstrate that the Na⁺-K⁺-ATPase ₁-isoform alone is able to maintain equilibrium K⁺ and Na⁺ gradients and to substitute for ₂-isoform in most cellular functions related to excitability and force. They further indicate that the ₂-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction. Na⁺-K⁺-ATPase ₂ catalytic subunit; heterozygous mice; knockout mice; resting potential     

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.     

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.     

Fatigue depresses maximal in vitro skeletal muscle Na(+)-K(+)-ATPase activity in untrained and trained individuals.

This study investigated whether fatiguing dynamic exercise depresses maximal in vitro Na(+)-K(+)-ATPase activity and whether any depression is attenuated with chronic training. Eight untrained (UT), eight resistance-trained (RT), and eight endurance-trained (ET) subjects performed a quadriceps fatigue test, comprising 50 maximal isokinetic contractions (180 degrees /s, 0.5 Hz). Muscle biopsies (vastus lateralis) were taken before and immediately after exercise and were analyzed for maximal in vitro Na(+)-K(+)-ATPase (K(+)-stimulated 3-O-methylfluoroscein phosphatase) activity. Resting samples were analyzed for [(3)H]ouabain binding site content, which was 16.6 and 18.3% higher (P < 0.05) in ET than RT and UT, respectively (UT 311 +/- 41, RT 302 +/- 52, ET 357 +/- 29 pmol/g wet wt). 3-O-methylfluoroscein phosphatase activity was depressed at fatigue by -13.8 +/- 4.1% (P < 0.05), with no differences between groups (UT -13 +/- 4, RT -9 +/- 6, ET -22 +/- 6%). During incremental exercise, ET had a lower ratio of rise in plasma K(+) concentration to work than UT (P < 0.05) and tended (P = 0.09) to be lower than RT (UT 18.5 +/- 2.3, RT 16.2 +/- 2.2, ET 11.8 +/- 0.4 nmol. l(-1). J(-1)). In conclusion, maximal in vitro Na(+)-K(+)-ATPase activity was depressed with fatigue, regardless of training state, suggesting that this may be an important determinant of fatigue.     

Mechanical stretching of alveolar epithelial cells increases Na(+)-K(+)-ATPase activity.

Alveolar epithelial cells effect edema clearance by transporting Na(+) and liquid out of the air spaces. Active Na(+) transport by the basolaterally located Na(+)-K(+)-ATPase is an important contributor to lung edema clearance. Because alveoli undergo cyclic stretch in vivo, we investigated the role of cyclic stretch in the regulation of Na(+)-K(+)-ATPase activity in alveolar epithelial cells. Using the Flexercell Strain Unit, we exposed a cell line of murine lung epithelial cells (MLE-12) to cyclic stretch (30 cycles/min). After 15 min of stretch (10% mean strain), there was no change in Na(+)-K(+)-ATPase activity, as assessed by (86)Rb(+) uptake. By 30 min and after 60 min, Na(+)-K(+)-ATPase activity was significantly increased. When cells were treated with amiloride to block amiloride-sensitive Na(+) entry into cells or when cells were treated with gadolinium to block stretch-activated, nonselective cation channels, there was no stimulation of Na(+)-K(+)-ATPase activity by cyclic stretch. Conversely, cells exposed to Nystatin, which increases Na(+) entry into cells, demonstrated increased Na(+)-K(+)-ATPase activity. The changes in Na(+)-K(+)-ATPase activity were paralleled by increased Na(+)-K(+)-ATPase protein in the basolateral membrane of MLE-12 cells. Thus, in MLE-12 cells, short-term cyclic stretch stimulates Na(+)-K(+)-ATPase activity, most likely by increasing intracellular Na(+) and by recruitment of Na(+)-K(+)-ATPase subunits from intracellular pools to the basolateral membrane.     

Na+,K+-ATPase trafficking in skeletal muscle: insulin stimulates translocation of both alpha 1- and alpha 2-subunit isoforms

We determined insulin-stimulated Na(+),K(+)-ATPase isoform-specific translocation to the skeletal muscle plasma membrane. When rat muscle plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha(2)- but not alpha(1)-subunits was detected. However, using cell surface biotinylation techniques, an insulin-induced membrane translocation of both alpha(1) and alpha(2)-subunits in rat epitrochlearis muscle and cultured human skeletal muscle cells was noted. Na(+),K(+)-ATPase alpha-subunit translocation was abolished by the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, as well as by the protein kinase C inhibitor GF109203X. Thus, insulin mediates Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunit translocation to the skeletal muscle plasma membrane via a PI 3-kinase-dependent mechanism.