The kallikrein-kinin system, activated during inflammatory conditions and the regulation of specific cardiovascular and renal functions, includes two G protein-coupled receptors for bradykinin (BK)-related peptides. 9 -BK (1-100 nM) rapidly concentrated the receptor-associated fluorescence into multiple aggregates that remained associated with the plasma membrane (no significant internalization) and colocalized with caveolin-1. This reaction was slowly reversible upon agonist washing at 37°C and prevented pretreatment with a B 1 R antagonist. ␤-Cyclodextrin treatment, which extracts cholesterol from membranes and disrupts caveolae-related rafts, prevented agonist-induced redistribution of B 1 R-YFP but not the PLA 2 activation mediated by this receptor. The agonist radioligand copurified with caveolin-1 to a greater extent than the tritiated antagonist in buoyant fractions of HEK 293 cells treated with the ligands. Agonistinduced cellular translocation of the kinin B 1 R to caveolaerelated rafts without endocytosis is a novel variation on the theme of G protein-coupled receptor adaptation.

Components of the kallikrein kinin system have been associated with the pathophysiology of hypertension in animal and human studies. In this study, we examined the distribution of four different polymorphisms of the kinin B1 and B2 receptor genes in a population of 120 normotensive and 77 hypertensive African-Americans. Allelic frequencies for three of the four polymorphisms were significantly different from those previously reported in Caucasian populations. Among the polymorphisms analyzed, a potentially functionally significant polymorphism in the core promoter of the kinin B2 receptor (C-58-->T transition) displayed an increased prevalence of the C-58 allele in the hypertensive patients as compared with the controls (0.75 v. 0.62, P = .009). Thus, this B2 receptor promoter polymorphism may represent a susceptibility marker for essential hypertension in African-Americans.

1 The rabbit AT 1 receptor (AT 1 R) for angiotensin II (A II ) has been conjugated to the yellow fluorescent protein (YFP) in order to establish the pharmacological profile of such a fusion protein and to facilitate the study of ligand-induced regulation. 2 A II bound AT 1 R -YFP (K D 8.1 nM in transiently transfected cells) and stimulated HEK 293 cells expressing the fusion protein at concentration ranges similar to the ones that stimulate the contraction of the isolated rabbit aorta. Antagonists found to be insurmountable in the latter assay (candesartan and EXP-3174 being the most extreme cases) were also insurmountable in the phospholipase A 2 assay applied to cells expressing AT 1 R -YFP, whereas losartan appeared to be surmountable in both assays. 3 Cells expressing AT 1 R -YFP exhibited a membrane-associated fluorescence that was partly and reversibly translocated into intracellular structures upon A II stimulation (confocal microscopy); the nonpeptide antagonists were not active in this respect, but prevented the effect of the agonist. 4 A II treatment increased the quantity of the fusion protein in cells, and phorbol 12-myristate 13-acetate (PMA) treatment even more so (immunoblot, confocal microscopy) but, unlike the agonist, the latter drug did not induce receptor endocytosis. A protein kinase C (PKC) inhibitor prevented the effect of either A II or PMA on AT 1 R -YFP abundance. 5 The conjugate AT 1 R -YFP retains the pharmacological properties of the parent rabbit AT 1 R. Agonist-induced downregulation was not documented using this system; to the contrary, we have observed a PKC-mediated increased expression AT 1 R -YFP likely to be the result of a decreased breakdown rate of the fusion protein.