IMMUNOLABELING OF THE RAT INTESTINAL-TRACT WITH ANTIBODIES SPECIFIC TO THE LONG FORM OF THE 5-HYDROXYTRYPTAMINE(3) RECEPTOR

The mouse 5-hydroxytryptamine(3) (5-HT3) type of serotonin receptors i s expressed as two forms, 5-HT3R-A(L) and 5-HT3R-A(S), generated by al ternative splicing of its primary transcript, that differ by a stretch of six amino acids in the second intracellular loop domain. Because t his sis-amino acid region contains a putative phosphorylation site tha t may be important for the Function and/or regulation of 5HT(3)R-A(L) receptor, specifically, we developed polyclonal antibodies as appropri ate tools for studies relevant to this question. Antibodies against a 20-amino acid peptide corresponding to the sequence of 5-HT3R-A(L) at the level of this six-amino acid region were obtained as soon as one m onth after injection of this synthetic peptide to rabbits. Immunocytoc hemistry with these antibodies led to a strong positive labelling of p lasma membrane, reticulum and Golgi apparatus of COS-7 cells expressin g cloned murine 5-HT3R-A(L), whereas COS-7 cells expressing similar le vels of 5-HT3R-A(S) exhibited only a very weak labelling. Immunoblots of fusion proteins combining glutathion-S-transferase and the second c ytoplasmic loop of 5-HT3R-A(L) or 5-HT3R-A(S) revealed a c. 20-fold se lectivity of the antibodies for the first, long form, as evaluated by densitometric analysis of enhanced chemiluminescence detection. Simila rly, immunoblots of COS-7 cells transfected with cloned 5-MT3 receptor s showed that the anti-peptide antibodies detected a band at 53.000 me l. wt only in cells transfected with 5-HT3R-A(L). finder optimal condi tions, antibodies immunoprecipitated 52% of 5-HT3R-A(L), but only 11% of 5-HT3R-A(L), solubilized from COS-7 cells transfected with the resp ective encoding plasmids. In the rat, no immunoautoradiographic labell ing by the anti-peptide antibodies could be detected in brain structur es which had previously been described to express preferentially a sho rt form of the 5-HT3 receptor. In contrast, a strong immunolabelling w as found in the intestinal mucosa: especially in the rat fetus (at the 17th embryonic day). suggesting the possible participation of the 5-H T3R-A(L) isoform in the development of this tissue. These results show that specific antibodies are useful tools for the Visualization of th e least abundant 5-HT3 receptor isoform in rat tissue. (C) 1998 IBRO. Published by Elsevier Science Ltd.