Bluelight-induced,flavin-mediated transport of redox equivalents across artificial bilayer membranes

Summary This paper continues our studies of physico-chemical properties of vesicle-bound flavins. Based on previous results, an advanced model system was designed in order to study the mechanisms underlying bluelight-induced redox transport across artificial membranes. The lumen of single-shelled vesicles was charged with cytochromec, and amphiphilic flavin (AF1 3, AF1 10) was bound to the membrane. Upon bluelight irradiation redox equivalents are translocated from exogeneous 1e ^–(EDTA)-and 2e ^–(BH₃CN^–) donors across the membrane finally reducing the trapped cytochromec both under aerobic and anaerobic conditions. The mechanisms involved are explored and evidence for the involvement of various redox states of oxygen, dihydroflavin and flavosemiquinone is presented.     

Cation stimulation of the proton-translocating redox activity at the plasmalemma of Catharanthus roseus cells

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system which may be associated with proton translocation. Evidence shows that endogenous pyridine nucleotides serve as hydrogen donors for the reaction. The proton translocating function of the redox system is confirmed, in intact cells and isolated protoplasts, by the ability of Ca²⁺ and other cations to increase both the redox activity and the efflux of protons. The role of the cations is seen to be not a simple general charge screening phenomenon as already described. By using ionic surfactants (CP⁺, SDS^–) it was shown that the net surface charge of the membrane can interact in the activation process via a cation attraction effect. It is proposed that specific binding of cations to the plasma membrane could alter the conformation of the redox system facilitating its interaction with NADH.Abbreviations CP⁺ cetylpyridinium - EGTA ethylene glycol bis (-aminoethyl)-N,N-tetraacetic acid - FeCN potassium ferricyanide - SDS^– sodium dodecyl sulfate - SHAM salicylhydroxamic acid     

Calcium ions in the presence of exogenous phosphatidylserine interfere with GABA diffusion through the Deiters' neuron membrane

Diffusion of GABA through the plasma membrane of GABA-acceptive neurons might be a mechanism of importance for the termination of its synaptic action. In the present investigation we studied the effects of phosphatidylserine (PS) (10^–4–10^–3 M), Ca²⁺ 2 mM and PS+2 mM Ca²⁺ on such a process. The method involved the use of single microdissected Deiters' membranes which were put between two small microchambers in order to study the passage of GABA across the membrane. The results show that whereas PS and Ca²⁺ by themselves have no effect on such a process, PS+2 mM CaCl₂ give a significant, although slight, inhibition. The hypothesis that Calcium ion + PS effect is due to a disturbance of the interaction between GABA and endogenous PS molecules of the membrane is discussed.     

Is the Drug-Responsive NADH Oxidase of the Cancer Cell Plasma Membrane a Molecular Target for Adriamycin?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC₅₀ for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.     

Adriamycin-induced leakage of lysosomal enzymes in vitro

Chronic treatment of rats with adriamycin has been shown to affect myocardial lysosomes as well as enzyme activities in the serum fraction. In this study, we examined in vitro effects of adriamycin (10^–6 to 10^–3 M) on the lysosomal fraction isolated from rat ventricular tissue. Morphological examination revealed that the isolated fraction was mainly vesicular in nature. Higher concentrations of adriamycin (10^–3 M) caused a significant loss of acid phosphatase and N-acetyl-B-d-glucosaminidase activity from the lyosomal vesicles. The enzyme leakage was not accompanied by any intravesicular localization of lanthanum, an extravesicular electron dense tracer. Preincubation of lysosomal vesicles with 10 g/ml superoxide dismutase did not protect against adriamycin-induced loss of lysosomal enzymes. The study shows that adriamycin induces loss of lysosomal enzymes in vitro and the superoxide radical may not be involved in this change.     

Effect of phosphatidylserine on the basal and GABA-activated Cl− permeation across single nerve membranes from rabbit Deiters' neurons

The permeation of labeled Cl^– ions across single plasma membranes from Deiter's neurons has been studied in the presence of various concentrations of phosphatidylserine (PS) on their extracellular side. PS reduces significantly basal Cl^– permeation only at 10^–5 M on the membrane exterior. No effect was found at other concentrations. GABA activable³⁶Cl^– permeation is heavily reduced and almost abolished at 10^–11–10^–5 M phosphatidylserine. This exogenous phosphatidylserine effect is difficult to interpret in relation to the function of the endogenous phospholipid. However, it may be involved in the epileptogenic effect in vivo of exogenous phosphatidylserine administration to rats.     

Transferrin-adriamycin conjugates which inhibit tumor cell proliferation without interaction with DNA inhibit plasma membrane oxidoreductase and proton release in K562 cells.

Conjugates of adriamycin crosslinked to transferrin with glutaraldehyde inhibit proliferation of transformed cells. Conjugates of this type inhibit oxidoreductase activity in the plasma membrane of K562 cells, and the inhibition of electron transport is found at concentrations ten times lower than concentrations of free adriamycin which inhibit electron transport and cell growth. The transferrin-adriamycin conjugate inhibits ferricyanide reduction, diferric transferrin reduction and plasma membrane NADH oxidase activity stimulated by transferrin. Activation of proton release from the K562 cells by diferric transferrin also is inhibited by the conjugate, and conjugate kills cells more effectively than free adriamycin. Since the conjugate does not transfer adriamycin to the nucleus, the growth control may be based on inhibition of the transferrin regulated redox system and Na+/H+ antiport activity at the plasma membrane.     

Stimulation of lymphocyte receptor capping by the ionophore monensin

Summary The carboxylic ionophore monensin has a biphasic effect on antibody-induced Thy-1 cap formation. At higher concentrations, 5×10^–6–5×10^–5 m monensin causes a significant inhibition of receptor capping similar to that previously found with the Ca²⁺ selective ionophore A23187. At lower concentrations, 5×10^–8–5×10^–7 m capping is stimulated. It is concluded that capping at lower ionophore concentrations is a secific response to the ability of monensin to induce a rise in intracellular Na⁺, which indirectly elevates intracellular Ca²⁺ activity. This in turn activates the contractile machinery required for the aggregation of surface receptors into capped structures. At higher concentrations monensin acts as a nonspecific detergent, which causes detrimental structural alterations in some of the membrane components involved in the capping process.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H₂O₂. The rate of H₂O₂ formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK _m value for NADH was found to be 3×10^–6 M. The H₂O₂-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H₂O₂ generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H₂O₂. In view of the growing evidence that H₂O₂ can be involved in metabolic control, the formation of H₂O₂ by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.     

Amino acid transport in the rat exocrine pancreas

Summary The two calcium antagonistic agents lanthanum and tetracaine cause severe disturbances in the secretory process of the exocrine pancreas, including inhibition of the rate of protein synthesis and exocytosis. The former effect resulted mainly from the inhibition of amino acid transport. Lanthanum in a concentration up to 1 mM inhibited transport of different species of amino acids in an unspecific way whereas tetracaine interfered specifically with the Na⁺-dependent transport system for neutral amino acids (¹⁴C--amino-isobutyric acid). Na⁺-independent transport of neutral amino acids (³H-leucine) was not affected. Transport inhibition was correlated to the activity of the Na⁺, K⁺-ATPase system which was measured in isolated plasma membrane fractions. At higher concentrations (5–10 mM) some uptake of lanthanum into the cells by limited endocytosis was observed. At lower concentrations lanthanum seemed to bind exclusively to certain components of the plasma membrane, mainly at the lateral and basal cell surface. Even at a concentration of 5–10 mM, no binding to the apical surface occurred. Similarly, no binding of lanthanum was observed to the limiting membrane of isolated zymogen granules, while mitochondria, contained in the same fraction, showed considerable binding affinity. The action of lanthanum and tetracaine on membrane carrier systems did not affect the interior organization of the plasma membrane. Particle density and distribution in freeze-fracture replicas as well as the submembrane microfilamentous-microtubular system and the junctional elements remained unaffected.Supported by a grant from the Deutsche Forschungsgemeinschaft (Ke 113/10). The expert technical assistance of Miss Helga Hollerbach and Miss Hiltraud Hosser and the editorial help of Mrs. Gisela Lesch is gratefully acknowledged     

Relationship between IAA-induced elongation growth and plasma membrane NADH oxidase in soybean (Glycine max Merr.) hypocotyls

A relationship between the activity of NADH oxidase of the plasma membrane and the IAA-induced elongation growth of hypocotyl segments in etiolated soybean (Glycine max Merr.) seedlings was investigated. The plasma membrane NADH oxidase activity increased in parallel to IAA effect on elongation growth in hypocotyl segments. Actually, NADH oxidase activity was stimulated 3-fold by 1 u,M IAA, and the elongation rate of segments was stimulated 10-fold by 10 iM IAA. The short-term elongation growth kinetics, however, showed that the IAA-induced elongation of hypocotyl segments was completely inhibited by plasma membrane redox inhibitors such as actinomycin D and adriamycin, at 80 μM and 50 μM respectively. In addition, 1 mM actinomycin D inhibited the IAA-stimulated NADH oxidase activity by about 80%. However, adriamycin had no effect on NADH oxidase activity of plasma membrane vesicles. Based on these results, the plasma membrane redox reactions seemed to be involved in IAA-induced elongation growth of hypocotyls, and the redox component responding to IAA was suggested to be NADH oxidase.     

Iodination (125I) of the apical plasma membrane of toad bladder epithelium: Electron-microscopic autoradiography and physiological effects

Summary The apical plasma membrane of toad bladder epithelial cells has been enzymatically iodinated, using lactoperoxidase, H₂O₂ (generated by a glucose-glucose oxidase system) and NaI. The site of labeling was demonstrated by electron-microscopic autoradiography; the silver grains (¹²⁵I) were found exclusively overlying the luminal plasma membranes of the epithelium. The iodination reaction reached completion in less than 5 min. The dependence of the degree of iodination on NaI concentrations (range=6.3×10^–8 to 6.3×10^–2 m) in the mucosal medium was determined. The results suggest that three classes of sites are iodinated within this concentration range. At concentrations of NaI of 6.3×10^–6 m or less, iodination of the apical membrane had no significant effect on either the fine structure of the epithelium or on electrophysiological properties. The baseline short-circuit current (SCC) remained steady and the response to vasopressin was unimpaired. At concentrations of 6.3×10^–5 m NaI and greater, the baseline SCC was depressed and the response to vasopressin was partially inhibited. The results indicate that¹²⁵I may serve as a covalent marker (specific for tyrosine and histidine residues) of the apical plasma membrane of epithelia.     

Gene amplification in Djungarian hamster cell lines possessing decreased plasma membrane permeability for colchicine and some other drugs

By multistep selection a set of clones and sublines possessing different levels of resistance to colchicine or adriablastin was obtained from the SV40-transformed Djungarian hamster cell lines, DM-15 and DM^cap. Resistance to both colchicine and adriablastin is associated with an alteration of plasma membrane permeability leading to a decreased uptake of various drugs (³H-colchicine, ³H-cytochalasin B, ³H-actinomycin D, ³H-puromycin, ³H-vinblastine, ¹⁴C-chloramphenicol). The DNA of cells highly resistant to cholchicine can transmit resistance only to low dosages of the drug. Comparison of DNAs from wild-type and resistant cells digested by restriction endonucleases revealed new classes of repeated DNA sequences in resistant cell lines. The degree of DNA repetition was correlated with the level of drug resistance. The repeated DNA sequences evidently represent parts of the genome that are amplified in resistant cells. The size of the amplified sequences is 200–250 kilobase pairs (kb). Cell lines highly resistant to colchicine contain amplified DNA, which like mitochondrial DNA replicate asynchronously with the main portion of the cellular DNA and related but not identical DNA sequences are amplified in independent cell lines selected for resistance to colchicine, adriablastin, and actinomycin D. These cell lines display similar patterns of alterations of plasma membrane permeability. The amplified DNA sequences may contain a gene or genes the overexpression of which leads to change in plasma membrane permeability and a development of resistance to various drugs.     

Reversed-phase liquid chromatographic determination of plasma levels of adriamycin and adriamycinol

A method is given for the determination of adriamycin and its main metabolite, adriamycinol in plasma from cancer patients after administration of adriamycin as the free drug or as a complex with DNA.Adriamycin and adriamycinol are extracted in a column from 1 ml of plasma (pH 8.6) using a mixture of chloroform—1-heptanol (8:2). After re-extraction into phosphate buffer pH 2.2, the separation is performed as reversed-phase liquid chromatography on a LiChrosorb RP-2 (5 μm) column with a mobile phase of acetonitrile—water, acidified with phosphoric acid.The precision by quantitation with photometric detection was better than 5% within the range 50–300 ng/ml. Plasma levels of adriamycin and adriamycinol in a cancer patient are presented in this paper.     

[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

Relationship of Transplasmalemma Redox Activity to Proton and Solute Transport by Roots of Zea mays

Transplasmalemma redox activity, monitored in the presence of exogenous ferricyanide stimulates net H⁺ excretion and inhibits the uptake of K⁺ and α-aminoisobutyric acid by freshly cut or washed, apical and subapical root segments of corn (Zea mays L. cv “Seneca Chief”). H⁺ excretion is seen only following a lag of about 5 minutes after ferricyanide addition, even though the reduction of ferricyanide occurs before 5 minutes and continues linearly. Once detected, the enhanced rate of H⁺ excretion is retarded by the ATPase inhibitors N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and vanadate. A model is presented in which plasmalemma redox activity in the presence of ferricyanide involves the transport only of electrons across the plasmalemma, resulting in a depolarization of the membrane potential and activation of an H⁺-ATPase. Such a model implies that this class of redox activity does not provide an additional and independent pathway for H⁺ transport, but that the activity may be an important regulator of H⁺ excretion. The 90% inhibition of K⁺ (⁸⁶Rb⁺) uptake within 2 minutes after ferricyanide addition can be contrasted with the 5 to 15% inhibition of uptake of α-aminoisobutyric acid. The possibility exists that a portion of the K⁺ and most of the α-aminoisobutyric acid uptake inhibitions are related to the ferricyanide-induced depolarization of the membrane potential, but that the redox state of some component of the K⁺ uptake system may also regulate K⁺ fluxes.     

Study of the redox properties of metallothionein in vitro by reacting with DsbA protein

Mammalian metallothionein (MT) contains 20 cysteine residues involved in the two metal clusters without a disulfide bond. The redox reaction of the Cys thiols was proposed to be associated with the metal distribution of MT. The E. coli DsbA protein is extremely active in facilitating thiol/disulfide exchange both in vivo and in vitro. To further investigate the redox properties of MT, reaction between MT and DsbA was carried out in vitro by fluorescence detection. Equilibrium characterization indicates that the reaction is stoichiometric (1:1) under certain conditions. Kinetic study gives a rate constant of the redox reaction of 4.42 × 10⁵ sec^–1 M^–1, which is 10³-fold larger than that of glutathione reacting with DsbA. Metal-free MT (apo-MT) shows a higher equilibrium reduction potential than MT, but exhibits an indistinguishable kinetic rate. Oxidation of MT by DsbA leads to metal release from the clusters. The characteristic fluorescence increase during reduction of DsbA may provide a sensitive probe for exploring the redox properties of some reductants of biological interest. The result also implies that oxidation of Cys thiols may influence the metal release or delivery from MT.