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Identification and characterization of a newly recognized population of high-Na⁺, low-K⁺ low-density sickle and normal red cells

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第14期97卷 8045-8050页

作者： Bookchin, RM Etzion, Z Sorette, M Mohandas, N Skepper, JN Lew, VL Yeshiva Univ Albert Einstein Coll Med Dept Med Bronx NY 10461 USA Univ Calif Berkeley Lawrence Berkeley Lab Berkeley CA 94720 USA Univ Cambridge Sch Biol Sci MultiImaging Ctr Cambridge CB2 3DY England Univ Cambridge Physiol Lab Cambridge CB2 3EG England

We describe a population of sickle cell anemia red cells (SS RBCs) (approximate to 4%) and a smaller fraction of normal RBCs (<0.03%) that fail to dehydrate when permeabilized to K+ with either valinomycin or elevated internal Ca2+. The nonshrinking, valinomycin-resistant (val-res) fractions, first detected by flow cytometry of density-fractionated SS RBCs, constituted up to 60% of the lightest, reticulocyte-rich (R1) cell fraction, and progressively smaller portions of the slightly denser R2 cells and discocytes. R1 val-res RBCs had a mean cell hemoglobin concentration of approximate to 21 g of Hb per dl, and many had an elongated shape like "irreversibly sickled cells," suggesting a dense SS cell origin. Of three possible explanations for val-res cells, failure of valinomycin to K+-permeabilize the cells, low co-ion permeability, or reduced driving K+ gradient, the latter proved responsible: Both SS and normal val-res RBCs were consistently high-Na+ and low-K+, even when processed entirely in Na-free media. Ca2+ + A23187-induced K+-permeabilization of SS R1 fractions revealed a similar fraction of cal-res cells, whose Rb-86 uptake showed both high Na/K pump and leak fluxes, val-res/calres RBCs might represent either a distinct erythroid genealogy, or an "end-stage" of normal and SS RBCs, This paper focuses on the discovery, basic characterization, and exclusion of artifactual origin of this RBC fraction. Many future studies will be needed to clarify their mechanism of generation and full pathophysiological significance.

关键词：贫血镰状细胞性/代谢布美他尼/药理学卡西霉素/药理学钙/药理学细胞膜通透性抗药性电子探针微区分析红细胞/药物作用红细胞/代谢离子载体/药理学哇巴因/药理学钾/代谢钾通道/代谢铷/代谢钠/代谢分光光度法原子缬氨霉素/药理学

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ACTIVATION OF 2,4-DINITROPHENOL-STIMULATED ATPASE ACTIVITY IN CAULIFLOWER AND CORN MITOCHONDRIA

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 1975年第2期168卷 358-368页

作者： JUNG, DW HANSON, JB UNIV ILLINOIS DEPT BOTURBANAIL 61801

Cauliflower mitochondria do not have a 2,4-dinitrophenol-stimulated ATPase unless they are permitted a brief period of respiration (respiratory priming) or are preincubated for an extensive period with ATP (self-priming). Both priming processes are dependent on Mg2+ , and are collapsed by 2,4-dinitrophenol in the absence of ATP. Corn mitochondria, which have an endogenous DNP-ATPase, contain significantly more Mg2+ and adenine nucleotides than cauliflower mitochondria. Primed cauliflower mitochondria have Mg2+ content comparable to corn mitochondria. Cauliflower mitochondria will actively accumulate adenine nucleotides through atractyloside-insensitive sites. It appears that priming consists of creating an electrochemical potential which is needed for accumulation of Mg2+ or adenine nucleotides or for charge compensation of the ATP 4− ADP 3-− exchange.

关键词：腺嘌呤核苷酸类/代谢腺苷三磷酸酶类/代谢腺苷三磷酸/药理学生物转运二硝基酚类/药理学酶激活镁/药理学线粒体/药物作用线粒体/酶学线粒体/代谢氧耗量磷酸盐类/药理学植物细胞植物/酶学氚缬氨霉素/药理学玉米

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STOICHIOMETRIC RELATIONSHIP BETWEEN ENERGY-DEPENDENT PROTON EJECTION AND ELECTRON-TRANSPORT IN MITOCHONDRIA

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1976年第2期73卷 437-441页

作者： BRAND, MD REYNAFARJE, B LEHNINGER, AL JOHNS HOPKINS UNIV SCH MEDDEPT PHYSIOL CHEM725 N WOLFE STBALTIMOREMD 21205

The number of protons ejected during electron transport per pair of electrons per energy-conserving site (the H+/site ratio) was measured in rat liver mitochondria by three different methods under conditions in which transmembrane movements of endogenous phosphate were minized or eliminated. (1) In the Ca2+ pulse method, between 3.5 and 4.0 molecules of 3-hydroxybutyrate and 1.75 to 2.0 Ca2+ ions were accumulated per 2 e- per site during Ca2+ induced electron transport in the presence of rotenone, when measured under conditions in which movements of endogenous phosphate were negligible. Since entry of 3-hydroxybutyrate requires its protonation to the free acid these data correspond to an H+/site ratio of 3.5-4.0 (2) In the oxygen pulse method addition of known amounts of oxygen to anaerobic mitochondria in the presence of substrate yielded H+/site ratios of 3.0 when phosphate transport was eliminated by addition of N-ethylmaleimide or by anaerobic washing to remove endogenous phosphate. In the absence of such measures the observed H+/site ratio was 2.0. (3) In the reductant pulse method measurement of the initial steady rates of H+ ejection and oxygen consumption by mitochondria in an aerobic medium after addition of substrate gave H+/site near 4.0 in the presence of N-ethylmaleimide; in the absence of the inhibitor the observed ratio was only 2.0. These and other experiments reported indicate that the values of 2.0 earlier obtained for the H+/site ratio by Mitchell and Moyle [Biochem J. (1967) 105, 1147-1162] and others were underestimates due to the unrecognized masking of H+ ejection by movements of endogenous phosphate. The results presented here show that the H+/site ratio of mitochondrial electron transport is at least 3.0 and may be as high as 4.0.

关键词：钙/代谢电子转运/药物作用乙基马来酰亚胺/药理学线粒体肝/代谢氧耗量/药物作用磷酸盐类/代谢质子鱼藤酮/药理学缬氨霉素/药理学动物大鼠

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ATP-sensitive K⁺ channel openers prevent Ca²⁺ overload in rat cardiac mitochondria

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JOURNAL OF PHYSIOLOGY-LONDON 1999年第2期519卷 347-360页

作者： Holmuhamedov, EL Wang, LW Terzic, A Mayo Clin & Mayo Fdn Dept Med & Pharmacol Div Cardiovasc Dis Rochester MN 55905 USA

1. Mitochondrial dysfunction, secondary to excessive accumulation of Ca2+, has been implicated in cardiac injury. We here examined the action of potassium channel openers on mitochondrial Ca2+ homeostasis, as these cardioprotective ion channel modulators have recently been shown to target a mitochondrial ATP-sensitive K+ channel. 2. In isolated cardiac mitochondria, diazoxide and pinacidil decreased the rate and magnitude of Ca2+ uptake into the mitochondrial matrix with an IC50 of 65 and 128 mu M, respectively. At all stages of Ca2+ uptake, the potassium channel openers depolarized the mitochondrial membrane thereby reducing Ca2+ influx through the potential-dependent mitachondrial uniporter. 3. Diazoxide and pinacidil, in a concentration-dependent manner, also activated release of Ca2+ from mitochondria. This was prevented by cyclosporin A, an inhibitor of Ca2+ release through the mitochondrial permeability transition pore. 4. Replacement of extramitochondrial K+ with mannitol abolished the effects of diazoxide and pinacidil on mitochondrial Ca2+, while the K+ ionophore valinomycin mimicked the effects of the potassium channel openers. 5. ATP and ADP, which block K+ flux through mitochondrial ATP-sensitive K+ channels, inhibited the effects of potassium channel openers, without presenting the action of valinomycin. 6. In intact cardiomyocytes, diazoxide also induced mitochondrial depolarization and decreased mitochondrial Ca2+ content. These effects mere inhibited by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. 7. Thus, potassium channel openers prevent mitochondrial Ca2+ overload by reducing the driving force for Ca2+ uptake and by activating cyclosporin-sensitive Ca2+ release. In this regard, modulators of an ATP-sensitive mitochondrial K+ conductance may contribute to the maintenance of mitochondrial Ca2+ homeostasis.

关键词：腺苷二磷酸/药理学腺苷三磷酸/药理学钙/代谢环孢菌素/拮抗剂和抑制剂环孢菌素/药理学二氮嗪/拮抗剂和抑制剂二氮嗪/药理学离子载体/药理学膜电位/药物作用膜电位/生理学膜蛋白质类/激动剂膜蛋白质类/代谢显微镜检查共焦线粒体心脏/药物作用线粒体心脏/代谢心肌/细胞学心肌/代谢氧耗量/药物作用通透性吡那地尔/拮抗剂和抑制剂吡那地尔/药理学钾通道大鼠 Sprague-Dawley 缬氨霉素/药理学动物大鼠

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INHIBITION OF DENITRIFICATION BY UNCOUPLERS OF OXIDATIVE-PHOSPHORYLATION

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BIOCHEMISTRY 1978年第15期17卷 3039-3045页

作者： WALTER, B SIDRANSKY, E KRISTJANSSON, JK HOLLOCHER, TC BRANDEIS UNIV DEPT BIOCHEM WALTHAM MA 02154 USA

In the denitrifiers, Pseudomonas denitrificans and P. aeruginosa, nitrogen oxide respirations were reversibly inhibited by several weak acid-type uncouplers of oxidative phosphorylation. Their effectiveness as uncouplers stood in about the same rank as their effectiveness as inhibitors. Uncouplers increased the rate of O2 respiration (release of respiratory control) in these 2 organisms and seemed not to inhibit even at rather high concentrations. The mechanism of inhibition of nitrogen oxide respiration by uncouplers was explored and several lines of evidence ruled out an inhibition which depended on collapse of the proton motive force per se. The concentrations of uncouplers required to inhibit nitrogen oxide respirations in P. denitrificans were at least 10 times higher than those required to inhibit respiration-driven amino acid uptake, to release respiratory control in the case of O2 respiration and to increase membrane proton conductivity to such levels that endergonic reactions dependent on proton motive force must, by calculation, be inhibited. Neither valinomycin/K+ (to collapse membrane potential) nor nigericin (to collapse .***) had an inhibitory effect on nitrogen oxide respirations, whether present separately or together. Nonuncoupling analogs of the nitrophenol and carbonyl cyanide phenylhydrazone uncouplers were effective in inhibiting respiration. The inhibitions are attributed in large part to the hydrophobic properties of these compounds. Spectrophotometric observations suggested that the site of inhibition lay on the reducing side of cytochromes b and c. In the steady states of nitrogen oxide respirations, cytochromes b and c remained nearly completely oxidized in the presence of u;ncouplers, and the transition from steady state to reduced state upon the exhaustion of nitrogen oxide was inhibited. Certain trivial explanations for the inhibitory effect of uncouplers, such as substrate starvation, were ruled out.

关键词：碳酰氰化物对三氟甲氧苯基腙/药理学二环己基碳二亚胺/药理学二硝基酚类/药理学动力学尼日利亚菌素/药理学氧化氮类/代谢氧耗量/药物作用假单胞菌属/药物作用假单胞菌属/代谢铜绿假单胞菌/药物作用铜绿假单胞菌/代谢解偶联药/药理学缬氨霉素/药理学

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PURIFICATION OF ACETYLCHOLINE-RECEPTOR FROM TORPEDO-CALIFORNICA AND ITS INCORPORATION INTO PHOSPHOLIPID VESICLES

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ANNALS OF THE NEW YORK ACADEMY OF SCIENCES 1975年第DEC30期264卷 175-182页

作者： MCNAMEE, MG WEILL, CL KARLIN, A COLUMBIA UNIV COLL PHYS & SURG DEPT NEUROL NEW YORK NY 10032 USA

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MODULATION OF LYMPHOCYTE RECEPTOR MOBILITY BY LOCALLY BOUND CONCANAVALIN-A

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1975年第4期72卷 1579-1583页

作者： YAHARA, I EDELMAN, GM ROCKEFELLER UNIV NEW YORKNY 10021

Binding of concanavalin A (con A) to the lymphocyte surface at room temperature leads to restriction of the mobility of a variety of cell surface receptors including those from immunoglobulin (Ig), O, H-2, beta2-microglobulin, Fc receptors results in "co-capping" of Ig, H-2, theta, Fc receptors, as well as receptors for other lectins. Addition of colchicine to the cell suspensions reverses this effect and allows Con A receptors as well as other receptors to form patches and caps. Capping of Con A receptors, and beta2-microglobulin in the absence of ligands specific for these receptors. Receptors binding Limulus hemagglutinin and wax bean agglutinin, as well as those interacting with carbohydrate-specific antibodies, were partially co-capped with Con A but receptors for wheat germ agglutinin were not co-capped, excluding the possibility that restriction of receptor mobility by Con A resulted simply from cross-linkage of mobile receptors to immobilized glycoproteins (Con A receptors). Latex beads and platelets coupled to Con A were bound to lymphocytes under the same conditions as free Con A. Binding of these particles to local regions of the cell surface resulted in restriction of the mobility of those receptors that could be co-capped with free Con A. In contrast to the findings with free Con A, however, addition of colchicine resulted in capping of the bound particles but did not cause co-capping of either the unbound Con A receptors or other receptors. These findings support the hypothesis that modulation occurs via a submembranous assembly containing microtubules, and they further suggest that the transitions of this assembly induced locally by Con A may be propagated via cooperative processes.

关键词：结合部位秋水仙碱/药理学伴刀豆球蛋白A/代谢免疫球蛋白类/代谢动力学胶乳淋巴细胞/药物作用淋巴细胞/代谢微球体蛋白质结合受体药物脾温度缬氨霉素/药理学动物小鼠

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Membrane hyperpolarization removes inactivation of Ca²⁺ channels, leading to Ca²⁺ influx and subsequent initiation of sperm motility in the common carp

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第5期97卷 2052-2057页

作者： Krasznai, Z Márián, T Izumi, H Damjanovich, S Balkay, L Trón, L Morisawa, M Univ Debrecen Med & Hlth Sci Ctr Dept Biophys & Cell Biol H-4012 Debrecen Hungary Univ Debrecen Med & Hlth Sci Ctr Positron Emiss Tomog Ctr H-4012 Debrecen Hungary Univ Tokyo Grad Sch Sci Misaki Marine Biol Stn Kanagawa 23802 Japan

Change of osmolality surrounding spawned sperm from isotonic to hypotonic causes the initiation of sperm motility in the common carp. Here we show that membrane-permeable cAMP does not initiate motility of carp sperm that is quiescent in isotonic solution, and that motility of the demembranated sperm can be reactivated without cAMP. Furthermore, the cAMP level does not change during the initiation of sperm motility, and inhibitors of protein kinase do not affect sperm motility, suggesting that no cAMP-dependent system is necessary for the regulation of sperm motility. Sperm motility could not be initiated in Ca2+-free hypoosmotic solutions, and significant increase in the intracellular Ca2+ level was observed by a Ca-sensitive fluorescence dye during hypoosmolality-induced active motion period. The demembranated sperm cells were fully reactivated in the solutions containing 10(-7) to 10(-5) M Ca2+. Ca2+ channel blockers such as verapamil and omega-conotoxin reversibly inhibited the initiation of sperm motility, suggesting that Ca2+ influx is the prerequisite for the initiation of carp sperm motility. Motility of intact sperm was completely blocked;however, that of the demembranated sperm was not inhibited by the calmodulin inhibitor W7, suggesting that the calmodulin bound close to the plasma membrane participated in the initiation of sperm motility. Flow cytometric membrane potential measurements and spectrophotometric measurements by using fluorescence dyes showed transient membrane hyperpolarization on hypoosmolality-induced motility. This article discusses the role of membrane hyperpolarization on removal of inactivation of Ca2+ channels, leading to Ca2+ influx at the initiation of carp sperm motility.

关键词：布拉地新/代谢布拉地新/药理学卡西霉素/药理学钙/代谢钙通道阻滞药/药理学钙通道/代谢鲤鱼类环AMP依赖性蛋白激酶类/拮抗剂和抑制剂环GMP依赖性蛋白激酶类双丁酰环GMP/代谢双丁酰环GMP/药理学离子载体/药理学蛋白激酶C/拮抗剂和抑制剂蛋白激酶抑制剂精子能动性/药物作用精子能动性/生理学缬氨霉素/药理学动物男(雄)性

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EFFECT OF IONOPHORES A23187 AND NIGERICIN ON LIGHT-INDUCED REDISTRIBUTION OF MG2+,K+ AND H+ ACROSS THYLAKOID MEMBRANE

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BIOCHIMICA ET BIOPHYSICA ACTA 1976年第1期449卷 48-58页

作者： BULYCHEV, AA VREDENBERG, WJ CTR AGROBIOL RES WAGENINGEN NETHERLANDS

Passive redistributions of Mg2+ and K+ ions across the thylakoid membranes, occurring in association with the light-driven electrogenic influx of H ions were examined in suspensions of broken spinach chloroplasts under a variety of conditions. In accord with results of Hind et al, at a low K/Mg concentration ratio in the medium the K-efflux is negligibly small, whereas a substantial Mg-efflux is observed. The converse is true when the K/Mg concentration ratio in the medium is high. In the presence of A23187, which causes approximately a 60% inhibition of the light-induced pH-gradient, a significant influx of Mg2+ was observed in the light at a high K/Mg concentration ratio. Conversely the Mg-influx was small in the presence of A23187 when the K/Mg concentration ratio in the medium was low. Under these conditions, the Mg-influx was considerably increased upon the addition of valinomycin. A23187 was found not to affect the K-efflux in the light. The light-induced K-influx observed in the presence of nigericin also was dependent on the concentration ratio of the monovalent and divalent cation. Its magnitude increased upon an increase in the K/Mg ratio. The results are interpreted in terms of a simplified model in which the total passive efflux of cations, driven the potential set by the electrogenic proton pump, is considered to be a constant fraction of the proton influx. An increase in the flux of an ion species, induced by raising its concentration, or by increasing its permeability through the membrane, cause a decrease in the flux of the other cations. The relevance of the results is discussed with respect to conclusions about the involvement and relative magnitudes of the passive K and Mg effluxes across the thylakoid membrane during energization of intact chloroplasts and chloroplasts in situ.

关键词：抗菌药/药理学卡西霉素/药理学叶绿体/代谢叶绿体/辐射效应依地酸/药理学电子转运氢/代谢氢离子浓度光镁/代谢膜/辐射效应模型生物学尼日利亚菌素/药理学氧耗量钾/代谢缬氨霉素/药理学

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Isoniazid accumulation in Mycobacterium smegmatis is modulated by proton motive force-driven and ATP-dependent extrusion systems

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第3期256卷 682-684页

作者： Choudhuri, BS Sen, S Chakrabarti, P Bose Inst Dept Chem Kolkata 700009 W Bengal India

Resistance to isoniazid (INH), a frontline, antituberculosis drug, presents a major problem in the chemotherapy of tuberculosis. Although several targets of INH have been identified, the mechanism of INH resistance remains incompletely understood. This report demonstrates that INH accumulation in Mycobacterium smegmatis is enhanced both upon addition of both a proton motive force (pmf) uncoupler, carbonyl-cyanide m-chlorophenylhydrazone (CCCP), and upon addition of ortho-vanadate, an inhibitor of ATP-dependent efflux pumps. Both the Delta psi and Delta pH components of the pmf are likely to be involved as judged by the effects of valinomycin and nigericin, respectively, Reserpine, an inhibitor of the human MDRI P-glycoprotein, enhances INH accumulation in a man ner similar too-vanadate. Verapamil, a calcium channel blocker, so enhances INH uptake. Taken together, the results provide evidence of the involvement of both pmf- and ATP-dependent extrusion systems in INH efflux in M. smegmatis, making it important to evaluate the role of such systems in INH resistance ire pathogenic mycobacteria. (C) 1999 Academic Press.

关键词：腺苷三磷酸/代谢抗结核药/代谢抗结核药/药理学生物转运主动/药物作用钙通道阻滞药/药理学碳酰氰化物间-氯苯基腙/药理学地尔硫|艹卓|/药理学抗药性微生物氢离子浓度离子载体/药理学异烟肼/代谢异烟肼/药理学分枝杆菌耻垢/药物作用分枝杆菌耻垢/生长和发育分枝杆菌耻垢/代谢硝苯地平/药理学尼日利亚菌素/药理学质子动力势/药物作用质子动力势/生理学利血平/药理学解偶联药/药理学缬氨霉素/药理学钒酸盐类/药理学维拉帕米/药理学

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