Expression cloning of a human B1 bradykinin receptor.

A cDNA clone encoding a human B1 bradykinin receptor was isolated from a human embryonic lung fibroblast cDNA library by expression cloning. The photoprotein aequorin was utilized as an indicator of the ability of the B1 receptor agonist [des-Arg10]kallidin to mediate Ca2+ mobilization in Xenopus laevis oocytes injected with RNA. A clone was isolated with a 1307-nucleotide insert which contains an open reading frame encoding a 353-amino acid protein with the characteristics of a G-protein-coupled receptor. The amino acid sequence of the B1 bradykinin receptor is 36% identical to the amino acid sequence of the B2 bradykinin receptor. The cloned B1 bradykinin receptor expressed in mammalian cells exhibits high affinity binding for 3H-labeled [des-Arg10]kallidin and low affinity for bradykinin. The B1 receptor antagonist [des-Arg10,Leu9]kallidin effectively displaces 3H-labeled [des-Arg10]kallidin from the cloned receptor, whereas the B2 receptor antagonist Hoe-140 (D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, where Thi is L-[3-(2-thienyl)alanyl], Tic is D-(1,2,3,4-tetrahydroisoquinolin-3-yl-carbonyl), and Oic is L-[(3aS, 7aS)-octahydroindol-2-yl-carbonyl]) does not. Therefore, the expressed receptor has the pharmacological characteristics of the B1 receptor subtype. The availability of both the cloned human B1 and B2 bradykinin receptors should allow the elucidation of the relative contributions of these two receptor subtypes in acute and chronic inflammatory processes.

Bradykinin B1 receptors in rabbit aorta smooth muscle cells in culture.

Kinin B1 receptors on rabbit aorta smooth muscle cells in culture were investigated. [3H]Des-Arg10-kallidin labeled a single site in cells at early passage with an equilibrium dissociation constant of 258 pM and a maximal binding density of approximately 680 sites/cell. Treatment of the same cells for 18 h with epidermal growth factor increased the binding density over 6-fold without affecting the ligand's affinity. At latter passages, the density of binding sites was found to increase and the growth factor had a much less pronounced effect. The rank order of potencies for agonist inhibition of binding (des-Arg10-kallidin > des-Arg9-BK = kallidin > bradykinin) was consistent with the specific labeling of a B1 receptor. Also, [3H]des-Arg10-kallidin binding was potently inhibited by the B1 receptor antagonist des-Arg9[Leu8]bradykinin but not by the B2 receptor antagonist Hoe 140. The agonists were found to stimulate phosphoinositide hydrolysis in the smooth muscle cells with an order of potencies that reflected their binding assay activities. Des-Arg9[Leu8] BK blocked the des-Arg10-kallidin response with a potency consistent with its known B1 receptor activity while Hoe 140 was inactive. These results demonstrate the presence of inducible B1 receptors on rabbit aorta smooth muscle cells in culture that couple to phospholipase C activation. These cells should be useful in future studies of the mechanisms and factors involved in the regulation of expression of the B1 receptor.

[125I]PIP HOE 140, a high affinity radioligand for bradykinin B2 receptors.

A high affinity radioligand for bradykinin B2 receptors was prepared by coupling an activated ester of [125I]4-iodobenzoic acid to the amino terminus nitrogen of the potent B2 antagonist HOE 140. The ligand, [125I]para-iodophenyl HOE 140 ([125I]PIP HOE 140), bound to a homogeneous set of sites in guinea pig ileal membranes with an equilibrium dissociation constant of 15 pM and a maximal binding density of 193 fmole/mg protein. Competition studies with a number of BK-related peptides indicated that the ligand specifically labeled B2 receptors in the preparation. The results suggest that [125I]PIP HOE 140 will be a useful tool for future studies of B2 receptors.

Evaluation of a rapid latex slide agglutination test for herpes simplex virus as a specimen screen and culture identification method.

A total of 449 clinical specimens and 199 culture fluids were tested using the Virogen Herpes Slide Test (Wampole Laboratories, Div. Carter-Wallace, Inc., Cranbury, N.J.), a rapid latex agglutination procedure. The results were compared with those obtained with isolation of herpes simplex virus in cell culture followed by identification using immunoperoxidase or fluorescent reagents. The sensitivity, specificity, and positive and negative predictive values of the direct test were 49.7, 93.4, 96.0, and 37.1%, respectively. The sensitivity and specificity of the latex agglutination test for culture confirmation were 75.9 and 100%, respectively.