The overall goal of this project is to explore signal transduction mechanisms for dopamine acting through D2-receptors, in the belief that this may elucidate the role played by these receptors in regulating neuronal activity. Consequently, the present proposal takes two approaches - first, to establish a model cell system in which dopamine's role in modulating the intracellular second messengers, cAMP and [Ca2+]i can be delineated and secondly, to investigate the electrophysiological consequences of a biochemical perturbation of D2-receptor mediated signal transduction in striatum. The model system to be evaluated will be a neuroblastoma x Chinese hamster embryonic brain explant hybrid cell line (NCB-20), which expresses receptors for dopamine, serotonin, bradykinin, opiates and acetylcholine. The first group of experiments will determine whether D2-receptors inhibit adenylate cyclase in NCB-20 cells and whether these receptors also may perturb [Ca2+]i mobilization in these cells, by measuring D2-receptor effects on phosphatidylinositol metabolism and [Ca2+]i mobilization. In the second group of experiments, conditions will be optimized for the intrastriatal injection of pertussis toxin, so that a significant portion of the pertussis toxin substrates are ADP-ribosylated; a determination will be made on the effectiveness with which this treatment eliminates receptor-mediated inhibition of adenylate cyclase; subsequently, receptor-mediated alterations in electrophysiological properties will be examined in these animals compared to sham-injected controls.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS028389-02
Application #
3414875
Study Section
Neurosciences Research Review Committee (BPN)
Project Start
1989-08-01
Project End
1992-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
Martin, Agnes C L; Willoughby, Debbie; Ciruela, Antonio et al. (2009) Capacitative Ca2+ entry via Orai1 and stromal interacting molecule 1 (STIM1) regulates adenylyl cyclase type 8. Mol Pharmacol 75:830-42
Willoughby, Debbie; Baillie, George S; Lynch, Martin J et al. (2007) Dynamic regulation, desensitization, and cross-talk in discrete subcellular microdomains during beta2-adrenoceptor and prostanoid receptor cAMP signaling. J Biol Chem 282:34235-49
Willoughby, Debbie; Cooper, Dermot M F (2006) Ca2+ stimulation of adenylyl cyclase generates dynamic oscillations in cyclic AMP. J Cell Sci 119:828-36
Willoughby, Debbie; Wong, Wei; Schaack, Jerome et al. (2006) An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics. EMBO J 25:2051-61
Gu, Chen; Cali, James J; Cooper, Dermot M F (2002) Dimerization of mammalian adenylate cyclases. Eur J Biochem 269:413-21
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Rich, T C; Tse, T E; Rohan, J G et al. (2001) In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J Gen Physiol 118:63-78
Fagan, K A; Schaack, J; Zweifach, A et al. (2001) Adenovirus encoded cyclic nucleotide-gated channels: a new methodology for monitoring cAMP in living cells. FEBS Lett 500:85-90
Fagan, K A; Graf, R A; Tolman, S et al. (2000) Regulation of a Ca2+-sensitive adenylyl cyclase in an excitable cell. Role of voltage-gated versus capacitative Ca2+ entry. J Biol Chem 275:40187-94
Rich, T C; Fagan, K A; Nakata, H et al. (2000) Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. J Gen Physiol 116:147-61

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