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.
Footnotes
This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants HL034215, HL046403, HL047073, HL089521]. A.J.M. is the recipient of a MERIT Review Grant from the Department of Veterans' Affairs and a grant from the Cancer Research and Treatment Fund.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.179994.
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ABBREVIATIONS:
- E-NTPDase1
- ecto-NTP diphosphohydrolase 1
- NE
- norepinephrine
- GFP
- green fluorescent protein
- bp
- base pair
- NGF
- nerve growth factor
- ANOVA
- analysis of variance
- WT
- wild type
- PPADS
- pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate
- PCR
- polymerase chain reaction
- sh
- short hairpin
- siRNA
- small interfering RNA
- ARL67156
- 6-N,N-diethyl-d-β,γ-dibromomethyleneATP trisodium salt
- DPCPX
- 8-cyclopentyl-1,3-dipropylxanthine
- HRP
- horseradish peroxidase
- WB
- Western blot
- TfR
- transferrin receptor
- EV
- empty vector.
- Received January 26, 2011.
- Accepted February 16, 2011.
- U.S. Government work not protected by U.S. copyright
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