Key Points
- To determine whether heterogeneous subpopulations of angiotensin AT2 receptors exist in murine neuroblastoma N1E-115 cells.
- Solubilized N1E-115 cell membranes using CHAPS detergent and fractionated the receptor proteins using heparin-Sepharose chromatography.
- Performed radioligand displacement assays with [125I]-AngII against AT2-selective ligands (CGP-42112A, PD-123319), AT1-selective Losartan, DTT, and GTPγS.
- Characterized receptor fractions using immunoblotting and immunoprecipitation with polyclonal antisera raised against the major eluted receptor peak.
- Heparin-Sepharose chromatography separated two binding peaks: Peak I (~80% of total binding) showed high affinity for PD-123319 (IC50 = 9.1 ± 4.1 nM; n = 3), whereas Peak III showed lower affinity (IC50 = 196 ± 27 nM; n = 3).
- Dithiothreitol increased [125I]-AngII specific binding in Peak III while decreasing it in Peak I, and GTPγS reduced high-affinity agonist binding only in Peak I.
- Peak I antisera recognized 110-kDa and 66-kDa proteins and successfully immunoprecipitated Peak I binding activity, but showed no immunoreactivity or immunoprecipitation toward Peak III.
Structured PICO
PPopulationMurine neuroblastoma N1E-115 cells
IInterventionHeparin-Sepharose chromatography and pharmacological analysis with angiotensin II receptor ligands
OOutcomeBiochemical, pharmacological, and immunological characterization of AT2 receptor populationssurrogate
The study demonstrates the existence of two pharmacologically, biochemically, and immunologically distinct populations of angiotensin AT2 receptors in murine neuroblastoma N1E-115 cells.