Abstract

Cholecystokinin (CCK) is a neuropeptide which display many properties of a neurotransmitter. It is thought to play an important role in the neurochemical balance of the brain and alterations in its expression or secretion may play a role in Neurological and Psychiatric Disease. Very little is known about the mechanism and regulation of CCK biosynthesis. An enzyme which generates CCK8 from CCK 33 (CCK 8 generating enzyme) has been isolated from rate brain synaptosome and cDNA clones have been identified by expression cloning which have identical properties to the isolated enzyme. Since CCK 8 is the major product of pro-CCK processing in the brain, this enzyme may be the rate limiting enzyme in the biosynthesis of CCK. A number of dibasic processing enzymes (PC1 and PC2) which have been cloned by other investigators may play a role in the processing of pro-CCK at dibasic sites. The following specific aims are designed to study in detail the possible role of CGE, PC1 and PC2 in the processing of pro-CCK: 1. Isolation and sequencing of full length CGE cDNA clones. 2. The distribution an molecular forms of CGE will be examined in rat brain, pituitary, intestine, and CCK-secreting tumor cells in culture using in situ hybridization, Northern analysis, radioimmunoassay, Western immunoblot analysis and immunocytochemistry. The possible co-localization of CGE and CCK will be examined by dual immunofluorescent staining. 3. The ability of CGE to cleave recombinant pro-CCK in vitro at monobasic and dibasic sites will be evaluated using both purified CGE produced by recombinant techniques. The ability of CGE, PC1 and PC2 to process pro- CCK in living cells will be evaluated by co-expressing them with pro-CCK in CV1 cells and analyzing the products. 4. The possibility that expression of CGE, PC1, PC2 and CCK mRNA are coordinately regulated in CCK-secreting tumor cells will be examined using Northern analysis. 5. The effect of inhibition of enzyme expression on pro-CCK processing will be evaluated in CCK-secreting tumor cells using CGE, PC1, and PC2 antisense cDNAs to block the expression of their mRNAs.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS031602-01A1
Application #
2269533
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1994-05-01
Project End
1998-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost

  Institution

Name
Saint Louis University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63103

  Publications

Beinfeld, Margery C; Funkelstein, Lydiane; Foulon, Thierry et al. (2009) Cathepsin L plays a major role in cholecystokinin production in mouse brain cortex and in pituitary AtT-20 cells: protease gene knockout and inhibitor studies. Peptides 30:1882-91
Reynolds, Nicole A; Blum, Alissa; Kitagawa, Kouki et al. (2006) Inhibition of PC5 expression decreases CCK secretion and increases PC2 expression. Peptides 27:901-4
Burgdorf, Jeffrey; Panksepp, Jaak; Beinfeld, Margery C et al. (2006) Regional brain cholecystokinin changes as a function of rough-and-tumble play behavior in adolescent rats. Peptides 27:172-7
Beinfeld, Margery C; Vishnuvardhan, Daesety; Blum, Alissa et al. (2006) Inhibition of prohormone convertase 1 (PC1) expression in cholecystokinin (CCK) expressing At-T20 cells decreased cellular content and secretion of CCK and caused a shift in molecular forms of CCK secreted. Peptides 27:905-10
Beinfeld, Margery C; Blum, Alissa; Vishnuvardhan, Daesety et al. (2005) Cholecystokinin levels in prohormone convertase 2 knock-out mouse brain regions reveal a complex phenotype of region-specific alterations. J Biol Chem 280:38410-5
Tagen, Michael B; Beinfeld, Margery C (2005) Recombinant prohormone convertase 1 and 2 cleave purified pro cholecystokinin (CCK) and a synthetic peptide containing CCK 8 Gly Arg Arg and the carboxyl-terminal flanking peptide. Peptides 26:2530-5
Cadel, Sandrine; Gouzy-Darmon, Cecile; Petres, Stephane et al. (2004) Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells. Protein Expr Purif 36:19-30
Cain, B M; Connolly, K; Blum, A C et al. (2004) Genetic inactivation of prohormone convertase (PC1) causes a reduction in cholecystokinin (CCK) levels in the hippocampus, amygdala, pons and medulla in mouse brain that correlates with the degree of colocalization of PC1 and CCK mRNA in these structures J Neurochem 89:307-13
Kleditzsch, Petra; Pratt, John; Vishnuvardhan, Daesety et al. (2003) Production, purification, and characterization of rat pro-CCK from serum-free adapted Drosophila cells. Protein Expr Purif 31:56-63
Cain, Brian M; Connolly, Kelly; Blum, Alissa et al. (2003) Distribution and colocalization of cholecystokinin with the prohormone convertase enzymes PC1, PC2, and PC5 in rat brain. J Comp Neurol 467:307-25

Showing the most recent 10 out of 30 publications