Utilization of a radioimmunoassay to detect the generation of Arg-Pro-Pro-Gly-Phe, a stable endproduct of bradykinin metabolism (from cultured rat aortic smooth muscle cells exposed to bradykinin)

Abstract

The peptide hormone bradykinin is a mediator in many physiological and pathological processes. The generation and, to a limited extent, metabolism occur at the sites of action. The short half-life of bradykinin (∼15 s) renders measurements of its concentration in bodily fluids difficult. As a result, recent methods utilizing either ELISA or HPLC/mass spectrometry, have been developed for the measurement of the stable metabolic endproduct of bradykinin, i.e., the pentapeptide Arg-Pro-Pro-Gly-Phe (RPPGF; BK[1–5]). Both have been successfully used to quantitate levels of RPPGF in plasma, pulmonary and nasal exudates. In this study, we demonstrate the characterization and utility of a radioimmunoassay for the measurement of RPPGF, using a newly synthesized radioiodinated analogue of RPPGF, i.e., RPPG( 125I)F. This radioimmunoassay shows an IC 50 of 80.5±7.4 pg/tube ( n=23) with a linear range of detection between 10 and 500 pg/tube. The assay was used to demonstrate the time-dependent generation of RPPGF by cultured rat aortic vascular smooth muscle cells exposed to bradykinin (100 ng/ml). Peak generation of RPPGF was 74.5±9.7 pmol/well ( n=5) after 24 h of incubation. Captopril, an inhibitor of angiotensin-converting enzyme (kininase II), inhibited generation in a concentration-dependent manner. The results characterize a new radioimmunoassay for the stable metabolic endproduct of bradykinin, RPPGF.