COMBINED IN-SITU HYBRIDIZATION, NORTHERN BLOT ANALYSIS, AND RECEPTOR-BINDING STUDIES IN CLONES EXPRESSING DIFFERENT LEVELS OF THE HUMAN 5-HT1A RECEPTOR

In order to set up the technique of semi-quantitative in situ hybridis ation to detect the serotonin receptor mRNA levels in brain tissue, a panel of three Swiss 3T3 cell clones (named clones 66, 53 and 47) expr essing the human 5-HT1A receptor at different densities were used as a model. The clones were generated by limiting dilution from pools of s tably transfected cells. In addition membranes were prepared from each clone to perform receptor binding studies. Clones 66, 53, and 47 show ed saturable binding for the agonist [H-3]-8-OH-DPAT, with receptor de nsities (B-max) of 227 +/- 86, 548 +/- 107 and 1505 +/- 212 fmol/mg pr otein respectively, and with corresponding affinity constants (pK(d)) of 8.8 +/- 0.1, 9.1 +/- 0.1, and 9.1 +/- 0.1 nM, respectively. Norther n blot analysis using a specific probe for the 5-HT1A receptor reveale d the presence of a single 1.56 kilobase mRNA species in the 5-HT1A re ceptor clones but not in control cells. In situ hybridisation studies were performed by measuring the 5-HT1A receptor mRNA levels in these t hree 5-HT1A transfectants using [S-35]alpha CTP labeled riboprobes (se nse and anti-sense). The following rank order of receptor mRNA express ion was found for clones 66, 53 and 47 respectively: 0.140 +/- 0.001, 0.365 +/- 0.045 and 0.835 +/- 0.115 (relative optical density units). With the sense probe no specific labelling was observed. In conclusion , a positive correlation was found between receptor density (B-max) an d receptor mRNA expression (semi-quantitative in situ hybridisation) u sing human 5-HT1A receptor clones with different expression levels.