L1210/VCR cell line (R) was obtained by adaptation of the L1210 mouse leukaemia cells (S) to vincristine and showed P-glycoprotein (P-gp) mediated multidrug resistance (MDR). R cells were observed to be more sensitive to high external calcium as parental S. More pronounced calcium uptake was observed for R cells. Moreover, differences in intracellular calcium cell localization between S and R cells were found ultrastructurally following a calcium precipitating cytochemical method. In S cells, calcium precipitates were found to be localized predominantly along the cell surface coat and within mitochondria delineating the cristae. In R cells, precipitates were also found inside nuclei, at the border of heterochromatin clumps, and scattered within the cytoplasm. High extracellular calcium did not influence the P-gp mediated extrusion of calcein/AM as P-gp substrate. These results indicate that calcium enters and consequently damages the MDR cells to a higher extent than parental cells.

Multidrug resistance (MDR) phenotype of L1210/VCR cell line, acquired by selection for vincristine (VCR), is predominantly mediated by P-glycoprotein (Pgp). Calcein/AM (Cal) was recently described as a fluorescent substrate for Pgp and may be used for measuring of transport activity of Pgp. Expression of Pgp in the cells prevents them to be loaded with the fluorescent marker. To detect the activity of Pgp, verapamil (Ver) or cyclosporine A (CsA) has to be used as Pgp inhibitors. Multidrug resistance protein (MRP), another drug efflux pump, may be inhibited by probenecid (Pro), i.e, the inhibitor of a wide variety of anion transporters. Ver, but not Pro, is able to induce the loading of L1210/CR cells by Cal that is measurable by fluorescence-activated cell sorter (FACS). Another dye, fluo-3/AM (F-3), has a similar behaviour like Cal. Using confocal microscopy we have proved that L1210/VCR cells, in contrast to parental sensitive cells, are not loaded with F-3. Marking of cells with the dye can be achieved using inhibitors of Pgp like Ver or CsA but not by Pro. These results indicate that F-3 is usable for detection of Pgp function in various MDR tissue cells.

The 45Ca2+ influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The 45Ca2+ equilibration occurred with t1/2 = 2.5 min and the steady-state was reached after 17 min with the level of 22 ± 2 μmol/Lpacked cells at 37 °C. The rate of the influx was 97 ± 17 μmol/Lpacked cells h. The 45Ca2+ influx was saturated with [Ca2+]0 at 4 mmol/L and was optimal at pH 6.5 and 30 °C. Divalent cations (10−4–10−6 mol/L), nifedipine (10−5–10−4 mol/L), DIDS (up to 10−4 mol/L), and quinidine (10−4–10−3 mol/L), inhibited the 45Ca2+ influx while uncoupler (10−6–10−5 mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the 45Ca2+ influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca2+ influx in intact RBC. The results show that the Ca2+ influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca2+ are coupled via the H+ homeostasis similarly as in intact RBC but with modified mechanism.

The basal (45)Ca(2+) influx in human red blood cells (RBC) into intact RBC was measured. (45)Ca(2+) was equilibrated with cells with t(1/2)=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5+/-0.2 micromol/l(packed cells) (n=6) at 37 degrees C. The average value of the Ca(2+) influx rate was 43.2+/-8.9 micromol/l(packed cells) hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 degrees C. The basal Ca(2+) influx was saturable with Ca(2+) up to 5 mmol/l but at higher extracellular Ca(2+) concentrations caused further increase of basal Ca(2+) influx. The (45)Ca(2+) influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3',4',5-tetrachloro-salicylanilide (TCS) 10(-6)-10(-5) mol/l) inhibited in part the Ca(2+) influx. The results show that the basal Ca(2+) influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca(2+) influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca(2+) are coupled via the RBC H(+) homeostasis.

The redox reaction of the neurotransmitter dopamine at the carbon fibre microelectrode was studied by several electrochemical methods. It was found that under conditions usual in a living body, the diffusion current fullfils, within experimental errors, the behavior theoretically predicted by the Cottrell equation. Nevertheless, attention should be paid to the fact that unsupported or weakly supported conditions give rise to a non-Cottrell response of diffusion current. Moreover, similar changes were observed if the dopamine concentration was either lower such as several units of μmol l-1, or about 100 μmol l-1 or higher. The non-Cottrell behavior of diffusion current involves the nonlinearity of the dopamine calibration curve obtained by pulse techniques. The present work is aimed at pointing out that such behavior of the measured data could lead to misinterpretation of the obtained dopamine concentration. Similar features could be also achieved for the other catecholamines.

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.

A boron-containing antibiotic, boromycin (BM), was found to influence the Ca2+ homeostasis in both excitable and non-excitable cells. In non-excitable cells (human erythrocytes and leucocytes) it inhibited the resting passive45Ca2+ transport in 10−6–10−5 mol/L concentrations. In human erythrocytes, the passive 45Ca2+ transport induced by the presence of 1 mmol/L NaVO3 was inhibited by boromycin (90% inhibition) as well. The inhibitory effect of BM on the NaVO3-induced passive 45Ca2+ transport was diminished in the presence of inhibitory concentrations of nifedipine (10 μmol/L – 60% inhibition) or of those of K+o (75 mmol/L – 20% inhibition). On the other hand, in rat brain synaptosomes, and rat cardiomyocytes, BM stimulated the passive 45Ca2+ transport in ‘resting’ cells at similar concentrations. In rat cardiomyocytes the stimulation was transient. The stimulatory effect on the passive 45Ca2+ transport in rat brain synaptosomes was accompanied with the increase of cytoplasmic Ca2+ concentration measured by means of the entrapped fluorescent Ca2+ chelator fura-2. The stimulatory effect of BM was diminished when synaptosomes were pre-treated with veratridine (10 μmol/L) which itself stimulated the passive 45Ca2+ transport. At saturating concentrations of veratridine, no stimulatory effect of BM was observed. These results could be explained by the indirect interaction of BM with both Ca2+ and Na+ transport systems via transmembrane ionic gradients of monovalent cations and could be useful in determining whether the cells belong to excitable, or non-excitable cells.

UNLABELLED:Clearance of human peptide endothelin-1 (ET-1) has been proposed to follow a receptor pathway involving a cascade of ET-1 receptor endocytosis and lysosomal degradation by a family of proteinases expressed constitutively by most cells. Genetically distinct endopeptidases produce ET-1 and degrade mature peptide. The ET-1 degradation products were considered to be inactive, however, recent evidence suggests that ET-1 fragments sustain most of the homeostatic response produced by parent peptides. The purpose of this study was to establish whether the overall structure of human ET-1 or the structure of its C-terminus is responsible for the subtype-selectivity, down-regulation and clearance of endothelin, and whether D-aminoacid substitution in the moiety of synthetic peptide is involved in effective ET-1 antagonism in coronary vascular smooth muscle. To characterize specific mechanism(s) leading to subtype-selective ET-receptor down-regulation and/or to ET-1 antagonism, ligand binding studies were accomplished with radioactive human (1-21)ET-1 and with C-terminal ET-1 fragments, both peptide agonists and antagonists, in adult male porcine coronary artery vascular smooth muscle (CVSM). The subcellular membranes of CVSM were isolated by isopycnic gradient centrifugation. Exposure of porcine coronary artery to exogenous ET-1 induced endothelin-ETB selective down-regulation. ETA-mediated subtype-ETB down-regulation was observed with distribution of ligand-ETB receptor complexes in light, endosomal, membranes. The ETA selective PD151242 significantly attenuated [3H]-thymidine incorporation, and the ETB selective antagonist BQ788 blocked down-regulation observed in porcine vascular fibroblasts (PF). Preincubation of coronary arteries with ETB selective BQ3020 was accompanied with a more intense down-regulation.CONCLUSION:our data are indicative of short-term ETB selective down-regulation of endothelin receptors in coronary vascular smooth muscle after exposure to ET-1. The presence in the carboxy-terminus of (Ala11,15) substitution in peptide fragments IRL1620 and BQ3020 determined the differential specificity of ETB-receptor coupling and was important for subtype-ETB-receptor down-regulation. The activation of the dominating ETA-receptor by ET-1 facilitated mitogenic responses to ET-1 in porcine vascular fibroblasts.

An alternative electroanalytical technique to the widely used differential pulse voltammetry (DPV) is presented in connection with monitoring proton redox reactions in strong acids (HCl, H2SO4, HNO3) and in l-ascorbic acid (AA) in the absence of any supporting electrolyte. Contrary to the DPV method, the current flowing through the working electrode in response to a double-step change of the applied potential is first integrated and subsequently processed by a three-channel correlator. Expressing the faradaic transient charge as Q(t)∝tβ, the ratio Rβ=[Q(t1)−2Q(5t1)+Q(9t1)/(Ilimt1), where t1 is the delay of the first sampling event with respect to the trailing edge of the potential double step and Ilim is the limiting current of the corresponding steady-state voltammetric wave, it is calculated and then compared to the values found experimentally. The sensitivity to the kinetics represented as dRβ/dβ has an optimum around β=0.5, a value consistent with the Cottrell equation. The experimental data point to a crucial role of CO2 (H2CO3) dissolved in the acid solution, envisaged as the reversed sign of the measured charge. After deaerating the solution by argon the sign became positive, nevertheless the experimental Rβ values were systematically higher than the predicted ones. The reaction of the protons of AA at negative potentials seems to be of the EC type when proceeding from negative to more positive potentials. Moreover, there is a dominant voltammetric wave of AA at positive potentials coming from an irreversible reaction, accompanied by a relatively weak peak of the correlated charge.

As an alternative to the numerous state-of-the-art versions of voltammetry, a kinetics-sensitive double-step voltcoulometry is introduced. The transient current flowing in response to a potential step across the electrochemical cell is integrated and simultaneously processed by a deliberately selected time-domain “cascade” filter, while scanning the applied potential. In contrast to the widely used sampling scheme of sampling the transient current just before and in the end of the excitation pulse, three values of the transient charge are sampled in the interval between subsequent excitation pulses. Each measuring period is preceded by a single measurement of the steady-state current with the excitation pulse being switched off. The latter measurement makes it possible to actively compensate the parasitic charge across the feedback capacitor of the integrator, due to the steady-state current, while storing the steady-state current data. The goal of introducing the third sampling event resides in discriminating the kinetics of the transient charge via the parameter β that enters the time dependence of the transient charge Δq∝tβ. In general, our filtering scheme is capable of eliminating both any constant or linear components in the transient charge (β=0, 1). Moreover, any superlinear transient charge (β>1) is detected as a peak of the Δq versus potential plot with its sign being opposite to that one of a sublinear redox reaction (0<β<1). This enhanced performance of our double-step voltcoulometry is documented by a series of experiments on aqueous solutions of ascorbic acid using a carbon fiber microelectrode as the working electrode. Finally, how to assess the degree of reversibility of the redox reaction and deduce the time constant RC of the double-layer charging current is shown.

1-Deoxy-1-nitro-D-mannitol (DN-Man) was used (femoral vein injection, approximate concentration in the blood 30 mmol.l-1) in pentobarbital anaesthetized rats as a promising marker detectable by differential pulse voltammetry (DPV) to study its transport across the blood-brain barrier (BBB) to the extra-cellular space of the frontoparietal cortex. DN-Man detection limit in in vitro calibrations (saline, blood) using DPV and carbon fiber microelectrodes was 0.5 mmol.l-1 with a good linearity (r = = 0.996) over the entire tested range (up to 30 mmol.l-1). The slow time-course of the rise of DN-Man signal (y = 106/(1 + (17.8/t)3)) in the cortex confirmed the functional BBB state. Electrical stimulation of the locus coeruleus (LC) (300 rectangular pulses at a frequency of 100 Hz, 1 mA, pulse duration 0.2 ms) elevated significantly DN-Man current in the cortex (to 168 +/- 59% of the control, mean +/- S.D., n = 8). The evoked permeation increase of the BBB to DN-Man was short-lasting (minutes), and the second LC stimulation (repeated 5 min after the first one) was ineffective. This fact was probably due to the reduction of DN-Man levels in blood and/or an altered response of microvessels to neurotransmitters. It was shown here that, under carefully controlled surgical and experimental conditions, DPV and DN-Man might be useful for the monitoring of the regional dynamics of BBB transport changes. The presented results also support the view that BBB transport can be influenced by LC neuronal activity.

The objective of this study was to determine the effect of angiotensin I (Ang I) treatmentin vivoon two major Ca-transport systems—thel-type voltage dependent calcium channel (l-VDCC) and the Na/Ca exchanger in rat heart. For our experiments we used four groups of rats, treated differently with saline, Ang I, the ACE inhibitor enalapril and/or combination of both for 6 days, every 24 h. We observed an increase in the activity, and also in mRNA expression of the Na/Ca exchanger, after repeated administration of Ang Iin vivo. The maximal binding capacity of Ca-antagonist PN 200-110, which binds to theα1subunit of thel-VDCC was elevated from 0.8–1.85 pg/mg protein. mRNA expression of the voltage-dependent calcium channels ofl-type system was also upregulated by Ang I administration, but not when enalapril was applied simultaneously with Ang I. These results demonstrate thatin vivoapplication of the Ang I significantly modulates not only the activity, but also expression of the Na/Ca exchanger and thel-VDCC in rat hearts through angiotensin II (Ang II). Since in thein vitroexperiments on the isolated cardiomyocytes, Ang II (100 nm) increased the calcium uptake after depolarization, and the AT1receptor agonist losartan prevented this increase, we assume that this regulation might involve the AT1receptors.

Intrafollicular luteinization stimulator was shown to be secreted by granulosa cells in culture with stimulatory effects on differentiation of immature granulosa cells. The purpose of this study was to evaluate the role of calcium ions in luteinization stimulator-enhanced luteinization process of granulosa cells. We examined the direct effect of ionophore A23187, voltage-sensitive Ca(2+)-channel blockers verapamil, nimodipine, nifedipine, niludipine and calmodulin antagonist trifluoroperazine on progesterone and cGMP levels in 3-day culture of small granulosa cells. It was shown that the dihydropyridine derivates of calcium blocker drugs (nimodipine, nifedipine, niludipine) and calmodulin antagonist (trifluoroperazine) in the micromolar range, significantly suppressed FSH-induced progesterone synthesis and cGMP accumulation in granulosa cells. On the contrary, phenylalkylamine calcium blocker verapamil and calcium ionophore A23187 had different effects on both processes. Calcium ionophore A23187 markedly enhanced cGMP formation, but simultaneously inhibited the FSH-induced progesterone synthesis. Verapamil at lower concentrations (10 microM) stimulated and at higher concentrations (50 microM) inhibited the formation of cGMP. To evaluate the role of extra- and intracellular calcium in luteinization stimulator-enhanced progesterone production by small granulosa cells, the effects of indicative agents on stimulatory activity of follicular fluid from large follicles, granulosa cells-conditioned media and granulosa cell extracts were tested. While verapamil is shown to be a less potent modulator, administration of other calcium antagonists as well as ionophore A23187 caused a significant decrease in stimulatory action of follicular fluid from large follicles, granulosa cells-conditioned media and extracts. These findings indicate that the stimulatory action of luteinization stimulator depends on the transport of calcium ions through voltage-sensitive calcium channels and is modulated by alteration of intracellular calcium levels.

The effect of chemically initiated (KCl) spreading cortical depression on catecholamine content in the extracellular cortical space was examined using voltammetry technique in chloralhydrate-anaesthetized rats. Correlation between alterations in catechol-oxidative current (CA.OC) and the time-course of the slow potential (SP) change (one of the chief features of the spreading depression) showed that spreading depression wave was accompanied by the significant increase in CA.OC content (up to 158 +/- 43%, mean +/- S.D., P < 0.001). The rise of the negative SP preceded significant CA.OC increase by 20 +/- 8 s. This fact provides the evidence that catecholamine overflow cannot participate in triggering spreading depression wave; nevertheless it can be an important link of the spreading depression mechanism.

Voltammetric techniques were used to compare the effects of K(+)-induced depolarization on catecholamine levels in in vitro synaptosomal preparations of the corpus striatum with those in the in vivo corpus striatum of anaesthetized animals. In vitro, the catechol-oxidation currents could be recorded only in dopamine-preloaded synaptosomes. In isolated synaptosomes prepared in the presence of elevated concentrations of Ca2+ (1 mmol.l-1) and Na+ (135 mmol.l-1), K(+)-induced depolarization had variable effects on catechol-oxidation current. The stimulatory effect of K(+)-induced depolarization (a short transient increase of catechol-oxidation current lasting for 30 s) could be observed after the addition of dopamine loaded synaptosomes in EGTA into the medium with elevated K+ concentration (90 mmol.l-1) and decreased concentrations of Na+ (75 mmol.l-1) and Ca2+ (0.75 mmol.l-1). These results suggest that experimental procedures and parameters of ionic composition of incubation media have to be carefully controlled, owing to an enhanced in vitro permeability of membranes of isolated synaptosomes for Ca2+ and Na+. In in vivo experiments, microinjection of KCl (3 microliters of 0.5 mol.l-1 KCl in 10 mmol.l-1 HEPES, pH 7.4) resulted in the appearance of several phases of catechol-oxidation current: the current increased (to severalfold of the control values) followed by a decrease or even total disappearance, with a gradual return to control values. Under conditions of depletion of extracellular calcium by EGTA (5 microliters of 0.5 mol.l-1 KCl + 0.25 mol.l-1 EGTA in 10 mmol.l-1 HEPES, pH 7.4) K(+)-induced depolarization confirmed the key role of calcium in the release of catecholamine transmitters as well as that in processes regulating the uptake and metabolism of these transmitters. The voltammetric techniques used in the present study may be a useful tool in extending of our knowledge about the cellular mechanisms of stimulus-response coupling in nerve cells.

Transport of 45Ca2+ into synaptosomes was measured at 30 degrees C by rapid membrane filtration. The transport was saturable in high (135.10(-3) mol/l-1) or medium (25.10(-3) mol/l-1) K+ solutions with apparent KM values for 45Ca2+ of 0.45 and 0.1.10(-3) mol/l-1 respectively. At low K+ concentration (5.10(-3) mol/l-1) the transport was not clearly saturable by substrates. Vanadate had no effect on 45Ca2+ transport at any K+ concentration tested. Replacement of Na+ by other ions stimulated the 45Ca2+ transport with biphasic dependence on K+ concentration. Upon the substitution of choline for Na+, the dependence was monophasic and corresponded to Na/Ca antiporter reversal. The first stimulatory phase observed only in the presence of K+ corresponded to depolarization-activated transport. The phase itself occasionally showed a biphasic pattern with an extreme case of bell-shaped dependence on K+ concentration. The first phase set in immediately, whereas the onset of the second phase varied between the preparations. It could be shown that high K+ concentrations (as a rule approx. 50 mmol/l-1) may not be suitable for the study of depolarization-induced 45Ca2+ transport since they may induce NaůCa antiporter reversal. Also, it has been suggested that transport ATPases do not affect directly the basic parameters of passive 45Ca2+ transport into rat brain synaptosomes.

The binding of Ca2+ antagonists to soluble proteins obtained by ammonium sulphate precipitation from cytosol fraction of rabbit skeletal muscles was studied. The KD values for 3H D-888 and 3H PN 200-110 binding to soluble proteins were 21.3 +/- 3.1 nmol.l-1 and 28.8 +/- 8.9 nmol.l-1 respectively. Photoaffinity labelling of the soluble proteins with the arylazide 1,4-dihydropyridine probe 3H azidopine resulted in labelling of the 85-95 K protein band as determined by SDS polyacrylamide gel electrophoresis. Partial purification of prelabelled soluble sample by gel filtration on Sephadex G-150 gave a more precise molecular weight of 90 +/- 2.5K. Polyclonal antibodies prepared against Ca2+ channel complex from rabbit muscle T-tubules inhibited the 3H PN 200-110 binding. Our results suggest that the soluble protein with Mr = 90K +/- 2.5K may be a precursor of the large subunit of the membrane bound L-type Ca2+ channel in rabbit skeletal muscle.

Proteins with Na+-Ca2+ exchange activity from the soluble fraction of crayfish striated muscle were inserted into asolectin proteoliposomes. A pH dependent calcium uptake with an optimum at the alkaline side and inhibition in the presence of sodium or strontium ions in the external medium was observed. When expressed per tissue wet weight the capacity for Na+-Ca2+ exchange of proteoliposomes with inserted soluble proteins was by one half higher than that of the membrane fraction and more than twice higher in comparison with the reconstituted membrane bound exchanger. Using polyacrylamide gel electrophoresis two most prominent proteins with Mr over 200 and 43 kDa could be detected in proteoliposomes with the highest Na+-Ca2+ exchange. It is assumed that protein(s) with Mr 43 kDa could represent the soluble Na+-Ca2+ exchanger in crayfish striated muscle soluble fraction.