Bone Marrow�Cderived Endothelial Progenitor Cells and Endothelial Cells May Contribute to Endothelial Repair in the Kidney Immediately After Ischemia�CReperfusion

In ischemic acute kidney injury, renal blood flow is decreased. We have previously shown that reperfused, transplanted kidneys exhibited ischemic injury to vascular endothelium and that preservation of peritubular capillary endothelial integrity may be critical to recovery from ischemic injury. We hypothesized that bone marrow–derived (BMD) endothelial progenitor cells (EPCs) might play an important role in renal functional recovery after ischemia. We tested this hypothesis in recipients of cadaveric renal allografts before and for 2 weeks after transplantation. We found that the numbers of circulating CD34-positive EPCs and CD146-positive endothelial cells (ECs) decreased immediately after ischemia–reperfusion. In renal allograft tissues obtained 1 hr after reperfusion, CD34-positive cells were more frequently observed along the endothelial lining of peritubular capillaries compared with non-ischemic controls. Moreover, 0–17.5% of peritubular capillary ECs were of recipient origin. In contrast, only 0.1–0.7% of tubule cells were of recipient origin. Repeat graft biopsy samples obtained 35 and 73 days after transplant did not contain capillary ECs of recipient origin, whereas 1.4% and 12.1% of tubule cells, respectively, were of recipient origin. These findings suggest that BMD EPCs and ECs may contribute to endothelial repair immediately after ischemia–reperfusion. (J Histochem Cytochem 58:687–694, 2010)     

Differential sensitivity of endothelial cells of various species to apoptosis induced by gene transfer of Fas ligand: role of FLIP levels

BACKGROUND: Fas ligand expression by cells of the vessel wall has been proposed to play a role in normal and pathologic conditions. Genetic engineering of vascularized organs for endothelial cell (EC) expression of FasL could protect the endothelium and underlying tissues from infiltrating Fas+ leukocytes. Nevertheless, the endogenous expression of FasL by ECs of different species and the potential deleterious effects of enforced FasL expression by ECs are largely unknown. In human ECs, levels of FLICE/caspase 8-inhibitory protein (FLIP) have been shown to control apoptosis mediated by Fas. MATERIALS AND METHODS: Cell surface expression of FasL in rat, mouse, human, and pig ECs was obtained using recombinant adenoviruses or transient plasmid transfection assays. FasL expression was evaluated by FACS analysis and cytotoxicity assays. Apoptosis was evaluated using annexin V, TUNEL, and cytotoxicity assays. FLIP levels were evaluated by Western blot analysis and overexpression was obtained by transient transfection. RESULTS: Analysis of ECs from different species showed that FasL was predominantly present in the cytoplasm, and depending on the species, little or no cell surface expression was detected. Enforced cell surface expression of FasL on rat or mouse ECs, either in culture or within the vessel wall resulted in massive apoptosis. In contrast, porcine or human ECs were completely resistant to apoptosis mediated by Fas-FasL interaction. Markedly reduced FLIP levels were observed in rat and mouse ECs compared to human and porcine ECs. Overexpression of FLIP in rat ECs conferred protection against cell surface expression of FasL. CONCLUSIONS: The consequences of FasL overexpression depend on the subcellular compartment and species in which FasL enforced expression is targeted and this is at least partially related to FLIP levels.     

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.     

Lipopolysaccharide induces a fibrotic‐like phenotype in endothelial cells

Endothelial dysfunction is crucial in endotoxaemia‐derived sepsis syndrome pathogenesis. It is well accepted that lipopolysaccharide (LPS) induces endothelial dysfunction through immune system activation. However, LPS can also directly generate actions in endothelial cells (ECs) in the absence of participation by immune cells. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge. However, the mechanism that confers endothelial resistance to LPS is not known. LPS‐resistant ECs exhibit a fibroblast‐like morphology, suggesting that these ECs enter a fibrotic programme in response to LPS. Thus, our aim was to investigate whether LPS is able to induce endothelial fibrosis in the absence of immune cells and explore the underlying mechanism. Using primary cultures of ECs and culturing intact blood vessels, we demonstrated that LPS is a crucial factor to induce endothelial fibrosis. We demonstrated that LPS was able and sufficient to promote endothelial fibrosis, in the absence of immune cells through an activin receptor–like kinase 5 (ALK5) activity–dependent mechanism. LPS‐challenged ECs showed an up‐regulation of both fibroblast‐specific protein expression and extracellular matrix proteins secretion, as well as a down‐regulation of endothelial markers. These results demonstrate that LPS is a crucial factor in inducing endothelial fibrosis in the absence of immune cells through an ALK5‐dependent mechanism. It is noteworthy that LPS‐induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxaemia‐derived sepsis syndrome and other inflammatory diseases.     

Endotoxin Induces Fibrosis in Vascular Endothelial Cells through a Mechanism Dependent on Transient Receptor Protein Melastatin 7 Activity

The pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of endothelial cells (ECs) into activated fibroblasts through endothelial­to­mesenchymal transition mechanism. Fibrogenesis is highly dependent on intracellular Ca²⁺ concentration increases through the participation of calcium channels. However, the specific molecular identity of the calcium channel that mediates the Ca²⁺ influx during endotoxin-induced endothelial fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However, the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs, we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7 expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca²⁺ levels were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis.     

Protection from lipopolysaccharide-induced pulmonary microvascular endothelial cell injury by activation of hedgehog signaling pathway

Pulmonary microvascular endothelial cells (PMVECs) are critically involved in the pathogenesis of acute lung injury. Hedgehog signaling pathway plays a fundamental role in embryonic development as well as adult morphogenesis and carcinogenesis. As the priming protein of hedgehog signaling pathway, sonic hedgehog (Shh) may recently be advantage for decreasing endothelial injury and promoting the repair of endothelial barrier function. To investigate the expression and role of hedgehog signal pathway in PMVECs injured by lipopolysaccharide (LPS), cells were divided into six groups: control group, LPS group, rhShh group, LPS + rhShh group, rhShh + cyclopamine group, and LPS + rhShh + cyclopamine group. Real time RT-PCR and Western blotting were used to detect the mRNA and protein expression of hedgehog signal molecules including Shh, Patched-1 (Ptc-1) and Gli1 in nucleus. The activity of PMVECs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In this study, we found that Shh, Ptch1, and Gli1 were expressed in rat PMVECs and their expression decreased when cells were treated by LPS. In the other hand, LPS inhibited the activity of rat PMVECs and caused the cells injury. Activation of Hedgehog signaling pathway by Shh could elevate the activity of PMVECs with pretreatment by LPS. Therefore, hedgehog signaling pathway should play a protective role on injury PMVECs by LPS.     

Myosin light-chain kinase regulates endothelial calcium entry and endothelium-dependent vasodilation.

Activation of smooth muscle myosin light-chain kinase (MLCK) causes contraction. Here we have proven that MLCK controls Ca2+ entry (CE) in endothelial cells (ECs): MLCK antisense oligonucleotides strongly prevented bradykinin (BK)- and thapsigargin (TG)-induced endothelial Ca2+ response, while MLCK sense did not. We also show that the relevant mechanism is not phosphorylation of myosin light-chain (MLC): MLC phosphorylation by BK required CE, but MLC phosphorylation caused by the phosphatase inhibitor calyculin A did not trigger Ca2+ response. Most important, we provide for the first time strong evidence that, in contrast to its role in smooth muscle cells, activation of MLCK in ECs stimulates the production of important endothelium-derived vascular relaxing factors: MLCK antisense and MLCK inhibitors abolished BK- and TG-induced nitric oxide production, and MLCK inhibitors substantially inhibited acetylcholine-stimulated hyperpolarization of smooth muscle cell membrane in rat mesenteric artery. These results indicate that MLCK controls endothelial CE, but not through MLC phosphorylation, and unveils a hitherto unknown physiological function of the enzyme: vasodilation through its action in endothelial cells. The study discovers a counter-balancing role of MLCK in the regulation of vascular tone.     

Lipopolysaccharide-stimulated responses in rat aortic endothelial cells by a systems biology approach

The vascular endothelium plays an important role in regulating immune and inflammatory responses to resist pathogens infection. Although it has been known that lipopolysaccharide (LPS) is a critical inducer of sepsis or endotoxemia, the systematic responses of LPS-stimulation in endothelial cells (ECs) are still unclear. The present study aims to analyze the late-phase responses of LPS-induced rat aortic ECs by using systematic biology approaches, including rat cDNA microarray, 2-DE and MALDI-TOF MS/MS, and cytokine protein array. Furthermore, to improve the efficiency of analysis of the bulk systematic data of rat, we designed a set of bioinformatic tools to convert and integrate these rat data into the corresponding human genes or proteins IDs based on BioCarta, KEGG, and Gene Ontology databases. Using the systematic analysis, it was shown that LPS could promote some signaling or metabolic pathways as well as pathophysiologic phenomena of proliferation, atherogenesis, inflammation, and apoptosis through activated nuclear factor-kappaB pathway in ECs. Interestingly, ECs also activated the mediators of anti-inflammation, antiapoptosis, and antioxidation to protect themselves. Moreover, the expressions of altered genes, proteins, and their involvement in the hypothetical signaling pathway can provide further understanding of inflammation associated responses in ECs.     

Non-neuronal nicotinic alpha 7 receptor, a new endothelial target for revascularization

Alpha 7 nicotinic acetylcholine receptor (alpha7 nAChR) is widely expressed in the central and peripheral nervous systems, and is also found in several non-neuronal tissues, such as endothelial cells (ECs), bronchial epithelial cells, skin keratinocytes and vascular smooth muscle cells. Recent evidence suggests that alpha7 nAChR is involved in angiogenesis. Here, we investigated the feasibility of alpha7 nAChR for revascularization in ischemic heart disease. RT-PCR and immunohistochemistry were used to examine the expression of alpha7 nAChR in human umbilical vein endothelial cell (HUVECs). The cellular function was examined using MTT, fluorescence confocal microscopy and angiogenesis assay in vitro. The capillary density in the rat model of myocardial infarction (MI) was investigated using immunohistochemistry. The results showed that alpha7 nAChR agonists choline increased the expression of alpha7 nAChR mRNA and protein, the intracellular Ca 2+ concentration, proliferation and tube formation of ECs. Reverse effects were observed by using alpha7 nAChR antagonist alpha-BTX. Furthermore, in the rat model of MI, alpha7 nAChR agonist enhanced the capillary density in ischemic tissues, whereas antagonist mecamylamine and alpha-BTX inhibited the effect. Our results suggest that alpha7 nAChR is involved in the regulation of cellular function in ECs, and capillary formation in MI, which are the important steps of angiogenesis. Therefore, alpha7 nAChR on ECs may be a new endothelium target for revascularization in therapeutic angiogenesis of ischemic heart disease.     

Endothelial barrier dysfunction caused by LPS correlates with phosphorylation of HSP27in vivo

Lung edema during sepsis is triggered by formation of gaps between endothelial cells followed by macrophage infiltration. Endothelial gap formation has been proposed to involve changes in the structure of the actin filament cytoskeleton. Heat shock protein 27 (HSP27) is believed to modulate actin filament dynamics or structure, in a manner dependent on its phosphorylation status. We hypothesized that HSP27 may play a role in endothelial gap formation, by affecting actin dependent events in endothelial cells. As there has been no report concerning HSP27 in lung edema in vivo, we examined induction and phosphorylation of HSP27 in lung following LPS injection, as a model of sepsis. In lung, HSP27 mainly localized in capillary endothelial cells of the alveolus, and in smooth muscle cells of pulmonary arteries. HSP27 became significantly more phosphorylated at 3 h after LPS treatment, while the distribution of HSP27 remained unchanged. Pre-treatment with anti-TNFalpha antibody, which has been shown to reduce lung injury, blocked increases in HSP27 phosphorylation at 3 h. HSP27 phosphorylation was also increased in cultured rat pulmonary arterial endothelial cells (RPAEC) by treatment with TNFalpha, LPS, or H2O2. This phosphorylation was blocked by pre-treatment with SB203580, an inhibitor of the upstream kinase, p38 MAP kinase. Increased endothelial permeability caused by H2O2 in vitro was also blocked by SB203580. The amount of actin associated with HSP27 was reduced after treatment with LPS, or H2O2. In summary, HSP27 phosphorylation temporally correlated with LPS induced pathological endothelial cell gap formation in vivo and in a cell culture model system. This is the first report of increased HSP27 phosphorylation associated with pathological lung injury in an animal model of sepsis.     

Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Summary Membrane-bound Ca²⁺-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells.The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca²⁺-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca²⁺-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca²⁺-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca²⁺-ATPase, postulating for the activities of Ca²⁺-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.     

Lipopolysaccharide inhibits activation of latent transforming growth factor-β in bovine endothelial cells

The activation of latent transforming growth factor-β (TGF-β) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF-β levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor-1 (PAI-1), we have tested whether LPS might suppress latent TGF-β activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 μM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF-β secreted into the culture modium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF-β expression as well as the suppression of surface activation of latent TGF-β. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF-β did not occur in cells maintained in LPS-contaminated culture medium. © 1995 Wiley-Liss, Inc.     

Ultracytochemical studies of capillary endothelial cells in the rat central nervous system

The ultracytochemical localization of eight hydrolytic enzymes (TMPase, 5'-NPase, TPPase, TTPase, Mg++-ATPase, Ca++-ATPase, ALPase and K+-NPPase) and one oxidative enzyme (MAO) was determined in rat brain capillary endothelial cells. In the somal plasma membrane, the enzymatic activity was mainly located in the antiluminal plasma membrane. This finding was appropriate for enzymes possessing the optimal pH at alkaline ranges, except for alkaline phosphatase. Most enzymes investigated showed a positive reaction on the pinocytotic vesicles of capillary endothelial cells. Differences in the intensity of the enzyme activities of the luminal and antiluminal plasma membranes may reflect the polarity in the capillary endothelial cells and relate to blood-brain barrier mechanisms.     

Epithelial properties of brain capillary endothelium

The specialized endothelial cells (ECs) in brain capillaries provide a blood-brain barrier to some solutes while facilitating transcapillary exchange of other solutes. In addition, brain capillaries may contribute to the secretion of spinal fluid, a process that is typically mediated by epithelial cells. This proposal is supported by the many epithelial properties of brain capillary ECs including the presence of 1) continuous tight junctions, 2) low transcellular permeability, 3) transcellular concentration gradients, 4) a transcellular potential difference, 5) a high transcellular resistance, and 6) an asymmetrical distribution of transport systems between the luminal and antiluminal plasma membranes. Thus, the brain capillary contains ECs that are structurally and perhaps functionally related to an epithelial cell. These unique features of brain ECs undoubtedly play an important role in regulating the formation and composition of the brain's interstitial fluid.     

Signal-regulatory protein alpha-CD47 interactions are required for the transmigration of monocytes across cerebral endothelium

Monocyte infiltration into inflamed tissue requires their initial arrest onto the endothelial cells (ECs), followed by firm adhesion and subsequent transmigration. Although several pairs of adhesion molecules have been shown to play a role in the initial adhesion of monocytes to ECs, the mechanism of transendothelial migration is poorly defined. In this study, we have investigated the role of signal-regulatory protein (SIRP)alpha-CD47 interactions in monocyte transmigration across brain ECs. CD47 expression was observed in vivo on cerebral endothelium of both control animals and animals suffering from experimental allergic encephalomyelitis. To investigate whether SIRPalpha-CD47 interactions are instrumental in the trafficking of monocytes across cerebral EC monolayers, in vitro assays were conducted in which the migration of monocytes, but not adhesion, was found to be effectively diminished by blocking SIRPalpha and CD47 on monocytes and ECs, respectively. In this process, SIRPalpha was found to interact solely with its counterligand CD47 on ECs. Overexpression of the CD47 molecule on brain ECs significantly enhanced monocytic transmigration, but did not affect adhesion. SIRPalpha-CD47-mediated transendothelial migration involved Gi protein activity, a known signaling component of CD47. Finally, cross-linking of CD47 on brain ECs induced cytoskeletal reorganization of the endothelium, a process that was Gi protein independent. These data provide the first evidence that the interaction of CD47 with its monocytic counterligand SIRPalpha is of importance in the final step of monocyte trafficking into the brain, a critical event in the development of neuroinflammatory diseases.     

Vascular dysfunction in aged mice contributes to persistent lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non‐resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro‐fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non‐resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ‐induced pro‐fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ‐induced lung fibroblast activation in 2D and 3D co‐cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.     

Elastase-like enzyme in the aorta of spontaneously hypertensive rats

In an attempt to obtain information regarding vascular elastase in arterial hypertension, we examined biochemical changes in elastase-like enzyme activity, and the intravascular localization of elastase by immunohistochemical techniques in the aorta of spontaneously hypertensive rats (SHR). In the biochemical study, aortic elastase-like enzyme activity was significantly higher in SHR than in controls. Using an antibody against rat pancreatic elastase raised in the rabbit, it was demonstrated immunohistochemically that the enzyme was localized in the endothelial cells and subendothelial spaces in the aorta of control animals. In SHR, elastase was also demonstrated in medial smooth muscle cells and particularly in the modified smooth muscle cells in areas of intimal thickening. Some vacuoles in the smooth muscle cells also showed positive enzyme staining. Elastase seems to play an important role in the development of hypertensive vascular changes.     

Lipopolysaccharide alters ecto-ATP-diphosphohydrolase and causes relocation of its reaction product in experimental intrahepatic cholestasis

Lipopolysaccharide (LPS), a bacterial endotoxin, exerts profound inflammatory actions toward various tissues and cells. We induced intrahepatic cholestasis in rats by administration of LPS and followed ecto-ATP-diphosphohydrolase (ecto-apyrase) activity in the liver. The activity of the enzyme had decreased to 77% 2 h after injection compared with the activity in control animals. The maximum decrease was detected 24 h after administration. The activity was found to have partially recovered 1 week after injection, but had yet to reach control levels. In contrast to the decrease in ecto-apyrase activity, there were increases in alkaline phosphatase activity and bilirubin concentration, markers of cholestasis. In response to LPS, the reaction product of ecto-apyrase was found to relocate from the canalicular domain of the plasma membrane of hepatocytes, its predominant localization in the liver of intact animals, to the basolateral and sinusoidal domains. The pattern of histochemical reaction indicated modulation of the enzyme activity and changes in trafficking of intracellular proteins. Taken together, our findings showed that LPS administration alters ecto-apyrase and causes relocation of its reaction product from the canalicular domain of the plasma membrane of hepatocytes in the rat. It is suggested that relocation of the reaction product may be a protective mechanism to enable the hepatocytes to withstand the cytokine-induced metabolic perturbations.