Major occurrence of the new a 2 h 1 isoform of NO-sensitive guanylyl cyclase in brain Evanthia Mergia a , Michael Russwurm a , Georg Zoidl b , Doris Koesling a, * a Pharmakologie und Toxikologie, Medizinische Fakulta ¨t MA N1/39, Ruhr-Universita ¨t Bochum, 44780 Bochum, Germany b Neuroanatomie, Medizinische Fakulta ¨t MA N6, Ruhr-Universita ¨t Bochum, 44780 Bochum, Germany Received 12 June 2002; accepted 24 July 2002 Abstract NO-sensitive guanylyl cyclase (GC) acts as the effector molecule for NO and therefore plays a key role in the NO/cGMP signalling cascade. Besides the long known GC isoform (a 1 h 1 ), another heterodimer (a 2 h 1 ) has recently been identified to be associated with PSD-95 in brain. Here, we report on the tissue distribution of all known guanylyl cyclase subunits to elucidate the isoform content in different tissues of the mouse. The guanylyl cyclase subunit levels were assessed with quantitative real-time PCR, and the most important results were verified in Western blots. We demonstrate the major occurrence of the a 2 h 1 heterodimer in brain, find a significant amount in lung and lower amounts in all other tissues tested. In brain, the levels of the a 2 h 1 and a 1 h 1 isoforms were comparable; in all other tissues, the a 1 h 1 heterodimer was the predominating isoform. The highest guanylyl cyclase content was found in lung; here the GC amounted to approximately twice as much as in brain. In sum, the major occurrence of the a 2 h 1 heterodimer suggests a special role in synaptic transmission; whether this isoform outside the brain also occurs in neuronal networks has to be addressed in future studies. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Real-time PCR; Quantitative PCR; Tissue distribution; NO-sensitive guanylyl cyclase; Nitric oxide 1. Introduction Nitric oxide has been shown to play an important role in the cardiovascular and the nervous system. Most effects of NO as a signalling molecule are mediated by the so-called soluble guanylyl cyclase (GC), the enzyme that catalyses the conversion of GTP to cGMP. NO binds to the prosthetic heme group of the enzyme and leads to an up to 200-fold increase in enzymatic activity [1,2]. The resulting rise in the cGMP concentration brings about changes in activity of the cGMP effector molecules (cGMP-dependent protein kinases, cGMP-dependent phosphodiesterases, cGMP gated-channels) thereby leading to alterations in cellular function [3]. NO-sensitive GC is a heterodimeric enzyme composed of two subunits, a and h; four subunits (a 1 , a 2 , h 1 , h 2 ) have been identified so far. The a 1 h 1 heterodimer corresponds to the enzyme purified from lung; the a 2 and h 2 subunits have been identified by homology screening [4,5]. In most laboratories, expression of the h 2 subunit did not yield a catalytically active enzyme; the specific activities reported by others were only marginal [6,7]. A mutation causing a frame shift that interfered with the production of the full- length protein has been identified in the human h 2 version [8]. In contrast to the h 2 subunit, coexpression of the a 2 subunit with the h 1 subunit resulted in a catalytically active NO-sensitive enzyme and the h 1 subunit was identified as the physiological dimerising partner [9]. Both a subunits share a highly conserved C-terminal catalytic part whereas the regulatory N-terminal domains differ considerably with only 30% identical amino acids. Despite these differences in primary structure, extensive analysis of the purified iso- forms (a 1 h 1 and a 2 h 1 ) did not reveal any differences as regards catalytic rates, sensitivity towards NO or the new YC-1-like NO-sensitisers. 0898-6568/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. PII:S0898-6568(02)00078-5 Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, guanylyl cyclase; NO, nitric oxide; PSD-95, postsynaptic density protein-95. * Corresponding author. Tel.: +49-234-322-6827; fax: +49-234-321- 4521. E-mail address: doris.koesling@ruhr-uni-bochum.de (D. Koesling). www.elsevier.com/locate/cellsig Cellular Signalling 15 (2003) 189 – 195