Research reportRegional and age-dependent expression of the nitric oxide receptor, soluble guanylyl cyclase, in the human brain
Introduction
Nitric oxide (NO) is an atypical inter- and intracellular messenger involved in several functions within the central nervous system [11], [16]. For example, NO regulates neurotransmitter release [27], blood flow and its coupling to brain metabolism [26], synaptic plasticity [6], learning, and memory formation [20], but can also induce neuronal cell death [10]. The most relevant intracellular receptor for NO, thus far identified, is soluble guanylyl cyclase (sGC), a heterodimeric hemoprotein to which NO binds and thereby triggers the synthesis of cyclic GMP (cGMP) [2], [17]. The second messenger molecule cGMP has a great variety of effector proteins, including cGMP-regulated phosphodiesterases, ion channels and protein kinases [16], [30]. Elevation of intracellular cGMP levels and subsequent activation of its effectors were shown to mediate many NO-dependent processes [20], [21], [27], [32].
Human sGC consists of a larger α (sGCα) and a smaller β (sGCβ) subunit [35]. The N-terminal domains of both subunits are essential for stimulating the enzyme by NO, although NO binds solely to the heme-containing sGCβ subunit [14]. The catalytic center of sGC is formed by association of the two C-terminal cyclase homology domains that are conserved in both subunits. It was found that only heterodimers are active [8], [19], in contrast to homodimers, which can be formed but are inactive [34].
Thus far, four sGC genes, encoding the subunits α1, α2, β1 and β2, have been identified. In adult brain, however, only α1 and β1 subunits have been described [15]. In rodent brain, sGCα1 and sGCβ1 mRNA [9], [23] and immunoreactivity [1], [25] as well as cGMP synthesizing activity [12] were found to be widely distributed. Expression of sGC in human brain has been investigated at the mRNA level [7], but hitherto no information is available about the regional distribution of sGC protein in human brain. Moreover, distribution and regulation of sGC in the human brain during ageing and age-related neurodegenerative diseases is incompletely understood. It was shown, however, that sGC activity is affected during Alzheimer’s disease [5].
Therefore, in the present study, we examined the regional distribution of sGCα1 and sGCβ1 subunits in the human brain and changes during the course of ageing. The distribution of sGC was compared with that of NOS-I [4], the main NOS (NO synthase) isoform in human brain.
Section snippets
Reagents
Horseradish peroxidase-conjugated polyclonal anti-rabbit antibody was obtained from Dako (Hamburg, Germany); an enhanced chemiluminescence (ECL) immunodetection kit and Hybond ECL nitrocellulose membrane and High Molecular Weight-SDS Calibration Kit, from Amersham Pharmacia Biotech (Freiburg, Germany); Roti-load sample buffer for electrophoresis, from Carl Roth (Karlsruhe, Germany). All other chemicals were of the highest purity available and obtained from Sigma (Deisenhofen, Germany). Water
Results
To determine the presence of sGCα1 and sGCβ1 in different regions of human brain during ageing, sGCα1 and sGCβ1 were detected by Western blot analysis using affinity-purified antibodies directed against different peptide sequences of both sGC subunits [35] (Fig. 1). Antibody directed against sGCα1 recognized a band of 80 kDa; sGCβ1 antibody, a band of 70 kDa. Both immunoreactive bands comigrated with the respective recombinant human sGC subunit standard. The sGCβ1 antibody recognized only a
Discussion
In the present study, we determined the regional distribution of the α1 and β1 subunits of sGC in the human brain and possible changes during ageing. We show that the expression levels of both subunits correlate with each other and with the distribution of the major enzymatic source of NO in the brain, NOS-I. Moreover, in the cortex and striatum, sGC expression was found to be affected by age.
A basic question in understanding the role of the NO/cGMP signaling pathway in the healthy and diseased
Acknowledgements
C.I. and P.I.N. received fellowships from the Deutscher Akademischer Austauschdienst (C.I., A/97/00418 and P.I.N., A/98/10074) and the Graduiertenkolleg ‘Molekulare Biologie und Pharmakologie’, Giessen (P.I.N.). This study was supported by the Deutsche Forschungsgemeinschaft (SFB547/C7), and by grants from the Bundesministerium für Bildung und Forschung (M.G., Würzburger Suchtforschungsverbund, 01EB9410). Helpful comments from Dr. Ulrike Zabel and technical assistance by Monika Weeger are
References (35)
- et al.
Characterization and regional distribution of nitric oxide synthase in the human brain during normal ageing
Brain Res.
(1999) - et al.
Reduced nitric oxide responsive soluble guanylyl cyclase activity in the superior temporal cortex of patients with Alzheimer’s disease
Neurosci. Lett.
(1995) - et al.
Tissue distribution of the human soluble guanylate cyclases
Biochem. Biophys. Res. Commun.
(1999) - et al.
Expression of soluble guanylate cyclase activity requires both enzyme subunits
Biochem. Biophys. Res. Commun.
(1991) - et al.
Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain
Neuroscience
(1998) - et al.
The distribution of nitric oxide synthase immunoreactivity in the human brain
Neuroscience
(1994) - et al.
The separation of the heme and apoheme forms of soluble guanylate cyclase
Biochem. Biophys. Res. Commun.
(1982) - et al.
Expression of soluble guanylyl cyclase. Catalytic activity requires two enzyme subunits
FEBS Lett.
(1990) - et al.
Protein measurement with Folin Phenol reagent
J. Biol. Chem.
(1951) - et al.
Tandem organization of medaka fish soluble guanylyl cyclase alpha1 and beta1 subunit genes. Implications for coordinated transcription of two subunit genes
J. Biol. Chem.
(1999)
Light and electron microscopic demonstration of guanylate cyclase in rat brain
Brain Res.
The nitric oxide and cGMP signal transduction system: regulation and mechanism of action
Biochim. Biophys. Acta
Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tag
J. Biol. Chem.
Immunohistochemical localization of guanylate cyclase within neurons of rat brain
Proc. Natl. Acad. Sci. USA
Nitric oxide activates guanylate cyclase and increases guanosine 3′:5′-cyclic monophosphate levels in various tissue preparations
Proc. Natl. Acad. Sci. USA
Assignment of GUCY1A3, a candidate gene for hypertension, to human chromosome bands 4q31.1→q31.2 by in situ hybridization
Cytogenet. Cell Genet.
Long-term depression in rat cerebellum requires both NO synthase and NO-sensitive guanylyl cyclase
Eur. J. Neurosci.
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2008, NeuropharmacologyCitation Excerpt :Thus, a complete pathway exists for cyclic GMP-mediated signal transduction regulation in the N18TG2 cell model. In Western blots to detect proteins, polyclonal antisera that had been made against peptides from both GC-α1 (aa 634-647) and GC-β1 (aa 593-614) (Calbiochem) detected bands from rat brain preparations that appeared at 80–85 kDa for GC-α1 and at 65–70 kDa for GCβ1 (Fig. 2A) (see also Ibarra et al., 2001; Russwurm et al., 2001). The 19-mer peptide immunogen recognized by the Calbiochem antisera was 84% identical in GC-α1 and GC-α2 proteins, with two conservative substitutions of cationic residues, and thus, the 80–85 kDa band probably recognizes both isoforms.
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Both authors contributed equally to this study.