Tunicamycin reduces Na(+)-K(+)-pump expression in cultured skeletal muscle.

The purpose of this study was to examine effects of tunicamycin (TM), which inhibits core glycosylation of the beta-subunit, on functional expression of the Na(+)-K+ pump in primary cultures of embryonic chick skeletal muscle. Measurements were made of specific-[3H]-ouabain binding, ouabain-sensitive 86Rb uptake, resting membrane potential (Em), and electrogenic pump contribution to Em (Ep) of single myotubes with intracellular microelectrodes. Growth of 4-6-day-old skeletal myotubes in the presence of TM (1 microgram/ml) for 21-24 hr reduced the number of Na(+)-K+ pumps to 60-90% of control. Na(+)-K+ pump activity, the level of resting Em and Ep were also reduced significantly by TM. In addition, TM completely blocked the hyperpolarization of Em induced in single myotubes by cooling to 10 degrees C and then re-warming to 37 degrees C. Effects of tunicamycin were compared with those of tetrodotoxin (TTX; 2 x 10(-7) M for 24 hr), which blocks voltage-dependent Na+ channels. TM produced significantly greater decreases in ouabain-binding and Em than did TTX, findings that indicate that reduced Na(+)-K+ pump expression was not exclusively secondary to decreased intracellular Na+, the primary regulator of pump synthesis in cultured muscle. Similarly, effects of TM were significantly greater than those of cycloheximide, which inhibits protein synthesis by 95%. These findings demonstrate that effects were not due to inhibition of protein synthesis. We conclude that glycosylation of the Na(+)-K+ pump beta-subunit is required for full physiological expression of pump activity in skeletal muscle.     

Protein kinase Cepsilon contributes to regulation of the sarcolemmal Na(+)-K(+) pump

A reduction in angiotensin II (ANG II) in vivo by treatment of rabbits with the angiotensin-converting enzyme inhibitor, captopril, increases Na(+)-K(+) pump current (I(p)) of cardiac myocytes. This increase is abolished by exposure of myocytes to ANG II in vitro. Because ANG II induces translocation of the epsilon-isoform of protein kinase C (PKCepsilon), we examined whether this isozyme regulates the pump. We treated rabbits with captopril, isolated myocytes, and measured I(p) of myocytes voltage clamped with wide-tipped patch pipettes. I(p) of myocytes from captopril-treated rabbits was larger than I(p) of myocytes from controls. ANG II superfusion of myocytes from captopril-treated rabbits decreased I(p) to levels similar to controls. Inclusion of PKCepsilon-specific blocking peptide in pipette solutions used to perfuse the intracellular compartment abolished the effect of ANG II. Inclusion of psiepsilonRACK, a PKCepsilon-specific activating peptide, in pipette solutions had an effect on I(p) that was similar to that of ANG II. There was no additive effect of ANG II and psiepsilonRACK. We conclude that PKCepsilon regulates the sarcolemmal Na(+)-K(+) pump.     

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 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.