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CCK-8S) of Protein Phosphorylation in the Neostriatum (Forskonln/N-Methyl-D-Aspartic Acid/Glutamate) GRETCHEN L

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CCK-8S) of Protein Phosphorylation in the Neostriatum (Forskonln/N-Methyl-D-Aspartic Acid/Glutamate) GRETCHEN L Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11277-11281, December 1993 Neurobiology Regulation by the neuropeptide cholecystokinin (CCK-8S) of protein phosphorylation in the neostriatum (forskonln/N-methyl-D-aspartic acid/glutamate) GRETCHEN L. SNYDER*, GILBERTO FISONE*, PATRIZIA MORINOt, VIDAR GUNDERSEN*, OLE PETTER OTTERSEN*, TOMAS HOKFELTt, AND PAUL GREENGARD*§ *Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, NY 10021; tDepartment of Histology and Neurobiology, Karolinska Institute, S-10401, Stockholm, Sweden; and *Department of Anatomy, University of Oslo, Blindern, N-0317 Oslo, Norway Contributed by Tomas Hokfelt, August 16, 1993 ABSTRACT Despite physiological evidence that cholecys- rons, apparently through a mechanism that involves the tokinin (CCK) is an excitatory neurotransmitter in the brain, release of an excitatory neurotransmitter and activation of little is known about its mechanism of action. CCK immuno- NMDA receptors. reactivity in the brain, including projections to the striatum, is primarily attributable to the sulfated octapeptide CCK-8S. We report here that CCK-8S abolishes cAMP-dependent phos- MATERIALS AND METHODS phorylation ofa dopamine- and cAMP-regulated 32-kDa phos- Materials. RPMI 1640 balanced salt solution, bovine serum phoprotein (DARPP-32) in striatal neurons. The effect of albumin, and 3-isobutylmethylxanthine were obtained from CCK-8S is prevented by antagonists of CCKB and N-methyl- Sigma; forskolin was from Calbiochem; NMDA and (+)-MK- D-aspartate receptors. Our results support a model in which 801 hydrogen maleate (MK-801) were from Research Bio- CCK-8S, originating from CCK or CCK/glutamate cortico- chemicals; CCK-8S was from Bachem; CI-988 was from J. striatal neurons, promotes the release of an excitatory neuro- Hughes; cAMP, RIA, and ECL Western blotting detection transmitter that causes the dephosphorylation and inactivation kits were from Amersham; and goat anti-mouse horseradish of DARPP-32, a potent protein phosphatase inhibitor, thereby peroxidase-linked antibody was from Pierce. Nitrocellulose modulating neuronal excitability. filters (pore size, 0.2 jum) were obtained from Schleicher & Schuell. A monoclonal antibody purification kit (Affi-Gel Numerous peptides are widely distributed in the brain (1), MAPSII) was obtained from Bio-Rad. and many of them coexist with classic transmitters in the Preparation and Incubation ofStriatal Slices. Male Sprague- same neurons (2). It was proposed that peptides may act as Dawley rats (150-200 g) were sacrificed by decapitation. neurotransmitters (2) but, with few exceptions (3-5), it has Brains were rapidly removed and placed in 50 ml of cold, been difficult to identify their physiological role in the central oxygenated RPMI 1640 medium. Striata were dissected on an nervous system. The gut hormone cholecystokinin (CCK) (6) ice-cold surface and placed in 5 ml of fresh, cold RPMI 1640 is present in the brain (7) mainly in the form of its sulfated medium. Coronal slices (350 ,um) were prepared from each C-terminal octapeptide (CCK-8S) (8). It has a wide distribu- striatum by using a Mcllwain tissue chopper (Brinkmann) and tion as shown by radioimmunoassay (9), immunohistochem- pooled; individual slices were placed (four per tube) in 5-nl istry (10, 11), and in situ hybridization (12). CCK-8S, like conical, polypropylene incubation tubes containing 1 ml of actions on neurons ice-cold, fresh RPMI 1640 medium. This solution was then glutamate, exerts excitatory target (13, replaced with 2 ml of fresh, oxygenated, Mg2+-free buffer 14) and there is evidence not only for glutamatergic (15) but containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.3 also for CCKergic (16-19) corticostriatal pathways in the mM CaCl2, 25 mM NaHCO3, 11.7 mM glucose, and 0.5 mM brain. 3-isobutylmethylxanthine. The tubes were transferred to a Medium-sized spiny neurons, which comprise the largest 30°C water bath and incubated under constant oxygenation cell population of the striatum, are excited by glutamate with 95% 02/5% CO2 for 30 min. Drug solutions were added acting through N-methyl-D-aspartate (NMDA)-type recep- to slices for 5 min. After drug treatment, tissue slices were tors (20, 21). These neurons are highly enriched in dopamine- rapidly transferred to polypropylene tubes, immediately fro- and cAMP-regulated 32-kDa phosphoprotein (DARPP-32) zen on dry ice, and stored at -70°C until assayed. (22). Dopamine, by acting on D1 receptors and increasing Immunoblotting. Frozen tissue samples were sonicated in cAMP, stimulates phosphorylation of DARPP-32 by cAMP- an aliquot of boiling 1% SDS and boiled for an additional 10 dependent protein kinase, converting it into a potent inhibitor min. Small aliquots of the homogenate were retained for of protein phosphatase 1 (23). Conversely, glutamate, by protein determination by the method of Lowry et al. (26) acting on NMDA receptors, and increasing calcium influx, using bovine serum albumin as a standard. Equal quantities stimulates the calcium/calmodulin-dependent protein phos- (250 ig) of each sample were loaded onto 12% acrylamide phatase calcineurin leading to dephosphorylation and inac- gels and proteins were separated by SDS/PAGE (27) and tivation ofDARPP-32 (24). These and other data indicate that then transferred to nitrocellulose membranes (0.2 ,im) by the the antagonistic effects of dopamine and glutamate on the method of Towbin et al. (28). The membranes were then excitability of striatal neurons may be expressed, at least in immunoblotted using a monoclonal antibody (mAB-23) part, through alterations in the state of phosphorylation of (1:100 dilution) (25) that selectively detects the phosphory- DARPP-32. We report here biochemical and immunocyto- lated form ofDARPP-32. In some experiments, a monoclonal chemical evidence that CCK, like glutamate, is able to antibody (C24-5a) (1:2000 dilution) (29) generated against regulate the phosphorylation of DARPP-32 in striatal neu- Abbreviations: DARPP-32, dopamine- and cAMP-regulated 32-kDa The publication costs ofthis article were defrayed in part by page charge phosphoprotein; CCK-8S, cholecystokinin octapeptide (sulfated); payment. This article must therefore be hereby marked "advertisement" NMDA, N-methyl-D-aspartic acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 11277 Downloaded by guest on September 27, 2021 11278 Neurobiology: Snyder et al. Proc. Natl. Acad. Sci. USA 90 (1993) DARPP-32, which is not phosphorylation state specific, was Postembedding Electron Micrographic Analysis of Gluta- used to estimate the total amount of DARPP-32 in samples. mate-like Immunoreactivity. Slices of rat striatum (30) were Antibody binding was revealed by incubation with goat incubated in oxygenated Krebs' solution, fixed in a mixture anti-mouse horseradish peroxidase-conjugated IgG (1:4000 of formalin/glutaraldehyde, treated with osmium tetroxide, dilution) and the ECL Western blotting detection system. dehydrated, and embedded in Durcupan (Fluka). Ultrathin Chemiluminescence was detected by autoradiography. Au- sections were stained by a postembedding immunogold toradiographic signals were then quantified by densitometry method (31). The grids were incubated with a rabbit antise- using a Bio-Rad model 620 video densitometer and Bio-Rad rum to glutamate (glutamate 03; 1:500) (32) to which were 1-D analyst software. Statistical significance of the data was added, to remove traces of crossreactivity, the amino acid- determined by one-directional Student's t test. glutaraldehyde complexes aspartate-G (100 ,uM) and gluta- cAMP Radjoimmunoassay. Striatal slices, prepared and mine-G (300 MM). After a brief rinse in polyethylene glycol, treated as for the phosphorylation experiments, were soni- the grids were'incubated for 2 hr with a goat anti-rabbit IgG cated in 4 mM Tris/EDTA buffer and assayed for cAMP coupled to colloidal gold particles (diameter, 10 nm; 1:20; using an Amersham cAMP RIA kit. Janssen). The sections were finally counterstained and ex- Immunocytochemical Preembedding Electron Microscopic amined as described above. In each of these experimental Analysis of CCK-like Immunoreactivity. Rats were perfused groups adsorption controls were included. with 1% paraformaldehyde followed by a mixture of 1% Immunofluorescence Microscopic Analysis ofAspartate-Hlke paraformaldehyde and 2.5% glutaraldehyde. The brains were Immunoreactivity. Sections (14 ,um) from formalin-fixed rat postfixed and cut in a vibrating microtome at 40 t&m. Sections brains were processed for the indirect immunofluorescence were treated with 1% sodium borohydrate for 1 min and then technique-i.e., incubated with a polyclonal rabbit an'tiserum left overnight in 20%o sucrose solution. After freeze-thaw to aspartate (1:400) (33), followed by fluorescein isothiocy- treatment, sections were incubated for 48 hr with a mono- anate-conjugated goat anti-rabbit antibodies (1:80) and ex- clonal antibody to gastrin-CCK (28.2; 1:5000; J. Walsh, amination in a'Nikon Microphot-FX microscope. personal communication), followed by a biotinylated donkey anti-mouse IgG (1:200; 2 hr) and avidin-biotin-peroxidase complex (1:100; 2 hr; Vector ABC kit). Peroxidase activity RESULTS was visualized by using diaminobenzidine (Sigma) as chro- Effect of CCK-8S on the Phosphorylation of DARPP-32. mogen. Sections were then treated with osmium tetroxide Forskolin, a drlig that activates adenylyl cyclase and in- (1%), dehydrated, and embedded'in
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