Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

Evidence for the involvement of (Cu-ATP)2− in the inhibition of human erythrocyte (Ca2+ + Mg2+-ATPase by copper

The effects of copper on the activity of erythrocyte $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ have been tested on membranes stripped of endogenous calmodulin or recombined with purified calmodulin. The interactions of copper with Ca²⁺, calmodulin and (Mg-ATP)^2? were determined by kinetic studies. The most striking result is the potent competitive inhibition exerted by (Cu-ATP)^2? against (Mg-ATP)^2?$\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 2.8 μ}\text{M}$), while free copper gives no characteristic inhibition. Our results also demonstrate that copper does not compete with calcium either on the enzyme or on calmodulin. The fixation of calmodulin on the enzyme is not altered in the presence of copper as shown by the fact that the dissociation constant remains unaffected. It may be speculated that (Cu-ATP)^2? is the active form of copper, which could plausibly be at the origin of some of the pathological features of erythrocytes observed in conditions associated with excess copper.     

Lobe-specific functions of Ca2+·calmodulin in alphaCa2+·calmodulin-dependent protein kinase II activation

N-methyl-D-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.     

A reappraisal of the role of Zn2+ in TGF-β1-induced apoptosis in primary hepatocytes

There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn²⁺ on transforming growth factor-β₁ (TGF-β₁)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-β₁ (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 μmol/L Zn²⁺ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-β₁-treated hepatocytes. Surprisingly, Zn²⁺ did not inhibit the formation of high-molecular-weight DNA fragments (30–50 kbp to 250–300 kbp). These data provide evidence that Zn²⁺ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-β₁-induced apoptosis in hepatocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Defining the roles of Ca2+ — permeable channels in sperm

Ion channels exert a vital role in the dialogue between male and female gametes and thus in the generation of new individuals in many species. Intracellular Ca²⁺ is possibly the key messenger between gametes. Different Ca²⁺-permeable channels have been detected in the plasma membrane and in the organelle-like acrosome membrane of sperm, which play vital roles in determining sperm fertilizing ability. Recent reports from several laboratories have adequately documented that the Ca²⁺-permeable channels of a sperm control a variety of functions ranging from motility to the acrosome reaction. In this article, we have reviewed the data from our and other laboratories, and have documented the mechanisms of different Ca²⁺-permeable channels involved in the fertilization event.     

Regulation of Ca2+ Sparks by Ca2+ and Mg2+ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 microM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4-40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10(-7) in the frog or 10(-8) in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.     

Degradation of DNA into 5′-Monodeoxyribonucleotides in the Presence of Mn2+ Ions

DNA is known to be aggregated by metal ions including Mn²⁺ ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn²⁺ ions ([Mn]/[P] = 46.3) at 70 °C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol–chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Cd²⁺, as transition element metal ions, were effective as to the degradation into dNMP. Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺, as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl^?, CH₃OO^?, and NO₃ ^? were found to increase the degradation rate. Sixty μg of the 120 μg of the starting DNA in 450 μl was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn²⁺ ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH^? ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5′-NMP, based on the H¹NMR spectra. This prosess should prove to be a new process for the production of 5′-dNMP in addtion to the exonuclease.     

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney α-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification

Summary NADH inhibition of bovine kidney -ketoglutarate dehydrogenase complex was compared at 10 m free Ca²⁺ or in the absence of Ca²⁺ (i.e., < 1.0 nM free Ca²⁺). In the presence of Ca^2–, NADH inhibition was appreciably decreased for a wide range of NADH : NAD⁺ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 m free Ca²⁺ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca²⁺ on the S_0.5 for -ketoglutarate which was decreased by Ca²⁺ with a half-maximal effect at a similar concentration. The effect of Ca²⁺ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the -ketoglutarate dehydrogenase complex is required to elicit the effect of Ca²⁺ on NADH inhibition.At a fixed -ketoglutarate concentration (50 m), removal of Ca² reduced the activity of the -ketoglutarate dehydrogenase complex by 8,5-fold (due to an increase in S_0.5 for -ketoglutarate) and, in the presence of different NADH : NAD⁺ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxP_i) or a combination of the phosphate potential and NADH :NAD⁺ ratio are also described. The possibility that the level of intramitochondrial free Ca²⁺ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.     

Role of Ca2+ and calmodulin-dependent enzymes in the regulation of glycine transport in Müller glia

Glycine (Gly) is considered an obligatory co-agonist at NMDA receptors. Müller glia from the retina harbor functional NMDA receptors, as well as low and high affinity Gly transporters, the later identified as GLYT1. We here studied the regulation of Gly transport in primary cultures of Müller glia, as this process could contribute to the modulation of NMDA receptor activity at glutamatergic synapses in the retina. We demonstrate that neither glutamate stimulation nor the activation or inhibition of protein kinases A or C modify transport. In order to assess a function for Ca2+ and calmodulin (CaM)-dependent processes in the regulation of Gly transport, we explored the participation of Ca2+ concentration, CaM and Ca2+/CaM-dependent enzymes on Gly transporter activity. ATP and carbachol, known to induce Ca2+ waves in Müller cells, as well as caffeine-induced Ca2+ release from intracellular stores stimulated transport, whereas Ca2+ chelation by BAPTA-AM markedly reduced transport. CaM inhibitors W-7, ophiobolin A, R-24571 and trifluoperazine, induced a specific dose-dependent inhibition of transport. The inhibition of CaMKII by the autocamtide-2-related inhibitory peptide or by KN62 caused a decrease in transport which, in the case of KN62, was due to the abolition of the high affinity component, ascribed to GLYT1. Our results further suggest that Gly transport is under cytoskeletal control, as activation of calpain by major increases in [Ca2+]i induced by ionophores, as well as actin destabilization clearly inhibit uptake. We here demonstrate for the first time the participation of CaM, CaMKII and the actin cytoskeleton in the regulation of Gly transport in glia. Ca2+ waves are induced in Müller cells by distinct neuroactive compounds released by neurons and glia, hence the regulation of [Gly] by this system may be of physiological relevance in the control of retinal excitability.     

Localization of Ca2+-binding proteins of rat cortex on two-dimensional gels—II. analysis of Ca2+ binding proteins in ammonium sulfate fractions of rat brain

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and ⁴⁵Ca²⁺-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by ⁴⁵Ca²⁺-autoradiography in this study are unknown.     

Zn2+ and l-isoleucine induce the expressions of porcine β-defensins in IPEC-J2 cells

The β-defensins, expressed in epithelial cells of multiple tissues including intestine, play a critical role in the mammalian innate immunity. However, it is little known about the role of functional nutrients in the regulation of porcine β-defensins’ expressions in intestinal epithelial cells. The present study was conducted to determine the hypothesis that zinc and l-isoleucine regulate the expressions of porcine β-defensins in IPEC-J2 cells. Cells were cultured in DMEM/F12 medium containing supplemental 0–500 μg/mL l-isoleucine or 0–500 μmol/mL zinc sulfate that was used to increase the concentration of Zn²⁺ in the medium. At 12 h after the treatment by the appropriate concentrations of l-isoleucine or Zn²⁺, the mRNA and protein expressions of porcine β-defensin 1, 2 and 3 were increased (P < 0.05), and reached their maximum after treatment with 25 or 100 μmol/mL zinc sulfate and 25 or 50 μg/mL isoleucine (P < 0.05). These results suggested that both Zn²⁺ and l-isoleucine could induce β-defensins’ expressions in porcine intestinal epithelial cells.     

Volume-induced increase of K+ and Cl− permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+

Summary Ehrlich ascites tumor cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but subsequently recover their volume within 5 to 10 min with an associated KCl loss. 1. The regulatory volume decrease was unaffected when nitrate was substituted for Cl^–, and was insensitive to bumetanide and DIDS. 2. Quinine, an inhibitor of the Ca²⁺-activated K⁺ pathway, blocked the volume recovery. 3. The hypotonic response was augmented by addition of the Ca²⁺ ionophore A23187 in the presence of external Ca²⁺, and also by a sudden increase in external Ca²⁺. The volume response was accelerated at alkaline pH. 4. The anti-calmodulin drugs trifluoperazine, pimozide, flupentixol, and chlorpromazine blocked the volume response. 5. Depletion of intracellular Ca²⁺ stores inhibited the regulatory volume decrease. 6. Consistent with the low conductive Cl^– permeability of the cell membrane there was no change in cell volume or Cl^– content when the K⁺ permeability was increased with valinomycin in isotonic medium. In contrast, addition of the Ca²⁺ ionophore A23187 in isotonic medium promoted Cl^– loss and cell shrinkage. During regulatory volume decrease valinomycin accelerated the net loss of KCl, indicating that the conductive Cl^– permeability was increased in parallel with and even more than the K⁺ permeability. It is proposed that separate conductive K⁺ and Cl^– channels are activated during regulatory volume decrease by release of Ca²⁺ from internal stores, and that the effect is mediated by calmodulin.     

The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 2000     

α-adrenergic receptor regulation of Ca2+/Mg2+-ATPase in brain synaptic membranes

The effects of ₁ and ₂ receptor ligands on Ca²⁺/Mg²⁺-ATPase have been studied using synaptosomal plasma membranes isolated from rat brain cortex. Both phenylephrine and clonidine inhibited Ca²⁺/Mg²⁺-ATPase, in a concentration-dependent fashion. IC₅₀ values for half-maximal inhibition for phenylephrine and clonidine were 29 M and 18 M, respectively. The inhibitory effect of phenylephrine was reversed by the alpha antagonist prazosin while yohimbine and rauwolscine reversed the inhibition of enzyme activity by clonidine. The two antagonist subtypes were effective only against the respective agonist subtypes, demonstrating distinct subtype preferences. Analysis of the kinetics of enzyme inhibition indicate both agonists to be noncompetitive. Some evidence suggests that yohimbine may exhibit mixed agonist/antagonist properties which depend on [Ca²⁺]. The present study provides biochemical evidence to support auto receptor adrenergic receptor regulation of neurotransmitter release.     

Stimulated γδ T cells increase the in vivo efficacy of trastuzumab in HER-2+ breast cancer

One fourth of women with HER-2(+) metastatic breast carcinoma are treated with a combination regimen with trastuzumab, but the frequent resistance to this Ab requires definition of new means to improve its bioactivity. The mechanisms of action of trastuzumab involve several pathways including Ab-dependent cellular cytotoxicity. Because human γδ T lymphocytes mediate Ab-dependent cellular cytotoxicity and can be activated further by phosphoantigens, these cells are prone to improve the efficacy of Abs, as recently demonstrated for CD20(+) B cell lymphomas. Whether this concept applies as well with carcinomas remained to be demonstrated in vivo, however. In this study, we asked whether a combination of trastuzumab and phosphoantigen-stimulated γδ lymphocytes increases the efficacy of trastuzumab against HER-2(+) breast carcinoma cell lines in vivo. We report that repeated infusions of this combination had a better efficacy than that of trastuzumab alone against HER-2(+) mammary carcinoma xenografts in mice. In these models, reduction of tumor growth was observed together with trastuzumab opsonization of HER-2(+) cells and tumor infiltration by γδ lymphocytes. In addition in humans, the mammary carcinomas of 27 of 30 patients showed significant γδ T cell infiltrates. Altogether, these findings indicate that combination of trastuzumab and stimulated γδ cells represents a new strategy to improve the efficacy of Herceptin (trastuzumab) in HER-2(+) breast cancer.     

Studies in quantum biology. II. The mobile electron characteristics of tryptophan+ in relation to those of FMN−, FMNH and FMNH 2 +

By the same method as in the previous paper by the author (Bull. Math. Biophysics,23, 55–68, 1961), the mobile electron system is studied in tryptophan which has donated an electron to another molecule. The obtained net charge distribution of tryptophan⁺ is compared with those of the semiquinones of riboflavin (FMN⁻, FMNH and FMNH ₂ ⁺ ). From this comparison, a suggestion is obtained for the structure of each of two charge transfer complexes between riboflavin and tryptophan in neutral and acid solution.     

Investigation of properties of the Ca2+ influx and of the Ca2+-activated K+ efflux (Gárdos effect) in vanadate-treated and ATP-depleted human red blood cells

In this study the properties of the 45Ca2+ influx in human red blood cells (RBC) induced by NaVO3 or ATP-depletion were compared. Both NaVO3-induced and ATP-depletion-induced 45Ca2+ influxes were in the range 10(-6)-10(-5) mol Ca2+ x l(-1)cells x h(-1). The saturatability of ATP-depletion-induced 45Ca2+ influx with Ca2+ was much less pronounced than that of NaVO3-induced 45Ca2+ influx. The NaVO3-induced Ca2+ influx was sensitive to nifedipine (IC50 = 50 micromol/l) and Cu2+ (IC50 = 9 micromol/l) but these inhibitors had only a marginal effect when ATP-depletion was used as the Ca2+ influx inducer. On the other hand, polymyxin B (PXB) (1-5 mg/ml) strongly stimulated the ATP-depletion-induced 45Ca2+ influx whereas its effect on the NaVO3-induced Ca2+ influx was biphasic, with about 10% stimulation at lower PXB concentrations and an inhibition of 40% at higher concentrations. SDS-PAGE revealed that both NaVO3 and PXB induced changes in the protein phosphorylation pattern in the presence of Ca2+. NaVO3 stimulated the phosphorylation of several proteins and this effect was counteracted by PXB. The comparison of the kinetics and temperature dependencies of the Gárdos effect induced by NaVO3 and the ATP-depletion showed marked differences. The ability of NaVO3 to induce the Gárdos effect dramatically increased in ATP-depleted cells. These findings indicate that the 45Ca2+ influxes preceding the activation of the Ca2+-activated K+ efflux (Gárdos effect) stimulated by NaVO3 and by ATP-depletion, are mediated by different transport pathways. In addition, obtained results demonstrate that ATP-depletion and NaVO3-treatment exert additive action in triggering the Gárdos effect.