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The expression level of ecto-NTP diphosphohydrolase1/CD39 modulates exocytotic and ischemic release of neurotransmitters in a cellular model of sympathetic neurons.

ABSTRACT: Once released, norepinephrine is removed from cardiac synapses via reuptake into sympathetic nerves, whereas transmitter ATP is catabolized by ecto-NTP diphosphohydrolase 1 (E-NTPDase1)/CD39, an ecto-ATPase. Because ATP is known to modulate neurotransmitter release at prejunctional sites, we questioned whether this action may be ultimately controlled by the expression of E-NTPDase1/CD39 at sympathetic nerve terminals. Accordingly, we silenced E-NTPDase1/CD39 expression in nerve growth factor-differentiated PC12 cells, a cellular model of sympathetic neuron, in which dopamine is the predominant catecholamine. We report that E-NTPDase1/CD39 deletion markedly increases depolarization-induced exocytosis of ATP and dopamine and increases ATP-induced dopamine release. Moreover, overexpression of E-NTPDase1/CD39 resulted in enhanced removal of exogenous ATP, a marked decrease in exocytosis of ATP and dopamine, and a large decrease in ATP-induced dopamine release. Administration of a recombinant form of E-NTPDase1/CD39 reproduced the effects of E-NTPDase1/CD39 overexpression. Exposure of PC12 cells to simulated ischemia elicited a release of ATP and dopamine that was markedly increased in E-NTPDase1/CD39-silenced cells and decreased in E-NTPDase1/CD39-overexpressing cells. Therefore, transmitter ATP acts in an autocrine manner to promote its own release and that of dopamine, an action that is controlled by the level of E-NTPDase1/CD39 expression. Because ATP availability greatly increases in myocardial ischemia, recombinant E-NTPDase1/CD39 therapeutically used may offer a novel approach to reduce cardiac dysfunctions caused by excessive catecholamine release.

SUBMITTER: Corti F

PROVIDER: S-EPMC3083107 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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The expression level of ecto-NTP diphosphohydrolase1/CD39 modulates exocytotic and ischemic release of neurotransmitters in a cellular model of sympathetic neurons.

Corti Federico F Olson Kim E KE Marcus Aaron J AJ Levi Roberto R

The Journal of pharmacology and experimental therapeutics 20110216 2

Once released, norepinephrine is removed from cardiac synapses via reuptake into sympathetic nerves, whereas transmitter ATP is catabolized by ecto-NTP diphosphohydrolase 1 (E-NTPDase1)/CD39, an ecto-ATPase. Because ATP is known to modulate neurotransmitter release at prejunctional sites, we questioned whether this action may be ultimately controlled by the expression of E-NTPDase1/CD39 at sympathetic nerve terminals. Accordingly, we silenced E-NTPDase1/CD39 expression in nerve growth factor-dif ...[more]

PMID: 21325440

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Project description:Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ecto-nucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound E-NTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either -2 or/and -3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism.

Project description:Cerebral ischemic stroke is a common neuron loss disease that is caused by the interruption of the blood supply to the brain. In order to enhance the CIS outcome, both identifying the treatment target of ischemic brain damage in the acute phase and developing effective therapies are urgently needed. Scutellarin had been found to be beneficial to ischemic injuries and has been shown to have potent effects in clinical application on both stroke and myocardial infarction. However, whether scutellarin improves ischemic brain damage in the acute phase remains unknown. In this study, the protective effects of scutellarin on ischemic brain damage in the acute phase (within 12 h) were illustrated. In middle cerebral artery occlusion and reperfusion (MCAO/R) modeling rats, the Z-Longa score was significantly down-regulated by 25% and 23.1%, and the brain infarct size was reduced by 26.95 ± 0.03% and 25.63 ± 0.02% when responding to high-dose and low-dose scutellarin treatments, respectively. H&E and TUNEL staining results indicated that the neuron loss of the ischemic region was improved under scutellarin treatment. In order to investigate the mechanism of scutellarin's effects on ischemic brain damage in the acute phase, changes in proteins and metabolites were analyzed. The suppression of scutellarin on the glutamate-inducing excitatory amino acid toxicity was strongly indicated in the study of both proteomics and metabolomics. A molecular docking experiment presented strong interactions between scutellarin and glutamate receptors, which score much higher than those of memantine. Further, by performing a parallel reaction monitoring-mass spectrometry (PRM-MS) study on both the cortex and hippocampus tissue of the ischemic region, we screened the scutellarin-regulating molecules that are involved in both the release and transportation of neurotransmitters. It was found that the aberrant levels of glutamate receptors, including EAAT2, GRIN1, GRIN2B, and GRM1, as well as of other glutamatergic pathway-involving proteins, including CAMKK2, PSD95, and nNOS, were significantly regulated in the ischemic cortex. In the hippocampus, EAAT2, GRIN1, nNOS, and CAM were significantly regulated. Taken together, scutellarin exerts potent effects on ischemic brain damage in the acute phase by regulating the activity of neurotransmitters and reducing the toxicity of excitatory amino acids in in neurons.

Dataset Information

The expression level of ecto-NTP diphosphohydrolase1/CD39 modulates exocytotic and ischemic release of neurotransmitters in a cellular model of sympathetic neurons.

Publications

The expression level of ecto-NTP diphosphohydrolase1/CD39 modulates exocytotic and ischemic release of neurotransmitters in a cellular model of sympathetic neurons.

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