DETECTION OF DOPAMINE-RECEPTOR D-1A SUBTYPE-SPECIFIC MESSENGER-RNA INRAT-KIDNEY BY IN-SITU AMPLIFICATION

In recent years, both molecular biological and immunohistochemical tec hniques, utilizing receptor subtype-specific probes and antibodies to cloned central nervous system dopamine receptors, have revealed their presence in a number of peripheral organs and tissues. Molecular techn iques have been hindered by the low abundance of receptor mRNA in thes e sites, and reverse transcription-polymerase chain reaction (RT-PCR) has been utilized to address this problem. However, RT-PCR is most oft en employed on either isolated mRNA or microdissected tissue samples, thereby limiting interpretation of whole tissue distribution. The pres ent paper describes the use of a novel self-sustained sequence replica tion system (3SR) to amplify a target; mRNA sequence in situ within th e tissue or cell of interest that is then detected with the use of an internal labeled probe, using standard nonisotopic in situ hybridizati on. Specifically, D-1A receptor mRNA was amplified and detected in kid ney sections of Wistar-Kyoto rats (WKY). The amplified D-1A receptor m RNA was localized to renal arterioles, juxtaglomerular apparatus, and both proximal and distal tubules. mRNA was colocalized to regions show n also to contain D-1A receptor protein. D-1A receptor mRNA was predom inantly localized in the cortex. Specificity of D-1A receptor mRNA det ection was confirmed by appropriate localization in rat brain sections known to express D-1A receptor mRNA. In addition, we confirmed the pr esence of renal D-1A receptor mRNA by RT-PCR. We conclude that D-1A re ceptor mRNA is expressed in a site-specific manner in the WKY kidney. The use of 3SR in situ permits elucidation of site specific mRNA local ization in a manner not reported previously.