Inge Paegelow

Kinins and kinin receptors: importance for the activation of leukocytes

In this article, we analyzed the role of kinins and kinin receptors with respect to the activation of leukocytes. In these cells, the biological effects of kinin peptides are mediated by kinin receptor subtypes B1, B2, or both, depending on species and cell type. In contrast to the other leukocytes, neutrophils contain the complete system for the synthesis and release of bioactive kinins. Consequently, very high concentrations of these peptides can be reached in the close neighborhood of the kinin receptors, in particular at the site of inflammation. Kinins are responsible for many effects in leukocytes including the release of other inflammatory mediators, such as cytokines, prostaglandins, leukotrienes, and reactive oxygen species. Obviously, the potency of kinins to stimulate leukocytes is dependent on the differentiation and especially on the activation stage of these cells. An upregulation of kinin receptors on neutrophils and macrophages appears to be involved in increasing the sensitivity of these cells to kinins at the site of inflammation.

Migratory Responses of Polymorphonuclear Leukocytes to Kinin Peptides

The present study examines the influence of kinins on the migratory capacity of human polymorphonuclear leukocytes under in vitro conditions using the Boyden chamber technique. By means of checkerboard analysis the migration of neutrophils induced by bradykinin could be characterized as true chemotaxis. The stimulation of human neutrophils with bradykinin, with the nonpeptide B₂ receptor agonist FR190997 as well as with des-Arg⁹-bradykinin and des-Arg¹⁰-kallidin results in a concentration-dependent migration. Pretreatment of the neutrophils with the B₂ receptor antagonist HOE-140 (icatibant) inhibited the bradykinin-induced migration but not that induced by B₁ receptor agonists, whereas the B₁receptor antagonist des-Arg¹⁰HOE-140 abolished the migration elicited by des-Arg⁹-bradykinin or des-Arg¹⁰-kallidin but not that evoked by bradykinin. Pretreatment of the neutrophils with the leukotriene B₄ (LTB₄) antagonist ZK158252 inhibited the LTB₄-induced chemotaxis as well as the chemotaxis produced by bradykinin and des-Arg¹⁰-kallidin. An involvement of interleukin-1β and of the chemokine IL-8 in the bradykinin-induced migration in vitro could be excluded during the migration time of the neutrophils. In conclusion, the present study provides pharmacological evidence showing that B₁ and B₂ kinin receptors are involved in the migration of human neutrophils in vitro, that LTB₄ participates in the downstream pathway and that the B₁ kinin receptor seems to be expressed already under physiological conditions.

RELEASE OF CYTOKINES FROM ISOLATED LUNG STRIPS BY BRADYKININ

Bradykinin as an inflammatory mediator was assayed for its ability to release cytokines from isolated lung tissue derived from guinea pigs, mice and in some cases from patients. Bradykinin elicited in concentrations, which were able to induce a contraction of isolated lung strips, a secretion of different cytokines from the tissue into organ baths as well as from lung tissue incubated in petri dishes (4h, 37°C). Using enzyme immuno assays and the tanned erythrocyte electrophoretic mobility (TEEM)-test in combination with monoclonal antibodies the cytokines could be identified preferably as interleukin(IL)-1, IL-2, sIL-2R and IL-6. Tyrode solution as a control and carbachol in a concentration causing also a contraction were not able to release cytokines in a significant amount. The bradykinin B2 receptor antagonist icatebant (HOE 140) inhibited the bradykinin-induced IL-2 and IL-6 release. The results show that bradykinin can elicit the secretion of the cytokine cascade via a receptor-mediated process.

Potentiation of bradykinin actions by analogues of the bradykinin potentiating nonapeptide BPP9α

Abstract Synthetic analogues of the bradykinin potentiating nonapeptide BPP9α indicate significantly different structural requirements for potentiation of the bradykinin (BK)-induced smooth muscle contraction (GPI) and the inhibition of isolated somatic angiotensin I-converting enzyme (ACE). The results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation. Our results indicate a stimulation of inositol phosphates (IP n ) formation independently from the B2 receptor. Furthermore, the results with La3+ support the role of extracellular Ca2+ and its influx through corresponding channels. The missing effect of calyculin on the GPI disproves the role of phosphatases in the potentiating action. These experimental studies should not only contribute to a better understanding of the potentiating mechanisms but also incorporate a shift in the research towards the immune system, in particular towards the immunocompetent polymorphonuclear leukocytes. The chemotaxis of these cells can be potentiated most likely by exclusive inhibition of the enzymatic degradation of bradykinin. Thus the obtained results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor.

Effects of bradykinin and bradykinin analogues on spleen cells of mice

The present study was undertaken to examine the effects of bradykinin and selected bradykinin analogues on mononuclear cells derived from mouse spleen. Bradykinin as well as des-Arg9-bradykinin, a bradykinin B1 receptor agonist, were able to induce the release of so-called charge-changing lymphokines, which could be identified as interleukin-1, interleukin-6, interleukin-2 and as interleukin-2 receptor. The cytokine release evoked by bradykinin and all analogues showed a bell-shaped dose dependence in a range of 10(-8) M to 10(-6) M and could be inhibited by the specific bradykinin receptor antagonist, D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE140), and by bradykinin analogues with N-methyl-phenylalanine at position 2 in concentrations as low as 10(-12) M and 10(-13) M, respectively. Obviously the N-terminus of bradykinin seems to be responsible for the interaction with the mononuclear cells concerning all peptides investigated.

Bradykinin receptors and signal transduction pathways in peritoneal guinea pig macrophages

The presence of a bradykinin receptor on guinea pig peritoneal macrophages was evidenced by binding studies and by the effect of bradykinin on activation of the phospholipase C and the increase in intracellular calcium concentration ([Ca2+]i). Binding studies demonstrated a specific, saturable binding for [3H]bradykinin inhibited by the bradykinin B2 (HOE 140) but not bradykinin B1 (des-Arg9[Leu8]bradykinin) receptor antagonist. Scatchard analysis revealed a single class of B2 bradykinin binding sites with a binding affinity (kd) of 0.8 nM and a receptor concentration (Bmax) of 35 fmol/5 × 106 cells, representing approximately 4000 bradykinin receptors per cell. Kinetic studies confirmed the presence of this single binding site by the determination of similar binding affinity. Activation of peritoneal macrophages by bradykinin resulted in a time-and dose-dependent release of inositol phosphates determined by anion exchang chromatography and intracellular calcium analyzed using fura-2/AM. The increase in [Ca2+]i induced by bradykinin was blocked by the specific bradykinin B2 receptor antagonist HOE 140 but not by the bradykinin B1 receptor antagonist des-Arg9[ These studies provide novel information regarding the nature of kinin receptors on guinea pig peritoneal macrophages and their signal transduction pathways.

Structure activity relationships for bradykinin antagonists on the inhibition of cytokine release and the release of histamine.

Highly potent bradykinin antagonists were found to inhibit bradykinin-induced release of cytokines but to stimulate histamine release. Both actions show structural requirements completely different from those for bradykinin B1 and B2 receptors, indicating that the release of some cytokines from spleen mononuclear cells and of histamine from rat mast cells is not mediated by these receptors. Most potent bradykinin antagonists release histamine at lower concentrations than does bradykinin itself. Dimers of bradykinin antagonists are the most potent compounds for histamine release. In contrast to enhanced histamine release, potent inhibition of cytokine release enhances the applicability of these compounds as anti-inflammatory drugs. Many of the peptides designed for high B2-receptor antagonism were found to be compared by their concentrations far more potent for inhibition of cytokine release than for smooth muscle contraction. Thus, for some antagonists inhibition of cytokine release was detected at concentrations as low as 10(-15) M. The rational design of peptide and nonpeptide bradykinin antagonists for therapeutic use requires not only knowledge about the potency but also knowledge about the structure-activity relationships of such important side effects as cytokine and histamine release.

Pharmacological and molecular actions of the bradykinin B2 receptor antagonist, Hoe 140, in the rat uterus

The type of antagonism exhibited by the potent bradykinin B2 receptor antagonist, Hoe 140, on the rat uterus was investigated using various approaches. In the isolated rat uterus the concentration-response curve of bradykinin was shifted to the right and the maximum effect was reduced after pretreatment with Hoe 140 for 5 min. Shorter times of antagonist pretreatment failed to decrease the maximum effect of bradykinin. In the [3H]BK binding assay Hoe 140 bound with pM affinity to a single site and did not discriminate multiple bradykinin receptors. Studying the bradykinin-induced G protein activation we could verify that Hoe 140 at subnanomolar concentrations selectively antagonized the low-affinity BK receptor in the rat myometrium. At nM concentrations Hoe 140 itself was able to stimulate G proteins. The results suggest that in the rat uterus, differently from guinea pig ileum, Hoe 140 possibly acts as a mixed competitive as well as functional antagonist.

Bradykinin action in the rat duodenum through the cyclic AMP system

Activators of the adenylate cyclase or inhibitors of the cAMP-phosphodiesterase, respectively, potentiate the bradykinin-induced relaxation of the rat duodenum, whereas imidazole as a stimulator of the cAMP-phosphodiesterase reduces the relaxation. The experiments indicate a linkage between the adenylate cyclase system with the biological action of bradykinin on the rat duodenum. In contrast, no similar effect has been observed on the rat uterus.

Degradation of bradykinin by peritoneal and alveolar macrophages of the guinea pig.

Peptidase inhibitors and identification of the peptide fragments were used for the characterization of the bradykinin metabolism by alveolar and peritoneal macrophages. Both cell types show differences in the rate of inactivation and in the quantity of the metabolites generated. BK(1-5), BK(1-8), and BK(1-7) are the predominant direct metabolites. Metalloendopeptidase 24.15, carboxypeptidase M, and an unidentified peptidase are responsible for their formation. Angiotensin-converting enzyme and neutral endopeptidase 24.11 do not play a crucial role in the degradation of bradykinin by macrophages. In the bronchoalveolar space, other cells than the macrophages are more important to the breakdown of this peptide.