Cell Surface Expression Of Na Research Articles

Effects of different magnitudes of cyclic stretch on Na+‐K+‐ATPase in skeletal muscle cells in vitro

The Na(+)-K(+)-ATPase, which plays a major role in modulation of skeletal muscle excitability and contractility, is one of the marker enzymes that senses the mechanical strain and adapts to the stimuli. Although many papers had been published on the effects of mechanical stress on Na(+)-K(+)-ATPase in aortic smooth muscle cells, little was known about the effects of different magnitudes of mechanical stretch on Na(+)-K(+)-ATPase in skeletal muscle cells. In the present study, we determined the effect of different magnitudes(6%, 12%, or 25% elongation) of cyclic stretch on the activity of the Na(+)-K(+)-ATPase and investigated possible mechanisms that might be involved in the action of stretch. The results showed the application of different magnitudes of cyclic stretch induced a magnitude-dependent increase of Na(+)-K(+)-ATPase activity in cultured skeletal muscle cells. Furthermore, inhibition of ionic fluxes through SACs prevented the action of stretch on Na(+)-K(+)-ATPase activity. The stretch-induced increase in Na(+)-K(+)-ATPase activity was not blocked by Actinomycin D. No significant changes in mRNA and total cell protein levels of Na(+)-K(+)-ATPase were detected after stretched continuous for 24 h. However, cyclic stretch increased cell surface expression of Na(+)-K(+)-ATPase alpha(1)- and alpha(2)-subunit proteins by 1.3- and 1.75-fold, respectively, and the increases in Na(+)-K(+)-ATPase activity and cell surface expression were abolished by LY-294002. These data indicated that cyclic stretch induced a "magnitude-dependent" increase of Na(+)-K(+)-ATPase activity in cultured skeletal muscle cells in vitro. The upregulation involved translocation of Na(+)-K(+)-ATPase alpha(1)- and alpha(2)-subunits to plasma membrane, not increased gene transcription. These results suggested a novel nontranscriptional mechanism for regulation of Na(+)-K(+)-ATPase in skeletal muscle cells by cyclic stretch.

Downregulation of voltage-gated sodium channels by dexamethasone in clonal rat pituitary cells

The effect of chronic dexamethasone (DEX) treatment (4–5 days) on Na + channel expression was examined in a clonal strain of rat pituitary cells secreting growth hormone (GH) and prolactin (GH3 cells). Using whole-cell patch clamp recording, we found that DEX (1 μM) induces an 80% decrease in Na + current density. No concomitant changes in current kinetics or voltage dependence of Na + channel function were detected. Instead, the decrease in current density was accompanied by a similar reduction in maximal Na + conductance, suggesting the loss of Na + channels from the plasma membrane. Accordingly, saxitoxin binding assays carried out on intact cells showed that the average number of Na + channels per cell is markedly decreased by DEX. Thus, this glucocorticoid inhibits the cell surface expression of Na + channels when chronically applied to GH3 cells.

Differential effects of bupivacaine enantiomers, ropivacaine and lidocaine on up-regulation of cell surface voltage-dependent sodium channels in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, (±)-bupivacaine inhibited veratridine-induced 22Na + influx (IC 50 6.8 μM). The IC 50 of (+)-bupivacaine (2.8 μM) was 6.2-, 7.4-, and 17.1-fold lower than those of (−)-bupivacaine (17.3 μM), (−)-ropivacaine (20.6 μM), and lidocaine (47.8 μM). Chronic (i.e. 3-h) treatment of cells with (±)-bupivacaine increased cell surface [ 3H]saxitoxin ([ 3H]STX) binding capacity by 48% (EC 50 of 233 μM; t 1/2=7.4 h), without changing the K d value. Treatment for 24 h with either (+)- or (−)-bupivacaine, or (−)-ropivacaine elevated [ 3H]STX binding, whereas 24-h treatment with lidocaine had no effect. The rise of [ 3H]STX binding by (±)-bupivacaine was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of cell surface vesicular exit from the trans-Golgi network; however, (±)-bupivacaine did not increase Na + channel α- and β 1-subunit mRNA levels. In cells subjected to (±)-bupivacaine treatment (1 mM for 24 h) followed by 3-h washout, veratridine-induced 22Na + influx was enhanced, even when measured in the presence of ouabain, an inhibitor of Na +,K +-ATPase. Ptychodiscus brevis toxin-3 potentiated veratridine-induced 22Na + influx by 2.3-fold in the (±)-bupivacaine-treated cells, as in non-treated cells. These results suggest that lipophilic bupivacaine enantiomers or (−)-ropivacaine acutely inhibit Na + channel gating, whereas its chronic treatment up-regulates cell surface expression of Na + channels via translational and externalization events.

C-peptide stimulates Na+,K+-ATPase activity via PKC alpha in rat medullary thick ascending limb.

C-peptide, the cleavage product of proinsulin processing exerts physiological effects including stimulation of Na(+),K(+)-ATPase in erythrocytes and renal proximal tubules. This study was undertaken to assess the physiological effects of connecting peptide on Na(+),K(+)-ATPase activity in the medullary thick ascending limb of Henle's loop. Na(+),K(+)-ATPase activity was measured as the ouabain-sensitive generation of (32)Pi from gamma[(32)P]-ATP and (86)Rb uptake on isolated rat medullary thick ascending limbs. The cell-surface expression of Na(+),K(+)-ATPase was evaluated by Western blotting of biotinylated proteins, and its phosphorylation amount was measured by autoradiography. The membrane-associated fraction of protein kinase C isoforms was evaluated by Western blotting. Rat connecting peptide concentration-dependently stimulated Na(+),K(+)-ATPase activity with a threshold at 10(-9) mol/l and a maximal effect at 10(-7) mol/l. C-peptide (10(-7) mol/l) already stimulates Na(+),K(+)-ATPase activity after 5 min with a plateau from 15 to 60 min. C-peptide (10(-7) mol/l) stimulated Na(+),K(+)-ATPase activity and (86)Rb uptake to the same extent, but did not alter Na(+),K(+)-ATPase cell surface expression. The stimulation of Na(+),K(+)-ATPase activity was associated with an increase in Na(+),K(+)-ATPase alpha-subunit phosphorylation and both effects were abolished by a specific protein kinase C inhibitor. Furthermore, connecting peptide induced selective membrane translocation of PKC-alpha. This study provides evidence that in rat medullary thick ascending limb, C-peptide stimulates Na(+),K(+)-ATPase activity within a physiological concentration range. This effect is due to an increase in Na(+),K(+)-ATPase turnover rate that is most likely mediated by protein kinase C-alpha phosphorylation of the Na(+),K(+)-ATPase alpha-subunit, suggesting that C-peptide could control Na(+) reabsorption during non-fasting periods.

L‐type calcium channel activity regulates sodium channel levels in rat pituitary GH3 cells

1. The effects of chronic pharmacological modulation of L-type Ca2+ channel activity on the cell surface expression of Na+ channels were examined in GH3 cells. 2. Prolonged inhibition (4-5 days) of L-channels with nimodipine caused a 50-60 % decrease in the peak amplitude of whole-cell Na+ currents recorded with the patch-clamp technique. On the contrary, prolonged exposure to the L-channel agonist Bay K 8644 induced an approximately 2.5-fold increase in peak Na+ current. In both cases, there were only minor changes in cell capacitance and no significant changes in Na+ channel gating properties. 3. Measurements of the specific binding of radiolabelled saxitoxin to intact cells showed that nimodipine treatment reduced the number of cell surface Na+ channels, whereas treatment with Bay K 8664 produced the opposite effect. The dual regulation of Na+ channel abundance explained the mentioned changes in Na+ current amplitude. 4. Plasma membrane Na+ channels had a half-life of approximately 17 h both in control cells and in cells treated with Bay K 8644, as estimated from the rate of decay of peak Na+ current after inhibition of protein synthesis with cycloheximide. Actinomycin D, an inhibitor of gene transcription, and also cycloheximide, occluded the stimulatory effect of Bay K 8644 on Na+ current density when measured over a 24 h period. 5. These findings indicate that the entry of Ca2+ through L-type channels influences in a positive way the number of functional Na+ channels in GH3 cells, and suggest that Ca2+ influx stimulates either Na+ channel gene expression or the expression of a regulatory protein that promotes translocation of pre-assembled Na+ channels into the plasma membrane.

Up‐Regulation of Sodium Channel Subunit mRNAs and Their Cell Surface Expression by Antiepileptic Valproic Acid: Activation of Calcium Channel and Catecholamine Secretion in Adrenal Chromaffin Cells

Treatment of cultured bovine adrenal chromaffin cells with a therapeutic concentration (0.6 mM) of valproic acid (VPA) for > 24 h caused a time-dependent (t1/2 = 74 h) increase in [3H]saxitoxin binding up to 1.4-fold without altering the KD value; it was prevented by the simultaneous treatment with cycloheximide (an inhibitor of protein synthesis). VPA also raised Na+ channel alpha- and beta 1-subunit mRNA levels 1.4- and 1.7-fold at 24 h, and 1.6- and 1.8-fold at 72 h, respectively. Chronic (but not acute) exposure to VPA enhanced 22Na+ influx caused by various concentrations of veratridine 1.4-2.1-fold, even when assayed in the presence of Na+,K(+)-ATPase inhibitor, but did not change the EC50 value of veratridine. Ptychodiscus brevis toxin-3 allosterically potentiated veratridine-induced 22Na+ influx by approximately 2-fold in VPA-treated cells as in nontreated cells. Long-term treatment with VPA augmented veratridine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels and catecholamine secretion, but had no effect on 45Ca2+ influx and catecholamine secretion caused by high K+ (a direct activation of voltage-dependent Ca2+ channels). Chronic treatment with VPA also enhanced nicotine-induced 22Na+ influx via the nicotinic receptor-ion channel complex 1.2-1.4-fold with little change in the EC50 value of nicotine, thereby increasing the nicotine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels and catecholamine secretion. These results suggest that chronic treatment with VPA up-regulates cell surface expression of Na+ channels via the transcription/translation-dependent mechanisms, and probably of nicotinic receptors, thereby resulting in the enhancement of Ca2+ channel gating and catecholamine secretion.

Arginine vasopressin and forskolin regulate apical cell surface expression of epithelial Na+ channels in A6 cells

Both arginine vasopressin (AVP) and forskolin regulate vectorial Na+ transport across high-resistance epithelia by increasing the Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. Pretreatment of A6 cells with brefeldin A partially inhibited the increase in Na+ transport in response to forskolin, suggesting recruitment of Na+ channels from an intracellular pool. The activation of Cl- secretion was not affected. Apical cell surface expression of Na+ channels was examined following activation of transepithelial Na+ transport across the epithelial cell line A6 by AVP or forskolin. Apical cell surface radioiodinated Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical plasma membrane and to determine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a potential mechanism by which AVP and forskolin increase apical membrane Na+ conductance. The activation of Na+ transport across A6 cells by AVP was accompanied by a significant increase in the biochemical pool of Na+ channels at the apical plasma membrane within 5 min after addition of hormone, which was sustained for at least 30 min. The increase in apical cell surface expression of Na+ channels was also observed 30 min after application of forskolin. No changes in the oligomeric subunit composition of the channel were noted. Brefeldin A inhibited the forskolin-stimulated increase in apical cell surface expression of Na+ channels. These results suggest that AVP and forskolin regulate Na+ transport, in part, via rapid recruitment of Na+ channels to the cell surface, perhaps from a pool of channels in the subapical cytoplasm.