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Localization of the Cgmp-Dependent Protein Kinases in Relation to Nitric Oxide Synthase in the Brain

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Localization of the Cgmp-Dependent Protein Kinases in Relation to Nitric Oxide Synthase in the Brain Journal of Chemical Neuroanatomy 17 (1999) 45–55 www.elsevier.com/locate/jchemneu Localization of the cGMP-dependent protein kinases in relation to nitric oxide synthase in the brain A.E.-D. El-Husseini a, J. Williams a, P.B. Reiner a, S. Pelech b, S.R. Vincent a,* a Graduate Program in Neuroscience, Department of Psychiatry, Di6ision of Neurological Sciences, The Uni6ersity of British Columbia, Vancou6er, Canada BC V6T 1Z3 b Department of Medicine, The Uni6ersity of British Columbia, Vancou6er, Canada BC V6T 1Z3 Received 17 February 1999; received in revised form 8 June 1999; accepted 27 June 1999 Abstract The distributions of the type I and type II isoforms of cGMP-dependent protein kinase were determined in the rat brain using immunohistochemistry and in situ hybridization, and compared with the localization of NO synthase determined with NADPH- diaphorase histochemistry. The type I cGMP-dependent protein kinase was highly expressed in the Purkinje cells of the cerebellar cortex, where it was closely associated with the NO synthase containing granule and basket cells. This kinase was also found in neurons in the dorsomedial nucleus of the hypothalamus, where it may be regulated by NO or atriopeptides. The type I kinase was not detected in other central neurons. In contrast, the type II kinase was widely distributed in the brain. In particular, it was highly expressed in the olfactory bulb, cortex, septum, thalamus, tectum and various brainstem nuclei. Many regions expressing this kinase also contained, or received innervation from NO synthase positive neurons. These results indicate that type I cGMP-dependent protein kinase may act as a downstream effector for NO only in the cerebellar cortex and the dorsomedial hypothalamus. The type II cGMP-dependent protein kinase appears to be a major mediator of NO actions in the brain. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Cyclic GMP; Nitric oxide; Protein kinase; Immunohistochemistry; In situ hybridization 1. Introduction There appear to be three main classes of intracellular targets for cGMP, cGMP-regulated phosphodi- Cyclic guanosine 5%-monophosphate (cGMP) medi- esterases, cGMP-gated ion channels and cGMP-depen- ates the physiological actions of two distinct intercellu- dent protein kinases. By altering phosphodiesterase lar messengers in the nervous system; the atriopeptides activity, cGMP can regulate the intracellular levels of and nitric oxide (NO). Atriopeptides act on a unique cAMP (Beavo, 1995). cGMP-gated ion channels are family of cell surface receptors, the particulate guanylyl well studied in photoreceptors and olfactory epithelium, cyclases, which have a single transmembrane domain and more recent studies have shown that they are also and an intracellular catalytic domain (Garbers, 1992). expressed in the brain (El-Husseini et al., 1995a). Fi- NO, which can diffuse freely across cell membranes, nally, by activating specific protein kinases, cGMP may acts on the soluble isoforms of guanylyl cyclase, bind- regulate the function of numerous substrate proteins to ing to the heme moiety of the protein, which causes its affect cell function in a manner analogous to that of activation (Ignarro, 1991; Hobbs, 1997). The resultant cAMP acting through cAMP-dependent protein kinase. increases in cGMP are thought to underlie the physio- Two distinct types of cGMP-dependent protein ki- logical actions of the atriopeptides and NO. nase have been identified in the brain, which differ in their catalytic and regulatory properties (Gamm et al., 1995). Type I (cGKI) occurs in two isoforms which arise from alternative splicing of a single gene which is * Corresponding author. Tel.:+1-604-822-7038; fax: +1-604-822- 7981. highly expressed in smooth muscle, lung and platelets E-mail address: [email protected] (S.R. Vincent) (Sandberg et al., 1989; Wernet et al., 1989). Biochemi- 0891-0618/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0891-0618(99)00023-X 46 A.E.-D. El-Husseini et al. / Journal of Chemical Neuroanatomy 17 (1999) 45–55 cal and immunohistochemical studies indicate that in bovine and human cGKIa and b was synthesised and the brain cGKI is concentrated in the Purkinje cells in coupled to keyhole limpet hemocyanin, and used to the cerebellum (Lohmann et al., 1981; De Camilli et al., immunize New Zealand white rabbits. The sera was 1984). applied onto an agarose column to which the immuno- A second gene, encoding a distinct cGMP-dependent gen was thio-linked. Antibody was eluted from the protein kinase (cGKII) was recently characterized from column with 0.1 M glycine (pH 2.5) and the antibody mouse brain and rat intestine (Uhler, 1993; Jarchau et solution was neutralized to pH 7.0 with saturated Tris al., 1994). More recently, others have cloned the human base. cGKII, and mapped it to chromosome 4 (Fujii et al., The antibody was tested using Western blotting. Ho- 1995; Orstavik et al., 1996). The various cAMP-depen- mogenates from rat cerebellum, cortex, thalamus and dent protein kinase isoforms are differentially expressed pons-medulla were separated on 8.5% SDS-PAGE gels, in the brain, indicating that functional differences in and transferred to nitrocellulose membranes which were cAMP responses may be mediated by specific kinases incubated in blocking solution of 50 mM Tris-buffered (Cadd and McKnight, 1989). Likewise we showed using saline (pH 7.4) containing 0.05% Tween-20 (TBST) and reverse transcriptase-PCR analysis and in situ hy- 5% milk powder. After rinsing, the membranes were bridization that the distribution of cGKII is much incubated with primary antibody overnight at 4°C in different from that of cGKI, with cGKII like NO TBST containing 1% bovine serum albumin (BSA). The synthase, being widely expressed in the brain (El-Hus- membranes were then rinsed and incubated with seini et al., 1995b). This led us to advance the hypothe- horseradish peroxidase-linked secondary antibodies sis that cGKII is a major target for NO/cGMP (1:5000 dilution; Amersham). After washing, im- signalling in the nervous system. In the present report munoreactive proteins were detected with enhanced we have determined the distribution of the two cGMP- chemilumenescence (ECL, Amersham). dependent protein kinases in the brain in detail. We For immunohistochemistry the ABC immunoperoxi- also compare the relationship of NO synthase-positive dase method was used as previously described (Vincent neurons to those expressing the cGMP-dependent et al., 1994). Brain sections were obtained from male protein kinases. Sprague–Dawley rats perfused with 4% paraformalde- hyde in 0.1 M PBS (pH 7.4). The brains were rinsed in 15% sucrose and 30 mm thick sections were cut on a 2. Materials and methods freezing microtome and washed in 0.02 M PBS. The antibody to cGKI was diluted 1:5000 in PBS containing All animal procedures were in strict accordance with 0.3% Triton X-100 (PBST) and 2% normal goat serum the NIH Guide for the Care and Use of Laboratory (NGS) for 24 h at 4°C. The sections were then rinsed Animals and were approved by the local Animal Care 3×20 min in PBST, and incubated with biotinylated Committee. Adult male Wistar rats were obtained from sheep anti-rabbit secondary antibody (Vector Labora- the Animal Care Centre of the University of British tories) diluted 1:1000 in PBST containing 2% NGS for Columbia. They were deeply anaesthestized with pento- 1 h at room temperature. The sections were again barbital and perfused through the ascending aorta with rinsed 3×20 min in PBST, and incubated for 1 h with 100 ml 0.9% NaCl, followed by 300 ml of a fixative an avidin-biotinylated horseradish peroxidase complex solution containing 4% paraformaldehyde in 0.1 M (ABC Elite, Vector Laboratories, Burlingame, CA) in sodium phosphate buffer (pH 7.4). The brain was dis- PBST. Following a final series of rinses in PBST, the sected out, immersed in fixative for2hat4°C, and then immunoreactivity was revealed using diaminobenzidine incubated in 15% sucrose in 0.1 M phosphate buffered with nickel ammonium sulphate (Vincent et al., 1994). saline (PBS) overnight at 4°C. Coronal and parasaggi- Control sections were processed in parallel with the tal sections were cut at 30 mm on a freezing microtome exception that normal rabbit serum was used in place and collected in PBS containing 0.02% sodium azide for of the cGKI antibody. Also, sections and Western blots immunohistochemistry. For in situ hybridization, 10 were examined using antibody preabsorbed overnight mm thick sections were thaw-mounted onto 3- with the synthetic peptide antigen (50 mg/ml antibody, aminopropyltriethoxysilane/chrome alum-coated slides diluted 1:10), and these showed no immunoreactivity. and stored at −80°C. All solutions were prepared with The expression of cGKI and NO synthase was exam- RNAse-free reagents and diethyl pyrocarbonate- ined on single sections using a double staining proce- treated, autoclaved water. dure (Vincent et al., 1994). The sections were first stained for NADPH-diaphorase histochemically to lo- 2.1. Immunohistochemistry and enzyme histochemistry calize NO synthase (Hope et al., 1991). The sections were incubated in PBS containing 1 mg/ml b-NADPH A 16 residue synthetic peptide (CDEPPPDDNSGW- and 0.1 mg/ml nitro blue tetrazolium, with 0.3% Triton DIDF) corresponding to the conserved C-terminal of X-100, at 37°C for 30–60 min. They were then rinsed A.E.-D. El-Husseini et al. / Journal of Chemical Neuroanatomy 17 (1999) 45–55 47 thoroughly in PBS and stained for cGKI-immunoreac- cGKI (Fig. 2A). The staining was found throughout the tivity using the method described above. This method dendritic tree in the molecular layer, and in the cell resulted in blue staining of NO synthase containing soma. The Purkinje cell nuclei were unstained.
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