LOCALIZATION OF cGMP-DEPENDENT PROTEIN KINASE TYPE II IN RAT BRAIN J. DE VENTE, a * E. ASAN, b S. GAMBARYAN, c M. MARKERINK-VAN ITTERSUM, a H. AXER, d K. GALLATZ, e S. M. LOHMANN c and M. PALKOVITS e a European Graduate School of Neuroscience (EURON), Department of Psychiatry and Neuropsychology, Maastricht University, UNS-50, P.O. Box 616, 6200 MD Maastricht, The Netherlands b Institute of Anatomy, University of Wu « rzburg, Koellikerstrasse 6, 97070 Wu « rzburg, Germany c Institute of Clinical Biochemistry and Pathobiochemistry, University of Wu « rzburg, Josef Schneider Strasse 2, 97080 Wu « rzburg, Germany d Department of Anatomy Rheinisch Westfalische Technische Hochschule, Aachen, Germany e Laboratory of Neuromorphology, Faculty of Medicine, Semmelweis University, Budapest, Hungary AbstractöIn brain, signaling pathways initiated by atrial natriuretic peptide, or transmitters which stimulate nitric oxide synthesis, increase cGMP as their second messenger. One important class of target molecules for cGMP is cGMP- dependent protein kinases, and in the present study, biochemical and immunocytochemical analyses demonstrate the widespread distribution of type II cGMP-dependent protein kinase in rat brain, from the cerebral cortex to the brainstem and cerebellum. Also, colocalization of cGMP-dependent protein kinase type II with its activator, cGMP, was found in several brain regions examined after in vitro stimulation of brain slices with sodium nitroprusside. In western blots, cGMP-dependent protein kinase type II was observed in all brain regions examined, although cerebellar cortex and pituitary contained comparatively less of the kinase. Immunocytochemistry revealed cGMP-dependent protein kinase type II in certain neurons, and occasionally in putative oligodendrocytes and astrocytes, however, its most striking and predominant localization was in neuropil. Electron microscopy examination of neuropil in the medial habenula showed localization of the kinase in both axon terminals and dendrites. As a membrane-associated protein, cGMP-dependent protein kinase type II often appeared to be transported to cell processes to a greater extent than being retained in the cell body. Thus, immunocytochemical labeling of cGMP-dependent protein kinase type II often did not coincide with the localization of kinase mRNA previously observed by others using in situ hybridization. We conclude that in contrast to cGMP-dependent protein kinase type I, which has a very restricted localization to cerebellar Purkinje cells and a few other sites, cGMP-dependent protein kinase type II is a very ubiquitous brain protein kinase and thus a more likely candidate for relaying myriad cGMP e¡ects in brain requiring protein phosphorylation. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: cGMP, protein kinase, nitric oxide, immunocytochemistry, colocalization, brain. Multiple signaling pathways which increase cGMP are present in brain and implicated in various aspects of brain physiological functions and pathophysiology. Glu- tamate receptors control the brain nitric oxide synthase (NOS)/soluble guanylate cyclase (sGC) pathway which increases cGMP, and other transmitters like GABA, ace- tylcholine and neuropeptides can do this as well, via polysynaptic circuits interacting with the glutamatergic system. These pathways have been implicated in diverse activities such as learning and memory, epilepsy, cerebral ischemia, and neurodegenerative diseases (Fedele and Raiteri, 1999). An important distinction, however, must be made between NO e¡ects which are cGMP-dependent and others which are cGMP-independent (Schmidt et al., 1993; Sausbier et al., 2000). In addition to NO/sGC, the atrial, brain, and C-type natriuretic peptides, ANP, BNP and CNP, also increase cGMP, however, via transmembrane receptor (particu- late) guanylate cyclases (pGC) (Koller and Goedel, 1992; Drewett and Garbers, 1994; Wedel and Garbers, 1997). Soluble GC activity has been demonstrated in almost every rat brain region (Ferrendelli, 1978; Schmidt et al., 1992; Southam and Garthwaite, 1993; De Vente et al., 1998), and pGC activity has been described in vari- ous parts of the rat brain in which the great majority of responding cells were characterized as astrocytes (De 27 *Corresponding author. Tel.: +31-43-3881022; fax: +31-43- 3671096. E-mail address : j.devente@np.unimaas.nl (J. de Vente). Abbreviations : ABC, avidin^biotin^peroxidase complex ; ANP, atrial natriuretic peptide ; BH 4 , tetrahydrobiopterin ; BSA, bovine serum albumin; CFTR, cystic ¢brosis transmembrane conduc- tance regulator ; cGK, cGMP-dependent protein kinase ; CNP, C-type natriuretic peptide ; DAB, 3,3P-diaminobenzidine ; DARPP-32, dopamine- and cAMP-regulated phosphoprotein of 32 kDa ; ECL, enhanced chemiluminescence ; FITC, £uorescein isothiocyanate ; IBMX, 3-isobutyl-1-methylxanthine ; LTD, long- term depression ; LTP, long-term potentiation ; NOS, nitric oxide synthase ; PBS, phosphate-bu¡ered saline ; pGC, particulate gua- nylate cyclase ; sGC, soluble guanylate cyclase ; SNP, sodium nitroprusside. NSC 5242 21-11-01 Cyaan Magenta Geel Zwart www.neuroscience-ibro.com Neuroscience Vol. 108, No. 1, pp. 27^49, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:S0306-4522(01)00401-8 0306-4522 / 01 $20.00+0.00