Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: implications for modulation of multiple homeostatic systems including feeding and sleep-wake behaviors

Three anti-peptide antisera were raised against three distinct amino acid sequences of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), characterized by Western blot analyses, and used to determine the distribution of NTPDase3 protein in adult rat brain. The three antisera all yielded similar immunolocalization data, leading to increased reliability of the results obtained. Unlike NTPDase1 and NTPDase2, NTPDase3 immunoreactivity was detected exclusively in neurons. Immunoreactivity was localized primarily to axon-like structures with prominent staining of presynaptic elements. Specific perikaryal immunostaining was detected primarily in scattered neurons near the lateral hypothalamic area and the perifornical nucleus. High densities of immunoreactive axon-like fibers were present in midline regions of the forebrain and midbrain. Highly scattered NTPDase3 positive fibers were observed in the cerebral cortex, the hippocampal formation, and the basal ganglia. Moreover, very high densities of immunostained fibers were detected in the mediobasal hypothalamus, with the overall mesencephalic pattern of staining associated closely with hormone responsive nuclei. High densities of NTPDase3 positive terminals were also associated with noradrenergic neurons. However, co-immunolocalization studies revealed clearly that NTPDase3 immunoreactivity was not localized within the noradrenaline cells or terminals. In contrast, nearly all of the NTPDase3 immunopositive hypothalamic cells, and most fibers in the mid- and hindbrain, also expressed hypocretin-1/orexin-A. The overall pattern of expression and co-localization with hypocretin-1/orexin-A suggests that NTPDase3, by regulating the extracellular turnover of ATP, may modulate feeding, sleep-wake, and other behaviors through diverse homeostatic systems.     

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/CD39L1) is the dominant ectonucleotidase expressed by rat astrocytes

Inflammatory and degenerative pathophysiological processes within the CNS are important causes of human disease. Astrocytes appear to modulate these reactions and are a major source of inflammatory mediators, e.g. extracellular adenine nucleotides, in nervous tissues. Actions following extracellular nucleotides binding to type 2 purinergic receptors are regulated by ectonucleotidases, including members of the CD39/ecto-nucleoside triphosphate diphosphohydrolase family. The ectonucleotidases of astrocytes expressed by rat brain rapidly convert extracellular ATP to ADP, ultimately to AMP. RT-PCR, immunocytochemistry as well as Western blotting analysis demonstrated expression of multiple ecto-nucleoside triphosphate diphosphohydrolase family members at both the mRNA and protein level. By quantitative real-time PCR, we identified Entpd2 (CD39L1) as the dominant Entpd gene expressed by rat hippocampal, cortical and cerebellar astrocytes. These data in combination with the elevated ecto-ATPase activity observed in these brain regions, suggest that NTPDase2, an ecto-enzyme that preferentially hydrolyzes ATP, is the major ecto-nucleoside triphosphate diphosphohydrolase expressed by rat astrocytes. NTPDase2 may modulate inflammatory reactions within the CNS and could represent a useful therapeutic target in human disease.     

Evidence that acute taurine treatment alters extracellular AMP hydrolysis and adenosine deaminase activity in zebrafish brain membranes

Taurine is one of the most abundant free amino acids in excitable tissues. In the brain, extracellular taurine may act as an inhibitory neurotransmitter, neuromodulator, and neuroprotector. Nucleotides are ubiquitous signaling molecules that play crucial roles for brain function. The inactivation of nucleotide-mediated signaling is controlled by ectonucleotidases, which include the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5′-nucleotidase. These enzymes hydrolyze ATP/GTP to adenosine/guanosine, which exert a modulatory role controlling several neurotransmitter systems. The nucleoside adenosine can be inactivated in extracellular or intracellular milieu by adenosine deaminase (ADA). In this report, we tested whether acute taurine treatment at supra-physiological concentrations alters NTPDase, ecto-5′-nucleotidase, and ADA activities in zebrafish brain. Fish were treated with 42, 150, and 400 mg L⁻¹ taurine for 1 h, the brains were dissected and the enzyme assays were performed. Although the NTPDase activities were not altered, 150 and 400 mg L⁻¹ taurine increased AMP hydrolysis (128 and 153%, respectively) in zebrafish brain membranes and significantly decreased ecto-ADA activity (29 and 38%, respectively). In vitro assays demonstrated that taurine did not change AMP hydrolysis, whereas it promoted a significant decrease in ecto-ADA activity at 150 and 400 mg L⁻¹ (24 and 26%, respectively). Altogether, our data provide the first evidence that taurine exposure modulates the ecto-enzymes responsible for controlling extracellular adenosine levels in zebrafish brain. These findings could be relevant to evaluate potential beneficial effects promoted by acute taurine treatment in the central nervous system (CNS) of this species.     

Neuroprotective effects of safflor yellow B on brain ischemic injury

The present study was conducted to investigate whether safflor yellow B (SYB) had a protective effect on cerebral ischemic injury and to determine the possible mechanisms in vivo and in vitro. In vivo, Male Wistar–Kyoto (WKY) rats were used to make the model of middle cerebral artery occlusion (MCAO). The behavioral test was used to measure neurological deficit scores for evaluation of the ischemic damage of brain. The infarction area of brain was assessed in brain slices stained with 2% solution of 2,3,5-triphenyl tetrazolium chloride (TTC). Spectrophotometric assay was used to determine the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx), contents of malondialdehyde (MDA) and adenosine triphosphate (ATP) of the brain. Furthermore, the respiratory control ratio (RCR = state 3/state 4) was assessed in the brain mitochondria. In vitro, the effect of SYB was tested in cultured fetal cortical cells exposed to glutamate to identify its neuroprotection against neurons damage. The results in vivo showed that SYB at doses of 3.0 and 6.0 mg kg⁻¹ markedly decreased the neurological deficit scores and the infarction area in MCAO rats. At the same time, SYB significantly improved mitochondrial energy metabolism, decreased MDA content, and increased SOD and GPx activities in ischemic brain. The results in vitro showed that SYB remarkably inhibited neuron damage induced by glutamate in cultured fetal cortical cells. These suggest that SYB might act as a potential neuroprotective agent against the cerebral ischemia-induced injury in rat brain through reducing lipid peroxides, scavenging free radicals, and improving the energy metabolism.     

Hypo-and hyperthyroidism affect the ATP, ADP and AMP hydrolysis in rat hippocampal and cortical slices

The presence of severe neurological symptoms in thyroid diseases has highlighted the importance of thyroid hormones in the normal functioning of the mature brain. Since, ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system (CNS), the ectonucleotidase cascade that hydrolyzes ATP to adenosine, is also involved in the control of brain functions. Thus, we investigated the influence of hyper-and hypothyroidism on the ATP, ADP and AMP hydrolysis in hippocampal and cortical slices from adult 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%) added to their drinking water for 14 days. Hypothyroid rats were hormonally replaced by daily injections of T4 (5 microg/100 g body weight, i.p.) for 5 days. Hyperthyroidism significantly inhibited the ATP, ADP and AMP hydrolysis in hippocampal slices. In brain cortical slices, hyperthyroidism inhibited the AMP hydrolysis. In contrast, hypothyroidism increased the ATP, ADP and AMP hydrolysis in both hippocampal and cortical slices and these effects were reverted by T4 replacement. Furthermore, hypothyroidism increased the expression of NTPDase1 and 5'-nucleotidase, whereas hyperthyroidism decreased the expression of 5'-nucleotidase in hippocampus of adult rats. These findings demonstrate that thyroid disorders may influence the enzymes involved in the complete degradation of ATP to adenosine and possibly affects the responses mediated by adenine nucleotides in the CNS of adult rats.     

The ectonucleotidases alkaline phosphatase and nucleoside triphosphate diphosphohydrolase 2 are associated with subsets of progenitor cell populations in the mouse embryonic, postnatal and adult neurogenic zones

Subventricular zone (SVZ)–derived adult neurospheres express two ectonucleotidases, nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and tissue non-specific alkaline phosphatase (TNAP). Agonists of the nucleotide receptors P2Y₁ and P2Y₂ as well as adenosine augment growth factor–mediated progenitor cell proliferation. NTPDase2 converts ATP and UTP to ADP and UDP, respectively, which are all P2Y receptor agonists. TNAP hydrolyzes nucleoside triphosphates and diphosphates and produces the P1 receptor agonist adenosine. In the SVZ, NTPDase2 is specifically expressed by type B cells. In order to further scrutinize the association of key molecules of the purinergic signaling pathway with neurogenic regions, we analyzed the expression of TNAP at the lateral ventricles of the adult and developing mouse brain. In the adult brain, TNAP was expressed by type B, type A and at least subsets of type C cells of the SVZ and throughout the rostral migratory stream. Almost 100% of the proliferating, Ki-67-positive cells of the adult SVZ stained for TNAP, supporting the notion of a ubiquitous association of TNAP with SVZ progenitors. In contrast, NTPDase2-positive progenitors of the dentate gyrus were TNAP-negative. Essentially all cells of the telencephalic vesicle at embryonic day (E) 14 revealed TNAP activity, including doublecortin-positive neuroblasts. During further embryonic development, enhanced TNAP activity became restricted to cells of the ventricular and SVZ. In contrast to TNAP, NTPDase2 was first expressed in the SVZ perinatally, in association with TNAP-positive SVZ border cells. During later development, NTPDase2-positive cells disappeared from the ventricular surface and began to form sheaths around clusters of subventricular doublecortin-positive cells, apparently transforming into type B cells. Our results identify TNAP and NTPDase2 as novel markers for subsets of progenitors in the adult and developing mouse brain. They further support the notion that signaling via extracellular nucleotides and nucleosides contributes to embryonic and adult neurogenesis.     

Kinetic study of NTPDase immobilization and its effect of haemocompatibility on polyethylene terephthalate

Poor haemocompatibility of material surfaces is a serious limitation that can lead to failure of blood-contacting devices and implants. In this work, we have improved the haemocompatibility of polyethylene terephthalate (PET) surfaces by immobilizing apyrase/ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) on to the carboxylated PET. NTPDase immobilized PET surfaces scavenge the ADP released by activated platelets, which prevents further platelet activation and aggregation. The surface properties of the modified PET were characterized by scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDAX), and contact angle measurement. The enzyme attachment and stability on the modified PET surfaces were evaluated. The kinetics of free enzyme and immobilized enzyme were studied and fitted using the Michaelis-Menten kinetic model. Both free and immobilized NTPDase followed Michaelis-Menten kinetics with similar Michaelis-Menten constants (K_m). This suggests that the activity of NTPDase was unchanged upon immobilization. Protein adsorption and %hemolysis was significantly reduced for carboxylated PET and NTPDase immobilized PET surfaces compared to unmodified PET. Lactate dehydrogenase assay showed that the number of adhered platelets reduced by more than an order of magnitude for the NTPDase immobilized PET surface compared to unmodified PET. These results clearly indicate that NTPDase immobilization significantly enhances the haemocompatibility of PET surfaces.     

Hyperbaric oxygen preconditioning reduces ischemia–reperfusion injury by inhibition of apoptosis via mitochondrial pathway in rat brain

This study examined the hypothesis that apoptotic inhibition via mitochondrial pathway was involved in hyperbaric oxygen preconditioning (HBO-PC)–induced neuroprotection on ischemia–reperfusion injury in rat brain. Male Sprague–Dawley rats (250∼280 g, n=144) were divided into control, middle cerebral artery occlusion (MCAO) for 90 min, and HBO-PC plus MCAO groups. HBO-PC was conducted four times by giving 100% oxygen at 2.5 atm absolute (ATA), for 1 h at 12 h intervals for 2 days. At 24 h after the last HBO-PC, MCAO was performed and at 24 h after MCAO, neurological function, brain water content, infarct volume, and cell death were evaluated. Enzymatic activity of capase-3 and −9, and expression of cytochrome c, Bcl-2 and Bax proteins were performed in the samples from hippocampus, ischemic penumbra and core of the brain cortex, respectively. HBO-PC reduced brain edema, decreased infarction volume, and improved neurological recovery. HBO-PC reduced cytoplasm cytochrome c levels, decreased caspase enzyme activity, upregulated the ratio of Bcl-2 and Bax expression, and abated the apoptosis of ischemic tissue. HBO-PC protects brain tissues from ischemia–reperfusion injury by suppressing mitochondrial apoptotic pathways.     

P2X_2和P2X_3受体在小鼠咽粘膜味蕾内的分布

应用免疫组织化学双标记技术研究ATP受体的P2X2和P2X3受体亚型在小鼠咽部粘膜味蕾和邻近组织内的分布,探究ATP在咽部味觉及一般感觉信号传导中的作用。在咽部各水平粘膜味蕾内均可观察到许多P2X2受体阳性味细胞和基细胞,未见P2X2受体阳性的神经纤维。许多降钙素基因相关肽(CGRP)阳性神经纤维围绕在味蕾周围,并发出分支与P2X2受体阳性味细胞和基细胞形成密切接触。P2X3受体多在咽部粘膜上皮味蕾内的神经终末表达,P2X3受体阳性纤维在粘膜基底部形成纤维束并与粘膜下P2X3受体阳性纤维相连,其分支在味蕾基底部形成神经丛,由神经丛发出神经终末到达顶部的味孔和味蕾各部,未见味蕾细胞表达P2X3受体。在无味蕾的粘膜上皮内,也观察到少量P2X3受体阳性神经终末。CGRP阳性纤维缠绕在味蕾周围,并发出分支与味蕾内的P2X3受体阳性纤维形成密切接触。上述结果表明ATP可能是咽粘膜内味觉信号感受与传导的神经递质。     

大鼠脑外伤时嘌呤受体P2X4受体的表达

目的：观察大鼠脑外伤后嘌呤受体亚型P2X4的表达水平。方法：免疫组织化学和半定量RT—PCR。结果：脑外伤后P2X4受体表达水平迅速升高，并在损伤后较长的时间内维持高的表达水平；脑外伤早期，损伤区P2X4免疫反应阳性细胞主要是圆形细胞，中后期主要以具有突起的细胞为主。结论：脑外伤早期，损伤区P2X4受体免疫反应阳性细胞主要为小胶质细胞，中后期可能星形胶质细胞为多；由此推测P2X4参与脑外伤时大脑生理或病理生理功能的调节。     

Delayed peripheral administration of the N-terminal tripeptide of IGF-1 (GPE) reduces brain damage following microsphere induced embolic damage in young adult and aged rats

We have previously reported that peripheral administration of GPE prevents neuronal injury after ischemic reperfusion injury in young adult rats. This study examined the ameliorating effects of GPE-treatment after embolic injury induced by microsphere injection in young adult and aged male rats. Unilateral injury was induced by injecting microspheres into the right internal carotid artery in both young adult (3–4 months) and aged (16–17 months) male rats. Either GPE (12 mg/kg) or the vehicle was infused intravenously over 1 h starting 3 h after embolic injury and the degree of brain injury, astrocytosis and vascular remodeling were examined using histological and immunohistochemical analysis 8 days later. Changes in core temperature, blood glucose concentration, oxygen saturation and heart rate were monitored. Microsphere injection induced multiple sites of focal damage in the ipsilateral subcortical regions. Massive numbers of microglia accumulated within the core of the tissue damage whereas astrocytes were located in the penumbra. There was no difference in the degree of brain injury between the young and aged control rats. However the aged rats showed less injury-induced astrocytosis and greater vascular remodeling. Intravenous infusion of GPE 3 h after the injury reduced overall damage scores in both young (p < 0.01) and aged rats (p < 0.05). GPE-treatment reduced astrocytosis in young, but not aged animals and did not significantly alter the vascular remodeling in either age group. The data suggested that the neuroprotection of the tripeptide is independent of cerebral reperfusion and is not age selective.     

Intraganglionic laminar endings in the rat esophagus contain purinergic P2X2 and P2X3 receptor immunoreactivity

Intraganglionic laminar endings (IGLEs) represent the most prominent vagal afferent terminal structures throughout the gastrointestinal tract. They are most prominent in the esophagus and stomach, but can be found down to the distal colon. Their role as mechanosensors as proposed on anatomical grounds was recently substantiated in elegant functional experiments. There is evidence that vagal mechanosensors in the esophagus and stomach respond to ATP. Thus, the present study aimed at detecting purinergic receptors on IGLEs. IGLEs in the rat esophagus were identified by immunohistochemistry for calretinin and sections were co-incubated with antibodies directed against P2X₂ or P2X₃ receptors. Also, double label immunocytochemistry for purinergic receptors and calcitonin gene-related peptide as a marker for spinal afferents was performed. Terminal nerve fibers immunoreactive for P2X₂ and P2X₃, respectively, were observed between outer and inner layers of the tunica muscularis, covering myenteric ganglia totally or partly. Both P2X₂ and P2X₃ receptor immunoreactivities were highly co-localized with calretinin positive IGLEs as shown by confocal laser scanning microscopy. Numerous calcitonin gene-related peptide immunostained fibers were found to closely approach and intermingle with P2X immunopositive IGLEs. However, there was never co-staining for either of the purinergic receptors and calcitonin gene-related peptide within the same fibers. P2X₃ but not P2X₂ immunoreactivity was also observed within nerve fiber arborizations in the mucosa of the pharynx. In the nodose ganglion, 8.9±1.1% of P2X₂ and 7.2±1.3% of P2X₃ immunopositive neurons, respectively, co-stained for calretinin. On the other hand, 63.4±4.6% and 60.1±5.3% of calretinin positive cell bodies contained P2X₂ and P2X₃ receptor immunoreactivity, respectively. These results indicate that IGLEs are equipped with both P2X₂ and P2X₃ receptors. Thus, they may act as chemosensors or their mechanosensory properties may be modulated by ATP. It is also suggested that spinal afferents innervating the esophagus are equipped with neither P2X₂ nor P2X₃ purinergic receptors.     

Administration of prosaposin ameliorates spatial learning disturbance and reduces cavity formation following stab wounds in rat brain.

The effectiveness of prosaposin as a neurotrophic factor was investigated using rats with bilateral stab wounds, injecting 240 ng per day of prosaposin for 3 days. In Morris water maze task, after 3 weeks postoperation, the stab-wounds rats show significant impairment in acquisition compared with the sham-operated rats. In the transfer test the mean number of crossings of the platform place in stab-wounds was significantly lower than that in sham-operated rats (P < 0.01). The stab-wounds rats treated with prosaposin showed significant improvement (P < 0.05). The cavities following stab wounds in the rats treated with prosaposin were significantly smaller than those in the rats treated with (P < 0.05). Our data support that prosaposin is likely to be a new agent for brain injury.     

Biochemical detection of enzymes NTPDase in tachyzoites of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> and possible functional correlations

This study aimed to investigate the presence and activity of NTPDase enzymes in tachyzoites of Toxoplasma gondii (RH strain), as well as to test an inhibitor of this enzyme. Initially, the strain was properly reactivated through successive passages in mice, followed by a final passage on 10 mice, which were monitored in 5 days. Then, all the animals were anesthetized and decapitated, and the peritoneal fluid containing T. gondii tachyzoites was collected. The parasites were separated from the other cells, and a pellet of tachyzoites was obtained. The protein concentration in the pellet each was determined as detailed in the text. The NTPDase activity in the parasite was tested, using 0 (control), 0.025, 0.05, 0.1, 0.2, 0.4, 0.7, and 1.0 mg mL^?1 concentrations. Activity of this enzyme in all concentrations tested was observed. The NTPDase activity (substrates ATP and ADP) increased progressively according to protein increase, up to a certain point, then immediately had reduced activity when higher concentrations of protein were tested. The NTPDase activity (hydrolysis of ATP and ADP) had variations depending on temperature, pH, and concentration of the substrate in the reaction. In tests with an NTPDase inhibitor (azide), it was observed an inhibition in the enzyme activity increased when the concentration of the inhibitor also increased. Based on these results, we conclude that the enzyme NTPDase is present in tachyzoites of T. gondii, and they could be detected biochemically by their activities. Azide is able to inhibit the enzyme detected in this study, which might be an option for chemotherapy.     

自发性高血压大鼠中脑导水管周围灰质nNOS阳性神经元的变化

目的观察自发性高血压大鼠(spontaneously hypertensiverats,SHR)中脑导水管周围灰质内神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)阳性神经元的变化。方法取SHR和京都种威斯特大鼠(Wistar-Koytorats,WKY)大鼠各30只,分别于3月(14周)龄,6月龄和12月龄测血压并处死,ABC免疫细胞化学方法显示nNOS阳性神经元。结果SHR血压随鼠龄的增长逐渐升高,于12～14周龄时血压在高位稳定,且均高于WKY大鼠(P<0.05)维持在[(20.8±1.1)～(26.3±1.0)]kPa(P<0.05);WKY大鼠各时期血压无明显差异,维持在[(13.7±1.6)～(15.1±1.7)]kPa。中脑导水管周围灰质nNOS阳性神经元以小细胞为主,突起有2-4个,许多朝中脑水管方向延伸。定量结果显示,SHR大鼠nNOS阳性神经元随着血压升高呈逐渐减少的趋势,12月龄与3月龄和6月龄相比均有显著差异(P<0.01),而各个时期WKY大鼠PAGnNOS阳性神经元均无明显变化。结论SHR中脑导水管周围灰质nNOS阳性神经元的减少可能通过影响延髓的血压调节中枢调控高血压的发生,并有可能与高血压的痛觉过敏有关。     

Inhibition of ecto-ATPase activity by curcumin in hepatocellular carcinoma HepG2 cells

Effects of curcumin, a major constituent of turmeric, on ecto-nucleotidases have not been clarified. Here, we investigated whether curcumin affects ecto-nucleotidase activities in human hepatocellular carcinoma HepG2 cells. In the cells, high levels of Mg²⁺-dependent activity of ecto-nucleotidases were observed in the presence of 1 mM adenosine triphosphate (ATP). The activity was inhibited by ecto-ATPase inhibitors such as suramin, ZnCl₂ and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. On the other hand, the activity was significantly decreased at alkaline pH (pH 9) and was not inhibited by levamisole, an inhibitor of alkaline phosphatase. In the presence of ATP, curcumin inhibited the activity in a concentration-dependent manner (IC₅₀ = 6.2 μM). In contrast, curcumin had no effects on ecto-nucleotidase activity in the presence of ADP (1 mM) or AMP (1 mM). The K _m value for ATP hydrolysis of curcumin-sensitive ecto-ATPase was similar to the value of NTPDase2, an isoform of ecto-nucleoside triphosphate diphosphohydrolase. These results suggest that curcumin is a potent inhibitor of ecto-ATPase and may affect extracellular ATP-dependent responses.