Identification of the ligands for Cardiac Orphan G protein -Coupled Receptors and Development of T herapy Modulating Cardiac Function

1. (A)为了鉴定在心肌组织中表达的孤儿G蛋白偶联受体（GPCR）的特异性配体，我们分析了已知在心肌细胞中表达的7个GPCR和我们证实其在心肌组织中表达的6个其他GPCR。用乙醚和C18柱从牛心肌组织中提取富肽组分，用反相色谱法分离。然后，我们通过荧光成像板读数器（FLIPR）测定（1-筛选）分析了这些肽级分在用GPCR转染的HEK 293 T细胞中的钙释放活性<st>。我们发现，有一个显着的钙释放在一个GPCR转染细胞与一定的分数。我们认为该馏分含有GPCR的特异性配体，因为我们确认了5次重现性。我们正在通过第二次筛选进一步纯化配体。(B)我们还用转染GPCR的黑素细胞分析了钙释放，在同样的GPCR转染细胞中发现了阳性反应，但我们不能重现该结果。2.由于下丘脑室管膜细胞似乎分泌一些体液因子，这些体液因子通过增强钙释放而特异性地刺激心肌细胞和垂体细胞，因此我们开始通过FLIPR测定和黑素细胞来纯化它们。我们发现，下丘脑室管膜细胞培养上清分离的反相色谱肽馏分刺激大鼠心肌细胞和鹌鹑心肌细胞系的钙释放。我们目前正在通过cAMP测定和反相色谱法进行纯化。

1. (A) To identify the specific ligand s for orphan G protein -coupled receptors (GPCRs) expressed in myocardial tissue, we analyzed 7 GPCRs known to be expressed in cardiac myocytes and 6 other GPCRs of which we confirmed their expression in myocardial tissue. We extracted peptide -rich fraction from bovine myocardial tisuue with ether and C18 column and separated it by reverse -phase chromatography. Then we analyzed calcium releasing activity of these peptide fractions in HEK293T cells transfected with GPCRs by fluorescent imaging plate reader (FLIPR) assay (1^<st> screening). We found that there was a significant calcium release in one of the GPCRs -transfected cells with a certain fraction. We thought that this fraction contained a specific ligand for the GPCR because we confirmed the reproducibility five times. We are further purifying the ligand by second screening. (B) We also analyzed calcium release by using melanophore cells transfected wit h GPCRs, and found positive reactions in the same GPCR -transfected cells, however, we could not reproduce the result. 2. Because hypothalamic ependymal cells seem to secrete some humoral factors which specifically stimulates cardiac myocytes and pituitary cells by enhancing calcium release, we started their purification by FLIPR assay and melanophore cells. We found that some peptide fractions from culture supernatant of hypothalamic ependymal cells separated by reverse-phase chromatography stimulated calcium release in rat cardiac myocytes and a quail myocardial cell line. We are currently proceeding the purification by cAMP assay and reverse-phase chromatography.