Combined in situ hybridisation, 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 hybridisation to detect the serotonin receptor mRNA levels in brain tissue, a panel of three Swiss 3T3 cell clones (named clones 66, 53 and 47) expressing the human 5-HT1A receptor at different densities were used as a model. The clones were generated by limiting dilution from pools of stably transfected cells. In addition membranes were prepared from each clone to perform receptor binding studies. Clones 66, 53, and 47 showed saturable binding for the agonist [3H]-8-OH-DPAT, with receptor densities (Bmax) of 227 +/- 86, 548 +/- 107 and 1505 +/- 212 fmol/mg protein respectively, and with corresponding affinity constants (pKd) of 8.8 +/- 0.1, 9.1 +/- 0.1, and 9.1 +/- 0.1 nM, respectively. Northern blot analysis using a specific probe for the 5-HT1A receptor revealed the presence of a single 1.56 kilobase mRNA species in the 5-HT1A receptor clones but not in control cells. In situ hybridisation studies were performed by measuring the 5-HT1A receptor mRNA levels in these three 5-HT1A transfectants using [35S]alphaCTP labeled riboprobes (sense and anti-sense). The following rank order of receptor mRNA expression 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 (Bmax) and receptor mRNA expression (semi-quantitative in situ hybridisation) using human 5-HT1A receptor clones with different expression levels.
Departments of Neuropharmacology and Biotechnology, Oss, The Netherlands.
P N
PN
Konings
M
M
Shahid
C
C
van Alebeek
W K
WK
Makkink
N J
NJ
Stam
G S
GS
Ruigt
P M
PM
Vanderheyden
D016475:3T3 Cells; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D014162:Transfection
D015347:RNA Probes; D012333:RNA, Messenger; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin