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).
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 (approximately 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 IC50 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.
Morinelli
Thomas A
TA
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Avenue, PO Box 250505, Charleston, SC 29425, USA. morinelt@musc.edu
Meier
G Patrick
GP
Webb
Jerry G
JG
Jaffa
Ayad A
AA
Privitera
Philip J
PJ
Margolius
Harry S
HS
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism; D001920:Bradykinin / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; C079000:bradykinin (1-5); D001920:Bradykinin