Human Neutrophil Leukotriene B4 Research Articles

(E)-3-[6-[[(2,6-dichlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2- pyridinyl]-2-propenoic acid: a high-affinity leukotriene B4 receptor antagonist with oral antiinflammatory activity.

An extensive structure-activity study based around the high-affinity leukotriene B4 (LTB4) receptor antagonist SB 201146 (1) led to the identification of (E)-3-[6-[[(2,6-dichlorophenyl)-thio]methyl]-3-(2-phenylethoxy)-2- pyridinyl]-2-propenoic acid (3). This compound displays high affinity for the human neutrophil LTB4 receptor (Ki = 0.78 nM), blocks LTB4-induced Ca2+ mobilization with an IC50 of 6.6 +/- 1.5 nM, and demonstrates potent oral and topical antiinflammatory activity in a murine model of dermal inflammation.

Chemotactic peptide down-regulation of calcium mobilization induced by platelet-activating factor and by leukotriene B4 in human neutrophils is uncovered by protein phosphatase inhibitors

When human neutrophils were incubated in the presence of the protein phosphatase inhibitors calyculin A or okadaic acid, the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) produced a sustained (> 5 min) inhibition of the Ca2+ mobilization from intracellular stores induced by platelet-activating factor (PAF) or by leukotriene B4 (LTB4). No effect on Ca2+ mobilization by PAF or LTB4 was observed 2 min after the addition of fMLP alone or only in the presence of phosphatase inhibitors, but a similar inhibition was produced by high (> 50 nM) concentrations of phorbol 12,13-dibutyrate (PDB). However, inhibition by PDB was sensitive to the protein kinase C (PKC) inhibitors staurosporin and Ro 31-8220, while inhibition by fMLP and calyculin A was not. These results suggest that fMLP induces a transient phosphorylation not mediated by PKC which interferes at some point with the transduction pathway leading from the plasma membrane receptors for PAF and LTB4 to the release of Ca2+ from the stores. Protein phosphatases 1 and/or 2A revert the inhibition effected by fMLP within less than 2 min. PAF and LTB4 were also able to activate this mechanism to a smaller extent. Phosphatase inhibitors also delayed by 1-2 s the start of agonist-induced rises in [Ca2+]i, and this delay was further increased by previous addition of any other agonist. Finally, given that both phosphatase inhibitors and low concentrations of PDB (2-10 nM) strongly inhibit Ca2+ entry, we conclude that phosphorylation down-regulates both agonist-induced Ca2+ entry and Ca2+ mobilization, but with different potency.

Cytoplasmic pH change induced by leukotriene B4 in human neutrophils

Leukotriene B 4 induced a biphasic change in the cytoplasmic pH of human neutrophils: an initial rapid acidification followed by an alkalinization. The acidification was slightly reduced by the removal of extracellular Ca 2+, but the subsequent alkalinization was not. The leukotriene B 4-induced alkalinization was dependent on extracellular Na + and pH, and was inhibited by amiloride and its more potent analogue, 5-( N,N-hexamethylene)amiloride. These characteristics indicate that the cytoplasmic alkalinization is mediated by the Na +-H + exchange. Oxidation products of leukotriene B 4, 20-hydroxyleukotriene B 4, 20-carboxyleukotriene B 4, and (5 S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) also stimulated the Na +-H + exchange, but higher concentrations were required. Treatment of the cells with pertussis toxin inhibited both phases of the leukotriene B 4 induced pH i change, while cholera toxin did not affect the pH i change. The alkalinization induced by leukotriene B 4 was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, but was not inhibited by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide which has a less inhibitory effect on protein kinase C. Acidification was not affected by the drugs. These findings suggest that a GTP-binding protein sensitive to pertussis toxin and protein kinase C are involved in the activation of the Na +-H + exchange stimulated by leukotriene B 4.