hexarelin and thiazolyl-blue

Loss of cardiomyocytes by apoptosis is proposed to cause heart failure. Angiotensin II (ANG II), an important neurohormonal factor during heart failure, can induce cardiomyocyte apoptosis. Inasmuch as hexarelin has been reported to have protective effects in this process, we examined whether hexarelin can prevent cardiomyocytes from ANG II-induced cell death. Cultured cardiomyocytes from neonatal rats were stimulated with ANG II. Apoptosis was evaluated using fluorescence microscopy, TdT-mediated dUTP nick-end labeling (TUNEL) method, flow cytometry, DNA laddering, and analysis of cell viability by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). It was found that incubation with 0.1 micromol/l ANG II for 48 h increased cardiomyocyte apoptosis. Administration of 0.1 micromol/l hexarelin significantly decreased this ANG II-induced apoptosis and DNA fragmentation and increased myocyte viability. To further investigate the underlying mechanisms, caspase-3 activity assay and mRNA expression of Bax, Bcl-2, and growth hormone secretagogue receptor (GHS-R; the supposed hexarelin binding site) were examined. GHS-R mRNA was abundantly expressed in cardiomyocytes and was upregulated after administration of hexarelin. These results suggest that hexarelin abates cardiomyocytes from ANG II-induced apoptosis possibly via inhibiting the increased caspase-3 activity and Bax expression induced by ANG II and by increasing the expression of Bcl-2, which is depressed by ANG II. Whether the upregulated expression of GHS-R induced by hexarelin is associated with this antiapoptotic effect deserves further investigation.

Topics: Angiotensin II; Animals; Animals, Newborn; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cells, Cultured; Coloring Agents; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Flow Cytometry; Growth Substances; In Situ Nick-End Labeling; Myocytes, Cardiac; Oligopeptides; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tetrazolium Salts; Thiazoles; Vasoconstrictor Agents

Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein-coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal-regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt.These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.

Topics: Animals; Apoptosis; Binding, Competitive; Blotting, Western; Cell Death; Cell Separation; Cells, Cultured; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Doxorubicin; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Ghrelin; Indoles; Inhibitory Concentration 50; Microscopy, Phase-Contrast; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myocardium; Oligopeptides; Peptide Hormones; Peptides; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Spiro Compounds; Swine; Tetrazolium Salts; Thiazoles; Time Factors