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

Mark A. Edwards, Rhonda A. Loxley, Kellysan Powers-Martin, Janusz Lipski, Douglas J. McKitrick, Leonard F. Arnolda, Jacqueline K. Phillips
Molecular Brain Research 129 (2004) 33-43

NMDA receptors are made up of one NR1 subunit and at least one NR2A-D subunit.  NR1 (of which there are several splice variants) is required for a functioning NMDA receptor, but none of the NR2 subunits are.  Different splice variants and NR2 subunits can be combined to produce NMDA receptors with different pharmacological properties.  SHR rats are known to be more sensitive to glutamate in the RVLM, and the authors of this paper are interested in seeing if the SHRs have different numbers or types of NDMA receptors than the WKYs do.  Also of interest is the action of NO, which is involved in regulating sympathetic output from the RVLM.  NO is produced by three different enzymes: neuronal NO synthase (nNOS), inducible NO synthase (iNOS), and endothelial NO synthase (eNOS), which have different stimuli (and thus different amounts of activity) and can produce different effects on sympathetic activity.  In addition, NMDA receptors can trigger the production of NO by nNOS when activated, and NO can cause modification of the NMDA receptor.  Conventional and RT-PCR were used to study the potential differences in gene expression related to these processes between the SHRs and WKYs.

Tissue punches were performed in the RVLM and RNA was isolated.  Reverse transcription was performed on the samples to obtain cDNA.  Conventional PCR was used to detect NR1 splice variants and these products were sequenced to confirm the presence of specific splice variants.  RT-PCR was done with Taqman probes to detect expression of NR1, NR2A-D, nNOS, and iNOS, which were normalized to neuronal specific enolase (NSE), the reference gene they chose.  Within each sample, NR2 subunits were compared to NR2A to determine relative abundance, because NR2A did not vary between SHRs and WKYs.  Finally, immunohistochemistry was used to detect nNOS in the RVLM.

There was no difference in the presence of NR1 splice variants between the two strains (but these were not quantified).  NR1 was found in highest concentration in both rat strains, and along with NR2A and NR2B, did not change significantly in concentration between the them.  NR2C had the highest expression among the NR2 subunits, and along with NR2D, was found in lower concentration in the SHRs than in the WKYs (NR2C was expressed only 0.37 times as much, and NR2D was expressed 0.26 times as much).  nNOS levels were higher in the WKYs than in the SHRs.  Because the composition of subunits affects the receptor's activity, it can be hypothesized that the difference in composition of NDMA receptors in the RVLMs of the two strains may contribute to the difference in sympathetic nerve activity that is seen (which would be a topic for another paper; this one did not address cause and effect).