Elsevier

Brain Research

Volume 907, Issues 1–2, 13 July 2001, Pages 54-60
Brain Research

Research report
Regional and age-dependent expression of the nitric oxide receptor, soluble guanylyl cyclase, in the human brain

https://doi.org/10.1016/S0006-8993(01)02588-4Get rights and content

Abstract

Nitric oxide (NO), synthesized by neuronal NO synthase (NOS-I), plays essential physiological roles in the brain. The major molecular target for NO is soluble guanylyl cyclase (sGC), a heterodimeric hemoprotein composed of a larger α and a smaller β subunit. Both subunits of sGC are needed to generate the second messenger cyclic GMP (cGMP). Here we show using subunit-specific antibodies and Western blot analysis that sGCα1 and sGCβ1 protein subunits are present in all examined human brain regions. The relative distribution of the two subunits was similar and also correlated well with the known distribution of NOS-I. The highest expression levels of sGC were found in cortex, basal ganglia and the limbic system. These regions display the most prominent biochemical and histological changes during ageing. In cortex, a negative correlation between the amounts of sGC and age was found, while sex and post-mortem delay time did not affect sGC levels significantly. Our data suggest that sGCα1 and sGCβ1 subunits are widely distributed in human brain, consistent with a major role in NO signaling. Moreover, the NO/cGMP pathway appears to be affected by ageing 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

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