Evidence for 5-HT1D beta but not 5-HT1D alpha receptor subtype expression in canine large coronary arteries and saphenous vein.

Abstract

5-Hydroxytryptamine1D (5-HT1D) receptors are believed to play a major role in the vasoconstriction of vascular smooth muscle in human coronary arteries. However, unequivocal evidence as to which subtype of this receptor (5-HT1D alpha or 5-HT1D beta) is involved in these vasoconstrictory effects is lacking. The aim of this study was to identify in the dog the 5-HT1D receptor subtype encoding mRNAs expressed in several large coronary arteries and in the saphenous vein. Degenerate oligonucleotide primers that selectively recognized only mammalian 5-HT1D alpha and 5-HT1D beta receptor sequences were used in RT-PCR experiments to study 5-HT1D receptor subtype expression in endothelium-denuded saphenous vein and large coronary arteries from beagle and alsatian dogs. Resulting PCR products were analysed and identified by Southern blots and sequencing. An identical PCR product whose sequence closely resembles that of the human 5-HT1D beta receptor (98% amino acid identity) was obtained from reverse-transcribed RNA isolated from either saphenous vein or coronary arteries, irrespective of dog race. Absence of 5-HT1D alpha expression was confirmed by Southern blot analysis. Control experiments using canine genomic DNA as template illustrated, nonetheless, that the primers chosen could amplify both 5-HT1D alpha and 5-HT1D beta sequences. Using RT-PCR, we isolated from dog vascular smooth muscle a cDNA fragment whose nucleotide sequence would encode a previously-unreported canine homologue of the 5-HT1D beta receptor. We illustrated that this subtype is the only 5-HT1D receptor subtype expressed in dog saphenous vein and large coronary arteries. The implications of these findings are discussed in light of results from functional studies of 5-HT1-like receptor-mediated effects in these canine blood vessels.