Identification of a novel 5-HT 4 receptor splice variant (r5-HT 4c1) and preliminary characterisation of specific 5-HT 4a and 5-HT 4b receptor antibodies

Abstract

The human 5-hydroxytryptamine (5-HT 4) receptor is encoded by a highly complex gene which gives rise to at least 10 distinct splice variants. However, the functional relevance of these variants is unknown. In rat, only three such variants have been identified, 5-HT 4a (r5-HT 4a), 5-HT 4b (r5-HT 4b) and 5-HT 4e (r5-HT 4e). In the current study we identify and characterise the pharmacology of a novel rat splice variant (r5-HT 4c1) and present the first comprehensive analysis of 5-HT 4 splice variant mRNA expression levels throughout the rat gastrointestinal tract. In addition, we describe preliminary characterisation of the first 5-HT 4 splice variant specific antibodies. In transfected cells, r5-HT 4c1 receptor exhibited similar binding properties to r5-HT 4a and r5-HT 4b. Functional studies showed that 5-HT 4 agonists prucalopride (4-amino-5-chloro-2,3-dihydro- N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofuran carboxamide monohydrochloride and renzapride (±)-endo-4-amino-5-chloro-2-methoxy- N-(1-azabicyclo[3.3.1]non-4-yl)benzamide monohydrochloride) acted as partial agonists at r5-HT 4c1, but full agonists at r5-HT 4a and r5-HT 4b. Moreover, in contrast to r5-HT 4a and r5-HT 4b, r5-HT 4c1 was not constitutively active. TaqMan mRNA analysis showed that r5-HT 4a expression in brain and dorsal root ganglion exceeded that in the gastrointestinal tract, whilst the reverse was true for r5-HT 4b and r5-HT 4c1. mRNA expression of each variant also increased distally throughout the gastrointestinal tract with the highest levels in the colon. r5-HT 4a and r5-HT 4b specific immunoreactivity was abundant on enteric neurons in jejunum, ileum and colon as well as neurons and satellite cells of the dorsal root ganglion. Only r5-HT 4b immunoreactivity was observed on endocrine cells in the duodenum. These data could have implications in rat models and aid understanding of 5-HT 4 splice variant function.