Peter Racay

Developmental Changes in Parvalbumin Regulate Presynaptic Ca2+ Signaling

Certain interneurons contain large concentrations of specific Ca2+-binding proteins (CBPs), but consequences on presynaptic Ca2+ signaling are poorly understood. Here we show that expression of the slow CBP parvalbumin (PV) in cerebellar interneurons is cell specific and developmentally regulated, leading to characteristic changes in presynaptic Ca2+ dynamics (Cai). Using whole-cell recording and fluorescence imaging, we studied action potential-evoked Cai transients in axons of GABA-releasing interneurons from mouse cerebellum. At early developmental stages [postnatal days 10-12 (P10-P12)], decay kinetics were significantly faster for basket cells than for stellate cells, whereas at P19-P21 both interneurons displayed fast decay kinetics. Biochemical and immunocytochemical analysis showed parallel changes in the expression levels and cellular distribution of PV. By comparing wild-type and PV(-/-) mice, PV was shown to accelerate the initial decay of action potential-evoked Cai signals in single varicosities and to introduce an additional slow phase that summates during bursts of action potentials. The fast initial Cai decay accounts for a previous report that PV elimination favors synaptic facilitation. The slow decay component is responsible for a pronounced, PV-dependent, delayed transmitter release that we describe here at interneuron-interneuron synapses after presynaptic bursts of action potentials. Numerical simulations account for the effect of PV on Cai kinetics, allow estimates for the axonal PV concentration (∼150 μm), and predict the time course of volume-averaged Cai in the absence of exogenous buffer. Overall, PV arises as a major contributor to presynaptic Cai signals and synaptic integration in the cerebellar cortex.

Mutational analysis of dendritic Ca2+ kinetics in rodent Purkinje cells: role of parvalbumin and calbindin D28k

The mechanisms governing the kinetics of climbing fibre‐mediated Ca2+ transients in spiny dendrites of cerebellar Purkinje cells (PCs) were quantified with high‐resolution confocal Ca2+ imaging. Ca2+ dynamics in parvalbumin (PV−/−) and parvalbumin/calbindin D28k null‐mutant (PV/CB−/−) mice were compared with responses in wild‐type (WT) animals. In the WT, Ca2+ transients in dendritic shafts were characterised by double exponential decay kinetics that were not due to buffered Ca2+ diffusion or saturation of the indicator dye. Ca2+ transients in PV−/− PCs reached the same peak amplitude as in the WT but the biphasic nature of the decay was less pronounced, an effect that could be attributed to PVs slow binding kinetics. In contrast, peak amplitudes in PV/CB−/− PCs were about two times higher than in the WT and the decay became nearly monophasic. Numerical simulations indicate that the residual deviation from a single exponential decay in PV/CB−/− is due to saturation of the Ca2+ indicator dye. Furthermore, the simulations imply that the effect of uncharacterised endogenous Ca2+ binding proteins is negligible, that buffered diffusion and dye saturation significantly affects spineous Ca2+ transients but not those in the dendritic shafts, and that neither CB nor PV undergoes saturation in spines or dendrites during climbing fibre‐evoked Ca2+ transients. Calbindins medium‐affinity binding sites are fast enough to reduce the peak amplitude of the Ca2+ signal. However, similar to PV, delayed binding by CB leads to biphasic Ca2+ decay kinetics. Our results suggest that the distinct kinetics of PV and CB underlie the biphasic kinetics of synaptically evoked Ca2+ transients in dendritic shafts of PCs.

Membrane ion transport systems during oxidative stress in rodent brain: protective effect of stobadine and other antioxidants.

The effect of oxidative stress in vitro induced by radical generating systems (RGS) (Fe2+-EDTA and Fe2+-EDTA plus H2O2) on synaptosomal and microsomal ion transport systems as well as on the membrane fluidity was investigated. Oxidative insult reduced Na+, K+-ATPase activity by 50.7% and Na+-dependent Ca2+ uptake measured in choline media by 46.7%. Membrane fluidity was also significantly reduced as observed with the fluorescent probe. Stobadine (ST) prevented the decrease in membrane fluidity and Na+-dependent Ca2+ uptake, however Na+, K+-ATPase activity was only partially protected, indicating a more complex mechanism of inhibition. Incubation of microsomes with RGS led to the loss of ability of membranes to sequester Ca2+, as well as to the decrease of Ca2+-ATPase activity and to the increase of Ca2+ permeability to 125.1%. The relative potency of the two RGS to decrease membrane fluidity correlated well with the systems potencies to induce lipid peroxidation. The extent of protection against depression of Ca2+ uptake values and Ca2+-ATPase activity by membrane soluble antioxidants (U-74500A, U-83836E, t-butylated hydroxytoluene-BHT and ST) was dependent on the experimental conditions and on the dose and nature of antioxidant used. ST seems to be at least as affective as BHT and 21-aminosteroids, and more potent than tocopherol acetate. Water soluble glutathione had no significant effect on the RGS induced inhibition of Ca2+-ATPase activity. Combination of ST with glutathione enhanced ST antioxidant efficacy, so drug combination might be beneficial therapeutically.

In vitro assays for the evaluation of drug resistance in tumor cells

Oncologic diseases are among leading cause of mortality in developed countries. Despite significant progress, the use of standard cytotoxic chemotherapy has reached a therapeutical plateau. Currently, the process of selecting chemotherapy represents a trial and error method neglecting biological individuality of tumor and its bearer. The improvement of treatment results is expected from ex vivo drug sensitivity testing which may allow to choose the most effective drug for individual patient and to exclude agents to which the tumor cells exert resistance. New techniques and rapidly increasing knowledge about the molecular basis of malignant diseases provide important opportunities for the future of chemotherapy. This paper reviews current methods used to test the resistance of tumor cells to a panel of anticancer agents in vitro. In addition, we focused on the in vitro MTT assay which represents one of major technique for testing of tumor cell resistance to anticancer agents.

Parvalbumin deficiency in fast-twitch muscles leads to increased 'slow-twitch type' mitochondria, but does not affect the expression of fiber specific proteins

Parvalbumin (PV), a small cytosolic protein belonging to the family of EF‐hand calcium‐binding proteins, is highly expressed in mammalian fast‐twitch muscle fibers. By acting as a ‘slow‐onset’ Ca2+ buffer, PV does not affect the rapid contraction phase, but significantly contributes to increase the rate of relaxation, as demonstrated in PV–/– mice. Unexpectedly, PV–/– fast‐twitch muscles were considerably more resistant to fatigue than the wild‐type fast‐twitch muscles. This effect was attributed mainly to the increased fractional volume of mitochondria in PV–/– fast‐twitch muscle, extensor digitorum longus, similar to levels observed in the slow‐twitch muscle, soleus. Quantitative analysis of selected mitochondrial proteins, mitochondrial DNA‐encoded cytochrome oxidase c subunit I and nuclear DNA‐encoded cytochrome oxidase c subunit Vb and F1‐ATPase subunit β revealed the PV–/–tibialis anterior mitochondria composition to be almost identical to that in wild‐type soleus, but not in wild‐type fast‐twitch muscles. Northern and western blot analyses of the same proteins in different muscle types and in liver are indicative of a complex regulation, probably also at the post‐transcriptional level. Besides the function in energy metabolism, mitochondria in both fast‐ and slow‐twitch muscles act as temporary Ca2+ stores and are thus involved in the shaping of Ca2+ transients in these cells. Previously observed altered spatio‐temporal aspects of Ca2+ transients in PV–/– muscles are sufficient to up‐regulate mitochondria biogenesis through the probable involvement of both calcineurin‐ and Ca2+/calmodulin‐dependent kinase II‐dependent pathways. We propose that ‘slow‐twitch type’ mitochondria in PV–/– fast muscles are aimed to functionally replace the slow‐onset buffer PV based on similar kinetic properties of Ca2+ removal.

Lipid peroxidation both inhibits Ca2 ‐ATPase and increases Ca2 permeability of endoplasmic reticulum membrane

Incubation of reticular membranes with Fe2+‐EDTA and H2O2 plus Fe2+‐EDTA at 37 °C for 30 min. led to the loss of membranes efficiency to sequester Ca2+ to 21.8 % and 3.6 % of control values, respectively. The incubation of microsomes with Fe2+‐EDTA and H2O2 plus Fe2+‐EDTA also caused decrease of Ca2+‐ATPase activity; to 44.9 % and 44.4 % (measured under the same conditions as Ca2+‐uptake) or to 79.6 % and 62.1 % (uncoupled from Ca2+ transport by detergent). In addition, incubation of membranes with Fe2+‐EDTA and H2O2 plus Fe2+‐EDTA at 37 °C for 30 min. led to the increase of Ca2+ permeability to 125.1 % and 124.2 %, respectively. Preincubation of membranes with membrane‐soluble antioxidants (U‐74500A, U‐83836E, t‐butyl hydroxytoluene and stobadine) protected the reticular membranes against depression of Ca2+ uptake values and Ca2+‐ATPase inhibition in a dose and an antioxidant nature dependent manner. Our results indicate that both processes, Ca2+‐ATPase inhibition and increase of endoplasmic reticulum membrane Ca2+ permeability, participate in the lipid peroxidation induced loss of membranes efficiency to sequester Ca2+.

Polymorphisms of glutathione-S-transferase M1, T1, P1 and the risk of prostate cancer: a case-control study

BackgroundIt has been suggested that polymorphisms in glutathione-S-transferases (GST) could predispose to prostate cancer through a heritable deficiency in detoxification pathways for environmental carcinogens. Yet, studies linking GST polymorphism and prostate cancer have so far failed to unambiguously establish this relation in patients. A retrospective study on healthy, unrelated subjects was conducted in order to estimate the population GST genotype frequencies in the Slovak population of men and compare our results with already published data (GSEC project-Genetic Susceptibility to Environmental Carcinogens). A further aim of the study was to evaluate polymorphisms in GST also in patients with prostate cancer in order to compare the evaluated proportions with those found in the control subjects.MethodsWe determined the GST genotypes in 228 healthy, unrelated subjects who attended regular prostate cancer screening between May 2005 and June 2007 and in 129 histologically verified prostate cancer patients. Analysis for the GST gene polymorphisms was performed by PCR and PCR-RFLP.ResultsWe found that the GST frequencies are not significantly different from those estimated in a European multicentre study or from the results published by another group in Slovakia. Our results suggest that Val/Val genotype of GSTP1 gene could modulate the risk of prostate cancer, even if this association did not reach statistical significance. We did not observe significantly different crude rates of the GSTM1 and GSTT1 null genotypes in the men diagnosed with prostate cancer and those in the control group.ConclusionUnderstanding the contribution of GST gene polymorphisms and their interactions with other relevant factors may improve screening diagnostic assays for prostate cancer. We therefore discuss issues of study feasibility, study design, and statistical power, which should be taken into account in planning further trials.

Ectopic parvalbumin expression in mouse forebrain neurons increases excitotoxic injury provoked by ibotenic acid injection into the striatum

A neuroprotective role for Ca(2+)-binding proteins in neurodegenerative conditions ranging from ischemia to Alzheimers disease has been suggested in several studies. A key phenomenon in neurodegeneration is the Ca(2+)-mediated excitotoxicity brought about by the neurotransmitter glutamate. To evaluate the relative ability to resist excitotoxicity of neurons containing the slow-onset Ca(2+)-binding protein parvalbumin (PV), we injected the glutamate agonist ibotenic acid (IBO) into the striatum of adult mice ectopically expressing PV in neurons. Striatal ibotenic acid injection results in local nerve cell loss and reactive astrogliosis. Light microscopic evaluation, carried out after a delay of 2 and 4 weeks, reveals an enlarged and accelerated neurodegenerative process in mice ectopically expressing neuronal PV. Thus, PV is not neuroprotective, it rather enhances nerve cell death. This result implicates that the increase in cytosolic Ca(2+)-buffering capacity in the transgenic mice impairs other systems involved in Ca2+ sequestration. In addition, ultrastructural morphometric analysis shows that in neurons the mitochondrial volume is reduced in mice ectopically expressing neuronal PV. This is paralleled by a reduction in the amount of the mitochondrial marker enzyme cytochrome c oxidase subunit I (COXI). We conclude that alterations in the Ca(2+) homeostasis present in mice ectopically expressing neuronal PV are more deleterious under excitotoxic stress and largely outweigh the potential benefits of an increased Ca(2+)-buffering capacity resulting from PV.

Oxidative modifications of cardiac mitochondria and inhibition of cytochrome c oxidase activity by 4-hydroxynonenal

Abstract 4-Hydroxynonenal (HNE) is a highly toxic product of lipid peroxidation (LPO). Its role in the inhibition of cytochrome c oxidase activity and oxidative modifications of mitochondrial lipids and proteins were investigated. The exposure of mitochondria isolated from rat heart to HNE resulted in a time- and concentration-dependent inhibition of cytochrome c oxidase activity with an IC50 value of 8.3 ± 1.0 μM. Immunoprecipitation-Western blot analysis showed the formation of HNE adducts with cytochrome c oxidase subunit I. The loss of cytochrome c oxidase activity was also accompanied by reduced thiol group content and increased HNE-lysine fluorescence. Furthermore, there was a marked increase in conjugated diene formation indicating LPO induction by HNE. Fluorescence measurements revealed the formation of bityrosines and increased surface hydrophobicity of HNE-treated mitochondrial membranes. Superoxide dismutase + catalase and the HO• radical scavenger mannitol partially prevented inhibition of cytochrome c oxidase activity and formation of bityrosines. These findings suggest that HNE induces formation of reactive oxygen species and its damaging effect on mitochondria involves both formation of HNE–protein adducts and oxidation of membrane lipids and proteins by free radicals.

Distribution of plasma membrane Ca2+ pump (PMCA) isoforms in the gerbil brain: effect of ischemia-reperfusion injury.

Non-species isoform-specific antibodies against three isoforms of the plasma membrane Ca2+ pump (PMCA) were used for immuno-localization of PMCA by Western blot analysis in membrane preparations isolated from different regions of gerbil brain. All three gene products were detected in the membranes from hippocampus, cerebral cortex and cerebellum. However, they showed a distinct distribution pattern. Two proteins were revealed in the case of PMCA1 with molecular masses 129 and 135 kDa. The antibody against PMCA2 recognized three proteins of about 130-137 kDa. Only one protein was detected with the anti-PMCA3 antibody. Levels of immuno-signal for the PMCA isoforms varied significantly among the different brain regions. The PMCA1 is the most abundant in the cerebro-cortical and hippocampal membrane preparations. The PMCA2 was detected in a lesser amount comparing to PMCA1 and was highest in the membrane preparations from cerebellum and in a slightly lesser amount from cerebral cortex. Anti-PMCA3 antibody stained weakly and was localized in the cerebellar and hippocampal membrane preparations. Transient forebrain ischemia (10 min) and reperfusion (for a prolonged period up to 10 d) leads to a significant decrease of PMCA immuno-signal. This decrease could be ascribed to the loss of PMCA1 signal, especially in hippocampal membrane preparations.