Proceedings of The Physiological Society

University of Leeds (2002) J Physiol 544P, S016


Light and electron microscopic localisation of the angiotensin type 1 receptor (AT1R) in regions of spinal cord of the rat that influence autonomic outflow

Zai Ahmad and Jim Deuchars

School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK

Intrathecal administration or microinjection of angiotensin II (angII) in the rat increases arterial pressure and heart rate via an increase in sympathetic nerve activity (Lewis & Coote, 1993). The AT1R appears to mediate such cardiovascular responses to angII since they are blocked by intrathecal losartan (Park & Henry, 1997). Consistent with a possible direct effect on sympathetic preganglionic neurones (SPNs), angiotensin II-like immunoreactivity was detected in synaptic contacts with neurones in the thoracic lateral horn (Galabov, 1992). To determine the site of the angII actions we examined the localisation of the AT1R in the spinal cord.

Male Wistar rats (100-150 g) were intraperitoneally injected with fluorogold (0.1 ml, 1 % in NaCl; Fluorochrome Inc.) and allowed to recover for 5 days prior to being humanely killed by anaesthesia with Sagatal (60 mg kg-1, I.P.) and transcardial perfusion with fixative containing 4 % paraformaldehyde/ 0-0.2 % glutaraldehyde in 0.1 M phosphate buffer. Transverse sections (50 mm) of thoracic spinal cord were cut on a vibrating microtome (Leica) and incubated in an affinity-purified antibody to the AT1 receptor (1:400, RDI). Sections were then washed and place in either donkey anti-rabbit IgG conjugated to Cy3 (1:1000, Jackson Immunochemicals) or to a biotinylated anti-rabbit IgG followed by peroxidase conjugated extravidin (Sigma), visualised with diaminobenzidine. Sections were then prepared for fluorescence or light and electron microscopic examination as appropriate.

In the intermediolateral cell column (IML) a subset of fluorogold SPNs contained immunoreactivity for the AT1R associated with their somatic membrane. In addition, punctate-like labelling was also apparent within this region. In lamina X, in the central autonomic area, AT1 receptor labelling was also occasionally detected in neurones dorsal to the central canal. The ependymal cells surrounding the central canal were strongly immunoreactive. Ultrastructural analysis revealed that in the IML AT1 receptor labelling was evident in neuronal somata where it was associated with the cytoplasm, and rough endoplasmic reticulum as well as the somatic membrane. Reaction product was also detected in myelinated axons and presynaptic terminals. In addition, immunoreactive glial cell processes were also apparent. The localisation to a subset of SPNs suggest that angII can act to directly influence sympathetic outflow. However, indirect actions are also possible via angII acting on presynaptic terminals or glial cells.

This work was supported by the BBSRC.

All procedures accord with current UK legislation.

Where applicable, experiments conform with Society ethical requirements