Biomedical subjects

Attila Tóth

Publications and source records attributed to Attila Tóth.

At least 19 recordsLinked to original sources

PubMed2006

Improved version of the printed circuit board (PCB) modular multi-channel microdrive for extracellular electrophysiological recordings.

The modular multi-channel PCB microdrive was described some years ago, since then several improvements were introduced while using these drives. Utilizing several years of experience with the original PCB microdrive we redesigned it to improve its stability and usability. The application of the printed circuit board technology and the extensive use of flexible fused silica capillaries for fabrication of the microdrive are described in detail. The improved design led to a low cost and light-weight multi-channel microdrive with outstanding modularity for extracellular field, single unit or multiunit tetrode recording up to 64/128 channels.

Electrodes, Implanted↗

PubMed2006

High-fat diet-induced reduction in nitric oxide-dependent arteriolar dilation in rats: role of xanthine oxidase-derived superoxide anion.

Obesity frequently leads to the development of hypertension. We hypothesized that high-fat diet (HFD)-induced obesity impairs the endothelium-dependent dilation of arterioles. Male Wistar rats were fed with normal (control) or HFD (60% of saturated fat, for 10 wk). In rats with HFD, body weight, mean arterial blood pressure, and serum insulin, cholesterol, and glucose were elevated. In isolated gracilis muscle arterioles (diameter: approximately 160 microm) of HFD, rat dilations to ACh (at 1 microM, maximum: 83 +/- 3%) and histamine (at 10 microM, maximum: 16 +/- 4%) were significantly (P < 0.05) decreased compared with those of control responses (maximum: 90 +/- 2 and 46 +/- 4%, respectively). Dilations to the NO donor sodium nitroprusside were similar in the two groups. Inhibition of NO synthesis by N(omega)-nitro-l-arginine methyl ester reduced ACh- and histamine-induced dilations in control arterioles but had no effect on microvessels of HFD rats. The superoxide dismutase mimetic Tiron or xanthine oxidase inhibitor allopurinol enhanced ACh (maximum: 90 +/- 2 and 93 +/- 2%, respectively)- and histamine (maximum: 30 +/- 7 and 37 +/- 8%, respectively)-induced dilations in HFD arterioles, whereas the NAD(P)H oxidase inhibitor apocynin had no significant effect. Correspondingly, in carotid arteries of HFD rats, an enhanced superoxide production was shown by lucigenin-enhanced chemiluminescence, in association with an increased xanthine oxidase, but not NAD(P)H oxidase activity. In addition, a marked xanthine oxidase immunostaining was detected in the endothelial layer of the gracilis arterioles of HFD, but not in control rats. These findings suggest that, in obese rats, NO mediation of endothelium-dependent dilation of skeletal muscle arterioles is reduced because of an enhanced xanthine oxidase-derived superoxide production. These alterations demonstrate substantial dysregulation of arteriolar tone by the endothelium in HFD-induced obesity, which may contribute to disturbed tissue blood flow and development of increased peripheral resistance.

Acetylcholine↗

PubMed2006

Effect of cortical spreading depression on basal forebrain neurons.

During natural sleep and anesthesia, rhythmic hypo- and hyperpolarizations alternate in cortical pyramidal cells and are reflected as slow (<1 Hz) cortical rhythm at the level of the electroencephalogram (EEG). Membrane potential changes in pyramidal neurons were initially attributed to the rhythmic fluctuation of the cholinergic input as the basal forebrain (BF) neurons fire in synchrony with cortical waves, but a more recent proposal suggested that the slow rhythm was of cortical origin. In the present experiments, interaction between the cortex and the BF was examined in urethane-anesthetized rats. BF neuronal activity was inhibited by local infusion of lidocaine into the substantia innominata in one group of rats, while in another group, the slow cortical rhythm was blocked by inducing spreading depression (SD) in the cortex. Slow cortical rhythm persisted after unilateral lidocaine injection, but rhythmic firing in BF neurons disappeared following SD induction. These findings support the view that slow cortical rhythm is generated in the cortex and transmitted to the BF through descending fibers. According to anatomical data, these fibers can excite cholinergic cells only indirectly as they terminate on non-cholinergic neurons. Thus, timing of activity changes in BF neurons during the slow cortical rhythm might give some clue regarding their transmitter specificity.

Action Potentials↗

PubMed2006

Kinetics of penetration influence the apparent potency of vanilloids on TRPV1.

Evidence that the ligand binding site of TRPV1 lies on the inner face of the plasma membrane and that much of the TRPV1 itself is localized to internal membranes suggests that the rate of ligand entry into the cell may be an important determinant of the kinetics of ligand action. In this study, we synthesized a BODIPY TR-labeled fluorescent capsaicin analog (CHK-884) so that we could directly measure ligand entry. We report that CHK-884 penetrated only slowly into Chinese hamster ovary (CHO) cells expressing rat TRPV1, with a t1/2 of 30 +/- 4 min, and localized in the endoplasmic reticulum and Golgi. Although CHK-884 was only weakly potent for TRPV1 binding (Ki = 6400 +/- 230 nM), it was appreciably more potent when assayed by intracellular calcium imaging and was 3.2-fold more potent with a 1-h incubation time (37 nM) than with a 5-min incubation time. Olvanil, a highly lipophilic vanilloid, yielded an EC50 of 4.3 nM upon intracellular calcium imaging with an incubation time of 1 h, compared with an EC50 value of 29.5 nM for calcium imaging assayed at 5 min. Likewise, the antagonist 5-iodo-resiniferatoxin (5-iodo-RTX) displayed a Ki of 4.2 pM if incubated with CHO-TRPV1 cells for 2 h before addition of capsaicin compared with 1.5 nM if added simultaneously. We conclude that some vanilloids may have slow kinetics of uptake; this slow uptake may affect assessment of structure activity relations and may represent a significant factor for vanilloid drug design.

Animals↗

PubMed2006

[Experiences with the first, Hungarian autologous bone marrow cell transplantation in acute myocardial infarction].

Intracoronary transfer of autologous bone marrow cells promotes recovery of left ventricular systolic function in patients with acute myocardial infarction. Although the exact mechanisms of stem cell therapy are still intensely debated, the concept of stem cell therapy has already been introduced into the clinical practice--at least as an adjunctive therapy in clinical trials. In this article the authors report their experiences about the first Hungarian phase I. trial in bone marrow stem cell transplantation after acute myocardial infarction. So far, four patients with acute ST elevation myocardial infarction were eligible and recruited into the trial. All patients received purified, autologous bone marrow stem cells into the re-opened infarct related artery via a second catheterisation. The primary end point of the study is ejection fraction, which is measured by cardiac MRI at the beginning and 6 months after recruitment. So far, cell transfer did not increase the risk of adverse clinical events or proarrhythmic effects.

Angina Pectoris↗

PubMed2006

Mistyping of angiotensinogen M235T alleles.

Conflicting results are to be found in the literature on the relationship between the M235T polymorphism of the angiotensinogen (AGT) gene and hypertension. The controversy may be due to insufficient numbers of subjects, the variability of the inclusion criteria and the different genotype analysis methods used. We have experienced that the most frequently used, original polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method involves significant uncertainties when the TT genotype is determined, independently of the restriction digestion. To make the determination more accurate, we improved the PCR by designing a new antisense primer containing only one mismatch instead of the two in the original protocol and also by adding DMSO to the PCR reaction mixture. The original and our improved methods were compared by using DNA from 123 patients: parallel determinations resulted in values of 33 MM, 90 MT and 0 TT with the original method and of 33 MM, 56 MT and 34 TT with the improved RFLP protocol. In summary, a plausible explanation for some of the conflicting data published on AGT M235T polymorphism may be that inaccuracies arose during the determination of the genotype.

Angiotensinogen↗

PubMed2006

Erdheim-Chester's disease of the heart: a diagnostic conundrum and collision with the same mass in the orbit.

Erdheim-Chester's disease is a rare multisystem xanthogranulomatosis, afflicting the skeletal system with the occasional involvement of soft tissues. We delineate an unusual case of a cardiac variant of Erdheim-Chester's disease presenting with pericardial effusion and as a collision with a synchronous orbital manifestation. We describe our diagnostic pathway and propose a novel treatment option involving nonsteroidal anti-inflammatory drugs. The role of cyclo-oxygenase in the disease process and inhibition thereof by NSAIDs is hypothesized and discussed.

Celecoxib↗

PubMed2005

Phosphorylation-dependent desensitization by anandamide of vanilloid receptor-1 (TRPV1) function in rat skeletal muscle arterioles and in Chinese hamster ovary cells expressing TRPV1.

It has been proposed that activation of vanilloid receptor-1 (TRPV1) affects the vasotone of resistance arteries. One of the endogenous activators of TRPV1 is anandamide. The effects of anandamide on TRPV1 responsiveness were tested on isolated, pressurized (80 mm Hg) skeletal muscle (m. gracilis) arterioles (179 +/- 33 microm in diameter). We found that the TRPV1 agonist capsaicin (1 microM) elicited a substantial constriction in isolated arterioles (51 +/- 12%). In contrast, anandamide (0-100 microM) did not affect arteriolar diameter significantly (3 +/- 5%). Isolated vessels were also preincubated with anandamide (30 microM for 20 min). This anandamide pretreatment completely blocked capsaicin-induced arteriolar constriction (response decreased to 1 +/- 0.6%), and this inhibition was reversed by a protein phosphatase-2B inhibitor (cyclosporin-A; 100 nM, 5 min) treatment (constriction, 31 +/- 1%). An exogenous TRPV1-expressing cell line [Chinese hamster ovary (CHO)-TRPV1] was used to specifically evaluate TRPV1-mediated effects of anandamide. The efficacy of anandamide in this system, as determined by 45Ca2+ uptake, was 65 +/- 8% of that of capsaicin. Upon treatment of the cells with cyclosporin-A or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), anandamide was transformed to a full agonist. Anandamide treatment caused an acute desensitization in these cells as measured by intracellular Ca2+ imaging. Application of cyclosporin-A or PMA reversed this desensitization. Our data suggest that anandamide may cause a complete (albeit phosphorylation-dependent) desensitization of TRPV1 in skeletal muscle arterioles and in CHO-TRPV1 cells, which apparently transforms the ligand-gated TRPV1 into a phosphorylation-gated channel. This property of anandamide may provide a new therapeutic strategy to manipulate TRPV1 activity.

Animals↗

PubMed2005

Transient alterations in neuronal and behavioral activity following bensultap and fipronil treatment in rats.

In the present multilevel study, neuromodulatory effect of two insecticides, bensultap and fipronil were investigated in rats. Although the new generation of insecticides shows greater affinity to invertebrate as compared to mammalian receptors, toxic effect of these compounds in vertebrates cannot be excluded. The aim of the study was to follow the course of neuronal changes in rats for 1 week after a high-dose insecticide exposure. Alterations in synaptic excitability, in sleep-wake pattern and in behavior were analyzed using conventional in vitro brain slice method, long-lasting EEG recordings, and open-field tests. The two chemicals examined in this study induced only weak and transient effects. Bensultap, acting on nicotinic acetylcholine receptors, caused a transient decrease in neuronal excitability. Sleep and behavioral changes demonstrated a similar time course. Fipronil, on the other hand, increased excitability and its effect lasted slightly longer. All effects were greatest on the first day following 'poisoning', and measured variables usually returned to normal within a week. These results suggest that the studied compounds do have some effects on the mammalian nervous system, but this effect is usually mild and temporary.

Animals↗

PubMed2005

Rewiring the exit from mitosis.

The mitotic cell cycle can be described as an alternation between two states. During mitosis, MPF (mitosis promoting factor) is high and keeps inactive its numerous molecular antagonists. In interphase, MPF is inactivated, and the antagonists prevail. The transition between the two states is ensured by 'helper' molecules that favor one state over the other. It has long been assumed that active MPF (a dimer of cyclin B and cyclin-dependent kinase 1) induces exit from mitosis by activating APC:Cdc20, a ubiquitin ligase responsible for cyclin B degradation. The molecular details have not been fully worked out yet, but recent results show that MPF and the ubiquitin ligase are not involved in a simple negative feedback loop. While it is proven that MPF activates APC, new data suggest that MPF inhibits Cdc20, i.e., that MPF and Cdc20 are antagonists. We introduce this new idea into a published model for cell cycle regulation in Xenopus laevis, and study its dynamical behavior. We show that the new wiring permits oscillations with a simpler and smaller network than previously envisaged and that the antagonism between MPF and Cdc20 suggests a new interpretation of the spindle checkpoint.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2005

Different vanilloid agonists cause different patterns of calcium response in CHO cells heterologously expressing rat TRPV1.

The vanilloid receptor subtype 1 (TRPV1 or VR1) is expressed in nociceptive primary afferents of the C-fiber 'pain' pathway and has attracted considerable attention as a therapeutic target. Here, using rat TRPV1 heterologously expressed in Chinese hamster ovary cells, we show that different agonists show different patterns of modulation of the intracellular Ca2+ concentration, monitored in individual cells by fura-2 Ca2+ imaging. We identified 5 parameters (potency, maximal response, latency of response, variability of latency of response among individual cells, and desensitization) which behaved differently for different compounds. The potencies of the compounds examined ranged from EC50 values of 80 pM to 9 microM. Peak levels of induced [Ca2+]i were observed either higher (RTX) or lower (anandamide) than for capsaicin. Significant latencies of response were observed for some (e.g. RTX) but not other derivatives, with great variation among individual cells in this latency. Marked desensitization after stimulation was detected in some cases (e.g. anandamide, capsaicin); for others, no desensitization was observed. We conclude that structurally diverse vanilloid agonists induce marked diversity in the patterns of Ca2+ response. This diversity of response may provide opportunities for pharmacological exploitation.

Animals↗

PubMed2005

Peroxynitrite-induced alpha-actinin nitration and contractile alterations in isolated human myocardial cells.

OBJECTIVE: Peroxynitrite-mediated myocardial protein nitration has been associated with a depressed cardiac pump function. In the present study, an attempt was made to elucidate the molecular background of peroxynitrite-evoked alterations in the human myocardium. METHODS: Isometric force generation was measured in permeabilized human ventricular myocytes and biochemical methods were employed to identify the proteins affected by peroxynitrite-induced nitrotyrosine formation. RESULTS: The maximal Ca(2+)-activated isometric force (pCa=4.75) decreased to zero with increasing concentrations of peroxynitrite in a concentration-dependent manner (IC50: 55+/-4 microM; based on a total of 75 myocytes). However, there were no differences before and after the application of 50 microM peroxynitrite in the Ca(2+)-sensitivity of force production (pCa50: 5.89+/-0.02 and 5.86+/-0.04), in the steepness of the Ca(2+)-force relationship (nHill: 2.22+/-0.11 and 2.42+/-0.25), and in the actin-myosin turnover kinetics (k(tr) at saturating [Ca2+]: 1.14+/-0.03 1/s and 1.05+/-0.07 1/s) (P>0.05). Nevertheless, 50 muM peroxynitrite greatly deteriorated the cross-striation pattern and induced a slight, but significant, increase in the passive force component (from 2.1+/-0.1 to 2.5+/-0.2 kN/m2; n=57 cells), reflecting ultrastructural alterations. Western immunoblots revealed that 50 microM peroxynitrite selectively induced the nitration of a protein with an apparent molecular mass of about 100 kDa. Subsequent immunoprecipitation assays identified this nitrated protein as alpha-actinin, a major Z-line protein. CONCLUSIONS: These results suggest alpha-actinin as a novel target for peroxynitrite in the human myocardium; its nitration induces a contractile dysfunction, presumably by decreasing the longitudinal transmission of force between adjacent sarcomeres.

Actinin↗

PubMed2005

EEG effect of basal forebrain neuropeptide Y administration in urethane anaesthetized rats.

Neuropeptide Y (NPY) is present both in local neurons as well as in fibers in the basal forebrain (BF), an area that plays an important role in the regulation of cortical activation. In previous studies, NPY axons were found to innervate corticopetal cholinergic cells in this area. In addition, identified NPY positive neurons have been shown to be silent during cortical activation, but active during slow EEG waves. However, no in vivo studies have shown the effect of local NPY release in the BF on the EEG. In the present experiments, the EEG was examined following NPY injection (0.5 microl, 300-500 pmol) into the BF of urethane-anaesthetized rats. Fronto-parietal EEG was recorded on both sides and relative EEG power was calculated in the delta (0-3 Hz), theta (3-9 Hz), alpha (9-16 Hz) and beta (16-48 Hz) frequency bands. We found a significant increase in relative delta power and a decrease in the power of all higher frequency bands (theta, alpha, beta) after NPY injection. These results suggest that NPY can inhibit cortical activation via the BF.

Anesthetics, Intravenous↗

PubMed2005

Expression and distribution of vanilloid receptor 1 (TRPV1) in the adult rat brain.

The vanilloid receptor (TRPV1 or VR1) is a molecular integrator of various painful stimuli, including capsaicin, acid, and high temperature. It can also be activated by endogenous ligands, like the cannabinoid 1 receptor (CB1) agonist anandamide. TRPV1 is well characterized at the terminals of sensory nerves involved in the pain pathway. There is also evidence that TRPV1 is expressed in the brain but little is known about its function. Here, using commercially available specific antibodies to investigate the localization of TRPV1 in the brain of the rat, we report that TRPV1 was expressed in hippocampus, cortex, cerebellum, olfactory bulb, mesencephalon and hindbrain. Immunohistochemical analyses showed high expression in the cell bodies and dendrites of neurons in the hippocampus and in the cortex. To address the question of subcellular localization, immunoelectronmicroscopy was used. TRPV1-like staining was detected in the synapses (mostly, but not exclusively in post-synaptic dendritic spines), on the end feet of astrocytes and in pericytes. In summary, TRPV1 expression shows wide distribution in the brain of the rat, being found in astrocytes and pericytes as well as in neurons. Its localization is consistent with multiple functions within the central nervous system, including the regulation of brain vasculature.

Animals↗

PubMed2005

Calpain-1-sensitive myofibrillar proteins of the human myocardium.

Calpain-1 is a ubiquitous intracellular Ca2+-activated protease, which has been implicated in the pathogenesis of reversible myocardial depression (i.e. myocardial stunning) that follows ischemia and reperfusion via myofibrillar protein degradation. However, the target proteins of this degradative process in the human myocardium have not yet been identified. In order to compare the levels of Calpain-1 susceptibility within a set of human myofibrillar proteins (titin, alpha-fodrin, desmin, troponin T (cTnT), troponin I (cTnI) and alpha-actinin), crude left ventricular tissue homogenates were incubated for 0.5, 15, 30, 60 or 120 min in the presence of Calpain-1 (1 U or 5 U). Differences in the kinetics and extents of protein degradation were subsequently evaluated by using silver-stained SDS-polyacrylamide gels and Western immunoblot analyses. These assays revealed myofibrillar proteins with high (titin and alpha-fodrin), moderate (desmin and cTnT), or low (cTnI and alpha-actinin) relative Calpain-1 susceptibilities. The level of phosphorylation of cTnI did not explain its relatively low Calpain-1 susceptibility. Moreover, the molecular mass distributions of the truncated alpha-fodrin, desmin and cTnI fragments resulting from Ca2+-dependent autoproteolysis exhibited marked similarities with those of their Calpain-1-clipped products. These in vitro results shed light on a number of structural (titin, alpha-fodrin, desmin and alpha-actinin) and regulatory (cTnT and cTnI) proteins within the contractile apparatus as potential targets of Calpain-1. Their degradation may contribute to the development of postischemic stunning in the human myocardium.

Actinin↗

PubMed2004

Design of a high-affinity competitive antagonist of the vanilloid receptor selective for the calcium entry-linked receptor population.

We describe the synthesis and characterization of N-(4-chlorobenzyl) -N'-(4-hydroxy-3-iodo-5-methoxybenzyl)thiourea (IBTU), a novel antagonist of the vanilloid receptor 1 (TRPV1 or VR1). IBTU competitively inhibited 45Ca2+ uptake into CHO cells heterologously expressing rat TRPV1, whether induced by capsaicin or resiniferatoxin (Ki = 99 +/- 23 and 93 +/- 34 nM, respectively). IBTU was thus somewhat more potent (5-fold) than capsazepine. In contrast to its antagonism of vanilloid-induced calcium uptake, IBTU (30 microM) inhibited [3H]resiniferatoxin binding to TRPV1 by less than 10%. We hypothesize that these dramatically distinct potencies reflect different fractions of TRPV1 in this system: namely, a minor plasma membrane fraction controlling 45Ca2+ uptake, and the predominant intracellular fraction that dominates the [3H]resiniferatoxin binding measurements. Intracellular Ca2+ imaging supports this explanation. IBTU antagonized the elevation in intracellular Ca2+ in response to 50 nM capsaicin with an IC50 of 106 +/- 35 nM. Likewise, 600 nM IBTU was able to antagonize the elevation in intracellular Ca2+ in response to 100 pM resiniferatoxin in the presence of normal (1.8 mM) extracellular Ca2+, where the increase in intracellular calcium reflects calcium influx. In contrast, in the absence of extracellular Ca2+, where in this system resiniferatoxin induces a modest increase in calcium from intracellular stores, IBTU was unable to block the response to resiniferatoxin, although the TRPV1 antagonist 5-iodoresiniferatoxin was able to do so. In summary, IBTU is a novel, potent TRPV1 antagonist with marked selectivity between subpopulations of TRPV1 and may permit the function of these distinct pools to be explored and potentially exploited.

Animals↗

PubMed2003

Arachidonyl dopamine as a ligand for the vanilloid receptor VR1 of the rat.

The vanilloid receptor VR1 is a nonspecific Ca(2+) channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO as a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [3H]RTX to VR1 with a K(d) value of 5.49 +/- 0.68 microM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K(d) (9.7 +/- 3.3 microM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated 45Ca(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC(50) values of 4.76 +/- 1.43 and 7.17 +/- 1.64 microM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of 45Ca(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca(2+) uptake assays (IC(50) = 3.24 +/- 0.84 microM, inhibition is 68.5 +/- 6.85%) as well as in Ca(2+) imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself. We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner.

Animals↗

PubMed2003

Division of the nucleolus and its release of CDC14 during anaphase of meiosis I depends on separase, SPO12, and SLK19.

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12delta and slk19delta mutants.

Anaphase↗