Dai Xiaoling, Galligan James J
Department of Pharmacology and Toxicology and Neuroscience Program, Life Science Building Room B308, Michigan State University, East Lansing, MI 48824, USA.
Exp Biol Med (Maywood). 2006 Jun;231(6):746-51.
Endothelin-1 (ET-1) is a vasoconstrictor peptide that acts on ET(A) and ET(B) receptors on smooth muscle cells (SMCs). Because vascular SMCs can express both receptors, it is difficult to study the localization and properties of each subtype. Therefore, we investigated the localization and function of ET(A) and ET(B) receptors transfected into HEK 293 cells. Immunocytochemistry was used to examine colocalization of ET receptors with the plasma membrane marker, pan cadherin. In cells transfected with ET(A) receptors, 83 +/- 2% of these receptors colocalized with pan cadherin. In ET(B) receptor-transfected cells, 54 +/- 2% of the receptor colocalized with pan cadherin. When ET(A) and ET(B) receptors were cotransfected, 97 +/- 1% of ET(B) receptors colocalized with ET(A) receptors and 84 +/- 2% of ET(B) receptors colocalized with pan cadherin. ET-1 and sarafotoxin 6c (S6c, ET(B) receptor agonist) increased [Ca2+]i in cells transfected with ET(A) or ET(B) receptors; 100 nM of ET-1 and S6c caused maximal responses. When stimulated with ET-1, ET(B) receptors desensitized faster (t(1/2) = 21 +/- 1 sec) than ET(A) receptors (t(1/2) = 48 +/- 1 sec). S6c-induced increases in [Ca2+]i desensitized in cells expressing ET(B) receptors only (t(1/2) = 17 +/- 1 s). Desensitization was eliminated in cells cotransfected with ET receptors. We conclude that ET(A) receptors localize to the cell membrane, whereas ET(B) receptors are in the membrane and intracellular compartments. Coexpressed ET receptors are in the membrane. ET(B) receptors desensitize faster than ET(A) receptors, but receptor coexpression eliminates desensitization. Finally, ET(A) and ET(B) receptors interact to change receptor trafficking which may modify ET receptor function in vascular SMCs coexpressing these receptors.
内皮素 -1（ET -1）是一种血管收缩肽，作用于平滑肌细胞（SMC）上的ET(A)和ET(B)受体。由于血管平滑肌细胞可同时表达这两种受体，因此很难研究每种亚型的定位和特性。为此，我们研究了转染至人胚肾293（HEK 293）细胞中的ET(A)和ET(B)受体的定位及功能。采用免疫细胞化学法检测ET受体与质膜标记物泛钙黏蛋白的共定位情况。在转染ET(A)受体的细胞中，83±2%的此类受体与泛钙黏蛋白共定位。在转染ET(B)受体的细胞中，54±2%的受体与泛钙黏蛋白共定位。当ET(A)和ET(B)受体共转染时，97±1%的ET(B)受体与ET(A)受体共定位，84±2%的ET(B)受体与泛钙黏蛋白共定位。ET -1和铃蟾毒素6c（S6c，ET(B)受体激动剂）可使转染ET(A)或ET(B)受体的细胞内钙离子浓度（[Ca2+]i）升高；100 nM的ET -1和S6c可引起最大反应。用ET -1刺激时，ET(B)受体脱敏速度比ET(A)受体快（半衰期t(1/2)=21±1秒）（ET(A)受体t(1/2)=48±1秒）。S6c诱导的[Ca2+]i升高仅在表达ET(B)受体的细胞中脱敏（t(1/2)=17±1秒）。在共转染ET受体的细胞中脱敏现象消失。我们得出结论，ET(A)受体定位于细胞膜，而ET(B)受体存在于膜和细胞内区室。共表达的ET受体位于膜上。ET(B)受体比ET(A)受体脱敏更快，但受体共表达可消除脱敏现象。最后，ET(A)和ET(B)受体相互作用以改变受体转运，这可能会改变共表达这些受体的血管平滑肌细胞中ET受体的功能。
