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L R Ballou

Publications and source records attributed to L R Ballou.

10 recordsLinked to original sources
PubMed1992

Interleukin-1-mediated PGE2 production and sphingomyelin metabolism. Evidence for the regulation of cyclooxygenase gene expression by sphingosine and ceramide.

We recently demonstrated that sphingosine enhances interleukin-1 beta (IL-1)-mediated prostaglandin E2 (PGE2) production in human dermal fibroblasts (Ballou, L. R., Barker, S. C., Postlethwaite, A. E., and Kang, A. H. (1990) J. Immunol. 145, 4245-4251). Because sphingosine and ceramide are interconvertable, we extended previous studies by treating cells with C2-ceramide (C2-cer), a membrane-soluble analogue of ceramide, and found that C2-cer stimulates IL-1-mediated PGE2 production to the same degree as sphingosine. In an effort to elucidate the mechanistic basis by which sphingosine and C2-cer affect PGE2 production, we examined the effect of these molecules on the expression of genes encoding cyclooxygenase (EC 1.14.99.1, Cox) and phospholipase A2 (EC 3.1.1.4, PLA2), the rate-limiting enzymes in PGE2 biosynthesis. We found that sphingosine and C2-cer treatment resulted in an 8-fold induction of Cox mRNA within 1-2 h which declined thereafter; concomitant changes in Cox protein were also observed. In contrast, expression of phospholipase A2 remained unaltered. We also found that IL-1-mediated PGE2 production was dramatically enhanced in cells treated simultaneously with sphingomyelinase which led us to directly test the effect of IL-1 on sphingomyelin turnover. IL-1 treatment induced the hydrolysis of a significant fraction of prelabeled sphingomyelin which was accompanied by increased levels of intracellular ceramide. Taken together, our results suggest that enhanced Cox expression may account for the observed enhancement of IL-1-mediated PGE2 production by sphingosine and C2-cer. These data also suggest that endogenous sphingomyelin metabolites, generated in response to IL-1, may play an important role in IL-1 signal transduction.

Base Sequence↗
PubMed1991

Sphingosine synergistically stimulates tumor necrosis factor alpha-induced prostaglandin E2 production in human fibroblasts.

Sphingosine is a biologically active derivative of sphingomyelin. It affects diverse cellular functions and its mechanism(s) of action is poorly defined. Tumor necrosis factor alpha (TNF alpha) has recently been shown to rapidly induce sphingomyelin turnover, implicating this metabolic pathway in TNF alpha signal transduction. Because TNF alpha is known to induce prostaglandin E2 (PGE2) production in human fibroblasts, we tested the effect of sphingosine on TNF alpha-induced PGE2 production. We found that sphingosine enhanced TNF alpha-induced PGE2 production by as much as 18-fold over TNF alpha alone. Sphingosine appeared to stimulate TNF alpha-induced PGE2 production independent of TNF alpha-mediated interleukin 1 (IL-1) production, because anti-IL-1 antibodies and IL-1 receptor antagonist protein (IRAP) did not inhibit TNF alpha-induced PGE2 production or the stimulatory effect of sphingosine. TNF alpha stimulated PGE2 production to the same degree in normal and protein kinase C (PKC) downregulated cells in the presence and absence of sphingosine, indicating that neither TNF alpha nor sphingosine require active PKC to elicit their respective effects. The sphingosine analogues stearylamine and stearoyl-D-sphingosine had little or no effect on TNF alpha-mediated PGE2 production, supporting a specific role for sphingosine in the activation process. Short-term (1 min) exposure of cells to sphingosine dramatically increased TNF alpha-induced PGE2 production. A potential mechanism by which sphingosine could increase TNF alpha-induced PGE2 production involves enhancement of phospholipase A2 (PLA2) and/or cyclooxygenase (Cox) activity, the rate-limiting enzymes in PGE2 production. We found that both TNF alpha and sphingosine alone enhanced these enzymatic activities, and that sphingosine additively increased the effect of TNF alpha on phospholipase A2 activity. It appears that sphingosine affects TNF alpha-induced PGE2 production via a mechanism that is independent of PKC involvement, and that sphingosine may function as an endogenous second messenger capable of modulating the responsiveness of the cell to external stimuli.

Cell Line↗
PubMed1991

Interleukin 1 stimulates phosphatidylinositol kinase activity in human fibroblasts.

IL-1 mediates multiple cellular immune and inflammatory responses, but little is known of the intracellular biochemical mechanisms involved in IL-1 actions. We studied the effects of IL-1 on phosphatidylinositol (PtdIns) metabolism and confirmed reports indicating that IL-1 does not stimulate increased PtdIns turnover; however, we observed the accumulation of PtdIns-4-phosphate (PtdInsP) in response to IL-1. Using a fibroblast membrane preparation, we were able to detect stimulated PtdInsP accumulation within 10 s of IL-1 addition. Increased PtdInsP accumulation was due to stimulated PtdIns kinase activity, not the inhibition of PtdInsP hydrolysis by phospholipase(s). PtdIns kinase activity was magnesium dependent, increased as a function of IL-1 concentration, and specifically phosphorylated the D4 position of inositol. Stimulated PtdIns kinase activity could be detected at 10(-12) M IL-1 in fibroblast membranes, a concentration within the physiological range for IL-1 action; half-maximal activity was reached at approximately 10(-10) M IL-1. Heat denaturation of IL-1 or treatment of IL-1 with anti-IL-1 antibody abrogated the IL-1 effect. These findings demonstrate the direct, IL-1-mediated, stimulation of PtdIns kinase. IL-1-stimulated PtdIns kinase activity represents an important physiological regulatory effect by IL-1 as it could control the synthesis and/or maintenance of phosphorylated derivatives of PtdIns which comprise only a very small pool of substrates for the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts.

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies.

Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin. Platelet aggregation induced by this antibody is preceded by and/or accompanied by accelerated phosphatidylinositol turnover, the generation of inositol phosphates and diacylglycerol (DAG), calcium mobilization, and protein phosphorylation. The production of inositol phosphate(s) was measurable within 15 s of either anti-p24/CD9 or thrombin addition. Within 10 s of antibody addition (10 micrograms/ml), the level of DAG was 200% over that of the control and similar to that observed with 2 units/ml thrombin (201% over that of the control). Therefore, as it appears to be true for thrombin, platelet response upon binding of anti-p24/CD9 is primarily mediated by the activation of phospholipase C. When platelets pretreated with aspirin (200 microM) and apyrase (1 mg/ml) were subsequently exposed to anti-p24/CD9, aggregation still occurred. This indicates that neither secreted ADP nor thromboxane generation is required for this aggregation response. Using indo-1 and ratio cytofluorometry, we observed that an increase in platelet cytosolic calcium is a relatively early event and occurs in either the presence or absence of calcium in the external media. Phosphorylation studies of platelet proteins showed that anti-p24/CD9 binding to platelets caused increased phosphorylation of four proteins with apparent molecular masses of 50,000, 47,000, 36,000, and 20,000 daltons. These studies suggest that platelet activation mediated by the surface protein p24/CD9 is mainly through the stimulation of a phospholipase C, the activation of which is responsible for the generation of second messengers inositol trisphosphate and DAG.

Adenylate Cyclase Toxin↗
PubMed1988

Demonstration of endogenous inhibitors for the formation of 14,15-oxido-5,8,11-eicosatrienoic acid from phosphatidylinositol in bovine lung extract.

Bovine lung extract contained an enzyme or enzyme system that catalyzed the formation of 14,15-oxido-5,8,11-eicosatrienoic acid from phosphatidylinositol. The enzyme activity increased markedly during the course of purification, apparently due to the removal of two endogenous inhibitors from the enzyme. Using membranes with known molecular weight cut-offs, we estimated the Mr of Inhibitor 1 to be between 10,000 and 100,000 and Inhibitor 2 less than 1,000. Inhibitor 1 appeared to be partially inactivated by trypsin and was heat labile, whereas Inhibitor 2 was resistant to trypsin and was heat resistant. Both inhibitors were hydrophilic.

8,11,14-Eicosatrienoic Acid↗
PubMed1988

Purification and initial characterization of a ferret Pneumocystis carinii surface antigen.

The purification and initial characterization of a ferret Pneumocystis carinii surface glycoprotein is described. Previous studies have demonstrated that passive administration of monoclonal antibody recognizing this glycoprotein reduces the severity of P. carinii pneumonitis in animal models of infection. This acidic glycoprotein (approximate isoelectric point, 5.0-5.7) contains both mannose (and/or glucose) and N-acetyl-glucosamine residues. The cross-reactive surface antigen on P. carinii of human origin is also a mannose- (and/or glucose-) containing glycoprotein. Initial biochemical characterization of these surface molecules should aid in understanding the immunobiology of this organism and in developing reliable diagnostic assays for P. carinii pneumonitis.

Animals↗
PubMed1987

Formation of cis-14,15-oxido-5,8,11-icosatrienoic acid from phosphatidylinositol in human platelets.

Human platelets contain a soluble enzyme or enzyme system that catalyzes the formation of lysophosphatidylinositol and a compound more polar than arachidonic acid (compound A) from 2-arachidonoyl sn-phosphatidylinositol. Arachidonic acid, 2-arachidonoyl sn-phosphatidylcholine, or 2-arachidonoyl sn-phosphatidylethanolamine did not serve as substrate for the production of compound A. The reaction required Ca2+ and was not affected by aspirin, indomethacin, or mepacrin. Enzyme activity was not enhanced in the presence of NADPH, but it was inhibited greater than 90% by CO or N2; inhibition was readily reversible by exposure to atmospheric air. Neither metapyrone (SKF 525A) nor cyanide, inhibitors of cytochrome P-450, inhibited compound A formation, suggesting that a cytochrome P-450 system was not involved. Thrombin stimulated the formation of compound A in whole platelets; ionophore A23187 did so much less effectively; and other agonists such as collagen, ADP, and epinephrine were ineffective. Compound A exhibited a fragmentation pattern by GC/MS identical to that of authentic cis-14,15-oxido-5,8,11-icosatrienoic acid. Collectively, these data indicate that human platelets may contain an enzyme system that catalyzes the epoxidation of the arachidonic acid moiety of phosphatidylinositol and its hydrolysis to liberate cis-14,15-oxido-5,8,11-icosatrienoic acid.

8,11,14-Eicosatrienoic Acid↗