Presentation Title

CHARACTERIZATION OF 125-I-ANGIOTENSIN (1-7) BINDING TO MOUSE FOREBRAIN AND LIVER AND RAT LIVER

Location

POSTER PRESENTATIONS

Format

Event

Start Date

12-2-2016 12:00 AM

Abstract

Objective. To assess the saturability of 125I-Ang 1-7 binding to mammalian tissue Background. Angiotensin II (Ang II) is a potent regulator of blood pressure and is pathogenetic for cardiovascular disease. Identification of the ACE homologue ACE2, which forms Ang 1-7 from Ang II, and the GPCR Mas as a functional Ang 1-7 receptor has provided biochemical and molecular tools to study the role of Ang 1-7 as a counterregulator of the pathogenesis of Ang II. Despite the physiological importance of Ang1-7, no one has demonstrated saturable 125I-Ang 1-7 binding in mammalian tissue. To date, only single concentration, specific binding of 125I-Ang 1-7 to mouse kidney has been reported. Methods. We prepared a low specific 41 activity 125/127I-Ang 1-7 by radioiodinating Ang 1-7 with 1 part 125I:19 parts 127I, using chloramine T at pH 7.5 to limit radioiodination to tyr4, and pH 8.5 to radioiodinate both tyr4 and his6, with subsequent purification of monoradioiodinated Ang 1-7 using reverse-phase HPLC. Forebrains from adult male athymic mice (nu/+) were dissected and homogenized in water and the membranes were precipitated by centrifugation at 48 kxG. Membranes were resuspended in Tris:EDTA (50:5 mM) pH 7.3 and incubated with 12 concentrations of 125/127I-Ang 1-7 ranging from 3-110 nM for 30 min at 22 C, after which bound 125/127I-Ang 1-7 was collected on GF/B filters and counted. Results. Both radioligands showed specific (100 μM Ang 1-7 displaceable) saturable binding: 35±8.3 and 35±4.2 fmol/mg initial wet weight (mean±SEM) with KD = 42.6±19.2 and 47.9±13.9 nM, for pH 7.5 and pH 8.5 preparations, respectively. This suggests that the presence of iodine on tyr4 or tyr4 + his6 does not differentially affect binding of Ang 1-7 to mouse brain membranes. Preliminary studies indicate that Ang II, Ang IV, Ang 3-7, Ang 4-7, Sar1,Ile8 Ang II, losartan and PD123319 have low affinity for the 125/127I-Ang 1-7 binding site in mouse brain membranes. 125/127I- Ang 1-7 (prepared at pH 8.5) binding to mouse liver using 50 mM Tris: 5 mM MgCl2, pH 7.2, also was saturable 179±10 fmol/g with KD = 20.4±4 nM. 125/127I-Ang 1-7 (prepared at pH 7.5) binding to rat liver membranes using 50 mM Tris: 5 mM MgCl2 pH 7.2 also was saturable 66.7±1.8 fmol/g with KD = 7.1±1.7 nM. No saturable binding was observed in rat heart. Conclusion. Future studies will assess whether this binding is to the mas protein using other rodent tissues, e.g., kidney, as well as mas knockout mice. If these studies document that 125/127I-Ang 1- 7 binds to mas, this radioligand can be used to assess the binding functionality of mas as well as to measure differences in the binding functionality of mas in animal models of cardiovascular disease. Grants. F.F. Conti: CAPES: 99999.008874/2014-00; M. Morris: DOD grant W81XWH-13-2-0085; R.C. Speth: NIH HL-11390, Pilot Award from the Translational Technologies Component of the Georgetown, Howard Universities Center for Clinical and Translational Science [UL1TR000101] and Cardiovascular Neuroscience Research Fund, Nova Southeastern University.

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Feb 12th, 12:00 AM

CHARACTERIZATION OF 125-I-ANGIOTENSIN (1-7) BINDING TO MOUSE FOREBRAIN AND LIVER AND RAT LIVER

POSTER PRESENTATIONS

Objective. To assess the saturability of 125I-Ang 1-7 binding to mammalian tissue Background. Angiotensin II (Ang II) is a potent regulator of blood pressure and is pathogenetic for cardiovascular disease. Identification of the ACE homologue ACE2, which forms Ang 1-7 from Ang II, and the GPCR Mas as a functional Ang 1-7 receptor has provided biochemical and molecular tools to study the role of Ang 1-7 as a counterregulator of the pathogenesis of Ang II. Despite the physiological importance of Ang1-7, no one has demonstrated saturable 125I-Ang 1-7 binding in mammalian tissue. To date, only single concentration, specific binding of 125I-Ang 1-7 to mouse kidney has been reported. Methods. We prepared a low specific 41 activity 125/127I-Ang 1-7 by radioiodinating Ang 1-7 with 1 part 125I:19 parts 127I, using chloramine T at pH 7.5 to limit radioiodination to tyr4, and pH 8.5 to radioiodinate both tyr4 and his6, with subsequent purification of monoradioiodinated Ang 1-7 using reverse-phase HPLC. Forebrains from adult male athymic mice (nu/+) were dissected and homogenized in water and the membranes were precipitated by centrifugation at 48 kxG. Membranes were resuspended in Tris:EDTA (50:5 mM) pH 7.3 and incubated with 12 concentrations of 125/127I-Ang 1-7 ranging from 3-110 nM for 30 min at 22 C, after which bound 125/127I-Ang 1-7 was collected on GF/B filters and counted. Results. Both radioligands showed specific (100 μM Ang 1-7 displaceable) saturable binding: 35±8.3 and 35±4.2 fmol/mg initial wet weight (mean±SEM) with KD = 42.6±19.2 and 47.9±13.9 nM, for pH 7.5 and pH 8.5 preparations, respectively. This suggests that the presence of iodine on tyr4 or tyr4 + his6 does not differentially affect binding of Ang 1-7 to mouse brain membranes. Preliminary studies indicate that Ang II, Ang IV, Ang 3-7, Ang 4-7, Sar1,Ile8 Ang II, losartan and PD123319 have low affinity for the 125/127I-Ang 1-7 binding site in mouse brain membranes. 125/127I- Ang 1-7 (prepared at pH 8.5) binding to mouse liver using 50 mM Tris: 5 mM MgCl2, pH 7.2, also was saturable 179±10 fmol/g with KD = 20.4±4 nM. 125/127I-Ang 1-7 (prepared at pH 7.5) binding to rat liver membranes using 50 mM Tris: 5 mM MgCl2 pH 7.2 also was saturable 66.7±1.8 fmol/g with KD = 7.1±1.7 nM. No saturable binding was observed in rat heart. Conclusion. Future studies will assess whether this binding is to the mas protein using other rodent tissues, e.g., kidney, as well as mas knockout mice. If these studies document that 125/127I-Ang 1- 7 binds to mas, this radioligand can be used to assess the binding functionality of mas as well as to measure differences in the binding functionality of mas in animal models of cardiovascular disease. Grants. F.F. Conti: CAPES: 99999.008874/2014-00; M. Morris: DOD grant W81XWH-13-2-0085; R.C. Speth: NIH HL-11390, Pilot Award from the Translational Technologies Component of the Georgetown, Howard Universities Center for Clinical and Translational Science [UL1TR000101] and Cardiovascular Neuroscience Research Fund, Nova Southeastern University.