Research report
Unique levels of expression of N-methyl-d-aspartate receptor subunits and neuronal nitric oxide synthase in the rostral ventrolateral medulla of the spontaneously hypertensive rat

https://doi.org/10.1016/j.molbrainres.2004.06.013Get rights and content

Abstract

The rostral ventrolateral medulla (RVLM) is the major brainstem region contributing to sympathetic control of blood pressure. We have compared the expression of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A–D), NR1 splice variants (NR1-1a/1b, -2a/2b, -3a/3b, -4a/4b), and the neuronal and inducible isoforms of NO synthase (nNOS and iNOS) in the RVLM of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), based on the hypothesis that altered NMDA receptor make-up or altered expression of endogenous NO may be associated with the increase in sympathetic output described from this site in hypertension. Total RNA was extracted and reverse transcribed from the RVLM of mature male WKY and SHR (16–23 weeks). Conventional polymerase chain reaction (PCR) indicated that only the NR1 splice variants NR1-2a, NR1-2b, NR1-4a and NR1-4b were expressed in the RVLM of either species. Quantitative real-time PCR indicated that for both strains of rat, mRNA for the NR1 subunit (all splice variants) was the most abundant (16.5-fold greater, P≤0.05, relative to the NR2A subunit). Amongst the NR2A–D subunits, NR2C was the most abundant (7- and 1.7-fold greater relative to the NR2A subunit, P≤0.05, WKY and SHR, respectively). Relative to WKY, mRNA levels for the NR2C and NR2D subunits in the SHR RVLM were significantly lower (0.3- and 0.25-fold less, P≤0.05), while nNOS was significantly higher (1.76-fold greater, P≤0.05). This was confirmed immunohistochemically for nNOS expression. These results demonstrate differential expression levels of NMDA receptor subunits and NOS isoforms in the RVLM region of SHR when compared to WKY rats.

Introduction

Brain pathways controlling arterial pressure are distributed throughout the neuraxis in discrete topographically arranged networks. Within the rostral ventrolateral medulla (RVLM), sympathoexcitatory neurons directly innervate sympathetic preganglionic neurons in the spinal cord and are critical to the tonic and reflex regulation of sympathetic tone and therefore of arterial blood pressure [24]. The activity of these RVLM neurons is regulated not only by baroreceptor input via the nucleus tractus solitarii and caudal ventrolateral medulla (CVLM) [27], but also by synaptic inputs from many other areas of the central nervous system. An increase in sympathoexcitatory output from the RVLM has been reported in different forms of hypertension, despite different initiating mechanisms [5], [40].

Within the RVLM, glutamate microinjections elicit large pressor responses, mediated in part by N-methyl-d-aspartate (NMDA) receptors [23]. Three gene families encoding NMDA receptor subunits have been characterized: NMDAR1 (NR1), NR2 and NR3. Alternative splicing of the NR1 gene results in eight functional variants, whereas the NR2A–D and NR3A–B subunits are encoded by different genes [14], [32]. To date, the role of the newly identified NR3 receptor subunits has not yet been clarified. All NMDA receptors appear to function as multimeric assemblies of the obligatory NR1 subunit in combination with at least one of the NR2 subunits. Each of the constituent subunits, including the particular NR1-splice variant, conveys distinct pharmacological properties to the receptor assembly [13]. Of relevance to the current study is work that demonstrates that cardiovascular responses to both glutamate and NMDA in the RVLM are augmented in the SHR [45] and that hypertension is associated with a change in the specific make-up of NMDA receptors in the region [4].

Recently, evidence for interactions between nitric oxide (NO) and sympathetic output via the RVLM has been presented [6], [9]. However, the role of this molecule is controversial, for although inhibitory effects evoked by NO within the RVLM have been described [28], excitatory effects have also been reported [17], [30]. Furthermore, it has been proposed that NO produced from different sources [i.e., neuronal (nNOS) vs. inducible (iNOS)] mediates differing effects on sympathetic output and blood pressure, being sympathoexcitation and sympathoinhibition, respectively [7], [9]. Overexpression of endothelial NOS (eNOS) within the RVLM causes sympathoinhibition [20], but recent work has shown that it is only expressed in blood vessels and does not contribute to basal sympathetic vasomotor activity in the RVLM [10].

One of the mechanisms by which NO can act is via a direct interaction with NMDA receptors. Calcium entry via the NMDA receptor stimulates NO production by nNOS [1], [11] and the NO produced exerts its effects by S-nitrosylation of proteins including the NMDA receptor [25] and the activation of guanylyl cyclase/cGMP [12].

Given the importance of NMDA receptor subunit/splice variant composition in regulating receptor activation, and the capacity for NO to modulate sympathetic output from the RVLM region, the present study was designed to investigate if differences in the expression of NMDA receptors or NOS isoforms exist between the normotensive Wistar Kyoto (WKY) rat and the spontaneously hypertensive rat (SHR).

Section snippets

Animals

All experiments were carried out with the approval of the Animal Ethics Committee of Murdoch University, Western Australia. Gene expression data was obtained from mature male WKY and SHR rats (n=9 each strain, 16–23 weeks old). Systolic arterial blood pressure was measured 1–2 weeks prior to tissue collection for mRNA analysis using the tail-cuff method. Blood pressure values for WKY and SHR were, respectively, 127.6±16.7 mm Hg (n=9) and 198.4±18.8 mm Hg (n=9). Prior to decapitation for tissue

Detection of NR1 splice variants in the RVLM

Conventional PCR for the NR1 splice variants on control whole brainstem cDNA produced all predicted product. Sequencing of the NR1-1 and NR1-2 products confirmed their identity, with results indicating 100% sequence homology to the published rat sequence. Gel electrophoresis of PCR products derived from RVLM was performed and results are summarized in Table 2. A typical gel is illustrated in Fig. 3A. In the SHR and WKY tissue samples, both NR1-a (exon-5-lacking) and NR1-b (exon-5-containing)

Discussion

This study has demonstrated differential expression of NMDA receptor subunits and neuronal NOS in the RVLM of SHR as compared to the WKY rat strain. Quantitative analysis showed greatest abundance of the NR1 subunit in both strains of animals, with the specific detection of the NR1-2a, NR1-2b and NR1-4a, NR1-4b splice variants (indicating an absence of exon 21). NR2C was the major NR2 subunit expressed, however, mRNA expression levels for both the NR2C and NR2D subunits were significantly less

Acknowledgements

The authors would like to thank Dr. Andrew Masel (Applied Biosystems, Australia) for assistance in the design of primers for the real-time PCR analysis and optimization of the PCR protocol, Ms. Paula Fuller for sequencing of the NR1-1 and NR1-2 PCR products, Dr. Vasyl Holobotovskyy for assistance with the blood pressure recordings and A/Prof. Paul Pilowsky and Dr. Ann Goodchild for their insightful comments during the implementation of these experiments. Financial support from the Australian

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