Tyrosine kinase activity, a determinant of Src homology domain interactions, has a prominent effect on cellular localization and catalysis by 5-lipoxygenase. Six separate inhibitors of tyrosine kinase each inhibited 5(S)-hydroxyeicosatetraenoic acid formation by HL-60 cells stimulated with calcium ionophore, in the presence or absence of exogenous arachidonic acid substrate, indicating that they modulated cellular 5-lipoxygenase activity. The tyrosine kinase inhibitors also blocked the translocation of 5-lipoxygenase from cytosol to membranes during cellular activation, consistent with their effects on its catalytic activity. These results fit a model which postulates that Src homology domain interactions are a molecular determinant of the processes which coordinate the subcellular localization and functions of 5-lipoxygenase. In addition, we demonstrate that activated leukocytes contain two molecularly distinct forms of 5-lipoxygenase: a phosphorylated form and a nonphosphorylated form. In activated HL-60 cells the pool of phosphorylated 5-lipoxygenase accumulates in the nuclear fraction, not with the membrane or cytosolic fractions. The amount of phosphorylated 5-lipoxygenase is a small fraction of the total. Overall, equilibrium reactions involving the nuclear localizing sequence, the proline-rich SH3 binding motif, and the phosphorylation state of 5-lipoxygenase may each influence its partnership with other cellular proteins and any novel functions derived from such partnerships.

Inflammatory infiltrates and endothelial cell proliferation have been appreciated in plexiform and concentric lesions, which characterize the vascular remodeling in primary pulmonary hypertension (PPH). Leukotriene production by perivascular and alveolar macrophages relies on activation of 5-lipoxygenase (5-LO), with translocation of the enzyme to the nuclear membrane, and association with the 5-LO activating protein (FLAP). Using immunohistochemical staining, we localized and semi-quantitatively estimated the abundance of 5-LO and FLAP in lungs obtained from patients with PPH, patients with interstitial lung disease (ILD), and normal control subjects. Expression of 5-LO and FLAP was prominent in alveolar macrophages in both the normal and PPH lungs; however, alveolar macrophages were more frequently clustered in the vicinity of remodeled blood vessel in PPH. Medium- and small-size pulmonary arteries in PPH showed more abundant FLAP expression than in control and ILD lungs. 5-LO expression in small arteries in PPH was more intense than in control and ILD patients. Endothelial cells in plexiform and concentric lesions in PPH expressed both 5-LO and FLAP. In situ hybridization confirmed the presence of 5-LO transcripts in macrophages and endothelial cells of the remodeled vessels in PPH. We propose that the overexpression of 5-LO and FLAP represents evidence for the participation of inflammation in the process of PPH vasculopathy or, alternatively, that the overabundance of the enzymes involved in generation of inflammatory mediators may themselves be related to vascular cell proliferation and cell growth.

Chronically elevated shear stress and inflammation are important in hypertensive lung vessel remodeling. We postulate that 5-lipoxygenase (5-LO) is a molecular determinant of these processes. Immunohistology localized the 5-LO to macrophages of normal and chronically hypoxic rat lungs and also to vascular endothelial cells in chronically hypoxic lungs only. In situ hybridization of normal and chronically hypoxic lungs demonstrated that 5-LO mRNA is expressed in macrophages. Rats hypoxic for 4 wk-developed pulmonary hypertension increased translocation of the lung 5-LO from the cytosol to the membrane fraction and increased levels of lung tissue 5-lipoxygenase-activating protein (FLAP). A FLAP ligand, 3-[1-(4-chlorobenzyl)-3-t -butyl-thio-t -isopropylindol-2-yl ]-2,2-dimethylpropanoic acid (MK-886), inhibited the acute angiotensin II and hypoxia-induced pulmonary vasoconstriction in vitro and

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