cyclosporin-h and t-butyloxycarbonyl-methionyl-leucyl-phenylalanine

In order to properly interpret receptor inhibition experiments, the precise receptor specificities of the employed antagonists are of crucial importance. Lately, a great number of agonists for various formyl peptide receptors have been identified using a selection of antagonists. However, some confusion exists as to the precise receptor specificities of many of these antagonists. We have investigated the effects of formyl peptide receptor family antagonists on the neutrophil response induced by agonists for the formyl peptide receptor (FPR) and the formyl peptide receptor like 1 (FPRL1). To determine FPR- and FPRL1-specific interactions, these antagonists should not be used at used at concentrations above 10 microM. Signaling through FPR was inhibited by low concentrations of the antagonists cyclosporin H, Boc-MLF (also termed Boc-1), and Boc-FLFLFL (also termed Boc-2), while higher concentrations also partly inhibited the signaling through FPRL1. The antagonist WRWWWW (WRW(4)) specifically inhibited the signaling through FPRL1 at low concentrations but at high concentrations also partly the signaling through FPR. Based on the difference in potency of cyclosporin H and the two Boc-peptides, we suggest using cyclosporin H as a specific inhibitor for FPR. To specifically inhibit the FPRL1 response the antagonist WRW(4) should be used.

Topics: Complement C5a; Cyclosporine; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Neutrophil Activation; Neutrophils; Oligopeptides; Receptors, Formyl Peptide; Receptors, Lipoxin; Signal Transduction; Time Factors

Cyclosporin A (CsA) binds with high affinity to cyclophilin, a critical step in the molecular mechanism of action of cyclosporins, where cyclosporin H (CsH) has extremely low affinity for cyclophilin. CsH differs from CsA by the substitution of the L-methyl valine at position 11 with it D-isomer.. We compared the effects of CsA and CsH on the release of performed (histamine) and de novo synthesized inflammatory mediators (peptide leukotriene C4) from peripheral blood basophils activated by N-formyl-methionyl-leucyl-phenylalanine (FMLP).. CsH (8 to 800 nmol/L) concentration-dependently inhibited histamine and leukotriene C4 release from purified and unpurified basophils activated by FMLP, whereas CsA (8 to 800 nmol/L) had little inhibitory effect on histamine release from basophils challenged with FMLP. Inhibition of histamine release from basophils challenged with FMLP was extremely rapid and was abolished by washing the cells (three times) before challenge. CsH (8 to 800 nmol/L) had no effect on the release of histamine caused by C5a, platelet activating factor, monocyte chemotactic activating factor, RANTES, IL-8, bryostatin 1, and phorbol myristate. Preincubation of basophils with granulocyte-macrophage colony-stimulating factor (30 and 100 pmol/L), but not IL-1 beta (30 and 100 ng/ml), concentration-dependently reversed the inhibitory effect of CsH on FMLP-induced histamine release. CsH competitively inhibited the effect of FMLP on histamine release from basophils. The dissociation constant (Kd) for the CsH-FMLP receptor complex was approximately 9 x 10(-8) mol/L, more than 10-fold lower than that (approximately equal to 1.3 x 10(-6) mol/L) of N-t-BOC-methionyl-L-leucyl-phenylalanine (BocMLP), a known formyl peptide receptor antagonist. CsH inhibited tritiated FMLP binding to human polymorphonuclear leukocytes with a concentration required to inhibit binding by 50% of approximately 5.4 x 10(-7) mol/L, whereas BocMLP was less potent with a concentration required to inhibit binding by 50% of approximately 9.1 x 10(-5) mol/L. Scatchard analysis revealed that the decreased tritiated FMLP binding caused by CsH was due to a decrease in the Bmax (0.22 +/- 0.04 nmol/L/5 x 10(6) cells vs 0.09 +/- 0.01 nmol/L/5 x 10(6) cells; p < 0.05), without a significant difference in the Kd (5.16 +/- 1.22 nmol/L vs 6.32 +/- 2.42 nmol/L; p = NS).. CsH is a potent and selective inhibitor of mediator release from basophils induced by activation of the formyl peptide receptor; it acts by interfering with agonist binding to FMLP receptors.

Topics: Basophils; Binding, Competitive; Bryostatins; Chemokine CCL2; Chemokine CCL5; Complement C5a; Cyclosporine; Histamine Release; Humans; Interleukin-1; Interleukin-8; Kinetics; Lactones; Leukotriene C4; Macrolides; N-Formylmethionine Leucyl-Phenylalanine; Oligopeptides; Platelet Activating Factor; Protein Binding; Tetradecanoylphorbol Acetate