Anica Horvat

Developmental profile of NTPDase activity in synaptic plasma membranes isolated from rat cerebral cortex

In the present study the developmental profile of ATP‐hydrolyzing activity promoted by NTPDase 1, its kinetic properties and the enzyme protein abundance associated with synaptic plasma membrane from rat cerebral cortex were characterized. NTPDase 1 activity increased from birth to day 30; afterwards it decreased and remained unchanged from adulthood (90 days) to senescence (365 days). Kinetic analysis revealed that enzyme exhibited the highest specific activity at day 30 and highest apparent affinity for ATP at day 365; however, Vmax/Km values remained unchanged for each age studied. Immunoblot analysis demonstrated that relative abundance of NTPDase 1 is highest at day 15 during ontogeny. The discrepancy between maximum enzyme activity and maximum enzyme protein abundance indicates that NTPDase 1 may have an additional role during development.

Low-Dose Dexamethasone Treatment Promotes the Pro-Survival Signalling Pathway in the Adult Rat Prefrontal Cortex

Synthetic glucocorticoid dexamethasone (DEX), a highly potent anti‐inflammatory and immunosuppressive agent, is widely used in the treatment of brain cancer, as well as for inflammatory and autoimmune diseases. The present study aimed to determine whether low‐dose subchronic DEX treatment (100 μg/kg for eight consecutive days) exerts long‐term effects on apoptosis in the adult rat prefrontal cortex (PFC) by examining the expression of cell death‐promoting molecules [poly(ADP‐ribose) polymerase (PARP), p53, procaspase 3, cleaved caspase 3, Bax] and cell‐survival molecules (AKT, Bcl‐2). The results obtained revealed that body, thymus and adrenal gland weights, as well corticosterone levels, in the serum and PFC were reduced 1 day after the last DEX injection. In the PFC, DEX caused activation of AKT, augmentation of pro‐survival Bcl‐2 protein and an enhanced Bcl‐2/Bax protein ratio, as well Bcl‐2 translocation to the mitochondria. An unaltered profile with respect to the protein expression of apoptotic molecules PARP, procaspase 3 and Bax was detected, whereas p53 protein was decreased. Reverse transcriptase ‐polymerase chain reaction analysis showed a decrease of p53 mRNA levels and no significant difference in Bcl‐2 and Bax mRNA expression in DEX‐treated rats. Finally, a DNA fragmentation assay and Fluoro‐Jade staining demonstrated no considerable changes in apoptosis in the rat PFC. Our findings support the concept that low‐dose DEX creates a hypocorticoid state in the brain and also indicate that subchronic DEX treatment activates the pro‐survival signalling pathway but does not change apoptotic markers in the rat PFC. This mechanism might be relevant for the DEX‐induced apoptosis resistance observed during and after chemotherapy of patients with brain tumours.

Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes

Ecto‐5′‐nucleotidase (CD73; EC 3.1.3.5, e‐5NT) is regarded as the key enzyme in the extracellular formation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor in the brain. In the present study, we have investigated e‐5NT activity, kinetic properties concerning AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM) preparations isolated from whole female rat brain at different ages. We observed pronounced increase in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15‐day‐old) and pre‐pubertal (30‐day‐old) rats. Immunodetection of e‐5NT protein in the SPM displayed the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum relative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme protein abundance in the SPM indicates that e‐5NT has additional roles in the synaptic compartment during postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic parameters, Km and Vmax, suggested that the increase in the enzyme activity with maturation was entirely due to the increase in the enzyme catalytic efficiency (Vmax/Km). Finally, double immunofluorescence staining against e‐5NT and presynaptic membrane marker syntaxin provided first direct evidence for the existence of this ecto‐enzyme in the presynaptic compartment. The results of the study suggest that e‐5NT may be a part of general scheme of brain development and synapse maturation and provide rationale for the previously reported inconsistencies between enzyme immunohistochemical and biochemical studies concerning localization of e‐5NT in the brain.

Radiation-Induced Hyposuppression of the Hypothalamic-Pituitary-Adrenal Axis is Associated with Alterations of Hippocampal Corticosteroid Receptor Expression

Abstract Veličković, N., Djordjević, A., Matić, G. and Horvat, A. Radiation-Induced Hyposuppression of the Hypothalamic-Pituitary-Adrenal Axis is Associated with Alterations of Hippocampal Corticosteroid Receptor Expression. Radiat. Res. 169, 397–407 (2008). Therapeutic brain irradiation in children can cause a progressive decline in cognitive functions through a diminished capability to learn and memorize. Because of the known involvement of the hippocampus in memory consolidation, this study was aimed at examining the late effects of γ radiation on hypothalamic-pituitary-adrenal (HPA) axis activity and hippocampal corticosteroid receptor expression in an animal model of cranial radiotherapy. In the late-response phase, the basal and stress-induced corticosterone levels were not affected by radiation, but the suppression of glucocorticoid negative feedback by dexamethasone was attenuated in irradiated rats. Western blot analyses showed that exposure to radiation led to a decrease of cytosolic glucocorticoid receptor (GR) levels and a concomitant elevation of mineralocorticoid receptor (MR). The results obtained were complemented by those of RT-PCR, since the ratio of GR/MR mRNA was also decreased after radiation exposure. Dexamethasone appeared to be much less effective in shifting GR to the nuclear compartment in irradiated rats than in sham-irradiated animals. However, the expression of chaperones that aid GR intracellular trafficking, Hsp90 and Hsp70, remained unaffected. In conclusion, our data suggest that the hallmark of the late response to γ radiation is a hyposuppressive state of the HPA axis that is associated with a decrease in both the GR/MR ratio and the nuclear accumulation of dexamethasone-activated GR in the hippocampus.

Effect of acute stress on NTPDase and 5'-nucleotidase activities in brain synaptosomes in different stages of development.

The aim of the present study was to examine the effect of acute restraint stress on rat brain synaptosomal plasma membrane (SPM) ecto‐nucleotidase activities at specific stages of postnatal development (15‐, 30‐, 60‐ and 90‐day‐old rats) by measuring the rates of ATP, ADP and AMP hydrolysis 1, 24 and 72 h post‐stress. At 1 h after stress NTPDase and ecto‐5′‐nucleotidase activities were decreased in rats aged up to 60 days old. In adult rats elevated enzyme activities were detected, which indicated the existence of different short‐term stress responses during development. A similar pattern of ATP and ADP hydrolysis changes as well as the ATP/ADP ratio in all developmental stages indicated that NTPDase3 was acutely affected after stress. The long‐term effect of acute stress on NTPDase activity differed during postnatal development. In juvenile animals (15 days old) NTPDase activity was not altered. However, in later developmental stages (30 and 60 days old rats) NTPDase activity decreased and persisted for 72 h post‐stress. In adult rats only ATP hydrolysis was decreased after 24 h, indicating that ecto‐ATPase was affected by stress. Ecto‐5′‐nucleotidase hydrolysing activity was decreased within 24 h in adult rats, while in 15‐ and 30‐day old rats it decreased 72 h post‐stress. At equivalent times in pubertal rats (60 days old) a slight activation of ecto‐5′‐nucleotidase was detected. Our results highlight the developmental‐dependence of brain ecto‐nucleotidase susceptibility to acute stress and the likely existence of different mechanisms involved in time‐dependent ecto‐nucleotidase activity modulation following stress exposure. Clearly there are differences in the response of the purinergic system to acute restraint stress between young and adult rats.

Estradiol Affects Calcium Transport across Mitochondrial Membrane in Different Brain Regions

Abstract: The in vitro effect of estradiol on flux of Ca2+ in the synaptosomal mitochondria from nucleus caudatus and hippocampus of chronically ovariectomized female rats was examined. No effect of estradiol on Ca2+ influx through ruthenium red‐sensitive channels was found. Estradiol, at a concentration of 0.05‐5 nmol/L for nucleus caudatus and 0.5‐5 nmol/L for the hippocampus, decreased Na‐dependent Ca2+ efflux about 25%.

Repeated low-dose 17β-estradiol treatment prevents activation of apoptotic signaling both in the synaptosomal and cellular fraction in rat prefrontal cortex following cerebral ischemia

Disturbance in blood circulation is associated with numerous pathological conditions characterized by cognitive decline and neurodegeneration. Activation of pro-apoptotic signaling previously detected in the synaptosomal fraction may underlie neurodegeneration in the prefrontal cortex of rats submitted to permanent bilateral common carotid arteries occlusion (two-vessel occlusion, 2VO). 17β-Estradiol (E) exerts potent neuroprotective effects in the brain affecting, among other, ischemia-induced pathological changes. As most significant changes in rats submitted to 2VO were observed on 7th day following the insult, of interest was to examine whether 7 day treatment with low dose of E (33.3 µg/kg/day) prevents formerly reported neurodegeneration and may represent additional therapy during the early post-ischemic period. Role of E treatment on apoptotic pathway was monitored on Bcl-2 family members, cytochrome c, caspase 3 and PARP protein level in the synaptosomal (P2) fraction of the prefrontal cortex. Furthermore, changes of these proteins were examined in the cytosolic, mitochondrial and nuclear fraction, with the emphasis on potential involvement of extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) activation and their role in nuclear translocation of transcriptional nuclear factor kappa B (NF-kB) associated with alteration of Bax and Bcl-2 gene expression. The extent of cellular damage was determined using DNA fragmentation and Fluoro-Jade B staining. The absence of activation of apoptotic cascade both in the P2 and cell accompanied with decreased DNA fragmentation and number of degenerating neurons clearly indicates that E treatment ensures the efficient protection against ischemic insult. Moreover, E-mediated modulation of pro-apoptotic signaling in the cortical cellular fractions involves cooperative activation of ERK and Akt, which may be implicated in the observed prevention of neurodegenerative changes.

17β-estradiol modulates mitochondrial Ca2+ flux in rat caudate nucleus and brain stem

The aim of this study was to examine the rapid non-genomic effect of 17β-estradiol (E2) on Ca(2+) transport in mitochondria isolated from the nerve terminals (synaptosomes) of caudate nuclei (NC) and brain stems (BS) of ovariectomised female rats. In physiological conditions no effect of E2 on Ca(2+) influx into synaptosomal mitochondria through ruthenium red (RR)-sensitive uniporter was observed. However, in the presence of uncoupling agent carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) (1μmol/l), pre-treatment with 0.5nmol/l E2 protected mitochondrial membrane potential and consequently increased Ca(2+) influx (2.3-fold in NC and 3.1-fold in BS). At the same time, 0.5nmol/l E2 by increasing the affinity of mitochondrial Na(+)/Ca(2+) exchanger for Na(+) inhibited mitochondrial Ca(2+) efflux in NC and BS by about 40%. Also, the specific binding of physiological E2 concentrations (0.1-10nmol/l) to isolated synaptosomal mitochondria was detected. Using membrane impermeable E2 bound to bovine serum albumin and selective inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, we obtained that E2s action on mitochondrial Ca(2+) efflux at least partially is due to the direct effects on the mitochondrial membrane and/or Na(+)/Ca(2+) exchanger located in inner mitochondrial membrane. Our results implicate E2 as a modulator of Ca(2+) concentration in mitochondrial matrix, and ultimately in the cytosol. Given the vital role of Ca(2+) in regulation of total nerve cells activity, especially energy metabolism, neurotransmission and directing the cells toward survival or cell death, the effects on mitochondrial Ca(2+) transport could be one of the important modes of E2 neuromodulatory action independent of the genome.

Effects of Metal Ions on Plasma Membrane Mg2+‐ATPase in Rat Uterus and Ovaries

Abstract: The in vitro effects of cadmium and mercury were investigated on the Mg2+‐ATPase activity of plasma membranes from the rat ovary and uterus. ATP hydrolyzing activities were significant and dose‐dependent‐inhibited in both plasma membrane preparations by both metals. According to the IC50 and apparent Ki, Cd2+ was most potent in the ovary, while Hg2+ was most potent in the uterus. In ovaries and uterus, Cd2+ inhibits competitively, while Hg2+ inhibits noncompetitively in both organs. The observed inhibition was a consequence of direct action of the chosen metal ions on the enzyme protein and by decreasing ATP hydrolysis, Hg2+ and Cd2+ may affect mammalian fertility.

Inhibition of mitochondrial Na+-dependent Ca2+ efflux by 17β-estradiol in the rat hippocampus

Our results, as well as those of others, have indicated that 17β-estradiol (E2) exerts its nongenomic effects in neuronal cells by affecting plasma membrane Ca(2+) flux. In neuronal cells mitochondria possess Ca(2+) buffering properties as they both sequester and release Ca(2+). The goal of this study was to examine the rapid non-genomic effect of E2 on mitochondrial Ca(2+) transport in hippocampal synaptosomes from ovariectomised rats. In addition, we aimed to determine if, and to what extent, E2 receptors participated in mitochondrial Ca(2+) transport modulation by E2 in vitro. E2-specific binding and Ca(2+) transport was monitored. At physiological E2 concentrations (0.1-1.5 nmol/L), specific E2 binding to mitochondria isolated from hippocampal synaptosomes was detected with a B(max.) and K(m) of 37.6±2.6 fmol/mg protein and 0.69±0.14 nmol/L of free E2, respectively. The main mitochondrial Ca(2+) influx mechanism is the Ruthenium Red-sensitive uniporter driven by mitochondrial membrane potential. Despite no effect of E2 on Ca(2+) influx, a physiological E2 concentration (0.5 nmol/L) protected mitochondrial membrane potential and consequently Ca(2+) influx from the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (1 μmol/L). In neuronal cells the predominant mitochondrial Ca(2+) efflux mechanism is the Na(+)/Ca(2+) exchanger. E2 caused Ca(2+) efflux inhibition (by 46%) coupled with increased affinity of the Na(+)/Ca(2+) exchanger for Na(+). Using E2 receptor (ERα and ERβ) antagonists and agonists, we confirmed ERβs involvement in E2-induced mitochondrial membrane potential protection as well as Ca(2+) efflux inhibition. In summary, our results indicate that the non-genomic neuromodulatory role of E2 in rat hippocampus is achieved by affecting mitochondrial Ca(2+) transport via, in part, mitochondrial ERβ.