Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations. (17/4381)

It has been established that the fusion of both biological membranes and phospholipid bilayers can be modulated by altering their lipid composition (Chernomordik et al., 1995 .J. Membr. Biol. 146:3). In particular, when added exogenously between apposing membranes, monomyristoylphosphatidylcholine (MMPC) inhibits membrane fusion, whereas glycerol monoleate (GMO), oleic acid (OA), and arachidonic acid (AA) promote fusion. This present study uses x-ray diffraction to investigate the effects of MMPC, GMO, OA, and AA on the bending and stability of lipid bilayers when bilayers are forced together with applied osmotic pressure. The addition of 10 and 30 mol% MMPC to egg phosphatidylcholine (EPC) bilayers maintains the bilayer structure, even when the interbilayer fluid spacing is reduced to approximately 3 A, and increases the repulsive pressure between bilayers so that the fluid spacing in excess water increases by 5 and 15 A, respectively. Thus MMPC increases the undulation pressure, implying that the addition of MMPC promotes out-of-plane bending and decreases the adhesion energy between bilayers. In contrast, the addition of GMO has minor effects on the undulation pressure; 10 and 50 mol% GMO increase the fluid spacing of EPC in excess water by 0 and 2 A, respectively. However, x-ray diffraction indicates that, at small interbilayer separations, GMO, OA, or AA converts the bilayer to a structure containing hexagonally packed scattering units approximately 50 A in diameter. Thus GMO, OA, or AA destabilizes bilayer structure as apposing bilayers are brought into contact, which could contribute to their role in promoting membrane fusion.  (+info)

Differential potentiation of arachidonic acid release by rat alpha2 adrenergic receptor subtypes. (18/4381)

CHO transfectants expressing the three subtypes of rat alpha2 adrenergic receptors (alpha2AR): alpha2D, alpha2B, alpha2C were studied to compare the transduction pathways leading to the receptor-mediated stimulation of phospholipase A2 (PLA2) in the corresponding cell lines CHO-2D, CHO-2B, CHO-2C. The alpha2B subtype stimulated the arachidonic acid (AA) release after incubation of the cells with 1 microM epinephrine, whereas alpha2D and alpha2C gave no stimulation. Calcium ionophore A23187 (1 microM) increased the release by a factor of 2-4 in the three strains. When cells were incubated with both epinephrine and Ca2+ ionophore, the AA release differed greatly between cell lines with strong potentiation in CHO-2B (2-3 times greater than Ca2+ ionophore alone), moderate potentiation in CHO-2D, and no potentiation in CHO-2C. The three cell lines each inhibited adenylylcyclase with similar efficiencies when 1 microM epinephrine was used as the agonist. The potentiation depended on both alpha2AR and Gi proteins since yohimbine and pertussis toxin inhibited the process. Pretreatment of CHO-2B cells with MAFP which inhibits both cytosolic and Ca2+-independent PLA2, reduced the release of AA induced by epinephrine+Ca2+ ionophore to basal value, whereas bromoenol lactone, a specific Ca2+-independent PLA2 inhibitor, had no effect. Preincubation of the cells with the intracellular calcium chelator BAPTA gave a dose-dependent inhibition of the arachidonic acid (AA) release. In CHO cells expressing the angiotensin II type 1 receptor, coupled to a Gq protein, the agonist (10-7 M) produced maximal AA release: there was no extra increase when angiotensin and Ca2+ ionophore were added together. There was no increase in the amount of inositol 1,4, 5-triphosphate following stimulation of CHO-2B, -2C, -2D cells with 1 microM epinephrine. Epinephrine led to greater phosphorylation of cPLA2, resulting in an electrophoretic mobility shift for all three cell lines, so inadequate p42/44 MAPKs stimulation was not responsible for the weaker stimulation of cPLA2 in CHO-2C cells. Therefore, the stimulation of cPLA2 by Gi proteins presumably involves another unknown mechanism. The differential stimulation of cPLA2 in these transfectants will be of value to study the actual involvement of the transduction pathways leading to maximal cPLA2 stimulation.  (+info)

Evidence of a cyclooxygenase-related prostaglandin synthesis in coral. The allene oxide pathway is not involved in prostaglandin biosynthesis. (19/4381)

Certain corals are rich natural sources of prostaglandins, the metabolic origin of which has remained undefined. By analogy with the lipoxygenase/allene oxide synthase pathway to jasmonic acid in plants, the presence of (8R)-lipoxygenase and allene oxide synthase in the coral Plexaura homomalla suggested a potential metabolic route to prostaglandins (Brash, A. R., Baertshi, S. W., Ingram, C.D., and Harris, T. M. (1987) J. Biol. Chem. 262, 15829-15839). Other evidence, from the Arctic coral Gersemia fruticosa, has indicated a cyclooxygenase intermediate in the biosynthesis (Varvas, K., Koljak, R., Jarving, I., Pehk, T., and Samel, N. (1994) Tetrahedron Lett. 35, 8267-8270). In the present study, active preparations of G. fruticosa have been used to identify both types of arachidonic acid metabolism and specific inhibitors were used to establish the enzyme type involved in the prostaglandin biosynthesis. The synthesis of prostaglandins and (11R)-hydroxyeicosatetraenoic acid was inhibited by mammalian cyclooxygenase inhibitors (indomethacin, aspirin, and tolfenamic acid), while the formation of the products of the 8-lipoxygenase/allene oxide pathway was not affected or was increased. The specific cyclooxygenase-2 inhibitor, nimesulide, did not inhibit the synthesis of prostaglandins in coral. We conclude that coral uses two parallel routes for the initial oxidation of polyenoic acids: the cyclooxygenase route, which leads to optically active prostaglandins, and the lipoxygenase/allene oxide synthase metabolism, the role of which remains to be established. An enzyme related to mammalian cyclooxygenases is the key to prostaglandin synthesis in coral. Based on our inhibitor data, the catalytic site of this evolutionary early cyclooxygenase appears to differ significantly from both known mammalian cyclooxygenases.  (+info)

Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells. (20/4381)

In Madin-Darby canine kidney D1 cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the beta-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2 indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Y receptor-promoted AA release, we did not find such inhibition for epinephrine (alpha1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2 activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components.  (+info)

Inhibitory effect of sulfur-containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets. (21/4381)

The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5'-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 microM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10(-4) M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane.  (+info)

Formation of reactive cyclopentenone compounds in vivo as products of the isoprostane pathway. (22/4381)

Cyclopentenone prostaglandins A2 and J2 are reactive compounds that possess unique biological activities. However, the extent to which they are formed in vivo remains unclear. In this study, we explored whether D2/E2-isoprostanes undergo dehydration in vivo to form A2/J2-isoprostanes. Oxidation of arachidonic acid in vitro generated a series of compounds that were confirmed to be A2/J2-isoprostanes by mass spectrometric analyses. A2/J2-isoprostanes were detected in vivo esterified to lipids in livers from normal rats at a level of 5. 1 +/- 2.3 ng/g, and levels increased dramatically by a mean of 24-fold following administration of CCl4. An A2-isoprostane, 15-A2t-isoprostane, was obtained and found to readily undergo Michael addition with glutathione and to adduct covalently to protein. A2/J2-isoprostanes could not be detected in the circulation, even following CCl4 administration, which we hypothesized might be explained by rapid formation of adducts. This was supported by finding that essentially all the radioactivity excreted into the urine following infusion of radiolabeled 15-A2t-isoprostane into a human volunteer was in the form of a polar conjugate(s). These data identify a new class of reactive compounds that are produced in vivo as products of the isoprostane pathway that can exert biological effects relevant to the pathobiology of oxidant injury.  (+info)

Regulation of 15-lipoxygenase expression and mucus secretion by IL-4 in human bronchial epithelial cells. (23/4381)

Our laboratory has recently shown that mucus differentiation of cultured normal human tracheobronchial epithelial (NHTBE) cells is accompanied by the increased expression of 15-lipoxygenase (15-LO). We used differentiated NHTBE cells to investigate the regulation of 15-LO expression and mucus secretion by inflammatory cytokines. Interleukin (IL)-4 and IL-13 dramatically enhanced the expression of 15-LO, whereas tumor necrosis factor-alpha, IL-1beta, and interferon (IFN)-gamma had no effect. These cytokines did not increase the expression of cyclooxygenase-2, with the exception of a modest induction by IL-1beta. The IL-4-induced 15-LO expression was concentration dependent, and mRNA and protein expression increased within 3 and 6 h, respectively, after IL-4 treatment. In metabolism studies with intact cells, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) were the major metabolites formed from exogenous arachidonic acid and linoleic acid. No prostaglandins were detected. IL-4 treatment dramatically increased the formation of 13-HODE and 15-HETE compared with that in untreated NHTBE cells, and several additional 15-LO metabolites were observed. Pretreatment of NHTBE cells with IFN-gamma or dexamethasone did not inhibit the IL-4-induced expression of 15-LO except at high concentrations (100 ng/ml of IFN-gamma and 10 microM dexamethasone). IL-4 treatment inhibited mucus secretion and attenuated the expression of the mucin genes MUC5AC and MUC5B at 12-24 h after treatment. Addition of 15-HETE precursor and 13-HODE precursor to the cultures did not alter mucin secretion or mucin gene expression. On the basis of the data presented, we conclude that the increase in 15-LO expression by IL-4 and attenuation of mucus secretion may be independent biological events.  (+info)

TGF-alpha reduces bradykinin-stimulated ion transport and prostaglandin release in human colonic epithelial cells. (24/4381)

The effect of chronic exposure to transforming growth factor-alpha (TGF-alpha) on bradykinin-stimulated acute prostanoid production and ion secretion in monolayers of HCA-7 colony 29 colonic epithelial cells has been studied. Monolayers synthesized prostaglandin E2 (PGE2) at a basal rate of 2.10 +/- 0.31 pg. monolayer-1. min-1 over 24 h. Bradykinin (10(-8)-10(-5) M) dose dependently increased acute PGE2 release by three orders of magnitude. This was associated with a rise in cAMP from 1.60 +/- 0.14 to 2.90 +/- 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC). When monolayers were primed by a 24-h exposure to TGF-alpha, basal PGE2 release rose to 6.31 +/- 0.38 pg. monolayer-1. min-1 (TGF-alpha concn 10 ng/ml; P = 0.001). However, the stimulation of acute prostaglandin release, intracellular cAMP, and increased SCC by bradykinin was significantly reduced by preincubation with TGF-alpha. Priming with PGE2 (10(-8)-10(-6) M) over 24 h mimicked the effect of TGF-alpha on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF-alpha may downregulate acute responses to bradykinin. In vivo, TGF-alpha could have an important modulatory function in regulating secretion under inflammatory conditions.  (+info)