Biomedical subjects

K Ondrias

Publications and source records attributed to K Ondrias.

At least 37 records · Page 2Linked to original sources

PubMed1996

Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation.

Tyrosine kinases indirectly raise intracellular calcium concentration ([Ca2+]i) by activating phospholipases that generate inositol 1,4,5-trisphosphate (IP3). IP3 activates the IP3 receptor (IP3R), an intracellular calcium release channel on the endoplasmic reticulum. T cell receptor stimulation triggered a physical association between the nonreceptor protein tyrosine kinase Fyn and the IP3R, which induced tyrosine phosphorylation of the IP3R. Fyn activated an IP3-gated calcium channel in vitro, and tyrosine phosphorylation of the IP3R during T cell activation was reduced in thymocytes from fyn-/- mice. Thus, activation of the IP3R by tyrosine phosphorylation may play a role in regulating [Ca2+]i.

Animals↗

PubMed1996

Single channel properties and calcium conductance of the cloned expressed ryanodine receptor/calcium-release channel.

The calcium-release channel/ryanodine receptor of the sarcoplasmic reticulum is a 2.3 million-D structure required for intracellular calcium release during excitation-contraction coupling in skeletal muscle. This structure is the largest ion channel characterized to date and is composed of four 565,000-D ryanodine receptors plus four molecules of FKBP12. In the present study we describe the single channel properties of the cloned expressed ryanodine receptor, with and without FKBP12, reconstituted into planar lipid bilayers with Ca as the charge carrier. The conductance for Ca (luminal, 53 mM/cytoplasmic, 10 microM) was 103 pS for the cloned expressed RyR and for the native channel from rabbit skeletal muscle. Conductance through the channel was Ca dependent: A decrease in the Ca gradient to luminal 10.6/cytoplasmic 10 microM reduced conductance to 68 pS for both the cloned and native RyR. The recombinant ryanodine receptor consistently behaved like the native skeletal muscle channel in terms of activation by caffeine, calcium, and ATP; inhibition by ruthenium red; and modulation by ryanodine. In the absence of FKBP12, the cloned expressed RyR exhibited multiple subconductance states and addition of FKBP12 reduced the frequency of subconductance states. These results show that with Ca as the charge carrier, the single channel properties of the cloned expressed RyR plus FKBP12 are essentially the same as those of the native channel.

Animals↗

PubMed1995

Increased cytotoxicity of 3-morpholinosydnonimine to HepG2 cells in the presence of superoxide dismutase. Role of hydrogen peroxide and iron.

3-Morpholinosydnonimine (SIN-1) is widely used to generate nitric oxide (NO(x).) and superoxide radical (O2-.). The effect of SOD on the toxicity of SIN-1 is complex, depending on what is the ultimate species responsible for toxicity. SIN-1 (< 1 mM) was only slightly toxic to HepG2 cells. Copper, zinc superoxide dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD) increased the toxicity of SIN-1. Catalase abolished, while sodium azide potentiated, this toxicity, suggesting a key role for H2O2 in the overall mechanism. Depletion of GSH from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD. Although Me2SO, sodium formate, and mannitol had no protective effect, iron chelators, thiourea and urate protected the cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose response being most pronounced at lower concentrations (10-100 units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of H2O2 in a concentration-dependent manner. In contrast, Cu,Zn-SOD increased H2O2 accumulation from SIN-1 at low but not high concentrations of the enzyme, suggesting that high concentrations of the Cu,Zn-SOD interacted with the H2O2. EPR spin trapping studies demonstrated the formation of hydroxyl radical from the decomposition of H2O2 by high concentrations of the Cu,Zn-SOD. The cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly enhanced by SOD; catalase had no effect. Thus, the oxidants responsible for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these conditions are different, with H2O2 derived from O2-. dismutation playing a major role with SIN-1. These results suggest that the potentiation of SIN-1 toxicity by SOD is due to enhanced production of H2O2, followed by site-specific damage of critical cellular sites by a transition metal-catalyzed reaction. These results also emphasize that the role of SOD as a protectant against oxidant damage is complex and dependent, in part, on the subsequent fate and reactivity of the generated H2O2.

Catalysis↗

PubMed1995

The inositol 1,4,5-trisphosphate receptor is essential for T-cell receptor signaling.

Antigen-specific activation of T lymphocytes, via stimulation of the T-cell antigen receptor (TCR) complex, is marked by a rapid and sustained increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). It has been suggested that the second messenger inositol 1,4,5-trisphosphate (IP3) produced after TCR stimulation binds to the IP3 receptor (IP3R), an intracellular Ca(2+)-release channel, and triggers the increase in [Ca2+]i that activates transcription of the gene for T-cell growth factor interleukin 2 (IL-2). However, the role of the IP3R in T-cell signaling and possibly in plasma membrane Ca2+ influx in T cells remains unproven. Stable transfection of T cells (Jurkat) with antisense type 1 IP3R cDNA prevented type 1 IP3R expression, providing a tool for dissecting the role of IP3 signaling during T-cell activation. T cells lacking type 1 IP3R failed to increase [Ca2+]i or produce IL-2 after TCR stimulation. Moreover, depletion of intracellular Ca2+ stores without TCR activation stimulated Ca2+ influx in cells lacking the type 1 IP3R. These results establish that the type 1 IP3R is required for intracellular Ca2+ release that triggers antigen-specific T-cell proliferation but not for plasma membrane Ca2+ influx.

Antigens, CD↗

PubMed1995

Expressed ryanodine receptor can substitute for the inositol 1,4,5-trisphosphate receptor in Xenopus laevis oocytes during progesterone-induced maturation.

Two structurally related forms of intracellular calcium release channels that can mediate the release of intracellular calcium have been identified: the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate receptors (IP3R). Each channel responds to distinct pathways for activation. The IP3R is activated by IP3 and the RyR is thought to be activated by calcium or by another second messenger cADP ribose. It has been proposed that each type of channel subserves a specialized pool of intracellular calcium, and it is not understood why some cell types require more than one form of intracellular calcium release channel. The present study was designed to examine whether the RyR can substitute for the IP3R during oocyte maturation. IP3R expression was inhibited in Xenopus laevis oocytes using antisense oligonucleotides. These oocytes, with reduced levels of IP3R, demonstrated a marked delay in the time course of progesterone-induced maturation. The cloned skeletal muscle RyR1 was then expressed in X. laevis oocytes that were deficient in IP3R. Functional studies showed that the properties of the cloned RyR1, expressed in oocytes, were comparable to those of the native RyR1. X. laevis oocytes deficient in IP3R, but expressing RyR1, were able to undergo progesterone-induced maturation with a time course comparable to that seen in wild-type oocytes when caffeine was used to activate RyR and induce intracellular calcium release. These studies show that RyR1 can substitute for the IP3R as the intracellular calcium release channel required for Xenopus oocyte maturation and that intracellular calcium release is important for controlling the rate of progesterone-induced maturation.

Animals↗

PubMed1994

Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein.

FK506-binding protein (FKBP12) was originally identified as the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. The cellular function of FKBP12, a ubiquitously expressed 12,000-dalton proline isomerase, has been unknown. FKBP12 copurifies with the 565,000-dalton ryanodine receptor (RyR), four of which form intracellular Ca2+ release channels of the sarcoplasmic and endoplasmic reticula. By coexpressing the RyR and FKBP12 in insect cells, we have demonstrated that FKBP12 modulates channel gating by increasing channels with full conductance levels (by > 400%), decreasing open probability after caffeine activation (from 0.63 +/- 0.09 to 0.04 +/- 0.02), and increasing mean open time (from 4.4 +/- 0.6 ms to 75 +/- 41 ms). FK506 or rapamycin, inhibitors of FKBP12 isomerase activity, reverse these stabilizing effects. These results provide the first natural cellular function for FKBP12, and establish that the functional Ca2+ release channel complex includes FKBP12.

Animals↗

PubMed1994

Comparison of antioxidant properties of nifedipine and illuminated nifedipine with nitroso spin traps in low density lipoproteins and phosphatidylcholine liposomes.

Illumination nifedipine, a calcium channel blocker, gives a nitroso-compound, 2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridine-dicarboxylic acid dimethyl ester (NTP), which has spin trapping properties. The antioxidant ability of NTP was tested in a model of lipid peroxidation in low density lipoproteins (LDL) and phosphatidylcholine liposomes, and was compared with parent nifedipine and with other nitroso spin traps such as 3,5-dibromo-4-nitrosobenzene-sulfonic acid (BNTB), nitrosobenzene (NTB) and 2-methyl-2-nitrosopropane (MNP). Nifedipine (20-200 mumol/l) did not inhibit lipid peroxidation either in LDL on in liposomes, whereas its photolytical product NTP was found to be very effective at the same concentrations. The average antioxidant potencies of the nitroso spin traps were similar in both models and decreased in the order: NTP > or = BNTB > NTB > or = MNP. As detected by EPR spectroscopy, the studied nitroso compounds formed stable nitroxide radicals in a pseudo-Diels-Alder reaction, as a result of their interaction with unsaturated bonds of lipids in LDL and liposomes. The relative concentrations of thus formed radicals were in the order: NTP >> BNTB >> NTB approximately MNP and were related to their antioxidant properties. Thus it seems that the ability of the nitroso-compounds to form nitroxide radicals with unsaturated lipids may play a role in the antioxidant effect of these compounds.

Antioxidants↗

PubMed1994

Reactive radical intermediates formed from illuminated nifedipine.

Nifedipine, (1,4-dihydro-2,6,dimethyl-4-(2-nitrophenyl)-3, 5-pyridinedicarboxylic acid dimethyl ester) a calcium channel blocker widely used in treatment of hypertension, is strongly photolabile. This may represent a problem in patients taking nifedipine and in handling of nifedipine samples. Reactive radical intermediates were determined and characterized in the process of nifedipine illumination using EPR spectroscopy. On illumination of nifedipine by daylight or by a mercury lamp, a nitroxide radical, RIIL-NIFNO.X was observed (in the first step), in various solvents like benzene, cyclohexane, methanol, acetonitrile, dimethylsulphoxide, or aqueous suspensions of liposomes. RIIL-NIF represents the nifedipine skeleton centered with phenyl group, and X is an EPR silent substituent. The generation of RIIL-NIFNO.X is coupled with the formation of nitroso compound, RIIL-NIFNO, as characterized by UV-visible spectroscopy. In a further step, RIIL-NIFNO abstracts hydrogen from nifedipine skeleton under the formation of RIIL-NIFNO.H radical. In addition to this, in system containing RIIL-NIFNO and unsaturated lipids, nitroxide radicals RIIL-NIFNO.RLIPIDS are formed probably via a pseudo Diels-Alder mechanism (RLIPIDS represents lipidic skeleton). The unusually easy photochemical activation of nifedipine is probably stimulated by photosensitization of its nitro group interacting with suitably positioned hydrogen or carboxylic methyl ester group from the pyridinyl ring.

Electron Spin Resonance Spectroscopy↗

PubMed1994

An unusual temperature dependence of malondialdehyde formation in Fe2+/H2O2-initiated lipid peroxidation of phosphatidylcholine liposomes.

Determination of malondialdehyde is a widely used procedure for measurement of lipid peroxidation. In this paper we report an unusual temperature dependence of malondialdehyde formation in egg yolk phosphatidylcholine liposomes oxidized by the Fenton system (0.1 mmol/l FeSO4 and 0.05 mmol/l H2O2). The amount of malondialdehyde formed was 37% higher in samples kept at 22 degrees C than at 50 degrees C. An alternative method for determination of lipid peroxidation, measurement of oxygen uptake, revealed complete consumption of dissolved oxygen to peroxidized lipids at 22 degrees C as well as 50 degrees C. Since oxygen is essential for the formation of cyclic peroxides--precursors of malondialdehyde--we conclude that the nature of the observed effect consists in limitation of oxygen availability at elevated temperatures.

Animals↗

PubMed1993

Activation of the calcium release channel (ryanodine receptor) by heparin and other polyanions is calcium dependent.

Heparin has been used as a potent competitive inhibitor of inositol 1,4,5-trisphosphate (IP3)-binding to IP3 receptors and to block IP3-gated calcium channels in bilayer experiments. In contrast to the effect on the IP3-gated channel, heparin (0.1-1 micrograms/ml) opened the Ca release channel (ryanodine receptor). Other polyanions such as pentosan polysulfate and polyvinyl sulfate also activated the Ca release channel. The effect of polyanions on the Ca release channel was Ca dependent. Polyanion addition activated the Ca release channel when free Ca was > 80 nM, but was ineffective when free Ca was < 20 nM. The level of channel activation could be altered by manipulating the free Ca concentration. These results suggest that the polyanions act by increasing the local concentration of Ca near regulatory sites on the channel complex. As most cells have both types of intracellular channels, the opposite effects of the polyanions on the two channel types suggests that addition of polyanions to intact cells may produce multiple effects.

Animals↗

PubMed1993

Reaction kinetics of alpha-tocopheroxyl radical with biologically and pharmacologically active substances.

The alpha-tocopheroxyl radical (alpha TR.)generated in the reaction with 1,1-diphenyl-2-picrylhydrazyl in n-butanol decayed according to second-order kinetics with a rate constant k alpha = 3 x 10(3) M-1s-1 as determined by EPR spectroscopy. Various biologically and pharmacologically active substances like isoprenaline (ISO), epinephrine (EPI), histamine (HIS), stobadine (STO), nafazatrom (NAF) and Kampo C medicine (KMC) accelerated the decay rate of alpha TR(.). The whole process is formally a third-order reaction with the rate constants (in 10(9) M-2s-1): ks(ISO) = 1.28, ks(NAF) = 1.25, ks(EPI) = 0.6, ks(HIS) = 0.4, and ks(STO) = 0.1. In the kinetics of the reaction mechanism, bimolecular intermediates are assumed and the rate constants of their formation were determined.

Antioxidants↗

PubMed1993

[Antioxidative properties of cardioactive agents].

Calcium entry blockers reduce the ischemia-reperfusion induced damage to the myocardium. Free oxygen radicals play a role in the pathogenesis of this injury. The mechanism of the protective action of calcium entry blockers is unknown in these conditions and appears to be independent of the blockade of slow calcium channels. As demonstrated in this paper, the calcium entry blockers nifedipine, verapamil and diltiazem are effective scavengers of free oxygen radicals and this characteristic may be importantly involved in their protective effect against myocardial damage induced by ischemia and reperfusion. The pro-oxidative capacity of the antineoplastic drug cisplatin is assumed to play a role in the mechanism of its renal toxicity action. The presented study showed that the new antioxidative substance with antiarrhythmic and antihypoxic properties-stobadine-succeeded in inhibiting the pro-oxidative effect of cisplatin. Our findings suggest that administration of cisplatin along with antioxidants may result in a protective effect against the renal toxicity of cisplatin. (Fig. 5, Ref. 38.)

Animals↗

PubMed1993

Incorporation of amphotericin B(Fungizone) in rat brain total lipid liposomes markedly decreases its i.v. toxicity in mice.

Toxicity of the clinically utilized formulation of amphotericin B (1; Fungizone) incorporated in different lipid liposomes was compared in mice. The relative toxicity of i.v. administration of 67 mg Fungizone/kg (30 mg 1/kg), was in the order: Fungizone > Fungizone in phosphatidylcholine liposomes > Fungizone in phosphatidylcholine/cholesterol liposomes > Fungizone in rat brain total lipid liposomes. The survival time of the mice after administration of Fungizone and of Fungizone in total lipid liposomes was less than 1 min and over 30 d, respectively. Using EPR spectroscopy of spin probes a relation between dynamics and disorder of the Fungizone liposomes and their toxicity was observed.

Amphotericin B↗

PubMed1992

Perturbation effect of the diheptanoyl phosphatidylcholine on rat brain total lipid liposomes. An electron paramagnetic resonance spectroscopy study.

Diheptanoyl phosphatidylcholine (DHPC) was reported to reduce inward sodium current in squid giant axons (Hendry et al., Biophys. J., 47, (1985) 841) and to decrease the frequency of the nicotinic acetylcholine-activated ion channel (Braun and Haydon, Pfügers Arch., 62, (1991) 418). To understand the DHPC effects, an influence of DHPC to increase dynamics/disorder (perturbation effect) in liposomes prepared from rat brain total lipids was studied at the 5th, 12th and 16th carbon membrane depths using the method of EPR spectroscopy of the spin labelled stearic acids and 1-palmitoyl-2-stearoyl phosphatidylcholines. The perturbation efficiency of DHPC in the liposomes was quantitated with the initial slope value of the order parameter S or inner splitting Amin versus DHPC concentration. DHPC at the DHPC/total lipid molar ratio of 1:10, 1:5, 1:3 and 1:2 increased dynamics/disorder of the liposomes. When the perturbation effect, compared at temperature scale, was normalized to 1 at the 5th carbon depth, the relative perturbation effect of DHPC was 1, 4.2, and 6.9 at the 5th, 12th and the 16th carbon depth, respectively, as detected by spin labelled stearic acids. Using the spin labelled lipids the perturbation effect was 1, 1.9 and 2.3, respectively. The differences of the perturbation effect of DHPC at different membrane depths correspond to the published perturbation effect of the local anesthetics lidocaine, tetracaine, dibucaine, heptacaine, IR-9 and carbisocaine on total lipid liposomes. The comparable perturbation properties of DHPC and of the local anesthetics support the hypothesis that the membrane perturbation caused by DHPC may play an important role in its effect on membrane function.

Animals↗

PubMed1992

Perturbation effect of eight calcium channel blockers on liposomal membranes prepared from rat brain total lipids.

EPR spectroscopy of phosphatidylcholine or stearic acid labeled at the doxyl group at the 16-carbon position was used to compare the perturbation effect of eight calcium channel blockers (CB) on overall dynamics/disorder of the hydrophobic part of liposome membranes prepared from rat brain total lipids at the drug/lipid molar ratio of 1/2. Nifedipine (NIF), nimodipine, niludipine and nitrendipine had a minor effect on the dynamics/disorder of the liposome membranes, whereas the disordering effect of verapamil (VER), mepamil, gallopamil and diltiazem was more pronounced. Concentration dependence of the overall disordering effect of VER on liposomal membranes was found at the VER/lipid ratio greater than 0.02 and for the tranquilizer thioridazine greater than 0.005. VER exerted a disordering effect at the hydrophobic part of synaptosomal membranes at concentrations greater than or equal to 0.32 mmol/l, whereas NIF did not exhibit a disordering effect even at concentrations of 10-20 mmol/l.

Animals↗

PubMed1992

Formation of stable free radicals from kampo medicines TJ-9, TJ-15, TJ-23, TJ-96, TJ-114 and their antioxidant effect on low density lipoproteins.

The Japanese herbal Kampo medicines TJ-9 (A), TJ-15 (B), TJ-23 (C), TJ-114 (D) and TJ-96 (E) were effective (2-5x less than alpha-tocopherol) in inhibiting a copper-induced peroxidation of low density lipoprotein. Kampo medicines dissolved in n-butanol formed stable free radical(s), detected by EPR spectroscopy as a single asymmetric line with g-value g = 2.005. The radical concentration increased in the order: C less than D approximately A approximately E less than B. When the Kampo medicines were oxidized in n-butanol by excess of PbO2 their radical concentration increased 7-15 fold and was in the order C less than D less than A approximately E much less than B. A relationship between the potency of the medicines to inhibit peroxidation of LDL and their ability to form stable free radicals upon oxidation was observed. The medicine which formed more radicals was more efficient in inhibiting peroxidation of LDL. In order to study whether Kampo medicines can reduce alpha-tocopherol radical, the alpha-tocopherol radical was generated by the reaction of alpha-tocopherol with UV irradiated di-tert-butylperoxide and by autooxidation of alpha-tocopherol in n-butanol (25 microliters ml-1) in air. In both systems vitamin-C greater than Kampo B decreased the concentration of the alpha-tocopherol radical and the EPR spectrum of Kampo B stable radical(s) appeared. The effect of other Kampo medicines was not clearly seen since their EPR spectra were superimposed with the spectrum of the alpha-tocopherol radical. The results indicate that Kampo medicines possess electron donor properties and ability to form stable radical(s). The results may contribute to understanding beneficial effects of Kampo medicines in diseases in which free radical damage is suggested.

Antioxidants↗

PubMed1992

Effect of cisplatin, carboplatin and stobadine on lipid peroxidation of kidney homogenate and phosphatidylcholine liposomes.

The effects of the nephrotoxic, anticancer agents cisplatin (CDDP) and carboplatin (CBDCA), and the free radical scavenger, stobadine, were investigated on lipid peroxidation (LPO) of rat kidney homogenates and phosphatidylcholine (PC) liposomes. Kidney homogenates were incubated in air at 37 degrees C for 6-48 h and lipid peroxidation was detected spectroscopically as absorbance (533 nm) of the thiobarbituric acid-malondialdehyde (TBA-MDA) complex. CDDP (0.3-10 mmol.l-1) increased LPO of the homogenate. CBDCA decreased the TBA-MDA absorbance, yet was found to interfere with MDA, TBA and/or with the TBA-MDA complex. Thus when CBDCA is involved, the TBA-MDA method for detection of LPO is not suitable. Stobadine (0.1 mmol.l-1 and 1 mmol.l-1) inhibited LPO either in the control homogenate and in the homogenate where peroxidation was increased by CDDP. The effect of CDDP and CBDCA on peroxidation of PC liposomes was monitored as oxygen consumption using a Clark-type oxygen electrode. CDDP increased but CBDCA decreased the rate of oxygen consumption during the peroxidation of liposomes induced by FeSO4. The results suggest that the effects of CDDP and CBDCA on LPO may be linked with their nephrotoxicity.

Animals↗

PubMed1992

A spin probe study of the effects of chlorpromazine and its derivatives on lipid-protein interactions in synaptosomal membranes.

The electron spin resonance spectra of 16-doxyl stearic acid (16-SA) incorporated into synaptosomes mostly showed a fluid lipid component and a minor motionally-restricted component (MRC) of the molar fraction of 10-20%, measured at 0 degree C. At 10 mmol/l concentration, thioridazine (TRZ), chlorpromazine (CPZ), chlorprothixene (CPT), perphenazine (PFZ) and levopromazine (LPZ) raised the MRC molar fraction in the synaptosomes to 100, 92, 65, 41 and 39%, respectively (as detected by the spin probe at 0 degrees C). At 4% concentration, TRZ, CPZ, CPT, PFZ, and LPZ the respective MRC percentages were 100, 75, 41, 24 and 17%. In synaptosomal membranes, AMRC splitting values of MRC, induced by TRZ and CPZ, were similar to those of the probe in human serum albumin. MRC induced by CPZ and TRZ was constant (+/- 15%) within the temperature range from 0 to 30 degrees C. At drug/lipid ratios > or = 2 : 1, TRZ and CPZ formed rigid complexes with total lipids isolated from the rat brain. The complexes melted upon increasing the temperature of the samples over 10-20 degrees C. The drugs decreased the lipid concentrations in synaptosomes in the order of potency TRZ > CPZ > CPT > PFZ > or = LPZ; this was similar to their effect on MRC increase. The drugs tested increased the membrane dynamics/disordering, and their potency fairly correlated with their MRC increasing effects. It is supposed that the drug-induced 16-SA probe MRC increase in synaptosomes was a result of mainly decreased lipid/protein ratio in the synaptosomal membranes, which in turn probably is connected with perturbation of lipid-protein interactions and/or membrane proteins. The perturbation of lipid-protein interactions and/or membrane proteins may be connected with the drug perturbation effect on the bulk lipid membrane part.

Animals↗