Effect of human defensins on the cytoplasmic Ca2+ content in platelets

The effect of the total fraction of human defensins (HNP-1, HNP-2, and HNP-3) on the cytoplasmic Ca²⁺ content ([Ca²⁺]_i) in the platelets of healthy donors was studied. At concentrations of 0.1–40 μg/ml and an incubation time of 10 min defensins have no effect on [Ca²⁺]_i in platelets labeled with Fura-2AM. However, at higher concentrations (100 μg/ml) they increased platelet [Ca²⁺]_i. In addition, defensins (40 μg/ml) inhibited the Ca²⁺ increase in platelets induced by thrombin, adenosine diphosphate, and the lipopolysaccharide ofS. typhimurium endotoxin. The most pronounced inhibitory effect was observed in a suspension of thrombin-stimulated platelets. It is shown that the effect of human defensins on the functional activity of platelets is due to the alterations in the intracellular Ca²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 600–603, December, 1994     

Effects of isoproterenol and suphan on the concentration of free calcium ions in isolated cardiomyocytes: a comparative study

Isoproterenol and suphan, two cardioactive drugs with different mechanisms of action, are studiedin vitro for their effects on calcium homeostasis in myocardial cells. Isoproterenol lowers the basal Ca²⁺ level in resting cardiomyocytes and potentiates its rise in these cells after their induction. Suphan stimulates reversible elevation of the diastolic Ca²⁺ concentration, causing increased calcium accumulation in the sarcoplasmic reticulum of cardiomyocytes. In anin vitro model of hypoxia, the Ca response to isoproterenol is significantly reduced, whereas that to dibutyryl cAMP is retained. The effect of suphan on the Ca²⁺ content of cardiomyocytes exposed to “chemical” hypoxia is 30–50% higher than its effect on the Ca²⁺ content of intact cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 8 pp. 170–172, August, 1996     

A study of the mechanism of action of befol on Ca2+ metabolism in cardiomyocytes using a FURA-2 fluorescent probe

The dynamics of the Ca-response of cardiomyocytes is studied and the efficiency of befol, verapamil, and amiodarone is compared using various experimental models of stimulation of [Ca²⁺]_i. Befol (1–5 μM) is shown to inhibit the caffeine-and strophanthin G-induced rise of [Ca²⁺]_i. Unlike verapamil and amiodarone, befol exhibits no Ca-blocking activity in modeled K-depolarization. It is concluded that the cardiotropic effect of befol is mediated through its primary action on Na⁺/Ca²⁺ exchange in cardiomyocytes, while the cardioplegic effect of verapamil and amiodarone is due to their ability to block the slow Ca²⁺ inward current. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 3, pp. 288–291, March, 1996     

Activation and inactivation kinetics of a Ca2+-activated Cl- current: photolytic Ca2+ concentration and voltage jump experiments

The activation kinetics of the endogenous Ca²⁺-activated Cl⁻ current (I _Cl,Ca) from Xenopus oocytes was investigated in excised “giant” membrane patches with voltage and Ca²⁺ concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca²⁺ concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca²⁺-dependent. The lag phase decreases from about 10 ms after a small Ca²⁺ concentration jump (0.1 μM) to less than 1 ms after a saturating concentration jump (55 μM). The rate constant of the rising phase is half-maximal at about 5 μM. At saturating Ca²⁺ concentrations, the rate constant is 400 to 500 s⁻¹. The Ca²⁺ dependence of the stationary current can be described by the Hill equation with n=2.3 and K _0.5=0.5 μM. The amplitude of the stationary current decreases after the excision of the membrane patch with t _1/2≈5 min (run-down). The activation kinetics of the current elicited by a Ca²⁺ concentration jump is not affected by the run-down phenomenon. At low Ca²⁺ concentration (0.3 μM), voltage jumps induce a slowly activating current with voltage-independent time-course. Activation is preceded by an initial transient of about 1-ms duration. At saturating Ca²⁺ levels (1 mM), the initial transient decays to a stationary current. The transient can be explained by a voltage-dependent inactivation process. The experimental data reported here can be described by a linear five-state reaction model with two sequential voltage-dependent Ca²⁺-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.     

Mobilization of intracellular Ca2+ induced by ADP and thrombin in platelets from diabetic patients with vascular complications

It is shown that the baseline level of cytoplasmic Ca²⁺ in platelets from diabetic patients is nearly 1.5 times as high as in healthy donors. The thrombin-induced increase of intracellular Ca²⁺ in patients with angiopathies is reliably lower than in the control, while in patients without angiopathies it is higher than that in donor platelets. The evevation of cytoplasmic Ca²⁺ induced by ADP is greater in both groups of patients. No Changes are found in the baseline level of intracellular Ca²⁺ or in the ADP-induced concentration of Ca²⁺ in platelets from diabetic patients during a 12-week course of insulin therapy. The intracellular Ca²⁺ does not rise after 2 weeks of insulin treatment in platelets from diabetic patients. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 1, pp. 112–115, January, 1996     

Effects of bioglycans isolated from birch fungiinonotus obliquus on electric activity of venous sinus cells in frog heart

Bioglycans isolated from chaga in a concentration of 0.0001% reduced frequency of action potential in venous sinus cells of frog heart during the first 15–30 min of exposure, then this parameter increased by 10% per hour over 3.5 h, and was 41±3 min⁻¹ from the 4th to the 20th hour of incubation. The frequency of action potentials in heart strips in the chaga extract was 40% higher than in Ringer's solution. The effect of chaga bioglycan is probably associated with adsorption on myocyte membranes. Binding of Ca²⁺ to bioglycans observed during the first 30 min inhibited efflux of intracellular Ca²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 264–266, September, 1999     

Comparative study of the effect of amiridine on biological membranes

The effects of amiridine and of the comparable drugs tacrine and piracetam on synaptosomes and membranes of sarcoplasmic reticulum were studied by electron paramagnetic resonance; in addition, the effects of these drugs on the activity of Ca²⁺, Mg²⁺-dependent ATPase regulating calcium transport in neurons were investigated. In concentrations of 10⁻⁷ to 10⁻⁵ M the drugs did not affect the structure of synaptosomal membranes of rat brain. Amiridine and tacrine in a concentration of 0.1 mM reduced the rate of calcium ion transport across the sarcoplasmic reticulum membrane by inhibiting the function of Ca²⁺, Mg²⁺-dependent ATPase and induced marked changes of the structural rigidity of the protein part of the membrane. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N№ 11, pp. 503–505, November, 1995 Presented by Yu. A. Romanov, Member of the Russian Academy of Medical Sciences     

Inhibition of platelet aggregation by monoclonal antibodies against glycoprotein IIb–IIIa complex

Monoclonal antibodies CRC64 are obtained against Ca²⁺-dependent glycoprotein IIb–IIIa complex of the platelet membrane which possess the ability to inhibit completely fibrinogen-dependent platelet aggregation. CRC64 is directed against the epitope formed by the glycoprotein IIb–IIIa complex and does not interact with proteins isolated after platelets are treated with ethylenediamine tetraacetate. Complete, reproducible blockade of platelet aggregation caused by 5 μM adenosine diphosphate is noted in an MCA concentration of 3 μg/ml, while in the case of a stronger inductor, namely 1 U/ml thrombin, platelet aggregation is inhibited in a concentration of 5 μg/ml. F(ab′)₂ fragments are also able to inhibit platelet aggregation completely and are usually effective in concentrations lower than native monoclonal antibodies. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 10, pp. 402–405, October, 1994 Presented by V. N. Smirnov, Member of the Russian Academy of Medical Sciences     

Effect of ricin and its B-subunit on calcium responses in human lymphocytes

The effect of ricin and its B-subunit on cytoplasmic Ca²⁺ concentration in human lymphocytes was studied using a fluorescence probe. Both agents dose-dependently increased cytosolic free Ca²⁺ by stimulating its release from of thapsigargin-sensitive store. It is suggested that this effect is associated with the increase in cytosolic content of inositol-1,4,5-trisphosphate. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 3, pp. 274–276, March, 2000     

Effects of intracellular calcium on GABAA receptors in mouse cortical neurons

 Using the patch-clamp technique, we studied the effect of intracellular Ca²⁺ on Cl^– current gated by type A γ-aminobutyric acid receptors (GABA_A) in mouse cortical neurons. When the rapid Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) was in the pipette solution, the GABA-activated Cl^– current amplitude decreased over time to 49 ± 7% of control. In contrast, equimolar replacement of BAPTA with ethylenebis(oxonitrilo)tetraacetate (EGTA) caused a 60 ± 10% increase in GABA current. An increased intracellular Ca²⁺ concentration caused a transient augmentation of the GABA current. This effect of Ca²⁺ was concentration dependent (10 nM to 34 μM). Ca²⁺ increased the amplitude of the current by enhancing the maximal response to GABA rather than by changing the affinity of the receptor to GABA (EC₅₀ = 5 ± 0.4 μM vs. 7 ± 0.3 μM). Both calmodulin (CaM) and a CaM kinase II inhibitor (200 μM) blocked the potentiating effect of Ca²⁺ suggesting that it was mediated by activation of CaM kinase II. We found that regulation of GABA_A receptors by intracellular Ca²⁺ in cortical neurons has important physiological implications since the potentiating effect of increasing the intracellular Ca²⁺ on responses to GABA was mimicked by activating excitatory receptors with 100 μM N-methyl-D-aspartate (NMDA). These findings suggest that modulation of GABA_A receptor activity by glutamate may be brought about via changes in intracellular Ca²⁺. Received: 20 May 1997 / Received after revision: 12 August 1997 / Accepted: 1 September 1997     

Antioxidant probucol as an effective scavenger of lipid radicals in low density lipoproteinsin vivo andin vitro

Probucol in concentrations of 10–15 μM effectively inhibits Cu²⁺-induced free radical oxidation of native low density lipoproteins and in concentration of 100 μM it inhibits lipoperoxide formation. The mean plasma concentration of probucol in patients receiving 250 mg of this drug is 25 μM. Both 250 and 1000 mg probucol daily during 3–6 month block the oxidation of isolated low density lipoproteins. Electron paramagnetic resonance spectrometry data showed that probucol incorporatedin vivo into lipoprotein particles interacts with lipid radicals yielding long-lived phenoxyradicals. Probucol can be used in complex therapy of atherosclerosis as an antioxidant drug and its dose required for lipoprotein protection against atherogenic modification can be decreased to 250 mg/day. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 8, pp. 186–189, August, 1999     

Effects of calcium and its antagonists on hemodynamics and respiration

Acute tests on cats under Nembutal anesthesia show that intravenous injection of Ca²⁺ causes pathological respiration of the apneustic type and slight rises in pulmonary and arterial pressures. The calcium channel blockers verapamil and nifedipine decrease the amplitude of respiratory movements, increase the respiration rate and pulmonary pulse pressure, and lower systemic pressure. The introduction of verapamil or nifedipine into the fourth ventricle of the brain does not alter respiration or hemodynamics, whereas the introduction of Ca²⁺ leads to irreversible respiratory standstill. Hemodynamic parameters decrease 2–3 min after the respiratory standstill. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 8, pp. 138–143, August, 1996     

Correlation between the inhibition of phosphorylation in platelet myosin light chains and the inhibition of platelet aggregation by a new calcium and calmodulin antagonist

The recently synthesized compound [1,2,5-trimethyl-4-phenyl-4-β-(N-methyl-N-4′-methoxybenzyl)-ethylamino]piperidine dihydrochloride (AR-3), which is a derivative of [1,2,5-trimethyl-4-phenyl-4-β-(N,N-disubstituted-ethylamino)]piperidines, was tested for its effects on platelet aggregation and phosphorylation of light myosin chains isolated from platelets. AR-3 caused 50% inhibition of platelet aggregation in concentrations of 25.5 to 32.2 μM (depending on the aggregation inducer used) and 50% inhibition of light myosin chain phosphorylation in a concentration of 70 μM or, when 1 μM calmodulin was added, 120 μM. The good correlation found between the inhibitory effects of AR-3 on platelet aggregation and the phosphorylation of light myosin chains from platelets indicates that this compound inhibits platelet aggregation largely by inhibiting the Ca²⁺-calmodulin-dependent phosphorylation of platelet myosin light chains, acting in this respect similarly to the well-known calmodulin antagonist W-7. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 7, pp. 40–42, July, 1996     

The calcium channel antagonist diltiazem effectively blocks two types of potassium channels in the neuronal membranes

Effect of the Ca²⁺-channel antagonist diltiazem on potential-operated Ca²⁺ and K⁺ currents was studied on isolated edible snail neurons by a two-microelectrode patch-clamp technique. Diltiazem in a concentration of 0.1 mM inhibits Ca²⁺ current, high-threshold Ca²⁺-dependent K⁺ current, and Ca²⁺-independent K⁺ current and has no effect on low-threshold K⁺ current and leakage current. It is suggested that therapeutic effect of diltiazem is mediated through blockade of Ca²⁺ and K⁺ channels. Tranlated fromByulleten' Eksperimental'noi biologii i Meditsiny, Vol. 124, No. 9, pp. 271–274. September, 1997     

Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca²⁺ channels (I _Ca) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak I _Ca by 42% and the late I _Ca by 55%. Pulses (10 s duration) with 70 mM K⁺/2.5 mM Ca²⁺ solution (70 K⁺/2.5 Ca²⁺), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K⁺/2.5 Ca²⁺ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca²⁺]_o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca²⁺ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca²⁺ entry through both channel types leads to similar increments of averaged [Ca²⁺]_i, the control of catecholamine release is dominated only by Ca²⁺ entering through L-type Ca²⁺ channels. This supports the idea of a preferential segregation of L-type Ca²⁺ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs.     

The effect of delta-sleep-inducing peptide on the Ca-transporting system of the sarcoplasmic reticulum and activity of the myocardial antioxidant enzymes

It is demonstrated that immobilization stress against the background of lowered catalase activity impairs the function of the sarcoplasmic reticulum Ca pump, particularly at high Ca²⁺ levels. the membranes of intracellular Ca²⁺ depots are destroyed much more rapidly than in the control, which results in Ca²⁺ release. Administration of delta sleep-inducing peptide to control animals results in a 30% increase in catalase activity for an unchanged level of superoxide dismusase and markedly improves the function of the Ca-transporting system at elevated levels of free Ca²⁺. A long-term stress after administration of the peptide not only causes no damage to the Ca-transporting system but actually increases its efficiency (compared with the control) at a high catalase level. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 3, pp. 248–251, March, 1996 Presented by G. N. Kryzhanovskii, Member of the Russian Academy of Medical Sciences     

Impact of 13(S)-HPODE,a lipoxygenase product of linoleic acid,on calcium transport and Na,K-ATPase activity in the myocardial sarcolemma

In thisin vitro study using a purified sarcolemmic fraction of guinea pig myocardium, the 13(S)-hydroperoxide of linoleic acid (13-HPODE) increased in a dose-dependent manner the permeability of myocardial sarcolemma to Ca ions in concentrations above 10 μmol/liter, stimulated Na/Ca exchange there in concentrations from 0.1 to 10 μmol/liter, and exerted a digitalis-like action on sarcolemmic Na,K-ATPase in concentrations between 0.1 and 100 μmol/liter (IC₅₀=20 μmol/liter). The results indicate that the linoleic acid hydroperoxide may be an effective modulator of sarcolemmic Ca²⁺ transport and of membrane-bound enzymes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 9, pp. 255–257, September, 1995 Presented by D. F. Chebotarev, Member of the Russian Academy of Medical Sciences     

Effect of chlorophos (dipterex,trichlorphon) on high-threshold potassium and calcium channels of the neuronal membrane

The effect of chlorophos (dipterex, trichlorphon) on high-threshold potassium and calcium currents is studied on isolated snail neurons using the patch-clamp technique. Chlorophos (10–1000 μmol/liter) is found to reversibly lower the peak amplitude of a high-threshold potassium current by 30% on average and exerts two independent effects on a high-threshold calcium current: reversible lowering of the peak amplitude by 35% on average and, in 30% of cases, reversible inhibition of its activation, inactivation, and deactivation. this effect is abolished by adding diltiazem (a calcium channel antagonist) in a concentration of 100 μmol/liter to the medium. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 1, pp. 59–62, January, 1996 Presented by Yu. A. Romanov, Member of the Russian Academy of Medical Sciences     

Chemiluminescence study of the contents of the products of Cu2+-induced lipid peroxidation in different fractions of human blood lipoproteins

It is demonstrated that the content of the primary products of lipid peroxidation reaches the maximum after about 1-h incubation with Cu²⁺ and then declines. At a Cu²⁺ concentration of about 10–15 μM, the content of lipid peroxidation products is maximal; it does not rise with a further increase in the Cu²⁺ concentration. Comparison of the kinetics of lipid peroxidation in different lipoprotein fractions shows that low density lipoproteins are much more strongly oxidized than high density lipoproteins. A strong positive correlation between the amplitude of the chemiluminescence burst and the diene conjugate content is established in 79 independent measurements. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 2, pp. 144–148, February, 1995     

Activation of nitric oxide pathway mediated by metabotropic glutamate receptors in primary cultures of cerebellar granule cells

Stimulation of L-glutamate receptors (GluRs) is thought to produce neuron death through the elevation of free intracellular Ca²⁺ levels, leading to activation of Ca²⁺/calmodulin-dependent synthesis of cytotoxic amounts of NO·. In the present study, NO synthase activation mediated by mGluR stimulation is investigated in primary cultures of granule cells (CGrC). It is found that a selective agonist of mGluRs, DL-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD), raises both the cGMP and nitrite (NO₂ ⁻) levels, which are used as a biochemical index to study the enzymatic NO· release from L-arginine. This effect is abolished by applying both Nω-nitro-L-arginine methyl ester (NAME) and DL-2-amino-4-phosphonobytyric acid (AP4), and is independent of Ca²⁺. In contrast, the α-amino-3-hydroxy-5-methylisoxarole-4-propionnate (AMPA)-induced increase in cGMP content is eliminated by the preincubation of CGrC with 4 mM EGTA-chelated Ca²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 7, pp. 46–49, July, 1995 Presented by Yu. A. Romanov, Member of the Russian Academy of Medical Sciences