Statin-induced changes in mitochondrial respiration in blood platelets in rats and human with dyslipidemia.

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used drugs for lowering blood lipid levels and preventing cardiovascular diseases. However, statins can have serious adverse effects, which may be related to development of mitochondrial dysfunctions. The aim of study was to demonstrate the in vivo effect of high and therapeutic doses of statins on mitochondrial respiration in blood platelets. Model approach was used in the study. Simvastatin was administered to rats at a high dose for 4 weeks. Humans were treated with therapeutic doses of rosuvastatin or atorvastatin for 6 weeks. Platelet mitochondrial respiration was measured using high-resolution respirometry. In rats, a significantly lower physiological respiratory rate was found in intact platelets of simvastatin-treated rats compared to controls. In humans, no significant changes in mitochondrial respiration were detected in intact platelets; however, decreased complex I-linked respiration was observed after statin treatment in permeabilized platelets. We propose that the small in vivo effect of statins on platelet energy metabolism can be attributed to drug effects on complex I of the electron transport system. Both intact and permeabilized platelets can be used as a readily available biological model to study changes in cellular energy metabolism in patients treated with statins.

Effect of Simvastatin, Coenzyme Q10, Resveratrol, Acetylcysteine and Acetylcarnitine on Mitochondrial Respiration.

Some therapeutic and/or adverse effects of drugs may be related to their effects on mitochondrial function. The effects of simvastatin, resveratrol, coenzyme Q10, acetylcysteine, and acetylcarnitine on Complex I-, Complex II-, or Complex IV-linked respiratory rate were determined in isolated brain mitochondria. The protective effects of these biologically active compounds on the calcium-induced decrease of the respiratory rate were also studied. We observed a significant inhibitory effect of simvastatin on mitochondrial respiration (IC50 = 24.0 µM for Complex I-linked respiration, IC50 = 31.3 µM for Complex II-linked respiration, and IC50 = 42.9 µM for Complex IV-linked respiration); the inhibitory effect of resveratrol was found at very high concentrations (IC50 = 162 µM for Complex I-linked respiration, IC50 = 564 µM for Complex II-linked respiration, and IC50 = 1454 µM for Complex IV-linked respiration). Concentrations required for effective simvastatin- or resveratrol-induced inhibition of mitochondrial respiration were found much higher than concentrations achieved under standard dosing of these drugs. Acetylcysteine and acetylcarnitine did not affect the oxygen consumption rate of mitochondria. Coenzyme Q10 induced an increase of Complex I-linked respiration. The increase of free calcium ions induced partial inhibition of the Complex I+II-linked mitochondrial respiration, and all tested drugs counteracted this inhibition. None of the tested drugs showed mitochondrial toxicity (characterized by respiratory rate inhibition) at drug concentrations achieved at therapeutic drug intake. Resveratrol, simvastatin, and acetylcarnitine had the greatest neuroprotective potential (characterized by protective effects against calcium-induced reduction of the respiratory rate).

Pig Brain Mitochondria as a Biological Model for Study of Mitochondrial Respiration.

Oxidative phosphorylation is a key process of intracellular energy transfer by which mitochondria produce ATP. Isolated mitochondria serve as a biological model for understanding the mitochondrial respiration control, effects of various biologically active substances, and pathophysiology of mitochondrial diseases. The aim of our study was to evaluate pig brain mitochondria as a proper biological model for investigation of activity of the mitochondrial electron transport chain. Oxygen consumption rates of isolated pig brain mitochondria were measured using high-resolution respirometry. Mitochondrial respiration of crude mitochondrial fraction, mitochondria purified in sucrose gradient, and mitochondria purified in Percoll gradient were assayed as a function of storage time. Oxygen flux and various mitochondrial respiratory control ratios were not changed within two days of mitochondria storage on ice. Leak respiration was found higher and Complex I-linked respiration lower in purified mitochondria compared to the crude mitochondrial fraction. Damage to both outer and inner mitochondrial membrane caused by the isolation procedure was the greatest after purification in a sucrose gradient. We confirmed that pig brain mitochondria can serve as a biological model for investigation of mitochondrial respiration. The advantage of this biological model is the stability of respiratory parameters for more than 48 h and the possibility to isolate large amounts of mitochondria from specific brain areas without the need to kill laboratory animals. We suggest the use of high-resolution respirometry of pig brain mitochondria for research of the neuroprotective effects and/or mitochondrial toxicity of new medical drugs.

Intracellular signalling pathways and mood disorders.

Findings are summarized about basic intracellular signalling pathways influencing neurotransmission and involved in neurodegenerative or neuropsychiatric disorders. Psychotropic drugs used in the therapy of a series of mental disorders, mood disorders especially, show neurotrophic or neuroprotective effects after long-term treatment. Thus, beyond adenylate cyclase, guanylate cyclase and calcium system, attention has been paid to the tyrosine kinase pathway and Wnt pathway. New neurochemical hypotheses of mood disorders are disclosed; they were formulated on the basis of known effects of antidepressants or mood stabilizers on intracellular signal transduction, i.e. on the function, plasticity and survival of neurons. These hypotheses focus on the constituents of intracellular signalling pathways that could be studied as biological markers of mood disorders: transcription factor CREB, neurotrophin BDNF and its trkB receptor, anti-apoptotic factor Bcl2, pro-apoptotic enzyme GSK3, caspases, calcium, and a number of mitochondrial functions related to brain energy metabolism.

Red blood cell triiodothyronine uptake as membrane parameter of depression.

We tested the hypothesis considering the role of hypothalamic-pituitary-thyroid axis (HPT), L-triiodothyronine (L-T3) uptake into erythrocytes, and the role of membrane lipids in the development and treatment of affective disorders. Changes in kinetic parameters (V(max), maximal velocity and K(M), apparent Michaelis constant) of L-T3 uptake into red blood cells (RBCs) and changes in membrane fluidity in a group of 24 patients with major depression were measured before treatment and after 1 month of treatment with citalopram. Parameters V(max) and K(M), as well as membrane microviscosity, were significantly increased in depressed patients both before and after treatment in comparison with healthy subjects. We concluded that the function of the membrane transporter for L-T3 in RBC is changed in depression. This change is probably connected with alteration of membrane fluidity and/or transporter-lipid interactions. We did not find any normalization of the measured parameters after 1 month of treatment. The results show the importance of composition and physical properties of the lipid bilayer for transmembrane transport of L-T3 and support the hypothesis that the HPT axis is in depression.

Effect of long-term administration of antidepressants on the lipid composition of brain plasma membranes.

The connection between changes in lipid pattern in brain plasma membranes and long-term administration of therapeutically effective doses of antidepressants has not been sufficiently demonstrated so far. Therefore, we analyzed effect of antidepressants that differ in pharmacological selectivity on membrane lipid composition in the rat brain tissue. Laboratory rats were given desipramine, maprotiline, citalopram, moclobemide or lithium for a 4-week period. We observed a significant decrease in phosphatidylethanolamine representation after administration of maprotiline, citalopram and moclobemide when compared with controls. Membrane cholesterol content was decreased after desipramine administration and increased after citalopram or lithium treatment. Electroneutral phospholipids were decreased after the administration of all tested antidepressants except for desipramine. Decrease in phosphatidylserine was found following long-term administration of maprotiline or desipramine; relative representation of phosphatidylinositol was reduced after lithium treatment. Statistically significant negative correlation between cholesterol and electroneutral phospholipids was discovered. Membrane microviscosity evaluated by fluorescence anisotropy of membrane probes was only slightly decreased after desipramine and increased after citalopram administration. Hypothesis was supported that changes in brain neurotransmission produced by antidepressants could be, at least partially, associated with adaptive changes in membrane cholesterol and phospholipids.

Interactions between tricyclic antidepressants and phospholipid bilayer membranes.

Participation of electrostatic and other noncovalent interactions in the binding of tricyclic antidepressants (TCAs) to the lipid bilayers was estimated from pH-dependencies of imipramine, desipramine, amitriptyline and nortriptyline binding to the lipid bilayers prepared from different phospholipids, both electroneutral and acidic. The binding was studied using a radioligand binding assay. It was found that the membrane phospholipid composition and methylation of the acyl side chain of TCA has a decisive effect on participation of particular noncovalent interactions in the binding. Apparent high-affinity binding of TCAs to the phosphatidylcholine or phosphatidylethanolamine membranes are achieved mainly by incorporation of uncharged drug molecules into the hydrophobic core of the bilayers. Van der Waals forces and hydrophobic effect are responsible for this binding. Both charged and uncharged drug molecules bind to phosphatidylserine membranes, therefore coulomb- or ion-induced dipole interactions play a role in these binding. Different spatial distribution of charged residues within the interface causes different electrostatic interactions between charged TCAs and vesicles formed from phosphatidylserine and phosphatidylinositol. The data supports the hypothesis under which TCAs could have effect on affective disorders partially via binding to the lipid part of the membrane and following changes of lipid-protein interactions.

Effect of pharmacologically selective antidepressants on serotonin uptake in rat platelets.

We tested a hypothesis that a long-term administration of antidepressants acting through different primary biochemical mechanisms is associated with changes in the platelet serotonin (5-hydroxytryptamine, 5-HT) transport. Laboratory rats were administered norepinephrine reuptake inhibitors (desipramine, maprotiline), selective 5-HT reuptake inhibitor (citalopram), reversible monoamine oxidase inhibitor (moclobemide), and lithium (inositol monophosphatase inhibitor among others) during a 4-week period. Apparent kinetic parameters of platelet 5-HT transport were analyzed. Significant decrease in apparent Michaelis constant (K(M)) was found after the administration of all tested antidepressants except for desipramine. There was certain increase in maximal velocity (V(max)) values following the administration of desipramine, maprotiline, and citalopram; however, the all V(max) changes were not significant. V(max)/K(M) ratio representing limiting permeability at low extracellular concentrations of 5-HT was systematically increased in all the tested drugs, but significant changes were occurred only in maprotiline- and citalopram-treated rats. Adaptive changes in platelet 5-HT transport induced by citalopram were opposite to the acute inhibitory effect of this drug on 5-HT transporter activity. An increase in limiting membrane permeability for 5-HT could be included in the common adaptive effect of the long-term administration of antidepressants that differ in pharmacologic selectivity.

Binding of imipramine to phospholipid bilayers using radioligand binding assay.

Binding of the tricyclic antidepressant imipramine (IMI) to neutral and negatively charged lipid membranes was investigated using a radioligand binding assay combined with centrifugation or filtration. Lipid bilayers were composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS). IMI binding isotherms were measured up to IMI concentration of 0.5 mmol/l. Due to electrostatic attraction, binding between the positively charged IMI and the negatively charged surfaces of PS membranes was augmented compared to binding to neutral PC membranes. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherms were described both by surface partition coefficients and by binding parameters (association constants and binding capacities). It was confirmed that binding of IMI to model membranes is strongly affected by negatively charged phospholipids and that the binding is heterogeneous; in fact, weak surface adsorption and incorporation of the drug into the hydrophobic core of lipid bilayer can be seen and characterized. These results support the hypothesis suggesting that the lipid part of biological membranes plays a role in the mechanism of antidepressant action.

In vitro early allogeneic reaction of murine brain cortex cells.

An allogeneic reaction among brain cortex cells (mixed reaction) was demonstrated previously by H-2 alloantigen-induced uncoupling of oxidative metabolism (Kováru Med. Biol. 58: 273, 1980). In the present study we have demonstrated that alloantigen already increased cell surface Na+,K+-ATPase activity after 100 min when the enzyme activation was highest at Mg2+/ATP ratio 4: 1. The allogeneic cell reaction was accompanied by an elevation of membrane lipid fluidity and probably also by a thermotropic lipid phase transition which might influence the membrane lipid-dependent Na+,K+-ATPase activity, while Mg2+-ATPase remained unaffected. Furthermore, the effects of proteins and peptides released into the supernatant during the allogeneic reaction were analyzed in brain cortex cells. One of the isolated active peptide fractions, FA (m.w. lower than 2.5 kD), was able to enhance Na+,K+-ATPase activity as well as to block K+-evoked O2 uptake by brain cortex cells. Thus the FA fraction simulated primary allorecognition events. The data indicate that various brain cell surface domains were influenced by a regulatory peptide fraction of the cytokine type during the early phase of allogeneic reaction. Allorecognition among brain cortex cells is directed against functionally important metabolic reactions.

[ELISA in the determination of antiphosphatidylserine antibodies]. / Metoda ELISA pro stanovení antifosfatidylserinových protilátek.

Antiphospholipid antibodies (APA) are a group of antibodies against various phospholipid antigens. In order to extend the spectrum of examined specificities of antiphospholipid antibodies the authors elaborated an ELISA method for assessment of antiphosphatidyl serine antibodies (APSA). As antigen they used phosphatidyl serine isolated from the white matter of cattle brain. The ELISA method was tested by examining APSA in 12 patients with rheumatic diseases, 24 women with reproductive disorders and 50 patients with testicular tumours and the results were compared with examinations of anticardiolipin antibodies. The concurrent presence of both types of antibodies was recorded in 20.8% women with reproductive disorders and in 14% of the patients with testicular tumours. In these groups antiphosphatidyl serine antibodies were found more frequently.

Imipramine distribution among red blood cells, plasma and brain tissue.

The relations between the concentrations of a drug in the blood (plasma, haemoglobin, red blood cells (RBCs), RBC membranes) and in the brain tissue (homogenate, membranes, cytosol) were investigated during chronic administration of imipramine. Radioimmunoassay was employed to measure the antidepressant concentrations. The concentrations were measured and analysed in 40 rats receiving various doses of imipramine. The concentration of total imipramine (imipramine + desipramine) in erythrocyte membranes (ghosts) amounted to 79.4 +/- 4.6% (mean +/- S.E.M., N = 40) of those measured in intact RBCs. Marked accumulation of the drug in the brain tissue, especially in brain cell membranes, was confirmed. The concentrations in brain tissue homogenate was found to be 14.8 times higher than that in RBCs. Values in brain membranes were 10.9 times higher than that in blood element membranes. There is a significant association between the concentrations measured in brain homogenate, the blood plasma and RBC membranes. Blood concentrations can be used to estimate imipramine concentrations in the brain.

[Anti-phosphatidylinositol antibodies and their determination using our ELISA method]. / Antifosfatidylinositolové protilátky a jejich stanovení vlastní ELISA metodou.

Antiphospholipid antibodies (APA) are autoantibodies to negatively charged phospholipids such as a cardiolipin, phosphatidylserine and phosphatidylinositol. The occurrence of APA is associated with arterial and venous thromboembolic manifestations, thrombocytopenia and recurrent fetal loss. To date most studies have concentrated on antibodies to cardiolipin specifically. In this study we present own modification of the ELISA method for assessment of antiphosphatidylinositol antibodies (API). The phosphatidylinositol isolated from small green peas was used as an antigen. To test ELISA method 151 serum samples of patients with various diseases and 22 serum samples of blood donors were examined and the results were compared with the results of anticardiolipin antibodies (ACA). The positivity of API was 22.5% and ACA 20.5% in a group of patients. The simultaneous of both types of APA was found in 15.2%.

[Ca(2+)-channel blockers and binding of tricyclic antidepressive agents]. / Blokátory Ca(2+)-kanálu a vazba tricyklických antidepresív.

The interactions between Ca(2+)-channel blockers (verapamil and gallopamil) and synaptic plasma membranes (SPM) from bovine brain or human lymphocyte and platelet plasma membranes were studied. Changes in binding parameters of [3H]imipramine, [3H]desmethylimipramine and [3H]gallopamil were determined after addition of unlabelled verapamil or imipramine and after addition of phosphatidylserine (PS) (PS-stimulation). Specific binding of [3H]imipramine to SPM was decreased and [3H]desmethylimipramine binding was increased by 1 microM verapamil. [3H]gallopamil binds specifically to SPM as well as to platelet and lymphocyte membranes. [3H]gallopamil binding to SPM or lymphocyte plasma membranes was PS-stimulated in contrast to platelet plasma membranes without PS effect on binding. Imipramine inhibited both [3H]gallopamil binding and PS-stimulated [3H]gallopamil binding to SPM or lymphocyte plasma membranes. Mutual effects of tricyclic antidepressants and Ca(2+)-channel blockers on their binding sites require relatively high drug concentrations. Mechanism of Ca(2+)-channel blockers action in the treatment of depression may be connected rather with changes in signal transduction through serotonin and catecholamine receptor systems than with direct interaction of drugs with binding sites for tricyclic antidepressants.

[The effect of cocaine on binding of tricyclic antidepressives in the synaptic plasma membranes in the brain]. / Vliv kokainu na vazbu tricyklických antidepresiv v SPM mozku.

Effect of cocaine on binding of 3H-imipramine, 3H-desmethylimipramine, 3H-didesmethylimipramine and 3H-amitriptyline to brain synaptic plasma membranes (SPM) was studied. Binding of methylated tricyclic antidepressants was more affected. Cocaine inhibits 3H-imipramine binding at concentrations higher than 10(-5) mol/l. Binding stimulated by phosphatidylserine was affected more significantly.

[Review of findings on binding of tricyclic antidepressants]. / Prehled poznatku o vazbe tricyklických antidepresív.

Authors summarize knowledge in the field of the high-affinity binding of tricyclic antidepressants on cell membranes within last 12 years. Some ideas about the significance of such binding are stated. Both the data from papers and our knowledge are discussed with regard to the possibility of their clinical application at depressed patients. Effects of the tricyclic antidepressants are linked with their influence on the active transport of neuromediators through the cell membrane but their molecular mechanisms are unknown. Present-day state of research is described and the orientation on the role of membrane phospholipids in the binding studies of the tricyclic antidepressants is emphasized.

[Adrenergic and serotonergic receptors during depression and its therapy]. / Adrenergní a serotonergní receptory pri depresi a behem její terapie.

It is supposed that adrenergic and serotonergic receptor systems can play a crucial role in pathophysiology of depressive disorder. The hypothesis are based on changes of levels of serotonin and noradrenaline and on changes of their binding sites during depression and its treatment. Study of adrenergic and serotonergic receptors by pharmacological and molecular biological methods give rise to their division to many types and subtypes. This division makes possible to study the depressive disorder and its treatment on well defined receptors systems. Decreased density of beta- and 5-HT1-receptors was found during depression. Antidepressants influence 5-HT2 and adrenergic receptors mainly. Consequently, interaction between adrenergic and serotonergic systems exerts both in the origin of depression and during the treatment. Mutual influence and cooperation of different receptors systems are the main topics of research in this field.

Airborne microorganism monitoring: a comparison of several methods, including a new direct counting technique.

Samples of aerosol from the surrounding air were collected by forcing them to impact onto a solid nutrient medium, onto membrane filters, or onto microscope slides on microcover slips. The samples were cultivated or viewed in a fluorescence microscope by using a technique developed by us, or investigated by scanning electron microscopy. The amounts of microorganisms found by cultivation method, i.e. those forming microcolonies (CFU), were on average 85% lower than the amounts determined by the fluorescence technique. Cultivation of microorganisms trapped on Synpor filters of various pore size resulted in lower counts than conventional cultivation. Among bacteria, the genera Micrococcus, Bacillus and Corynebacterium predominated; the genera Neisseria, Actinomyces, Pseudomonas and others were also found. Of micromycetes, the genera Penicillium, Aspergillus and Cladosporium occurred most frequently in air. In the summer season, yeasts and other micromycetes prevailed, whereas in the winter season the bacteria counts were higher. The total counts of all components of the aeroplankton were higher in summer than in winter. Dust particles bounded mainly bacteria.

Liposomes--model membranes to study the binding of tricyclic antidepressants.

Binding of four tricyclic antidepressants (TCAs)--imipramine, desipramine, didesmethylimipramine and amitriptyline--on the lipid part of biological membranes was studied. Heterogeneity in partitioning of these drugs in artificial lipid bilayers (liposomes) was quantified using a radioligand binding method. High-affinity binding sites on the liposomes were found and characterized by apparent dissociation constant (Kd) and by binding capacity (Bmax). Change in the membrane lipid composition affected the binding parameters of the high-affinity binding, while the ligand modification affected non-specific binding (low-affinity adsorption) of TCAs. The possible role and methodological importance of high-affinity binding to the lipid part of biological membranes are both discussed. Determination of radioligand binding on model lipid membranes is recommended to avoid certain problems in interpretation of receptor binding studies.

Ontogenetic analysis of some surface markers on pig lymphocytes using fluorescence-activated cell sorter.

Surface markers were demonstrated on pig lymphocytes using anti-T cell-IgG and anti-Helix pomatia (HP) IgG during prenatal and postnatal development. A fluorescence-activated cell sorter analysis of T-cell surface markers was accompanied by an image analysis to prove the association of T antigenic determinants with the plasma membrane only. We found development-dependent changes in both anti-T cell and HP surface markers in both primary and secondary lymphatic organs. The number of T-positive (T+) cells estimated by anti-T cell-IgG was very similar to the results obtained by spontaneous E-rosette forming tests. At all selected age intervals, changes in the number of T+ cells were not significant in the thymus, but a marked increase in T+ cells was found in both spleen and lymph nodes. The image analysis confirmed the expression of T cell markers on the cell surface. The distribution of T cell markers was uneven, i.e. various degree of fluorescence intensity on whole ring-pattern projection of the cell surface image was estimated. In second lymphatic organs especially, fluorescence intensity of cells, i.e. total number of T cell markers estimated by anti-T cell-IgG, increased with age. On fetal day 73, T cell markers were slightly expressed, but very high fluorescence intensity and heterogeneous distribution of T cell markers on lymphocytes were found on fetal day 107 and postnatal day 56. The results indicate the possibility of functional maturation of various T cell markers on T cell subsets, furthermore a different degree of expression of T cell markers on various T cell subsets can be suggested. The number of HP+ cells increased with age in both primary and secondary lymphatic organs. In the prenatal period, the expression of HP receptors was very weak in both primary and secondary organs in contrast to the marked increase in HP+ cells during the postnatal interval. Differences in fluorescence intensity of cells were found, representing the increase by 22% in thymus cells comparing to cells of secondary lymphatic organs. Heterogeneity of HP+ cell populations in thymus was shown by the Scatchard plot, indicating at least two subpopulations of HP+ cells with different avidity to HP. Cells with low HP avidity could include a subset with cytolytic activity.