Proceedings of The Physiological Society

Puerto de la Cruz, Tenerife (2003) J Physiol 548P, P138

Poster Communications

Subtypes and splice variants of GABAB receptors in the NTS of normotensive and spontaneously hypertensive rats

Emma J. Spary, Azhar Maqbool, Trevor F.C. Batten and Sikha Saha

Institute for Cardiovascular Research, University of Leeds, Leeds LS2 9JT, UK

Inhibitory transmission mediated via the metabotropic GABAB receptor is believed to be important in control of cardiovascular reflexes at the level of the nucleus of the solitary tract (NTS) and has been implicated in hypertension (Sved & Sved, 1989; Tsukamoto & Sved, 1993; Durgam et al. 1999). There are two subtypes of the receptor, GABABR1 and GABABR2, with numerous splice variants of the rat R1 subtype having been cloned (1a-1g). In this study, we investigated the expression of known GABAB receptor variants in the NTS of spontaneously hypertensive (SHR), normotensive Wistar-Kyoto (WKY) and Wistar adult (120-150 g) rats.

Rats were humanely killed by decapitation under halothane anaesthesia (5 % in O2) and tissue microdissected from the caudal NTS. RNA was reverse transcribed to cDNA. Primers designed to be specific for the GABAB subunit variants were co-amplified with the ubiquitous internal standard GAPDH in the presence of Taq DNA polymerase, for 28 cycles, determined to be within the exponential phase for all primers. PCR products were separated on 2 % agarose gels and densitometric analysis was made using NIH software Image J v1.2. Results were normalised to GAPDH and for the R1a, R1b and R2 subunits the SHR value was compared to its WKY control. The veracity of PCR products was verified by endonuclease digestion and by sequencing with a ABI Prism 3100 Genetic Analyser and Sea Scape software v1.1.

The data suggested that the R1a, b, c, d splice variants are abundantly expressed in the NTS of all strains of rats, along with R2, with R1e and g weakly expressed and R1f absent. As a positive control, all splice variants were detected in the cortex. The levels of expression of R1b was not significantly different between SHR and WKY, but R1a was significantly increased in SHR (P < 0.002, n = 6, Student's unpaired t test), as was R2 (P < 0.03, n = 6, Student's unpaired t test). These data suggest that there is an increase in the expression of GABAB receptors in the NTS associated with hypertension. This is in agreement with the results of previous pharmacological studies showing enhanced pressor responses to GABAB receptor activation in the NTS of SHR (Sved & Sved, 1989; Tsukamoto & Sved, 1993), and in acutely hypertensive kidney-wrap rats (Durgam et al. 1999). Furthermore, our data suggest a preferential upregulation in the R1a subtype, which we hypothesise may be localised presynaptically on baroreceptor nerve terminals in the NTS.

This work was supported by a Medical Research Council Studentship.

Where applicable, experiments conform with Society ethical requirements