calcimycin and geldanamycin

In resident mouse peritoneal macrophages, group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) mediates arachidonic acid (AA) release and eicosanoid production in response to diverse agonists such as A23187, phorbol myristate acetate, zymosan, and the enterotoxin, okadaic acid (OA). cPLA(2)alpha is regulated by phosphorylation and by calcium that binds to the C2 domain and induces translocation from the cytosol to membranes. In contrast, OA activates cPLA(2)alpha-induced AA release and translocation to the Golgi in macrophages without an apparent increase in calcium. Inhibitors of heat shock protein 90 (hsp90), geldanamycin, and herbimycin blocked AA release in response to OA but not to A23187, PMA, or zymosan. OA, but not the other agonists, induced activation of a cytosolic serine/threonine 54-kDa kinase (p54), which phosphorylated cPLA(2)alpha in in-gel kinase assays and was associated with cPLA(2)alpha in immunoprecipitates. Activation of the p54 kinase was inhibited by geldanamycin. The kinase coimmunoprecipitated with hsp90 in unstimulated macrophages, and OA induced its loss from hsp90, concomitant with its association with cPLA(2)alpha. The results demonstrate a role for hsp90 in regulating cPLA(2)alpha-mediated AA release that involves association of a p54 kinase with cPLA(2)alpha upon OA stimulation.

Topics: Animals; Arachidonic Acid; Benzoquinones; Calcimycin; Calcium; Carcinogens; Cytosol; Enzyme Activation; Enzyme Inhibitors; Female; Golgi Apparatus; Green Fluorescent Proteins; Group IV Phospholipases A2; HSP90 Heat-Shock Proteins; Immunoblotting; Immunoprecipitation; Ionophores; Lactams, Macrocyclic; Macrophages, Peritoneal; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinase 10; NIH 3T3 Cells; Okadaic Acid; Phosphoamino Acids; Phosphorylation; Protein Transport; Rifabutin; Signal Transduction; Tetradecanoylphorbol Acetate; Zymosan

The objective of this study was to investigate the effects of heat shock (HS) treatment and geldanamycin (GA) on the release of arachidonic acid (AA) from human peripheral blood mononuclear cells (PBMC), monocytes and lymphocytes. Mononuclear cells prepared from blood of healthy subjects were preincubated with (3)H-AA. The release of (3)H-AA incorporated into the membrane was studied after pretreatment of cells by HS (43 degrees C, 1 h) and GA. The activation of AA producing enzymes was achieved by the addition of phorbol 12-myristate 13-acetate (PMA) or by the combination of PMA+calcium ionophore A-23187. Treatment of cells by HS inhibited the release of AA. Furthermore, the release of AA by PBMC was dose dependently inhibited by GA. The combination of treatments by HS and GA augmented the inhibition of AA release. The HS response involves a diminished release of AA from PBMC. The inhibitory effect of GA on the AA release is a new element in the antiinflammatory pharmacological ability of this drug.

Topics: Arachidonic Acid; Benzoquinones; Calcimycin; Cells, Cultured; Enzyme Inhibitors; Heat-Shock Response; Hot Temperature; Humans; Ionophores; Lactams, Macrocyclic; Leukocytes, Mononuclear; Quinones; Tetradecanoylphorbol Acetate