Extracellular Purine Metabolism-Potential Target in Multiple Sclerosis.

The purinergic signaling system comprises a complex network of extracellular purines and purine-metabolizing ectoenzymes, nucleotide and nucleoside receptors, ATP release channels, and nucleoside transporters. Because of its immunomodulatory function, this system is critically involved in the pathogenesis of multiple sclerosis (MS) and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). MS is a chronic neuroinflammatory demyelinating and neurodegenerative disease with autoimmune etiology and great heterogeneity, mostly affecting young adults and leading to permanent disability. In MS/EAE, alterations were detected in almost all components of the purinergic signaling system in both peripheral immune cells and central nervous system (CNS) glial cells, which play an important role in the pathogenesis of the disease. A decrease in extracellular ATP levels and an increase in its downstream metabolites, particularly adenosine and inosine, were frequently observed at MS, indicating a shift in metabolism toward an anti-inflammatory environment. Accordingly, upregulation of the major ectonucleotidase tandem CD39/CD73 was detected in the blood cells and CNS of relapsing-remitting MS patients. Based on the postulated role of A2A receptors in the transition from acute to chronic neuroinflammation, the association of variants of the adenosine deaminase gene with the severity of MS, and the beneficial effects of inosine treatment in EAE, the adenosinergic system emerged as a promising target in neuroinflammation. More recently, several publications have identified ADP-dependent P2Y12 receptors and the major extracellular ADP producing enzyme nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) as novel potential targets in MS.

Expression of Ectonucleoside Triphosphate Diphosphohydrolase 2 (NTPDase2) Is Negatively Regulated Under Neuroinflammatory Conditions In Vivo and In Vitro.

Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, which is the ligand for P2Y1,12,13 receptors. The present study describes the distribution of NTPDase2 in adult rat brains in physiological conditions, and in hippocampal neurodegeneration induced by trimethyltin (TMT). The study also describes the regulation of NTPDase2 by inflammatory mediators in primary astrocytes and oligodendroglial cell line OLN93. In physiological conditions, NTPDase2 protein was most abundant in the hippocampus, where it was found in fibrous astrocytes and synaptic endings in the synaptic-rich hippocampal layers. In TMT-induced neurodegeneration, NTPDase2-mRNA acutely decreased at 2-dpi and then gradually recovered to the control level at 7-dpi and 21-dpi. As determined by immunohistochemistry and double immunofluorescence, the decrease was most pronounced in the dentate gyrus (DG), where NTPDase2 withdrew from the synaptic boutons in the polymorphic layer of DG, whereas the recovery of the expression was most profound in the subgranular layer. Concerning the regulation of NTPDase2 gene expression, proinflammatory cytokines IL-6, IL-1ß, TNFα, and IFNγ negatively regulated the expression of NTPDase2 in OLN93 cells, while did not altering the expression in primary astrocytes. Different cell-intrinsic stressors, such as depletion of intracellular energy store, oxidative stress, endoplasmic reticulum stress, and activation of protein kinase C, also massively disturbed the expression of the NTPDase2 gene. Together, our results suggest that the expression and the activity of NTPDase2 transiently cease in neurodegeneration and brain injury, most likely as a part of the acute adaptive response designed to promote cell defense, survival, and recovery.

Agmatine Mitigates Inflammation-Related Oxidative Stress in BV-2 Cells by Inducing a Pre-Adaptive Response.

Neuroinflammation and microglial activation, common components of most neurodegenerative diseases, can be imitated in vitro by challenging microglia cells with Lps. We here aimed to evaluate the effects of agmatine pretreatment on Lps-induced oxidative stress in a mouse microglial BV-2 cell line. Our findings show that agmatine suppresses nitrosative and oxidative burst in Lps-stimulated microglia by reducing iNOS and XO activity and decreasing O2- levels, arresting lipid peroxidation, increasing total glutathione content, and preserving GR and CAT activity. In accordance with these results, agmatine suppresses inflammatory NF-kB, and stimulates antioxidant Nrf2 pathway, resulting in decreased TNF, IL-1 beta, and IL-6 release, and reduced iNOS and COX-2 levels. Together with increased ARG1, CD206 and HO-1 levels, our results imply that, in inflammatory conditions, agmatine pushes microglia towards an anti-inflammatory phenotype. Interestingly, we also discovered that agmatine alone increases lipid peroxidation end product levels, induces Nrf2 activation, increases total glutathione content, and GPx activity. Thus, we hypothesize that some of the effects of agmatine, observed in activated microglia, may be mediated by induced oxidative stress and adaptive response, prior to Lps stimulation.

Antenatal Dexamethasone Treatment Induces Sex-dependent Upregulation of NTPDase1/CD39 and Ecto-5'-nucleotidase/CD73 in the Rat Fetal Brain.

Dexamethasone (DEX) is frequently used to treat women at risk of preterm delivery, but although indispensable for the completion of organ maturation in the fetus, antenatal DEX treatment may exert adverse sex-dimorphic neurodevelopmental effects. Literature findings implicated oxidative stress in adverse effects of DEX treatment. Purinergic signaling is involved in neurodevelopment and controlled by ectonucleotidases, among which in the brain the most abundant are ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5'-nucleotidase (e5'NT/CD73), which jointly dephosphorylate ATP to adenosine. They are also involved in cell adhesion and migration, processes integral to brain development. Upregulation of CD39 and CD73 after DEX treatment was reported in adult rat hippocampus. We investigated the effects of maternal DEX treatment on CD39 and CD73 expression and enzymatic activity in the rat fetal brain of both sexes, in the context of oxidative status of the brain tissue. Fetuses were obtained at embryonic day (ED) 21, from Wistar rat dams treated with 0.5 mg DEX/kg/day, at ED 16, 17, and 18, and brains were processed and used for further analysis. Sex-specific increase in CD39 and CD73 expression and in the corresponding enzyme activities was induced in the brain of antenatally DEX-treated fetuses, more prominently in males. The oxidative stress induction after antenatal DEX treatment was confirmed in both sexes, although showing a slight bias in males. Due to the involvement of purinergic system in crucial neurodevelopmental processes, future investigations are needed to determine the role of these observed changes in the adverse effects of antenatal DEX treatment.

The Function of the Hypothalamic-Pituitary-Adrenal Axis During Experimental Autoimmune Encephalomyelitis: Involvement of Oxidative Stress Mediators.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease with an unknown origin. Previous studies showed the involvement of the hypothalamic-pituitary-adrenal (HPA) axis to susceptibility to autoimmune diseases, including MS, and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). During MS/EAE, innate immune cells are activated and release cytokines and other inflammatory mediators, leading to a vicious cycle of inflammation. In response to inflammation, the activated HPA axis modulates immune responses via glucocorticoid activity. Because the mechanisms involving oxidative stress to the HPA axis are relatively unrevealed, in this study, we investigate the inflammatory and oxidative stress status of HPA axis during EAE. Our results reveal an upregulation of Pomc gene expression, followed by POMC and ACTH protein increase at the peak of the EAE in the pituitary. Also, prostaglandins are well-known contributors of HPA axis activation, which increases during EAE at the periphery. The upregulated Tnf expression in the pituitary during the peak of EAE occurred. This leads to the activation of oxidative pathways, followed by upregulation of inducible NO synthase expression. The reactive oxidant/nitrosative species (ROS/RNS), such as superoxide anion and NO, increase their levels at the onset and peak of the disease in the pituitary and adrenal glands, returning to control levels at the end of EAE. The corticotrophs in the pituitary increased in number and volume at the peak of EAE that coincides with high lipid peroxidation levels. The expression of MC2R in the adrenal glands increases at the peak of EAE, where strong induction of superoxide anion and malondialdehyde (MDA), reduced total glutathione (GSH) content, and catalase activity occurred at the peak and end of EAE compared with controls. The results obtained from this study may help in understanding the mechanisms and possible pharmacological modulation in MS and demonstrate an effect of oxidative stress exposure in the HPA activation during the course of EAE.

Induction of NTPDase1/CD39 by Reactive Microglia and Macrophages Is Associated With the Functional State During EAE.

Purinergic signaling is critically involved in neuroinflammation associated with multiple sclerosis (MS) and its major inflammatory animal model, experimental autoimmune encephalomyelitis (EAE). Herein, we explored the expression of ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) in the spinal cord, at the onset (Eo), peak (Ep), and end (Ee) of EAE. Several-fold increase in mRNA and in NTPDase1 protein levels were observed at Eo and Ep. In situ hybridization combined with fluorescent immunohistochemistry showed that reactive microglia and infiltrated mononuclear cells mostly accounted for the observed increase. Colocalization analysis revealed that up to 80% of Iba1 immunoreactivity and ∼50% of CD68 immunoreactivity was colocalized with NTPDase1, while flow cytometric analysis revealed that ∼70% of mononuclear infiltrates were NTPDase1+ at Ep. Given the main role of NTPDase1 to degrade proinflammatory ATP, we hypothesized that the observed up-regulation of NTPDase1 may be associated with the transition between proinflammatory M1-like to neuroprotective M2-like phenotype of microglia/macrophages during EAE. Functional phenotype of reactive microglia/macrophages that overexpress NTPDase1 was assessed by multi-image colocalization analysis using iNOS and Arg1 as selective markers for M1 and M2 reactive states, respectively. At the peak of EAE NTPDase1 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype. Additionally, in ∼80% of CD68 positive cells NTPDase1 was coexpressed with Arg1 compared to negligible fraction coexpresing iNOS and ∼15% coexpresing both markers, additionally indicating prevalent association of NTPDase1 with M2-like microglial/macrophages phenotype at Ep. Together, our data suggest an association between NTPDase1 up-regulation by reactive microglia and infiltrated macrophages and their transition toward antiinflammatory phenotype in EAE.

Voltage Gated Potassium Channel Kv1.3 Is Upregulated on Activated Astrocytes in Experimental Autoimmune Encephalomyelitis.

Kv1.3 is a voltage gated potassium channel that has been implicated in pathophysiology of multiple sclerosis (MS). In the present study we investigated temporal and cellular expression pattern of this channel in the lumbar part of spinal cords of animals with experimental autoimmune encephalomyelitis (EAE), animal model of MS. EAE was actively induced in female Dark Agouti rats. Expression of Kv1.3 was analyzed at different time points of disease progression, at the onset, peak and end of EAE. We here show that Kv1.3 increased by several folds at the peak of EAE at both gene and protein level. Double immunofluorescence analyses demonstrated localization of Kv1.3 on activated microglia, macrophages, and reactive astrocytes around inflammatory lesions. In vitro experiments showed that pharmacological block of Kv1.3 in activated astrocytes suppresses the expression of proinflammatory mediators, suggesting a role of this channel in inflammation. Our results support the hypothesis that Kv1.3 may be a therapeutic target of interest for MS and add astrocytes to the list of cells whose activation would be suppressed by inhibiting Kv1.3 in inflammatory conditions.

Down-regulation of NTPDase2 and ADP-sensitive P2 Purinoceptors Correlate with Severity of Symptoms during Experimental Autoimmune Encephalomyelitis.

The present study explores tissue and cellular distribution of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and the gene and protein expression in rat spinal cord during the course of experimental autoimmune encephalomyelitis (EAE). Given that NTPDase2 hydrolyzes ATP with a transient accumulation of ADP, the expression of ADP-sensitive P2 purinoceptors was analyzed as well. The autoimmune disease was actively induced in Dark Agouti female rats and the changes were analyzed 10, 15 and 29 days after the induction. These selected time points correspond to the onset ( Eo ), peak ( Ep ) and recovery ( Er ) from EAE. In control animals, NTPDase2 was confined in the white matter, in most of the glial fibrillary acidic protein (GFAP)-immunoreactive (ir) astrocytes and in a considerable number of nestin-ir cells, while the other cell types were immunonegative. Immunoreactivity corresponding to NTPDase2 decreased significantly at Eo and Ep and then returned to the baseline levels at Er . The preservation of the proportion of GFAP single-labeled and GFAP/NTPDase2 double-labeled elements along the course of EAE indicated that changes in NTPDase2-ir occurred at fibrous astrocytes that typically express NTPDase2 in normal conditions. Significant downregulation of P2Y1 and P2Y12 receptor proteins at Eo and several-fold induction of P2Y12 and P2Y13 receptor proteins at Ep and/or Er were observed implying that the pathophysiological process in EAE may be linked to ADP signaling. Cell-surface expression of NTPDase2, NTPDase1/CD39 and ecto-5'-nucleotidase (eN/CD73) was analyzed in CD4+ T cells of a draining lymph node by fluorescence-activated cell sorting. The induction of EAE was associated with a transient decrease in a number of CD4+ NTPDase2+ T cells in a draining lymph node, whereas the recovery was characterized by an increase in NTPDase2+ cells in both CD4+ and CD4- cell populations. The opposite was found for NTPDase1/CD39+ and eN/CD73+ cells, which slightly increased in number with progression of the disease, particularly in CD4- cells, and then decreased in the recovery. Finally, CD4+ NTPDase2+ cells were never observed in the spinal cord parenchyma. Taken together, our results suggest that the process of neuroinflammation in EAE may be associated with altered ADP signaling.

Extracellular ATP induces graded reactive response of astrocytes and strengthens their antioxidative defense in vitro.

It is widely accepted that adenosine triphosphate (ATP) acts as a universal danger-associated molecular pattern with several known mechanisms for immune cell activation. In the central nervous system, ATP activates microglia and astrocytes and induces a neuroinflammatory response. The aim of the present study was to describe responses of isolated astrocytes to increasing concentrations of ATP (5 µM to 1 mM), which were intended to mimic graded intensity of the extracellular stimulus. The results show that ATP induces graded activation response of astrocytes in terms of the cell proliferation, stellation, shape remodeling, and underlying actin and GFAP filament rearrangement, although the changes occurred without an apparent increase in GFAP and actin protein expression. On the other hand, ATP in the range of applied concentrations did not evoke IL-1ß release from cultured astrocytes, nor did it modify the release from LPS and LPS+IFN-γ-primed astrocytes. ATP did not promote astrocyte migration in the wound-healing assay, nor did it increase production of reactive oxygen and nitrogen species and lipid peroxidation. Instead, ATP strengthened the antioxidative defense of astrocytes by inducing Cu/ZnSOD and MnSOD activities and by increasing their glutathione content. Our current results suggest that although ATP triggers several attributes of activated astrocytic phenotype with a magnitude that increases with the concentration, it is not sufficient to induce full-blown reactive phenotype of astrocytes in vitro. © 2016 Wiley Periodicals, Inc.

Low-dose ribavirin treatments attenuate neuroinflammatory activation of BV-2 Cells by interfering with inducible nitric oxide synthase.

Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 µM) modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 µM) to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS) stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 µM ribavirin promoted LPS induced apoptosis. We determined that 1 µM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 µM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation.

Extracellular ATP Selectively Upregulates Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 and Ecto-5'-Nucleotidase by Rat Cortical Astrocytes In Vitro.

Extracellular ATP (eATP) acts as a danger-associated molecular pattern which induces reactive response of astrocytes after brain insult, including morphological remodeling of astrocytes, proliferation, chemotaxis, and release of proinflammatory cytokines. The responses induced by eATP are under control of ecto-nucleotidases, which catalyze sequential hydrolysis of ATP to adenosine. In the mammalian brain, ecto-nucleotidases comprise three enzyme families: ecto-nucleoside triphosphate diphosphohydrolases 1-3 (NTPDase1-3), ecto-nucleotide pyrophosphatase/phospodiesterases 1-3 (NPP1-3), and ecto-5'-nucleotidase (eN), which crucially determine ATP/adenosine ratio in the pericellular milieu. Altered expression of ecto-nucleotidases has been demonstrated in several experimental models of human brain dysfunctions. In the present study, we have explored the pattern of NTPDase1-3, NPP1-3, and eN expression by cultured cortical astrocytes challenged with 1 mmol/L ATP (eATP). At the transcriptional level, eATP upregulated expression of NTPDase1, NTPDase2, NPP2, and eN, while, at translational and functional levels, these were paralleled only by the induction of NTPDase2 and eN. Additionally, eATP altered membrane topology of eN, from clusters localized in membrane domains to continuous distribution along the cell membrane. Our results suggest that eATP, by upregulating NTPDase2 and eN and altering the enzyme membrane topology, affects local kinetics of ATP metabolism and signal transduction that may have important roles in the process related to inflammation and reactive gliosis.

Benfotiamine attenuates inflammatory response in LPS stimulated BV-2 microglia.

Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.

Effect of stab injury in the rat cerebral cortex on temporal pattern of expression of neuronal cytoskeletal proteins: an immunohistochemical study.

Compelling evidence now points to the critical role of the cytoskeleton in neurodegeneration. In the present study, using an immunohistochemical approach, we have shown that cortical stab injury (CSI) in adult Wistar rats significantly affects temporal pattern of expression of neurofilament proteins (NFs), a major cytoskeleton components of neurons, and microtubule-associated proteins (MAP2). At 3 days post-injury (dpi) most of the NFs immunoreactivity was found in pyknotic neurons and in fragmentized axonal processes in the perilesioned cortex. These cytoskeletal alterations became more pronounced by 10dpi. At the subcellular level CSI also showed significant impact on NFs and MAP-2 expression. Thus, at 3dpi most of the dendrites disappeared, while large neuronal somata appeared like open circles pointing to membrane disintegration. Conversely, at 10dpi neuronal perikarya and a few new apical dendrites were strongly labeled. Since aberrant NF phosphorylation is a pathological hallmark of many human neurodegenerative disorders, as well as is found after stressor stimuli, the present results shed light into the expression of neurofilaments after the stab brain injury.

Brain Injury Alters Ectonucleotidase Activities and Adenine Nucleotide Levels in Rat Serum.

BACKGROUND: Cortical stab injury (CSI) induces changes in the activity, expression and cellular distribution of specific ectonucleotidases at the injury site. Also, several experimentally induced neuropathologies are associated with changes in soluble ectonucleotidase activities in the plasma and serum, whilst various insults to the brain alter purine compounds levels in cerebrospinal fluid, but also in serum, indicating that insults to the brain may induce alterations in nucleotides release and rate of their hydrolysis in the vascular system. Since adenine nucleotides and adenosine regulate diverse cellular functions in the vascular system, including vascular tone, platelet aggregation and inflammatory responses of lymphocytes and macrophages, alterations of ectonucleotidase activities in the vascular system may be relevant for the clinical outcome of the primary insult. METHODS: We explored ectonucleotidase activities using specific enzyme assays and determined adenine nucleotides concentrations by the UPLC method in the rat serum after cortical stab injury. RESULTS: At 4-h post-injury, ATP and AMP hydrolysis increased by about 60% and 40%, respectively, while phosphodiesterase activity remained unchanged. Also, at 4-h post-injury a marked decrease in ATP concentration and more than 2-fold increase in AMP concentration were recorded. CONCLUSIONS: CSI induces rapid up-regulation of nucleotide catabolizing soluble ectonucleotidases in rat serum, which leads to the observed shift in serum nucleotide levels. The results obtained imply that ectonucleotidases and adenine nucleotides participate in the communication between the brain and the vascular system in physiological and pathological conditions and thereby may be involved in the development of various human neuropathologies.

Expression of a second ecto-5'-nucleotidase variant besides the usual protein in symptomatic phase of experimental autoimmune encephalomyelitis.

Ecto-5'-nucleotidase/cluster of differentiation 73 (CD73) (eN) is a 70-kDa glycoprotein expressed in several different mammalian tissues and cell types. It is the rate-limiting enzyme of the purine catabolic pathway, which catalyzes the hydrolysis of AMP to produce adenosine with known anti-inflammatory and immunosuppressive actions. There is strong evidence for lymphocyte and endothelial cell eN having a role in experimental autoimmune encephalomyelitis (EAE), but the role of eN in cell types within the central nervous system is less clear. We have previously shown that eN activity significantly increased in the lumbar spinal cord during EAE. The present study is aimed to explore molecular pattern of the eN upregulation over the course of the disease and cell type(s) accountable for the induction. EAE was induced in Dark Agouti (DA) rats by immunization with the spinal cord tissue homogenate and adjuvant. Animals were sacrificed 8, 15, and 28 days following immunization (D8, D15, and D28), i.e., at time points which corresponded to the presymptomatic, symptomatic, and postsymptomatic phases of the disease, respectively. Significant increase in eN activity and its upregulation at the gene and the protein levels were demonstrated at D15 and less prominently at D28 in comparison to control. Additionally, reactive astrocytes abundantly present in the lumbar spinal cord parenchyma were identified as principal cell type with significantly elevated eN expression. In all experimental groups, eN was expressed as a 71-kDa protein band of uniform abundance, whereas the overexpression of eN at D15 and D28 was associated with the expression of a second 75-kDa eN variant. The possible outcome of eN upregulation during EAE as a part of protective astrocyte repertoire contributing to the resolution of the disease is discussed.

The effect of pH on the activity of soluble peroxidase in needles of Serbian spruce (Picea omorika (Panc.) Purkinye): application of a mathematical model.

We studied the dependence of peroxidase (POD) activity on pH in crude extract of Picea omorika (Panc.) Purkinye needles and in its acidic and basic fractions, obtained by ion exchange chromatography. Nonlinear regression was applied on the activity data with pH as the explaining variable, using the Levenberg-Marquardt algorithm. Studying crude extract at three different temperatures, the shape of the simulated activity/pH dependences indicated an existence of two components, which was confirmed by mathematical modeling. The kinetic parameters Act0, KEH and KEOH of both components are presented. The curves and pH optima shifted under increasing temperatures towards lower pH values, which was verified after decomposition. Nonlinear regression detected the presence of two components for both fractions, and there is no considerable difference between their pH optima. Our results show for the first time that the sum of components, each described by the mathematical model employed, can be used to explain the complex pH-related POD activity in the extract with two or more enzyme forms simultaneously active.

Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum.

Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K (m) for p-Nph-5'-TMP hydrolysis of 61.8 +/- 5.2 microM. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5'-TMP hydrolysis with K (i) values ranging 13-43 microM. Nucleotide analogs, alpha,beta-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas alpha,beta-metADP induced mixed inhibition, with K (i) ranging from 2 to 20 microM. Chromatographic analysis revealed that alpha,beta-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and alpha,beta-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application.

Regional changes in ectonucleotidase activity after cortical stab injury in rat.

During a variety of insults to the brain adenine nucleotides are released in large quantities from damaged cells, triggering local cellular and biochemical responses to injury. Different models of brain injury reveal that the local increase in adenine nucleotides levels is followed by a compensatory up-regulation of ectonucleotidase enzymes that catalyze sequential hydrolysis of ATP to ADP, AMP and adenosine. However, recent studies imply that changes in adenine nucleotides release may also occur in the areas distant from the site of direct damage. Therefore, in the present study we have used the model of cortical stab injury to analyze extracellular ATP, ADP and AMP hydrolysis in the membrane preparations obtained from the brain regions that were not subjected to direct tissue damage. The brain regions analyzed were contralateral cortex, hippocampus, caudate nucleus, thalamus and hypothalamus. It was evidenced that cortical stab injury induced early widespread decrease in AMP hydrolysis in all brain areas tested, except in the hypothalamus, without changes in ATP hydrolysis. These findings imply that brain injury affects global extracellular adenine nucleotide and nucleoside levels, consequently affecting neuronal function in the regions distant to the primary damage.

Kinetic parameters for thermal inactivation of soluble peroxidase from needles of Serbian spruce Picea omorika (Pancic) Purkyne.

Thermal inactivation of peroxidase (POD) in an extract of Picea omorika (Pancic) Purkyne needles initiated by heat treatment was studied. This is the first study of this kind on a conifer species. Non-linear regression analysis was applied on the inactivation rate data, combining Mitscherlich and Arrhenius equations, treating time and temperature simultaneously as explaining variables. We determined the inactivation rate constant k, the Arrhenius energy of inactivation E and the remaining activity C(min) for the crude extract and for separated acidic and basic enzyme fractions, as well as for individual isoenzymes separated electrophoretically. A comparison of inactivation parameters for acidic and basic fractions shows that the thermal inactivation rate of the basic fraction is higher. The obtained value of inactivation energy for crude extract was between the values for acidic and basic isoenzyme fractions. One of the three analysed individual isoenzymes was characterised by a lower inactivation rate constant and higher inactivation energy. Another isoenzyme showed considerably higher level of remaining activity compared to the others, which identified it as the most resistant to high temperatures. The acquired values of Arrhenius energy of inactivation for POD in crude extract were intermediate, considering a range of POD values for various other plant species.