Neuroprotective effects of berberine on recognition memory impairment, oxidative stress, and damage to the purinergic system in rats submitted to intracerebroventricular injection of streptozotocin.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease. The present study investigated the effects of 50 and 100 mg/kg berberine (BRB) on recognition memory, oxidative stress, and purinergic neurotransmission, in a model of sporadic dementia of the Alzheimer's type induced by intracerebroventricular (ICV) injection of streptozotocin (STZ) in rats. Rats were submitted to ICV-STZ 3 mg/kg or saline, and 3 days later, were started on a treatment of BRB or saline for 21 days. The results demonstrated that BRB was effective in protecting against memory impairment, increased reactive oxygen species, and the subsequent increase in protein and lipid oxidation in the cerebral cortex and hippocampus, as well as δ-aminolevulinate dehydratase inhibition in the cerebral cortex. Moreover, the decrease in total thiols, and the reduced glutathione and glutathione S-transferase activity in the cerebral cortex and hippocampus of ICV-STZ rats, was prevented by BRB treatment. Besides an antioxidant effect, BRB treatment was capable of preventing decreases in ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase (EC-5'-Nt), and adenosine deaminase (ADA) activities in synaptosomes of the cerebral cortex and hippocampus. Thus, our data suggest that BRB exerts a neuroprotective effect on recognition memory, as well as on oxidative stress and oxidative stress-related damage, such as dysfunction of the purinergic system. This suggests that BRB may act as a promising multipotent agent for the treatment of AD.

A ketogenic diet did not prevent effects on the ectonucleotidases pathway promoted by lithium-pilocarpine-induced status epilepticus in rat hippocampus.

A Ketogenic Diet (KD) mimics the anticonvulsant effects of fasting, which are known to suppress seizures. The purinergic system has been investigated in the matter of epilepsy development, especially the nucleoside adenosine, which has been considered a natural brain anticonvulsant. During epileptic seizures, extracellular adenosine concentration rises rapidly to micromolar levels. Adenosine can exert its anticonvulsant functions, after its release by nucleoside bidirectional transport, or by production through the sequential catabolism of ATP by ectonucleotidases, such as E-NTPDases (ectonucleoside triphosphate diphosphohydrolases) and ecto-5'-nucleotidase. Here, we have investigated the effect of a ketogenic diet on the nucleotide hydrolysis and NTPDases expression in the lithium-pilocarpine (Li-Pilo) model of epilepsy. For the induction of Status Epileticus (SE), 21-day-old female Wistar rats received an i.p. injection of lithium chloride (127 mg/kg) and 18-19 h later an i.p. injection of pilocarpine hydrochloride (60 mg/kg). The control groups received an injection of saline. After induction of SE, the control and Li-Pilo groups received standard or ketogenic diets for 6 weeks. The lithium-pilocarpine exposure affected the ATP (a decrease of between 8 % and 16 %) and ADP (an increase of between 18 % and 22 %) hydrolysis in both groups whereas the diet did not impact the nucleotide hydrolysis. NTPDase2 and 3 mRNA expressions decreased in the Li-Pilo group (41 % and 42 %). This data highlights the participation of the purinergic system in the pathophysiology of this model of epilepsy, since nucleotide hydrolysis and NTPDase expressions were altered by Li-Pilo exposure, with no significant effects of the ketogenic diet. However, the interaction between purinergic signaling and a ketogenic diet on epilepsy still needs to be better elucidated.

Leishmania chagasi: an ecto-3'-nucleotidase activity modulated by inorganic phosphate and its possible involvement in parasite-macrophage interaction.

In this work we showed that living cells of Leishmania chagasi was able to hydrolyse 3'AMP 10 times more than 5'AMP. When parasites were grown in a low phosphate concentration (2 mM) the cellular proliferation decreased by 50% compared to cells grown in the presence of a high phosphate concentration (80 mM). However, the ecto-3'nucleotidase activity was 2-fold higher when L. chagasi was grown in a low phosphate concentration. This modulation observed on ecto-3'nucleotidase activity was not observed on ecto-5'nucleotidase activity. These results suggest that low concentration of Pi in the culture medium modulates ecto-3'nucleotidase activity that may lead to modulation of important processes for the cell. Interestingly, the macrophage-parasite interaction increased by 45% when L. chagasi were grown at low phosphate concentration compared to the parasites grown in the presence of high phosphate source. Altogether, the results described here suggest that 3'nucleotidase activity modulated by external stimuli, Pi concentration, could be involved on parasite-macrophage interaction.

The effect of aluminium on NTPDase and 5'-nucleotidase activities from rat synaptosomes and platelets.

Aluminium (Al), a neurotoxic compound, has been investigated in a large number of studies both in vivo and in vitro. In this study, we investigated the effect in vivo of long-term exposure to Al on NTPDase (nucleoside triphosphate diphosphohydrolase) and 5'-nucleotidase activities in the synaptosomes (obtained from the cerebral cortex and hippocampus) and platelets of rats. Here, we investigated a possible role of platelets as peripheral markers in rats. Rats were loaded by gavage with AlCl(3) 50 mg/(kg day), 5 days per week, totalizing 60 administrations. The animals were divided into four groups: (1) control (C), (2) 50 mg/kg of citrate solution (Ci), (3) 50 mg/kg of Al plus citrate (Al+Ci) solution and (4) 50 mg/kg of Al (Al). ATP hydrolysis was increased in the synaptosomes from the cerebral cortex by 42.9% for Al+Ci and 39.39% for Al, when compared to their respective control (p<0.05). ADP hydrolysis was increased by 13.15% for both Al and Al+Ci, and AMP hydrolysis increased by 32.7% for Al and 27.25% for Al+Ci (p<0.05). In hippocampal synaptosomes, the hydrolysis of ATP, ADP and AMP, was increased by 58.5%, 28.5% and 25.92%, respectively, for Al (p<0.05) and 36.7%, 22.5% and 37.64% for Al+Ci, both when compared to their respective controls. ATP, ADP and AMP hydrolysis, in platelets, was increased by 172.3%, 188.52% and 92.1%, respectively in Al+Ci, and 317.9%, 342.8% and 177.9%, respectively, for Al, when compared to their respective controls (p<0.05). Together, these results indicate that Al increases NTPDase and 5'-nucleotidase activities, in synaptosomal fractions and platelets. Thus, we suggest that platelets could be sensitive peripheral markers of Al toxicity of the central nervous system.

Dexamethasone inhibits proliferation and stimulates ecto-5'-nucleotidase/CD73 activity in C6 rat glioma cell line.

Malignant gliomas are the most common and devastating primary tumors of the adult central nervous system. Dexamethasone, a synthetic glucocorticoid, is commonly co-administered to control edema in the management of brain tumors during chemotherapy and radiotherapy. In the present study, the effect of dexamethasone on proliferation and ectonucleotidase activities in rat C6 glioma cell line was investigated. Dexamethasone concentrations ranging from 0.001 to 10 microM induced a time- and concentration-dependent inhibition of C6 rat glioma cell proliferation after 24, 48 and 72-h treatment. The tetrazolium reduction assay (MTT) indicated a reduction of in cell viability (44 +/- 7.6%) after 48-h treatment with 1 microM dexamethasone. Pretreatment with 10 microM of RU38486, an antagonist of glucocorticoid receptors, abolished the effect of 1 microM dexamethasone by 78 +/- 9.8% after 48 h of treatment, indicating that this action is mediated via the glucocorticoid receptor. Members of the E-NTPDase family and ecto-5'-nucleotidase/CD73 can modulate extracellular ATP degradation and adenosine formation, both of which have been described as proliferation factors. Treatment of C6 glioma cells for 48 h with 1 microM dexamethasone increased in 38 +/- 8.09% the AMP hydrolysis and in 3.7-fold the ecto-5'-nucleotidase/CD73 expression, suggesting an increase in adenosine formation and, therefore, a possible modulatory role in the elicitation of cell death responses. In addition, pretreatment with 5 microM GF 109203X, a protein kinase C (PKC) inhibitor, abolished the effect of dexamethasone on cell proliferation and on ecto-5'-NT activity, suggesting that dexamethasone could exert this action via PKC. The alterations in the catabolism of extracellular purines induced by dexamethasone treatment in glioma C6 cells could be related to its pharmacological effects.

Increased serum adenosine deaminase activity in schizophrenic receiving antipsychotic treatment.

Adenosine is an important modulator of the nervous system that has been implicated in the pathophysiology of schizophrenia. We studied peripheral adenosine metabolism by determining the activity of serum adenosine deaminase, which converts adenosine into inosine, and 5'-nucleotidase, which converts AMP into adenosine, in 26 DSM-IV male schizophrenic patients under antipsychotic monotherapy and 26 healthy volunteers balanced for age and race. Schizophrenic patients treated either with typical antipsychotics or clozapine showed increased serum adenosine deaminase activity compared to controls (controls=18.96+/-4.61 U/l; typical=25.09+/-10.98 U/l; clozapine=30.32+/-10.83 U/l; p<0.05, ANOVA) and 5'-nucleotidase activity was also increased in patients on clozapine. After adjusting for confounding factors, adenosine deaminase, but not 5'-nucleotidase, alterations remained significant particularly in the clozapine group. This result suggests that either altered adenosine metabolism is present in schizophrenic patients or is influenced by treatment with antipsychotics, particularly clozapine.

5'-nucleotidase activity is altered by hypo- and hyperthyroidism in platelets from adult rats.

Thyroid disorders are associated to a number of vascular diseases that involve processes such as platelet aggregation and vascular tone control. Since, these processes can be also affected by ATP, ADP and adenosine levels, we investigate the hydrolysis of these nucleotides in platelets from hyperthyroid, hypothyroid, and hypothyroid with hormonal replacement rats. Hyperthyroidism was induced by daily injections of L-thyroxine (T4) 25 microg/100 g body weight for 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) for 14 days. In the hormonal replacement group, hypothyroid rats were injected with T4 (5 microg/100 g body weight, i.p.) for 5 days. The AMP hydrolysis by platelets was increased 49% in hyperthyroid rats and decreased 50% in response to hypothyroidism, while the ATP and ADP hydrolysis was not altered in both groups. Besides, the T4 replacement significantly reversed the inhibition of the AMP hydrolysis observed in hypothyroid rats. Our findings indicate that the thyroid disorders affect the 5'-nucleotidase activity and consequently can alter the adenosine levels in a reversible manner in platelet fraction. Since, adenosine is able to inhibit platelet aggregation and acts as a potent vasodilator, these results can contribute to a better comprehension of the vascular events described in thyroid disorders.

Changes in nucleotide hydrolysis in rat blood serum induced by pentylenetetrazol-kindling.

There is growing pharmacological evidence from several animal models of seizure disorders that adenosine possesses endogenous anticonvulsant activity. Apart from being released from cells, adenosine can be produced by the degradation of adenine nucleotides by ectoenzymes or soluble nucleotidases. These enzymes constitute an important mechanism in synaptic modulation, as they hydrolyze ATP, an excitatory neurotransmitter, to adenosine, a neuroprotective compound. We recently demonstrated an increase in ectoenzyme activity in rat brain synaptosomes after pentylenetetrazol-kindling in rats resistant to kindling, suggesting a role for ectonucleotidases in the seizure control. The present work investigates the effect of seizures induced by pentylenetetrazol kindling on the enzymes that could be playing a role in ATP, ADP and AMP hydrolysis to adenosine in rat blood serum. Animals received injections of PTZ (30 mg/kg, i.p., dissolved in 0.9% saline) once every 48 h, totaling 10 stimulations and the controls animals were injected with saline. The hydrolysis of ATP, ADP and AMP were significantly increased (42, 40, and 45%, respectively), while phosphodiesterase activity was unchanged. These results suggest once more that an increase in the ATP diphosphohydrolase and 5'-nucleotidase activities and, possibly, in adenosine levels, could represent an important compensatory mechanism in the development of chronic epilepsy. Moreover, the fact that this increase can also be measured in serum could mean that these enzymes might be useful as plasma markers of seizures in epilepsy.

Polyunsaturated fatty acid deficiencies: effects on hepatic plasma membrane fatty acid composition and enzyme activity.

Research on dietary polyunsaturated fatty acids (PUFA), on the activity of 5'nucleotidase and adenylate cyclase are largely contradictory due, mostly, to the absence of adequate control group. In this study; four different diets have been evaluated on the 5'nucleotidase and adenylate cyclase activities in rat liver plasma membranes. Wistar rats were given a semisynthetic diet in which lipids were supplied by 5% of either peanut oil (n-3 PUFA deficient diet), cod liver oil (n-6 PUFA deficient diet) partially hydrogenated palm oil (total PUFA deficient diet) and a mixture of peanut and rapeseed oil (control group). Liver plasma membranes were separated by using a Percoll gradient in a Beckman JA 20 centrifuge. 5'nucleotidase and adenylate cyclase activities were measured in a liquid scintilation detector by following the degradation of 3HAMP (adenosine monophosphate) and production of 3HcAMP (cyclic adenosine monophosphate) respectively. Animals fed the total PUFA deficient diet exhibited significant lower body weight and lower liver weight than did the control group. Low cholesterol concentrations were observed in animals deficient either in n-3 or total PUFA in relation to the control group. All dietary deficiencies studies provoked reduced phospholipid levels. Phosphatidylcholine and phosphatidylethanolamine were not modified whatever the deficiency studied. Phospholipids fatty acid composition was significantly modified by the diets studied. The specific activity of 5'nucleotidase in hepatic plasma membrane was independent of dietary PUFA. The catalytic unit of adenylate cyclase complex in totally deficient animals was augmented. The unit of the enzyme stimulated by the guanydyl imidodiphosphate (GppNHp) in n-3 PUFA deficient animals was augmented and reduced in animals receiving the n-6 PUFA deficient diet. In conclusion, our results show that each dietary PUFA deficiency modifies differently the proportions of phospholipid classes and their fatty acid composition. The mechanisms responsible for these modification remain to be elucidated. However, the phospholipid fatty acid changes did not influence the 5'nucleotidase activity except in the case of extreme excess which concerns more toxicology than nutritional modifications. Finally, the catalytic unit (Forskoline + GDP beta s) of adenylate cyclase complex and the regulatory unit (GppNHp) may be sensitive to alterations in PUFA composition.

Effect of an essential fatty acid deficient diet on experimental infection with Trypanosoma cruzi in germfree and conventional mice.

1. To investigate the possible role of essential fatty acid deficiency in host cell/parasite interaction, weanling germfree (GF) and conventional (CV) CFW mice maintained on an essential fatty acid-deficient (-) or a control (+) diet for 110 days were infected with Trypanosoma cruzi. 2. Blood parasitemia indicated that the disease was milder in the animals maintained on the essential fatty acid-deficient diet than in the GF and CV controls (maximum parasitemia: GF+ 33,300, GF-26,200, CV+ 17,100 and CV- 6,400 trypomastigotes/ml blood). 3. Survival 30 days after infection was 12% for GF+, 28% for GF-, 37% for CV+ and 65% for CV- mice. 4. Linoleic and arachidonic acid levels were significantly lower in animals kept on the essential fatty acid-deficient diet (GF-: 28.0 +/- 9.3, 23.4 +/- 8.6; CV-: 37.6 +/- 5.8, 19.9 +/- 3.6) than in controls (GF+: 164.4 +/- 48.8, 162.6 +/- 45.8; CV+: 147.1 +/- 26.5, 107.5 +/- 23.6) confirming the deficiency. 5. Before the infection, succinic dehydrogenase levels were higher in liver of all CV mice (4.52 micrograms phosphate/mg fresh tissue) than in GF mice (0.84 micrograms phosphate/mg fresh tissue), whereas the opposite was true for 5'-nucleotidase levels in brain and liver, respectively (GF: 2.84 and 3.18 micrograms phosphate/mg fresh tissue; CV: 1.25 and 1.54 micrograms phosphate/mg fresh tissue). 6. The disease was milder in deficient than in control animals in both the GF and CV groups on the basis of parasitemia and survival, indicating that fatty acid-deficient mice are partially protected against Chagas' disease. The mechanism underlying this phenomenon requires further investigation.