HPD Research Day | February 16, 2018

13 Melnick Auditorium 11:45 a.m. – 12:15 p.m. Development of a Novel Radioligand for Characterization of the Mas Receptor for Angiotensin 1-7 Robert Charles Speth, Ph.D., Professor, College of Pharmacy, Nova Southeastern University Hong Weng Pang, College of Pharmacy, Nova Southeastern University Christopher T. Neagra, Palmetto General Hospital, Hialeah, FL Samantha Bergoine, College of Pharmacy, Nova Southeastern University Andrea Linares, College of Pharmacy, Nova Southeastern University Filipe F. Stoyell-Conti, College of Pharmacy, Nova Southeastern University Objective. To use radioligand binding to characterize Mas receptor binding in tissue. Background. Mas is the receptor for angiotensin 1-7 (Ang 1-7), however, no studies have characterized Mas receptor radioligand binding in tissue membranes. While the presence of a single iodine-125 molecule on the tyrosine of AngII does not impair its binding to the AT 1 and AT 2 receptors, it is not known whether iodine-125 on the tyrosine of Ang 1-7 is equally innocuous. Methods. To address this question, we prepared a novel analog Tyr 0 -Ang 1-7, to provide an alternate site for radioiodination of Ang 1-7. Tyr 0 -Ang 1-7 was radioiodinated using chloramine T (Hunter and Greenwood, 1962) with a 7-times excess of peptide to iodine to minimize di-iodination of the peptide. Two radioiodinated peaks, presumed to be mono 125 I-Y 0 -Ang 1-7 and mono 125 I-Y 4 -Ang 1-7, were resolved from uniodinated peptide by HPLC (C 18 reverse phase column, 14.5% acetonitrile:triethylamine phosphate, pH 3.0 mobile phase). Saturation radioligand binding assays were run for both peaks using rat liver membranes ± 10 μM Ang 1-7. Results. Both 125 I- Y-Ang 1-7 peaks displayed high affinity, saturable binding to rat liver membranes: early peak: K D =7.7±2.3 nM, B max =3.3±0.72 fmol/mg wet weight; and late peak K D =11±2, B max =3.6±0.4 fmol/mg wet weight. Conclusion. These results suggest that addition of a tyrosine at the amino terminus of Ang 1-7 increases its binding affinity for Mas and that the presence of an iodine-125 on either Y 0 or Y 4 of Ang 1-7 does not preclude high affinity, saturable binding to Mas. Melnick Auditorium 2:15 – 2:45 p.m. GRK5 Protects the Heart Against Excessive Aldosterone by Phosphorylating and Inhibiting the Cardiac Mineralocorticoid Receptor Anastasios Lymperopoulos, Ph.D., Associate Professor, College of Pharmacy, Nova Southeastern University Yanelys Duarte, College of Pharmacy, Nova Southeastern University Victoria L. Desimine, College of Pharmacy, Nova Southeastern University Uma Markan, Halmos College of Natural Sciences and Oceanography, Nova Southeastern University Samhitha Pasupuleti, Halmos College of Natural Sciences and Oceanography, Nova Southeastern University Shelby L. Wertz, College of Pharmacy, Nova Southeastern University Katie Anne McCrink, College of Pharmacy, Nova Southeastern University Jennifer Maning, College of Pharmacy, Nova Southeastern University Objective. To study the role of GRK5 in cardiac MR regulation. Background. Aldosterone (Aldo) is one of several increased cardio-toxic hormones in chronic heart failure (HF), contributing to its morbidity & mortality. Most of Aldo`s cardiotoxic effects are mediated by the mineralocorticoid receptor (MR). G protein-coupled receptor (GPCR)-kinases (GRKs) are a family of seven serine/threonine kinases that primarily phosphorylate and desensitize GPCRs. GRK2 and GRK5, the most abundant GRKs in the heart, are also known to phosphorylate non-GPCR substrates. The MR is known to get phosphorylated at various serines, which diminishes its capacity to either translocate to the nucleus or to activate transcription (inhibitory phosphorylation). Methods. We used the cardiomyocyte cell line H9c2 and adult rat cardiomyocytes. We performed co-immunoprecipitation experiments for GRK interactions with MR. We measured MR phosphorylation via western blotting and MR transcriptional activity via the luciferase reporter assay. We also measured cellular apoptosis via TUNEL. Results. GRK5, but not GRK2, phosphorylates the MR in H9c2 cardiomyocytes. This effect is constitutive and is enhanced by beta2-adrenoceptor (but not beta1-adrenoceptor) stimulation. The GRK5-phosphorylated MR is incapable of activating gene transcription in cardiomyocytes, since MR transcriptional activity is markedly suppressed upon GRK5 overexpression. Conversely, CRISPR-mediated GRK5 gene deletion augments cardiac MR transcriptional activity. Finally, GRK5 is absolutely necessary for the anti-apoptotic effects of the MR antagonist drug eplerenone in cardiomyocytes. Conclusions. GRK5 blocks the cardio-toxic MR-dependent effects of Aldo. Thus, cardiac GRK5