Visualisation of non-glycosylated somatostatin receptor two (ngsst2) immunoreactivity in the rat central nervous...

The biological actions of the neuropeptides somatostatin-14 and -28 are receptor-mediated. To date, five G protein-coupled receptors sst1 to sst5 have been characterised pharmacologically and their genes have been cloned. In this study, we used an affinity-purified polyclonal antibody (AS-68) raised against a specific N-terminal peptide sequence of sst2 to localise N-terminal sst2-immunoreactive regions in the rat brain and the cervical spinal cord. The specificity of the antiserum was demonstrated by Western and slot blotting experiments using a N-terminal sst2 fusion protein. Further blotting experiments with a sst2(A)-transfected cell line and rat CNS membrane proteins showed that the antibody detected the non-glycosylated and/or non-sialated receptor. A strong signal using an sst2(A)-transfected CHO-K1 cell line was obtained only if the cells had been treated with N-Glycosidase F prior to the immunochemical detection. Two variants of sst2 (sst2(A) and sst2(B)) have been identified by cloning procedures and gene expression studies in the rodents. They differ in their carboxy-termini: AS-68 would, however, be able to recognise the non-glycosylated form of both these variants. We present here the central nervous system distribution of non-glycosylated sst2-immunoreactivity in the rat using this N-terminal antibody. The sst2 non-glycosylated N-terminal like immunoreactivity was distributed throughout the brain with cells and processes labelled in the cerebral cortex and the basal ganglia (neostriatum, substantia nigra), in the limbic system (hippocampal formation, amygdala), in the diencephalon (epithalamus, thalamus, hypothalamus), the superior colliculus, the periaqueductal grey matter and some of the reticular formation nuclei. The distribution of the non-glycosylated sst2-like immunoreactivity detected here was consistent with that predicted from the localisation of sst2 mRNA and SRIF-ligand binding studies.