The formation of constitutive transport vesicles involves the association of non-clathrin coat proteins to transport organelles. Here we report that IgE receptors and protein kinase C (PKC) regulate the GTP-dependent binding of the two coat proteins ADP-ribosylation factor (ARF) and beta-COP to Golgi membranes in rat basophilic leukaemia cells. Activation of IgE receptors and PKC prevented the ARF and beta-COP dissociation from Golgi membranes that occurs in permeabilized cells in the absence of GTP and potentiated the association-promoting effects of GTP and the G protein activator fluoroaluminate. In contrast, PKC downregulation and PKC inhibition abolished the activity of GTP and fluoroaluminae in promoting ARF binding to the Golgi complex. Studies of ARF binding to isolated Golgi membranes gave similar results. These findings suggest that coat assembly on Golgi membranes, and thus possibly constitutive secretory traffic, is modulated by membrane receptors and second messengers.

Brefeldin A (BFA) is a fungal metabolite that exerts profound and general Inhibitor actions on membrane transport. At least some of the BFA effects are due to inhibition of the GDP-GTP exchange on the ADP-ribosylation factor (ARF) catalyzed by membrane protein(s). ARF activation is likely to be a key event The fungal toxin brefeldin A (BFA) has been widely used to analyze the mechanisms of membrane transport. The effects of BFA include the disappearance of non-clathrin-coated buds and transport vesicles, the inhibition of constitutive secretion, and a series of changes in'shape, location, and function of the organelles of the exocytic and endocytic pathways (1-4). These changes are preceded and probably, at least in part, caused by the release ofa set ofproteins from the Golgi complex including two major non-clathrin coat proteins, the ADP-ribosylation factor (ARF, a small Ras-like GTPase) and 8COP, a component of the cytosolic protein complex "coatomer" (5-7). Moreover, BFA inhibits the GDP-GTP exchage on ARF catalyzed by a Gojgi protein and the binding of ARF to Golgi membranes, suggesting that the components involved in ARF association to transport organelles may be the primary targets of BFA and a key site of regulation of vesicular transport pathways (8, 9).ARF, in addition to being involved in membrane transport, has long been known as a cofactor in the ADP-ribosylation of the a subunit of the GTP-binding protein Gs by cholera toxin and to be able to interact directly with cholera toxin (10-12). ADP-ribosylation is a posttranslational modification of proteins produced by the transfer of ADP-ribose from NAD to specific amino acid residues, which can be catalyzed by both bacterial toxins and eukaryotic enzymes (13). Analogous to its role in the activation of the exogenous ADP-ribosyltransferase cholera toxin, one of the physiological functions of ARF may be to activate an endogenous cellular mono(ADPribosyl)transferase (11). Indeed, a family of brain mono(ADP-ribosyl)transferases has been reported to be sensitive to ARF (14). These considerations prompted us to determine whether BFA, possibly by perturbing ARF binding, might affect cellular ADP-ribosylations. Here we report that BFA markedly stimulates the ADP-ribosylation of two cytosolic proteins of38 and 50 kDa (p38 and p50). p38 appears to be identical with an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme and a multifunctional protein that has been implicated in several cellular processes (15)(16)(17)(18)(19)(20)(21)(22) 1114The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Supplementation with pharmacological doses of vitamin E has no detectable effects on lipid peroxidation and thromboxane biosynthesis in vivo in healthy subjects with a mild degree of oxidant stress. These findings are consistent with the hypothesis that the basal rate of lipid peroxidation is a major determinant of the response to vitamin E supplementation and have implications for the use of vitamin E in healthy subjects as well as for the design and interpretation of clinical trials of antioxidant intervention.

1 The isoprostane 8-epi-prostaglandin (PG)F2a is produced by free radical-catalyzed peroxidation of arachidonic acid. It may also be formed as a minor product of the cyclo-oxygenase activity of platelet PGH synthase (PGHS)-l. We investigated 8-epi-PGF2. production associated with induction of the human monocyte PGHS-2 and its pharmacological modulation. 2 Heparinized whole blood samples were drawn from healthy volunteers, 48 h following oral dosing with aspirin 300 mg to suppress platelet cyclo-oxygenase activity. One ml aliquots were incubated with lipopolysaccharide (LPS: 0.1-50 ug ml-') for 0-24 h at 37'C. PGE2 and 8-epi-PGF2a were measured in separated plasma by radioimmunoassay and enzyme immunoassay techniques. 3 Levels of both eicosanoids were undetectable (i.e. <60 pg ml-') at time 0. LPS induced the formation of PGE2 and 8-epi-PGF2, in a time-and concentration-dependent fashion, coincident with the induction of PGHS-2 detected by Western blot analysis of monocyte lysates. After 24 h at 10 mig ml-' LPS, immunoreactive PGE2 and 8-epi-PGF2, averaged 10,480+4,643 and 295+ 140 pg ml-' (mean + s.d., n = 6), respectively. 4 Dexamethasone-and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-l-indanone (L-745,337), a selective inhibitor of the cyclo-oxygenase activity of PGHS-2, reduced PGE2 and 8-epi-PGF2. production in response to LPS.5 Isolated monocytes produced PGE2 and 8-epi-PGF2, in response to LPS (10 Mg ml-') in a timedependent fashion. Monocyte PGE2 and 8-epi-PGF2a production was largely prevented by dexamethasone (2 gM) and cycloheximide (10 Mg ml-') in association with suppression of PGHS-2 but not of PGHS-1 expression. 6 We conclude that the induction of PGHS-2 in human monocytes is associated with cyclo-oxygenasedependent generation of the vasoconstrictor and platelet-agonist 8-epi-PGF2,.

1 We have evaluated the selectivity of ketoprofen and two novel nonsteroidal anti-inflammatory drugs, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulphonamide and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indanone (L-745,337), in inhibiting the cyclo-oxygenase activity of prostaglandin endoperoxide synthase-2 (PGHS-2) vs PGHS-1 in human blood monocytes and platelets, respectively. 2 Heparinized whole blood samples were drawn from healthy volunteers pretreated with aspirin, 300 mg 48 h before sampling, to suppress the activity of platelet PGHS-1 and incubated at 37°C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 yig ml-'). Immunoreactive PGE2 levels were measured in plasma by a specific radioimmunoassay as an index of the cyclo-oxygenase activity of LPS-induced monocyte PGHS-2. 3 The effects of the same inhibitors on platelet PGHS-1 activity were assessed by allowing whole blood samples, drawn from the same subjects in aspirin-free periods, to clot at 37°C for 1 h in the presence of the compounds and measuring immunoreactive thromboxane B2 (TXB2) levels in serum by a specific radioimmunoassay.4 Under these experimental conditions, ketoprofen enantioselectively inhibited the cyclo-oxygenase activity of both PGHS-1 and PGHS-2 with equal potency (IC50 ratio: approx. 0.5 for both enantiomers), while L-745,337 and NS-398 achieved selective inhibition of monocyte PGHS-2 (IC50 ratio: > 150). L-745,337 and NS-398 did not affect LPS-induced monocyte PGHS-2 biosynthesis to any detectable extent. 5 We conclude that L-745,337 and NS-398 are selective inhibitors of the cyclo-oxygenase activity of human monocyte PGHS-2. These compounds may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease.