Maria Beatriz Moretto

New Benzodiazepines Alter Acetylcholinesterase and ATPDase Activities

This study examines the effect of new 1,5 benzodiazepines on acetylcholinesterase (AChE) and ATPDase (apyrase) activities from cerebral cortex of adult rats. Simultaneously, the effects of the classical 1,4-benzodiazepine on these enzymes were also studied for comparative purpose. The compounds 2-trichloromethyl-4-phenyl-3H-1,5-benzodiazepin and 2-trichloromethyl-4-(p-methyl-phenyl)-3H-1,5-benzodiazepin significantly inhibited acetylcholinesterase activity (p < 0.01) when tested in the range of 0.18–0.35 mM. The inhibition caused by these two new benzodiazepines was noncompetitive in nature. Similarly, at concentrations ranging from 0.063 to 0.25 mM, the 1,5 benzodiazepines inhibited ATP and ADP hydrolysis by synaptosomes from cerebral cortex (p < 0.01). However, the inhibition of nucleotide hydrolysis was uncompetitive in nature. Our results suggest that, although diazepam and the new benzodiazepines have chemical differences, they both presented an inhibitory effect on acetylcholinesterase and ATPDase activities.

Syzygium cumini inhibits adenosine deaminase activity and reduces glucose levels in hyperglycemic patients

Syzigium cumini (L.) Skeels from the Myrtaceae family is among the most common medicinal plants used to treat diabetes in Brazil. Leaves, fruits, and barks of S. cumini have been used for their hypoglycemic activity. Adenosine deaminase (ADA) is an important enzyme that plays a relevant role in purine and DNA metabolism, immune responses, and peptidase activity. ADA is suggested to be an important enzyme for modulating the bioactivity of insulin, but its clinical significance in diabetes mellitus (DM) has not yet been proven. In this study, we examined the effect of aqueous leaf extracts of S. cumini (L.) (ASC) on ADA activity of hyperglycemic subjects and the activity of total ADA, and its isoenzymes in serum and erythrocytes. The present study indicates that: (i) the ADA activity in hyperglycemic serum was higher than normoglycemic serum and ADA activity was higher when the blood glucose level was more elevated; (ii) ASC (60–1000 μg/mL) in vitro caused a concentration‐dependent inhibition of total ADA activity and a decrease in the blood glucose level in serum; (iii) ADA1 and 2 were reduced both in erythrocytes and in hyperglycemic serum. These results suggest that the decrease of ADA activity provoked by ASC may contribute to control adenosine levels and the antioxidant defense system of red cells and could be related to the complex ADA/DPP‐IV‐CD26 and the properties of dipeptidyl peptidase IV (DPP‐IV) inhibitors which serve as important regulators of blood glucose.

Evidence that 3-hydroxyglutaric acid interacts with NMDA receptors in synaptic plasma membranes from cerebral cortex of young rats.

Neurological symptoms are common in patients with glutaric acidemia type I (GA-I). Although the pathophysiology of this disorder is not yet fully established, 3-hydroxyglutaric acid (3-HGA), which accumulates in affected patients, has recently been demonstrated to be excitotoxic to embryonic chick and neonatal rat neurons probably via NMDA glutamate receptors. In the present study, we investigated the in vitro effects of 3-HGA on the [(3)H]glutamate and [(3)H]MK-801 (dizocilpine) binding to rat synaptic plasma membranes from cerebral cortex of young rats in order to elucidate the interactions of 3-HGA with glutamate receptors and its possible contribution to the in vitro excitotoxic properties of 3-HGA. 3-HGA (10-100 microM) significantly decreased Na(+)-dependent (up to 62%) and Na(+)-independent (up to 30%) [(3)H]glutamate binding to synaptic membranes, reflecting a possible competition between glutamate and 3-HGA for the glutamate transporter and receptor sites, respectively. Since a decrease in Na(+)-independent glutamate binding might represent an interaction of 3-HGA with glutamate receptors, we next investigated whether 3-HGA interacts with NMDA receptors by adding NMDA alone or combined with 3-HGA and measuring Na(+)-independent [(3)H]glutamate binding to synaptic membranes (binding to receptors). We verified that 3-HGA and NMDA, at 10 and 100 microM concentrations, decreased glutamate binding by up to 20 and 45%, respectively, and that the simultaneous addition of both substances did not provoke an additive effect, implying that they bind to NMDA receptors at the same site. Furthermore, the binding of the NMDA-channel blocker [(3)H ]MK-801 was significantly increased (approximately 32-40%) by 10 and 100 microM 3-HGA, implying that 3-HGA was able to open the NMDA channel allowing MK-801 binding, which is a characteristic of NMDA agonists. On the other hand, glutamate had a much higher stimulatory effect on this binding (180% increase), reflecting its strong NMDA agonist property. Furthermore, the simultaneous addition of 3-HGA and glutamate provoked an additive stimulatory effect on [(3)H]MK-801 binding to the NMDA receptor. These data indicate that, relatively to glutamate, 3-HGA is a weak agonist of NMDA receptors. Finally, we demonstrated that 3-HGA provoked a significant increase of extracellular calcium uptake by cerebral cortex slices, strengthening therefore, the view that 3-HGA activates NMDA receptors. The present study therefore, demonstrates at the molecular level that 3-HGA modulates glutamatergic neurotransmission and may explain previous findings relating the neurotoxic actions of this organic acid with excitotoxicity.

The activity and expression of NTPDase is altered in lymphocytes of multiple sclerosis patients

BACKGROUND Multiple sclerosis (MS) is a demyelinating neurological disease, which is presumed to be a consequence of infiltrating lymphocytes that are autoreactive to myelin proteins. ATP and adenosine contribute to fine-tuning immune responses and NTPDase (CD39) and adenosine deaminase (ADA) are important enzymes in the control of the extracellular levels of these molecules at the site of inflammation. We evaluated the activity and expression of NTPDase and adenosine deaminase (ADA) activity in lymphocytes from patients with the relapsing-remitting form of MS (RRMS). METHODS This study involved 22 patients with RRMS and 22 healthy subjects as a control group. The lymphocytes were isolated from blood and separated on Ficoll density gradients and after isolation the NTPDase and ADA activities were determined. RESULTS The NTPDase activity and expression were increased in lymphocytes from RRMS patients when compared with the control group (p<0.05). In addition, a decrease in ADA activity was observed in lymphocytes from these patients when compared to the control group (p<0.05). CONCLUSIONS The regulation of ATP and adenosine levels by NTPDase and ADA activities may be important to preserve cellular integrity and to modulate the immune response in MS.

Assessment of Inflammatory and Oxidative Biomarkers in Obesity and Their Associations with Body Mass Index

The aim of this study was to evaluate the inflammatory and oxidative biomarkers’ levels in obese subjects and their associations with body mass index (BMI), in order to investigate the role of these biomarkers in obesity. Fasting glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, apolipoprotein A, apolipoprotein B, albumin, urinary albumin, creatinine, glomerular filtration rate, interleukin-6 (IL-6), nitrate/nitrite (NOx), and ischemia-modified albumin (IMA) were measured in 93 subjects divided according to different BMI. IL-6, urinary albumin, and IMA levels were significantly higher in obese subjects. However, the levels of NOx were significantly lower in this population. Significant correlations between BMI and IL-6 (r = 0.326, P = 0.002), NOx (r = −0.249, P = 0.021), urinary albumin (r = 0.270, P = 0.008), and IMA (r = 0.286, P = 0.005) were reported. We have shown an increase of IL-6, urinary albumin, and IMA combined with lower levels of NOx in obese patients and an association between of these biomarkers with BMI, suggesting a possible interplay of oxidative stress, inflammation, and endothelial dysfunction state in obesity.

Activity of the enzyme adenosine deaminase in serum, erythrocytes and lymphocytes of rats infected with Trypanosoma evansi.

In Trypanosoma evansi infections changes in the haemogram are commonly observed, and the enzyme adenosine deaminase (ADA) plays an important role in the production and differentiation of blood cells. Thus, the aim of this study was to evaluate the activity of ADA in serum, erythrocytes and lymphocytes of rats infected with T. evansi compared to non-infected rats. Thirty adult rats were used, divided into 3 uniform groups. The animals in groups A and B were infected intraperitoneally with 2 x 10⁶ trypomastigotes/rat. Rodents from group C (control group), were not-infected. Blood collection was performed on days 4 and 20 post-infection (p.i.) in order to obtain acute and chronic infection stages of disease. The blood was used to assess the activity of ADA. In the blood, reduced haematocrit and increased lymphocytes were correlated with ADA activity in erythrocytes and lymphocytes. We observed reduction of ADA activity in serum and erythrocytes in rats infected with T. evansi compared to non-infected rats (P < 0.05). ADA activity in lymphocytes was decreased after 4 days, when the parasitaemia was high and increased after 20 days, when the number of circulating parasites was low. In conclusion, our results showed that the ADA activity was altered in serum, lymphocytes and erythrocytes of rats, concomitantly with haematological parameters, in experimental infection by T. evansi.

Urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin as indicators of tubular damage in normoalbuminuric patients with type 2 diabetes.

OBJECTIVES Renal dysfunction has been reported in normoalbuminuric patients, demonstrating the necessity to improve the diagnostic and prognostic tools for diabetic kidney disease (DKD) investigation. Therefore, the aim of this study was to investigate whether the urinary levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are increased in type 2 diabetes mellitus (DM) patients with normal or mildly increased albuminuria. DESIGN AND METHODS In this study, 117 type 2 DM patients classified into three groups according to urinary albumin/creatinine ratio (uACR): uACR<10mg/g creatinine, uACR 10-30mg/g creatinine and uACR>30mg/g creatinine were enrolled. Urinary concentrations of KIM-1 (uKIM-1) and NGAL (uNGAL) were measured. RESULTS uKIM-1 levels increased progressively from uACR<10mg/g creatinine (69.0±20.8pg/ml) to uACR 10-30mg/g creatinine (106.1±41.2pg/ml) and to uACR>30mg/g creatinine (166.0±31.9pg/ml) (P<0.001). In addition, uNGAL levels increased progressively from uACR<10mg/g creatinine (29.5±8.8ng/ml) to uACR 10-30mg/g creatinine (51.7±10.9ng/ml) and to uACR>30mg/g creatinine (71.0±9.6ng/ml) (P<0.001) patients. Similarly, both uKIM-1 and uNGAL adjusted by urinary creatinine were increased in patients with uACR 10-30mg/g creatinine. Significant and positive correlations were observed between uACR, uKIM-1 and uNGAL. CONCLUSIONS uKIM-1 and uNGAL were increased in type 2 DM patients with normal or mildly increased albuminuria, which indicates that tubular and glomerular injuries may be occurring even at the earliest stage of DKD.

Activity of ectonucleotidases and adenosine deaminase in rats exposed to cigarette smoke

Cigarette smoke is a complex mixture of various toxic substances that are capable of initiating oxidative damage and promoting blood platelet alterations. In this study, we investigated the activities of the ectoenzymes NTPDase (ectonucleoside triphosphate diphosphohydrolase, CD39) and 5′-nucleotidase (CD73) in platelets as well as adenosine deaminase (ADA) in the plasma of rats exposed to aged and diluted sidestream smoke during 4 weeks. The rats were divided into two groups: I (control) and II (exposed to smoke). After the exposure period, blood was collected and the platelets and plasma were separated for enzymatic assay. The results demonstrated that NTPDase (with ATP as substrate) and 5′-nucleotidase (AMP as substrate) activities were significantly higher in group II (p < 0.05) as compared to group I, while no significant difference was observed for NTPDase with ADP as substrate. The ADA activity was significantly reduced in group II (p < 0.05) as compared with group I. Platelet aggregation was significantly increased in group II (p < 0.05) as compared with group I. We suggest that these alterations in the activity of enzymes from the purinergic system are associated with an increase in platelet aggregation. However, our study has demonstrated that the organism tries to compensate for this enhanced aggregation by increasing hydrolysis of AMP and reducing hydrolysis of adenosine, a potent inhibitor of aggregation and an important modulator of vascular tone.

Syzygium cumini extract decrease adenosine deaminase, 5'nucleotidase activities and oxidative damage in platelets of diabetic patients.

Diabetes mellitus, a chronic metabolic disorder, has assumed epidemic proportions and its long-term complications can have devastating consequences. The oxidative stress in diabetes was greatly increased due to prolonged exposure to hyperglycemia and impairment of oxidant/antioxidant equilibrium. Syzygium cumini is being widely used to treat diabetes by the traditional practitioners over many centuries. Adenosine deaminase (ADA) and 5′-Nucleotidase (5′NT) are enzymes of purine nucleoside metabolism that play an important role in the regulation of adenosine (Ado) levels. In this study, we investigated the effect of Syzygium cumini aqueous leaves extract (ASc) on ADA and 5′NT activities and on parameters of oxidative stress under in vitro conditions, using platelets of patients with Type 2 diabetes mellitus. Platelet-Rich Plasma (PRP) was assayed by ADA, 5′NT, Catalase (CAT), Superoxide Dismutase (SOD) activities and Thiobarbituric acid reactive substances (TBARS) levels. We observed that ADA, 5′NT activities and TBARS levels were significantly higher when compared to the control group, and ASc (100 and 200 µg/mL) prevented these effects. Our study demonstrates that ASc was able to remove oxidant species generated in diabetic conditions and modulates in the Ado levels. Then, ASc may promote a compensatory response in platelet function, improving the susceptibility-induced by the diabetes mellitus.

Comparative Evaluation of Adenosine Deaminase Activity in Cerebral Cortex and Hippocampus of Young and Adult Rats: Effect of Garlic Extract ( Allium sativum L.) on Their Susceptibility to Heavy Metal Exposure

Adenosine plays an important neuromodulatory role in the central nervous system, and adenosine deaminase is an important enzyme in the degradation of adenine nucleotides. Methylmercury is the most prevalent form of mercury found in the environment. Methylmercury neurotoxicity has been correlated to the production of reactive oxygen species. In this study, its potential pathogenic effects were investigated in vitro in cerebral cortex and hippocampus of rats. We first observed that adenosine deaminase activity was higher in young rat brains when compared to the 60-day-old rats and was higher in hippocampus when compared to the cortex. Methylmercury (0.1, 1.0, 20 microM) inhibited adenosine deaminase activity in 7- and 60-day-old rats in a concentration-dependent manner. We have demonstrated that methylmercury-induced inhibition was antagonized by garlic alcoholic extract, but sodium selenate did not alter enzyme activity. In addition, glutathione and dithiothreitol restored the methylmercury-induced decrease of adenosine deaminase activity. These results demonstrated that there are age-related changes in adenosine deaminase activity and that thiol agents may contribute to the maintenance of adenosine deaminase activity and may be important in the neuromodulation of adenosine. Garlic alcoholic extract may be effective in reducing the effect of methylmercury-induced adenosine deaminase, which may be due to its sulphur-containing compounds.