Snježana Petrović

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.

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%.

Trans fatty acid content in Serbian margarines: Urgent need for legislative changes and consumer information.

This study examined the fatty acid (FA) composition of 13 (7 soft and 6 hard) Serbian margarines. Significantly higher amounts of trans fatty acids (TFA) were found in hard margarines (up to 28.84% of total FA), than in soft ones (0.17-6.89%). Saturated FA (SFA) were present with 22.76-51.17%. Oleic acid ranged from 26.78% to 43.78%. The proportion of polyunsaturated FA (PUFA) was 22.15-49.29% in soft margarines, but only 8.02-15.28% in hard margarines, probably due to the hydrogenisation process. The atherogenic and thrombogenic indexes (AI and TI, respectively) in soft margarines were relatively low (AI 0.23-0.63 and TI 0.44-0.97), but in hard margarines AI and particularly TI were high (1.03-1.67 and 1.96-3.04, respectively). These findings suggest that FA composition of Serbian margarines should be improved by replacing atherogenic TFA and SFA with beneficial ones, in order to avoid adverse effects on health. Therefore legislative changes and consumer information are urgently needed.

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.

Modulation of hepatic inflammation and energy-sensing pathways in the rat liver by high-fructose diet and chronic stress

PurposeHigh-fructose consumption and chronic stress are both associated with metabolic inflammation and insulin resistance. Recently, disturbed activity of energy sensor AMP-activated protein kinase (AMPK) was recognized as mediator between nutrient-induced stress and inflammation. Thus, we analyzed the effects of high-fructose diet, alone or in combination with chronic stress, on glucose homeostasis, inflammation and expression of energy sensing proteins in the rat liver.MethodsIn male Wistar rats exposed to 9-week 20% fructose diet and/or 4-week chronic unpredictable stress we measured plasma and hepatic corticosterone level, indicators of glucose homeostasis and lipid metabolism, hepatic inflammation (pro- and anti-inflammatory cytokine levels, Toll-like receptor 4, NLRP3, activation of NFκB, JNK and ERK pathways) and levels of energy-sensing proteins AMPK, SIRT1 and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α).ResultsHigh-fructose diet led to glucose intolerance, activation of NFκB and JNK pathways and increased intrahepatic IL-1β, TNFα and inhibitory phosphorylation of insulin receptor substrate 1 on Ser307. It also decreased phospho-AMPK/AMPK ratio and increased SIRT1 expression. Stress alone increased plasma and hepatic corticosterone but did not influence glucose tolerance, nor hepatic inflammatory or energy-sensing proteins. After the combined treatment, hepatic corticosterone was increased, glucose tolerance remained preserved, while hepatic inflammation was partially prevented despite decreased AMPK activity.ConclusionHigh-fructose diet resulted in glucose intolerance, hepatic inflammation, decreased AMPK activity and reduced insulin sensitivity. Chronic stress alone did not exert such effects, but when applied together with high-fructose diet it could partially prevent fructose-induced inflammation, presumably due to increased hepatic glucocorticoids.

Effects of acute gamma-irradiation on extracellular adenine nucleotide hydrolysis in developing rat brain

Cell membrane is highly sensitive to irradiation which, acting directly or indirectly, may disturb functions of constitutive proteins including membrane enzymes. Plasma membrane surface-located enzyme chain of ecto-nucleotide triphospho diphosphohydrolases (NTPDases) and 5′-nucleotidase are involved in termination of cell purinergic signalization by hydrolyzing extracellular, excitatory adenosine triphosphate (ATP), as well as nucleotide di-, and mono-phosphate (ADP and AMP) to neuroprotective adenosine. Extracellular ATP, ADP, and AMP hydrolyzes were examined in purified synaptic plasma membranes after whole-body acute irradiation. All measurements were done 24 h after irradiation of developing (15-, 30-day-old) and adult (90-day-old) rats with low (50 cGy) and high (2 Gy) dose of gamma-rays. Both, high and low doses inhibited nucleotide hydrolyses in 15-day-old rats; in 30-day-old rats low dose of radiation inhibited ADP and AMP hydrolyses while high dose inhibited only ATP hydrolyse. In adult rats high dose induced no effects, while low dose stimulated nucleotides hydrolyses. According to obtained results it was concluded that ecto-nucleotidases of young rats are more sensitive to irradiation, since even low dose induces inhibition of ecto-nucleotidases activities. Ionizing radiation, by decreasing brain nucleotide hydrolyses in developing rats, induces accumulation of ATP and decreases production of adenosine in synaptic cleft which could be neurocytotoxic. On the contrary, in adult rats low dose of radiation stimulates NTPDase and 5′-nucleotidase activity and protective adenosine production which indicates protective and adaptive mechanisms developed in adult brain neuronal cells.

Involvement of glucocorticoid prereceptor metabolism and signaling in rat visceral adipose tissue lipid metabolism after chronic stress combined with high-fructose diet

Both fructose overconsumption and increased glucocorticoids secondary to chronic stress may contribute to overall dyslipidemia. In this study we specifically assessed the effects and interactions of dietary fructose and chronic stress on lipid metabolism in the visceral adipose tissue (VAT) of male Wistar rats. We analyzed the effects of 9-week 20% high fructose diet and 4-week chronic unpredictable stress, separately and in combination, on VAT histology, glucocorticoid prereceptor metabolism, glucocorticoid receptor subcellular redistribution and expression of major metabolic genes. Blood triglycerides and fatty acid composition were also measured to assess hepatic Δ9 desaturase activity. The results showed that fructose diet increased blood triglycerides and Δ9 desaturase activity. On the other hand, stress led to corticosterone elevation, glucocorticoid receptor activation and decrease in adipocyte size, while phosphoenolpyruvate carboxykinase, adipose tissue triglyceride lipase, FAT/CD36 and sterol regulatory element binding protein-1c (SREBP-1c) were increased, pointing to VAT lipolysis and glyceroneogenesis. The combination of stress and fructose diet was associated with marked stimulation of fatty acid synthase and acetyl-CoA carboxylase mRNA level and with increased 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase protein levels, suggesting a coordinated increase in hexose monophosphate shunt and de novo lipogenesis. It however did not influence the level of peroxisome proliferator-activated receptor-gamma, SREBP-1c and carbohydrate responsive element-binding protein. In conclusion, our results showed that only combination of dietary fructose and stress increase glucocorticoid prereceptor metabolism and stimulates lipogenic enzyme expression suggesting that interaction between stress and fructose may be instrumental in promoting VAT expansion and dysfunction.