Synthesis and properties of three fluorescent derivatives of gastrin

Summary As a first step in the study of hormone interaction with gastrin receptor-expressing cells, three fluorescent derivatives of heptagastrin were synthesized, characterized and tested for specificity and affinity towards gastrin/CCK_B receptor by means of confocal laser scanning microscopy (CLSM). Cyanine dye Cy3.29 and borfluoropyrromethene (BODIPY) derivatives of the hormone were found to be absorbed into the cells and concentrated in perinuclear organelles by a non-receptor mediated process. The BODIPY derivative turned out to be chemically unstable and was bleached by the laser beam very rapidly. Rhodamine Green-heptagastrin retained a high affinity toward the gastrin receptor (K_d=45 nm in displacement of ¹²⁵I-labeled cholecystokinin-8) and showed specific binding to NIH/3T3 cells stably transfected with human gastrin/CCK_B receptor cDNA, but not to nontransfected 3T3 cells. The fluorescent signal of all three dyes was sufficiently intense for localization of the compounds in cells by means of CLSM. Rhodamine Green derivative was found to be a useful tool for the study of endocytosis of the hormone. It can also be utilized for quantitative estimation of binding and determination of K_d instead of the traditionally used radiolabeled derivatives of gastrin.Abbreviations BODIPY borfluoropyrromethene - CCK cholecystokinin - CCK-8 CCK octapeptide - RG-7G Rhodamine Green heptagastrin - BSA bovine serum albumin - DMEM Dulbecco's modified Eagle's medium - TFA trifluoroacetic acid - DMSO dimethylsulfoxide - EDTA ethylenediamino tetraacetic acid - CLSM confocal laser scanning microscopy