A fluorimetric binding assay for angiotensin II and kinin receptors.

Angiotensin II (AngII) and kinins (bradykinin (BK) and des-Arg9-bradykinin (DBK)), are potent agents involved in the maintenance of blood pressure and several biological activities, and their better understanding is important to produce new drugs aimed to control arterial blood pressure. Previous studies on ligand-receptor binding have been based on radioactive methods, which led us to study a new method based on the fluorimetric method. A lanthanide attached to the N-terminal segment of the peptide (AngII, BK and DBK), which produces a time-resolved-fluorescent ligand, was used in a binding test with CHO cells expressing the AT1, AT2, B1 or B2 receptors in comparison with the same cell line tested with the radioactive ligand. Our findings indicated that the non-radioactive method provided a comparable result for the angiotensin receptors. On the other hand, the kinin receptors showed a slight reduction in the binding affinity, probably due to the linkage at the N-terminal segment and/or to the lower biological stability associated to the high temperature (37°C) used for the fluorimetric method, while the radioactive one was at 4°C. We can conclude that a time-resolved fluorescence assay would provide a sensitive method as an alternative tool for receptor studies.

The role of N-terminal and C-terminal Arg residues from BK on interaction with kinin B2 receptor.

Bradykinin (BK) is a nonapeptide important for several physiological processes such as vasodilatation, increase in vascular permeability and release of inflammatory mediators. BK performs its actions by coupling to and activating the B2 receptor, a family A G-protein coupled receptor. Using a strategy which allows systematical monitoring of BK R1 and R9 residues and B2 receptor acidic residues Glu5.35(226) and Asp6.58(298), our study aims at clarifying the BK interaction profile with the B2 receptor [receptor residue numbers are normalized according to Ballesteros and Weinstein, Methods Neurosci. 25 (1995), pp. 366-428) followed by receptor sequence numbering in brackets]. N- and C-terminal analogs of BK (-A1, -G1, -K1, -E1 and BK-A9) were tested against wild type B2, Glu5.35(226)Ala and Asp6.58(298)Ala B2 mutant receptors for their affinity and capability to elicit responses by mechanical recordings of isolated mice stomach fundus, measuring intracellular calcium mobilization, and competitive fluorimetric binding assays. BK showed 2- and 15-fold decreased potency for Glu5.35(226) and Asp6.58(298) B2 mutant receptors, respectively. In B2-Glu5.35(226)Ala BK analogs showed milder reduction in evaluated parameters. On the other hand, in the B2-Asp6.58(298)Ala mutant, no N-terminal analog was able to elicit any response. However, the BK-A9 analog presented higher affinity parameters than BK in the latter mutant. These findings provide enough support for defining a novel interaction role of BK-R9 and Asp6.58(298) receptor residues.

Evidence that kinin B2 receptor expression is upregulated by endothelial overexpression of B1 receptors.

Bradykinin (BK) and des-Arg(9)-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B2 (B2R) and B1 (B1R) receptors, respectively. It was already shown that the deletion of kinin B1R or of B2R induces upregulation of the remaining receptor subtype. However studies on overexpression of B1R or B2R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B1R (TGR(Tie2B1)) exclusively in the endothelium. In another study, mice overexpressing B1R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B2R was investigated in the thoracic aorta isolated from TGR(Tie2B1) rats overexpressing the B1R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B2R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie2B1) rats could be due to the enhanced expression of B2R associated with the overexpression of the B1R.

Distinct binding mode of 125I-AngII to AT1 receptor without the Cys18-Cys274 disulfide bridge.

Previous studies on angiotensin II (AngII) AT(1) receptor function have revealed that the N-terminal residues of AngII may modulate receptor activation by binding at the receptor extracellular site. A remarkable feature of this site is an insertion of 8 amino acids in the middle of the EC-3 loop including the Cys(274) residue that supposedly makes a disulfide bond with N-terminal Cys(18). As demonstrated by assays with Del(267-275)AT(1), the role of the Cys(18)-Cys(274) disulfide bridge is to keep a conformation of the inserted residues that allows a normal binding of the AngII N-terminal residues. C18S AT(1) receptor mutant, supposedly having a dissociated disulfide bridge, but an intact residue insertion, is constitutively activated and can less efficiently bind AngII. Similar results were observed when the S-S disulfide bond was disrupted in (C18S,C274S) AT(1) receptor. The importance of the free N-terminal amino group of Asp(1) and of the Arg(2) guanidino group for the binding of AngII to C18S mutant with EC-3 loop insertion was investigated by means of assays using AngII peptide analogues bearing a single mutation of Asp(1) for Sar(1) or Arg(2) for Lys(2), as ligands. This study showed that like AngII, [Sar(1)]-AngII can bind the C18S mutant receptor with low affinity whereas [Lys(2)]-AngII binding is still more reduced. Interestingly, when (125)I-AngII instead of (3)H-AngII was used, no significant binding of this mutant was observed although wild type AT(1) receptor was shown to bind all AngII analogues.

Cross talk between kinin and angiotensin II receptors in mouse abdominal aorta.

Bradykinin (BK) is a vasorelaxant, algesic and inflammatory agent. Angiotensin II (AngII) is known to control vascular tone and promote growth, inflammation and artherogenesis. There is evidence for cross talking between BK and AngII receptors. Therefore, the effect of lack of kinin receptors was assessed in mice with genetic disruption of B(1) or B(2) and both receptors. Responsiveness of abdominal aortic rings to BK and AngII as well as the receptor gene expression of both peptides were analysed. Although no specific phenotype was displayed in the normotensive and healthy mice lacking the kinin receptors, a decreased expression level of the remaining kinin receptor mRNA was observed. AT(1) receptor mRNA level was also reduced, indicating that kinin receptors regulate AngII receptors. Downregulation of the receptors was well correlated with reduction in the reactivity of both agonists to induce contraction of aortic rings, but other signal regulations must be sought in these transgenic mice. We conclude that cross talk between kinin and AngII receptors occurs in mouse abdominal aorta and that both peptides may regulate the initiation and progression of important pathophysiological processes, such as hypertension and inflammation.