Ouabain-sensitive 86Rb Research Articles

Involvement of Na/K-ATPase in hydrogen peroxide-induced activation of the Src/ERK pathway in LLC-PK1 cells

We have shown that Na/K-ATPase interacts with Src. Here, we test the role of this interaction in H2O2-induced activation of Src and ERK. We found that exposure of LLC-PK1 cells to H2O2 generated by the addition of glucose oxidase into the culture medium activated Src and ERK1/2. It also caused a modest reduction in the number of surface Na/K-ATPases and in ouabain-sensitive Rb+ uptake. These effects of H2O2 seem similar to those induced by ouabain, a specific ligand of Na/K-ATPase, in LLC-PK1 cells. In accordance, we found that the effects of H2O2 on Src and ERK1/2 were inhibited in α1 Na/K-ATPase-knockdown PY-17 cells. Whereas expression of wild-type α1 or the A420P mutant α1 defective in Src regulation rescued the pumping activity in PY-17 cells, only α1, and not the A420P mutant, was able to restore the H2O2-induced activation of protein kinases. Consistent with this, disrupting the formation of the Na/K-ATPase/Src complex with pNaKtide attenuated the effects of H2O2 on the kinases. Moreover, a direct effect of H2O2 on Na/K-ATPase-mediated regulation of Src was demonstrated. Finally, H2O2 reduced the expression of E-cadherin through the Na/K-ATPase/Src-mediated signaling pathway. Taken together, the data suggest that the Na/K-ATPase/Src complex may serve as one of the receptor mechanisms for H2O2 to regulate Src/ERK protein kinases and consequently the phenotype of renal epithelial cells.

Enhanced insulin action following subcutaneous co‐administration of insulin and C‐peptide in rats

This study was undertaken to examine if C-peptide (C) may interact with hexameric insulin and facilitate its disaggregation into the physiologically active monomeric form. Regular insulin (I) or an insulin analogue (IA) were injected s.c. in rats together with C or its C-terminal pentapeptide (PP). I or IA and C or PP were administered either as a physical mixture or into two separate s.c. depots. Whole body glucose utilization was evaluated using the euglycemic clamp technique. Phosphorylation of Akt/PKB and GSK in liver and skeletal muscles and ⁸⁶Rb⁺ uptake by L6 cells were measured. S.c. injection of a mixture of I and C or I and PP resulted in a 30-55% greater (P < 0.01-0.001) and 15-27% (P < 0.05-0.001) longer stimulation of whole body glucose utilization than after separate injections. Insulin-stimulated phosphorylation of Akt/PKB in liver increased 35% more after injection of I and C in mixture compared with after separate injections. Phosphorylation of GSK3 was augmented by 50% (P < 0.05) following the injection of I and C in mixture compared with separate injections. Stimulation of myotubes with premixed I and C (1 nM) elicited 20% additional increase in ouabain-sensitive ⁸⁶Rb⁺ uptake (P < 0.05) in comparison with the effect when I and C were added separately. Subcutaneous co-administration of insulin and C results in augmented insulin bioactivity at the level of tissue glucose uptake, intracellular signalling, and enzyme activation. These effects may be attributed to augmented C mediated disaggregation of hexameric insulin into its physiologically active monomeric form.

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.

Long-term regulation of Na,K-ATPase pump during T-cell proliferation

The aim of the study was to elucidate the mechanism responsible for the proliferation-related regulation of Na,K-ATPase pump. Our data demonstrate that in mitogen-stimulated human blood lymphocytes, enhanced ouabain-sensitive Rb(K) fluxes in the middle/late stage of G(0)/G(1)/S transit are associated with the increased number of Na,K-ATPase pumps expressed at the cell surface (as determined by the [(3)H]ouabain binding). Analysis of total RNA (reverse transcription-polymerase chain reaction) and protein (Western blotting) showed a threefold increase in the level of Na,K-ATPase alpha1-subunit and beta1-subunit mRNAs and significant increase in the Na,K-ATPase alpha1-subunit protein during the first day of mitogen-induced proliferation. The elevated K transport as well as the increased expression of Na,K-ATPase is closely associated with the IL-2-dependent stage of T-cell response. The pharmacological inhibition of IL-2-induced MEK/ERK or JAK/STAT cascades suppressed the IL-2-induced proliferation and reduced the functional and protein expressions of Na,K-ATPase. It is concluded that during the lymphocyte transition from resting stage to proliferation, (1) long-term activation of Na,K-ATPase pump is due to the enhanced expression of Na,K-ATPase protein and mRNA, and (2) the cytokine signaling via the IL-2 receptor is necessary for the cell cycle-associated upregulation of Na,K-ATPase.

Alterations of Na +/K +-ATPase function in caveolin-1 knockout cardiac fibroblasts

Recent studies have demonstrated that the Na +/K +-ATPase is not only an ion pump, but also a membrane receptor that confers the ligand-like effects of cardiotonic steroids (CTS) such as ouabain on protein kinases and cell growth. Because CTS have been implicated in cardiac fibrosis, this study examined the role of caveolae in the regulation of Na +/K +-ATPase function and CTS signaling in cardiac fibroblasts. In cardiac fibroblasts prepared from wild-type and caveolin-1 knockout [Cav-1(−/−)] mice, we found that the absence of caveolin-1 did not affect total cellular amount or surface expression of Na +/K +-ATPase α1 subunit. However, it did increase ouabain-sensitive 86Rb + uptake. While knockout of caveolin-1 increased basal activities of Src and ERK1/2, it abolished the activation of these kinases induced by ouabain but not angiotensin II. Finally, ouabain stimulated collagen synthesis and cell proliferation in wild type but not Cav-1(−/−) cardiac fibroblasts. Thus, we conclude that caveolae are important for regulating both pumping and signal transducing functions of Na +/K +-ATPase. While depletion of caveolae increases the pumping function of Na +/K +-ATPase, it suppresses CTS-induced signal transduction, growth, and collagen production in cardiac fibroblasts.

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.

Endothelin-1 Impairs Alveolar Epithelial Function via Endothelial ETB Receptor

Endothelin-1 (ET-1) is increased in patients with high-altitude pulmonary edema and acute respiratory distress syndrome, and these patients have decreased alveolar fluid reabsorption (AFR). To determine whether ET-1 impairs AFR via activation of endothelial cells and nitric oxide (NO) generation. Isolated perfused rat lung, transgenic rats deficient in ETB receptors, coincubation of lung human microvascular endothelial cells (HMVEC-L) with rat alveolar epithelial type II cells or A549 cells, ouabain-sensitive 86Rb+ uptake. The ET-1-induced decrease in AFR was prevented by blocking the endothelin receptor ETB, but not ETA. Endothelial-epithelial cell interaction is required, as direct exposure of alveolar epithelial cells (AECs) to ET-1 did not affect Na,K-ATPase function or protein abundance at the plasma membrane, whereas coincubation of HMVEC-L and AECs with ET-1 decreased Na,K-ATPase activity and protein abundance at the plasma membrane. Exposing transgenic rats deficient in ETB receptors in the pulmonary vasculature (ET-B(-/-)) to ET-1 did not decrease AFR or Na,K-ATPase protein abundance at the plasma membrane of AECs. Exposing HMVEC-L to ET-1 led to increased NO, and the ET-1-induced down-regulation of Na,K-ATPase was prevented by the NO synthase inhibitor l-NAME, but not by a guanylate cyclase inhibitor. We provide the first evidence that ET-1, via an endothelial-epithelial interaction, leads to decreased AFR by a mechanism involving activation of endothelial ETB receptors and NO generation leading to alveolar epithelial Na,K-ATPase down-regulation in a cGMP-independent manner.

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.

Long-term regulation of Na+/K+-ATPase pump in human lymphocytes: Role of JAK/STAT and MAPK signaling pathways

In interleukin-2 (IL-2)-induced human blood lymphocytes, the Na+/K+ pump function (assessed by ouabain-sensitive Rb+ influx), the abundance of Na+, K+-ATPase alpha1-subunit (determined by Western blotting) and the alpha1- and beta1-subunits mRNA of Na+, K+-ATPase (RT-PCR), as well as the phosphorylation of STAT5 and STAT3 family proteins and ERK1/2 kinase have been examined. A 3.5-4.0-fold increase in the expression of alpha1- and beta1-subunits mRNA of Na+, K+-ATPase was found at 24 h of IL-2 stimulation. The inhibitors of JAK3 kinase (B-42, WHI-P431) was shown to decrease both the phosphorylation of STATs and the rise in the oubain-sensitive rubidium influx as well as the increased abundance of Na+, K+-ATPase alpha1-subunit. The inhibition of the protein kinases ERK1/2 by PD98059 (20 microM) suppressed the alpha1-subunit accumulation. All the kinase inhibitors tested did not alter the intracellular content ofmonovalent cations in resting and IL-2-stimulated lymphocytes. It is concluded that MAPK and JAK/STAT signaling pathways mediate the IL-2-dependent regulation of the Na+, K+-ATPase expression during the lymphocyte transition from resting stage to proliferation.

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.

Na+–K+–2Cl− cotransporter is implicated in gender differences in the response of the rat aorta to phenylephrine

Inhibition of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) with bumetanide reduced contractile responses to phenylephrine (PE) in male rat aortas (129+/-4% of 60 mM KCl-induced contraction control vs 108+/-7% bumetanide; PE 10(-5) M; P<0.01) but did not change equivalent responses in female rat aortas. Removal of the endothelium blunted the effect of NKCC1 inhibition on the response to PE (10(-5) M) in males, whereas in denuded aorta from female rats, bumetanide reduced this response (162+/-5% control vs 146+/-3% bumetanide; P<0.05). NKCC1 basal activity did not show gender differences in intact aortic rings, but in the presence of PE, bumetanide-sensitive (86)Rb(+)/K(+) uptake increased more in male than female aortas (179+/-8 in males vs 158+/-5 nmol (86)Rb(+)/K(+) min(-1) (g aorta)(-1) in females; P<0.05). PE did not stimulate NKCC1 activity in denuded aorta from male rats. However, in female rats, PE increased NKCC1 activity similarly in both denuded (169+/-11 nmol (86)Rb(+)/K(+) min(-1) (g aorta)(-1)) and intact aortas. Ovariectomy increased the bumetanide-sensitive (86)Rb(+)/K(+) uptake increase elicited by PE (223+/-17 nmol (86)Rb(+)/K(+) min(-1) (g aorta)(-1)) and hormone replacement with 17beta-estradiol prevented this effect (159+/-29 nmol (86)Rb(+)/K(+) min(-1) (g aorta)(-1)). Na(+),K(+)-ATPase basal activity, measured as ouabain-sensitive (86)Rb(+)/K(+) uptake, was similar in male and female rats, but the effect of PE was significantly less in intact male aortas (232+/-16 in males vs 296+/-25 nmol (86)Rb(+)/K(+) min(-1) (g aorta)(-1) in females; P<0.05). Our results suggest that PE induced activation of NKCC1 and Na(+),K(+)-ATPase in the rat aorta in a gender-dependent way.

Endothelin decreases lung edema clearance and Na,K‐ATPase activity in alveolar epithelial cells via ET‐B receptor and Nitric Oxide generation

RATIONALE ALI is characterized by high levels of endothelin-1 (ET-1). Administration of ET-1 is associated with a rapid increase in edema formation. We hypothesized that ET-1 may impair the ability of the lung to reabsorb fluid from the alveoli by inhibiting the Na,K-ATPase. METHODS A) Isolated-perfused rat lung model: Alveolar fluid clearance was assessed in an isolated-perfused rat lung model by determining the concentration of Evans blue-tagged albumin in the airspace instillate as a function of time. B) Isolated rat alveolar epithelial cells (AEC) were treated with ET-1 to assess Na,K-ATPase activity by a ouabain-sensitive 86Rb+ uptake and protein analysis by Western blotting. RESULTS Isolated rat lungs perfused for 60 min with different concentrations of ET-1 had a dose dependent decrease in alveolar fluid reabsorption. Non-selective ET A/B receptor antagonist blocked the decrease in lung edema clearance, but not by a selective ET-A antagonist. ET-1 decreased alveolar fluid clearance was blocked by a nitric oxide antagonist (L-NAME). Based on ouabain-sensitivity curves, the endothelin-mediated decrease in edema clearance is due predominantly to AEC type I (AECI). CONCLUSIONS These data support the hypothesis that ET-1 impairs the ability of the lungs to clear fluid by decreasing the Na,K-ATPase activity in AECI cells via ET-B receptor activation and nitric oxide production, thus impairing epithelial lung recovery from lung injury. Funding: Supported by HL-48129, T32-HL076139 and 5F32HL071421

Interaction with the Na,K-ATPase and Tissue Distribution of FXYD5 (Related to Ion Channel)

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,K-ATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365-370). The goal of this study was to determine whether FXYD5 can modulate the Na,K-ATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henle's loop. FXYD5 was effectively immunoprecipitated by antibodies to the alpha subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates alpha. Co-expressing FXYD5 with the alpha1 and beta1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabain-blockable outward current or as ouabain-sensitive (86)Rb(+) uptake. Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.

Moderate-to-severe ischemic conditions increase activity and phosphorylation of the cerebral microvascular endothelial cell Na+-K+-Cl− cotransporter

Brain edema that forms during the early stages of stroke involves increased transport of Na+ and Cl- across an intact blood-brain barrier (BBB). Our previous studies have shown that a luminal BBB Na+-K+-Cl- cotransporter is stimulated by conditions present during ischemia and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema formation in the rat middle cerebral artery occlusion model of stroke. The present study focused on investigating the effects of hypoxia, which develops rapidly in the brain during ischemia, on the activity and expression of the BBB Na+-K+-Cl- cotransporter, as well as on Na+-K+-ATPase activity, cell ATP content, and intracellular volume. Cerebral microvascular endothelial cells (CMECs) were assessed for Na+-K+-Cl- cotransporter and Na+-K+-ATPase activities as bumetanide-sensitive and ouabain-sensitive 86Rb influxes, respectively. ATP content was assessed by luciferase assay and intracellular volume by [3H]-3-O-methyl-D-glucose and [14C]-sucrose equilibration. We found that 30-min exposure of CMECs to hypoxia ranging from 7.5% to 0.5% O2 (vs. 19% normoxic O2) significantly increased cotransporter activity as did 7.5% or 2% O2 for up to 2 h. This was not associated with reduction in Na+-K+-ATPase activity or ATP content. CMEC intracellular volume increased only after 4 to 5 h of hypoxia. Furthermore, glucose and pyruvate deprivation increased cotransporter activity under both normoxic and hypoxic conditions. Finally, we found that hypoxia increased phosphorylation but not abundance of the cotransporter protein. These findings support the hypothesis that hypoxia stimulation of the BBB Na+-K+-Cl- cotransporter contributes to ischemia-induced brain edema formation.

Relationships between membrane lipids and ion transport in red blood cells of Dahl rats

Distinct changes of membrane lipid content could contribute to the abnormalities of ion transport that take part in the development of salt hypertension in Dahl rats. The relationships between lipid content and particular ion transport systems were studied in red blood cells (RBC) of Dahl rats kept on low- and high-salt diets for 5 weeks since weaning. Dahl salt-sensitive (SS/Jr) rats on high-salt diet had increased blood pressure, levels of plasma triacylglycerols and total plasma cholesterol compared to salt-resistant (SR/Jr) rats. Furthermore, RBC of SS/Jr rats differed from SR/Jr ones by increased content of total membrane phospholipids, but membrane cholesterol was not changed significantly. SS/Jr rats had higher RBC intracellular Na + (Na i +) content and enhanced bumetanide-sensitive Rb + uptake. RBC membrane content of cholesterol and phospholipids correlated positively with RBC Na i + content, with the activity of Na +–K + pump and Na +–K +–2Cl − cotransport and also with Rb + leak. The content of phosphatidylserines plus phosphatidylinositols was positively associated with RBC Na i + content, with the activity of Na +–K + pump and Na +–K +–2Cl − cotransport and with Rb + leak. The content of sphingomyelins was positively related to Na +–K +–2Cl − cotransport activity and negatively to ouabain-sensitive Rb +–K + exchange. We can conclude that observed relationships between ion transport and the membrane content of cholesterol and/or sphingomyelins, which are known to regulate membrane fluidity, might participate in the pathogenesis of salt hypertension in Dahl rats.

Interleukin-2-dependent regulation of Na/K pump in human lymphocytes

The present study provides the first evidence that the abundance of catalytic α1-subunit of Na,K-ATPase increases in the course of T cell blast transformation. Immunodepressant cyclosporin A at anti-proliferative doses diminished the induction of α1 protein in activated lymphocytes. Furthermore, in competent T cells, IL-2 increases both the transport activity of Na/K pump and the content of Na,K-ATPase α1 protein in a time-dependent manner. A correlation was found between the long-term elevation in ouabain-sensitive Rb influxes and the increase in α1 protein content in late activated T cells. These results suggest that (1) the increased expression of Na,K-ATPase proteins underlie the cell cycle-dependent upregulation of ion pump during T cell transformation, and (2) IL-2 is involved in the regulated expression of Na,K-ATPase in human lymphocytes.

The Effect of the Gamma Modulator on Na/K Pump Activity of Intact Mammalian Cells

This study concerns the modulatory effects of the gamma modulator of the Na/K pump, in particular whether the effects seen in previous experiments with isolated membranes are relevant to Na/K pump behavior in intact mammalian cells. For this purpose, HeLa cells previously transfected with the rat Na/K catalytic subunit were used. The results show that both variants of the regulator, gammaa and gammab, decrease the apparent affinity of the pump for Na(+) and cause a modest increase in apparent ATP affinity as seen in measurements of ouabain-sensitive (86)Rb(K(+)) influx into cells in which ATP was varied using antimycin A and glucose. Equivalent results had been obtained previously in our analyses of Na,K-ATPase activity of membrane fragments, i.e., an increase in K(0.5(Na)) at high K(+) concentration and a decrease in K'(ATP). Comparison of clones of gamma-transfected and mock-transfected cells (with similar V(max) values) indicated that gamma causes a modest approximately 30% increase in the steady-state concentration of intracellular Na(+). Furthermore, for both gammaa and gammab, values of intracellular Na(+) were similar to those predicted from the kinetic constants, K(0.5(Na)) and V(max). Finally, there was a gamma-mediated increase in apparent affinity for extracellular K(+), which had not been detected in assays of permeabilized membranes.

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.