The overall goal of this research project is to better understand the structure, function and regulation of muscarinic cholinergic receptors. It appears from our studies and studies from other laboratories, that at least in rat CNS in situ, two distinct binding sites can be distinguished that have the properties expected of a muscarinic receptor. One of these sites appears to mediate the breakdown of phosphoinositides while the other appears to mediate the inhibition of adenylate cyclase activity. The possibility that these binding sites and their associated responses can be regulated independently will be examined. Chronic alterations in the degree of receptor stimulation caused by the administration of receptor antagonists and agonists or by selective lesions will be made and their effects on the density and properties of each binding site and its associated response will be determined. Additionally, the ontogenetic profile for each binding site and its associated biochemical response will be determined to investigate the possibility that they do not develop concomitantly. Finally, quantitative autoradiographic techniques will be developed and used to examine the normal distribution of each site throughout the rat brain at 20 Mum resolution. The effects of lesions on the density of each site will be studied at high anatomical resolution with this technique. To determine if these binding sites are physically separate entities, a series of compounds will synthesized and utilized for affinity chromatographic purification of each site. Functional reconstitution of purified binding site preparations into clonal cells which have either a strong receptor-mediated inhibition of adenylate cyclase activity or stimulation of phosphoinositide breakdown but do not possess muscarinic receptors will be attempted. Additionally, functional reconstitution of purified binding sites with the purified guanine nucleotide-binding regulatory proteins that stimulate (Ns) and inhibit (Ni) adenylate cyclase will be performed in defined phospholipid vesicles.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031155-07
Application #
3279088
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1982-08-01
Project End
1989-03-31
Budget Start
1988-08-01
Budget End
1989-03-31
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Luo, J; Wolfe, B B (1995) Use of insoluble fusion proteins to purify antibodies. Biotechniques 19:544-6, 548, 550
Schachter, J B; Yasuda, R P; Wolfe, B B (1995) Adenosine receptor activation potentiates phosphoinositide hydrolysis and arachidonic acid release in DDT1-MF2 cells: putative interrelations. Cell Signal 7:659-68
Wall, S J; Wolfe, B B; Kromer, L F (1994) Cholinergic deafferentation of dorsal hippocampus by fimbria-fornix lesioning differentially regulates subtypes (m1-m5) of muscarinic receptors. J Neurochem 62:1345-51
Yasuda, R P; Ciesla, W; Flores, L R et al. (1993) Development of antisera selective for m4 and m5 muscarinic cholinergic receptors: distribution of m4 and m5 receptors in rat brain. Mol Pharmacol 43:149-57
Rothberg, B S; Yasuda, R P; Satkus, S A et al. (1993) Effects of chronic ethanol on cholinergic actions in rat hippocampus: electrophysiological studies and quantification of m1-m5 muscarinic receptor subtypes. Brain Res 631:227-34
Schachter, J B; Wolfe, B B (1992) Cyclic AMP differentiates two separate but interacting pathways of phosphoinositide hydrolysis in the DDT1-MF2 smooth muscle cell line. Mol Pharmacol 41:587-97
Schachter, J B; Ivins, J K; Pittman, R N et al. (1992) Competitive regulation of phospholipase C responses by cAMP and calcium. Mol Pharmacol 41:577-86
Wall, S J; Yasuda, R P; Hory, F et al. (1991) Production of antisera selective for m1 muscarinic receptors using fusion proteins: distribution of m1 receptors in rat brain. Mol Pharmacol 39:643-9
Wall, S J; Yasuda, R P; Li, M et al. (1991) Development of an antiserum against m3 muscarinic receptors: distribution of m3 receptors in rat tissues and clonal cell lines. Mol Pharmacol 40:783-9
Li, M; Yasuda, R P; Wall, S J et al. (1991) Distribution of m2 muscarinic receptors in rat brain using antisera selective for m2 receptors. Mol Pharmacol 40:28-35

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