Heterocyclic thioureylenes protect from calcium-dependent neuronal cell death.

Calcium-dependent cell death occurs in neurodegenerative diseases and ischemic or traumatic brain injury. We analyzed whether thioureylenes can act in a neuroprotective manner by pharmacological suppression of calcium-dependent pathological pathways. In human neuroblastoma (SK-N-SH) cells, thioureylenes (thiopental, carbimazole) inhibited the calcium-dependent neuronal protein phosphatase (PP)-2B, the activation of the proapoptotic transcription factor nuclear factor of activated T-cells, BAD-induced initiation of caspase-3, and poly-(ADP-ribose)-polymerase cleavage. Caspase-3-independent cell death was attenuated by carbimazole and the protein kinase C (PKC) delta inhibitor rottlerin by a PP-2B-independent mechanism. Neuroprotective effects were mediated by the redox-active sulfur of thioureylenes. Furthermore, we observed that the route of calcium mobilization was differentially linked to caspase-dependent or independent cell death and that BAD dephosphorylation did not necessarily induce intrinsic caspase activation. In addition, a new 30- to 35-kDa caspase-3 fragment with an unknown function was identified. In organotypic hippocampal slice cultures, thioureylenes inhibited caspase-3 activation or reduced N-methyl-d-aspartate and kainic acid receptor-mediated cell death that was independent of caspase-3. Because prolonged inhibition of caspase-3 resulted in caspase-independent cellular damage, different types of cell death must be taken under therapeutic consideration. Here we show that thioureylenes in combination with PKCdelta inhibitors might represent a promising therapeutic approach to attenuate neuronal damage.

Heme oxygenase-1 inhibits the proliferation of pancreatic stellate cells by repression of the extracellular signal-regulated kinase1/2 pathway.

Activation of pancreatic stellate cells (PSCs) is the key process in the development of pancreatic fibrosis, a common feature of chronic pancreatitis and pancreatic cancer. In recent studies, curcumin has been shown to inhibit PSC proliferation via an extracellular signal-regulated kinase (ERK)1/2-dependent mechanism. In addition, curcumin is a potent inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1) in other cell types. Therefore, the aims of this study were to 1) characterize the effect of curcumin on HO-1 gene expression in PSCs, 2) explore whether HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation, and 3) clarify the involvement of the mitogen-activated protein kinase (MAPK) family in this context. Cultured rat PSCs were incubated with curcumin and assessed for HO-1 up-regulation by Northern blot analysis, immunoblotting, and activity assays. The effect of HO-1 on platelet-derived growth factor (PDGF)-induced PSC proliferation and MAPK activation was determined by immunoblotting, cell proliferation assays, and cell count analyses. Curcumin induced HO-1 gene expression in PSCs in a time- and dose-dependent manner and inhibited PDGF-mediated ERK1/2 phosphorylation and PSC proliferation. These effects were blocked by treatment of PSCs with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA. Our data provide evidence that HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation. Therefore, therapeutic up-regulation of HO-1 could represent a mode for inhibition of PSC proliferation and thus may provide a novel strategy in the prevention of pancreatic fibrosis.

Carbon monoxide inhalation reduces pulmonary inflammatory response during cardiopulmonary bypass in pigs.

BACKGROUND: Cardiopulmonary bypass (CPB) is associated with pulmonary inflammation and dysfunction. This may lead to acute lung injury and acute respiratory distress syndrome with increased morbidity and mortality. The authors hypothesized that inhaled carbon monoxide before initiation of CPB would reduce inflammatory response in the lungs. METHODS: In a porcine model, a beating-heart CPB was used. The animals were either randomized to a control group, to standard CPB, or to CPB plus carbon monoxide. In the latter group, lungs were ventilated with 250 ppm inhaled carbon monoxide in addition to standard ventilation before CPB. Lung tissue samples were obtained at various time points, and pulmonary cytokine levels were determined. RESULTS: Hemodynamic parameters were largely unaffected by CPB or carbon monoxide inhalation. There were no significant differences in cytokine expression in mononuclear cells between the groups throughout the experimental time course. Compared with standard CPB animals, carbon monoxide significantly suppresses tumor necrosis factor-alpha and interleukin-1beta levels (P < 0.05) and induced the antiinflammatory cytokine interleukin 10 (P < 0.001). Carbon monoxide inhalation modulates effector caspase activity in lung tissue during CPB. CONCLUSIONS: The results demonstrate that inhaled carbon monoxide significantly reduces CPB-induced inflammation via suppression of tumor necrosis factor alpha, and interleukin-1beta expression and elevation of interleukin 10. Apoptosis induced by CPB was associated with caspase-3 activation and was significantly attenuated by carbon monoxide treatment. Based on the observations of this study, inhaled carbon monoxide could represent a potential new therapeutic modality for counteracting CPB-induced lung injury.

Protective role of heme oxygenase-1 in pancreatic microcirculatory dysfunction after ischemia/reperfusion in rats.

OBJECTIVES: Microcirculatory derangements caused by ischemia and reperfusion (I/R) play a pivotal role in acute and graft pancreatitis. The inducible enzyme heme oxygenase 1 (HO-1) has been shown to decrease I/R injury by modulation of capillary perfusion in other organs. It was the aim of this study to evaluate the effect of HO-1 induction on pancreatic microcirculation after I/R. METHODS: Rats were randomized into 4 groups: (1) sham controls; (2) 1-hour ischemia and 2-hour reperfusion (I/R); (3) I/R + cobalt protoporphyrin (CoPP), an HO-1 inducer; and (4) I/R + CoPP + tin protoporphyrin, an HO inhibitor. Functional capillary density (FCD) and leukocyte endothelium interaction were analyzed using intravital microscopy during reperfusion. Expression of HO-1 mRNA, HO-1 protein, and HO activity were assessed by Northern blot, Western blot, and an HO activity assay. RESULTS: Functional capillary density decreased significantly in the I/R group as compared with sham controls. Cobalt protoporphyrin treatment increased FCD to control values. In contrast, HO inhibition in CoPP-pretreated animals lowered FCD and increased leukocyte endothelium interaction significantly. Cobalt protoporphyrin administration increased HO-1 mRNA, protein, and HO activity, whereas activity of the enzyme was reduced after injection of tin protoporphyrin. CONCLUSIONS: Heme oxygenase 1 plays a beneficial role in pancreatic microcirculatory derangements after I/R. This could be of therapeutic relevance after pancreas transplantation and other forms of postischemic pancreatitis.

Sevoflurane-mediated activation of p38-mitogen-activated stresskinase is independent of apoptosis in Jurkat T-cells.

BACKGROUND: Modulation of the inflammatory stress response by anesthesia may be responsible for an increased susceptibility to infectious complications, such as wound infection or pneumonia. Sevoflurane, a specific inhibitor of activator protein-1, an immediate early transcription factor, induces apoptosis in T-cells. Because p38 can be involved either in pro- or antiapoptotic processes, we examined whether the sevoflurane-induced apoptosis is mediated by p38 activation in Jurkat T-cells. METHODS: Jurkat T-cells were exposed to different concentrations of sevoflurane, isoflurane, or desflurane in vitro. Phosphorylation of mitogen-activated protein (MAP) kinases, upstream kinases, downstream activating transcription factor 2 ATF-2, and caspase-3 processing were evaluated by Western blot. p38 kinase activity was evaluated after immunoprecipitation and phosphorylation of the substrate ATF-2 using Western blot. Apoptosis was assessed using flow cytometry after staining with green fluorescent protein-annexin V. RESULTS: While desflurane had no effect, sevoflurane and isoflurane induced p38 phosphorylation with sevoflurane inducing p38 kinase activity. Sevoflurane did not affect the MAP kinases ERK and JNK. Sevoflurane exposure also induced phosphorylation of apoptosis signal-regulating kinase-1 (ASK1), MAP kinase kinases 3 and 6 (MKK3/MKK6), and ATF-2. Pretreatment of cells with the general caspase inhibitor Z-VAD.fmk did not prevent the sevoflurane-induced phosphorylation of p38. Isoflurane- and sevoflurane-mediated caspase-3 processing and apoptosis could not be abolished by pretreatment with the specific p38 inhibitors SB202190 and SB203580. CONCLUSIONS: Sevoflurane is a specific activator of the apoptosis signal-regulating kinase-1-, MKK3/MKK6-p38 MAP kinase cascade in Jurkat T-cells. Our data suggest that sevoflurane-induced p38 activation is not affected by caspase activation. Furthermore, sevoflurane-induced apoptosis is not dependent on p38 MAP kinase activation.

Thiopental protects human T lymphocytes from apoptosis in vitro via the expression of heat shock protein 70.

Barbiturates, which are used for the treatment of intracranial hypertension after severe head injury, have been associated with anti-inflammatory side effects. Although all barbiturates inhibit T-cell function, only thiobarbiturates markedly reduce the activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Various pharmacologic inhibitors of the NF-kappaB pathway are concomitant nonthermal inducers of the heat shock response (HSR), a cellular defense system that is associated with protection of cells and organs. We hypothesize that thiopental mediates cytoprotection by inducing the HSR. Human CD3(+) T lymphocytes were incubated with thiopental, pentobarbital, etomidate, ketamine, midazolam, or propofol. Human Jurkat T cells were transfected with small interfering RNA (siRNA) targeting heat 70-kDa shock protein (hsp 70) before thiopental incubation. Apoptosis was induced by staurosporine. DNA binding activity of HSF-1 was analyzed by electrophoretic mobility shift assay; mRNA expression of hsp27, -32, -70, and -90 was analyzed by Northern blot, and protein expression of hsp70 was analyzed by Western blot and flow cytometry after fluorescein isothiocyanate (FITC)-hsp70-antibody staining. Apoptosis was assessed by flow cytometry after annexin V-FITC or annexin V-phycoerythrin staining. Activity of caspase-3 was measured by fluorogenic caspase activity assay. Thiopental induced hsp27, -70, and -90 but not hsp32 mRNA expression as well as hsp70 protein expression. Thiopental dose-dependently activated the DNA binding activity of HSF-1, whereas other substances investigated had no effect. In addition, pretreatment with thiopental significantly attenuated staurosporine-induced apoptosis and caspase-like activity. Transfection with hsp70-siRNA before thiopental treatment reduced this attenuation. Thiopental specifically and differentially induces a heat shock response, and it mediates cytoprotection via the expression of hsp70 in human T lymphocytes.

Nitric oxide-deficiency regulates hepatic heme oxygenase-1.

Nitric oxide plays a crucial role in the maintenance of liver function and integrity. During stress, the inducible heme oxygenase-1 protein and its reaction products, including carbon monoxide, also exert potent hepatoprotective effects. We investigated a potential relationship between endogenous nitric oxide synthesis and the hepatic regulation of heme oxygenase-1. Inhibition of nitric oxide synthesis in vivo by injection of l-NAME led to a dose-dependent induction of heme oxygenase-1 mRNA, protein and activity in the rat liver, whereas did not affect the expression of other heat shock proteins. The effect of l-NAME was demonstrated by hemodynamic changes within the liver circulation as measured by ultrasonic flow probes. Inhibition of nitric oxide synthase led to a decline in hepatic arterial and portal venous blood flow, and subsequently caused liver cell damage. In contrast, the combined administration of l-NAME and the nitric oxide-independent intestinal vasodilator dihydralazine completely restored portal venous flow, abolished the liver cell damage, and prevented the upregulation of heme oxygenase-1, despite inhibition of nitric oxide production. In conclusion, nitric oxide deficiency upregulates hepatic heme oxygenase-1, which is reversible by maintaining hepatic blood flow. This interdependence has important implications for the development of therapeutic strategies aimed at modulating the activity of these hepatoprotective mediator systems.

Thionamides inhibit the transcription factor nuclear factor-kappaB by suppression of Rac1 and inhibitor of kappaB kinase alpha.

Thionamides, inhibitors of the thyroid peroxidase-mediated iodination, are clinically used in the treatment of hyperthyroidism. However, the use of antithyroid drugs is associated with immunomodulatory effects, and recent studies with thionamide-related heterocyclic thioderivates demonstrated direct anti-inflammatory and immunosuppressive properties. Using primary human T-lymphocytes, we show that the heterocyclic thionamides carbimazole and propylthiouracil inhibit synthesis of the proinflammatory cytokines tumor necrosis factor (TNF)alpha and interferon (IFN)gamma. In addition, DNA binding of nuclear factor (NF)-kappaB, a proinflammatory transcription factor that regulates both TNFalpha and IFNgamma synthesis, and NF-kappaB-dependent reporter gene expression were reduced. Abrogation of NF-kappaB activity was accompanied by reduced phosphorylation and proteolytic degradation of inhibitor of kappaB (IkappaB)alpha, the inhibitory subunit of the NF-kappaB complex. Carbimazole inhibited NF-kappaB via the small GTPase Rac-1, whereas propylthiouracil inhibited the phosphorylation of IkappaBalpha by its kinase inhibitor of kappaB kinase alpha. Methimazole had no effect on NF-kappaB induction, demonstrating that drug potency correlated with the chemical reactivity of the thionamide-associated sulfur group. Taken together, our data demonstrate that thioureylenes with a common, heterocyclic structure inhibit inflammation and immune function via the NF-kappaB pathway. Our results may explain the observed remission of proinflammatory diseases upon antithyroid therapy in hyperthyroid patients. The use of related thioureylenes may provide a new therapeutic basis for the development and application of anti-inflammatory compounds.

Repression of T-cell function by thionamides is mediated by inhibition of the activator protein-1/nuclear factor of activated T-cells pathway and is associated with a common structure.

Treatment of hyperthyroidism by thionamides is associated with immunomodulatory effects, but the mechanism of thionamide-induced immunosuppression is unclear. Here we show that thionamides directly inhibit interleukin-2 cytokine expression, proliferation, and the activation (CD69 expression) of primary human T lymphocytes. Inhibition of immune function was associated with a repression of DNA binding of the cooperatively acting immunoregulatory transcription factors activator protein 1 (AP-1) and nuclear factor of activated T-cells (NFAT). Likewise, thionamides block the GTPase p21Ras, the mitogen-activated protein kinases, and impair the calcineurin/calmodulin-dependent NFAT dephosphorylation and nuclear translocation. The potency of inhibition correlated with the chemical reactivity of the thionamide-associated sulfur group. Taken together, our data demonstrate that thio-derivates with a common heterocyclic thioureylene-structure mediate a direct suppression of immune functions in T-cells via inhibition of the AP-1/NFAT pathway. Our observations may also explain the clinical and pathological resolution of some secondary, calcineurin, and mitogen-activated protein kinase-associated diseases upon thionamide treatment in hyperthyroid patients. This offers a new therapeutic basis for the development and application of heterocyclic thio-derivates.

The mitogen-activated protein kinase p38 regulates activator protein 1 by direct phosphorylation of c-Jun.

The involvement of p38 in fundamental physiological processes and the fact that deregulation often leads to disease indicates the potential impact of p38 dependent mechanisms. Here we demonstrate a new pathway that includes the induction of the mitogen activated protein kinase p38 by protein kinase C and results in a specific phosphorylation of c-Jun in T-lymphocytes. P38 directly phosphorylates c-Jun within its transactivation domain at serine 63 and serine 73 and thus posttranscriptionally affects the presence of DNA-bound phosphorylated c-Jun, a prerequisite for activator protein 1 dependent gene transcription. Moreover, DNA-binding activity of c-Fos, FosB, and JunB were also dependent on the p38 protein kinase activity, whereas JunD, Fra-1 and Fra-2 were not affected. Although we show that stress induced mitogen activated protein kinases share c-Jun as a substrate for phosphorylation, p38 mediated effects could not be rescued by the c-Jun N-terminal kinases. This demonstrates that the protein kinase p38 plays a unique and non-redundant role in posttranslational c-Jun regulation. The induction of a p38 dependent c-Jun phosphorylation was comparable in both CD4(+) and CD8(+) T-cells, proposing a ubiquitous pathway that is not linked to T-cell subtype and effector function. In contrast, ATF-2 was predominantly phosphorylated in CD8(+) T-cells. Different cell lines show p38-dependent c-Jun phosphorylation upon phorbol ester induction but there is evidence that the simian virus 40 large T-antigen may interfere with this pathway.

Heme oxygenase-1 induction by the clinically used anesthetic isoflurane protects rat livers from ischemia/reperfusion injury.

OBJECTIVE: It was the aim of this study to characterize the influence of isoflurane-induced heme oxygenase-1 (HO-1) expression on hepatocellular integrity after ischemia and reperfusion. SUMMARY BACKGROUND DATA: Abundant experimental data characterize HO-1 as one of the most powerful inducible enzymes that contribute to the protection of the liver and other organs after harmful stimuli. Therapeutic strategies aimed at utilizing the protective effects of HO-1 are hampered by the fact that most pharmacological inducers of this enzyme perturb organ function by themselves and are not available for use in patients because of their toxicity and undesirable or unknown side effects. METHODS: Rats were pretreated with isoflurane before induction of partial hepatic ischemia (1 hour) and reperfusion (1 hour). At the end of each experiment, blood and liver tissue were obtained for molecular biologic, histologic, and immunohistochemical analyses. RESULTS: Isoflurane pretreatment increased hepatic HO-1 mRNA, HO-1 protein, HO enzyme activity, and decreased plasma levels of AST, ALT, and alpha-GST. Histologic analysis of livers obtained from isoflurane-pretreated rats showed a reduction of necrotic areas, particularly in the perivenular region, the predominant site of isoflurane-induced HO-1 expression. In addition, sinusoidal congestion that could otherwise be observed after ischemia/reperfusion was inhibited by the anesthetic. Furthermore, isoflurane augmented hepatic microvascular blood flow and lowered the malondialdehyde content within the liver compared with control animals. Administration of tin protoporphyrin IX inhibited HO activity and abolished the isoflurane-induced protective effects. CONCLUSIONS: This study provides first evidence that pretreatment with the nontoxic and clinically approved anesthetic isoflurane induces hepatic HO-1 expression, and thereby protects rat livers from ischemia/reperfusion injury.

Dihydralazine treatment limits liver injury after hemorrhagic shock in rats.

OBJECTIVE: Impaired hepatic perfusion after hemorrhagic shock frequently results in hepatocellular dysfunction associated with increased mortality. This study characterizes the effect of the vasodilators dihydralazine and urapidil on hepatocellular perfusion and integrity after hemorrhagic shock and resuscitation. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University experimental laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: To register systemic and regional hepatic hemodynamics, rats (n=6 per group) were instrumented and randomly assigned to the following groups: shock+vehicle; shock+dihydralazine (1.5 mg/kg); or shock+urapidil (3 mg/kg). After 1 hr of hemorrhagic shock, animals were resuscitated for 5 hrs and mean arterial pressure was maintained at 70+/-5 mm Hg by administration of dihydralazine or urapidil. To evaluate hepatic heme oxygenase-1 expression and liver injury (determination of levels of alanine and aspartate aminotransferase [ALT, AST] and histology), an additional series of experiments with six animals per group was performed. At the end of each experiment, animals were killed and blood and liver tissue was obtained for subsequent analyses. MEASUREMENTS AND MAIN RESULTS: Dihydralazine increased cardiac output and portal and hepatic microvascular flow (p<.05) and reduced liver injury after shock (lower ALT and AST levels [p<.05]; improvement of histopathological changes). In contrast, urapidil had no effect on portal flow or liver injury. Hepatic heme oxygenase-1 mRNA expression was upregulated in animals subjected to hemorrhagic shock but did not differ among experimental groups. CONCLUSIONS: Dihydralazine increases nutritive portal and hepatic microvascular flow and limits liver injury after hemorrhagic shock. This protective effect appears to be the result of increased cardiac output and increased portal flow. These findings may offer a new strategy for hepatic protection after hemorrhagic shock.

Mechanism of hepatic heme oxygenase-1 induction by isoflurane.

BACKGROUND: The heme oxygenase pathway represents a major cell and organ protective system in the liver. The authors recently showed that isoflurane and sevoflurane up-regulate the inducible isoform heme oxygenase 1 (HO-1). Because the activating cascade remained unclear, it was the aim of this study to identify the underlying mechanism of this effect. METHODS: Rats were anesthetized with pentobarbital intravenously or with isoflurane per inhalation (2.3 vol%). Kupffer cell function was inhibited by dexamethasone or gadolinium chloride. Nitric oxide synthases were inhibited by either N(omega)-nitro-L-arginine methyl ester or S-methyl thiourea. N-acetyl-cysteine served as an antioxidant, and diethyldithiocarbamate served as an inhibitor of cytochrome P450 2E1. Protein kinase C and phospholipase A2 were inhibited by chelerythrine or quinacrine, respectively. HO-1 was analyzed in liver tissue by Northern blot, Western blot, immunostaining, and enzymatic activity assay. RESULTS: In contrast to pentobarbital, isoflurane induced HO-1 after 4-6 h in hepatocytes in the pericentral region of the liver. The induction was prevented in the presence of dexamethasone (P < 0.05) and gadolinium chloride (P < 0.05). Inhibition of nitric oxide synthases or reactive oxygen intermediates did not affect isoflurane-mediated HO-1 up-regulation. In contrast, chelerythrine (P < 0.05) and quinacrine (P < 0.05) resulted in a blockade of HO-1 induction. CONCLUSION: The up-regulation of HO-1 by isoflurane in the liver is restricted to parenchymal cells and depends on Kupffer cell function. The induction is independent of nitric oxide or reactive oxygen species but does involve protein kinase C and phospholipase A2.

Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro.

BACKGROUND: Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. METHODS: Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3 T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti-caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti-cytochrome c Western blotting and enzyme-linked immunosorbent assays. RESULTS: Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3-like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. CONCLUSION: Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.

Isoflurane pretreatment lowers portal venous resistance by increasing hepatic heme oxygenase activity in the rat liver in vivo.

BACKGROUND/AIMS: The heme oxygenase (HO) system contributes to the maintenance of hepatic perfusion and integrity. It was the objective of this study to determine the influence of isoflurane (ISO) on hepatic HO-1 induction and its impact on hepatic hemodynamics. METHODS: Rats were pretreated with or without ISO for 5h. After hemodynamic measurements by pressure-, laser doppler-, and ultrasound based techniques, the liver was harvested. HO-1 was analyzed by an HO activity assay, Northern- and Western blotting. RESULTS: ISO pretreatment induced hepatic HO-1 mRNA and protein resulting in an increase of HO activity. No effect on hsp-27, hsp-70 and hsp-90 mRNA could be observed. ISO lowered portal resistance. HO inhibition by tin protoporphyrine IX increased portal resistance in ISO pretreated animals up to control levels. This was associated with an increase in portal pressure and a reduction of portal flow. Microvascular flux was also impaired after HO blockade and ISO. However, hepatic arterial and systemic hemodynamics remained unchanged, indicating a specific effect within the portal vascular bed. CONCLUSIONS: ISO pretreatment induces hepatic HO-1 mRNA and protein followed by an increase in HO activity, thereby reducing portal resistance. These findings indicate a beneficial effect of ISO on hepatic hemodynamics in vivo.

Dobutamine inhibits phorbol-myristate-acetate-induced activation of nuclear factor-kappaB in human T lymphocytes in vitro.

Adrenergic drugs are often used for hemodynamic support of cardiac output and vasomotor tone in critically ill patients. Recent evidence shows that the administration of these vasoactive drugs may affect cytokine release and could influence the inflammatory response. However, the mechanism of this immunomodulatory effect remains unknown. The nuclear transcription factor-kappaB (NF-kappaB) regulates the expression of many cytokines and plays a central role in the immune response. Therefore, we examined the effects of various adrenergic drugs (dobutamine, xamoterol, clenbuterol, epinephrine, norepinephrine, and phenylephrine) on the activation of NF-kappaB, on the NF-kappaB-driven reporter gene activity, and on the expression of the NF-kappaB target gene interleukin (IL)-8. In addition, we quantified the amount of the NF-kappaB inhibitors IkappaBalpha and IL-10. Here we report that dobutamine inhibited the activation of NF-kappaB in primary human CD3(+) T lymphocytes. Suppression of NF-kappaB involved the stabilization of its inhibitor, IkappaBalpha. The effect appears to be beta(2)-receptor specific, because beta(1)-adrenergic and alpha-adrenergic substances (i.e., xamoterol, epinephrine, norepinephrine, and phenylephrine) did not affect NF-kappaB activation and because dobutamine-mediated inhibition of NF-kappaB could be prevented by a specific beta(2)-antagonist. Our results demonstrate that dobutamine is a potent and specific inhibitor of NF-kappaB, and they thus provide a possible molecular mechanism for the immunomodulation associated with beta-adrenergic therapy.

Sevoflurane inhibits phorbol-myristate-acetate-induced activator protein-1 activation in human T lymphocytes in vitro: potential role of the p38-stress kinase pathway.

BACKGROUND: Modulation of immune defense mechanisms by volatile anesthetics during general anesthesia may compromise postoperative immune competence and healing reactions and affect the infection rate and the rate of tumor metastases disseminated during surgery. Several mechanisms have been suggested to account for these effects. The current study was undertaken to examine the molecular mechanisms underlying these observations. METHODS: Effects of sevoflurane, isoflurane, and desflurane were studied in vitro in primary human CD3 T-lymphocytes. DNA-binding activity of the transcription factor activator protein-1 (AP-1) was assessed using an electrophoretic mobility shift assay. Phorbol-myristate-acetate-dependent effects of sevoflurane on the phosphorylation of the mitogen-activated protein kinases were studied using Western blots, the trans-activating potency of AP-1 was determined using reporter gene assays, and the cytokine release was measured using enzyme-linked immunosorbent assays. RESULTS: Sevoflurane inhibited activation of the transcription factor AP-1. This effect was specific, as the activity of nuclear factor kappabeta, nuclear factor of activated T cells, and specific protein-1 was not altered and several other volatile anesthetics studied did not affect AP-1 activation. Sevoflurane-mediated suppression of AP-1 could be observed in primary CD3 lymphocytes from healthy volunteers, was time-dependent and concentration-dependent, and occurred at concentrations that are clinically achieved. It resulted in an inhibition of AP-1-driven reporter gene activity and of the expression of the AP-1 target gene interleukin-3. Suppression of AP-1 was associated with altered phosphorylation of p38 mitogen-activated protein kinases. CONCLUSION: The data demonstrate that sevoflurane is a specific inhibitor of AP-1 and may thus provide a molecular mechanism for the antiinflammatory effects associated with sevoflurane administration.

Inhibition of activator protein 1 by barbiturates is mediated by differential effects on mitogen-activated protein kinases and the small G proteins ras and rac-1.

Barbiturates are known to suppress protective immunity, and their therapeutic use is associated with nosocomial infections. Although barbiturates inhibit T cell proliferation, differentiation, and cytokine synthesis, only thiobarbiturates markedly reduce the activation of immune regulatory transcription factors such as nuclear factor-kappaB and nuclear factor of activated T cells. In this study, we investigated barbiturate-mediated effects on the regulation of the transcription factor activator protein 1 (AP-1) in primary T lymphocytes. We show that both thiobarbiturates and their oxy-analogs inhibit AP-1-dependent gene expression and AP-1 complex formation at clinically relevant doses. Furthermore, mitogen-activated protein (MAP) kinase activity, which transcriptionally and posttranslationally regulates AP-1 complex formation, is suppressed by most barbiturates. CD3/CD28- or phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced p38 and extracellular signal-regulated kinase 1/2 phosphorylation or c-jun NH2-terminal kinase (JNK) 1/2 kinase activity was significantly diminished by pentobarbital, thiamylal, secobarbital, or methohexital treatment. These barbiturates also inhibited the initiators of the MAP kinase cascade, the small G proteins ras and rac-1, and prevented binding to their partners raf-1 and PAK, respectively. Thiopental, unlike the other barbiturates, only reduced ras and JNK activity upon direct CD3/CD28 receptor engagement. Contrarily, upon PMA/ionomycin stimulation, thiopental blocked AP-1-dependent gene expression independently of the small G protein ras and MAP kinases, thus suggesting an additional, unknown mechanism of AP-1 regulation. In conclusion, our results contribute to the explanation of a clinically manifested immune suppression in barbiturate-treated patients and support the idea of a MAP kinase-independent regulation of AP-1 by PKC and calcium in human T cells.

Barbiturates directly inhibit the calmodulin/calcineurin complex: a novel mechanism of inhibition of nuclear factor of activated T cells.

Barbiturates are frequently used for the treatment of intracranial hypertension after brain injury but their application is associated with a profound increase in the infection rate. The mechanism of barbiturate-induced failure of protective immunity is still unknown. We provide evidence that nuclear factor of activated T cells (NFAT), an essential transcription factor in T cell activation, is a target of barbiturate-mediated immunosuppression in human T lymphocytes. Treatment of primary CD3+ lymphocytes with barbiturates inhibited the PMA and ionomycin induced increase in DNA binding of NFAT, whereas the activity of other transcription factors, such as Oct-1, SP-1, or the cAMP response element-binding protein, remained unaffected. Moreover, barbiturates suppressed the expression of a luciferase reporter gene under control of NFAT (stably transfected Jurkat T cells), and of the cytokine genes interleukin-2 and interferon-gamma that contain functional binding motifs for NFAT within their regulatory promotor domains (human peripheral blood CD3+ lymphocytes). Neither GABA receptor-initiated signaling nor direct interactions of barbiturates with nuclear proteins affected the activity of NFAT. In contrast, barbiturates suppressed the calcineurin-dependent dephosphorylation of NFAT in intact T cells and also inhibited the enzymatic activity of calcineurin in a cell-free system, excluding upstream regulation. Thus, our results demonstrate a novel mechanism of direct inhibition of the calcineurin/calmodulin complex that may explain some of the known immunosuppressive effects associated with barbiturate treatment.

Thiopental inhibits tumor necrosis factor alpha-induced activation of nuclear factor kappaB through suppression of kappaB kinase activity.

BACKGROUND: Thiopental is frequently used for the treatment of intracranial hypertension after severe head injury and is associated with immunosuppressive effects. The authors have recently reported that thiopental inhibits activation of nuclear factor (NF) kappaB, a transcription factor implicated in the expression of many inflammatory genes. Thus, it was the aim of the current study to examine the molecular mechanism of this inhibitory effect. METHODS: The authors tested gamma-aminobutyric acid (GABA), the GABA(A) antagonist bicuculline, and the GABA(B) antagonist dichlorophenyl-methyl-amino-propyl-diethoxymethyl-phosphinic acid (CGP 52432) in combination with thiopental for their influence on the activation of NF-kappaB. In addition, they investigated the direct effect of thiopental on activated NF-kappaB DNA binding activity. These experiments were conducted in Jurkat T lymphocytes using electrophoretic mobility shift assays. The presence of the phosphorylated and dephosphorylated NF-kappaB inhibitor IkappaBalpha (Western blotting) and IkappaB kinase activity were studied in Jurkat T cells and human CD3+ T lymphocytes. In addition, the authors tested the effect of the structural barbiturate analog pairs thiopental-pentobarbital and thiamylal-secobarbital and of thiopental in combination with the thio-group containing chemical dithiothreitol on the activation of NF-kappaB. RESULTS: GABA did not inhibit NF-kappaB activation, and the GABA(A) and GABA(B) antagonists bicuculline and CGP did not diminish the thiopental-mediated inhibitory effect on NF-kappaB activation. Thiopental did not inhibit activated NF-kappaB directly in a cell-free system. The phosphorylation of IkappaBalpha was prevented after incubation with 1,000 microg/ml thiopental. The same concentration of thiopental also inhibited IkappaB kinase activity in tumor necrosis factor-stimulated Jurkat T cells and human CD3+ T lymphocytes (60% suppression, P < 0.05 vs. tumor necrosis factor alpha alone). Thiobarbiturates (4 x 10(-3) m) inhibited NF-kappaB activity, whereas equimolar concentrations of the structural oxyanalogs did not. Preincubation of thiopental with dithiothreitol diminished the inhibitory effect. CONCLUSION: Thiopental-mediated inhibition of NF-kappaB activation is due to the suppression of IkappaB kinase activity and depends at least in part on the thio-group of the barbiturate molecule.