Loss of claudin-1 in lipopolysaccharide-treated periodontal epithelium.

BACKGROUND AND OBJECTIVE: The epithelial barrier is a critical component of innate immunity and provides protection against microbial invasion. Claudin-1, a tight junction protein, is known to contribute to the epithelial cell barrier. An experimentally induced rat periodontal disease model was used to study the effects of lipopolysaccharide (LPS) on the expression of tight junction-associated molecule genes in the junctional epithelium. MATERIAL AND METHODS: LPS was applied for 8 wk in the gingival sulcus, and junctional epithelium was collected by laser-capture microdissection and subjected to microarray analysis. RESULTS: Microarray analysis identified that expression of the claudin-1 gene was decreased in the epithelium by chronic LPS challenge. Immunohistochemical analysis confirmed the expression of claudin-1 protein in junctional epithelium and that 8 wk of chronic LPS topical application significantly reduced claudin-1 expression. The effect of LPS on claudin-1 protein expression was validated using a porcine junctional epithelial cell culture Transwell model. The epithelial barrier, as measured using transmembrane resistance, was significantly reduced after 3 wk of LPS challenge and this was associated with a decreased level of expression of claudin-1 protein. CONCLUSION: These results confirm that the initiation of experimental periodontal disease is associated with reduction in the expression of claudin-1 gene and protein. This decreased level of a critical tight junction protein may result in the disruption of barrier function and may play an important role in the initiation of periodontal disease.

Regulation of epithelial cell growth factor receptor protein and gene expression using a rat periodontitis model.

BACKGROUND AND OBJECTIVE: Regulation of epithelial cell behavior associated with periodontitis is not well elucidated but many responses will ultimately be regulated by growth factor receptors. Using a rat experimental periodontitis model, protein and gene expression of select growth factor receptors in junctional and pocket epithelium were examined. MATERIAL AND METHODS: Periodontal disease was induced by daily topical application of lipopolysaccharide using an established protocol. Animals were killed at time 0 (control), and at 2 and 8 wk. Frozen tissue samples were collected from the right palatal gingival soft tissue, and the left periodontal tissues were decalcified and embedded in paraffin. Laser microdissection and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify keratinocyte growth factor receptor (KGFR), hepatocyte growth factor receptor (HGFR), epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) gene expression, and in situ RT-PCR localized these increases to specific epithelial cells. Receptor protein expression was examined immunohistochemically. In cell culture, induction of HGFR and KGFR protein expression by serum, lipopolysaccharide and pro-inflammatory cytokines were examined using flow cytometry. RESULTS: Eight-week tissue samples exhibited histological changes consistent with periodontitis. KGFR and HGFR gene and protein expression were significantly induced at the 8 wk time point. KGFR expression was significantly up-regulated in basal and parabasal pocket epithelial cells, but HGFR was up-regulated throughout the pocket epithelium. In cell culture serum, lipopolysaccharide and pro-inflammatory cytokines, interleukin-1beta and tumour necrosis factor-alpha significantly induced KGFR protein receptor expression, but HGFR expression was only induced by serum. CONCLUSION: KGFR and HGFR are highly up-regulated in this model of periodontal disease and may play a significant role in regulating the proliferation and migration of pocket epithelium.

Matrilysin (matrix metalloproteinase-7) expression in human junctional epithelium.

Matrilysin is a matrix metalloproteinase expressed in exocrine and mucosal epithelium in many human tissues. Immunohistochemical staining showed that matrilysin is expressed in suprabasal cells of junctional epithelium facing the teeth and in epithelial cell rests of Malassez. No matrilysin expression was seen in the periodontal pocket tissue. In a tissue culture model mimicking junctional epithelium, matrilysin expression was also observed in suprabasal epithelial cells. Of 13 anaerobic oral bacterial species tested, F. nucleatum, F. necrophorum, P. endodontalis, and P. denticola stimulated matrilysin expression in porcine periodontal ligament epithelial cells from 2.5- to 5.7-fold, compared with untreated cells. The enzyme was localized in intracytoplasmic vesicles that also reacted with antibodies against lysosomal membrane protein h-lamp-1. The results indicate that matrilysin may play an important role in the normal physiology of junctional epithelium.

Exogenous phospholipase C stimulates epithelial cell migration and integrin expression in vitro.

Phospholipase C secreted by bacterial pathogens has been identified as a virulence factor in several human diseases and has been implicated in impeding wound healing. The role of phospholipase C in the intracellular signal control of epithelial growth was studied in normal human skin keratinocytes cultured in conditions simulating aspects of wound healing. Bacillus cereus phospholipase C decreased cell-cell contact and increased cell migration resulting in disruption of the advancing epithelial sheet. Phospholipase C-induced migration was blocked by inhibitor of the phosphoinositol signal transduction pathway neomycin sulfate and protein kinase C inhibitor RO-31-8220. Induced migration was associated with elevated levels of matrix metalloproteinase-9 which, when blocked by tissue inhibitor of metalloproteinase-1, was accompanied by a loss of migration. Adhesion studies showed that phospholipase C treatment enhanced cell binding to fibronectin, vitronectin and collagen IV. Immunostained phospholipase C-stimulated cells cultured on fibronectin showed enhanced expression and relocation of the integrin subunits alpha(v), alpha5 and beta1. Confocal microscopy showed that phospholipase C-induced levels of integrin subunit beta1 were predominantly deposited on the basal surface of the cell apparently in focal contacts and associated with actin stress fibers. These results indicate that exogenous phospholipase C signaling from a bacterial source may play an important role in perturbing normal reepithelialization via altered expression of integrins and matrix metalloproteinase-9.

Oral mucosal Langerhans' cells as target, effector and vector in HIV infection.

The mechanism underlying a transition of the oral cavity mucosal epithelium towards susceptibility to opportunistic infections in HIV-seropositive patients was investigated. Phenotypic markers CD1a, HLA-DR, and CD86 of oral mucosal Langerhans' cells (LCs), p17 core protein of human immunodeficiency virus (HIV), and CD45RO of memory T cells were labeled on oral hairy leukoplakia lesional biopsies and clinically normal autologous tissue of HIV-infected patients. HIV p17 protein was detected in association with mucosal LCs, mainly within the lesional epithelium. There were significant correlations between the detection of HIV p17 and the depletion of LCs, and between the depletion of LCs and the presence of hairy leukoplakia lesions. Conjugates of activated LCs and memory T cells were also evident in the submucosal area of lesional biopsies. The findings from this study support the hypothesis that oral mucosal LCs are also the target of HIV infection. Cytopathic changes of LCs caused by productive HIV infection may contribute to selective depletion of LCs, which may impair the mucosal immunologic protection against colonization by microorganisms causing HIV-associated oral mucosal lesions.

Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen and fibronectin.

Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.

Collagenase-3 (matrix metalloproteinase-13) expression is induced in oral mucosal epithelium during chronic inflammation.

Increased proliferation of mucosal epithelium during inflammation is associated with degradation of subepithelial connective tissue matrix and local invasion of the epithelial cells. Here we have studied, whether collagenase-3 (MMP-13), a collagenolytic matrix metalloproteinase with an exceptionally wide substrate specificity, is expressed in the epithelium of chronically inflamed mucosa. Examination of human gingival tissue sections from subjects with chronic adult periodontitis with in situ hybridization revealed marked expression of MMP-13 in basal cells of some epithelial rete ridges expanding into connective tissue. Immunohistochemical staining demonstrated that these cells also expressed strongly laminin-5, suggesting that they are actively migrating cells. A strong signal for MMP-13 mRNA was occasionally also noted in the suprabasal epithelial cells facing the gingival pocket, whereas no collagenase-1 (MMP-1) mRNA was detected in any areas of the epithelium. MMP-13 expression was also detected in fibroblast-like cells associated with collagen fibers of the inflamed subepithelial connective tissue. In organ culture of human oral mucosa, MMP-13 mRNA expression was observed in epithelial cells growing into connective tissue of the specimens. Regulation of MMP-13 expression was examined in cultured normal nonkeratinizing epithelial cells isolated from porcine periodontal ligament. In these cells, MMP-13 expression at the mRNA and protein level was potently enhanced (up to sixfold) by tumor necrosis factor-alpha, transforming growth factor-beta(1), and transforming growth factor-alpha and by keratinocyte growth factor in the presence of heparin. In addition, plating periodontal ligament epithelial cells on type I collagen stimulated MMP-13 expression (sevenfold) as compared with cells grown on tissue culture plastic. The results of this study show, that expression of MMP-13 is specifically induced in undifferentiated epithelial cells during chronic inflammation due to exposure to cytokines and collagen. Thus, it is likely that MMP-13 expression is instrumental in the subepithelial collagenolysis and local invasion of the activated mucosal epithelium into the connective tissue.

Effects of titanium substratum and grooved surface topography on metalloproteinase-2 expression in human fibroblasts.

The chemical and topographic effects of commercially pure titanium on cell morphology and the regulation of matrix metalloproteinase-2 (MMP-2) gene expression, synthesis, and activity were investigated in early passage human gingival fibroblasts. Scanning electron microscopy showed that on smooth titanium (Ti), fibroblasts remained well spread and randomly oriented throughout the culture period. In contrast, cells on V-shaped grooved titanium (VTi) were oriented along the grooves by 16 h and proliferated in this organization throughout the culture period. The effects of substratum surface chemistry on MMP-2 expression were found to be distinct from those of topography. Northern hybridization analysis of fibroblasts cultured on Ti revealed an MMP-2 mRNA time-course expression pattern parallel to that observed on the tissue culture plastic (TCP) dishes, but at significantly lower levels at each time-point. The Ti mRNA levels were decreased by 34% at 16 h, 55% at 40 h, and 45% at 90 h relative to TCP. In contrast, MMP-2 mRNA expression on VTi showed both an altered time-course expression pattern and altered levels compared to Ti and TCP. Relative to TCP, VTi MMP-2 mRNA levels were approximately 80% less at 16 h and approximately 50% less at 40 h, but not significantly different at 90 h. Relative to Ti, VTi MMP-2 mRNA levels were approximately 75% less at 16 h, but approximately 40% greater at 40 h and approximately 70% greater at 90 h. These differences may be explained in part by the observed changes in MMP-2 mRNA half-life which decreased by approximately 40% on Ti but increased by over fourfold on VTi relative to TCP. The smooth Ti also showed an approximate twofold increase of MMP-2 secretion in the late cultures over TCP controls. These results indicate that substratum surface chemistry and topography-induced changes in cell shape can alter MMP-2 expression in normal fibroblasts. The molecular approach to investigating the major molecules involved in tissue degradation may provide sensitive indicators of tissue remodeling at the tissue-biomaterial interface.

Bacterial phospholipase C upregulates matrix metalloproteinase expression by cultured epithelial cells.

Phospholipase C (PLC) is a putative virulence factor of several pathogenic bacteria. We studied if exogenous PLC would perturb epithelial behavior in infected tissues. Gelatin and casein zymography of cell culture medium indicated that the broad-spectrum PLC of Bacillus cereus induced matrix metalloproteinase (MMP) production in epithelial cells of human skin (NHEK), human gingiva (HGE), and porcine periodontal ligament (PLE). In all three cell types, the strongest increase (ninefold) at 0.1 U/ml was seen in the MMP-9 (92-kDa gelatinase) activity, and the effect was dose dependent in the range of 0.1 to 1.0 U/ml. A relatively weaker increase (twofold) in MMP-2 (72-kDa gelatinase) was also observed in each cell type. PLC induction of MMP-3 (48-kDa stromelysin) was also seen in NHEK and HGE on gelatin and more sensitively for PLE by casein zymography (fivefold). Total gelatinolytic activity as measured by degradation of 14C-labeled denatured type I collagen increased by about 18-fold (NHEK), 12-fold (HGE), and 14-fold (PLE). Northern analysis showed a clear increase in the MMP-9, and a minor increase in MMP-3 mRNA levels but no significant increase in MMP-2 mRNA levels. Further studies with PLE revealed that MMP-9 induction by PLC progressively increased with the length of cell culture time in the absence of serum. PLC induction of MMPs was polar, with MMP-9 and MMP-3 secreted primarily in the apical direction and MMP-2 secreted mainly in the basal direction. The PLC effect was blocked by neomycin, an inhibitor of the phosphoinositol signal pathway. No significant effects were observed in MMP expression with the calcium ionophore A23187 or phospholipase A2. Morphologically, PLC treatment resulted in reduced contacts between the cultured cells and loss of the cell surface microvilli. These results suggest that PLC secreted by bacterial pathogens may disrupt epithelium of infected tissue and increase the subepithelial tissue destruction through induction of MMPs.

Targeting gene expression to hypoxic tumor cells.

Solid tumors with areas of low oxygen tension (hypoxia) have a poor prognosis, as cells in this environment often survive radiation and chemotherapy. In this report we describe how this hypoxic environment can be used to activate heterologous gene expression driven by a hypoxia-responsive element (HRE), which interacts with the transcriptional complex hypoxia-inducible factor-1 (HIF-1). Our results demonstrate that the HIF-1/HRE system of gene regulation is active in hypoxic tumor cells and show the potential of exploiting tumor-specific conditions for the targeted expression of diagnostic or therapeutic genes in cancer therapy.

Randomised controlled trial of steroid withdrawal in renal transplant recipients receiving triple immunosuppression.

BACKGROUND: The combination of cyclosporin, azathioprine, and prednisolone (triple immunosuppression) is the most commonly used immunosuppressive regimen early after renal transplantation, but the risks and benefits of maintaining the steroid component of this regimen in the long term are uncertain. METHODS: A randomised controlled trial of steroid withdrawal was conducted among renal transplant patients receiving triple immunosuppression. Between one and six years after transplantation, 100 such patients were randomised either to reduce prednisolone treatment to zero over about four months or to maintain their triple immunosuppression unchanged. Outcome was analysed according to "Intention-to-treat". FINDINGS: In 42 (86%) of 49 patients allocated to steroid withdrawal, complete steroid withdrawal was achieved. Although these patients did not experience defined acute rejection episodes, insidious increases in plasma creatinine were observed more frequently in this group than in the controls. In 97 patients surviving one year after trial entry, plasma creatinine exceeded the baseline by more than 25% at some time in the first year in 25 (53%) of 47 in the steroid withdrawal group compared with 9 (18%) of 50 in the control group (p < 0.001, chi-square test). In some cases these increases were transient. However, when corrected for the baseline (entry) value significant differences between groups were apparent in both mean plasma creatinine and mean creatinine clearance; mean (SD) plasma creatinine values at entry, immediately after withdrawal, and at one year were 138 (27), 151 (36), and 150 (36) mumol/L in the steroid withdrawal group versus 138 (34), 140 (51), and 139 (47) mumol/L in the control group (p = 0.017, analysis of covariance). Steroid withdrawal patients showed a further rise in mean plasma creatinine to 160 (44) and 161 (65) mumol/L at two and three years from trial entry. Changes in several clinical and metabolic indices were also observed in association with steroid withdrawal. Blood pressure declined but the reduction was incompletely sustained, being more evident immediately after steroid withdrawal than at one year. Total cholesterol declined about 1 mmol/L in the steroid withdrawal group. Other changes associated with steroid withdrawal were reductions in white cell count and haemoglobin and increases in plasma phosphate and alkaline phosphatase. INTERPRETATION: Late steroid withdrawal is feasible in most patients with stable graft function on triple immunosuppression and has potentially beneficial metabolic effects. However, a substantial proportion of patients show a reduction in graft function, indicating a need for caution in considering the long term outcome.

Chymotrypsin-like enzyme secretion is stimulated in cultured epithelial cells during proliferation and in response to Actinobacillus actinomycetemcomitans.

A chymotrypsin-like enzyme was partially purified from culture medium of epithelial cells of human skin, human gingiva and porcine periodontal ligament by aprotinin-affinity chromatography. The enzyme levels from all three cell types were low in quiescent cultures but increased markedly when the cells were allowed to proliferate. The biphasic elution profile of the enzyme from the affinity column closely matched that of alpha-chymotrypsin and the protein comigrated with it on polyacrylamide gels at 27,000 ML. Synthetic substrate tests of purified fractions showed strong chymotrypsin-like but no trypsin-like or elastase-like activity. Inhibition of protease activity and pH optimum in the range of 7.5-8.0 were consistent with chymotrypsin-like enzymes. Secreted activity was found to be significantly increased by phorbol myristate acetate treatment in a time-course that differed from that of elastase-like activity. Keratinocyte growth factor and epidermal growth factor but not transforming growth factor-beta increased the chymotrypsin-like activity in a concentration-dependent manner. The enzyme secretion by epithelial cells was strongly elevated by exposure to 5 of 6 Actinobacillus actinomycetemcomitans strains isolated from plaque samples of juvenile periodontitis patients. These results indicate that chymotrypsin-like enzymes are secreted by proliferative phenotypes of normal epithelial cells. This enzyme may, therefore, play a role in epithelial physiology and in cell response to certain pathogenic bacteria.

Endothelin-1 is involved in norepinephrine-induced ventricular hypertrophy in vivo. Acute effects of bosentan, an orally active, mixed endothelin ETA and ETB receptor antagonist.

BACKGROUND: Endothelin-1 (ET-1) has potent effects on cell growth and induces hypertrophy of cultured ventricular myocytes. Catecholamines increase expression of ET-1 mRNA by cultured myocytes. We investigated the role of endogenous ET-1 in catecholamine-induced hypertrophy in vivo by studying the effects of continuous norepinephrine infusion on physical and molecular markers of ventricular hypertrophy, ventricular and noncardiac expression of ET-1 mRNA, and the acute effects of bosentan, an orally active ETA and ETB receptor antagonist. METHODS AND RESULTS: Seventy male Sprague-Dawley rats (175 to 200 g) were divided into four groups: (1) sham-operated rats, (2) norepinephrine-infused rats (600 micrograms.kg-1.h-1 by subcutaneous osmotic pump, up to 7 days), (3) sham-operated rats given bosentan, and (4) norepinephrine-infused rats given bosentan. Bosentan (100 mg/kg once daily) was administered by gavage for 6 days starting 1 day before operation. Norepinephrine caused increases in absolute ventricular weight and ratios of ventricular weight to body weight and ventricular RNA to protein. Ventricular expression of mRNAs for atrial natriuretic factor, skeletal alpha-actin, and beta-myosin heavy chain, which in adult rat ventricle are indicators of hypertrophy, also increased. Ventricular expression of ET-1 mRNA was elevated in the norepinephrine group at 1, 2, and 3 days. By 5 days, this had fallen to control levels. In lung, kidney, and skeletal muscle, norepinephrine did not significantly increase expression of ET-1 mRNA. Bosentan attenuated norepinephrine-induced increases in ventricular weight, ratio of RNA to protein, and expression of skeletal alpha-actin mRNA and beta-myosin heavy chain mRNA at 5 days, but it did not attenuate increased ventricular expression of atrial natriuretic factor mRNA. CONCLUSIONS: These data suggest that endogenous ET-1 plays a direct role in mediating norepinephrine-induced ventricular hypertrophy in vivo.

Effects of titanium on transcriptional and post-transcriptional regulation of fibronectin in human fibroblasts.

The effects of commercially pure titanium (Ti) on the regulation of fibronectin gene expression and synthesis were investigated in early-passage human gingival fibroblasts. The fibroblasts were cultured on 50 nm Ti-coated silicon wafers treated with radio-frequency glow discharge prior to use and on Falcon tissue culture plastic (TCP) dishes as a control. Northern hybridization analysis revealed that fibroblasts cultured on Ti reduced the fibronectin mRNA level by 58% at 16 h, but increased it by 2.6-fold at 90 h, although the cell numbers and house-keeping gene GAPD mRNA levels on these two surfaces were essentially the same. The amount of total RNA was slightly less on the Ti surface. While the total [35S]methionine incorporation was essentially unaltered, the amount of [35S]methionine-labeled fibronectin was significantly increased in cells cultured on a Ti surface in early cultures but decreased in the late cultures. The apparent discrepancy between the increased fibronectin mRNA levels and decreased translation could be explained by a 30% reduction in fibronectin mRNA half life in cells cultured on Ti. The distribution of fibronectin between the medium and the cell layer also was altered on Ti surfaces, with a approximately 100-fold increase of fibronectin assembled in extracellular matrix at 16 h, but a 36% reduction at 90 h. In contrast, the amount of fibronectin recovered in the medium was essentially unchanged. The total amount of protein assembled into the extracellular matrix by cells on Ti increased 2.1-fold at 16 h but decreased by 19% in 90-h cultures. These significant changes in fibronectin gene activity and gene product distribution by cells cultured on Ti surfaces demonstrate that the surface chemistry of biomaterials can selectively regulate the cellular behavior at the molecular level and, conversely, that molecular biological techniques provide sensitive indicators of the molecular biocompatibility of implant materials.

Stimulation of collagenase (matrix metalloproteinase-1) synthesis in histiotypic epithelial cell culture by heparin is enhanced by keratinocyte growth factor.

The role of heparin and heparan sulfate in the control of epithelial collagenase production was investigated utilizing a histiotypic cell culture model. The effect of keratinocyte growth factor (KGF), a heparin-binding growth factor, on collagenase secretion was also examined. Heparin, and, to a lesser extent, heparan sulfate induced release of a 58-kDa, gelatin-degrading enzyme which was subsequently identified as the collagenase, matrix metalloproteinase-1. The increase in collagenase secretion by heparin was further enhanced by the addition of KGF. KGF alone did not have any effect. Analysis of secreted radiolabelled proteins showed that the increase in collagenase activity was not due to a general increase in protein synthesis. Synthesis of collagenase protein was specifically increased by heparin and further increased by KGF plus heparin. Heparin and heparan sulfate in combination with KGF may thus have important roles in the regulation of epithelial cell collagenase under conditions such as inflammation and wound healing.

Influence of hypoxia on hepatic and renal endothelin gene expression.

This study aimed to investigate the influence of different forms of tissue hypoxia on the expression of the endothelin genes in kidneys and livers. Tissue hypoxia in rats was induced by five different manoeuvres, namely hypoxia (8% O2), functional anaemia (0.1% CO), haemorrhage (haematocrit, hct = 0.12), cobalt treatment (60 mg/kg) for 6 h each and renal artery stenosis (0.2-mm clips) for 2 days. Endothelin-1 (ET-1) mRNA levels in the kidneys were increased by 200% with renal artery stenosis, 70% by hypoxia, 50% by anaemia, 30% by CO, but were not changed by cobalt. ET-3 mRNA in the kidneys decreased during renal artery clipping and cobalt treatment and were not significantly changed under the other conditions. ET-2 mRNA was not detected in the kidneys and livers. The abundance of ET-1 in the livers of normoxic animals was about 15% of that found in the kidney. Hypoxia increased ET-1 mRNA by 200%, haemorrhage by 400%, whilst CO and cobalt did not change hepatic ET-1 gene expression. The abundance of ET-3 mRNA in the livers of normoxic animals was about 6% of that found in the kidneys. The expression of the ET-3 gene in the livers was decreased by CO, but was not changed by any of the other experimental conditions used. These findings suggest that hyoxaemia and tissue hypoxia are moderate stimuli for the expression of the ET-1 gene but not for the ET-3 gene in the kidney and more potent stimuli in the liver, whilst cobalt does not activate ET-1 gene expression in the kidneys nor the livers.

Tissue expression of endothelin-1 mRNA in endotoxaemia.

Circulating endothelin-1 concentrations are elevated in animal models of sepsis. The major actions of endothelin-1 appear to be as a local autocrine and paracrine factor, rather than as a circulating hormone, and plasma concentrations may not reflect local tissue concentrations. We therefore measured tissue expression of mRNA encoding pre-pro-endothelin-1 by RNase protection assays, as an indicator of local production of ET-1 in an in vivo rat model of endotoxaemia. The effects of dexamethasone pre-treatment were also examined. There was a tissue-specific increase in pre-pro-endothelin-1 mRNA in endotoxaemia, apparent at 6h after endotoxin challenge in heart and lung. No significant changes in expression were seen in kidney or skeletal muscle. Dexamethasone pre-treatment significantly attenuated the rise in pre-pro-endothelin-1 mRNA in heart at 6h. Therefore, we have demonstrated tissue-specific differences in the effect of endotoxin upon pre-pro-endothelin-1 mRNA expression and in sensitivity to dexamethasone. This could account for some of the inter-tissue differences seen in local vascular response to endotoxin.