This project will characterize biochemical aspects of acetylcholine (ACh) storage by mammalian brain synaptic vesicles. Bovine brain will be used as starting material for isosmotic isolation of highly purified synaptic vesicles of mixed neurotransmitter type using biochemical separation techniques routinely used for purification of Torpedo electric organ synaptic vesicles. In Torpedo vesicles it is known that the ACh storage system is composed of an ATPase, s transporter for ACh and a receptor for the inhibitory compound 2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Binding of (3H)vesamicol to the brain receptor in purified vesicles will be characterized, including determining whether receptor can exist in a cryptic form as in Torpedo vesicles. The measurements will be obtained using a vacuum assisted filtration technique. Radiolabeled ACh active transport by the purified brain vesicles will be studied also by the filter method. The linkage between (3H)vesamicol binding and (14C)ACh active transport inhibition in the same samples will be characterized to see if it is as complex as in Torpedo vesicles. The vesamicol receptor of brain vesicles will be detergent solubilized and purified to homogeneity to determine how similar its composition is to that of the Torpedo receptor. Finally, the ACh transporter in brain vesicles will be photoaffinity-labeled with a radioactive ACh analogue and the labeled subunit identified by gel electrophoresis and fluorography. The overall aim of this project is to determine the extent of similarity of the ACh storage system in brain to that of the electric organ. The long term aim is to understand the biochemistry of ACh transport and storage, and to determine the relationship of ACh transport to ACh release in the mammalian cholinergic nerve terminal. This includes an understanding of all the regulatory features in the system so that they might be pharmacologically or otherwise manipulated to increase ACh storage in and release from compromised cholinergic terminals. Since many disease or toxic states impact selectively on the cholinergic nervous system of humans, for example, Alzheimer's disease, 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 #
5R01NS026168-02
Application #
3411861
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1988-04-01
Project End
1991-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
2
Fiscal Year
1989
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