Presentation Title

Searching for the Elusive Angiotensin-1-7 Receptor

Speaker Credentials

Professor

Speaker Credentials

Ph.D.

College

College of Pharmacy

Location

Nova Southeastern University, Davie, Florida, USA

Format

Podium Presentation

Start Date

21-2-2020 8:30 AM

End Date

21-2-2020 4:00 PM

Abstract

Robert C. Speth, Ph.D. Professor, College of Pharmacy Filipe Stoyell-Conti, Research Associate, College of Pharmacy Alesa Chabbra, B.S. candidate, Halmos College of Natural Sciences and Oceanography Objective. To use radioligand binding assays to characterize the receptor(s) for angiotensin-1-7, a metabolite of the primary agonist of the renin-angiotensin system. Background. Angiotensin-1-7 (Ang- 1-7) was first reported to be a hormone in 1987. However, subsequent attempts to define its physiological function for this peptide were problematic. In 2003, we, Santos et al. reported that Ang-1-7 was an endogenous ligand for the protooncogene/orphan G protein-coupled receptor protein Mas. While we demonstrated 125I-Ang-1-7 binding using receptor autoradiography, it is necessary to demonstrate that this binding meets pharmacological criteria for a receptor. Methods. Classical radioligand binding assays of tissue membrane suspensions are incubated with a range of concentrations of 125/127I-Ang-1-7 or 3H-Ang-1-7 with and without non-radiolabeled Ang-1-7 or with different Ang peptides, e.g., AngII, AngIII, Ang-2-7, etc. “Specific” receptor binding of 125/127I-Ang-1-7 is evaluated using non-linear regression analysis to fit the Langmuir isotherm. Additionally, HPLC analysis of metabolism of Ang 1-7 in the assay is determined. Results. By varying assay medium constituents and peptidase inhibitors we can approximate the concentration and dissociation constant for 125/127I-Ang-1-7 binding in testis and liver of spontaneously hypertensive rats. However, other Ang peptides compete for this 125/127I-Ang-1-7 receptor equivalent to Ang-1-7, calling into question the pharmacological specificity of this binding site. Additionally, there is significant metabolism of Ang-1-7 which compromises the ability of the binding assays to obtain accurate values. Conclusion. We are continuing to validate this receptor binding assay to definitively characterize the receptor(s) for Ang-1-7. Grants. This study was partially funded by a PFRDG.

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Feb 21st, 8:30 AM Feb 21st, 4:00 PM

Searching for the Elusive Angiotensin-1-7 Receptor

Nova Southeastern University, Davie, Florida, USA

Robert C. Speth, Ph.D. Professor, College of Pharmacy Filipe Stoyell-Conti, Research Associate, College of Pharmacy Alesa Chabbra, B.S. candidate, Halmos College of Natural Sciences and Oceanography Objective. To use radioligand binding assays to characterize the receptor(s) for angiotensin-1-7, a metabolite of the primary agonist of the renin-angiotensin system. Background. Angiotensin-1-7 (Ang- 1-7) was first reported to be a hormone in 1987. However, subsequent attempts to define its physiological function for this peptide were problematic. In 2003, we, Santos et al. reported that Ang-1-7 was an endogenous ligand for the protooncogene/orphan G protein-coupled receptor protein Mas. While we demonstrated 125I-Ang-1-7 binding using receptor autoradiography, it is necessary to demonstrate that this binding meets pharmacological criteria for a receptor. Methods. Classical radioligand binding assays of tissue membrane suspensions are incubated with a range of concentrations of 125/127I-Ang-1-7 or 3H-Ang-1-7 with and without non-radiolabeled Ang-1-7 or with different Ang peptides, e.g., AngII, AngIII, Ang-2-7, etc. “Specific” receptor binding of 125/127I-Ang-1-7 is evaluated using non-linear regression analysis to fit the Langmuir isotherm. Additionally, HPLC analysis of metabolism of Ang 1-7 in the assay is determined. Results. By varying assay medium constituents and peptidase inhibitors we can approximate the concentration and dissociation constant for 125/127I-Ang-1-7 binding in testis and liver of spontaneously hypertensive rats. However, other Ang peptides compete for this 125/127I-Ang-1-7 receptor equivalent to Ang-1-7, calling into question the pharmacological specificity of this binding site. Additionally, there is significant metabolism of Ang-1-7 which compromises the ability of the binding assays to obtain accurate values. Conclusion. We are continuing to validate this receptor binding assay to definitively characterize the receptor(s) for Ang-1-7. Grants. This study was partially funded by a PFRDG.