High affinity bradykinin binding to human inflammatory cells

Abstract

Specific direct bradykinin (BK) binding and competitive inhibition was detected in human neutrophil and peripheral blood mononudear celt (PBMC) detergent solubilized extracts and purified plasma membranes using in vitro radioreceptor ligand binding. Scatchard analyses of [~2sI]-BK binding revealed an equilibrium dissociation constant (K a ) of 2.9x10 ~' M for neutrophils and 5.6x10 ~ M for PBMC using [des-argg]-BK a B1 agonist; 2.6x10 ~a M for neutrophils, 6.2x10 ~' M for PBMC with BK a B2 agonist; 5.4x10 ~ M for PBMC using Lys-BK a B2 agonist. The number of binding sites (B) was calculated to be 0.113 fM/p.g protein (720 receptors per cell) for neutrophils and 0.200 fM/p.g protein (1289 receptors per ceil) for PBMC with the B1 agonist while with the B2 agonists the values were 0.128 fM/~g protein (818 receptors per cell) for neutrophils and 0.157 fM/~tg protein (1005 receptors per cell) for PBMC with BK, and 0.293 fM/~tg protein (1870 receptors per cell) with Lys-BK for PBMC.In a competitive binding inhibition assay using neutrophil and PBMC glycerol purified plasma membranes, high affinity binding in the nanomolar range was detected to Lys-BK and BK but with [des-argg]-BK a 10-100 fold lower order affinity was observed this being indicative of pharmacologically defined B2 characteristics.

Comparison of kinin B1 and B2 receptor expression in neutrophils of asthmatic and non-asthmatic subjects

“…Neutrophils therefore have the capacity for localized formation of biologically active kinin peptides that could produce autocrine and paracrine effects by binding to their respective receptors [20,21]. However until recently, evidence that kinin receptors are present on human neutrophils was restricted to a radioligand binding study demonstrating binding sites for kinin B 1 and B 2 agonists in purified plasma membrane preparations [22]. The recently published study of Ehrenfeld et al [21] provided more definitive evidence for the presence of kinin B 1 receptor mRNA and protein in peripheral blood neutrophils of healthy subjects.…”

Migratory Responses of Polymorphonuclear Leukocytes to Kinin Peptides

“…1). The relatively low migratory response of PMN to bradykinin could depend on the low number of receptors on these cells (818 per cell) [12].…”

“…The kinin B 2 receptor is constitutively present, and the B 1 receptor is not widely expressed in normal tissue but is highly inducible during a host inflammatory response [9]. Bradykinin receptors have been localized on the plasma membrane of PMN using antibodies and indirect labeling techniques [10,11]; a specific molecular bradykinin receptor variant on PMN has been characterized by ligand binding using bradykinin B 1 and B 2 agonists [12,13]. In 2001, the expression of the kinin B 1 receptor in murine neutrophils could be evidenced by means of the RT-PCR [14].…”

Stimulated human neutrophils form biologically active kinin peptides from high and low molecular weight kininogens

“…Ligand blot assays suggest that tissue prokallikrein may attach to the cell membrane also by interacting with H-kininogen [18]. In addition, binding experiments using purified cell membranes have shown that neutrophils display kinin-binding sites that have been characterized as B1 and B2 kinin receptor subtypes [19].…”