Expression of rat L-FABP in mouse fibroblasts: role in fat absorption

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose level is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigatedin vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accomodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtainedin vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.Abbreviations FABP fatty acid-binding protein - L-FABP liver fatty acid-binding protein - I-FABP intestinal fatty acid-binding protein - H-FABP heart fatty acid-binding protein - A-FABP adipocyte fatty acid-binding protein - PMFABP plasma membrane fatty acid-binding protein - SCP-2 sterol carrier protein-2 - Dehydroergosterol (DHE) d-5,7,9(11),22-ergostatetraene-3b-ol - cis-parinaric acid-9Z, 11E, 13E, 15Z-octatetraenoic acid - trans parinaric acid, 9E, 11E, 13E, 14E-octatetraenoic acid - BSA bovine serum albumin - KRH Krebs-Ringer-Henseleit buffer     

Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein

The potential role of liver fatty acid binding protein (L-FABP) in modulating cellular sterol distribution was examined in mouse L-cell fibroblasts transfected with cDNA encoding L-FABP. L-cells were chosen because they contain only a small amount of endogenous FABP which does not bind [3H]cholesterol, does not enhance intermembrane sterol transfer, and whose content is unaltered by the expression of L-FABP. Transfected L-cells expressed 0.34% of cytosolic protein as L-FABP. Transfection alone with low expression of L-FABP (0.008% of cytosolic protein) had no effect on any of the parameters tested. Three aspects of cellular sterol transfer were examined. First, cellular sterol uptake, monitored by [3H]cholesterol and the fluorescent sterol, delta-5,7,9(11),22-ergostatetraen-3 beta-ol, was increased 21.5 +/- 2.6% (p less than 0.001) in L-cells expressing L-FABP. This increase was not accounted for by increased sterol esterification in the cells expressing L-FABP. Inhibition of both cholesterol transfer and esterification with 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide from Sandoz abolished the L-FABP related enhancement of both [3H]cholesterol uptake and esterification. Second, plasma membrane transbilayer distribution of sterol, determined by fluorescence methods indicated that the majority of sterol was in the inner leaflet of the plasma membrane. In transfected cells expressing L-FABP, twice as much sterol (28 +/- 4%) was present in the exofacial leaflet of the plasma membrane as compared to that of control cells (15 +/- 2%). Third, expression of L-FABP enhanced sterol transfer from the plasma membrane to microsomes in intact cells. Treatment of [3H]cholesterol or [3H]oleate-loaded cells with sphingomyelinase resulted in increased formation of radiolabeled cholesterol ester, consistent with enhanced microsomal esterification of plasma membrane derived cholesterol. Concomitantly, plasma membrane [3H]cholesterol became less accessible to oxidation by cholesterol oxidase. Sphingomyelinase-stimulated cholesterol esterification was 21 +/- 3% greater in transfected cells. Concomitantly, accessibility of plasma membrane [3H]cholesterol to cholesterol oxidase was decreased 18 +/- 3% in cells expressing L-FABP. These differences are consistent with the ability of L-FABP to influence sterol transport and plasma membrane transbilayer sterol distribution in intact cells.     

Synthesis and assembly of functional mammalian Na,K-ATPase in yeast.

The yeast Saccharomyces cerevisiae was investigated as an in vivo protein expression system for mammalian Na,K-ATPase. Unlike animal cells, yeast cells lack endogenous Na,K-ATPase. Expression of high affinity ouabain binding sites, ouabain-sensitive ATPase activity, or ouabain-sensitive p-nitrophenylphosphatase activity in membrane fractions of yeast cells was observed to require the expression of both alpha subunit and beta subunit polypeptides of Na,K-ATPase in the same cell. High affinity ouabain binding sites are also expressed at the cell surface of intact yeast cells containing both the alpha subunit and the beta subunit of Na,K-ATPase. These observations demonstrate that both the alpha subunit and the beta subunit of Na,K-ATPase are required for the expression of functional Na,K-ATPase activity and that yeast cells can correctly assemble this oligomeric membrane protein and transport it to the cell surface.     

Altered membrane structure in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein

Mouse L cell fibroblasts were transfected with cloned cDNA encoding rat liver fatty acid binding protein (L-FABP) also known as sterol carrier protein. Stable transfectant cell lines were selected and expression of L-FABP determined using Western blot analysis. The nontransfected controls and low expression cells did not differ significantly in any of the properties examined. All cell lines showed similar doubling times but cells expressing high levels of L-FABP attained 2-fold higher cell saturation density and differed significantly in their lipid metabolism as indicated by 1) higher cholesterol ester and phospholipid content, and 2) decreased sterol/phospholipid ratio. The observed changes in the lipid composition predicted a lower degree of membrane-lipid order (higher fluidity) in the plasma membranes of cells expressing high levels of L-FABP. Therefore, fluorescent molecule, 1,6-diphenyl-1,3,5-hexatriene, and multifrequency (1-250 MHz) phase and modulation fluorometry were used to probe the effect of L-FABP expression on membrane structure. Steady-state polarization and limiting anisotropy of diphenylhexatriene were significantly lower in the isolated plasma membrane vesicles from the high expression clones. The observed changes in L-cells as a result of de novo expression of L-FABP are consistent with the ability of this protein to bind sterols and fatty acids, stimulate sterol esterification, and stimulate phospholipid biosynthesis. This evidence is supportive of a physiologic role for L-FABP in modulating cellular lipid metabolism and membrane structure.     

Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na+ and K+ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic alpha-subunit (four isoforms) and an ancillary beta-subunit (three isoforms). Mutations in the alpha2-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase alpha2-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [3H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. 86Rb+-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase alpha2-isoform.     

Inhibition of Na,K-ATPase and sodium pump by protein kinase C regulators sphingosine, lysophosphatidylcholine, and oleic acid

The effects and modes of action of certain lipid second messengers and protein kinase C regulators, such as sphingosine, lysophosphatidylcholine (lyso-PC), and oleic acid, on Na,K-ATPase and sodium pump were examined. Inhibition of purified rat brain synaptosome Na,K-ATPase by these lipid metabolites, unlike that by ouabain, was subject to membrane dilution (i.e. inhibition being counteracted by increasing amounts of membrane lipids). Kinetic analysis, using the purified enzyme, indicated that sphingosine and lyso-PC were likely to interact, directly or indirectly, with Na+-binding sites of Na,K-ATPase located at the intracellular face of plasma membranes, a conclusion also supported by studies on Na,K-ATPase and 22Na uptake using the inside-out vesicles of human erythrocyte membranes. The studies also showed that ouabain (but not sphingosine and lyso-PC) increased the affinity constant (K0.5) for K+, whereas sphingosine and lyso-PC (but not ouabain) increased K0.5 for Na+. Sphingosine and lyso-PC inhibited 86Rb uptake by intact human leukemia HL-60 cells at potencies comparable to those for inhibitions of purified Na,K-ATPase and protein kinase C. It is suggested that Na,K-ATPase (sodium pump) might represent an additional target system, besides protein kinase C, for sphingosine and possibly other lipid second messengers.     

Ouabain resistance of a human trophoblast cell line is not related to its reactivity to ouabain

Ouabain is a specific inhibitor of sodium, potassium-dependent adenosine triphosphatase (Na,K-ATPase), a P-type ion-transporting ATPase which is essential for the maintenance of adequate concentrations of intracellular Na+ and K+ ions. The present study describes the establishment of a ouabain-resistant mutant, TLouaR, from a human trophoblast cell line TL. Morphologically TL and TLouaR are indistinguishable, but, TLouaR is about 1000 times more resistant to the cytotoxic effect of ouabain and > 2000 times to that of bufalin and yet ouabain can retard the growth of the TLouaR cells and in parallel reduce its cloning efficiency in a time- and dose-dependent manner. Furthermore, Na,K-ATPase activity from TLouaR cells is inhibitable by ouabain albeit with lower efficiency. [3H]ouabain binding studies reveal that TLouaR cells have less (P < 0.05) ouabain binding sites (1.7 +/- 0.15 x 10(4)/cell vs. 2.3 +/- 0.115 x 10(4)/cell in the control). However, affinities (dissociation constants Kd) to ouabain for TL and TLouaR cells are not significantly different. Lastly, Na,K-ATPase activity (1.375 +/- 0.25 micromole ATP/min mg protein) of TLouaR cells is significantly higher (P < 0.05) than that of the TL cells (0.895 +/- 0.12 micromole ATP/min x mg protein). These studies show that the interactions between ouabain and Na,K-ATPase can be mediated through different pathways resulting in diverse phenotypic characteristics. In addition, ouabain resistance does not necessarily reflect the lack of response to the digitalis drug. The exact mechanisms of ouabain resistance observed in the present study remain to be determined but the TLouaR cells may be the best tool to uncover the many functional characteristics of Na,K-ATPase.     

The steroid-binding subunit of the Na/K-ATPase as a progesterone receptor on the amphibian oocyte plasma membrane

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.     

低浓度哇巴因对人白血病细胞株Jurkat 的生长调控作用及其分子 机制初步研究

目的:观察低浓度哇巴因对人白血病细胞株Jurkat生长的影响并初步探讨哇巴因特异性调节Jurkat细胞生长的机制。方法:分别用不同低浓度哇巴因作用于人白血病细胞株Jurkat后,采用四甲基偶氮唑盐MTT法检测细胞增殖情况、流式细胞学FCM技术检测细胞凋亡情况以及细胞内线粒体膜电位变化情况,Western blot法观察哇巴因对Jutkat细胞膜表面钠钾ATP酶的表达调节作用。结果:MTT及FCM检测结果表明随着哇巴因浓度的增高,哇巴因对Jurkat细胞的增殖抑制及促凋亡作用越明显。WesternBlot结果显示30nM及50nM哇巴因作用于Jurkat细胞株48h后引起细胞钠钾ATP酶表达下调,[3H]-哇巴因结合实验结果显示在Jurkat细胞株随着哇巴因作用浓度升高,细胞膜钠泵对哇巴因的亲和力逐渐下降。结论:低浓度哇巴因即可抑制白血病细胞株Jurkat增殖并诱导其凋亡。这种特异性细胞生长调控作用与哇巴因引起的细胞膜钠钾ATP酶表达变化相关,最终引起细胞内线粒体膜电位发生变化,释放相关凋亡蛋白,诱导细胞凋亡。     

Ankyrin B modulates the function of Na,K-ATPase/inositol 1,4,5-trisphosphate receptor signaling microdomain

Na,K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappaB activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na,K-ATPase/IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na,K-ATPase and IP3R. We identify the N terminus tail of the Na,K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na,K-ATPase and IP3R, reduced the number of cells responding to pm doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappaB. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,K-ATPase in the plasma membrane.     

Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na⁺ and K⁺ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic α-subunit (four isoforms) and an ancillary β-subunit (three isoforms). Mutations in the α₂-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase α₂-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [³H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. ⁸⁶Rb⁺-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase α₂-isoform.     

Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition

Administration of high-dose ethinylestradiol to rats decreases bile flow, Na,K-ATPase specific activity, and liver plasma membrane fluidity. By use of highly purified sinusoidal and bile canalicular membrane fractions, the effect of ethinylestradiol administration on the protein and lipid composition and fluidity of plasma membrane fractions was examined. In sinusoidal fractions, ethinylestradiol (EE) administration decreased Na,K-ATPase activity (32%) and increased activities of alkaline phosphatase (254%), Mg2+-ATPase (155%), and a 160-kDa polypeptide (10-fold). Steady-state and dynamic fluorescence polarization was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH) was significantly higher in canalicular compared to sinusoidal membrane fractions. Ethinylestradiol (5 mg/kg per day for 5 days) selectively increased sinusoidal polarization values. Similar changes were demonstrated with the probes 2- and 12-anthroyloxystearate. Time-resolved fluorescence polarization measurements indicated that EE administration for 5 days did not change DPH lifetime but increased the order component (r infinity) and decreased the rotation rate (R). However, 1 and 3 days after EE administration and with low doses (10-100 micrograms/kg per day for 5 days) the Na,K-ATPase, bile flow, and order component were altered, but the rotation rate was unchanged. Vesicles prepared from total sinusoidal membrane lipids of EE-treated rats, as well as phospholipid vesicles, demonstrated increased DPH polarization, as did intact plasma membrane fractions. Liver plasma membrane fractions showed no change in free cholesterol or cholesterol/phospholipid molar ratio, while esterified cholesterol content was increased with high-dose but not low-dose ethinylestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Na,K-ATPase and sodium pump by anticancer ether lipids and protein kinase C inhibitors ET-18-OCH3 and BM 41.440

The anticancer ether lipid analogs ET-18-OCH3 and BM 41.440 inhibited Na, K-ATPase in the purified rat brain membrane fragments, with a potency comparable to that of their inhibition of protein kinase C. They also inhibited Na,K-ATPase in the crude membrane fraction of HL60 cells. Kinetic analysis indicated that the lipids had a mode of action different from that of ouabain, a classic inhibitor of the ATPase. The lipids also blocked 22Na uptake in the inside-out membrane vesicles of human erythrocytes. It is suggested that Na,K-ATPase might represent an additional site with which certain protein kinase C inhibitors can interact to alter cellular activities.     

Functional interaction between nicotinic cholinergic receptors and Na, K-ATPase in the skeletal muscles

Acetylcholine (ACh) hyperpolarized the rat diaphragm muscle fibers by 4.5 +/- 0.8 mV (K0.5 = = 36 +/- 6 nmol/l). The AC-induced hyperpolarization was blocked by d-tubocurarine and ouabain in nanomolar concentrations. This effect of ACh was not observed in cultured C2C12 muscle cells and in Xenopus oocytes with expressed embryonic mouse muscle nicotinic acetylcholine receptors (nAChR) or with neuronal alpha 4 beta 2 nAChR. In membrane preparations from the Torpedo californica electric organ, containing both nAChR and Na, K-ATPase, 10 nmol/l ouabain modulated the binding kinetics of the cholinergic ligand dansyl-C6-choline to the nAChR. These results suggest that in-sensitive alpha 2 isoform) and nAChR in a state with high affinity to Ach and d-tubocurarine may form a functional complex in which binding of ACh to nAchR is coupled to activation of the Na, K-ATPase.     

Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes

Summary Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant,K _m (1.5×10^–5 m), similar to the inhibitory constantK _I (3×10^–5 m), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabain binding at 22°C was 1.3×10³ m ^–1 sec^–1 and is similar to the association rate constants reported for other tissues and species. The high dissociation rate constant, 3.6×10^–2 sec^–1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represent a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.This paper is dedicated to the memory of Professor David H. Smyth, FRS, who died on September 10, 1979.     

Fatty Acid-Induced Modulation of Ouabain Responsiveness of Rat Na,K-ATPase Isoforms

Membrane phospholipids represent a potential influence on the enzymatic properties of the Na,K-ATPase. Little is known concerning the effects of the fatty acid environment surrounding the enzyme on the kinetic properties of the Na,K-ATPase. We used the most obvious difference among the α isoforms of rat, their affinities for digitalis glycosides, to examine the relationship between the lipid environment and the Na,K-ATPase. Specific membrane environments that differ in their fatty acid composition were produced by drug-induced diabetes, as well as variations in diet. The α1 isoforms in various tissues were then characterized by their resistance to ouabain in Na,K-ATPase-enriched membrane microsomal fractions. The Na,K-ATPase activity in nerves and hearts were altered by diabetes and partially restored in nerves after a fish oil diet. Evaluation of enzyme kinetics (dose-response curves for ouabain) in membrane preparations allowed us to correlate the ouabain affinity of α1 isoform with fatty acid composition. The affinity of the α1 isoform for ouabain was significantly increased with accretions in the total amount of fatty acids of the n-6 series (P < 0.0001). Our observations provide a partial explanation for the observed difference in isoform properties among tissues. Moreover, these results underline the interaction between membrane fatty acids and the glycoside binding site of the Na,K-ATPase α1 subunit. Received: 15 June 1998/Revised: 18 November 1998     

Omegacoeur, a Mediterranean nutritional complement, stimulates Na,K-ATPase activity in human endothelial cells.

Fatty acids are known as modulators of the vasoactive properties of the vessel wall and can influence the physical and functional properties of cell membrane. The membrane-bound enzyme Na,K-ATPase plays a central role in endothelial function such as vasoconstriction. In a previous study, we have shown that omega3 fatty acids inhibited Na,K-ATPase activity in human endothelial cells. As Mediterranean diet is known to protect from cardiovascular diseases, we have investigated the effects of Omegacoeur, a Mediterranean nutritional complement consisting of omega3, omega6, omega9 fatty acids, garlic and basil, on Na,K-ATPase activity in human endothelial cells (HUVECs). Cells were incubated for 18 hr with pure lecithin liposomes or Omegacoeur-enriched emulsions (4 mg lecithin/ml). Na,K-ATPase and 5'-nucleotidase activities were determined using coupled assay methods on microsomal fractions obtained from HUVECs. Cell fatty acid composition was evaluated by gas chromatography after extraction of lipids and fatty acids methylation. The results showed that Omegacoeur (0.1 mM) increased Na,K-ATPase activity by 40% without changes in 5'-nucleotidase activity. Cells incubated with Omegacoeur preferentially incorporated linoleic acid. Therefore, linoleic acid or others constituents of Omegacoeur could be responsible of the stimulation of the Na,K-ATPase activity that might be related to changes in endothelial membrane fluidity.     

Effects of detergents on Na+ + K+-dependent ATPase activity in plasma-membrane fractions prepared from frog muscles. Studies of insulin action on Na+ and K+ transport.

The increase in Na+/K+ transport activity in skeletal muscles exposed to insulin was analysed. Plasma-membrane fractions were prepared from frog (Rana catesbeiana) skeletal muscles, and examination of the Na,K-ATPase (Na+ + K+-dependent ATPase) activity showed that it was insensitive to ouabain. In contrast, plasma-membrane fractions prepared from ouabain-pretreated muscles, by the same procedures, showed extremely low Na,K-ATPase activity. On adding saponin to the membrane suspension, the Na,K-ATPase activity increased, according to the detergent concentration. The maximum activity was about twice the control value, at 0.33 mg of saponin/mg of protein. Thus saponin makes vesicle membranes leaky, allowing ouabain in assay solutions to reach receptors on the inner surface of vesicles. Addition of insulin to saponin-treated membrane suspensions had no effect on the Na,K-ATPase activity, whereas the maximum activity of Na,K-ATPase in whole muscles was stimulated by exposure to insulin. The results show that the stimulation of Na+/K+ transport by insulin is not directly due to insulin binding to receptors on the cell surface, but rather support the view that the increase in the Na,K-ATPase induced by insulin requires an alteration of intracellular events.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

Effects of ouabain on the rotational dynamics of renal Na,K-ATPase studied by saturation-transfer EPR.

The interaction of a nitroxide spin-labeled derivative of ouabain with sheep kidney Na,K-ATPase and the motional behavior of the ouabain spin label-Na,K-ATPase complex have been studied by means of electron paramagnetic resonance (EPR) and saturation-transfer EPR (ST-EPR). Spin-labeled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per mole of ATP-dependent phosphorylation sites, even after repeated centrifugation and resuspension of the purified ATPase-containing membrane fragments. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance at 0 degrees C, characteristic of a tightly bound spin label which is strongly immobilized by the protein backbone. Saturation-transfer EPR measurements of the spin-labeled ATPase preparations yield effective correlation times for the bound labels significantly longer than 100 microseconds at 0 degrees C. Since the conventional EPR measurements of the ouabain spin-labeled Na,K-ATPase indicated the label was strongly immobilized, these rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements of ouabain spin-labeled Na,K-ATPase (a) cross-linked with glutaraldehyde and (b) crystallized in two-dimensional arrays indicated that the observed rotational correlation times predominantly represented the motion of large Na,K-ATPase-containing membrane fragments, as opposed to the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The results suggest that the binding of spin-labeled ouabain to the ATPase induces the protein to form large aggregates, implying that cardiac glycoside induced enzyme aggregation may play a role in the mechanism of action of the cardiac glycosides in inhibiting the Na,K-ATPase.