Ozono R, O'Connell D P, Vaughan C, Botkin S J, Walk S F, Felder R A, Carey R M
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Hypertension. 1996 Mar;27(3 Pt 2):693-703. doi: 10.1161/01.hyp.27.3.693.
The subtype 1A dopamine receptor (D1A) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D1A receptor in Sprague-Dawley and Wistar Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D1A receptor expressed in stably transfected LTK- cells. D1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription-polymerase chain reaction. D1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold method demonstrated the D1A receptor along the cellular membranes of coronary smooth muscle cells and ventricular myocytes and in the myosin thick filaments and M-lines. D1A receptor mRNA was present in coronary vessels and myocardium in amplified but not in unamplified sections. Western blot analysis showed specific D1A bands in transfected LTK- cells and the atrium but not in nontransfected LTK- cells and the ventricle. The selective D1-like receptor agonist SKF38393 stimulated adenylyl cyclase in ventricular myocardial plasma membranes in a dose-related fashion, and the response was abolished by the selective D1-like receptor antagonist SCH23390. These results demonstrate that the D1A receptor gene and protein are expressed in normal rat heart. The physiological and pathophysiological roles and predominant cell signaling mechanism or mechanisms of this receptor remain to be determined.
最近在大鼠肾脏中检测到了1A亚型多巴胺受体（D1A）。在本研究中，我们运用光学显微镜免疫组织化学、电子显微镜免疫细胞化学以及mRNA原位扩增技术，证实了D1A受体在斯普拉格-道利大鼠和威斯塔-京都大鼠心脏中的存在。对于免疫组织化学和免疫细胞化学，抗肽多克隆抗体针对天然受体的第三个细胞外和细胞内结构域的氨基酸序列。通过识别稳定转染的LTK-细胞中表达的D1A受体来验证其选择性。使用一种新型的基于转录的等温原位扩增系统以及逆转录-聚合酶链反应检测到了D1A受体mRNA。D1A受体蛋白分布于整个心房和心室心肌。免疫前和吸收前对照均为阴性。使用蛋白A金法的电子显微镜免疫细胞化学显示，D1A受体存在于冠状动脉平滑肌细胞和心室肌细胞的细胞膜上以及肌球蛋白粗丝和M线中。在扩增的切片中冠状动脉血管和心肌中存在D1A受体mRNA，而未扩增的切片中则没有。蛋白质印迹分析显示，在转染的LTK-细胞和心房中有特异性的D1A条带，而在未转染的LTK-细胞和心室中则没有。选择性D1样受体激动剂SKF38393以剂量相关的方式刺激心室心肌质膜中的腺苷酸环化酶，并且该反应被选择性D1样受体拮抗剂SCH23390消除。这些结果表明，D1A受体基因和蛋白在正常大鼠心脏中表达。该受体的生理和病理生理作用以及主要的细胞信号传导机制仍有待确定。
