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 B₁ receptor 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.

For further studies on the structural and conformational requirements of positions 2,3, and 7 in the bradykinin sequence, we replaced the proline residues by the more hydrophobic and conformationally restricted N-methyl-L- and D-phenylalanine (NMF). The biological activities of the new analogs were evaluated on rat uterus, guinea pig ileum, and guinea pig lung strip. Receptor binding of the analogs was studied in membranes from rat uterus and guinea pig ileum. Influence of bradykinin analogs on the release of cytokines from mouse spleen cell cultures was also measured. Bradykinin analogs were synthesized by the solid phase method, using Boc strategy on PAM or Merrifield resins. The best results in the formation of the N-methylamide bond were obtained with the coupling reagent PyBrop. In position 7 the substitution of D-Phe by D-NMF, retaining the configuration of the amino acid, converts bradykinin antagonists into agonists. The bradykinin analogs with D-NMF at position 7 gave the highest known tissue selectivity for rat uterus among agonists. [L-NMF(2)]bradykinin has moderate agonist activity on rat uterus but antagonist activity on guinea pig lung strip. It represents a new antagonist for B(2) receptors without any replacement at position 7. The same analog completely inhibits bradykinin-evoked cytokine expression by mononuclear cells.

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 degrees 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.

Synthetic analogues of the bradykinin potentiating nonapeptide BPP9alpha 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 (IPn) 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.