Transport studies through liquid membranes of ciprofloxacin and norfloxacin

Ciprofloxacin and norfloxacin, the widely used drugs have been shown to generate liquid membranes in series with a supporting membrane (Sartorius celluose acetate microfiltration membrane). Transport of dextrose and ions, such as NH4+, Mg2+, Ca2+, K+ and PO4(3-) has been studied in the presence of liquid membranes generated by these drugs. The data obtained on the modification in the permeability of dextrose and ions in the presence of liquid membrane indicate the significance of liquid membranes in passive transport.     

The effect of membrane-bound calcium on the activity of adenosine triphosphatase from erythrocytes and erythrocyte permeability for monovalent cations]

The activities of Ca2+, Mg2+-ATPase and Na+, K+-ATPase and the permeability of reconstituted human erythrocytes for Na and K ions were measured, using Ca2+-EGTA, Ca2+ATP and Ca2+-sodium citrate buffers. It was found that the increase in the Ca2+/chelate ratio caused stimulation of Ca2+, Mg2+- and Na+, K+-Atpases and an increase in the rate constants of ouabain--dependent 42K+ influx and 22Na+ efflux from the erythrocytes. The use of the Ca2+-sodium citrate system as a calcium buffer did not change the parameters of the functional state of erythrocyte membranes. The data obtained are discussed in terms of a possible role of calcium ions, which are bound to the inner surface of the erythrocyte membrane, in the regulation of the systems of active and passive transport of cations.     

外源EBR对NaCl胁迫下燕麦幼苗无机离子吸收、运输和分配的影响

为了探讨外源2,4-表油菜素内酯(2,4-epibrassinolide,EBR)诱导燕麦(Avena sativa L.)幼苗抗盐性的效果及其生理调节机制,以"青引2号"和"加燕2号"燕麦为材料,研究NaCl胁迫下施用外源EBR对燕麦幼苗无机离子吸收、运输和分配的影响。结果表明:100mmol·L-1NaCl胁迫下,"青引2号"和"加燕2号"燕麦幼苗叶片和根系中的Na+、Cl-含量均显著升高,对阳离子的吸收产生了拮抗作用,导致燕麦幼苗叶片和根系中的K+、Ca2+、Mg2+、Mn2+、Fe2+、Zn2+、Cu2+含量显著降低,离子稳态平衡被打破;100 mmol·L-1NaCl胁迫下,施用0.01μmol·L-1外源EBR后,"青引2号"和"加燕2号"燕麦幼苗叶片和根系中的Na+和Cl-含量显著降低,促进了燕麦幼苗根系对K+、Ca2+、Mg2+、Fe2+、Mn2+、Cu2+和Zn2+的吸收,叶片和根系中K+/Na+、Cl-/Na+、Ca2+/Na+、Mg2+/Na+、Fe2+/Na+、Mn2+/Na+、Cu2+/Na+和Zn2+/Na+显著升高,并且有效调控燕麦幼苗体内无机离子的运输比和阳离子的运输选择性比率,离子稳态重新达到平衡状态;说明外源EBR能够缓解NaCl胁迫下Na+和Cl-对燕麦幼苗所造成的离子毒害作用,有效调控燕麦幼苗对无机离子的选择性吸收、运输和分配,对维持燕麦幼苗体内的离子稳态平衡具有正向调控作用。     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

Method for determination of sodium, potassium, calcium, magnesium, chloride, and phosphate in the rat choroid plexus by flame atomic absorption and visible spectroscopy

A rapid simple technique for the measurement of Na+, K+, Mg2+, Ca2+, PO4(3-), and Cl- was developed to analyze ion contents in the choroid plexus of the rat. The technique involves digestion in piperidine, precipitation of proteins with HClO4, and analysis of Na+, K+, Ca2+, and Mg2+ by atomic absorption spectroscopy and Cl- and PO4(3-) by visible spectroscopy. The coefficient of variation for the measurement of eight replicates was 1-3% for all ions. Analysis of choroid plexuses from eight rats yielded coefficients of variation of about 6% and the values for Na+, K+, and Cl- compared favorably to previous works. The analytical procedure described in this paper allows the determination of six major physiologic ions in rat choroid plexus (4 mg wet wt).     

Review. Peering into an ATPase ion pump with single-channel recordings

In principle, an ion channel needs no more than a single gate, but a pump requires at least two gates that open and close alternately to allow ion access from only one side of the membrane at a time. In the Na+,K+-ATPase pump, this alternating gating effects outward transport of three Na+ ions and inward transport of two K+ ions, for each ATP hydrolysed, up to a hundred times per second, generating a measurable current if assayed in millions of pumps. Under these assay conditions, voltage jumps elicit brief charge movements, consistent with displacement of ions along the ion pathway while one gate is open but the other closed. Binding of the marine toxin, palytoxin, to the Na+,K+-ATPase uncouples the two gates, so that although each gate still responds to its physiological ligand they are no longer constrained to open and close alternately, and the Na+,K+-ATPase is transformed into a gated cation channel. Millions of Na+ or K+ ions per second flow through such an open pump-channel, permitting assay of single molecules and allowing unprecedented access to the ion transport pathway through the Na+,K+-ATPase. Use of variously charged small hydrophilic thiol-specific reagents to probe cysteine targets introduced throughout the pump's transmembrane segments allows mapping and characterization of the route traversed by transported ions.     

The structure of CorA: a Mg(2+)-selective channel

The crystal structure of a closed form of the CorA Mg2+ transporter from Thermatoga maritima completes a set of representative structures of transport systems for all of the major biological elements, Mg2+, Ca2+, Na+, K+ and Cl-. The CorA monomer has a C-terminal membrane domain containing two transmembrane segments and a large N-terminal cytoplasmic soluble domain. In the membrane, CorA forms a homopentamer shaped like a funnel. Comparison of the structure of CorA with that of other ion channels and transporters suggests numerous common features, but, as might be predicted from the unique chemistry of the Mg2+ cation, the structure of CorA has several unusual features. Among these are initial binding in the periplasm of a fully hydrated Mg2+ ion; a ring of positive charge external to the ion-conduction pathway at the cytosolic membrane interface; and highly negatively charged helices in the cytosolic domain that appear capable of interacting with the ring of positive charge to facilitate Mg2+ entry. Finally, there are bound Mg2+ ions in the cytosolic domain that are well placed to control the interaction of the ring of positive charge and the negatively charged helices, and thus control Mg2+ entry.     

Effect of anthelmintics and phenothiazines on adenosine 5'-triphosphatases of filarial parasite Setaria cervi

S. cervi showed particulate bound Ca2+ ATPase and Na+,K(+)-ATPase activities while Mg2+ ATPase was detected in traces. ATPase of S. cervi was also differentiated from the nonspecific p-nitrophenyl phosphatase activity. Female parasite and microfilariae exhibited higher Ca2+ ATPase and Na+,K(+)-ATPase activities than the male adults and the enzyme Na+,K(+)-ATPase was mainly concentrated in the gastrointestinal tract of the filarial parasite. Na+,K(+)-ATPase of the filariid was ouabain-sensitive while Ca2(+)-ATPase activity was regulated by concentration of Ca2+ ions and inhibited by EGTA. Phenothiazines, viz. trifluoperazine, promethazine and chlorpromazine caused significant inhibition of Ca2+ ATPase and Na+,K(+)-ATPase. Diethylcarbamazine was a potent inhibitor of these ATPases. Mebendazole, levamisole and centperazine also caused significant inhibition of the ATPases indicating this enzyme system as a common target for the action of anthelmintic drugs.     

Nantenine and papaverine differentially modify synaptosomal membrane enzymes.

Papaverine (1-[(3,4-Dimethoxyphenyl) methyl]-6,7-dimethoxyisoquinoline) and nantenine (O-methyldomesticine) are chemically related isoquinoline alkaloids displaying similar dose-dependent sedative or convulsant effects, but seem to act differentially on synaptosomal membrane enzymes. Na+, K+-, Mg2+- and Ca2+-ATPase activities were inhibited by nantenine but not by papaverine, whereas acetylcholinesterase activity remained unchanged by nantenine but slightly enhanced by papaverine. Nantenine inhibited roughly both 20-50% Ca2+- and Mg2+-ATPase activities but 40-90% Na+, K+-ATPase activity. Kinetic analysis indicated that nantenine interacts with the substrate ATP for Ca2+-ATPase activity but that it competes with K+ for Na+, K+-ATPase activity. Given the roles of Na+, K+-ATPase and Ca2+-ATPase in cation transport and [Ca2+]i regulation, respectively, the inhibitory effect of nantenine upon these enzymes may explain its convulsant effect though not its sedative activity. The sedative action of both nantenine and papaverine is hardly attributable to an effect on the synaptosomal membrane enzymes assayed.     

Rapid release of 45Ca from an occluded state of the Na,K-pump

45Ca is bound to the occluded state of the Na,K-pump, apparently at K+ sites. Only one 45Ca ion is bound in place of two K+ ions, with an affinity approximately 0.08 mM; K+ competes with an apparent affinity approximately 0.04 mM. 45Ca is released rapidly from Na,K-ATPase in the presence of ATP or ADP, presumably to the intracellular medium. The rate constant of 45Ca release with ATP is greater than 100 s-1 at 20 degrees C, more than twice as fast as the rate of release of 42K from the occluded state. Phosphorylation of Na,K-ATPase with MgPi, which would lead to release of occluded K+ or Rb+ to the extracellular face of the membrane, stabilizes occluded 45Ca. 45Ca release is slower immediately after exposure to MgPi than after a rinse in the absence of Pi indicating that in the former circumstance the rate of 45Ca release is limited by dephosphorylation; 45Ca release is even slower after exposure to Mg2+ arsenate, consistent with dearsenylation being slower than dephosphorylation. When limited by dephosphorylation, the rate of 45Ca release is dependent on the species of monovalent cation present, increasing in the order N-methylglucamine less than Cs+ less than Li+ less than Na+ less than Rb+ less than K+. When the 45Ca occluded state is exposed to K + Mg + Pi and then to Na+ + Mg2+ + ATP, the exposure to K+ is "remembered," indicating simultaneous occlusion of 45Ca and K+. The apparent affinity for K+ in formation of this state is 10-50 mM, and the rate of release of K+ is approximately 2 s-1. Ca2+ has effects on the release of 86Rb from the occluded state: With ATP, Ca2+ acts like Mg2+ by stimulating 86Rb release at low concentrations and inhibiting at high concentrations; with MgPi, Ca2+ inhibits 86Rb release, presumably by preventing phosphorylation. Thus, Ca2+ has two actions on the Na,K-pump as studied here: one as a Mg2+ congener, and another as a K+ congener at transport sites. In the latter role Ca2+ is unusual in that it appears to be able to bind to the transport sites from the intracellular face of the pump and to become occluded, but unable to be released from extracellular sites.     

Effect of intracellular free Ca2+ on leucine transport in Chang liver cells

Addition of Ca2+ ionophore (A23187) to the medium stimulated the Na+-independent leucine transport in Chang liver cells, increasing the cytoplasmic free Ca2+ concentration, irrespective of the presence or absence of extracellular Ca2+. Anticalmodulin drugs, such as chlorpromazine, trifluoperazine, and W-7, significantly inhibited the leucine transport in the cells. The stimulatory effect of A23187 on leucine transport was completely blocked in the presence of the anticalmodulin drug. Two microtubule disrupting drugs, colchicine and colcemid, significantly stimulated leucine transport. On the other hand, taxol, a microtubule stabilizing agent, decreased the stimulatory effect of colchicine on the leucine transport. These results strongly suggest the involvement of Ca2+ and calmodulin in regulation of Na+-independent leucine transport, possibly through control of assembly and disassembly of the microtubule network.     

Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena thermophila acts as an enzyme, catalyzing sequence-specific cleavage of RNA substrates. We have now examined the metal ion requirements of this reaction. Mg2+ and Mn2+ are the only metal ions that by themselves give RNA enzyme activity. Atomic absorption spectroscopy indicates that Zn, Cu, Co, and Fe are not present in amounts equimolar to the RNA enzyme and when added to reaction mixtures do not facilitate cleavage. Thus, these ions can be eliminated as cofactors for the reaction. While Ca2+ has no activity by itself, it alleviates a portion of the Mg2+ requirement; 1 mM Ca2+ reduces the Mg2+ optimum from 2 to 1 mM. These results, combined with studies of the reactivity of mixtures of metal ions, lead us to postulate that two classes of metal ion binding sites are required for catalysis. Class 1 sites have more activity with Mn2+ than with Mg2+, with the other divalent ions and Na+ and K+ having no activity. It is not known if ions located at class 1 sites have specific structural roles or are directly involved in active-site chemistry. Class 2 sites, which are presumably structural, have an order of preference Mg2+ greater than or equal to Ca2+ greater than Mn2+ and Ca2+ greater than Sr2+ greater than Ba2+, with Zn2+, Cu2+, Co2+, Na+, and K+ giving no detectable activity over the concentration range tested.     

Measurement of total, intracellular and surface bound cations in animal cells grown in culture.

A procedure is described in which animal cells grown in culture on a dish are rapidly rinsed in situ with 0.25 M sucrose solutions for subsequent measurement of total, intracellular and rapidly exchangeab le Na+, K+, Mg2+ and Ca2+ by atomic absorption spectrophotometry. Repeated rinses with CO2-free (pH similar to 7) 0.25 M sucrose solution produced essentially no loss of cellular protein or cations. One 10-second rinse with CO2-saturated (pH 4) 0.25 M sucrose solution removed a rapidly proton exchangeable cellular cation fraction which is interpreted as being externally (membrane) bound. Rinses with physiological electrolyte solutions are shown to produce loss of cellular protein as well as displacement of surface exchangeable cations. Thus, isotonic sucrose solution is more satisfactory than electrolytic media for rinsing cultured cells prior to measurement of cellular cations. The technique employing sucrose rinse media is very rapid and reproducible and permits measurement of total, intracellular or surface bound Na+, K+, Mg2+ and Ca2+ in the same sample.     

Electron-cytochemical study of the localization and properties of ATPases in the isolated nuclei of rabbit skeletal muscle under normal conditions and in experimental muscular dystrophy]

The properties and localization of ATPase system in nuclei of skeletal muscle of normal rabbit and of those with experimental muscle dystrophy were studied by electron cytochemistry. The product of cytochemical reaction of ATP hydrolysis, which is a marker of ATPase activity localization in nuclear ultrastructures, was detected on the nuclear membrane, in chromatin and in the nucleolus, ATPase activity in the nuclei was detected in the presence of both, Mg2+ and Ca2+. Addition to the incubation medium, originally containing Mg2+, Na+ and K+, resulted in an increased formation of the product reaction in all the nuclear ultrastructures in both in the norm and under experimental muscle dystrophy. However, specific inhibitor of Mg2+, Na+, K+-ATPase--ouabain--suggests the absence in the nuclei of skeletal muscles of rabbit of transport ATPase working in the "Na-pump" system. The results of experiments with a specific complex of Ca2+--EGTA allow to suppose that Mg2+, Ca2+-ATPase of skeletal muscle nuclei of normal rabbits is localized in the nucleoplasm, whereas Mg2+-ATPase is found on the nuclear membrane. Using EGTA we failed to detected the localization of Mg2+, Ca2+-ATPase in nuclear ultrastructures upon experimental muscular dystrophy.     

Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore

We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by repulsive interaction with Ba2+. Ca ions reduced the amplitude of unitary Ba currents (50% inhibition at approximately 10 mM [Ca]o with 50 mM [Ba]o) without detectable flicker, presumably because Ca ions exit the pore very rapidly following Ba entry. However, Ca2+ entry and exit rates could be resolved when micromolar Ca blocked unitary Li+ fluxes through the Ca channel. The blocking rate was essentially voltage independent, but varied linearly with Ca concentration (rate coefficient, 4.5 X 10(8) M-1s-1); evidently, the initial Ca2+-pore interaction is outside the membrane field and much faster than the overall process of Ca ion transfer. The unblocking rate did not vary with [Ca]o, but increased steeply with membrane hyperpolarization, as if blocking Ca ions were driven into the cell. We suggest that Ca is both an effective permeator and a potent blocker because it dehydrates rapidly (unlike Mg2+) and binds to the pore with appropriate affinity (unlike Cd2+). There appears to be no sharp dichotomy between "permeators" and "blockers," only quantitative differences in how quickly ions enter and leave the pore.     

盐胁迫对药用蒲公英生长特性及有效成分积累与离子吸收分配效应的影响

以药用蒲公英(Taraxacum officinale)为试材,研究不同浓度盐胁迫对其生长特性、有效成分积累和离子吸收分配的影响。结果表明,低盐胁迫(0.1% NaCl)对药用蒲公英生长和菊苣酸含量无显著影响,叶中Na+含量与对照无显著差异,K+含量及K+/Na+显著升高;高盐胁迫(≥0.2% NaCl)下其生长受到显著抑制,菊苣酸含量显著降低,类囊体膜结构随着盐胁迫加剧趋于紊乱,光合能力减弱,叶片Na+含量显著上升,而K+、Ca2+和Mg2+含量下降,K+/Na+、Ca2+/Na+和Mg2+/Na+显著降低。离子运输选择性系数(SCa,Na、SMg,Na、SK,Na)随着盐胁迫加剧呈先升后降趋势。相关性分析表明,盐胁迫下蒲公英叶片Na+含量与叶片生理指标呈极显著负相关。因此,叶片Na+富集是药用蒲公英遭受盐害导致生长受抑制的主要原因之一。     

Effects of pH, temperature and Ca2+ content on the conformation of alpha-lactalbumin in a medium modelling physiological conditions

Data obtained by the intrinsic protein fluorescence technique showed that, in addition to Ca2+ and Mg2+ ions, bovine alpha-lactalbumin also binds physiologically significant Na+ and K+ ions, the nucleotides ATP, ADP, UTP, UDP and UDP-galactose. The release of the bound Ca2+ ions from the protein in a medium modelling physiological conditions (containing Mg2+, Na+, K+, ATP and ADP in physiological concentrations) induced a transition of the protein from the native state of the Ca2+-loaded form to a state which is a mixture of native and and thermally changed states of the apo- and metal bound forms. Any variations in temperature result in changes in the populations of these states. This may be associated with some Ca2+ and temperature dependent regulation of the protein function. Variations of pH within the physiological limits had little influence on the conformation of both Ca2+-loaded and Ca2+-free alpha-lactalbumin.     

The effects of storage of sarcoplasmic reticulum fragments on the Ca2+, Mg2+-ATPase.

The effects of K+ and Na+ on the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum fragments (SRF) were investigated at 1 mM ATP. There was an alteration of the sensitivity of the ATPase to the monovalent cations during storage of the SRF preparation. The Ca2+, Mg2+-ATPase of freshly prepared SRF was slightly activated by 5-10 mM K+ and Na+. Mg2+-ATPase was inhibited by both the monovalent cations to the same extent, and this response to the ions was independent of the freshness of the preparations. After storage of SRF, however, the Ca2+,Mg2+-ATPase was markedly activated by higher concentrations of K+ and Na+ (0.2-0.3 M). K+ and Na+ reduced the Ca uptake at the steady state in freshly prepared SRF, but did not affect pre-steady state uptake. In the presence of oxalate, the rate of Ca accumulation both in fresh and stored preparations was activated by 0.1-0.2 M K+ and Na+. The Ca2+, mg2+-ATPase with oxalate, so-called "extra ATPase," showed the same response to the ions as did the activity without oxalate during storage.