Stefan Uhlig

ICE-PROTEASE INHIBITORS BLOCK MURINE LIVER INJURY AND APOPTOSIS CAUSED BY CD95 OR BY TNF-ALPHA

The two apoptosis receptors of mammalian cells, i.e. the 55 kDa TNF receptor (TNF-R1) and CD95 (Fas/APO1) are activated independently of each other, however, their signaling involves a variety of ICE-related proteases [I]. We used a cell-permeable inhibitor of ICE-like protease activity to examine in vivo whether post-receptor signaling of TNF and CD95 are fully independent processes. Mice pretreated with the inhibitor, Z-VAD-fluoromethylketone (FMK) were dose-dependently protected from liver injury caused by CD95 activation as determined by plasma alanine aminotransferase and also from hepatocyte apoptosis assessed by DNA fragmentation (ID50 = 0.1 mg/kg). A dose of 10 mg/kg protected mice also from liver injury induced by TNF-alpha. Similar results were found when apoptosis was initiated via TNF-alpha or via CD95 in primary murine hepatocytes (IC50 = 1.5 nM) or in various human cell lines. In addition to prevention, an arrest of cell death by Z-VAD-FMK was demonstrated in vivo and in vitro after stimulation of apoptosis receptors. These findings show in vitro and in vivo in mammals that CD95 and the TNF-alpha receptor share a distal proteolytic apoptosis signal.

An improved setup for the isolated perfused rat lung

This article will describe a method for perfusing and ventilating rat lungs which allows assessment of lung mechanics, segmental vascular resistance, gas exchange, and vascular permeability. Isolated perfused rat lungs (IPL) were ventilated by negative pressure ventilation and were perfused at constant pressure with recirculating, albumin-containing Krebs-Henseleit buffer. A newly constructed respirator pump facilitated well-defined ventilation of the lungs and permitted switching between negative and positive pressure ventilation. Using this setup lungs were ventilated with positive pressure during both surgery and measurement of lung weight. Once the lungs were prepared, electronic control circuits ensured stability of the perfusate pH and perfusate pressure. Such preparations were stable for at least 3 hr. Breathing mechanics, for example, tidal volume, pulmonary compliance, and pulmonary resistance, perfusate characteristics, such as pulmonary vascular resistance, pulmonary pre- and postcapillary resistance, perfusate pH, PO2, and PCO2, and the capillary filtration coefficient were determined. All measured parameters were within normal physiological range, and the lungs were biochemically active, and responded to various stimuli.

Quinine inhibits release of tumor necrosis factor, apoptosis, necrosis and mortality in a murine model of septic liver failure

We investigated the effect of quinine on liver injury induced by lipopolysaccharide in mice sensitized with D-galactosamine. This model is characterized by high systemic release of tumor necrosis factor, which mediates hepatic apoptosis and necrosis. Pretreatment with quinine, a K+ channel blocker, prevented formation of tumor necrosis factor (TNF) as well as the subsequent hepatic DNA fragmentation and liver enzyme leakage. Thus, inhibition of K+ channels may be a novel therapeutic approach in cytokine-related organ damage.

Glutathione enhancement in various mouse organs and protection by glutathione isopropyl ester against liver injury

Intraperitoneal administration of glutathione isopropyl ester to fasted, male NMRI mice dose dependently increased the glutathione concentration in various organs. Administration of 1 g/kg glutathione isopropyl ester led to the following increases: liver 166%; lung 164%; heart 121% after 4 hr; and brain 133% after 6 hr. Spleen, kidney, muscle, serum and blood cell glutathione were not affected by the treatment. Pretreatment with glutathione isopropyl ester was found to protect against paracetamol- or allyl alcohol-induced liver damage. Following treatment with the ester a significant correlation between protection against liver damage and enhancement of liver glutathione content was obtained. The dose dependence of this protection was studied.

Measuring the weight of the isolated perfused rat lung during negative pressure ventilation

Weight measurement represents a means of quantitating edema formation in isolated lungs and following its time course. During ventilation by negative pressure measurements with commonly used weight transducers are impossible because the pressure changes inside the artificial thoracic chamber affect the weight reading. Therefore, we have developed a weight transducer that can be used during negative pressure ventilation. Its design and applicability for assessing weight gain induced by various agents in the negative-pressure-ventilated isolated perfused rat lung is presented here.

Determination of vascular compliance, interstitial compliance, and capillary filtration coefficient in rat isolated perfused lungs.

Gravimetric methods are useful for investigating mechanisms of edema formation. In isolated lungs perfused under isogravimetric conditions, important information may be gained by analyzing the weight changes induced by a sudden change in capillary pressure. In the present study, we investigated the possibility to analyze this weight change by a biexponential equation and use the coefficients obtained to derive vascular compliance (Cv), interstitial compliance (Ci) and the capillary filtration coefficient (Kf,c). Fitting the data of the weight gain to a biexponential curve explained the data significantly better than fitting it to a monoexponential curve, suggesting that two phases can be separated. The first phase is thought to represent vascular filling and was completed for 95% after 0.57 +/- 0.21 min (n = 30). In contrast, 95% completion of phase 2, which relates to interstitial filling, took 18.4 +/- 13.9 min (n = 30). The values obtained for Cv, Ci and Kf,c were 0.064 +/- 0.018 ml/cm H2O, 0.067 +/- 0.030 ml/cm H2O, and 0.588 +/- 0.206 ml/min/cm H2O/100 g lung wet weight, respectively. Treatment of the lungs with platelet activating factor caused no changes in Cv or Ci, but increased Kf,c suggesting increased endothelial permeability. We conclude that analysis of weight changes by a biexponential equation offers a quick and reliable way to analyze factors related to edema formation.

Isolation and characterization of rat primary lung cells

SummaryLung cell culture may be useful as anin vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2×108 cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of >70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC50=5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC50=0.6 mM). All cell types were equally sensitive to the more potent toxicanttert-butylhydroperoxide (EC50=0.1 mM). Paraquat was more toxic to lung cells (EC50=0.03 mM) than to rat (EC50=2.8 mM) and mouse (EC50=0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC50=2.6 mM) compared to rat (EC50=0.2 mM) and mouse (EC50=0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC50=0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC50=0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants.

The interaction of endothelin receptor responses in the isolated perfused rat lung

To gain more insight into the complex pulmonary interactions of endothelins (ET), we studied airway and vascular responses to endothelins in isolated perfused rat lungs in the presence of the novel ETB-receptor antagonist BQ788. In particular we focused on airway responses and on prostacyclin release. The effectiveness of BQ788 in our system was shown by its ability to concentration-dependently prevent vasoconstriction (IC50 0.1μM), bronchoconstriction (IC50 0.1μM) and prostacyclin production (IC50<0.1μM) induced by the ETB-receptor agonist IRL1620 (1nmol). Airway responses to ET-1: ET-1-induced bronchoconstriction was aggravated by BQ123 (1 or 8μM), while BQ788 pretreatment (1 or 8μM) showed no significant effect. Simultaneous treatment with 8μM BQ123 and BQ788 attenuated the ET-1-induced bronchoconstriction. Vascular responses to ET-1: ET-1 (1nmol)-induced vasoconstriction was potentiated by BQ788 (1 or 8μM), but attenuated by the ETA-receptor antagonist BQ123 (1μM). In the presence of BQ788 diminished amounts of the stable prostacyclin metabolite 6-keto-PGF1α were detected in the perfusate. Simultaneous treatment with 8μM BQ123 and BQ788 completely prevented the ET-1-induced vasoconstriction. Conclusions: Both ETA- and ETB-receptors contribute to ET-1-induced vasoconstriction and bronchoconstriction. The ET-1-induced vasoconstriction is attenuated by stimulation of ETB-receptors, a response that is partly mediated by prostacyclin. Due to the mutual interactions between ETA- and ETB-receptors, simultaneous inhibition of both receptors is required to prevent the deleterious effects of ET-1 on lung functions.

Quantification of apoptotic and lytic cell death by video microscopy in combination with artificial neural networks

Apoptosis is characterized by chromatin condensation and DNA fragmentation in the absence of release of cytosolic enzymes such as lactate dehydrogenase (LDH). In contrast, necrosis is characterized by cell swelling, membrane disintegration with cytosolic enzyme release, and absence of chromatin condensation. Staining of cells with Hoechst H33342 dye is a routine method for identifying apoptotic nuclei. However, this process is tedious and prone to individual bias. Therefore, we investigated the suitability of an artificial neural network (ANN) to recognize and distinguish between apoptosis and necrosis. Using a human endothelial cell line (ECV304), we trained an ANN with DNA-stained apoptotic and necrotic nuclei obtained from cells exposed for 8 h to cycloheximide (Chx; 100 microM)/tumour necrosis factor-alpha (TNF; 50 ng/ml) or tert-butylhydroperoxide (t-BH; 2 mM), respectively. After this training step, the ANN correctly assigned necrosis induced by t-BH and apoptosis induced via Chx/TNF, Chx/CD95 activation, Pseudomonas exotoxin A/TNF, or X-rays. In all cases, apoptosis and necrosis as assigned by the ANN correlated with DNA fragmentation and LDH release.

Tolerance against tumor necrosis factor α (TNF)-induced hepatotoxicity in mice: the role of nitric oxide

D-Galactosamine-sensitized mice challenged with tumor necrosis factor alpha (TNF) developed severe apoptotic and secondary necrotic liver injury as assessed by histology, measurement of cytosolic DNA fragments and determination of liver-specific enzymes in plasma. Pretreatment of mice with interleukin-1 beta (IL-1) resulted in elevated levels of nitrite/nitrate in serum and rendered mice insensitive towards TNF toxicity. Pharmacological doses of the nitric oxide (NO) donor sodium nitroprusside (SNP) also conferred complete protection against TNF toxicity, suggesting a possible link between IL-1- and NO-induced protection. However, NO-synthesis inhibition by NG-monomethyl-L-arginine failed to abrogate IL-1-induced tolerance against TNF toxicity. We conclude that IL-1 and NO protect against TNF-induced liver injury through distinct pathways.