Activity Of Na Pump Research Articles

Concept article: Antidepressant-induced destabilization in bipolar illness mediated by serotonin 3 receptor (5HT3).

Antidepressants used by patients with bipolar disorder have been associated with destabilization with an increase in mania, depression, and cycling. The most commonly proposed mechanism, that antidepressants 'overshoot' their antidepressant effect to create a manic or mixed state, is unlikely since antidepressants have actually been found to be ineffective in treating bipolar depression. Beginning with known bipolar-specific pathophysiologic abnormalities provides the greatest likelihood of insight. PubMed was queried with 'bipolar', 'sodium', 'intracellular sodium', 'serotonin 3', '5HT3', '5-hydroxytryptamine type 3 receptors', and 'antidepressant' either individually or in combination. Pathologic mood states (both mania and depression) are associated with increased intracellular sodium (Na) concentrations that depolarize the resting membrane potential to increase cellular excitability (mania) or cause depolarization block (depression). Stimulation of the serotonin (5HT) receptors depolarizes the post-synaptic neuron. Stimulation of 5HT3 may be of particular importance since it is coupled to a cation channel that directly depolarizes the membrane. These effects directly impact the physiology of patients with bipolar disorder to alter neuronal excitability in a fashion that worsens both mania and depression. The most consistently observed biological abnormality in individuals going through mania or bipolar depression involves a decline in Na pump activity, with consequent elevation of intracellular Na levels. Antidepressant treatment potentiates this, particularly by activation of 5HT3. This hypothesis can be tested by coadministering a 5HT3 antagonist (e.g., vortioxetine or ondansetron) to achieve blockade of that receptor while treating bipolar depression with a serotoninergic antidepressant.

Oxidative inhibition of erythrocyte sodium pump: A functionally relevant circulating marker of oxidative stress

O stress plays critical roles in the pathogenesis of diabetes and heart disease. The activity of Na pump has been shown to be depressed from the membrane of erythrocyte preparations of these patients coupled with alterations in membrane protein composition. We had previously discovered that the β1 subunit of the Na pump undergoes oxidative modification by oxidative stress in vitro (β1-GSS) and this mediates Na pump inhibition. This study aims to develop and validate erythrocyte β1-GSS as an oxidative stress biomarker in the premier emerging tool for prognosis of pathophysiological oxidative stress in patients with or at risk of cardiovascular disease (CVD) and diabetes. The marker is quickly and easily tested from blood using ELISA; and could be packaged and marketed as a simple kit. The eβ1-GSS biomarker is modified in heart attack, heart failure and diabetes, and exhibits potential to predict disease progression. With further investment, the eβ1-GSS biomarker could be developed initially as a versatile CVD prognosis tool for universal pre-hospital diagnostics, CVD-severity risk-stratification, and as a Companion Diagnostic (Dx) for CVD medications. Subsequently it could be adapted for diabetes.

Cardiac Glycoside Activities Link Na+/K+ ATPase Ion-Transport to Breast Cancer Cell Migration via Correlative SAR

The cardiac glycosides ouabain and digitoxin, established Na+/K+ ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na+/K+ ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure–activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na+/K+ ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein–small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na+/K+ transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1–M6 correlates with the Na+ pump activity and cell migration. Other Na+/K+ ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na+/K+ ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.

Substrate recognition by the cell surface palmitoyl transferase DHHC5.

The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼ 120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.

Wfs1-deficient animals have brain-region-specific changes of Na+, K+-ATPase activity and mRNA expression of α1 and β1 subunits.

Mutations in the WFS1 gene, which encodes the endoplasmic reticulum (ER) glycoprotein, cause Wolfram syndrome, a disease characterized by juvenile-onset diabetes mellitus, optic atrophy, deafness, and different psychiatric abnormalities. Loss of neuronal cells and pancreatic β-cells in Wolfram syndrome patients is probably related to the dysfunction of ER stress regulation, which leads to cell apoptosis. The present study shows that Wfs1-deficient mice have brain-region-specific changes in Na(+),K(+)-ATPase activity and in the expression of the α1 and β1 subunits. We found a significant (1.6-fold) increase of Na-pump activity and β1 subunit mRNA expression in mice lacking the Wfs1 gene in the temporal lobe compared with their wild-type littermates. By contrast, exposure of mice to the elevated plus maze (EPM) model of anxiety decreased Na-pump activity 1.3-fold in the midbrain and dorsal striatum and 2.0-fold in the ventral striatum of homozygous animals compared with the nonexposed group. Na-pump α1 -subunit mRNA was significantly decreased in the dorsal striatum and midbrain of Wfs1-deficient homozygous animals compared with wild-type littermates. In the temporal lobe, an increase in the activity of the Na-pump is probably related to increased anxiety established in Wfs1-deficient mice, whereas the blunted dopamine function in the forebrain of Wfs1-deficient mice may be associated with a decrease of Na-pump activity in the dorsal and ventral striatum and in the midbrain after exposure to the EPM.

Knockout Mice Reveal Key Roles for Claudin 18 in Alveolar Barrier Properties and Fluid Homeostasis

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased β-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase β1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.

Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs

Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg(-1)·min(-1) compared with only 9.9 ± 1.1 AFU·mg(-1)·min(-1) in controls (P < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% (P < 0.043) greater in tubules from ANG II-treated than vehicle rats. ANG II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg(-1)·day(-1)) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

Adrenaline and triiodothyronine modify the iron handling in the freshwater air-breathing fish Anabas testudineus Bloch: Role of ferric reductase in iron acquisition

The effects of in vivo adrenaline and triiodothyronine (T3) on ferric reductase (FR) activity, a membrane-bound enzyme that reduces Fe(III) to Fe(II) iron, were studied in the organs of climbing perch (Anabas testudineus Bloch). Adrenaline injection (10ngg−1) for 30min produced significant inhibition of FR activity in the liver and kidney and that suggests a role for this stress hormone in iron acquisition in this fish. Short-term T3 injection (40ngg−1) reduced FR activity in the gills of fed fish but not in the unfed fish. Similar reduction of FR activity was also obtained in the intestine and kidney of fed fish after T3 injection. Feeding produced pronounced decline in FR activity in the spleen but T3 challenge in fed and unfed fish increased its activity in this iron storing organ and that point to the sensitivity of FR system to feeding activity. The in vitro effects of Fe on FR activity in the gill explants of freshwater fish showed correlations of FR with Na+, K+-ATPase and H+-ATPase activities. Substantial increase in the FR activity was found in the gill explants incubated with all the tested doses of Fe(II) iron (1.80, 3.59 and 7.18μM) and Fe(III) iron (1.25, 2.51 and 5.02μM) and this indicate that FR and Na pump activity are positively correlated. On the contrary, substantial reduction of gill H+-ATPase activity was found in the gill explants incubated with Fe(II) iron and Fe(III) iron indicating that perch gills may not require a high acidic microenvironment for the reduction of Fe(III) iron. Accumulation of iron in the gill explants after Fe(III) iron incubation implies a direct relationship between Fe acquisition and FR activity in this tissue. The inverse correlation between FR activity and H+-ATPase activity in Fe(II) or Fe(III) loaded gills and the significant positive correlations of FR activity with total [Fe] content in the Fe(III) loaded gills substantiate that FR which shows sensitivity to sodium and proton pumps, has a vital role in Fe(II) and Fe(III) iron handling in this fish. Our data also provide evidence that adrenaline, T3 and the feeding status are the vital factors that can regulate the storage and handling of iron in fish.

Express Method for Determination of Low Value of Trans-membrane Potential of Living Cells with Fluorescence Probe: Application on Haemocytes at Immune Responses

The method for measurement of trans-membrane potential of cell membrane was evaluated for the case of low potential value using fluorescence probe 4-(4-dimethylaminostyryl)-1-methylpyridinium, DSM. The method is based on comparative titration of cells with probe in buffers containing Na(+) or K(+). The apparent trans-membrane potential obtained with this way is a result of K(+)-Na(+) pump activity. The presented approach allowed measuring the low value of potential with 1-2 mV of accuracy without additional calibration procedures. The method was applied for investigation of potential of cell membrane of haemocytes of Galleria mellonella larvae. The value of potential of intact insect's haemocytes was found in the range from -10 to -20 mV. The change of potential value of haemocytes was investigated under model immune response and natural envenomation and parasitizing. The obtained deviations of cell membrane potential were in good correlation with changes of activity of main immune reactions, described in literature and obtained by us earlier.

Lactoferrin Stimulates Erythrocyte Na+/K+-Adenosine Triphosphatase: Effect of Some Modulators of Membrane Phosphorylation

We studied the effect of some modulators of signal transduction on the erythrocyte Na+/ K+-ATPase. Go6976 and Go6983 (protein kinase C inhibitors) showed a stimulatory effect and calyculin A (protein phosphatase inhibitor) exerted an inhibitory effect on the Na pump activity. Some of the tested modulators of cell-signaling [protein phosphatase(s), phosphodiesterase, calmodulin and some protein kinases] interfered with the lactoferrin (Lf) stimulatory effect on the sodium pump. Lf itself was able to modulate the effect of some agents upon the pump activity. Moreover, an additive effect of stimulation was found when Lf and some agents were used simultaneously. The summarized results showed that: (i) Lf upregulates the Na+/K+-ATPase in erythrocytes and facilitates the K+ influx into the erythrocytes; (ii) the effect of pump stimulation is mediated by phosphorylation processes. These results suggest a potential opportunity for using Lf alone or together with other agents as a stimulator of the erythrocyte Na+/K+-ATPase.

Fiber specific differential phosphorylation of the α1-subunit of the Na+,K+-ATPase in rat skeletal muscle: the effect of aging

In skeletal muscle, the Na(+),K(+)-ATPase maintains the Na(+) and K(+) gradients and modulates contractile functions. The different fibers of the skeletal muscle possess diverse properties and functions, and thus, the demands for the Na-pump activity might be different. Because phosphorylation of the alpha1-subunit of the Na(+),K(+)-ATPase appears to serve a regulatory role in the activity of Na(+),K(+)-ATPase, we postulated that a difference in the phosphorylation of the alpha1-subunit may be found among the fibers. We utilized two well-characterized specific antibodies for the alpha1-subunit, namely the McK1 and alpha6F, to determine, by immunofluorescence, if the alpha1-subunit in rat skeletal muscle fiber is differentially phosphorylated. McK1 has the unique property that its binding to the alpha1-subunit is greatly reduced when Ser-18 is phosphorylated. Our data show that, in red gastrocnemius muscle, only a small number of the fibers were stained on the sarcolemmal membrane by McK1, while other fibers were almost completely devoid of any staining. By contrast, the staining pattern by McK1 in the white gastrocnemius muscle was mostly uniform. Immunostaining of serial sections using the alpha6F antibody showed that the alpha1-subunit is expressed in all fibers. Dephosphorylation of the tissue sections by phosphatase partially restored immunostaining of the alpha1-subunit by McK1. Fiber typing results showed that, in red gastrocnemius, those fibers stained positive for alpha1-subunit by McK1 are the Type I fibers, whereas those stained negative are the Type IIA, IID, and IIB fibers. With age, the number of fibers in red gastrocnemius stained positive for McK1 increased markedly in 30-month old rats compared to 6-month old rats. In conclusion, our result suggests that, in rats, the alpha1-subunit of the Na(+),K(+)-ATPase is differentially phosphorylated in the fibers of the red gastrocnemius muscle. Furthermore, advanced age is associated with an apparent decrease in the phosphorylation of the alpha1-subunit, in addition to the previously demonstrated increase in the levels of expression of the subunit.

Sodium pump α2 subunits control myogenic tone and blood pressure in mice

A key question in hypertension is: How is long-term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain-like compound (EOLC) and induces salt-dependent hypertension mediated by Na(+)/Ca(2)(+) exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na(+) pump alpha1 (alpha1(+/-)) or alpha2 (alpha2(+/-)) catalytic subunits. Both low-dose ouabain (1-100 nm; inhibits only alpha2) and high-dose ouabain (> or =1 microm; inhibits alpha1) elevate myocyte Ca(2)(+) and constrict arteries from alpha1(+/-), as well as alpha2(+/-) and wild-type mice. Nevertheless, only mice with reduced alpha2 Na(+) pump activity (alpha2(+/-)), and not alpha1 (alpha1(+/-)), have elevated blood pressure. Also, isolated, pressurized arteries from alpha2(+/-), but not alpha1(+/-), have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB-R7943) normalize myogenic tone in ouabain-treated arteries. Only the NCX blockers normalize the elevated myogenic tone in alpha2(+/-) arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt-dependent and ouabain-induced hypertension. Thus, chronically reduced alpha2 activity (alpha2(+/-) or chronic ouabain) apparently regulates myogenic tone and long-term blood pressure whereas reduced alpha1 activity (alpha1(+/-)) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt-dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing alpha2 Na(+) pump activity and promoting Ca(2)(+) entry via NCX in myocytes.

Lung Fluid Balance During Development

After completing this article, readers should be able to: 1. Describe the major driving forces for liquid formation in and liquid removal from the lumen of the fetal lung. 2. Delineate the role of a normal volume of liquid within the lumen of the fetal lung for normal lung growth during development. 3. Describe the effects of labor on Na,K-ATPase activity in lung epithelial cells and clearance of lung liquid after birth. 4. Explain the route by which most residual liquid drains from the lung. 5. List the conditions associated with preterm birth that may contribute to delayed removal of fetal lung liquid. Curious students of mammalian development recognized more than a century ago that the lungs are filled with liquid during fetal life, (1) but the origin of that liquid remained obscure until 1948, when an unexpected discovery by two French scientists dispelled the prevailing view that fetal lung liquid derived from intrauterine inhalation of amniotic fluid. In studies designed to explore the ontogeny of the pituitary-adrenal axis, tracheal ligation of fetal rabbits for 9 days resulted in fluid distention of the lungs. (2) This serendipitous finding, subsequently confirmed and embellished by others, (3)(4)(5) established that the fetal lung itself, rather than the amniotic sac, is the source of the liquid that fills the lung during development. This liquid forms a slowly expanding structural template that prevents collapse and promotes growth of the fetal lung. Subsequent studies, conducted mostly with sheep, showed that liquid in the lumen of the fetal lung forms as a result of chloride secretion in the respiratory epithelium, (6)(7) a process that can be inhibited by diuretics that block Na,K-2Cl cotransport. (8)(9) In vitro experiments using cultured explants of lung tissue and monolayers of epithelial cells harvested from human fetal lung have …

Angiotensin II Regulation of the Na+ Pump Involves the Phosphatidylinositol-3 Kinase and p42/44 Mitogen-Activated Protein Kinase Signaling Pathways in Vascular Smooth Muscle Cells

This investigation used primary cultured rat vascular smooth muscle cells to examine angiotensin II (Ang II) regulation of Na(+), K(+)-ATPase (Na(+) pump) activity, and Na(+) pump alpha(1)- and beta(1)-subunit gene transcription. This regulation was mediated through both phosphatidylinositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44(MAPK)) signaling pathways. Both acute (10 min) and prolonged (24 h) treatment with Ang II stimulated Na(+) pump activity. Also, prolonged exposure to Ang II (24 h) increased promoter transcription of the Na(+) pump alpha(1)- and beta(1)-subunits. Furthermore, PI3K activities because well because p42/44(MAPK) phosphorylation were increased within 10 min after Ang II treatment. To determine whether these stimulatory activities of Ang II are acting through Ang II receptors 1 and/or 2 (AT(1), AT(2)), cells were pretreated with either AT(1) receptor blocker losartan or the AT(2) receptor blocker PD 123,319. Indeed, these treatments prevented the stimulatory effect of Ang II on Na(+) pump activity at both acute and 24-h time points. Furthermore, the Ang II-stimulated alpha(1)-subunit promoter transcription was inhibited by losartan but not by the AT(2) receptor blocker. These results indicate that Ang II acts through both the AT(1) and AT(2) receptor to up-regulate Na(+) pump activity; however, Ang II regulates alpha(1)-gene transcription through AT(1) but not AT(2) receptors. It was also observed that the Ang II-stimulated beta(1)-subunit gene transcription is not mediated through either AT(1) or AT(2) receptors. To examine whether the Na(+)/H(+) exchanger is involved in Ang II-stimulated Na(+) pump activity, cells were pretreated with amiloride, a specific inhibitor of the Na(+)/H(+) exchanger. This pretreatment prevented 24 h, but not acute, Ang II-stimulated Na(+) pump activity. The 24-h Ang II-stimulated alpha(1)-subunit promoter transcription was also inhibited by amiloride. This suggests that the prolonged effect of Ang II on Na(+) pump activity is dependent on increased Na(+)/H(+) exchange. Because Ang II treatment for 10 min increased PI3K activity because well because p42/44(MAPK) phosphorylation, studies were performed to determine the involvement of PI3K and p42/44(MAPK) signaling pathways in both Ang II-stimulated Na(+) pump activity and alpha(1)- and beta(1)-gene transcription. Cells were pretreated with either the PI3K inhibitor wortmannin or the p42/44(MAPK) inhibitor PD 98059. Ang II-stimulated PI3K or p42/44(MAPK) activity was inhibited by these pretreatments. Furthermore, pretreatment of cells with the PI3K inhibitors wortmannin and LY29404 or the MAPK inhibitors U0126 and PD 98059 were all observed to inhibit Ang II-stimulated Na(+) pump activity. To more specifically determine the role of PI3K in Ang II-regulation of alpha(1)-and beta(1)-gene transcription, cells were cotransfected with a dominant-negative p85 construct. Cotransfection with dominant-negative p85 reduced Ang II-stimulated alpha(1)-but not beta(1)-gene transcription in vascular smooth muscle cells. These results indicate that Ang II acts through PI3K/p42/44(MAPK) signaling pathways to up-regulate Na(+) pump activity and alpha(1)-gene transcription and that Ang II-regulated beta(1)-gene transcription is not mediated through either PI3K or p42/44 (MAPK) signaling pathways.

Regulation of Ca2+ signaling by Na+ pump alpha-2 subunit expression.

The role of the Na(+) pump alpha2 subunit in Ca(2+) signaling was examined in primary cultured astrocytes from wild type (WT) mouse fetuses and those with a null mutation in one (Het) or both (KO) alpha2 genes. Cytosol Na(+) and Ca(2+) concentrations ([Na(+)](CYT) and [Ca(2+)](CYT)) were measured with benzofuran isophthalate (SBFI) and Fura-2. Het astrocytes express approximately 50% of normal alpha2; KO cells express none. Resting [Na(+)](CYT) = 6.5 mM in WT, 6.8 mM in Het, and 8.0 mM in KO cells; 500 nM ouabain (which inhibits only alpha2) equalized [Na(+)](CYT) at 8 mM in all genotypes. Resting [Ca(2+)](CYT) = 132 nM in WT, 162 nM in Het and 196 nM in KO cells. Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) Ca(2+) pumps, induces elevation of [Ca(2+)]CYT. These Ca(2+) responses are augmented in Het and KO cells. This correlates with alpha2 Na(+) pump and Na(+)/Ca(2+) exchanger (NCX) localization in plasma membrane (PM) microdomains that overlie the ER. Selective reduction of alpha2 Na(+) pump activity apparently elevates local [Na(+)] and, via NCX, [Ca(2+)] in the tiny cytosolic space between the PM and ER. This augments adjacent ER Ca(2+) stores and amplifies Ca(2+) signaling without elevating bulk [Na(+)](CYT).

P427 The activity of the sarcolemmal Na pump determines the functional consequence of adenoviral Na/Ca exchanger overexpression

P427 The activity of the sarcolemmal Na pump determines the functional consequence of adenoviral Na/Ca exchanger overexpression

Short-term stretch translocates the alpha-1-subunit of the Na pump to plasma membrane.

Short-term (2 30 min) cyclic stretch activates the Na pump in cultured aortic smooth muscle cells (ASMCs). This effect of stretch involves the phosphotidylinositol 3-kinase (PI 3-kinase) participation. Presently, we investigated whether this stimulation is the result of translocation of Na(+),K(+)-ATPase from endosomes to the plasma membrane. ASMCs were stretched 20% for 5 min using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na(+),K(+)-ATPase alpha-1-subunit protein. Membrane marker enzyme, 5' nucleotidase activity, and the early and recycling endosome markers Rab4 and Rab11 were used to verify the enrichment of these fractions. Stretch increased Na(+),K(+)-ATPase alpha-1 expression in plasma membrane fractions and decreased it in endosomes. PI 3-kinase inhibitors LY294002 and wortmannin blocked the stretch-induced translocation of the Na(+),K(+)-ATPase alpha-1-subunit. Rab4 and Rab11 were enriched in the endosomal fraction, whereas 5' nucleotidase activity was enriched in the plasma membrane fraction. We conclude that stimulation of the Na pump activity by shortterm cyclic stretch is the result, at least in part, of transport of the alpha-subunit of the enzyme from endosomes to the plasma 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.

Long-term modulation of glucose utilization by IL-1 alpha and TNF-alpha in astrocytes: Na+ pump activity as a potential target via distinct signaling mechanisms.

Interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) markedly stimulate glucose utilization in primary cultures of mouse cortical astrocytes. The mechanism that gives rise to this effect, which takes place several hours after application of cytokine, has remained unclear. Experiments were conducted to identify the major signaling cascades involved in the metabolic action of cytokine. First, the selective IL-1 receptor antagonist (IL-1ra) prevents the effect of IL-1alpha on glucose utilization in a concentration-dependent manner, whereas it has no effect on the action of TNF-alpha. Then, using inhibitors of three classical signaling cascades known to be activated by cytokines, it appears that the PI3 kinase is essential for the effect of both IL-1alpha and TNF-alpha, whereas the action of IL-1alpha also requires activation of the MAP kinase pathway. Participation of a phospholipase C-dependent pathway does not appear critical for both IL-1alpha and TNF-alpha. Inhibition of NO synthase by L-NAME did not prevent the metabolic response to both IL-1alpha and TNF-alpha, indicating that nitric oxide is probably not involved. In contrast, the Na(+)/K(+) ATPase inhibitor ouabain prevents the IL-1alpha- and TNF-alpha-stimulated 2-deoxyglucose (2DG) uptake. When treatment of astrocytes with a cytokine was followed 24 h later by an acute application of glutamate, a synergistic enhancement in glucose utilization was observed. This effect was greatly reduced by ouabain. These data suggest that Na(+) pump activity is a common target for both the long-term metabolic action of cytokines promoted by the activation of distinct signaling pathways and the enhanced metabolic response to glutamate.

Segmental localization of mRNAs encoding Na(+)-K(+)-ATPase alpha(1)- and beta(1)-subunits in diabetic rat kidneys using RT-PCR.

The present study evaluated renal Na(+)-K(+)-ATPase activity and mRNA in rats with diabetes mellitus. To localize the segmental alpha(1)- and beta(1)-mRNAs of Na(+)-K(+)-ATPase 1 and 8 days after induction of diabetes, we used the polymerase chain reaction after reverse transcription of the mRNA in microdissected nephron segments. Na(+)-K(+)-ATPase activity in the proximal convoluted tubule (PCT) rose on days 1 and 8 by 42 and 23%, respectively. In the medullary thick ascending limb (MTAL), it remained unchanged on day 1 and increased on day 8 by 55%. In the cortical collecting duct (CCD), activity rose by 81 and 45% on days 1 and 8, respectively. In parallel, alpha(1)-mRNA in the PCT increased by 52 and 22% on days 1 and 8, respectively. In the MTAL, alpha(1)-mRNA remained unchanged on day 1 and rose by 47% on day 8. In the CCD, alpha(1)-mRNA increased by 140 and 110% on days 1 and 8, respectively. beta(1)-mRNA was unchanged in the PCT throughout the study and was elevated in the MTAL and CCD on days 1 and 8. Thus there was a temporal dissociation between alpha(1)- and beta(1)-subunit expression. There was a highly significant linear correlation between Na(+)-K(+)-ATPase activity and alpha(1)-mRNA in all nephron segments throughout the experiment. It appears that microdissection of nephron tubules combined with reverse transcription-polymerase chain reaction defines the molecular identity of the amplified gene product and its segmental distribution in the nephron. We propose that altered gene expression may be the mechanism underlying enhanced Na(+) pump activity along the nephron in diabetic rats.