Dexamethasone-induced reduction of phospholipase D activity in the rat. Possible role of lipocortin

Subcutaneous injection of dexamethasone resulted in a reduction of demonstrable phospholipase D activity of rat brain and liver microsomes. Partially purified rat lung lipocortin inhibited the activity of both microsomal and partially purified rat brain phospholipase D. These results show that phospholipase D activity is suppressed by dexamethasone and one of the possible mechanisms of inhibition may be a phospholipase inhibitory protein, lipocortin.     

Enhancement of ATPase Activity by a Lipid Peroxide of Arachidonic Acid in Rat Brain Microvessels

The effects of 15-hydroperoxyarachidonic acid (15-HPAA) on Na+, K+- and Mg+-ATPase activities in the blood-brain barrier (BBB) were examined using rat brain microvessels (MV). 15-HPAA markedly stimulated these ATPase activities in MV at low concentrations whereas the synaptosomal Na+, K+-ATPase activity was inhibited in a dose-dependent manner. Further neurochemical analysis revealed that this stimulatory effect of 15-HPAA in MV was not due to a simple detergent-like action of the compound on the membranes but rather to stimulation of the phospholipase A2 and lipoxygenase activity within MV. In addition, it was shown that free radical reactions were involved in the mechanism. Since such anti-edema drugs as 1,2-bis(nicotinamido)propane were proved to be potent suppressors of the enhanced ATPase activity, further speculations on the role of this effect for ischemic brain edema are offered.     

Treatment with the NK1 Antagonist Emend Reduces Blood Brain Barrier Dysfunction and Edema Formation in an Experimental Model of Brain Tumors

The neuropeptide substance P (SP) has been implicated in the disruption of the blood-brain barrier (BBB) and development of cerebral edema in acute brain injury. Cerebral edema accumulates rapidly around brain tumors and has been linked to several tumor-associated deficits. Currently, the standard treatment for peritumoral edema is the corticosteroid dexamethasone, prolonged use of which is associated with a number of deleterious side effects. As SP is reported to increase in many cancer types, this study examined whether SP plays a role in the genesis of brain peritumoral edema. A-375 human melanoma cells were injected into the right striatum of male Balb/c nude mice to induce brain tumor growth, with culture medium injected in animals serving as controls. At 2, 3 or 4 weeks following tumor cell inoculation, non-treated animals were perfusion fixed for immunohistochemical detection of Albumin, SP and NK1 receptor. A further subgroup of animals was treated with a daily injection of the NK1 antagonist Emend (3 mg/kg), dexamethasone (8 mg/kg) or saline vehicle at 3 weeks post-inoculation. Animals were sacrificed a week later to determine BBB permeability using Evan''s Blue and brain water content. Non-treated animals demonstrated a significant increase in albumin, SP and NK1 receptor immunoreactivity in the peritumoral area as well as increased perivascular staining in the surrounding brain tissue. Brain water content and BBB permeability was significantly increased in tumor-inoculated animals when compared to controls (p<0.05). Treatment with Emend and dexamethasone reduced BBB permeability and brain water content when compared to vehicle-treated tumor-inoculated mice. The increase in peritumoral staining for both SP and the NK1 receptor, coupled with the reduction in brain water content and BBB permeability seen following treatment with the NK1 antagonist Emend, suggests that SP plays a role in the genesis of peritumoral edema, and thus warrants further investigation as a potential anti-edematous treatment.     

Regulation of guinea pig macrophage collagenase production by dexamethasone and colchicine

Previous studies have demonstrated that exposure of guinea pig macrophages to a primary signal, such as lipopolysaccharide (LPS), stimulates the synthesis of prostaglandin E2 (PGE2) which, in turn, elevates cAMP levels resulting in the production of the enzyme, collagenase. The potential of regulating the biochemical events in this activation sequence was examined with the anti-inflammatory agents dexamethasone and colchicine, which suppress the destructive sequelae in chronic inflammatory lesions associated with the degradation of connective tissue. The addition of dexamethasone with LPS to macrophage cultures resulted in a dose-dependent inhibition of PGE2 and collagenase production, which was reversed by the exogenous addition of phospholipase A2. Collagenase production was also restored in dexamethasone-treated cultures by the addition of products normally produced as a result of phospholipase action, such as arachidonic acid, PGE2 or dibutyryl-cAMP. Since the effect of dexamethasone was thus linked to phospholipase A2 inhibition, mepacrine, a phospholipase inhibitor, was also tested. Mepacrine, like dexamethasone, caused a dose-dependent inhibition of PGE2 and collagenase. In addition to corticosteroid inhibition, colchicine was also found to block collagenase production. However, this anti-inflammatory agent had no effect on PGE2 synthesis. Colchicine was effective only when added at the onset of culture and not 24 h later, implicating a role for microtubules in the transmission of the activation signal rather than enzyme secretion. The failure of lumicolchicine to inhibit collagenase activity provided additional evidence that microtubules are involved in the activation of macrophages. These findings demonstrate that dexamethasone and colchicine act at specific steps in the activation sequence of guinea pig macrophages to regulate collagenase production.     

The selective release of phospholipase A2 by resident mouse peritoneal macrophages.

Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a phosphatidylinositol-specific phospholipase C activity. When macrophages are exposed to zymosan in culture, the cellular activity of pH-4.5 phospholipase A2 is diminished in a manner dependent on zymosan concentration and time of exposure, whereas the cellular activities of pH-8.5 phospholipase A2 and phospholipase C remain unchanged. The depletion of pH-4.5 phospholipase A2 activity from the cell is paralleled by a quantitative recovery of this activity in the culture medium in a manner similar to the cellular depletion and extracellular recovery of two lysosomal enzymes. This release is specifically elicited by an inflammatory substance such as zymosan, since macrophages incubated with 6 micrometer latex spheres retain pH-4.5 phospholipase A2 activity and lysosomal enzyme activities intracellularly.     

Phospholipase A2 inhibitory activity in thymocytes of dexamethasone-treated mice--possible implication of lipocortins

The cellular phospholipase A2 activity of mouse thymocytes was estimated in vitro by the release of [3H]-Arachidonic acid from labeled and calcium ionophore A23187-stimulated cells. This activity was decreased in thymocytes from dexamethasone-treated mice. Thus, the presence of phospholipase A2 inhibitory proteins in mouse thymus was investigated. Three main proteins (36 kDa I, 36 kDa II, 73 kDa) were purified. These proteins were able to inhibit both phospholipase A2 in vitro, and the release of [3H]-Arachidonic acid from labeled and stimulated mouse thymocytes. Biochemical analysis revealed that the three proteins were lipocortin-like proteins. Our results show that in vivo dexamethasone treatment induces a phospholipase A2 inhibitory activity in mouse thymus, such an inhibition can be reproduced on isolated thymocytes by purified thymic lipocortins, known as glucocorticosteroid-inducible proteins.     

The pharmacological profile of ovalbumin-induced paw oedema in rats

Rats are commonly used in anaphylaxis models, mainly in intestinal anaphylaxis. Hypersensitivity mechanisms are complex and they are not clearly defined. Ovalbumin (OVA) is commonly used for studies on the hypersensitivity mechanism. However, the potential pro-inflammatory mediators induced by this antigen in the model of paw oedema in immunized rats are still not completely understood. This work examines the pharmacological modulation of several mediators involved in rat hind paw immune oedema induced by OVA. Wistar rats were previously immunized (14-18 days) with OVA (30 microg, intraperitoneally) or sham-sensitized with aluminum hydroxide (control). The paw volumes were measured before the antigenic stimuli and 1, 2, 3 and 4 h after the intraplantar injection of OVA (10 microg/paw). Subcutaneous injection of dexamethasone, diphenhydramine, cyproheptadine, chlorpromazine or methysergide significantly inhibited (p < 0.05) the allergic paw oedema. The dual inhibitor of cyclooxygenase and lipoxygenase (NDGA), the cyclooxygenase inhibitor (indomethacin), the lipoxygenase inhibitor (MK-886), the PAF antagonist (WEB 2086), the mast cell stabilizer (ketotifen), and the anti-histamine (meclizine) did not inhibit the immune oedema. In addition, thalidomide and pentoxifylline (anti-tumour necrosis factor drugs) were ineffective against OVA-induced oedema. The fact that indomethacin, MK-886, NDGA and WEB 2086 are unable to inhibit this allergic oedema indicates that the dexamethasone action seems not to be via phospholipase A2, but possibly due to the synthesis and/or the inhibitory activity of cytokines. The paw oedema inhibition by diphenhydramine, but not by meclizine, may suggest a different mechanism, which is independent of the effect of histamine. These data indicate that allergic oedema is more sensitive to anti-serotonin drugs, mainly anti-5-HT2, suggesting that the principal mediator of this inflammatory response is serotonin.     

Role of prostaglandin H synthase isoforms in murine ear edema induced by phorbol ester application on skin.

Topical application of TPA to a murine ear induced an edema that was accompanied by eicosanoid biosynthesis and an early enhancement of prostaglandin H synthase 2 (PGHS-2) expression. PGHS-2 induction may be correlated with the time-course of TPA-induced edema formation. Treatment with drugs that inhibit AA mobilization such as dexamethasone or manoalide or inhibitors of leukotriene formation such as zileuton or baicalein, reduced TPA-induced edema development and PGHS-2 levels. On the other hand, arachidonic acid (AA) application on the murine ear induced rapid expression of PGHS-2. This effect was not reproduced by other fatty acids such as oleic, linoleic, eicosatetraynoic or eicosapentaenoic acids. PGHS-2 expression induced by AA application was independent of PGHS and lipoxygenase metabolite synthesis. However, topical application of PGE2 on skin induced PGHS-2 overexpression. This study suggests that AA release and/or subsequent metabolism by PGHS may be involved in the induction of PGHS-2 expression in murine TPA- and AA-induced ear oedema.     

Differential effects of aspirin and dexamethasone on phospholipase A2 and C activities and arachidonic acid release from endothelial cells in response to bradykinin and leukotriene D4

The CPAE bovine endothelial cell line may be stimulated to produce eicosanoids. Leukotriene D4 increased the release of arachidonic acid primarily by activating phospholipase A2 while bradykinin activated the phospholipase C pathway. Cells pretreated with dexamethasone, a phospholipase A2 inhibitor, no longer responded to stimulation by LTD4 but did release arachidonic acid when treated with bradykinin. Aspirin blocked bradykinin-stimulated production of arachidonic acid but left the response to LTD4 unaffected. We conclude that these cells produce eicosanoids by activation of both PLA2 and PLC, and that the two different methods of arachidonic acid release can be distinguished by using the common anti-inflammatory drugs aspirin and dexamethasone.     

Excitotoxin induction of ornithine decarboxylase in cerebral cortex is reduced by phospholipase A2 inhibition.

The enzyme ornithine decarboxylase (ODC) has been shown to be induced by a number of conditions such as cold-injury, kindling, ischaemia and excitotoxin injection. In previous studies we have characterised the cortical response to kainate injection into the nucleus basalis and shown a substantial increase in both ODC mRNA and enzyme activity which reaches a maximum at 8h. This response is completely prevented by treatment with MK-801, indicating the involvement of NMDA receptors in mediating this response. Whilst NMDA receptors are known to gate a cation channel leading to increased calcium entry, an additional effect on the release of arachidonic acid has been reported. The possibility that NMDA receptor mediated activation of phospholipase A2 and release of arachidonic acid might mediate this ODC response was investigated in this study by treatment with the phospholipase inhibitors quinacrine and dexamethasone. Treatment of animals with quinacrine (100 mg/kg) at the time of injection of kainate into the nucleus basalis caused a significant attenuation of the induction of ODC in cerebral cortex of 43%. No further attenuation was seen at higher doses. A similar reduction in ODC induction was seen after treatment with dexamethasone (1 mg/kg) but a greater effect could be obtained (65% attenuation) at higher doses. The possible involvement of arachidonic acid derivatives in mediating ODC induction was further investigated by treatment with the cyclo-oxygenase inhibitor indomethacin and the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Indomethacin was able to significantly attenuate the induction of ODC (greater than 60%) whilst NDGA (30 mg/kg) was ineffective. These results indicate the possible role of arachidonic acid derivatives in the regulation of the expression of ODC in cerebral cortex after excitotoxin injection.     

In vivo activity of pseudoguaianolide sesquiterpene lactones in acute and chronic inflammation

The pseudoguaianolide sesquiterpene lactones 4-alpha-O-acetyl-pseudoguaian-6beta-olide (1), hymenin (2), ambrosanolide (3), tetraneurin A (4), parthenin (5), hysterin (6) and confertdiolide (7) were evaluated for their ability to affect the inflammation responses induced by different agents. All the compounds showed activity against the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse ear edema. The ethyl phenylpropiolate (EPP)-induced mouse ear edema was inhibited by compounds 3, 5 and 7. However, when sesquiterpene-lactones were assayed on the arachidonic acid (AA)-induced mouse ear edema, none of them were active. The only sesquiterpene lactone orally active against the paw mouse edema induced by carrageenan was 7, which gave a 46% edema inhibition after 3 h. On the other hand, compounds 3, 5 and 7 reduced the serotonin-induced paw edema in mice, although compound 7 was inactive in presence of cycloheximide. In addition, the sesquiterpene lactones were assayed against the chronic inflammation induced by repeated application of TPA on mouse ear. Confertdiolide was the most active compound; it reduced the edema by 87% and had a more moderate effect against the leukocyte recruitment (36% reduction in myeloperoxidase (MPO) levels). A histological study of ear the samples treated with 7 presented no detectable morphological lesions such as those treated with dexamethasone. On the oxazolone-induced delayed type hypersensitivity (DTH) only compounds 4 and 5 were active 24 h after the challenge. Compound 5 affected polymorphonuclear leukocyte infiltration (69% reduction in MPO levels). The results suggest that the especial chemical structure and spatial conformation of confertdiolide may facilitate its anti-inflammatory effect.     

Effect of Dexamethasone on Transport of α-Aminoisobutyric Acid and Sucrose Across the Blood-Brain Barrier

The effect of glucocorticoids on the blood-brain barrier (BBB) was studied in rats following a single injection or 3 days of dexamethasone administration. Tracers with a low permeability across the intact endothelium, [14C]sucrose and alpha-[3H]aminoisobutyric acid ([3H]AIB), were simultaneously injected intravenously in untreated rats or in rats treated with dexamethasone. Unidirectional blood-to-brain transfer constants (Ki) in 14 regions of the rat brain were determined. In regions of control brain, average Ki values for AIB and sucrose were approximately 0.0020 and 0.00060 ml g-1 min-1, respectively. The lowest transfer constants were found in caudate nucleus, hippocampus, white matter, and cerebellum. In dexamethasone-treated animals, Ki values for both sucrose and AIB markedly decreased by 30-50% in almost all brain regions. These results indicate that a single injection or 3 days of treatment with dexamethasone causes an apparent reduction in the normal BBB permeability, and dexamethasone may greatly interfere with drug delivery into brain. These observations may have an importance for the administration of drugs in brain disease in the presence of steroids.     

5-Aminosalicylate stimulates phospholipase D activity in macrophages

5-Aminosalicylate, which is considered to be the active moiety of sulfasalazine, is one of the most widely used agents for treatment of inflammatory bowel disease. However, its mechanism of action is unclear. In this report, we provide evidence that the phospholipase D pathway is a target for this drug in macrophages. Activation of phospholipase D leads to the generation of important second messengers such as phosphatidic acid, lysophosphatidic acid and diacylglycerol, all of which can regulate cellular responses involved in inflammation. Murine peritoneal macrophages were labeled with [(3)H]myristate, incubated with various drugs, agonists, or inhibitors, and phospholipase D activity was assayed. 5-Aminosalicylate or sulfasalazine stimulated phospholipase D in a time- and concentration-dependent manner. Chelation of extracellular Ca(2+) inhibited phospholipase D activation by either of these drugs whereas pretreatment of macrophages with the tyrosine kinase inhibitor genistein had no effect. Downregulation of protein kinase C by prolonged incubation with phorbol ester completely blocked the activation of phospholipase D. Pertussis toxin decreased the activation of phospholipase D. The levels of inositol 1,4,5-trisphosphate increased by 260% after treatment of macrophages with 5-aminosalicylate. A phosphoinositide-specific phospholipase C inhibitor U73122 blocked phospholipase D activation completely. Interestingly, long-term preincubation of the macrophages with a relatively low concentration of 5-aminosalicylate that did not stimulate phospholipase D activity by itself, potentiated the effect of phorbol ester-induced activation of phospholipase D. Taken together, these results show that 5-aminosalicylate activates phospholipase D via a pathway involving inositol 1,4,5-trisphosphate generation, calcium fluxes, and Gi/Go. Although the mechanisms by which phospholipase D activation by 5-aminosalicylate or sulfasalazine might attenuate inflammatory responses in the intestine remain to be defined, these results highlight a novel potential mechanism of action for these drugs.     

Dexamethasone inhibits receptor-activated phosphoinositide breakdown in rat basophilic leukemia (RBL-2H3) cells

The activation of rat basophilic leukemia cells for histamine release is accompanied by Ca2+ influx and arachidonic acid release. IgE receptor but not A23187 ionophore stimulation of these cells also resulted in phosphoinositide breakdown. In these experiments, the culture of these cells with dexamethasone inhibited IgE- and ionophore-mediated histamine release. The concentration for 50% of maximal inhibition was 12 nM, and prolonged exposure to the drug was required, with maximal effect observed in 8 to 15 hr. The inhibitory effect of dexamethasone was reversible (t1/2 for recovery was 16 hr). Dexamethasone blocked the IgE-mediated 45Ca2+ influx and the release of [14C]-arachidonic acid (IC50 of 1 nM and 10 nM respectively). Dexamethasone inhibited the IgE receptor-mediated phosphoinositide breakdown (IC50 of 5 nM). It also decreased arachidonic acid release after A23187 stimulation demonstrating an effect on phospholipase A2. Therefore, exposure of the cells to dexamethasone results in the inhibition of both phospholipase A2 and phospholipase C pathways of arachidonic acid generation.     

Inflammatory factors stimulate expression of group II phospholipase A2 in rat cultured astrocytes. Two distinct pathways of the gene expression

Inflammatory factors such as tumor necrosis factor (TNF), interleukin 1 (IL-1), and lipopolysaccharide (LPS) greatly enhance the expression of group II phospholipase A2 (PLA2-II) mRNA, leading to increased secretion of PLA2-II enzyme from rat-cultured astrocytes. The potent antiinflammatory agent dexamethasone suppressed the PLA2-II expression induced by LPS. In vivo studies also demonstrated that the level of PLA2-II mRNA in the brain increased with intravenous injection of LPS. These results suggest that PLA2-II in the brain plays important roles in the inflammatory response. Agents which increase intracellular cAMP concentration did not stimulate PLA2-II expression by themselves but selectively enhanced TNF-induced PLA2-II expression about 5-fold. Phorbol ester, a well known protein kinase C activator, increased the PLA2-II expression. H-7, a protein kinase C inhibitor, inhibited the LPS-induced PLA2-II expression, but did not inhibit the TNF-induced one. Therefore, we conclude that the TNF-activated pathway differs from the LPS-activated one: the former is enhanced by cAMP and the latter involves protein kinase C.     

Cytokine- and forskolin-induced synthesis of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by dexamethasone

We have previously described that treatment of rat glomerular mesangial cells with interleukin-1 beta, tumor necrosis factor or forskolin stimulates the synthesis and secretion of prostaglandin E2 and group II phospholipase A2. We now report that pretreatment of the mesangial cells with dexamethasone dose-dependently suppresses the cytokines- and forskolin-induced synthesis of prostaglandin E2 as well as the induced synthesis and secretion of group II phospholipase A2. These observations implicate that the inhibition of the cellular or secreted phospholipase A2 activity by dexamethasone in rat mesangial cells is not due to induced synthesis of phospholipase A2 inhibitory proteins but caused by direct inhibition of phospholipase A2 protein expression.     

The efficacy of anti-inflammatory agents with respect to extracellular phospholipase A2 activity

The effects of steroidal and non-steroidal anti-inflammatory agents on extracellular phospholipase A2 (PLA2) activity were investigated. Enzyme release was inhibited by 5 x 10(-8) M dexamethasone but not by indomethacin, whereas the soluble extracellular enzyme was inactivated by mepacrine but not by dexamethasone or indomethacin. PLA2, released into the interstitium by activated macrophages is both pro-inflammatory and vasoactive. The anti-inflammatory efficacy of steroidal and non-steroidal drugs may partially reside in their ability to inhibit the release of PLA2, or inactivate preformed extracellular PLA2 in chronically inflamed sites.     

Stimulation of phospholipase A2 activity by oxygen-derived free radicals in isolated brain capillaries

An exogenous free radical generating system added to isolated brain capillaries induces degradation of phospholipids. This inductive effect reflects increased phospholipase activities as measured by fatty acid composition of various phospholipid fractions. The correlation of phospholipid degradation with stimulation of phospholipases was further investigated by using cationic amphiphilic agents, which are known to be phospholipase A2 inhibitors. The breakdown of phospholipids was inhibited by the pretreatment of isolated capillaries with these drugs.