Silk Fibroin-Modified Disulfiram/Zinc Oxide Nanocomposites for pH Triggered Release of Zn2+ and Synergistic Antitumor Efficacy.

Disulfiram (DSF) is an FDA-approved anti-alcoholic drug that has recently proven to be effective in cancer treatment. However, the short half-life in the bloodstream and the metal ion-dependent antitumor activity significantly limited the further application of DSF in the clinical field. To this end, we constructed a silk fibroin modified disulfiram/zinc oxide nanocomposites (SF/DSF@ZnO) to solubilize and stabilize DSF, and, more importantly, achieve pH triggered Zn2+ release and subsequent synergistic antitumor activity. The prepared SF/DSF@ZnO nanocomposites were spherical and had a high drug loading. Triggered by the lysosomal pH, SF/DSF@ZnO could induce the rapid release of Zn2+ under the acidic conditions and caused nanoparticulate disassembly along with DSF release. In vitro experiments showed that cytotoxicity of DSF could be enhanced by the presence of Zn2+, and further amplified when encapsulated into SF/DSF@ZnO nanocomposites. It was confirmed that the significantly amplified cytotoxicity of SF/DSF@ZnO was resulted from pH-triggered Zn2+ release, inhibited cell migration, and increased ROS production. In vivo study showed that SF/DSF@ZnO nanocomposites significantly increased the tumor accumulation and prolonged the retention time. In vivo antitumor experiments in the xenograft model showed that SF/DSF@ZnO exerted the highest tumor-inhibition rate among all the drug treatments. Therefore, this exquisite study established silk fibroin-modified disulfiram/zinc oxide nanocomposites, SF/DSF@ZnO, where ZnO not only acted as a delivery carrier but also served as a metal ion reservoir to achieve synergistic antitumor efficacy. The established DSF nanoformulation displayed excellent therapeutic potential in future cancer treatment.

Application of Polymeric Nanoparticles for CNS Targeted Zinc Delivery In Vivo.

A dyshomeostasis of zinc ions has been reported for many psychiatric and neurodegenerative disorders including schizophrenia, attention deficit hyperactivity disorder, depression, autism, Parkinson's and Alzheimer's disease. Furthermore, alterations in zinc-levels have been associated with seizures and traumatic brain injury. Thus, altering zinclevels within the brain is emerging as a new target for the prevention and treatment of psychiatric and neurological diseases. However, given the restriction of zinc uptake into the brain by the blood-brain barrier, methods for controlled regulation and manipulation of zinc concentrations within the brain are rare. Here, we performed in vivo studies investigating the possibility of brain targeted zinc delivery using zinc-loaded nanoparticles which are able to cross the blood-brain barrier. After injecting these nanoparticles, we analyzed the regional and time-dependent distribution of zinc and nanoparticles within the brain. Moreover, we evaluated whether the presence of zinc-loaded nanoparticles alters the expression of zinc sensitive genes and proteins such as metallothioneins and zinc transporters and quantified possible toxic effects. Our results show that zinc loaded g7 nanoparticles offer a promising approach as a novel non - invasive method to selectively enrich zinc in the brain within a small amount of time.

[Mechanisms of action, pharmacology and interactions of dolutegravir]. / Mecanismo de acción, farmacología e interacciones de dolutegravir.

Dolutegravir is a second-generation integrase strand transfer inhibitor (INSTI), whose potential and binding half-life in the integrase are far superior to those of raltegravir and elvitegravir, conferring it with unique characteristics in terms of its genetic barrier to resistance and activity against viruses with one or more mutations in the integrase. The pharmacokinetic properties of dolutegravir allow once-daily dosing (50 mg), with or without food, maintaining concentrations far above those effective against wild-type viruses. If integrase resistance mutations are present, the recommended dosing regimen is 50 mg/12 h. The distribution of dolutegravir in cerebrospinal fluid is good and effective concentrations are also reached in the male and female genital tracts. Dolutegravir is metabolized by UGT1A1 and, to a lesser extent, by CYP3A4, without being an inducer or inhibitor of the usual metabolic systems. It has a very low potential for drug interactions and can be administered in routine doses with most drugs. Dose adjustment is not required, even in patients with renal insufficiency or mild or moderate liver failure. Increasing the dose of dolutegravir (50 mg/12 h) is only recommended when administered with efavirenz, nevirapine, fosamprenavir/r, tipranavir/r, rifampicin, carbamazepine, phenytoin and phenobarbital. Coadministration of dolutegravir with etravirine is not recommended without a protease inhibitor or with Hypericum perforatum. Dolutegravir should be administered 2 h before or 6 h after antacids or products with polyvalent cations. Dolutegravir can reduce renal tubule secretion of substances excreted via OCT2, with a slight initial increase in creatinine, with no risk of renal toxicity. The drug can also increase metformin concentrations and consequently monitoring is recommended in case dose adjustment is required. In summary, dolutegravir has excellent pharmacokinetic and drug interaction profiles.

Influence of hardness on the bioavailability of silver to a freshwater snail after waterborne exposure to silver nitrate and silver nanoparticles.

The release of Ag nanoparticles (AgNPs) into the aquatic environment is likely, but the influence of water chemistry on their impacts and fate remains unclear. Here, we characterize the bioavailability of Ag from AgNO(3) and from AgNPs capped with polyvinylpyrrolidone (PVP AgNP) and thiolated polyethylene glycol (PEG AgNP) in the freshwater snail, Lymnaea stagnalis, after short waterborne exposures. Results showed that water hardness, AgNP capping agents, and metal speciation affected the uptake rate of Ag from AgNPs. Comparison of the results from organisms of similar weight showed that water hardness affected the uptake of Ag from AgNPs, but not that from AgNO(3). Transformation (dissolution and aggregation) of the AgNPs was also influenced by water hardness and the capping agent. Bioavailability of Ag from AgNPs was, in turn, correlated to these physical changes. Water hardness increased the aggregation of AgNPs, especially for PEG AgNPs, reducing the bioavailability of Ag from PEG AgNPs to a greater degree than from PVP AgNPs. Higher dissolved Ag concentrations were measured for the PVP AgNPs (15%) compared to PEG AgNPs (3%) in moderately hard water, enhancing Ag bioavailability of the former. Multiple drivers of bioavailability yielded differences in Ag influx between very hard and deionized water where the uptake rate constants (k(uw), l g(-1) d(-1) ± SE) varied from 3.1 ± 0.7 to 0.2 ± 0.01 for PEG AgNPs and from 2.3 ± 0.02 to 1.3 ± 0.01 for PVP AgNPs. Modeling bioavailability of Ag from NPs revealed that Ag influx into L. stagnalis comprised uptake from the NPs themselves and from newly dissolved Ag.

Probing the structural basis of Zn2+ regulation of the epithelial Na+ channel.

Extracellular Zn(2+) activates the epithelial Na(+) channel (ENaC) by relieving Na(+) self-inhibition. However, a biphasic Zn(2+) dose response was observed, suggesting that Zn(2+) has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn(2+), we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn(2+) activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn(2+) activating site, γH239A diminished Na(+) self-inhibition and thus concealed the activating effects of Zn(2+). Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn(2+) inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp(516) was predicted to be in close proximity to γHis(88). Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn(2+). Our results suggest that external Zn(2+) regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His(193), His(200), and His(202) and (ii) a low-affinity Zn(2+) inhibitory site within the palm subdomain that includes His(88) and Asp(516).

Ca²+ sorption on regenerated cellulose fibres.

High calcium content in cellulose materials can cause considerable problems in pulp processing, textile chemical treatment and consumer use, e.g. dyeing operations or household laundry. The Ca(2+) binding capacity of cellulose also is of relevance in food and medical applications. Through their carboxyl group content regenerated cellulose fibres can act as weak anion exchangers, thus all types of regenerated cellulose fibres such as lyocell, viscose and modal fibres, show a distinct ability to bind Ca(2+) ions. The binding capacity is limited by the carboxyl group content, which was determined with 15 mmol/kg for lyocell fibres and 20 mmol/kg for viscose fibres, using the Methylene Blue sorption method. The presence of bound Ca(2+) also was demonstrated by complex formation with alizarin. The molar ratio between carboxylic group content and bound Ca(2+) ions was one Ca(2+) ion for a single carboxyl group. As a result of Ca(2+) sorption a positive net charge of the cellulose results and another anion has to be bound as counter ion for reasons of charge neutralisation. Results of potentiometric titrations indicate HCO(3)(-) to be present as counter ion in the Ca(2+) cellulose system. Thus under the experimental conditions studied, bound Ca(2+) is proposed to be present in the form COO(-)Ca(2+)HCO(3)(-).

Physiological metal uptake by Nostoc punctiforme.

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+) and the non-essential divalent cation Cd(2+) in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5 µM Cd(2+), 2 µM Co(2+), 0.5 µM Cu(2+), 500 µM Mn(2+), 1 µM Ni(2+), and 18 µM Zn(2+). Cells exposed to these non-toxic concentrations with combinations of Zn(2+) and Cd(2+), Zn(2+) and Co(2+), Zn(2+) and Cu(2+) or Zn(2+) and Ni(2+), had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500 µM Mn(2+) showed similar growth compared to the untreated controls. Metal levels were measured after one and 72 h for whole cells and absorbed (EDTA-resistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using (65)Zn showed that after 72 h of exposure Zn(2+) uptake was reduced by most metals particularly 0.5 µM Cd(2+), while 2 µM Co(2+) increased Zn(2+) uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals.

Strontium modified biocements with zero order release kinetics.

Strontium-substituted beta-TCP with the general formula Ca((3-x))Sr(x)(PO(4))(2) (0

Induction of catalytic activity of plasminogen by monoclonal antibody IV-Ic in the presence of divalent metal cations and alpha2-antiplasmin.

Investigation of the influence of divalent metal cations on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic showed that the presence of metal cations in the reaction medium changes the induction by slowing down or accelerating the process. Ions of Zn(2+), Mn(2+), and Cu(2+) completely inhibit activation. Ions of Co(2+) and Ni(2+) decrease the rate of the first and second phases of the reaction more than 2 times. Ca(2+) ions do not have any effect on the activation rate. Ions of Mg(2+), Ba(2+), and Sr(2+) increase the rate of the first phase of the reaction by 1.5, 2.0, and 2.0 times and the rate of the second phase by 2.0, 3.8, and 4.7 times, correspondingly. Sr(2+) ions have the strongest stimulating effect on plasminogen activation by monoclonal antibody IV-Ic. Investigation of the dose dependent effect of Sr(2+) on the rate of plasminogen activation by monoclonal antibody IV-Ic showed stimulating effect of Sr(2+) at concentrations from 0.1 to 1.0 mM with half maximum at 0.6 mM. However, Sr(2+) ions do not affect amidolytic activity of plasmin and activation of plasminogen by streptokinase. Sr(2+) ions also do not affect monoclonal antibody IV-Ic binding to plasminogen. The effect of Sr(2+) is specific and mediated by the IV-Ic component. The presence of metal cations affects conformational changes in the process of active site formation. Metal cations also affect structure of the plasminogen molecule active site in the complex with monoclonal antibody IV-Ic and enzyme-substrate interaction. The effect of alpha(2)-antiplasmin on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic in range of concentrations from 5 to 30 nM has been studied. alpha(2)-Antiplasmin at concentration 30 nM almost completely inhibits induction of plasminogen catalytic activity by monoclonal antibody IV-Ic at the ratio plasminogen/alpha(2)-antiplasmin of 3 : 1. This can be explained by competition of alpha(2)-antiplasmin and monoclonal antibody IV-Ic for the lysine-binding sites of plasminogen and inhibition of the active center in activated complex plasminogen*-mAB IV-Ic. Divalent metal cations and alpha(2)-antiplasmin are important factors in induction of plasminogen catalytic activity by monoclonal antibody IV-Ic.

Equilibrium and kinetic studies for the biosorption system of copper(II) ion from aqueous solution using Tectona grandis L.f. leaves powder.

The biosorption of copper(II) ions from aqueous solution by Tectona grandis L.f. was studied in a batch adsorption system as a function of pH, metal ion concentration, adsorbent concentration and adsorbent size. The biosorption capacities and rates of copper(II) ions onto T. grandis L.f. were evaluated. The Langmuir, Freundlich, Redlich-Peterson and Temkin adsorption models were applied to describe the isotherms and isotherm constants. Biosorption isothermal data could be well interpreted by the Langmuir model with maximum adsorption capacity of 15.43 mg/g of copper(II) ion on T. grandis L.f. leaves powder. The kinetic experimental data properly correlated with the second-order kinetic model. Various thermodynamic parameters such as deltaG(o), deltaH(o), and deltaS(o) were calculated indicating that this system was a spontaneous and exothermic process.

The adsorption of Cd(II) ions on sulphuric acid-treated wheat bran.

The adsorption of Cd(II) ions which is one of the most important toxic metals by using sulphuric acid-treated wheat bran (STWB) was investigated. The effects of solution pH and temperature, contact time and initial Cd(II) concentration on the adsorption yield were studied. The equilibrium time for the adsorption process was determined as 4 h. The adsorbent used in this study gave the highest adsorption capacity at around pH 5.4. At this pH, adsorption capacity for an initial Cd(II) ions concentration of 100 mg/L was found to be 43.1 mg/g at 25 degrees C for contact time of 4 h. The equilibrium data were analysed using Langmuir and Freundlich isotherm models to calculate isotherm constants. The maximum adsorption capacity (qmax) which is a Langmuir constant decreased from 101.0 to 62.5 mg/g with increasing temperature from 25 to 70 degrees C. Langmuir isotherm data were evaluated to determine the thermodynamic parameters for the adsorption process. The enthalpy change (deltaH(o)) for the process was found to be exothermic. The free energy change (deltaG(o)) showed that the process was feasible. The kinetic results indicated that the adsorption process of Cd(II) ions by STWB followed first-order rate expression and adsorption rate constant was calculated as 0.0081 l/min at 25 degrees C. It was observed that the desorption yield of Cd(II) was highly pH dependent.

Removal of Cu(II) ions by activated poplar sawdust (Samsun clone) from aqueous solutions.

In this work, adsorption of Cu(II) ions on sawdust (SD) and activated sawdust (ASD) has been studied by using batch adsorption techniques. The equilibrium adsorption level was determined to be a function of the pH, initial Cu(II) concentration, and adsorbent dosage. The equilibrium nature of Cu(II) adsorption has been described by the Freundlich and Langmuir isotherms. The experimental adsorption data were fitted to the Langmuir adsorption model both sawdust and activated sawdust. The equilibrium capacity of sawdust and activated sawdust were 5.432 and 13.495 mg copper per g adsorbent, respectively at room temperature and natural pH. The maximum adsorption capacity was obtained at the maximum zeta potential value that -74.5 mV (pH 5) for activated sawdust and at -48.4 mV (pH 4) for sawdust. It was observed that activated sawdust was a suitable adsorbent than sawdust for removal of Cu(II) from aqueous solutions.

Inhaled cationic amphiphilic drug-induced pulmonary phospholipidosis in rats and dogs: time-course and dose-response of biomarkers of exposure and effect.

This study compares the pulmonary response to an inhaled highly soluble hydrochloride (CAD-HCl) with a low soluble sulphate salt (CAD-SO4) of a dicationic amphiphilic drug (CAD). These salts are known to accumulate in the lung. The bioavailability through gastrointestinal uptake is poor. Wistar rats and beagle dogs received repeated 1 h/day inhalation exposures over 1-4 weeks. The focus of this analysis is to appraise the correlation of markers of effects related to pulmonary phospholipidosis and cytotoxicity relative to the concentration of CAD in the lung tissue, alveolar macrophages and serum. Rats and dogs are known to experience remarkable differences in their respiratory minute volumes and respiratory patterns. In order to facilitate dosimetric comparisons, identical exposure paradigms and methodological procedures were selected. Over a wide range of cumulative dosages, the CADs bound to lung tissue and cells in bronchoalveolar lavage (BAL) paralleled, whilst no clear association existed in terms of plasma concentrations. This suggests that analysis of the fractional loading of BAL-cells (mainly alveolar macrophages) with CAD or CAD-surfactant complexes is amenable to monitor the accumulation of CADs in the lung. In terms of the comparative phospholipidosis-inducing potency, the CAD-HCl was more potent as compared to CAD-SO4. Following dosimetric adjustments, rats and dogs appeared to be equally susceptible to phospholipidosis. In summary, when exposed to equivalent concentrations of CADs, dogs did not demonstrate a markedly different susceptibility than rats. With regard to the relative intensity of changes, the increase of phospholipids in BAL-fluid and especially BAL-cells correlated with the cumulative exposure dose. Thus, with regard to probing the extent of CAD-induced 'overloading' of alveolar macrophages pharmacokinetic determinations in BAL-cells are considered superior to determinations in plasma. Additional advantages of using the alveolar macrophage as denominator to normalize pulmonary drug concentrations include comparisons across species, and exposure regimens are feasible based on almost readily available endpoints in both pre-clinical and selected clinical studies.

Depolarization-induced 65zinc influx into cultured cortical neurons.

Toxic Zn(2+) influx may be a key mechanism underlying selective neuronal death after transient global ischemia in rats. To identify routes responsible for neuronal Zn(2+) influx, we measured the accumulation of (65)Zn(2+) into cultured murine cortical cells under depolarizing conditions (60 mM K(+)) associated with severe hypoxia-ischemia in brain tissue. Addition of 60 mM K(+) or 300 microM kainate substantially increased (65)Zn(2+) accumulation into mixed cultures of neurons and glia, but not glia alone. (65)Zn(2+) accumulation was attenuated by increasing concentrations of extracellular Ca(2+) or trypsin pretreatment, but not by late trypsinization, and corresponded to an increase in atomic Zn(2+). Confirming predominantly neuronal entry, K(+)-induced (65)Zn(2+) accumulation was reduced by prior selective destruction of neurons with NMDA. K(+)-induced (65)Zn(2+) influx was not sensitive to glutamate receptor antagonists, but was attenuated by Gd(3+) and Cd(2+) as well as 1 microM nimodipine; it was partially sensitive to 1 microM omega-conotoxin-GVIA, and insensitive to 1 microM omega-agatoxin-IVA. K(+)-induced, Gd(3+)-sensitive (45)Ca(2+) accumulation but not (65)Zn(2+) accumulation was sharply attenuated by lowering extracellular pH to 6.6.

The effect of external divalent cations on spontaneous non-selective cation channel currents in rabbit portal vein myocytes.

1. The effects of external divalent cations on spontaneous single non-selective cation channel currents were studied in outside-out patches from rabbit portal vein smooth muscle cells in K+-free conditions. 2. In an external medium containing 1.5 mM Ca2+ (Ca2+o) the majority of spontaneous channel currents had a unitary conductance of 23 pS, reversal potential (Vr) of +10 mV and a low open probability (Po) at negative patch potentials. Some channels opened to a lower conductance state of about 13 pS suggesting that the cation channels have two conductance states. Open time and burst duration distributions could both be described by two exponentials with time constants of about of 1 ms and 7 ms for open times and 3 ms and 16 ms for burst durations. 3. In 0 Ca2+o the majority of spontaneous cation channels had a unitary conductance of 13 pS and Vr was shifted to +4 mV. Moreover the longer open time and longer burst duration time constants were both reduced to approximately half the values in 1.5 mM Ca2+o. 4. Compared to 0 Ca2+o the single channel currents in 3 microM and 100 microM Ca2+o had a 5- to 6-fold increase in Po which was accompanied by increases in both open times and burst durations. In 3 microM and 100 microM Ca2+o the unitary conductance of the single channel currents was between 22 and 26 pS. 5. At positive membrane potentials the single channel currents had an increased Po compared to negative potentials which was associated with increased open times and burst durations but these values were similar in 3 microM, 100 microM and 1.5 mM Ca2+o. 6. In 1.5 mM Sr2+o and 1.5 mM Ba2+o channels opened to the higher conductance state of about 22-25 pS and had a 3- to 7-fold greater Po than in 0 Ca2+o. 7. In conclusion, external divalent cations have marked effects on the unitary conductance and kinetic behaviour of non-selective cation channels in rabbit portal vein smooth muscle cells.

Cadmium biosorption by a cadmium resistant strain of Bacillus thuringiensis: regulation and optimization of cell surface affinity for metal cations.

A marine bacterial strain putatively identified as Bacillus thuringiensis strain DM55, showed multiple heavy metal resistance and biosorption phenotypes. Electron microscopic studies revealed that DM55 cells are encased in anionic cell wall polymers that can immobilize discrete aggregates of cations. Factors affecting cell surface affinity for metal cations, monitored by means of Cd2+ binding capability, are investigated. The mechanisms of cadmium resistance and Cd2+ biosorption by the bacterium appeared to be inducible and coincident. Medium components affecting metal removal under cadmium-stressed growth conditions were explored based on the application of two sequential multi-factorial statistical designs. Concentrations of potassium phosphates and peptone were the most significant variables. Optimized culture conditions allowed DM55 cells grown in the presence of 0.25 mM CdCl2 to remove about 79% of the metal ions within 24 h with a specific biosorption capacity of 21.57 mg g(-1) of biomass. Both fresh and dry cells of DM55 prepared under cadmium-free optimal nutrient condition were also able to biosorb Cd2+. In addition to the concentration of phosphate in the medium, KinA, a major phosphate provider in the phosphorelay of Bacillus cells, was also demonstrated to regulate the magnitude of cell surface affinity for cadmium ions.

Uptake and intracellular distribution of labile and total Zn(II) in C6 rat glioma cells investigated with fluorescent probes and atomic absorption.

The uptake, intracellular distribution and cytotoxicity of high doses of extracellular zinc was investigated in C6 rat glioma cells. Net zinc uptake occurred only above certain thresholds in time and concentration, below them no alterations of the intracellular zinc level were observed. These results were obtained by measurements with the fluorescent dye Zinquin and by atomic absorption spectrometry, yielding similar results with both methods. Sequestration of zinc in intracellular vesicles was observed by fluorescence microscopy. A protective effect of vesicular sequestration is indicated, because increased levels of intracellular zinc located in vesicles did not necessarily lead to an increase in cytotoxicity. We were able to show that in C6 cells, in contrast to other cell lines, zinc that is released from proteins by the NO donor SNOC is also sequestered in vesicular structures. These zinc-carrying vesicles showed to be constitutive and are assumed to have a function in the maintainance of the cytosolic content of Zn2+ ions.

Divalent cation permeability and blockade of Ca2+-permeant non-selective cation channels in rat adrenal zona glomerulosa cells.

1. The effects of the divalent cations Ca2+, Mg2+ and Ni2+ on unitary Na+ currents through receptor-regulated non-selective cation channels were studied in inside-out and cell-attached patches from rat adrenal zona glomerulosa cells. 2. External Ca2+ caused a concentration-dependent and voltage-independent inhibition of inward Na+ current, exhibiting an IC50 of 1.4 mM. The channel was also Ca2+ permeant and external Ca2+ shifted the reversal potential as expected for a channel exhibiting a constant Ca2+ : Na+ permeability ratio near to 4. 3. External and internal 2 mM Mg2+ caused voltage-dependent inhibition of inward and outward Na+ current, respectively. Modelling Mg2+ as an impermeant fast open channel blocker indicated that external Mg2+ blocked the pore at a single site exhibiting a zero voltage Kd of 5.1 mM for Mg2+ and located 19 % of the distance through the transmembrane electric field from the external surface. Internal Mg2+ blocked the pore at a second site exhibiting a Kd of 1.7 mM for Mg2+ and located 36% of the distance through the transmembrane electric field from the cytosolic surface. 4. External Ni2+ caused a voltage- and concentration-dependent slow blockade of inward Na+ current. Modelling Ni2+ as an impermeant slow open channel blocker indicated that Ni2+ blocked the pore at a single site exhibiting a Kd of 1.09 mM for Ni2+ and located 13.7% of the distance through the transmembrane electric field from the external surface. 5. External 2 mM Mg2+ increased the Kd for external Ni2+ binding to 1.27 mM, consistent with competition for a single binding site. Changing ionic strength did not substantially affect Ni2+ blockade indicating the absence of surface potential under physiological ionic conditions. 6. It is concluded that at least two divalent cation binding sites, separated by a high free energy barrier (the selectivity filter), are located in the pore and contribute to Ca2+ selectivity and permeability of the channel.

Time-dependent modulation of capacitative Ca2+ entry signals by plasma membrane Ca2+ pump in endothelium.

In vascular endothelial cells, depletion of intracellular Ca2+ stores elicited capacitative Ca2+ entry (CCE) that resulted in biphasic changes of intracellular Ca2+ concentration ([Ca2+]i) with a rapid initial peak of [Ca2+]i followed by a gradual decrease to a sustained plateau level. We investigated the rates of Ca2+ entry, removal, and sequestration during activation of CCE and their respective contributions to the biphasic changes of [Ca2+]i. Ca2+ buffering by mitochondria, removal by Na+/Ca2+ exchange, and a fixed electrical driving force for Ca2+ (voltage-clamp experiments) had little effect on the CCE signal. The rates of entry of Mn2+ and Ba2+, used as unidirectional substitutes for Ca2+ entry through the CCE pathway, were constant and did not follow the concomitant changes of [Ca2+]i. Pharmacological inhibition of the plasma membrane Ca2+ pump, however, abolished the secondary decay phase of the CCE transient. The disparity between the biphasic changes of [Ca2+]i and the constant rate of Ca2+ entry during CCE was the result of a delayed, Ca(2+)-dependent activation of the pump. These results suggest an important modulatory role of the plasma membrane Ca2+ pump in the net cellular gain of Ca2+ during CCE.