Ouabain-sensitive 86Rb Uptake Research Articles

Nitric Oxide Regulation of Na, K‐ATPase Activity in Ocular Ciliary Epithelium Involves Src Family Kinase

The nitric oxide (NO) donor sodium nitroprusside (SNP) is known to reduce aqueous humor (AH) secretion in the isolated porcine eye. Previously, SNP was found to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE), AH-secreting cells, through a cGMP/protein kinase G (PKG)-mediated pathway. Here we show Src family kinase (SFK) activation in the Na,K-ATPase activity response to SNP. Ouabain-sensitive (86) Rb uptake was reduced by >35% in cultured NPE cells exposed to SNP (100 µM) or exogenously added cGMP (8-Br-cGMP) (100 µM) and the SFK inhibitor PP2 (10 µM) prevented the response. Ouabain-sensitive ATP hydrolysis was reduced by ~40% in samples detected in material obtained from SNP- and 8-Br-cGMP-treated cells following homogenization, pointing to an intrinsic change of Na,K-ATPase activity. Tyrosine-10 phosphorylation of Na,K-ATPase α1 subunit was detected in SNP and L-arginine-treated cells and the response prevented by PP2. SNP elicited an increase in cell cGMP. Cells exposed to 8-Br-cGMP displayed SFK activation (phosphorylation) and inhibition of both ouabain-sensitive (86) Rb uptake and Na,K-ATPase activity that was prevented by PP2. SFK activation, which also occurred in SNP-treated cells, was suppressed by inhibitors of soluble guanylate cyclase (ODQ; 10 µM) and PKG (KT5823; 1 µM). SNP and 8-Br-cGMP also increased phosphorylation of ERK1/2 and p38 MAPK and the response prevented by PP2. However, U0126 did not prevent SNP or 8-Br-cGMP-induced inhibition of Na,K-ATPase activity. Taken together, the results suggest that NO activates guanylate cyclase to cause a rise in cGMP and subsequent PKG-dependent SFK activation. Inhibition of Na,K-ATPase activity depends on SFK activation.

Activation of AMP-activated Protein Kinase Stimulates Na+,K+-ATPase Activity in Skeletal Muscle Cells

Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.

Dopamine regulation of Na+-K+-ATPase requires the PDZ-2 domain of sodium hydrogen regulatory factor-1 (NHERF-1) in opossum kidney cells

Na(+)-K(+)-ATPase activity in renal proximal tubule is regulated by several hormones including parathyroid hormone (PTH) and dopamine. The current experiments explore the role of Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) in dopamine-mediated regulation of Na(+)-K(+)-ATPase. We measured dopamine regulation of ouabain-sensitive (86)Rb uptake and Na(+)-K(+)-ATPase α1 subunit phosphorylation in wild-type opossum kidney (OK) (OK-WT) cells, OKH cells (NHERF-1-deficient), and OKH cells stably transfected with full-length human NHERF-1 (NF) or NHERF-1 constructs with mutated PDZ-1 (Z1) or PDZ-2 (Z2) domains. Treatment with 1 μM dopamine decreased ouabain-sensitive (86)Rb uptake, increased phosphorylation of Na(+)-K(+)-ATPase α1-subunit, and enhanced association of NHERF-1 with D1 receptor in OK-WT cells but not in OKH cells. Transfection with wild-type, full-length, or PDZ-1 domain-mutated NHERF-1 into OKH cells restored dopamine-mediated regulation of Na(+)-K(+)-ATPase and D1-like receptor association with NHERF-1. Dopamine did not regulate Na(+)-K(+)-ATPase or increase D1-like receptor association with NHERF-1 in OKH cells transfected with mutated PDZ-2 domain. Dopamine stimulated association of PKC-ζ with NHERF-1 in OK-WT and OKH cells transfected with full-length or PDZ-1 domain-mutated NHERF-1 but not in PDZ-2 domain-mutated NHERF-1-transfected OKH cells. These results suggest that NHERF-1 mediates Na(+)-K(+)-ATPase regulation by dopamine through its PDZ-2 domain.

Analysis of exercise‐induced Na+–K+ exchange in rat skeletal muscle in vivo

We aimed to quantify the Na(+)-K(+) exchange occurring during exercise in rat skeletal muscle in vivo. Intracellular Na(+) and K(+) content, Na(+) permeability ((22)Na(+) influx), Na(+)-K(+) pump activity (ouabain-sensitive (86)Rb(+) uptake) and Na(+)-K(+) pump alpha(2) subunit content ([(3)H]ouabain binding) were measured. Six-week-old rats rested (control animals) or performed intermittent running for 10-60 min and were then killed or were killed at 15 or 90 min following 60 min exercise. In the soleus muscle, intracellular Na(+) was 80% higher than in control rats after 60 min exercise, was still elevated (38%) after 15 min rest and returned to control levels after 90 min rest. Intracellular K(+) showed corresponding decreases after 15-60 min exercise, returning to control levels 90 min postexercise. Exercise induced little change in Na(+) and K(+) in the extensor digitorum longus muscle (EDL). In soleus, the exercise-induced rise in Na(+) and reduction in K(+) were augmented by pretreatment with ouabain or by reducing the content of muscular Na(+)-K(+) pumps by prior K(+) depletion of the animals. Fifteen minutes after 60 min exercise, ouabain-sensitive (86)Rb(+) uptake in the soleus was increased by 30% but was unchanged in EDL, and there was no effect of exercise on [(3)H]ouabain binding measured in vitro or in vivo in either muscle. In conclusion, in the soleus, in vivo exercise induces a rise in intracellular Na(+), which reflects the excitation-induced increase in Na(+) influx and leads to augmented Na(+)-K(+) pump activity without apparent change in Na(+)-K(+) pump capacity.

Nanomolar concentrations of ouabain stimulate Na‐K ATPase through sodium hydrogen exchanger‐1 (NHE1) dependent mechanism.

Recent investigations have shown increased Na‐H exchanger activity and plasma levels of endogenous cardiotonic glycosides, such as ouabain in spontaneously hypertensive rats. At high concentrations, ouabain inhibits pump activity, and at lower concentrations, it increases pump activity and induces cell proliferation. Stimulation of NHE1 and Na‐K ATPase activity involve protein kinase B (AKT), suggesting that both NHE1 and Na‐K ATPase are mediated by the same proteins. We hypothesize that the activity of NHE1 and Na‐K ATPase are linked. In the present study we aim to 1) determine the role of activation of NHE1 in ouabain‐sensitive stimulation of Na‐K ATPase, 2) determine if NHE1 and Na‐K ATPase association increases when cells were treated with NH4Cl or ouabain in various kidney proximal tubule cell lines (OK, HK2, HK5, and HK11). Our data suggests that NH4Cl stimulation of NHE1 increases ouabain sensitive 86Rb uptake in all cell lines examined.Treatment with ethylisopropylamiloride (EIPA) a NHE1 specific inhibitor decreased Na‐K ATPase activity in HK5 and HK11 cells and prevented nanomolar ouabain‐stimulated Na‐K ATPase activity in OK cells. Immunoblot analysis indicated that NH4Cl increased membrane expression of NHE1 and Na‐K ATPase in OK and HK11 cells. Surprisingly, treatment with EIPA alone increased membrane expression of NHE1 and Na‐K ATPase. The reason for increased presence of these proteins in the membrane is unknown, however, tyrosine phosphorylation of the catalytic subunit of Na‐K ATPase, believed to be an indication of Na‐K ATPase activation, is decreased under these same conditions. These results suggest that activation of NHE1 is critical for ouabain stimulated increase in Na‐K ATPase activity, and that upon activation, NHE1 and Na‐K ATPase associate, perhaps to form a signalosome.

ANP Differentially Modulates Marinobufagenin-Induced Sodium Pump Inhibition in Kidney and Aorta

NaCl loading and plasma volume expansion stimulate 2 natriuretic systems, vasoconstrictor, digitalis-like Na/K-ATPase inhibitors and vasorelaxant ANP peptides. Several hormones, including ANP, regulate activity of the Na/K-ATPase by modulation of its phosphorylation state. We studied effects of ANP on Na/K-ATPase phosphorylation and inhibition by an endogenous sodium pump ligand, marinobufagenin, in the aorta and renal medulla from male Sprague-Dawley rats. Marinobufagenin dose-dependently inhibited the Na/K-ATPase in renal and vascular membranes at the level of higher (nanomolar) and lower affinity (micromolar) binding sites. Marinobufagenin (1 nmol/L) inhibited Na/K-ATPase in aortic sarcolemma (18%) and in renal medulla (19%). prepro-ANP 104 to 123 (ppANP) and alpha-human ANP ([alpha-hANP] both 1 nmol/L) potentiated marinobufagenin-induced Na/K-ATPase inhibition in the kidney, but reversed the effect of marinobufagenin in the aorta. Similarly, ppANP and alpha-hANP modulated the sodium pump (ouabain-sensitive (86)Rb uptake) inhibitory effects of marinobufagenin in the aorta and renal medulla. In renal medulla, ppANP and alpha-hANP induced alpha-1 Na/K-ATPase phosphorylation, whereas in aorta, both peptides dephosphorylated Na/K-ATPase. The effect of ppANP on Na/K-ATPase phosphorylation and inhibition was mimicked by a protein kinase G activator, 8-Br-PET-cGMP (10 micromol/L), and prevented by a protein kinase G inhibitor, KT5823 (60 nmol/L). Our results suggest that alpha-1 Na/K-ATPase inhibition by marinobufagenin in the kidney is enhanced via Na/K-ATPase phosphorylation by ANP, whereas in the aorta, ANP exerts an opposite effect. The concurrent production of a vasorelaxant, ANP, and a vasoconstrictor, marinobufagenin, potentiate each other's natriuretic effects, but ANP peptides may offset the deleterious vasoconstrictor effect of marinobufagenin.

13 Effect of Erythropoietin on Fluid Movement Across the Respiratory Epithelium.

Erythropoietin has a wide range of nonhematopoietic effects in the body, including the regulation of certain enzymes in the lung. Clinically, its administration has been associated with a decrease in the number of days in oxygen for sick premature infants. If erythropoietin has a favorable effect on respiratory illness, one mechanism could be a reduction in pulmonary edema. In previous studies, we demonstrated that erythropoietin increases cellular uptake of sodium, a necessary first step in ion transport-linked fluid clearance (Pediatr Res 58:817A, 2005). To further characterize the effect on ion transport, we studied the effect of erythropoietin on the Na-K-ATPase, an enzyme important in sodium translocation. To accomplish this goal, we treated A549 cells, an immortal adenocarcinoma cell line with features of Type II cells, with erythropoietin and measured the uptake of 86Rb+ (a mimic of K+) in the presence and absence of ouabain, an inhibitor of Na-K-ATPase. We found that after 24h, erythropoietin (35 U/ml) increased ouabain-sensitive Rb uptake on average by 25% (n=8, control: 1.6 ± 0.6 vs erythropoietin: 2.0 ± 0.8 nmol Rb/million cells/min, p<0.05), indicating an increase in Na-K-ATPase activity. To confirm this finding, we disrupted the cell membrane and assessed Na-K-ATPase activity by an alternative method in which we measured the release of inorganic phosphate in the presence and absence of ouabain. Similar to our results in intact cells, erythropoietin (24h, 35 U/ml) increased the activity of the Na-K-ATPase (n=7, control: 0.9 ± 0.4 vs erythropoietin: 2.1 ± 1.3 mg phosphate/mg protein, p<0.05). In additional studies, we grew A549 cells on a semipermeable membrane and measured fluid movement across the monolayer by measuring the change in protein concentration in the supernatant as a reflection of fluid loss. After 24 h, erythropoietin increased by nearly 25% the volume of fluid loss from the supernatant (n=6, control: 132 ± 26 vs erythropoietin: 164 ± 34 uL/24 h, p<0.05). We then added amiloride (1mM), an inhibitor of Na channels important in the transepithelial movement of liquid, to monolayers treated with erythropoietin and found that fluid loss from the supernatant decreased to that of control cells (n=6, control: 126 ± 26 uL/24 h). Our findings show that erythropoietin increases Na-K-ATPase activity and transepithelial liquid movement in vitro. We speculate that if erythropoietin were to increase sodium transport and Na-K-ATPase activity in vivo, then lung water balance and respiratory difficulty might improve as well.

Segmental Heterogeneity in the Mechanism of Sodium Nitroprusside-Induced Relaxation in Ovine Pulmonary Artery

Segmental heterogeneity in relaxation response to nitric oxide (NO) was examined using NO donor sodium nitroprusside (SNP) in second- (medium) and fourth-generation (small) ovine isolated intralobar pulmonary arteries. In vessels precontracted with serotonin, NO donors SNP and S-nitroso-N-acetylpenicillamine (SNAP) were more potent in relaxing medium, in comparison to the small, arteries. Soluble guanylyl cyclase (sGC) inhibitor [1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one (ODQ 3 microM) caused a profound inhibition of SNP relaxation in small as compared with medium-sized arteries. However, both basal and SNP (10 microM)-stimulated intracellular cyclic guanosine monophosphate (cGMP) content was identical in these 2 arterial segments. The Na,K-ATPase inhibitor ouabain (1 microM) had a marked inhibitory effect on SNP-mediated relaxation in both segments. There was no segmental difference in SNP (10 microM)-stimulated plasma membrane Na,K-ATPase activity and ouabain-sensitive Rb-uptake. 4-AP (1 mM), a relatively selective inhibitor of Kv channels, decreased the potency of SNP relaxation by about 10-fold in the medium-sized vessels. On the other hand, 4-AP was without effect on the vasodilator potency of SNP in small vessels. Interestingly, in the presence of 4-AP, SNP was equipotent in dilating both medium (pD2 = 5.80 +/- 0.07; Emax = 84 +/- 1.6%, n = 7) and small (pD2 = 5.74 +/- 0.15; Emax = 83 +/- 2.5%, n = 7) pulmonary arteries. In conclusion, the results of the present study suggest that Kv channels determine the segmental heterogeneity of NO-mediated relaxation in ovine pulmonary artery.

C-peptide stimulates Na+, K+-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells

Proinsulin-connecting peptide (C-peptide) exerts physiological effects partially via stimulation of Na(+), K(+)-ATPase. We determined the molecular mechanism by which C-peptide stimulates Na(+), K(+)-ATPase in primary human renal tubular cells (HRTCs). Incubation of the cells with 5 nM human C-peptide at 37 degrees C for 10 min stimulated (86)Rb(+) uptake by 40% (p<0.01). The carboxy-terminal pentapeptide was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive (86)Rb(+) uptake, C-peptide increased alpha subunit phosphorylation and basolateral membrane (BLM) abundance of the Na(+), K(+)-ATPase alpha(1) and beta(1) subunits. The increase in BLM abundance of the Na(+), K(+)-ATPase alpha(1) and beta(1) subunits was accompanied by depletion of alpha(1) and beta(1) subunits from the endosomal compartments. C-peptide action on Na(+), K(+)-ATPase was ERK1/2-dependent in HRTCs. C-peptide-stimulated Na(+), K(+)-ATPase activation, phosphorylation of alpha(1)-subunit and translocation of alpha(1) and beta(1) subunits to the BLM were abolished by a MEK1/2 inhibitor (20 muM PD98059). C-peptide stimulation of (86)Rb(+) uptake was also abolished by preincubation of HRTCs with an inhibitor of PKC (1 muM GF109203X). C-peptide stimulated phosphorylation of human Na(+), K(+)-ATPase alpha subunit on Thr-Pro amino acid motifs, which form specific ERK substrates. In conclusion, C-peptide stimulates sodium pump activity via ERK1/2-induced phosphorylation of Thr residues on the alpha subunit of Na(+), K(+)-ATPase.

ERK1/2 Mediates Insulin Stimulation of Na,K-ATPase by Phosphorylation of the α-Subunit in Human Skeletal Muscle Cells

Insulin stimulates Na(+),K(+)-ATPase activity and induces translocation of Na(+),K(+)-ATPase molecules to the plasma membrane in skeletal muscle. We determined the molecular mechanism by which insulin regulates Na(+),K(+)-ATPase in differentiated primary human skeletal muscle cells (HSMCs). Insulin action on Na(+),K(+)-ATPase was dependent on ERK1/2 in HSMCs. Sequence analysis of Na(+),K(+)-ATPase alpha-subunits revealed several potential ERK phosphorylation sites. Insulin increased ouabain-sensitive (86)Rb(+) uptake and [(3)H]ouabain binding in intact cells. Insulin also increased phosphorylation and plasma membrane content of the Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunits. Insulin-stimulated Na(+),K(+)-ATPase activation, phosphorylation, and translocation of alpha-subunits to the plasma membrane were abolished by 20 microm PD98059, which is an inhibitor of MEK1/2, an upstream kinase of ERK1/2. Furthermore, inhibitors of phosphatidylinositol 3-kinase (100 nm wortmannin) and protein kinase C (10 microm GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X in HSMCs. Insulin also stimulated phosphorylation of alpha(1)- and alpha(2)-subunits on Thr-Pro amino acid motifs, which form specific ERK substrates. Furthermore, recombinant ERK1 and -2 kinases were able to phosphorylate alpha-subunit of purified human Na(+),K(+)-ATPase in vitro. In conclusion, insulin stimulates Na(+),K(+)-ATPase activity and translocation to plasma membrane in HSMCs via phosphorylation of the alpha-subunits by ERK1/2 mitogen-activated protein kinase.

Clathrin-mediated Endocytosis of Na+,K+-ATPase in Response to Parathyroid Hormone Requires ERK-dependent Phosphorylation of Ser-11 within the α1-Subunit

Parathyroid hormone (PTH) inhibits Na(+),K(+)-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the alpha(1)-subunit. To determine whether specific serine phosphorylation sites within the Na(+),K(+)-ATPase alpha(1)-subunit are involved in the Na(+),K(+)-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na(+),K(+)-ATPase alpha(1)-subunit (WT), serine 11 to alanine mutant alpha(1)-subunit (S11A), or serine 18 to alanine mutant alpha(1)-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na(+),K(+)-ATPase alpha(1)-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na(+),K(+)-ATPase alpha(1)-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na(+),K(+)-ATPase alpha(1)-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive (86)Rb uptake and Na(+),K(+)-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of (86)Rb uptake, Na(+),K(+)-ATPase activity, alpha(1)-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na(+),K(+)-ATPase alpha(1)-subunit, ERK was shown to be capable of causing phosphorylation of Na(+),K(+)-ATPase alpha(1)-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na(+),K(+)-ATPase by PKC and ERK-dependent alpha(1)-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na(+),K(+)-ATPase alpha(1)-subunit.

The molecular chaperone alpha-crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress.

Alpha-crystallin, a molecular chaperone and lens structural protein protects soluble enzymes against heat-induced aggregation and inactivation by a variety of molecules. In this study we investigated the chaperone function of alpha-crystallin in a more physiological system in which alpha-crystallin was incorporated into red cell 'ghosts'. Its ability to protect the intrinsic membrane protein Na/K-ATPase from external stresses was studied. Red cell ghosts were created by lysing the red cells and removing cytoplasmic contents by size-exclusion chromatography. The resulting ghost cells retain Na/K-ATPase activity. alpha-Crystallin was incorporated in the cells on resealing and the activity of Na/K-ATPase assessed by ouabain-sensitive 86Rb uptake. Incubation with fructose, hydrogen peroxide and methylglyoxal (compounds that have been implicated in diabetes and cataract formation) were used to test inactivation of the Na/K pump. Intracellular alpha-crystallin protected against the decrease in ouabain sensitive 86Rb uptake, and therefore against inactivation induced by all external modifiers, in a dose-dependent manner.

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

The transport activity of the Na,K-ATPase (a 3 Na+ for 2 K+ ion exchange) is electrogenic, whereas the closely related gastric and non-gastric H,K-ATPases perform electroneutral cation exchange. We have studied the role of a highly conserved serine residue in the fifth transmembrane segment of the Na,K-ATPase, which is replaced with a lysine in all known H,K-ATPases. Ouabain-sensitive 86Rb uptake and K+-activated currents were measured in Xenopus oocytes expressing the Bufo bladder H,K-ATPase or the Bufo Na,K-ATPase in which these residues, Lys800 and Ser782, respectively, were mutated. Mutants K800A and K800E of the H,K-ATPase showed K+-stimulated and ouabain-sensitive electrogenic transport. In contrast, when the positive charge was conserved (K800R), no K+-induced outward current could be measured, even though rubidium transport activity was present. Conversely, the S782R mutant of the Na,K-ATPase had non-electrogenic transport activity, whereas the S782A mutant was electrogenic. The K800S mutant of the H,K-ATPase had a more complex behavior, with electrogenic transport only in the absence of extracellular Na+. Thus, a single positively charged residue in the fifth transmembrane segment of the alpha-subunit can determine the electrogenicity and therefore the stoichiometry of cation transport by these ATPases.

Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C.

Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein kinase A or C, the density of co-localizing Na,K-EGFP and transferrin vesicles was increased 3-4-fold, while the ouabain-sensitive 86Rb uptake was reduced by 22%. Simultaneous activation of PKA and PKC had additive effects with a 6-fold increase of co-localization and a 38% reduction of 86Rb uptake. Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation or protein phosphatase inhibition.

Interaction of Cofilin with Triose-phosphate Isomerase Contributes Glycolytic Fuel for Na,K-ATPase via Rho-mediated Signaling Pathway

We reported previously that cofilin, an actin-binding protein, interacts with Na,K-ATPase and enhances its activity (Lee, K., Jung, J., Kim, M., and Guidotti, G. (2001) Biochem. J. 353, 377-385). To understand the nature of this interaction and the role of cofilin in the regulation of Na,K-ATPase activity, we searched for cofilin-binding proteins in the rat skeletal muscle cDNA library using the yeast two-hybrid system. Several cDNA clones were isolated, some of which coded for triose-phosphate isomerase, a glycolytic enzyme. The interaction of cofilin with triose-phosphate isomerase as well as Na,K-ATPase was confirmed by immunoprecipitation and confocal microscopy in HeLa cells. Cofilin was translocated to the plasma membrane along with triose-phosphate isomerase by the Rho activator lysophosphatidic acid but not by the p160 Rho-associated kinase inhibitor Y-27632, suggesting that the phosphorylated form of cofilin bound to TPI interacts with Na,K-ATPase. Ouabain-sensitive (86)Rb(+) uptake showed that Na,K-ATPase activity was increased by the overexpression of cofilin and lysophosphatidic acid treatment, but not by the overexpression of mutant cofilin S3A and Y-27632 treatment. Pretreatment with the glycolytic inhibitor iodoacetic acid caused a remarkable reduction of Na,K-ATPase activity, whereas pretreatment with the oxidative inhibitor carbonyl cyanide m-chlorophenylhydrazone caused no detectable changes, suggesting that the phosphorylated cofilin is involved in feeding glycolytic fuel for Na,K-ATPase activity. These findings provide a novel molecular mechanism for the regulation of Na,K-ATPase activity and for the nature of the functional coupling of cellular energy transduction.

Ionic mechanisms of regulatory volume increase (RVI) in the human hepatoma cell-line HepG2.

We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na(+)/H(+) antiport were determined fluorometrically; the rates of Na(+)-K(+)-2Cl(-) symport and Na(+)/K(+)-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain-sensitive (86)Rb(+) uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd(3+), flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl-amiloride (EIPA). Most likely, this cation conductance was non-selective for Na(+) over K(+). Hypertonic stress did not change K(+) conductance, whereas possible changes in Cl(-) conductance remain ambiguous. The contribution of Na(+)/H(+)antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of Na(+)-K(+)-2Cl(-)symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na(+)/K(+)-ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K(+) conductance, Na(+)/H(+) antiport, Na(+)-K(+)-2Cl(-) symport, and Na(+)/K(+)-ATPase to this process.

In rat hepatocytes, the hypertonic activation of Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is mediated by protein kinase C.

1. The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an import of extracellular Na(+) via Na(+) conductance, Na(+)-K(+)-2Cl(-) symport, and Na(+)-H(+) antiport. 2. Here, the protein kinase inhibitors staurosporine (100 nmol l(-1)) and bis-indolyl-maleimide I (400 nmol l(-1)) were used to test for a possible contribution of protein kinase C (PKC) to the hypertonic activation of these transporters in confluent primary cultures. 3. Stimulation of Na(+) conductance was monitored: (i) by use of a differential approach based on Na(+) fluxes, (ii) by means of cable analysis, and (iii) in experiments with low Na(+) pulses. All three experimental protocols in concert demonstrated a block of the activation of Na(+) conductance by staurosporine and bis-indolyl-maleimide I. 4. In addition, both compounds significantly reduced the hypertonic activation of Na(+)-K(+)-2Cl(-) symport (quantified on the basis of furosemide-sensitive (86)Rb(+) uptake) to approximately 30 %. 5. In contrast, neither staurosporine nor bis-indolyl-maleimide I had any detectable effect on the hypertonicity-induced alkalinization of cell pH via Na(+)-H(+) antiport (determined fluorometrically). 6. Staurosporine and bis-indolyl-maleimide I completely blocked the RVI of rat hepatocytes (quantified by means of confocal laser-scanning microscopy). The high efficiency of the block suggests an additional inhibitory effect of both compounds on the activity of Na(+)/K(+)-ATPase (determined as ouabain-sensitive (86)Rb(+) uptake). 7. It is concluded that the hypertonic activation of rat hepatocyte Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is probably mediated by PKC.

Inhibition of Na+,K+ pump affects nucleic acid synthesis and smooth muscle cell proliferation via elevation of the [Na+]i/[K+]i ratio: possible implication in vascular remodelling.

Na+,K+ pump inhibition is known to delay the development of apoptosis in vascular smooth muscle cells (VSMC). This study examines Na+,K+ pump involvement in the regulation of VSMC macromolecular synthesis and proliferation. DNA, RNA and protein synthesis in VSMC from the rat aorta was studied by the incorporation of [3H]-labelled thymidine, uridine and leucine. Cell cycle progression was estimated by flow cytometry. Intracellular Na+ and K+ content and Na+,K+ pump activity were quantified as the steady-state distribution of 22Na and 86Rb and the rate of ouabain-sensitive 86Rb uptake in Na+-loaded cells, respectively. Ouabain inhibited the Na+,K+ pump with a Ki of 0.1 mmol/l. At concentrations less than 0.1 mmol/l, neither [Na+]i nor [K+]i was affected by ouabain; elevation of ouabain concentration sharply increased the [Na+]i/[K+]i ratio with a K0.5 of approximately 0.3 mmol/l. At concentrations higher than 0.1 mmol/l, ouabain time- and dose-dependently activated RNA and DNA syntheses in serum-deprived VSMC and inhibited cell cycle progression triggered by serum. In quiescent VSMC, ouabain did not affect protein synthesis, total cell number, but slightly increased the percentage of cells in the S-phase (4.25 versus 1.46%) and attenuated cell death assessed by staining with trypan blue and lactate dehydrogenase release. Elevation of the [Na+]i/[K+]i ratio caused by Na+,K+ pump inhibition markedly enhances nucleic acid synthesis in quiescent VSMC and blocks cell cycle progression in serum-supplied VSMC. The relative contribution of this phenomenon as well as the anti-apoptotic action of increased [Na+]i/[K+]i ratio to vascular remodelling under augmented content of endogenous Na+,K+ pump inhibitors, seen in volume-expanded hypertension, should be investigated by in-vivo studies.

Bradykinin increases Na(+)-K(+) pump activity in cultured guinea-pig tracheal smooth muscle cells.

1. The effect of bradykinin on the Na+-K+ pump of airway smooth muscle was investigated by measuring ouabain-sensitive (86)Rb(+) uptake in cultured guinea-pig tracheal smooth muscle cells. 2. Bradykinin induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake, with an EC(50) of 3 nM (pD(2) = 8.50+/-0.10). Stimulation was not affected by indomethacin (1 microM) suggesting that it is not mediated by cycloxygenase products of arachidonic acid. 3. The B(1) receptor agonists Lys-des-Arg(9)-bradykinin and des-Arg(9)-bradykinin had no effect on ouabain-sensitive (86)Rb(+) uptake. In contrast, the B(1) and B(2) receptor agonist Lys-bradykinin induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake with an EC(50) of 6 nM (pD(2) = 8.21 +/- 0.20). 4. The B(1) receptor antagonist des-Arg(10)-HOE 140 (1 microM) had no effect on bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake. The B(2) receptor antagonists HOE 140 and WIN 64338 antagonized bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake with pK(B) values (-log M) of 8.20 +/- 0.08 and 8.11 +/- 0.20 respectively. 5. Reducing extracellular Na+ from 146 mM to 11 mM caused a 53.5% decrease in basal ouabain-sensitive (86)Rb+ uptake and abolished bradykinin-induced uptake. Two inhibitors of the Na(+)-H(+) exchanger, methylisobutyl-amiloride (MIA; 1 - 100 microM) and ethylisopropyl-amiloride (EIPA; 0.1 - 10 microM), inhibited bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake without affecting basal uptake. 6. These results suggest that bradykinin increases Na+-K+ pump activity of guinea-pig tracheal smooth muscle via stimulation of B(2) receptors and activation of the Na+-H+ exchanger.

Canrenone competes for the same site of ouabain on Na+/K+-ATPase

Recently several studies suggested the existence in human plasma of an endogenous digitalis-like factors (EDLF) similar to ouabain which could have a role in the pathogenesis of some forms of hypertension. Canrenone is a metabolic product of the antialdosteronic drug spironolactone, used in hypertensive therapy and recently described as a blocker of ouabain effects. The aim of this study was to evaluate the effect of canrenone on Na+/K+-ATPase in relation to ouabain investigating its interaction with the ouabain receptor and with the sodium-potassium pump activity. For this purpose we employed a3H ouabain receptor assay in human placental membranes and a 86Rb uptake assay in human erythrocytes. Increasing concentration of canrenone (0-180 μM) partially inhibited the ouabain sensitive 86Rb uptake in red blood cells until 40%. Moreover canrenone partially competed for 30% with 3H ouabain binding in placental membrane receptors in absence and in presence of 5 mM K+, respectively at a concentration of 350 μM and 70 μM. When the displacement of ouabain (10-7M) by canrenone (180 μM) was valuated, measuring ouabain sensitive 86Rb uptake, a recovery of 86Rb uptake was obtained only in presence of 5 mM K+. Finally Scatchard plot from radioreceptor assay showed that ouabain in absence and presence of canrenone intersected at a common point on the abscissal axis. These results provide evidence that canrenone is a partial competitive agonist of ouabain and interacts with the same receptor site suggesting that it could be used in hypertensive states with high levels of endogenous ouabain.