Prostanoid Formation Research Articles

Effects of synthetic sphingosine-1-phosphate analogs on arachidonic acid metabolism and cell death

Sphingolipid metabolites such as sphingosine regulate cell functions including cell death and arachidonic acid (AA) metabolism. d-erythro-C18-Sphingosine-1-phosphate ( d- e-S1P), a sphingolipid metabolite, acts as an intracellular messenger in addition to being an endogenous ligand of some cell surface receptors. The development of S1P analogs may be useful for studying and/or regulating S1P-mediated cellular responses. In the present study, we found that several synthetic S1P analogs at pharmacological concentrations stimulated AA metabolism and cell death in PC12 cells. d- erythro- N, O, O-Trimethyl-C18-S1P ( d- e-TM-S1P), l- threo- O, O-dimethyl-C18-S1P ( l- t-DM-S1P) and l- threo- O, O-dimethyl-3 O-benzyl-C18-S1P ( l- t-DMBn-S1P) at 100 μM stimulated [ 3H]AA release from the prelabeled PC12 cells. l- t-DMBn-S1P at 20 μM increased prostanoid formation in PC12 cells. l- t-DMBn-S1P-induced AA release was inhibited by d- e-sphingosine, but not by the tested PLA 2 inhibitors. l- t-DMBn-S1P did not stimulate the activity of cytosolic phospholipase A 2α (cPLA 2α) in vitro and the translocation of cPLA 2α in the cells, and caused AA release from the cells lacking cPLA 2α. These findings suggest that l- t-DMBn-S1P stimulated AA release in a cPLA 2α-independent manner. In contrast, d- e-S1P and d- erythro- N-monomethyl-C18-S1P caused cell death without AA release in PC12 cells, and the effects of d- e-TM-S1P, l- t-DM-S1P and l- t-DMBn-S1P on cell death were limited. Synthetic S1P analogs may be useful tools for studying AA metabolism and cell death in cells.

Receptor-activating peptides for PAR-1 and PAR-2 relax rat gastric artery via multiple mechanisms

Receptor-activating peptides for protease-activated receptors (PARs) 1 or 2 enhance gastric mucosal blood flow (GMBF) and protect against gastric mucosal injury in rats. We thus examined and characterized the effects of PAR-1 and PAR-2 agonists on the isometric tension in isolated rat gastric artery. The agonists for PAR-2 or PAR-1 produced vasodilation in the endothelium-intact arterial rings, which was abolished by removal of the endothelium. The mechanisms underlying the PAR-2- and PAR-1-mediated relaxation involved NO, endothelium-derived hyperpolarizing factor (EDHF) and prostanoids, to distinct extent, as evaluated by use of inhibitors of NO synthase, cyclo-oxygenase and Ca 2+-activated K + channels. The EDHF-dependent relaxation responses were significantly attenuated by gap junction inhibitors. These findings demonstrate that endothelial PAR-1 and PAR-2, upon activation, dilate the gastric artery via NO and prostanoid formation and also EDHF mechanisms including gap junctions, which would enhance GMBF.

Cyclosporine A attenuates the natriuretic action of loop diuretics by inhibition of renal COX-2 expression

It is known that inhibition of cyclooxygenase (COX) impairs the renal actions of loop diuretics. Recently, we found that cyclosporine A (CsA) inhibits renal COX-2 expression. Therefore, we examined the interferences of CsA with the renal actions of loop diuretics. We investigated the renal effects of furosemide administration (12 mg/day subcutaneously) in male Sprague-Dawley rats receiving in addition vehicle, CsA (15 mg/kg x day), rofecoxib (10 mg/kg x day), or a combination of both. CsA, rofecoxib, and their combination lowered the furosemide-induced increase of prostaglandin E(2) (PGE(2)) and of 6-keto prostaglandin F(1 alpha) (6-keto PGF(1 alpha)) excretion by 55% and by 70%. They also lowered furosemide stimulated renal excretion of sodium and water by about 65% and 60%. Basal as well as furosemide-induced stimulation of plasma renin activity (PRA) and of renal renin mRNA was further enhanced by CsA. In contrast, rofecoxib attenuated the furosemide-induced rise of PRA and of renin mRNA, both in the absence and in the presence of CsA. In addition, the increase in plasma 6-keto PGF(1 alpha) levels by furosemide was further enhanced by CsA and was attenuated by rofecoxib. Taken together, our data suggest that CsA acts as an antinatriuretic, likely by the inhibition of COX-2-mediated renal prostanoid formation. Since the furosemide-induced stimulation of the renin system is not attenuated by CsA but by COX-2 inhibition, we speculate that extrarenal COX-2-derived prostanoids may be involved in the stimulation of the renin system by CsA and by loop diuretics.

NOS II Inhibition Restores Attenuation of Endothelium-Dependent Hyperpolarization in Rat Mesenteric Artery Exposed to Lipopolysaccharide

The aim of this study was to assess the effects of lipopolysaccharide (LPS) exposure on the endothelium-dependent hyperpolarization in the rat mesenteric artery using isometric tension recordings and electrophysiological studies. Mesenteric arterial rings of male Sprague-Dawley rats were incubated with LPS for 6 hours. All experiments were performed in the presence of indomethacin to inhibit the formation of vasoactive prostanoids. Contraction to phenylephrine was significantly reduced in rings incubated with LPS, which was restored in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME). L-NAME resistant relaxation to acetylcholine was attenuated in LPS-treated rings. LPS exposure hyperpolarized resting membrane potentials of arterial smooth muscle cells, which was repolarized by incubation with either L-NAME or 1400W, a selective inhibitor of nitric oxide synthase II (NOS II). Endothelium-dependent hyperpolarization to acetylcholine was attenuated in arteries incubated with LPS, while incubation with LPS and 1400W restored EDHF-mediated hyperpolarization. LPS-induced membrane potential change was mimicked by incubation with either SIN-1 or diethylamine NONOate, a donor of nitric oxide. These data suggest that LPS exposure attenuates EDHF-mediated both relaxation and hyperpolarization in the rat mesenteric artery. The possible mechanisms underlying decreased EDHF-mediated responses might be due to, at least in some part, massive nitric oxide induced by NOS II.

Essential role of EDHF in the initiation and maintenance of adrenergic vasomotion in rat mesenteric arteries.

The possible roles of endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)), nitric oxide (NO), arachidonic acid (AA) metabolites, and Ca(2+)-activated K(+) (K(Ca)) channels in adrenergically induced vasomotion were examined in pressurized rat mesenteric arteries. Removal of the endothelium or buffering [Ca(2+)](i) selectively in endothelial cells with BAPTA eliminated vasomotion in response to phenylephrine (PE; 10.0 microM). In arteries with intact endothelium, inhibition of NO synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME; 300.0 microM) or N(omega)-nitro-l-arginine (l-NNA; 300.0 microM) did not eliminate vasomotion. Neither inhibition of cGMP formation with 10.0 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor inhibition of prostanoid formation (10.0 microM indomethacin) eliminated vasomotion. Similarly, inhibition of AA cytochrome P-450 metabolism with an intraluminal application of 17-octadecynoic acid (17-ODYA) or 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) failed to eliminate vasomotion. In contrast, intraluminal application of the K(Ca) channel blockers apamin (250.0 nM) and charybdotoxin (100.0 nM), together, abolished vasomotion and changed synchronous Ca(2+) oscillations in smooth muscle cells to asynchronous propagating Ca(2+) waves. Apamin, charybdotoxin, or iberiotoxin (100.0 nM) alone did not eliminate vasomotion, nor did the combination of apamin and iberiotoxin. The results show that adrenergic vasomotion in rat mesenteric arteries is critically dependent on Ca(2+)-activated K(+) channels in endothelial cells. Because these channels (small- and intermediate-conductance K(Ca) channels) are a recognized component of EDHF, we conclude therefore that EDHF is essential for the development of adrenergically induced vasomotion.

A protective role of protease-activated receptor 1 in rat gastric mucosa

Background & Aims: On activation, protease-activated receptor (PAR)-2 modulates multiple gastric functions and exerts mucosal protection via activation of sensory neurons. The role of PAR-1, a thrombin receptor, in the stomach remains unknown. We thus examined if the PAR-1 agonist could protect against gastric mucosal injury in rats. Methods: Gastric mucosal injury was created by oral administration of ethanol/HCl or absolute ethanol in conscious rats. Gastric mucosal blood flow and acid secretion were determined in anesthetized rats. Immunohistochemical analyses of PAR-1 and cyclooxygenase (COX)-1 were also performed in rat and human stomach. Results: The PAR-1 agonist TFLLR-NH2, administered intravenously in combination with amastatin, protected against the gastric mucosal injury induced by ethanol/HCl or absolute ethanol. The protective effect of TFLLR-NH2 was abolished by indomethacin or a COX-1 inhibitor but not by ablation of sensory neurons with capsaicin. TFLLR-NH2 produced an NO-independent increase in gastric mucosal blood flow that was partially inhibited by blockade of the endothelium-derived hyperpolarizing factor pathway. This inhibitory effect was promoted by indomethacin. TFLLR-NH2 suppressed carbachol-evoked acid secretion in an indomethacin-reversible manner. Immunoreactive PAR-1 and COX-1 were expressed abundantly in rat gastric muscularis mucosae and smooth muscle, and the former protein was also detectable in blood vessels. Similar staining was observed in human gastric muscularis mucosae. Conclusions: The PAR-1 agonist, given systemically, protects against gastric mucosal injury via COX-1-dependent formation of prostanoids, modulating multiple gastric functions. Our data identify a novel protective role for PAR-1 in gastric mucosa, and the underlying mechanism is entirely different from that for PAR-2.

Role of COX-1 and -2 in prostanoid generation and modulation of angiotensin II responses.

The role of cyclooxygenase (COX)-1 and -2 in prostanoid formation and modulation of pressor responses to ANG II was investigated in the pulmonary and systemic vascular beds in the rat. In the present study, selective COX-1 and -2 inhibitors attenuated increases in pulmonary arterial pressure and decreases in systemic arterial pressure in response to arachidonic acid but did not alter responses to PGE1 or U-46619. The selective COX-1 and -2 inhibitors did not modify systemic pressor responses to injections or infusions of ANG II or pulmonary pressor responses to injections of the peptide. COX-2 inhibitors did not alter, whereas a COX-1 inhibitor depressed, arachidonic acid-induced platelet aggregation. These data provide evidence in support of the hypothesis that prostanoid synthesis occurs by way of the COX-1 and -2 pathways in the pulmonary and systemic vascular beds but that pressor responses to ANG II are not mediated or modulated by these pathways in the rat.

Nonsteroidal antiinflammatory drugs inhibiting prostanoid efflux: as easy as ABC?

Nonsteroidal antiinflammatory drugs (NSAIDs) and prostanoids continue to be fascinating research targets. Humans have been using NSAIDs in one form or another, from folk remedies through to the products of modern pharmaceutical research, for thousands of years. However, we still have not characterized all of the systems through which these drugs produce their beneficial and harmful effects (1, 2). We are certain, from seminal work carried out in the 1960s and early 1970s, that NSAIDs have the common property of inhibiting the activity of cyclooxygenase (COX), the enzyme that initiates the formation of prostanoids (1). An explosion of research in the last decade or so has led us to understand that COX exists in two forms, COX-1 and -2, and that drugs targeted against COX-2 still retain the antiinflammatory properties of traditional NSAIDs (1, 2). Because the traditional NSAIDs generally inhibit both isoforms of COX, it is the ability of NSAIDs to inhibit COX-1 that has become associated with their deleterious side effects, particularly within the gastrointestinal tract. However, this dualist approach, COX-1 = good prostanoids and COX-2 = bad prostanoids, does not appear to supply a full understanding of the effects of NSAIDs in vivo . For that reason, researchers have continued to look for other targets at which NSAIDs may exert effects. For example, NSAIDs have been shown to inhibit factors within inflammatory pathways, such as nuclear factor κ-B (3), and to elevate the production of antiinflammatory adenosine (4). Other researchers have suggested that there could be yet more COX isoforms on which the NSAIDs act (see ref. 2). Experiments with chiral NSAIDs have also shown that enantiomers inactive on COX duplicate some of the therapeutic effects of NSAIDs. For example, whereas for ketoprofen and flurbiprofen only the S- and not the R-enantiomers inhibit COX (5 …

Effect of aspirin on constitutive nitric oxide synthase and the biovailability of NO

Introduction: Aspirin decreases the activity of iNOS and the formation of prostanoids. Constitutive nitric oxide synthase (cNOS) is present in endothelial cells, platelets, leukocytes and neurons, yet no data are available on the effect of aspirin on cNOS and the bioavailability of NO produced by this enzyme. Materials and methods: Human umbilical vein endothelial cells (HUVECs), rat adrenal gland pheocromocytoma cells (PC-12) and human platelets were incubated with different aspirin concentrations. The kinetics of NO, O2− and ONOO− release were measured simultaneously in single cells or platelet suspensions using tandem electrochemical nanosensors. The NO, O2− and ONOO− release from cells and platelets was stimulated with calcium ionophore and collagen, respectively. cNOS expression was estimated by Western blot analysis. Results: Incubation of HUVECs and PC-12 with 10−5 mol/l of aspirin increased cNOS expression by 70±7% and 50±5, respectively. However, the NO concentration increased only by 33% in HUVECs incubated with the same aspirin concentration. Incubation of HUVECs with aspirin also increased the O2− and ONOO− production. Therefore the bioavailability of NO increased only slightly in endothelium and did not reflect the increase in eNOS. This was in contrast to platelets, where maximal NO bioavailability almost doubled after incubation with aspirin. Conclusions: Aspirin did not have a significant effect on the NO bioavailability in endothelial cells. However, aspirin highly improved the NO production in platelets. The high NO production in platelets may counteract the effect of thromboxane, inhibit platelet aggregation, and compensate for the reduction of prostacycline concentration by aspirin.

Proinflammatory Cytokines Induce Cyclooxygenase-2 mRNA and Protein Expression in Human Pulp Cell Cultures

The increased release of prostaglandins (PG) within pulpal tissues is considered to play a pathogenic role during pulpal disease progression. The rate-limiting step in the formation of PG from arachidonic acid is catalyzed by cyclooxygenase (COX). COX-2 is an inducible enzyme believed to be responsible for PG synthesis at site of inflammation. The effect of proinflammatory cytokines on human pulp cells with special reference to COX-2 expression has not been reported earlier. The aim of the present study was to investigate the effects of interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha) on the expression of COX-2 mRNA gene and protein in cultured human pulp cells. Investigations of the time dependence of COX-2 mRNA expression in proinflammatory cytokines-treated human pulp cells revealed a rapid accumulation of the transcript, a significant signal first detectable 1 h after exposure. In addition, both IL-1alpha and TNF-alpha up-regulated COX-2 protein expression by human pulp cells. The kinetics of this response showed that COX-2 was detectable in cell lysates as early as 2 h post proinflammatory cytokines challenge and remained elevated throughout the 24-h incubation period. This suggests that one of the pathogenic mechanisms of pulpal inflammation in vivo may be the synthesis of COX-2 by resident cells in response to a proinflammatory cytokines challenge. COX-2 may play an important role in the regulation of prostanoid formation in the pathogenesis of pulpal inflammation. Taken together, we propose that the use of selective COX-2 inhibitors might provide a valuable tool in the control of pulpal inflammation.

Conjugated linoleic acid exhibits stimulatory and inhibitory effects on prostanoid production in human endothelial cells and platelets

In addition to their reported antitumorigenic properties, various conjugated linoleic acid (CLA) isomers have also been shown to decrease prostanoid synthesis as a result of inhibiting the cyclooxygenase (COX) enzyme. We have previously reported that several CLA isomers inhibited both platelet aggregation and formation of thromboxane A 2 (TXA 2), a proaggregatory and vasoconstrictive agent. Since the interaction between platelets and vascular endothelial cells is essential to maintaining vascular homeostasis, we decided to investigate the effects of various CLA isomers on the production of endothelial prostacyclin (PGI 2), a potent vasodilator and inhibitor of platelet function. Using interleukin 1-β (IL1-β)-stimulated human umbilical vein endothelial cells (HUVECs), we initially established that HUVECs of passage #2 should be used since these cells were most responsive to thrombin-induced conversion of endogenous arachidonic acid to PGI 2, as monitored by the formation of its stable, inactive metabolite, 6-ketoPGF 1α. In the first part of the study, the effects of CLA isomers in the free fatty acid form were tested. The 10( E), 12( Z)- and 9( Z), 11( E)-CLA isomers inhibited thrombin-induced 6-ketoPGF 1α formation with I 50's of 2.6 and 5.5 μM, whereas the 9( Z), 11( Z)- and 9( E), 11( E)-CLA were ineffective at concentrations up to 60 μM. The inhibitory effect of the 10( E), 12( Z)-CLA was irreversible. Next, the effects of CLA incorporation into HUVECs on PGI 2 generation was determined. An average 8-fold stimulation of 6-ketoPGF 1α formation was obtained with quiescent IL1-β-exposed HUVECs pretreated for 18 h with 25 μM 9( Z), 11( Z)-CLA, whereas cells preincubated with the 10( E), 12( Z) isomer enhanced this eicosanoid 3-fold. Such IL1-β-treated HUVECs prelabeled with 25 μM 9( Z), 11( Z)-CLA became refractory to thrombin stimulation, as measured by 6-ketoPGF 1α production, whereas a small, statistically insignificant, inhibition was observed upon thrombin treatment of HUVECs prelabeled with the 10( E), 12( Z) isomer. Qualitative similar results were obtained with resting or thrombin-stimulated platelets containing these esterified CLA isomers indicating that these effects occur with cells that contain either the COX-1 or COX-2 isozymes. The results of this in vitro study indicate that the effects of CLA on cellular prostanoid formation in endothelial cells and platelets can be either inhibitory or stimulatory, and this seems to depend not only on the specific CLA isomer and whether or not the CLA is in the free fatty acid form or esterified into cellular lipids, but also whether cells are in the resting or stimulated state. These findings suggest that in vivo, CLA might have multiple, complex effects on vascular homeostasis.

Multiple roles for protease-activated receptor-2 in gastric mucosa

Protease-activated receptor-2 (PAR-2), a G-protein-coupled receptor, is activated by proteolytic unmasking of the N-terminal cryptic tethered ligand. In gastric mucosa, activation of PAR-2 expressed by sensory neurons triggers release of CGRP and tachykinins, leading to mucus secretion via activation of CGRP1 and NK2 receptors, respectively. The PAR-2 agonist reveals mucosal cytoprotective effects in several gastric injury models. Further, PAR-2 activation inhibits gastric acid secretion, independently of sensory neurons or prostanoid formation. The PAR-2 agonist also induces increase in gastric mucosal blood flow, an effect being independent of endogenous CGRP or NO. Endothelium-derived hyperpolarizing factor (EDHF) appears to be involved in the PAR-2-mediated enhancement of mucosal blood flow. In contrast, mucosal chief cells are abundant in immunoreactive PAR-2, and PAR-2 stimulation triggers pepsinogen secretion. Taken together, primarily, PAR-2 plays protective roles in gastric mucosa through multiple mechanisms. Considering PAR-2-mediated pepsinogen secretion, PAR-2 might function as a double-edged sword in gastric mucosa.

Adenosine Up-Regulates Cyclooxygenase-2 in Human Granulocytes: Impact on the Balance of Eicosanoid Generation

Polymorphonuclear neutrophils (granulocytes; PMNs) are often the first blood cells to migrate toward inflammatory lesions to perform host defense functions. PMNs respond to specific stimuli by releasing several factors and generate lipid mediators of inflammation from the 5-lipoxygenase and the inducible cyclooxygenase (COX)-2 pathways. In view of adenosine's anti-inflammatory properties and suppressive impact on the 5-lipoxygenase pathway, we addressed in this study the impact of this autacoid on the COX-2 pathway. We observed that adenosine up-regulates the expression of the COX-2 enzyme and mRNA. Production of PGE(2) in response to exogenous arachidonic acid was also increased by adenosine and correlated with COX-2 protein levels. The potentiating effect of adenosine on COX-2 could be mimicked by pharmacological increases of intracellular cAMP levels, involving the latter as a putative second messenger for the up-regulation of COX-2 by adenosine. Specific COX-2 inhibitors were used to confirm the predominant role of the COX-2 isoform in the formation of prostanoids by stimulated PMNs. Withdrawal of extracellular adenosine strikingly emphasized the inhibitory potential of PGE(2) on leukotriene B(4) formation and involved the EP(2) receptor subtype in this process. Thus, adenosine may promote a self-limiting regulatory process through the increase of PGE(2) generation, which may result in the inhibition of PMN functions. This study identifies a new aspect of the anti-inflammatory properties of adenosine in leukocytes, introducing the concept that this autacoid may exert its immunomodulatory activities in part by modifying the balance of lipid mediators generated by PMNs.

Gene expression of prostanoid forming enzymes along the rat nephron

To obtain information about the general capability of nephron segments to elaborate prostanoids, we determined the gene expression of key enzymes for prostanoid formation. For this goal mRNAs were assayed for cyclooxygenases-1 and -2 as well as for the synthases of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostacyclin (PGI2) and thromboxane A2 (TXA2) in microdissected rat nephron segments by RT-PCR. Cyclooxygenase-1 (COX-1) mRNA was strongly expressed in all segments of the collecting ducts and to a lesser extent in glomeruli. COX-2 mRNA was found in the cortical thick ascending limb of Henle, and weaker expression also was detected in glomeruli. The lipocalin-type PGD synthase mRNA displayed a broad expression pattern in the cortex and outer medulla, including proximal convoluted tubule, thick ascending limb of Henle, distal convoluted tubule, and cortical and outer medullary collecting duct. The hematopoietic PGD synthase mRNA was restricted to the outer medullary collecting duct, and the membrane-associated PGE-synthase mRNA was exclusively expressed in the whole collecting duct system. Prostacylin-synthase mRNA was found in the whole kidney, but not in any microdissected nephron segment analyzed in this study. TXA-synthase mRNA was expressed in glomeruli. Given that the existence of cyclooxygenase in combination with the different PG-synthases is a prerequisite for the formation of prostanoids, our data suggest that PGD2 is mainly formed in the thick ascending limb and in the collecting duct, while PGE2 appears to be mainly generated by the collecting ducts. Probably no formation of PGI2 occurs within the nephron. Whether TXA2 can be formed by nephron segments remains questionable.

Translational Regulation of Prostaglandin Endoperoxide H Synthase-1 mRNA in Megakaryocytic MEG-01 Cells: SPECIFIC PROTEIN BINDING TO A CONSERVED 20-NUCLEOTIDE CISELEMENT IN THE 3′-UNTRANSLATED REGION

Prostaglandin endoperoxide H synthase-1 (PGHS-1) is an abundant enzyme in platelets, where it plays a key role in the cascade of prostanoid formation. In platelets, the primary site of PGHS-1 synthesis is in precursor megakaryocytic cells. We have previously shown that in megakaryocytic MEG-01 cells, TPA induces an increase of PGHS-1 mRNA within a few hours, whereas protein increase occurs after several days of treatment. We now report that the delayed increase in PGHS-1 protein is caused by translational regulation. De novo PGHS-1 synthesis, measured using [(35)S]methionine pulse labeling followed by immunoprecipitation, was detected at day 4 after TPA treatment but not at day 1. To identify a potential element of PGHS-1 mRNA controlling translation, we compared the 3'-untranslated region from different species and identified a 20-nt segment perfectly conserved. The 20-nt segment was used as a probe in RNA gel mobility-shift assays using MEG-01 extracts from control cells or from TPA-treated cells. Four complexes were formed with extracts from control cells or cells treated with TPA for 1 day but were not observed with extracts from cells treated for 4 days. Of the 4 complexes, one was sequence-specific and binding involved uridylate residues and interactions with a 45-kDa protein and a protein doublet of 116 kDa. Binding of this 45/116-kDa complex to the 20-nt conserved cis element most likely regulates negatively PGHS-1 protein accumulation. We have provided evidence that the PGHS-1 gene is regulated at the translational level.

The effects of a high-potency topical steroid on cutaneous healing of burns in pigs.

Burns are dynamic injuries that tend to progress over the course of several days. Steroids inhibit the formation of vasoconstrictive prostanoids that may contribute to this progression of injury. The authors hypothesized that adding topical steroids to a standard antimicrobial agent would reduce the progression of burns and accelerate reepithelialization without increasing infection rates. This was a prospective, blinded, controlled, experimental trial. Forty-eight standardized second-degree burns were created by applying an aluminum bar preheated to 80 degrees C to the flanks of isoflurane-anesthetized young pigs for 20 seconds. Three equal sets of 16 burns were randomly treated with silver sulfadiazine cream (SSD), clobetasol propionate 0.05% (CP), or both (SSD+CP). Daily dressing changes were performed for 14 days. Full-thickness biopsies were taken after injury and at one, two, seven, ten, and 14 days for blinded histopathological evaluation using hematoxylin and eosin (H&E) staining. The primary outcome was the % reepithelialization (REP) calculated by dividing the length of the neoepidermis by the section's total length (interobserver correlation = 0.99). Depth of injury was measured for each dermal element (collagen; epithelial, mesenchymal, and vascular cells; and vessel thrombosis). Comparisons across groups were performed using one-way analysis of variance (ANOVA). A repeated-measures ANOVA was used to compare injury depths over time. This study had 80% power to detect a 33-percentage point difference in REP across groups (two-tailed alpha = 0.05). Pretreatment burn depths were similar across groups. While burn depth changed over time, there was no difference between the groups in burn injury progression. There was no difference across the groups in REP or infection rates at all times. Addition of a potent topical steroid to standard antimicrobial topical agents does not reduce burn depth or accelerate reepithelialization after burns.

The Effects of a High‐potency Topical Steroid on Cutaneous Healing of Burns in Pigs

Abstract Objective: Burns are dynamic injuries that tend to progress over the course of several days. Steroids inhibit the formation of vasoconstrictive prostanoids that may contribute to this progression of injury. The authors hypothesized that adding topical steroids to a standard antimicrobial agent would reduce the progression of burns and accelerate reepithelialization without increasing infection rates. Methods: This was a prospective, blinded, controlled, experimental trial. Forty‐eight standardized second‐degree burns were created by applying an aluminum bar preheated to 80°C to the flanks of isoflurane‐anesthetized young pigs for 20 seconds. Three equal sets of 16 burns were randomly treated with silver sulfadiazine cream (SSD), clobetasol propionate 0.05% (CP), or both (SSD+CP). Daily dressing changes were performed for 14 days. Full‐thickness biopsies were taken after injury and at one, two, seven, ten, and 14 days for blinded histopathological evaluation using hematoxylin and eosin (H&E) staining. The primary outcome was the % reepithelialization (REP) calculated by dividing the length of the neoepidermis by the section's total length (interobserver correlation = 0.99). Depth of injury was measured for each dermal element (collagen; epithelial, mesenchymal, and vascular cells; and vessel thrombosis). Comparisons across groups were performed using oneway analysis of variance (ANOVA). A repeated‐measures ANOVA was used to compare injury depths over time. This study had 80% power to detect a 33‐percentage point difference in REP across groups (two‐tailed alpha = 0.05). Results: Pretreatment burn depths were similar across groups. While burn depth changed over time, there was no difference between the groups in burn injury progression. There was no difference across the groups in REP or infection rates at all times. Conclusions: Addition of a potent topical steroid to standard antimicrobial topical agents does not reduce burn depth or accelerate reepithelialization after burns.

Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets.

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A(2). We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34(+) hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34(+) cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34(+) cells synthesized more PGE(2) than TXB(2) (214 +/- 50 vs. 30 +/- 10 pg/10(6) cells), whereas the reverse was true in mature megakaryocytes (TXB(2) 8,440 +/- 2,500 vs. PGE(2) 906 +/- 161 pg/10(6) cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE(2) and TXB(2) to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE(2) and TXA(2) may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.

Low tonicity mediates a downregulation of cyclooxygenase-1 expression by furosemide in the rat renal papilla.

It is well known that loop diuretics enhance the renal excretion of prostanoids; therefore, this study aimed to characterize the influence of loop diuretics on the intrarenal expression of cyclooxygenases, which are the key enzymes for prostanoid formation. Male Sprague-Dawley rats were infused with furosemide (12 mg/kg per d) for 6 d, and the expression of cyclooxygenase-1 and -2 (Cox-1 and Cox-2) was analyzed in the different kidney zones. Furosemide increased Cox-2 mRNA expression approximately twofold in the cortex, but it left Cox-1 mRNA expression unaltered there. In the outer medulla, furosemide changed neither Cox-1 nor Cox-2 mRNA expression. In the inner medulla, however, furosemide decreased Cox-1 and Cox-2 mRNA levels to approximately 30% and 60% of their control levels, respectively. The downregulation of mRNA was paralleled by a decrease of Cox protein in the collecting ducts and interstitial cells. Moreover, tissue prostaglandin E(2) (PGE(2)) concentrations in the papilla were markedly decreased by furosemide to about 30% of the control level. Furosemide lowered urine osmolality from 1550 mosmol/kg to 480 mosmol/kg; therefore, further consideration was given to the influence of tonicity as a possible mediator of the effects of furosemide on the Cox expression. Water loading was therefore used to reduce the medullary tonicity by a second maneuver. Water loading led to a similar reduction in papillary Cox mRNA expression and PGE(2) content like furosemide. To investigate the influence of the osmolarity on the expression of Cox and the production of PGE(2) under defined in vitro conditions, inner medullary collecting duct cells were incubated with culture medium containing graded amounts of NaCl ranging from 200 mmol/L to 600 mmol/L, and Cox-1 and Cox-2 mRNA abundance were determined after 24 h an 48 h. Cox-1 and Cox-2 mRNA abundance changed in parallel with the osmolarity. The data suggest that loop diuretics decrease the expression of cyclooxygenases and consequently tissue PGE(2) concentrations in the kidney inner medulla. This effect could be related to the breakdown of the papillary osmotic gradient induced by loop diuretics.

LPS-induced synthesis and release of PGE2 in liver macrophages: regulation by CPLA2, COX-1, COX-2, and PGE2 synthase.

Liver macrophages respond to a variety of agents with the release of biologically active mediators including cytokines, nitric oxide, oxygen radicals and prostanoids. A rapid formation of prostanoids (within minutes) is induced by phagocytic stimuli, and by agents activating protein kinase C or increasing cytosolic calcium. A delayed formation of prostanoids (after a lag phase of hours) occurs after stimulation of liver macrophages with lipopolysaccharide (LPS), viruses, cytokines and muramyl tripeptides1.