Penicillic acid as Na+,K+ and Ca2+ channel blocker in isolated frog's heart at toxic levels

The effect of penicillic acid on isolated frog's heart has been studied along with ions of Na+,K+ and Ca2+. Penicillic acid has been found to inhibit the entry of these ions into cardiac tissue thereby arresting the action of the heart. The blockage can be washed away by perfusion with Ringer's solution.     

Na+,K+-ATPase and Ca2+-ATPase isozymes in malignant neoplasms

A current state of researches on mechanisms of ion homeostasis regulation in the specific conditions of the uncontrolled malignant tumor growth (mainly in carcinomas) concerning the contribution of Na+,K+-ATPase, plasma membrane and sarco(endo)plasmic reticulum Ca2+-ATPases has been reviewed. Particular attention has been focused on the molecular and biochemical links providing the redistribution of the transporting ATPases isozyme pattern for the regulatory requirements of the cell signaling pathways at stable proliferation and viability in malignancy.     

Transport of H+, K+, Na+ and Ca++ in Streptococcus

Summary The streptococci differ from other bacteria in that cation translocations (with the possible exception of one of the K⁺ uptake systems) occur by primary transport systems, i.e., by cation pumps which use directly the free energy released during hydrolysis of chemical bonds to power transport. Transport systems in other bacteria, especially for Na⁺ and Ca⁺⁺, are often secondary, using the free energy of another ion gradient to drive cation transport. In streptococci H⁺ efflux occurs via the F₁F₀-ATPase. This enzyme is composed of eight distinct subunits. Three of the subunits are embedded in the membrane and form a H⁺ channel; this is called the F₀ portion of the enzyme. The other five subunits form the catalytic part of the enzyme, called F₁, which faces the cytoplasm and can easily be stripped from the membrane. Physiologically, this enzyme functions as a H⁺-ATPase, pumping protons out of the cell to form an electrochemical proton gradient, . The F₁F₀-ATPase, however, is fully reversible and if supplied with P_i, ADP and a + of sufficient magnitude (ca –200 mv) catalyzes the synthesis of ATP. Streptococcus faecalis can accumulate K⁺ and establish a gradient of 50 000:1 (in>out) under some conditions. Uptake occurs by two transport systems. The dominant, constitutive system requires both an electrochemical proton gradient and ATP to operate. The minor, inducible K⁺ transport system, which has many similarities to the K⁺-ATPase of the Kdp transport system found in Escherichia coli, requires only ATP to power K⁺ uptake.Sodium extrusion occurs by a Na⁺/H⁺-ATPase. Exchange is electroneutral and there is no requirement for a . The possibility that the Na⁺/H⁺-ATPase may consist of two parts, a catalytic subunit and a Na⁺/H⁺ antiport subunit, is suggested by the finding that damage to the Na⁺ transport system either through mutation or protease action leads to the appearance of -requiring Na⁺/H⁺ antiporter activity.Ca⁺⁺ like Na⁺ is extruded from metabolizing, intact cells. Transport requires no but does require ATP. Reconstitution of Ca⁺⁺ transport activity with accompanying Ca⁺⁺-stimulated ATPase activity into proteoliposomes suggests that Ca⁺⁺ is transported by a Ca⁺⁺-translocating ATPase.Where respiring organelles and bacteria use secondary transport systems the streptococci have developed cation pumps. The streptococci, which are predominantly glycolyzing bacteria, generate a much inferior to that of respiring organisms and organelles. The cation pumps may have developed simply in response to an inadequate .Abbreviations electrochemical potential of protons - membrane potential - pH pH gradient - p proton-motive force - DCCD N,Na¹-dicyclohexlcarbodiimide - TCS tetrachlorosalicylanilide - FCCP carbonylcyanide-p-trifluoromethylphenylhydrazone - CCCP carbonylcyanie-m-chlorophenylhydrazone - TPMP⁺ triphenylmethyl phosphonium ion - DDA⁺ dibenzyldimethylammonium ion - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA ethyleneglycol-bis (amino-ethyl-ether)-N,N-tetraacetic acid     

Competitive membrane adsorption of Na+, K+, and Ca2+ in smooth muscle cells

Summary A theory for Na⁺, K⁺ and Ca²⁺ competitive adsorption to a charged membrane is used to explain a number of experimental observations in smooth muscle. Adsorption is described by Langmuir isotherms for mono- and divalent cations which in turn are coupled in a self-consistent way to the bulk solution through the diffuse double layer theory and the Boltzman equations. We found that the dissociation constants for binding of Na⁺, K⁺ and Ca²⁺ in guinea pig taenia coli areca. 0.009, 1.0, and 4×10^–8 m, respectively. Furthermore, the effect of a Ca²⁺ pump that maintains free surface Ca²⁺ concentration constant is investigated. A decrease in intracellular Na⁺ content results in an increased Ca²⁺ uptake; part of this uptake is due to an increase in surface-bound Ca²⁺ in an intracellular compartment which is in contact with the myofilaments. Variations in the amount of charge available to bind Ca²⁺ and the surface charge density are studied and their effect interpreted in terms of different pharmacological agents.     

盐胁迫下杨树根际系统盐分离子分布特性

在温室条件下,采用盆栽根箱培养的方法研究盐胁迫下I 69杨(PopulusdeltoidesBartr.cv.'Lux')和NL 1381杨〔PopulusdeltoidesBartr.cv.'Lux'×P.euramericana(Dode)GeninierCL'I 45 51'〕根际、非根际土壤盐分分布特征。盐处理浓度共设3个水平:CK(NaCl0g kg)、处理A(NaCl1g kg)和处理B(NaCl2g kg),采用完全随机设计。结果表明,2个杨树无性系根际水溶性K+亏缺,水溶性Na+、Ca2+和Mg2+富集。K+的亏缺率及Na+的富集率随NaCl处理浓度的增大而减小,Ca2+和Mg2+的富集率在非盐渍条件下最低,处理A达最高,处理B较处理A略有下降。在盐胁迫下,无性系NL 1381杨根际土壤Na+的浓度和电导率均低于无性系I 69杨,可以有效减轻盐分对根系的渗透胁迫,相对而言具有较强的抗盐性。     

Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states

Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the crystals had p1 symmetry. A large number of helical symmetries were observed, so a three-dimensional structure was calculated by averaging both Fourier-Bessel coefficients and real-space structures of data from the different symmetries. The resulting structure clearly reveals cytoplasmic, transmembrane, and extracellular regions of the molecule with densities separately attributable to alpha and beta subunits. The overall shape bears a remarkable resemblance to the E2 structure of rabbit sarcoplasmic reticulum Ca2+-ATPase. After aligning these two structures, atomic coordinates for Ca2+-ATPase were fit to Na+,K+-ATPase, and several flexible surface loops, which fit the map poorly, were associated with sequences that differ in the two pumps. Nevertheless, cytoplasmic domains were very similarly arranged, suggesting that the E2-to-E1 conformational change postulated for Ca2+-ATPase probably applies to Na+,K+-ATPase as well as other P-type ATPases.     

The accumulation ratio of K+, Na+, Ca2+ and tetrapropylammonium in steady-state Mitochondria.

The accumulation ratio of a permeant cation under steady-state conditions after active uptake, is defined as: ($\text{{cat}{}_{\mathrm{i}}\text{}}\text{{cat}{}_0\text{}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>z</mtext>}}$, where Z is the valence of the cation, and {cat_i} and {cat₀} are the internal and external cation activities, respectively. The electrogenic proton pump predicts that the accumulation ratio should be independent of (i) the chemical structure of the cation and (ii) the degree of permeability of the membrane to cations. Furthermore the accumulation ratio should be the same for all permeant cation species simultaneously present. In the present study it is found that under steady-state conditions: (i) the accumulation ratio is not the same for potassium in the presence of valinomycin, for tetrapropylammonium in the presence of tetraphenylboron, and for calcium in the presence of acetate; (ii) the accumulation ratio is not identical for two cations such as potassium and sodium present simultaneously in the presence of gramicidin; (iii) the accumulation ratio is dependent on the external carrier concentration, such as valinomycin or tetraphenylboron. It is concluded that the cation distribution ratios under steady-state conditions are not compatible with the predictions of the electrogenic proton pump model.     

SIMS Investigation of the Distribution of K+, Na+, Mg2+ and Ca2+ Cations in Birch Pollen

Two microanalytical techniques were used to investigate the inorganic cation content and distributions in birch (Betula verrucosa Ehrh.) pollen. With intact pollen grains. X-ray microanalysis (EDX) could only give a mean ionic composition. Secondary Ion Microscopy and Spectrometry (SIMS) appeared to be a more suitable technique to image ion distributions in the different pollen structures. This was carried out with samples prepared using a new vapour phase technique designed to improve ion retention. Transmission electron microscopy (TEM)showed good structural preservation of the samples. Monovalent ion (K⁺, Na⁺) distribution showed features different from those of the divalent cations (Ca²⁺, Mg²⁺). In the vegetative cell, the alkaline cations were mainly distributed in the most internal part of the cytoplasm and they were probably associated with starch grains or concentrated in dry vacuoles. Calcium distribution correlated well with the areas in the cytoplasm of the vegetative cell containing a dense network of mitochondria and endoplasmic reticulum. Within the pollen grain, the sperm cell appeared to contain the most calcium. Calcium was also abundant in the sporoderm. These results reveal the potential of SIMS for pollen studies that include germination, the monitoring of air pollutants and the allergens-ion interactions.     

Two components of extracellularly-recorded photoreceptor potentials in the cephalopod retina: Differential effects of Na+, K+ and Ca2+

The ERG of the isolated, superfused half-eye of the cephalopod Sepiola atlantica, evoked by a brief (10 s) light flash, has been studied by recording intraretinal potentials with glass microelectrodes. The intensity-response characteristics of the potentials recorded at an electrode fixed at the surface (V _s ) can be fitted by a simple equation derived from an equivalent circuit model based on a sodium conductance increase mechanism. Raising the external potassium level reduces the maximal response (V _m ), but does not alter the half-saturation intensity value (I ₀). Reducing external sodium does not affect (V _m ), but increases I ₀. Reducing external calcium also does not affect (V _m ), but decreases I ₀. These effects are adequately described by the model if it is also assumed (a) that changing the external sodium does not significantly alter the transmembrane sodium gradient, and (b) that sodium and calcium ions compete for the sensitivity control mechanism.Differential-depth recording between the fixed electrode at the surface and another electrode that could be moved into the retina revealed that the two component appearance of the transretinal ERG arose from the superposition of two vitreal-negative waveforms. An initial fast component was mainly recorded in the photoreceptive distal segments while a slow component was prominent in the more proximal regions of the retina. Perfusion with high K⁺ salines resulted in a decrease in the amplitudes of both fast and slow components of the response whereas reducing external Na⁺ reduced the amplitude of the fast component at all light intensities but reduced the amplitude of the slow component only at low intensities. The amplitudes of both the fast and slow components increased on reducing external calcium, but the rate of rise and fall of the fast component was independent of external calcium. The rate of rise of the slow component was also independent of the external Ca²⁺ level but a minimum in the recovery time (t _F ) was shifted to a lower intensity value at lower calcium concentrations. The shift of the minimum was to a higher intensity value with lowered sodium perfusing solutions. On the basis of the differential sensitivity of the two components to ion changes, as well as stimulus intensity and intraretinal distribution of the components, it is suggested that they reflect two distinct processes in the light-evoked potential of the photoreceptor cells.List of the more important symbols V _s Potential of fixed electrode at the surface of retina relative to grounded electrode - V _p Potential of moveable electrode relative to ground - V _D V _s - V _p - V _m Maximum value of V _s produced by saturating light intensities - I _o Stimulus required to evoke a response of half-maximal amplitude - g _K Membrane potassium conductance - g _Na Membrane sodium conductance - E _K Nernst potential for potassium - E _Na Nernst potential for sodium - G Coefficient connecting light intensity and light-induced change of conductance - t _R Time, measured from the stimulus onset, for the response to reach 50% of peak amplitude - t _F Time, measured from the time to peak, for the response to fall to 50% of its maximal value     

Evaluation of a role for intracellular Na+, K+, Ca2+, and Mg2+ in hyperthermic cell killing

A dose of heat which renders 98% of a population of Chinese hamster ovary cells reproductively dead has no significant effect on their Na+, K+, or Mg2+ content by 28 h postheat. In contrast, the cellular Ca2+ content increases in a dose-dependent manner as observed at 22 h after heating for 15-35 min at 45 degrees C. However, the rates of both influx and efflux of Ca2+ were reduced by heating. Increasing the cellular Ca2+ content by incubating the cells in high extracellular Ca2+, either at the time of heating or for a period of 22 h following heat, does not potentiate the lethal effect of heat. Completely blocking the heat-induced increase in Ca2+ content by incubating the cells in medium containing a low Ca2+ concentration does not protect the cells. Therefore, we conclude that heat does not produce any significant changes in the Na+, K+, or Mg2+ content of cells and that the heat-induced increase in Ca2+ does not play an important role in hyperthermic cell killing.     

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.     

Modulation of free Ca oscillations in single hepatocytes by changes in extracellular K+, Na+ and Ca2+

Single rat hepatocytes, microinjected with the calcium-sensitive photoprotein aequorin, when stimulated with either phenylephrine or arg8-vasopressin exhibit agonist-specific oscillations in cytosolic free calcium levels (free Ca). In the majority of the cells examined adding excess potassium chloride, sodium chloride or choline chloride abolished transient behaviour. However, in cells that continued to oscillate the transient parameters were subtly modified by these treatments. In experiments using phenylephrine as the agonist, adding excess potassium chloride to the superfusate significantly reduced transient length, increased the rate of transient rise and reduced the smoothed peak free Ca level without significantly altering the intertransient resting free Ca level or the falling time constant. The possible mechanisms by which these alterations may occur are discussed.     

Vasoconstriction triggered by hydrogen sulfide: Evidence for Na+,K+,2Cl-cotransport and L-type Ca2+ channel-mediated pathway

ObjectivesThis study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC).MethodsIsometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na⁺/K⁺-pump and Na⁺,K⁺,2Cl^-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the ⁸⁶Rb influx, respectively.ResultsNaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K⁺]_o=30 mM). At 10^?4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10^?3 M it decreased contractile responses by more than two-fold. Contractions evoked by 10^?4 M NaHS were completely abolished by bumetanide, a potent inhibitor of Na⁺,K⁺,2Cl^-cotransport, whereas the inhibition seen at 10^?3 M NaHS was suppressed in the presence of K⁺ channel blocker TEA. In cultured SMC, 5×10^?5 M NaHS increased Na⁺,K⁺,2Cl^- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, ⁴⁵Ca influx was enhanced in the presence of 10^?4 M NaHS and suppressed under elevation of [NaHS] up to 10^?3 M. ⁴⁵Ca influx triggered by 10^?4 M NaHS was abolished by bumetanide and L-type Ca²⁺ channel blocker nicardipine.ConclusionsOur results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na⁺,K⁺,2Cl^-cotransport and Ca²⁺ influx via L-type channels.     

Iron-Induced Inhibition of Na+, K+-ATPase and Na+/Ca2+ Exchanger in Synaptosomes: Protection by the Pyridoindole Stobadine

The effect of oxidative stress, induced by Fe²⁺-EDTA system, on Na⁺,K⁺-ATPase, Na⁺/Ca²⁺ exchanger and membrane fluidity of synaptosomes was investigated. Synaptosomes isolated from gerbil whole forebrain were incubated in the presence of 200 M FeSO₄-EDTA per mg of protein at 37°C for 30 min. The oxidative insult reduced Na⁺,K⁺-ATPase activity by 50.7 ± 5.0 % and Na⁺/Ca²⁺ exchanger activity measured in potassium and choline media by 47.1 ± 7.2 % and 46.7 ± 8.6 %, respectively. Membrane fluidity was also significantly reduced as observed with the 1,6-diphenyl-1,3,5-hexatriene probe. Stobadine, a pyridoindole derivative, prevented the decrease in membrane fluidity and in Na⁺/Ca²⁺ exchanger activity. The Na⁺,K⁺-ATPase activity was only partially protected by this lipid antioxidant, indicating a more complex mechanism of inhibition of this protein. The results of the present study suggest that the Na⁺/Ca²⁺ exchanger and the Na⁺,K⁺-ATPase are involved in oxidation stress-mediated disturbances of intracellular ion homeostasis and may contribute to cell injury.     

Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger

The properties of the Na-Ca exchanger in the plasma membrane of rod outer segments isolated from bovine retinas (ROS) were studied. Unidirectional Ca2+, Na+, and K+ fluxes were measured with radioisotopes and atomic absorption spectroscopy. We measured K+ fluxes associated with the Ca-Ca self-exchange mode of the Na-Ca exchanger to corroborate our previous conclusion that the ROS Na-Ca exchanger differs from Na-Ca exchangers in other tissues by its ability to transport K+ (Schnetkamp, P. P. M., Basu, D. K. & Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-C157). The Na-Ca-K exchanger was the only functional cation transporter in the plasma membrane of bovine ROS with an upper limit of a flux of 10(5) cations/ROS/s or a current of 0.01 pA contributed by other cation channels, pumps, or carriers; cation fluxes via the Na-Ca-K exchanger amounted to 5 x 10(6) cations/ROS/s or a current of 1 pA. Ca2+ efflux via the forward mode of the Na-Ca-K exchanger did not operate with a fixed single stoichiometry. 1) The Na/Ca coupling ratio was increased from three to four when ionophores were added that could provide electrical compensation for the inward Na-Ca exchange current. 2) The K/Ca coupling ratio could vary by at least 2-fold as a function of the external Na+ and K+ concentration. The results are interpreted in terms of a model that can account for the variable Ca/K coupling ratio: we conclude that the Ca2+ site of the exchanger can translocate independent of translocation of the K+ site, whereas translocation of the K+ site requires occupation of the Ca2+ site, but not its translocation. The results are discussed with respect to the physiological role of Na-Ca-K exchange in rod photoreceptors.     

α2 isoform of Na+,K+-ATPase via Na+,Ca2+ exchanger modulates myelin basic protein synthesis in oligodendrocyte lineage cells in vitro

Oligodendrocytes in the CNS myelinate neuronal axons, facilitating rapid propagation of action potentials. Myelin basic protein (MBP) is an essential component of myelin and its absence results in severe hypomyelination. In oligodendrocyte lineage cell (OLC) monocultures MBP synthesis starts at DIV4. Ouabain (10 nM), a Na⁺,K⁺-ATPase (NKA) blocker, stimulates MBP synthesis. As OLCs express the α2 isoform of NKA (α2-NKA) that has a high affinity for ouabain, we hypothesized that α2-NKA mediates this effect. Knockdown of α2-NKA with small interfering (si)RNA (α2-siRNA) significantly potentiated MBP synthesis at DIV4 and 5. This effect was completely blocked by KB-R7943 (1 μM), a Na⁺,Ca²⁺ exchanger (NCX) antagonist. α2-NKA ablation increased the frequency of NCX-mediated spontaneous Ca²⁺ transients ([Ca²⁺]t) at DIV4, whereas in control OLC cultures comparable frequency of [Ca²⁺]t was observed at DIV5. At DIV6 almost no [Ca²⁺]t were observed either in control or in α2-siRNA-treated cultures. Immunocytochemical analyses showed that α2-NKA co-localizes with MBP in proximal processes of immature OLCs but is only weakly present in MBP-enriched membrane sheets. Knockdown of α2-NKA in cortical slice cultures did not change MBP levels but reduced co-localization of neurofilament- and MBP-positive compartments. We conclude that α2-NKA activity in OLCs affects NCX-mediated [Ca²⁺]t and the onset of MBP synthesis. We suggest therefore that neuronal activity, presumably in form of local extracellular [K⁺] changes, might locally influence NCX-mediated [Ca²⁺]t in OLC processes thus triggering local MBP synthesis in the vicinity of an active axon.