Piracetam Prevents Scopolamine-Induced Memory Impairment and Decrease of NTPDase, 5′-Nucleotidase and Adenosine Deaminase Activities

Piracetam improves cognitive function in animals and in human beings, but its mechanism of action is still not completely known. In the present study, we investigated whether enzymes involved in extracellular adenine nucleotide metabolism, adenosine triphosphate diphosphohydrolase (NTPDase), 5′-nucleotidase and adenosine deaminase (ADA) are affected by piracetam in the hippocampus and cerebral cortex of animals subjected to scopolamine-induced memory impairment. Piracetam (0.02 μmol/5 μL, intracerebroventricular, 60 min pre-training) prevented memory impairment induced by scopolamine (1 mg/kg, intraperitoneal, immediately post-training) in the inhibitory avoidance learning and in the object recognition task. Scopolamine reduced the activity of NTPDase in hippocampus (53 % for ATP and 53 % for ADP hydrolysis) and cerebral cortex (28 % for ATP hydrolysis). Scopolamine also decreased the activity of 5′-nucleotidase (43 %) and ADA (91 %) in hippocampus. The same effect was observed in the cerebral cortex for 5′-nucleotidase (38 %) and ADA (68 %) activities. Piracetam fully prevented scopolamine-induced memory impairment and decrease of NTPDase, 5′-nucleotidase and adenosine deaminase activities in synaptosomes from cerebral cortex and hippocampus. In vitro experiments show that piracetam and scopolamine did not alter enzymatic activity in cerebral cortex synaptosomes. Moreover, piracetam prevented scopolamine-induced increase of TBARS levels in hippocampus and cerebral cortex. These results suggest that piracetam-induced improvement of memory is associated with protection against oxidative stress and maintenance of NTPDase, 5′-nucleotidase and ADA activities, and suggest the purinergic system as a putative target of piracetam.     

Heparin and chondroitin sulfate inhibit adenine nucleotide hydrolysis in liver and kidney membrane enriched fractions

The inhibition of adenine nucleotide hydrolysis by heparin and chondroitin sulfate (sulfated polysaccharides) was studied in membrane preparations from liver and kidney of adult rats. Hydrolysis was measured by the activity of NTPDase and 5′-nucleotidase. The inhibition of NTPDase by heparin was observed at three different pH values (6.0, 8.0 and 10.0). In liver, the maximal inhibition observed for ATP and ADP hydrolysis was about 80% at pH 8.0 and 70% at pH 6.0 and 10.0. Similarly to the effect observed in liver, heparin caused inhibition of ATP and ADP hydrolysis that reached a maximum of 70% in kidney (pH 8.0). Na⁺, K⁺ and Rb⁺ changed the inhibitory potency of heparin, suggesting that its effects may be related to charge interaction. In addition to heparin, chondroitin sulfate also caused a dose-dependent inhibition in liver and kidney membranes. The maximal inhibition observed for ATP and ADP hydrolysis was about 60 and 50%, respectively. In addition, the hepatic and renal activity of 5′-nucleotidase was inhibited by heparin and chondroitin sulfate, except for kidney membranes where chondroitin sulfate did not alter AMP hydrolysis. On this basis, the findings indicate that glycosaminoglycans have a potential role as inhibitors of adenine nucleotide hydrolysis on the surface of liver and kidney cell membranes in vitro.     

Trypanosoma evansi: Activities of adenine nucleotide degradation enzymes in cerebral cortex of infected rats

This study aimed to evaluate the activities of the ectoenzymes NTPDase and 5′-nucleotidase in synaptosomes from cerebral cortex of rats experimentally infected with Trypanosoma evansi. The animals were divided in four groups (n = 10) according to the time and degree of parasitemia (groups A, B, C and D). The animals from group A were euthanized on day 3 (low parasitemia), group B on day 5 (high parasitemia) and group C on day 15 (low parasitemia). Group D consisted of healthy rats (not-infected, n = 15) and were divided in three periods (n = 5) in order to compare with the infected groups. After euthanasia, cerebral cortex was removed for the preparation of synaptosomes and enzymatic assays. Group A showed no changes in enzymatic activities compared with control. The hydrolysis of ATP, ADP and AMP by the enzymes NTPDase and 5′-nucleotidase were increased (P < 0.05) in group B (38%, 140% and 61%, respectively) when compared with control. In the group C it was observed a decreased (22%) hydrolysis of ATP when compared with control group. The activities of NTPDase and 5′-nucleotidase in synaptosomes alters the acute phase of the disease when the number of circulating parasites is high, thus the change observed is probably due to the parasitemia.     

Antiepileptic Drugs Prevent Changes Induced by Pilocarpine Model of Epilepsy in Brain Ecto-Nucleotidases

Ecto-nucleotidases, one of the main mechanisms involved in the control of adenosine levels in the synaptic cleft, have shown increased activities after the pilocarpine model of epilepsy. Here we have investigated the effect of the antiepileptic drugs (AEDs) on ecto-nucleotidase activities from hippocampal and cerebral cortical synaptosomes of rats at seven days after the induction of the pilocarpine model. Expression of these enzymes were investigated as well. Our results have demonstrated that phenytoin (50 mg/kg) and carbamazepine (30 mg/kg) were able to prevent the increase in ecto-nucleotidase activities elicited by pilocarpine in brain synaptosomes. However, sodium valproate (at 100 mg/kg) was only able to avoid the increase on ATP and ADP hydrolysis in hippocampal synaptosomes. Increase on ATP hydrolysis in hippocampal synaptosomes was also prevented by sodium valproate at 286 mg/kg, which corresponds to ED50 for pilocarpine model. NTPDase1, NTPDase2, NTPDase3, and ecto-5′-nucleotidase expressions were not affected by pilocarpine in cerebral cortex. However, expressions of NTPDase2, NTPDase3, and ecto-5′-nucleotidase were increased by pilocarpine in hippocampus. Our results have indicated that previous treatment with AEDs was able to prevent the increase in hippocampal ecto-nucleotidases of pilocarpine-treated rats. These findings have shown that anticonvulsant drugs can modulate plastic events related to the increase of nucleotidase expression and activities in pilocarpine-treated rats.     

Hypothyroidism Enhanced Ectonucleotidases and Acetylcholinesterase Activities in Rat Synaptosomes can be Prevented by the Naturally Occurring Polyphenol Quercetin

Thyroid hormones have an influence on the functioning of the central nervous system. Furthermore, the cholinergic and purinergic systems also are extensively involved in brain function. In this context, quercetin is a polyphenol with antioxidant and neuroprotective properties. This study investigated the effects of (MMI)-induced hypothyroidism on the NTPDase, 5′-nucleotidase, adenosine deaminase (ADA), and acetylcholinesterase (AChE) activities in synaptosomes of rats and whether the quercetin can prevent it. MMI at a concentration of 20 mg/100 mL was administered for 90 days in the drinking water. The animals were divided into six groups: control/water (CT/W), control/quercetin 10 mg/kg, control/quercetin 25 mg/kg, methimazole/water (MMI/W), methimazole/quercetin 10 mg/kg (MMI/Q10), and methimazole/quercetin 25 mg/kg (MMI/Q25). On the 30th day, hormonal dosing was performed to confirm hypothyroidism, and the animals were subsequently treated with 10 or 25 mg/kg quercetin for 60 days. NTPDase activity was not altered in the MMI/W group. However, treatment with quercetin decreased ATP and ADP hydrolysis in the MMI/Q10 and MMI/Q25 groups. 5′-nucleotidase activity increased in the MMI/W group, but treatments with 10 or 25 mg/kg quercetin decreased 5′-nucleotidase activity. ADA activity decreased in the CT/25 and MMI/Q25 groups. Furthermore, AChE activity was reduced in all groups with hypothyroidism. In vitro tests also demonstrated that quercetin per se decreased NTPDase, 5′-nucleotidase, and AChE activities. This study demonstrated changes in the 5′-nucleotidase and AChE activities indicating that purinergic and cholinergic neurotransmission are altered in this condition. In addition, quercetin can alter these parameters and may be a promising natural compound with important neuroprotective actions in hypothyroidism.     

NTPDase3 and ecto-5′-nucleotidase/CD73 are differentially expressed during mouse bladder cancer progression

According to the World Health Organization, bladder cancer is the seventh most common cancer among men in the world. The current treatments for this malignancy are not efficient to prevent the recurrence and progression of tumors. Then, researches continue looking for better therapeutic targets which can end up in new and more efficient treatments. One of the recent findings was the identification that the purinergic system was involved in bladder tumorigenesis. The ectonucleotidases, mainly ecto-5′-nucleotidase/CD73 have been revealed as new players in cancer progression and malignity. In this work, we investigated the NTPDase3 and ecto-5′-nucleotidase/CD73 expression in cancer progression in vivo. Bladder tumor was induced in mice by the addition of 0.05 % of N-butyl-N-(hydroxybutyl)-nitrosamine (BBN) in the drinking water for 4, 8, 12, 18, and 24 weeks. After this period, mice bladders were removed for histopathology analysis and immunofluorescence assays. The bladder of animals which has received BBN had alterations, mainly inflammation, in initial times of tumor induction. After 18 weeks, mice’s bladder has developed histological alterations similar to human transitional cell carcinoma. The cancerous urothelium, from mice that received BBN for 18 and 24 weeks, presented a weak immunostaining to NTPDase3, in contrast to an increased expression of ecto-5′-nucleotidase/CD73. The altered expression of NTPDase3 and ecto-5′-nucleotidase/CD73 presented herein adds further evidence to support the idea that alterations in ectonucleotidases are involved in bladder tumorigenesis and reinforce the ecto-5′-nucleotidase/CD73 as a future biomarker and/or a target for pharmacological therapy of bladder cancer.     

Immunohistological Determination of Ecto-nucleoside Triphosphate Diphosphohydrolase1 (NTPDase1) and 5′-nucleotidase in Rat Hippocampus Reveals Overlapping Distribution

Distribution of two enzymes involved in the ectonucleotidase enzyme chain, ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5′-nucleotidase, was assessed by immunohistochemistry in the rat hippocampus. Obtained results have shown co-expression of the enzymes in the hippocampal region, as well as wide and strikingly similar cellular distribution. Both enzymes were expressed at the surface of pyramidal neurons in the CA1 and CA2 sections, while cells in the CA3 section were faintly stained. The granule cell layer of the dentate gyrus was moderately stained for NTPDase1, as well as for ecto-5′-nucleotidase. Glial association for ecto-5′-nucleotidase was also observed, and fiber tracts were intensively stained for both enzymes. This is the first comparative study of NTPDase1 and ecto-5′-nucleotidase distribution in the rat hippocampus. Obtained results suggest that the broad overlapping distribution of these enzymes in neurons and glial cells reflects the functional importance of ectonucleotidase actions in the nervous system.     

Neuroprotective effect of quercetin in ectoenzymes and acetylcholinesterase activities in cerebral cortex synaptosomes of cadmium-exposed rats

This study investigated the effect of quercetin on nucleoside triphosphate diphosphohydrolase (NTPDase), 5′-nucleotidase, adenosine deaminase (ADA), and acetylcholinesterase (AChE) activities in synaptosomes from cerebral cortex of adult rats exposed to cadmium (Cd). Rats were exposed to Cd (2.5 mg/Kg) and quercetin (5, 25 or 50 mg/Kg) by gavage for 45 days. Rats were randomly divided into eight groups (n = 8–10): saline/ethanol, saline/Querc 5 mg/kg, saline/Querc 25 mg/kg, saline/Querc 50 mg/kg, Cd/ethanol, Cd/Querc 5 mg/kg, Cd/Querc 25 mg/kg, and Cd/Querc 50 mg/kg. Results demonstrated that AChE activity increased in the Cd/ethanol group when compared to saline/ethanol group. Treatment with quercetin prevented the increase in AChE activity when compared to Cd/ethanol group. Quercetin treatment prevented the cadmium-induced increase in NTPDase, 5-nucleotidase, and ADA activities in Cd/ethanol group when compared to saline/ethanol group. Our data showed that quercetin have a protector effect against Cd intoxication. This way, is a promising candidate among the flavonoids to be investigated as a therapeutic agent to attenuate neurological disorders associated with Cd intoxication.     

Ectonucleotidase and acetylcholinesterase activities in silver catfish (Rhamdia quelen) exposed to different salinities

The purpose of this study was to investigate whether salinity adaptation can alter the purinergic (ecto-nucleoside triphosphate diphosphohydrolase; NTPDase and, 5′-nucleotidase) and cholinergic (acetylcholinesterase; AChE) systems in whole brain and blood tissue of the silver catfish, Rhamdia quelen. Silver catfish were gradually adapted to salinities of 0, 4 or 8 ppt and maintained at the experimental salinity for 10 days before brain and blood samples were collected. Blood AChE activity decreased significantly at 8 ppt and significant decreases in AChE activity were observed in the brain with salinity increases. ATP hydrolysis did not change between the groups. In contrast, ADP and AMP hydrolysis in silver catfish maintained at salinities of 4 and 8 ppt were significantly higher than those kept at 0 ppt. In conclusion, this study showed that there is an enhancement in the NTPDase (ADP hydrolysis) and 5′-nucleotidase activities in the brains of silver catfish exposed to increased salinity. Therefore, the activities of these enzymes can act as markers of salinity changes.     

Inhibition of 5′-nucleotidase activity after growth of Dictyostelium discoideum

The specific activity of 5′-nucleotidase activity in cell-free extracts of Dictyostelium discoideum at both exponential and stationary growth phases was determined. The 5′-nucleotidase activity of both membrane and soluble fractions was determined. The results show that at exponential growth more activity is found in the soluble fraction. Furthermore, the results show that stationary phase cells contain about 10-fold less activity than cells at exponential growth. To determine if stationary phase cells contained an inhibitor of 5′-nucleotidase, purified membranes were incubated with a high speed supernatant (S-100) prepared from cells at this stage. The results showed not only a time and concentration dependent loss of membrane bound activity, but also that most of the lost activity could be recovered in a soluble form. This result suggested that the 5′-nucleotidase was being released by a factor in the S-100. Additional studies showed inactivation of the releasing factor by a protease and further, that this inactivation could be prevented by serine protease inhibitors. The specificity of releasing factor with respect to two other membrane bound activities was determined. The results indicated no loss of either 3′5′-cyclic phosphodiesterase or adenylate cyclase. In addition, the results of a comparison of the activity of the releasing factor at two stages of growth showed similar values at both exponential and stationary growth phase. This latter finding suggests that the loss of 5′-nucleotidase activity at stationary phase is not due to modulation of the releasing factor activity. An alternative mechanism is proposed.     

Extracellular adenine nucleotides metabolism in astrocyte cultures from different brain regions

Primary astrocyte cultures from hippocampus, cortex and cerebellum presented different extracellular pattern of adenine nucleotide hydrolysis. The ATP/ADP hydrolysis ratio was 8:1 for hippocampal and cortical astrocytes and 5:1 for cerebellar astrocytes. The AMP hydrolysis in cerebellar astrocytes was seven-fold higher than in cortical or hippocampal cells. No accumulation of extracellular adenosine in all structures studied was observed. Dipyridamol increased significantly inosine levels in the extracellular medium of hippocampal and cortical, but not in cerebellar astrocytes medium. A higher expression of ecto-5′-nucleotidase was identified by RT-PCR in cerebellum. The differences observed may indicate functional heterogeneity of nucleotides in the brain.     

Effects In Vitro of Guanidinoacetate on Adenine Nucleotide Hydrolysis and Acetylcholinesterase Activity in Tissues from Adult Rats

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. The aim of this work was to investigate the in vitro effect of guanidinoacetate in NTPDase, 5′-nucleotidase and acetylcholinesterase activities in the synaptosomes, platelets and blood of rats. The results showed that in synaptosomes the NTPDase and 5′-nucleotidase activities were inhibited significantly in the presence of GAA at concentrations of 50, 100, 150 and 200 μM (P < 0.05). However, in platelets GAA at the same concentrations caused a significant increase in the activities of these two enzymes (P < 0.05). In relation to the acetylcholinesterase activity, GAA caused a significant inhibition in the activity of this enzyme in blood at concentrations of 150 and 200 μM (P < 0.05), but did not alter the acetylcholinesterase activity in synaptosomes from the cerebral cortex. Our results suggest that alterations caused by GAA in the activities of these enzymes may contribute to the understanding of the neurological dysfunction of GAMT-deficient patients.     

Acetylcholinesterase activation and enhanced lipid peroxidation after long-term exposure to low levels of aluminum on different mouse brain regions

Aluminum (Al), oxidative stress and impaired cholinergic functions have all been related to Alzheimer's disease (AD). The present study evaluates the effect of aluminum on acetylcholinesterase (AChE) and lipid peroxidation in the mouse brain. Mice were loaded by gavage with Al 0.1 mmol/kg/day 5 days per week during 12 weeks. The mice were divided into four groups: (1) control; (2) 10 mg/mL of citrate solution; (3) 0.1 mmol/kg of Al solution; (4) 0.1 mmol/kg of Al plus 10 mg/mL of citrate solution. AChE activity was determined in the hippocampus, striatum, cortex, hypothalamus and cerebellum and lipid peroxidation was determined in the hippocampus, striatum and cortex. An increase of AChE activity was observed in the fourth group (Al + Ci) in the hippocampus (36%), striatum (54%), cortex (44%) and hypothalamus (22%) (p<0.01). The third group (Al) presented a decrease of AChE activity in the hypothalamus (20%) and an enhancement in the striatum (27%). Lipid peroxidation, measured by TBARS (thiobarbituric acid reactive substances), was elevated in the hippocampus and cerebral cortex when compared with the control (p < 0.01). The effect of aluminum on AChE activity may be due to a direct neurotoxic effect of the metal or perhaps a disarrangement of the plasmatic membrane caused by increased lipid peroxidation.     

In vitro and in vivo interactions of aluminum on NTPDase and AChE activities in lymphocytes of rats

Al adjuvants are used in vaccines to increase the immune response. NTPDase and AChE play a pivotal role and act in the regulation of the immune system. The effect of Al exposure in vitro and in vivo on NTPDase and AChE activities in the lymphocytes of rats was determined. In vitro, ATP hydrolysis was decreased by 20.4% and 17.3% and ADP hydrolysis was decreased by 36.5% and 34.8%, in groups D and E, respectively, when compared to the control. AChE activity was increased by 157.3%, 152.5%, 74.7% and 90.8% in groups B, C, D, and E, respectively, when compared to the control. In vivo, ATP hydrolysis was increased by 85% and 86% and ADP hydrolysis was increased by 104.2% and 74%, in Al plus citrate and Al groups, respectively, when compared to the control. AChE activity was increased by 50.7% in Al plus citrate and by 28.6% in Al groups, when compared to the control. Our results show that Al exposure both in vitro and in vivo altered NTPDase and AChE activities in lymphocytes. These results may demonstrate the ability of Al to elicit the immune system, where NTPDase and AChE activities can act as purinergic and cholinergic markers in lymphocytes.     

迷走神经刺激对难治性癫痫大鼠海马神经炎性反应及α7nAChR表达的影响*

目的: 探讨迷走神经刺激(VNS)对难治性癫痫(IE)模型大鼠海马神经炎性反应及α7nAChR表达的影响。方法: 80只成年雄性SD大鼠,SPF级,随机分为对照组、模型组、VNS组、甲基牛扁亭(MLA)+VNS组,其中对照组与MLA+VNS组分别20只,模型组与VNS组因模型制作失败与动物死亡,分别剩下15只和14只。除对照组之外,其余各组皆通过腹腔注射皮罗卡品建立氯化锂-皮罗卡品IE大鼠模型。对照组仅分离迷走神经,不采取电刺激;模型组不采取任何干预措施;VNS组在模型制作成功后7 d采取VNS,连续4周;MLA+VNS组先侧脑室给药MLA(3.4 μg/μl,5 μl),然后给予VNS,连续4周。观察并记录各组大鼠癫痫发作的次数与持续时间的变化;然后断头处死大鼠,快速分离海马并制备10%组织匀浆,离心并提取上清液,通过分光光度法测定上清液中AChE、ChAT活性;ELISA法检测TNF-ɑ、IL-6和IL-1β表达;Western blot检测海马组织α7nAChR蛋白表达;免疫荧光染色法检测海马组织α7nAChR与小胶质细胞共表达。结果: ①通过VNS治疗4周后,大鼠癫痫发作的频率以及持续的时间都明显低于模型组(P<0.01);MLA阻断后在给予VNS,大鼠癫痫发作的频率以及持续的时间也明显低于模型组,但高于VNS组(P<0.01)。②与对照组比较,模型组大鼠海马组织ChAT表达明显下降,AChE表达明显升高(P<0.01);与模型组比较,VNS组与MLA+VNS组大鼠海马组织ChAT表达明显升高,AChE表达明显降低(P< 0.01);与VNS组比较,MLA+VNS组大鼠海马组织ChAT、AChE表达无明显变化(P>0.05)。③与对照组比较,模型组大鼠海马组织TNF-ɑ、IL-6和IL-1β表达明显升高(P<0.01);与模型组比较,VNS组大鼠海马组织TNF-ɑ、IL-6和IL-1β表达明显降低(P<0.01);与VNS组比较,MLA+VNS组大鼠海马组织TNF-ɑ、IL-6和IL-1β表达明显升高(P<0.01)。④与对照组比较,模型组大鼠海马组织以及小胶质细胞上α7nAChR表达明显降低(P<0.01);与模型组比较,VNS组大鼠海马组织以及小胶质细胞上α7nAChR表达明显上调(P<0.01);与VNS组比较,MLA+VNS组海马小胶质细胞上共表达α7nAChR数量明显减少(P<0.01)。结论: VNS对IE大鼠有明显的治疗作用,其机制可能是通过直接激活海马小胶质细胞CAP,抑制海马神经炎性反应来实现的。     

Previous treatment with ebselen and vitamin E alters adenine nucleotide hydrolysis in platelets from adult rats experimentally demyelinated with ethidium bromide

Many aspects of the relationship between the demyelinating pathology and platelet function need to be elucidated. Thus, the activity of NTPDase and 5'-nucleotidase enzymes was analyzed in platelets from rats demyelinated with ethidium bromide (EB) and previously treated with ebselen (Ebs) and vitamin E (Vit. E). The animals were divided into four groups: for ebselen, the groups were: I-control (saline), II-(saline and Ebs), III-(EB) and IV-(EB and Ebs); and for vitamin E, the groups were: I - control (saline), II-(saline and Vit. E), III-(EB) and IV-(EB and Vit. E). After 3 and 21 days, the blood was collected and the platelets were separated for enzymatic assays. For the treatment with Ebs, the NTPDase activity for ATP substrate was significantly lower in groups II, III and IV (p < 0.05) after 3 days, while after 21 days, a reduction was observed in group III (p < 0.05). ADP hydrolysis was reduced in group II (p < 0.05) and increased in group IV (p < 0.05) after 3 days, while after 21 days there was an increase in group IV (p < 0.05). In the treatment with Vit. E, ATP hydrolysis was lower in groups II, III and IV (p < 0.05) after 3 and 21 days. ADP hydrolysis was increased in group II (p < 0.05) after 3 days, and in group IV (p < 0.05) after 21 days. However, 5'-nucleotidase activity was not altered by the treatments. These findings demonstrate that NTPDase activity in platelets is diminished in demyelinating events and the treatments with Ebs and Vit. E modulated adenine nucleotide hydrolysis.     

NTPDase and 5′-nucleotidase Activities of Synaptosomes from Hippocampus of Rats Subjected to Hyperargininemia

ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system. Since the ecto-nucleotidase cascade that hydrolyzes ATP to adenosine is involved in the control of brain functions and previous studies realized in our laboratory have recently reported that acute administration of Arg decreases the NTPDase and 5′-nucleotidase activities of rat blood serum, in the present study we investigated the effect of arginine administration on NTPDase and 5′-nucleotidase activities by synaptosomes from hippocampus of rats. First, sixty-days-old rats were treated with a single or a triple intraperitoneal injection of arginine (0.8 g/Kg) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. Second, rats received an intracerebroventricular injection of 1.5 mM arginine solution or saline (5 μL) and were killed 1 h later. We also tested the in vitro effect of arginine (0.1–1.5 mM) on nucleotide hydrolysis in synaptosomes from rat hippocampus. Results showed that intraperitoneal arginine administration did not alter nucleotide hydrolysis. On the other hand, arginine administered intracerebroventricularly reduced ATP (32%), ADP (30%) and AMP (21%) hydrolysis, respectively. In addition, arginine added to the incubation medium, provoked a decrease on ATP (19%), ADP (17%) and AMP (23%) hydrolysis, respectively. Furthermore, kinetic studies showed that the inhibitory effect of arginine was uncompetitive in relation to ATP, ADP and AMP. In conclusion, according to our results it seems reasonable to postulate that arginine alters the cascade involved in the extracellular degradation of ATP to adenosine.     

NTPDase and 5′-nucleotidase activities in platelets of human pregnants with a normal or high risk for thrombosis

The nucleotide degrading enzymes, ectonucleotidases, present on the platelet surface of human pregnant with a normal (without complications) or high risk for thrombosis (hypertension and gestational diabetes) were studied. NTPDase (E.C. 3.6.1.5, CD39) and 5′-nucleotidase (E.C. 3.1.3.5, CD73) activities of four patient groups, non-pregnant (NP, n = 18), pregnant without complications (P, n = 25), pregnant with hypertension (HP, n = 15) and pregnant with gestational diabetes mellitus (GDP, n = 10), were analyzed. Increased NTPDase activities were observed in the groups P (37.0%, S.D. = 2.03 and 34.0%, S.D. = 3.19), HP (40.0%, S.D. = 3.32 and 56.0%, S.D. = 3.25) and GDP (23.0%, S.D. = 2.30 and 42.0%, S.D. = 2.26) in comparison to the control group NP (p < 0.01, S.D. = 1.92 and S.D. = 2.48) when ATP and ADP were used as substrate, respectively. AMP was used as substrate to determine the 5′-nucleotidase activities, which showed to be elevated in the groups P (45.0%, S.D. = 1.73), HP (54.0%, S.D. = 2.64) and GDP (68.0%, S.D. = 1.69) when compared to the control group NP (p < 0.01, S.D. = 1.26). However, no statistically significant differences were observed between the groups P, HP and GDP. As a consequence, the enhanced ATP, ADP and AMP hydrolysis was ascribed to the pregnancy itself, independent of a normal or high risk for thrombosis. The enhanced NTPDase and 5′-nucleotidase activities in platelets suggest that these enzymes are involved in the thromboregulation process in the pregnancy.     

The measurement of cyclic GMP and cyclic AMP phosphodiesterases

Methods are described for measuring phosphodiesterases for cGMP and cAMP in the range of activity yielding 10⁻¹² to 10⁻⁸ mol of product. The 5′-GMP formed is measured by conversion to GDP with guanylate kinase. Amounts of GDP greater than 10⁻¹⁰ mol are measured directly with an enzyme system which results in stoichiometric oxidation of NADH. This is either determined by the decrease in fluorescence or the excess NADH is destroyed with acid and the NAD⁺ measured by its fluorescence in strong NaOH. With smaller amounts of GDP, sensitivity is amplified 1000-fold with the succinic thiokinase-pyruvate kinase cycle. In the case of cAMP diesterase, larger amounts of 5′-AMP are measured in the same way as 5′-GMP, except that adenylate kinase is substituted for guanylate kinase. With smaller amounts, the 5′-AMP is converted to ATP, and sensitivity is amplified with the adenylate kinase-pyruvate kinase cycle. As little as 20 ng dry weight of average brain is sufficient for accurate assay of the diesterase activity toward either cAMP or cGMP. When there is danger of significant destruction of AMP or GMP by tissue 5′-nucleotidase, this is prevented by adding GMP to the cAMP reagent, AMP to the cGMP reagent, or 5′-UMP to either reagent.     

RanBP9 Overexpression Accelerates Loss of Pre and Postsynaptic Proteins in the APΔE9 Transgenic Mouse Brain

There is now compelling evidence that the neurodegenerative process in Alzheimer’s disease (AD) begins in synapses. Loss of synaptic proteins and functional synapses in the amyloid precursor protein (APP) transgenic mouse models of AD is well established. However, what is the earliest age at which such loss of synapses occurs, and whether known markers of AD progression accelerate functional deficits is completely unknown. We previously showed that RanBP9 overexpression leads to robustly increased amyloid β peptide (Aβ) generation leading to enhanced amyloid plaque burden in a mouse model of AD. In this study we compared synaptic protein levels among four genotypes of mice, i.e., RanBP9 single transgenic (Ran), APΔE9 double transgenic (Dbl), APΔE9/RanBP9 triple transgenic (Tpl) and wild-type (WT) controls. We found significant reductions in the levels of synaptic proteins in both cortex and hippocampus of 5- and 6-months-old but not 3- or 4-months-old mice. Specifically, at 5-months of age, rab3A was reduced in the triple transgenic mice only in the cortex by 25% (p<0.05) and gap43 levels were reduced only in the hippocampus by 44% (p<0.01) compared to wild-type (WT) controls. Interestingly, RanBP9 overexpression in the Tpl mice reduced gap43 levels by a further 31% (p<0.05) compared to APΔE9 mice. RanBP9 also further decreased the levels of drebrin in the hippocampus by 32% (p<0.01) and chromogranin in the cortex by 24% (p<0.05) compared to APΔE9 mice. At 6-months of age, RanBP9 expression in the cortex led to further reduction of rab3A by 30% (p<0.05) and drebrin by 38% (p<0.01) compared to APΔE9 mice. RanBP9 also increased Aβ oligomers in the cortex at 6 months. Similarly, in the hippocampus, RanBP9 expression further reduced rab3A levels by 36% (p<0.01) and drebrin levels by 33% (p<0.01). Taken together these data suggest that RanBP9 overexpression accelerates loss of synaptic proteins in the mouse brain.