Growth hormone-releasing hormone: not only a neurohormone. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-10 and TNF- compared to placebo. LDN-192960 Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and pro-fibrotic systems, and by elevation of bone morphogenetic proteins. Conclusions Treatment with GHRH agonists appears to reduce the inflammatory responses post-MI and may consequently improve mechanisms of healing and cardiac remod eling by regulating pathways involved in fibrosis, apoptosis and cardiac repair. Patients with cardiac dysfunction could benefit from treatment with novel GHRH agonists. and to accelerate wound healing . Recently, Granata and activities of these highly potent new GHRH analogs, and elucidate their mechanisms of action in promoting and/or enhancing cardiac repair. RESULTS Presence of GHRH ligand and GHRH receptor in H9c2 rat cardiomyoblast cell line Reverse-transcribed mRNA from H9c2 cells and rat pituitary was subjected to RT-PCR. The amplicons for GHRH (195 bp), GHRH-R (110 bp), and -actin (133 bp) were detected at their expected sizes (Figure ?(Figure11). Open in a LDN-192960 separate window Figure 1 Expression of GHRH-R and GHRH mRNA in H9c2 cardiomyoblast cell lineThe PCR products of GHRH-R, GHRH and -actin were detected at their expected sizes: at 110 bp, 195 bp and 133 bp, respectively. Abbreviations: Pit: rat pituitary, Car: H9c2 cells, NEC: no enzyme (reverse transcriptase) control, M: marker. Effect of GHRH agonists on H9c2 cell survival in a nutritionally deprived environment H9c2 cells were cultured in a nutritionally deprived environment for 72 hours, their media containing various GHRH-agonists at 100 nmol/L concentration. Cells in a serum free medium without any hormonal additions served as controls. All the tested GHRH-agonists, except JI-38 and MR-502, significantly improved the viability of the cardiac cells after 72 hours of starvation, compared to their control. The survival of H9c2 cells was increased from 67.8% to 87.3% after the treatment with MR-361, from 67.8% to 85.8% with MR-367, from 74.5% to 87.6% with MR-403, and from 74.5% to 85.7% with MR-409 (Figure ?(Figure2A2A). Open in a separate window Figure 2 LDN-192960 Effect of GHRH agonists at 100 nmol/L concentration on A. survival, and at 1 mol/L concentration on B. Ca++-influx in H9c2 cardiomyoblast cells cultured in a nutrition-deprived mediumAll values represent means SEM. * 0.05, ? 0.01, ? 0.001, vs. placebo positive control and 0.001 vs. vehicle control. Effect of GHRH agonists on calcium mobilization in a nutritionally deprived environment Calcium influx is associated with cell death, and increase in intracellular calcium indicates ensuing apoptosis and necrosis. H9c2 cells were kept in a serum free medium for 72 hours, while they were exposed to various GHRH agonists at 1 mol/L concentrations. Cells cultured in a medium containing FBS served as negative control, and cardiac cells in a nutritionally deprived medium, without any treatment, were the positive control. When compared to the positive control, all the tested GHRH-agonists significantly decreased the calcium influx in the H9c2 cells, 175.6% increase in the positive control vs. 146.3%, F2rl3 119.1%, 147.9%, 141.3%, 105.1%, 90.2%, and 137.9%, in the cells treated with JI-38, MR-356, MR-361, MR-367, MR-403, MR-409, and MR-502, respectively (Figure ?(Figure2B).2B). However, two of these analogs, MR-403 and MR-409, which almost completely inhibited calcium influx, showed no significant difference when compared to the negative control. Effect of GHRH agonists on the expression of genes responsible for signal transduction activation and inhibition in H9c2 cell line We investigated the actions of GHRH and its signaling in H9c2 cell line to determine mechanisms LDN-192960 responsible for the survival observed in the treated cells. The Rat Signal Transduction Pathway Finder PCR array used in our study provided a simple and sensitive tool for profiling LDN-192960 the expression of 84 key genes responsible for signal transduction pathway activation or inhibition. We identified important functional molecules affected by treatment with the GHRH agonists and selected genes potentially related to cell death, senescence and cardiac remodeling. After treatment with MR-409 more than 20 genes in the H9c2 cells exhibited significant transcriptional change in mRNA expression relative to control, and also relative to cells cultured in a nutritionally deprived environment without exposure to GHRH agonists (Table.