Sgard F, Faure C, Graham D
Synthélabo Recherche, Rueil Malmaison, France.
Cardiovasc Res. 1996 May;31(5):793-9. doi: 10.1016/0008-6363(96)00014-4.
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.
5-羟色胺1D（5-HT1D）受体被认为在人类冠状动脉血管平滑肌的血管收缩中起主要作用。然而，关于该受体的哪种亚型（5-HT1Dα或5-HT1Dβ）参与这些血管收缩作用，尚无明确证据。本研究的目的是在犬中鉴定在几条大冠状动脉和隐静脉中表达的编码5-HT1D受体亚型的mRNA。
使用仅选择性识别哺乳动物5-HT1Dα和5-HT1Dβ受体序列的简并寡核苷酸引物，在逆转录聚合酶链反应（RT-PCR）实验中研究来自比格犬和阿尔萨斯犬的内皮剥脱隐静脉和大冠状动脉中5-HT1D受体亚型的表达。通过Southern印迹和测序分析并鉴定所得的PCR产物。
从隐静脉或冠状动脉分离的逆转录RNA中获得了相同的PCR产物，其序列与人类5-HT1Dβ受体的序列非常相似（氨基酸同一性为98%），与犬种无关。Southern印迹分析证实不存在5-HT1Dα表达。然而，以犬基因组DNA为模板的对照实验表明，所选择的引物可以扩增5-HT1Dα和5-HT1Dβ序列。
使用RT-PCR，我们从犬血管平滑肌中分离出一个cDNA片段，其核苷酸序列将编码一种以前未报道的犬5-HT1Dβ受体同源物。我们证明该亚型是在犬隐静脉和大冠状动脉中表达的唯一5-HT1D受体亚型。根据这些犬血管中5-HT1样受体介导作用的功能研究结果，讨论了这些发现的意义。
