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R Blostein

Publications and source records attributed to R Blostein.

At least 19 recordsLinked to original sources
PubMed1992

Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K(+)-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle.

Unlike glucose transport, where translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane is the principal mechanism underlying insulin stimulation, no consensus exists presently for the mechanism by which insulin activates the Na+/K(+)-ATPase. We have investigated (i) the subunit isoforms expressed and (ii) the effect of insulin on the subcellular distribution of the alpha beta isoforms of the Na+/K(+)-ATPase in plasma membranes (PM) and internal membranes (IM) from rat skeletal muscle. Western blot analysis, using isoform-specific antibodies to the various subunits of the Na+/K(+)-ATPase, revealed that skeletal muscle PM contains the alpha 1 and alpha 2 catalytic subunits and the beta 1 and beta 2 subunits of the Na+ pump. Skeletal muscle IM were enriched in alpha 2, beta 1, and beta 2; alpha 1 was barely detectable in this fraction. After insulin treatment, alpha 2 content in the PM increased, with a parallel decrease in its abundance in the IM pool; insulin did not have any effect on alpha 1 isoform amount or subcellular distribution. The beta 1 subunit, but not beta 2, was also elevated in the PM after insulin treatment, but this increase originated from a sucrose gradient fraction different from that of the alpha 2 subunit. Our findings suggest that insulin induces an isoform-specific translocation of Na+ pump subunits from different intracellular sources to the PM and that the hormone-responsive enzyme in rat skeletal muscle is an alpha 2:beta 1 dimer.

Animals↗
PubMed1992

Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells.

Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.

Animals↗
PubMed1992

Rat kidney Na-K pumps incorporated into low-K+ sheep red blood cell membranes are stimulated by anti-Lp antibody.

A genetic dimorphism of sheep red blood cells characterized by differences in the intracellular K+ concentration of mature red blood cells (low-K+ or high-K+ cells) reflects differences in their Na-K pumps and is known to be linked to the ML blood group system. We investigated the relationship of Na-K pumps in red blood cells from sheep of the low-K+ phenotype with an antigen, Lp, that is restricted to low-K+ cells. Anti-Lp antibody stimulates the Na-K pumps in these cells presumably by relieving inhibition of the pumps by Lp. The questions addressed were as follows: is Lp a molecular entity distinct from pumps and, if so, can it interact with pumps of exogenous origin? Rat kidney Na-K pumps were incorporated by fusion of microsomes into either low-K+ or high-K+ sheep red blood cells. The activity of the exogenous kidney pumps was distinguished from that of the endogenous red blood cell pumps by the low sensitivity of rodent pumps to ouabain. Anti-Lp stimulated by > 50% rat kidney pumps incorporated into immature low-K+ sheep cells. This indicates that Lp is a distinct molecular entity free to dissociate from endogenous pumps and inhibit exogenous pumps. Anti-Lp did not stimulate kidney pumps incorporated into mature low-K+ cells but did stimulate kidney pumps following in vitro maturation of microsome fused reticulocytes, probably reflecting restriction of lateral movement of pumps and antigens by the cytoskeleton in mature cells.

Animals↗
PubMed1992

Altered stoichiometry of the Na,K-ATPase.

With inside-out membrane vesicles derived from human red cells and incubated at pH > or = 6.6, an altered sodium pump stoichiometry (1Na+:2K+) associated with altered charge transfer is observed when the cytoplasmic Na+ concentration is reduced to very low levels (0.2 mM). With increased proton concentration (approximately pH 6.0), protons can substitute for Na+ or K+ ions such that the Na,K-ATPase can effect either electroneutral Na+/H+ exchange (K+ absent), H+/K+ exchange (Na+ absent), or H(+)-plus-Na+ cotransport in exchange for K+ (low Na+ concentration). Evidence that the stoichiometries of these exchanges are 3Na+/3H+, 2H+/2K+ and 1H(+)-plus-1Na+/2K+, respectively, is presented.

Erythrocytes↗
PubMed1990

Na,K-ATPase isoform expression in sheep red blood cell precursors.

Isoform expression of mammalian red cell Na,K-ATPase was analyzed using messenger RNA isolated from red cell precursor-enriched bone marrow of anemic sheep. Expression of the catalytic alpha subunit was analyzed using rat isoform-specific cDNA probes and expression of the beta 1 subunit, using a sheep beta 1-specific cDNA probe. RNA isolated from sheep kidney and brain were analyzed concurrently. In the red cell, as in the kidney, messenger RNA encoding only one isoform (alpha 1) of the catalytic subunit is detected; neither of the other isoforms (alpha 2 or alpha 3) could be detected. This holds true for bone marrow of sheep of either the low potassium or high potassium phenotype. Relative to the expression of alpha 1, beta subunit-specific message (beta 1) was extremely low in the red cell compared to either kidney (less than 5%) or brain (less than 3%). Using a rat cDNA probe specific for a beta 1-like subunit, beta 2, message was detected in brain but not in either kidney or bone marrow.

Animals↗
PubMed1990

Decrease in Na(+)-K(+)-ATPase associated with maturation of sheep reticulocytes.

Na(+)-K(+)-ATPase of immature and mature sheep red blood cells of both the high-K+ and low-K+ genotype and of immature cells matured in vitro was detected using polyclonal antiserum to purified sheep kidney Na(+)-K(+)-ATPase. This antiserum detects both alpha (alpha 1)- and alpha + (alpha 2 and/or alpha 3)-isoforms of the catalytic subunit as well as the beta-subunit of brain and kidney Na(+)-K(+)-ATPase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, a single major immunologically reactive component corresponding to the alpha-subunit was detected in membranes of immature and mature cells from sheep of both genotypes. Cells separated according to increasing density showed a corresponding decrease in ouabain binding sites on the cells and Na(+)-activated ATP hydrolysis of membranes isolated from the cells. A progressive decrease in immunologically reactive alpha-subunit was also observed. After in vitro culture of reticulocytes, reduction in ouabain binding to the cells was also associated with loss in alpha-subunit. As well, appearance of immunologically reactive alpha-subunit was detected in membranous material shed into the incubation medium, accounting for a fraction (less than or equal to 30%) of the material lost from the cells. Proteolytic sensitivity of the alpha-subunit indicates that, in this material, the cytoplasmic surface of the enzyme is exposed to the medium. The shed material was largely devoid of function as evidenced in little, if any, Na(+)-dependent phosphorylation of Na(+)-K(+)-ATPase. The existence in reticulocytes of an intracellular pool of ouabain binding sites was indicated by the transient appearance on the cell surface of ouabain binding sites after rapid ATP depletion and also after addition of chloroquine to cells during culture. Taken together, these findings indicate that the maturation-associated loss of sodium pump protein involves, at least partly, energy-dependent endocytosis and, presumably, processing whereby inactivation of function occurs as well as release of pump protein into the extracellular milieu.

Anemia↗
PubMed1989

Sodium ions as substitutes for protons in the gastric H,K-ATPase.

In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes.

Adenosine Triphosphatases↗
PubMed1989

Effects of cytoplasmic sodium concentration on the electrogenicity of the sodium pump.

Inside-out membrane vesicles were prepared from human red blood cells pretreated with diisothiocyano-2,2'-disulfonic stilbene to inhibit anion fluxes. The pH-sensitive probe fluorescein isothiocyanate-dextran was incorporated inside the vesicles. Formation of pH gradients due to proton transport by the sodium pump was distinguished from pH gradients formed in response to transmembrane electrical potentials generated by the pump by virtue of their insensitivity and sensitivity, respectively, to dissipation by lipophilic cations. Under the conditions used (pH 6.6), proton transport by the Na,K-ATPase was minimized, and the formation of pH gradients in response to electrical potentials was detected. Thus, the generation of a strophanthidin-sensitive, ATP-dependent electrical potential, inside positive (approximately 1 mV) upon addition of 4 meq of sodium to potassium-filled inside-out vesicles is consistent with the well documented stoichiometry of three sodium ions exchanging with two potassium ions. In contrast, when the cytoplasmic sodium concentration is reduced to less than or equal to 0.4 mM, the potential generated is of the opposite sign, i.e. inside negative, consistent with the decreased Na:K coupling ratio reported previously, i.e. Na:K(Rb) coupling ratios of approximating 1:2 when the sodium concentration is reduced to 0.2 mM (Blostein, R. (1983) J. Biol. Chem. 258, 12228-12232).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Ion pumps.

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Adenosine Triphosphatases↗
PubMed1988

Protons as substitutes for sodium and potassium in the sodium pump reaction.

The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Human placental Na+,K+-ATPase alpha subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization.

A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na+,K+-ATPase alpha subunit was cloned from human placenta and its sequence was identical to that encoding the alpha subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na+,K+-ATPase gene from various human tissues and cell lines revealed only one band (approximately 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine greater than placenta greater than liver greater than pancreas, and in the cell lines the levels were human erythroleukemia greater than butyrate-induced colon greater than colon greater than brain greater than HeLa cells. mRNA was undetectable in reticulocytes, consistent with our failure to detect positive clones in a size-selected (greater than 2 kilobases) lambda gt11 reticulocyte cDNA library. DNA analysis revealed a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the alpha subunit is on the short arm (band p11-p13) of chromosome 1.

Amino Acid Sequence↗
PubMed1987

Characteristics of membrane transport losses during reticulocyte maturation.

The decline in activity of distinct membrane transport systems was followed during in vitro maturation of sheep reticulocytes, namely the sodium pump (measured as specific ouabain binding sites), Na+-glycine cotransport, and the nucleoside transporter (measured as specific nitrobenzylthioinosine binding sites). Certain features of this maturation-associated decline in membrane transport are clarified. Thus, the apparent retardation of loss by metabolic (ATP) depletion, reported previously for the sodium pump and Na+-glycine cotransport, is applicable also to the decline in nucleoside transport. The absolute losses, as well as relative effects of ATP depletion, are different for the three distinct systems. Inhibitors of membrane recycling and (or) intracellular processing, such as chloroquine, as well as ATP depletion, prevent not only the loss but also cause a transient increase in nucleoside transport sites apparent at the surface. Proteolytic processing, at least in the case of the nucleoside transporter, is probably also involved since leupeptin retards the loss in binding sites. Protection against the decline in transporters can also be affected by specific ligands as evidenced in ouabain protection of sodium pump sites. The results provide evidence that membrane transporter recycling is a fundamental process underlying the energy-dependent, maturation-associated loss in membrane transport functions.

Animals↗
PubMed1987

Factors affecting transport changes associated with reticulocyte maturation.

Maturation and aging of the mammalian red blood cell is characterized by the loss in many cytoplasmic and membrane functions including ion and solute transport. We have used long-term in vitro incubation of sheep reticulocytes to examine the nature of several membrane transport changes. Three distinct transport systems have been studied, namely the Na,K-pump measured as specific [3H]ouabain binding sites, Na+/glycine cotransport and the nucleoside transporter measured as specific [3H]nitrobenzylthioinosine binding. As reported previously for the Na,K-pump and Na+/glycine cotransport (Weigensberg, A.M. and Blostein, R., Proc. Nat. Acad. Sci. 80:4978-4982, 1983), the losses of all three functions are diminished markedly by energy (ATP) depletion. Furthermore, for at least one of the transport systems, the Na,K-pump, the maturation-associated loss is modulated by ligand-induced perturbations which are known to effect specific protein conformational changes.

Adenosine Triphosphate↗