Infrared-emitting, peptidase-resistant fluorescent ligands of the bradykinin B2 receptor: application to cytofluorometry and imaging
Infrared-emitting, peptidase-resistant fluorescent ligands of the bradykinin B2 receptor: application to cytofluorometry and imaging
复制标题
缓激肽 B2 受体的红外发射、肽酶抗性荧光配体：在细胞荧光测定和成像中的应用
作者：
L. Gera;Xavier Charest;Mélissa Jean;H. Bachelard;F. Marceau
我们以前曾报道缓激肽（BK）B2受体（B2 R）配体的设计，药理学性质和成像应用与荧光团，如荧光素衍生物在其N-末端共轭。为了利用红外光对活组织的高穿透性及其在光谱的该区域中的低自发荧光，合成并表征了与花青染料7（Cy 7）缀合的另外的探针。拮抗剂B-9430（D-Arg-[Hyp 3，Igl 5，D-Igl 7，Oic 8]-BK）和激动剂B-9972（D-Arg-[Hyp 3，Igl 5，Oic 7，Igl 8]-BK）用红外荧光团Cy 7进行N-末端延伸，分别产生肽B-10665和B-10666。药理学研究表明，与亲本肽相比，激动剂B-10666丧失了对B2 R的许多亲和力，而与B-9430相比，拮抗剂B-10665更好地保留了其效力（[3 H]BK与人B2 R结合的竞争，人分离的脐静脉的收缩性，在每种情况下，效力损失对其更重要）。两种探针均以特定方式（共聚焦显微镜）染色表达B2 R-绿色荧光蛋白（GFP）构建的HEK 293细胞，并且在任一情况下绿色和红外荧光均具有非常广泛的共定位。100 nM的激动剂B-10666促进活细胞中B2 R-GFP的内吞作用，但10-25 nM的拮抗剂版本则不。Cy 7标记的肽不标记表达β2-肾上腺素受体-GFP构建体的细胞。低纳摩尔浓度的B-10665在细胞荧光测定法和细胞威尔斯孔的宏观成像（用于红外荧光检测的IVIS成像系统）中是重组B2 R的有效探针。尽管在高浓度和有限的灵敏度下使用时有非特异性结合的倾向，但Cy 7缀合的肽酶抗性B2 R配体支持原始成像和细胞荧光测定应用。
We have previously reported the design, pharmacological properties and imaging application of bradykinin (BK) B2 receptor (B2R) ligands conjugated with fluorophores such as fluorescein derivatives at their N-terminus. To take advantage of the high penetration of infrared light into living tissues and their low autofluorescence in this region of the spectrum, additional probes conjugated with cyanine dye 7 (Cy7) were synthesized and characterized. The antagonist B-9430 (D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-BK) and the agonist B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-BK) were N-terminally extended with the infrared fluorophore Cy7, producing the peptides B-10665 and B-10666, respectively. Pharmacological studies indicated that the agonist B-10666 lost much affinity for the B2R vs. the parent peptide, whereas the antagonist B-10665 better retained its potency vs. B-9430 (competition of [3H]BK binding to human B2R, contractility of the human isolated umbilical vein for which potency losses were more important in each case). Both probes stained HEK 293 cells that expressed the B2R-green fluorescent protein (GFP) construction in a specific manner (confocal microscopy) and with very extensive co-localization of the green and infrared fluorescence in either case. The agonist B-10666 at 100 nM promoted the endocytosis of B2R-GFP in live cells, but not the antagonist version at 10–25 nM. The Cy7-labeled peptides did not label cells expressing the β2-adrenoceptor-GFP construction. B-10665 at low nanomolar concentrations was an effective probe for the recombinant B2Rs in cytofluorometry and macroscopic imaging of cell wells (IVIS imaging system operated for infrared fluorescence detection). Despite a propensity for non-specific binding when used at high concentrations and limited sensitivity, Cy7-conjugated peptidase-resistant B2R ligands support original imaging and cytofluorometric applications.
缓激肽的竞争性拮抗剂。
影响因子：
3
作者：
Vavrek,RJ;Stewart,JM
通讯作者：
Stewart,JM