Article ; Online: Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes.
2014 Volume 54, Page(s) 81–88
Abstract: Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed ... of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin ... in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys(19)(Cy5 ...
Abstract | Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr(3)(octanoyl), Lys(19)(Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys(19)(Cy5)]ghrelin (1-19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues. |
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MeSH term(s) | Amino Acid Sequence ; Animals ; Cell Differentiation ; Female ; Ghrelin/analogs & derivatives ; Ghrelin/metabolism ; HEK293 Cells/metabolism ; Humans ; Male ; Mice, Inbred C57BL ; Molecular Imaging/methods ; Molecular Sequence Data ; Molecular Structure ; Myocardium/cytology ; Myocardium/metabolism ; Myocytes, Cardiac/cytology ; Myocytes, Cardiac/metabolism ; Peptide Fragments ; Receptors, Ghrelin/analysis ; Receptors, Ghrelin/metabolism |
Chemical Substances | (Dpr3(octanoyl)Lys19(Cy5))ghrelin(1-19) ; Ghrelin ; Peptide Fragments ; Receptors, Ghrelin ; ghrelin, des-n-octanoyl |
Language | English |
Publishing date | 2014-04 |
Publishing country | United States |
Document type | Journal Article ; Research Support, Non-U.S. Gov't |
ZDB-ID | 769028-9 |
ISSN | 1873-5169 ; 0196-9781 |
ISSN (online) | 1873-5169 |
ISSN | 0196-9781 |
DOI | 10.1016/j.peptides.2014.01.011 |
Database | MEDical Literature Analysis and Retrieval System OnLINE |
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