This project will characterize biochemical and pharmacological aspects of acetylcholine (ACh) storage by rat brain synaptic vesicles. The storage system is composed of a proton-pumping ATPase and a separate ACh transporter that is associated with an allosteric receptor site for the inhibitory drug vesamicol. The previously characterized ACh transporter- vesamicol receptor (AChT-VR) complex of the electric ray, surprisingly, is strongly associated with synaptic vesicle proteoglycan. This project will determine how similar the rat brain AChT-VR is to the electric ray AChT-VR. Rat brain synaptic vesicles will be prepared either in free form (that is, a P3 preparation) or inside metabolically active synaptosomes. Active transport of radiolabeled ACh and binding of radio-labeled vesamicol or a more tightly binding analog to these preparations will be studied using filter assays. Other biochemical, bioorganic and immunochemical techniques also will be used as appropriate.
The specific aims are to (1) study the mechanism and specific ion dependence of the rat brain ACh active transport system, (2) study the relationship of vesamicol analog binding to transport inhibition, (3) test whether the AChT-VR in rat vesicles is linked to proteoglycan, (4) identify the rat brain AChT-VR by photo-affinity labeling with a tritiated high affinity ACh analog, (5) develop an assay for the AchT-VR inside of rat brain synaptosomes based on binding of a high affinity, radioactive analog of vesamicol, and (6) subject the synaptosomes to different pharmacological manipulations that alter the metabolism and that might affect the properties of the AChT-VR. The long term goal of the project is to understand the complete biochemistry of ACh storage by synaptic vesicles in mammals, whether it is subject to regulation, and whether storage and release are coupled. This might lead to development of a pharmacology that would increase ACh storage in and release from compromised cholinergic terminals. Since many disease and toxic states impact on the cholinergic nervous system, this capability could be of significant clinical benefit.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS026168-05
Application #
3411863
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1988-04-01
Project End
1994-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California Santa Barbara
Department
Type
Schools of Arts and Sciences
DUNS #
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
Haigh, J R; Noremberg, K; Parsons, S M (1994) Acetylcholine active transport by rat brain synaptic vesicles. Neuroreport 5:773-6
Rogers, G A; Stone-Elander, S; Ingvar, M et al. (1994) 18F-labelled vesamicol derivatives: syntheses and preliminary in vivo small animal positron emission tomography evaluation. Nucl Med Biol 21:219-30
Ingvar, M; Stone-Elander, S; Rogers, G A et al. (1993) Striatal D2/acetylcholine interactions: PET studies of the vesamicol receptor. Neuroreport 4:1311-4
Ingvar, M; Eriksson, L; Rogers, G A et al. (1991) Rapid feasibility studies of tracers for positron emission tomography: high-resolution PET in small animals with kinetic analysis. J Cereb Blood Flow Metab 11:926-31
Hicks, B W; Rogers, G A; Parsons, S M (1991) Purification and characterization of a nonvesicular vesamicol-binding protein from electric organ and demonstration of a related protein in mammalian brain. J Neurochem 57:509-19