In tissue, mechanical cell-to-cell interactions may contribute to cardiomyocyte injury in anoxia-reoxygenation. In the present study, the disturbance of energy metabolism and cell injury were investigated in isolated cardiomyocytes, free of external mechanical constraints. Cardiomyocytes from adult rat, attached to culture dishes, were exposed to 120 min of anoxia and 15 min of reoxygenation in a substrate-free modified Tyrode solution. The energetic state of the cells in anoxia-reoxygenation was characterized by the free-energy change of ATP hydrolysis (delta GATP), amounting to 57 kJ/mol ATP in normoxia. After 120 min of anoxia, all cells were contracted to 65% of their length and delta GATP decreased to 41 kJ/mol. No lactate dehydrogenase was released. Reoxygenation caused a partial oxygen paradox: immediate hypercontracture of the cells, but no release of lactate dehydrogenase. delta GATP recovered to 51 kJ/mol within 15 min. The results demonstrate that anoxic cardiomyocytes can be energy depleted without losing sarcolemmal integrity. They can undergo hypercontracture, elicited by reoxygenation, and yet an almost normal delta GATP can be reestablished.

We have devised a technique for monitoring cytoplasmic ATP continuously in single hepatocytes. Single isolated rat hepatocytes were injected with the ATP-dependent luminescent protein firefly luciferase, and then superfused with 45 microM luciferin in air-equilibrated medium. Signals of approx. 10-200 photoelectron counts per second could be recorded from individual healthy cells for up to 3 h. The response of the luminescent signal to chemical hypoxia (2-5 mM CN- and 5-10 mM 2-deoxyglucose) was monitored. We found a great cell-to-cell variability in the time course of the ATP decline in response to CN-, 2-deoxyglucose or to their combination; the time for the signal to fall to 10% of the original (corresponding to approx. 100 microM ATP) ranged from approx. 20 to 75 min. This resistance of the cytoplasmic ATP concentration to depletion after blockade of oxidative phosphorylation and glycolysis could be abolished by pretreatment of the cells with etomoxir, which blocks mitochondrial beta-oxidation. Etomoxir alone had no effect on the luciferase signal, but etomoxir-pre-treated cells showed a prompt fall in the luciferase signal starting within 1-2 min of application of cyanide and 2-deoxyglucose and falling to 10% of the original signal in approx. 6-10 min. The technique allows cytoplasmic ATP changes to be monitored in single hepatocytes at concentrations of 1 mM or lower, but more precise calibration of the signal will require correction for the effects of cytoplasmic pH changes.

The inhibitor of jB kinase e (IKKe) is pivotal for an efficient innate immune response to viral infections and has been recognized as breast cancer oncogene. The antiviral function of IKKe involves activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-jB, thus inducing the expression of type I IFN. Here, we have identified two novel splice variants of human IKKe, designated IKKe-sv1 and IKKe-sv2, respectively. Interestingly, RT-PCR revealed quantitatively different isoform expression in PBMC from different individuals. Moreover, we found cell type-and stimulus-specific protein expression of the various splice variants. Overexpression of full-length wt IKKe (IKKe-wt) leads to the activation of NF-jB-as well as IRF3-driven luciferase reporter genes. Although none of the splice variants activates IRF3, IKKe-sv1 still activates NF-jB, whereas IKKe-sv2 is also defective in NF-jB activation. Both splice variants form dimers with IKKe-wt and inhibit IKKe-wt-induced IRF3 signaling including the antiviral activity in a dominant-negative manner. The lack of IRF3 activation is likely caused by the failure of the splice variants to interact with the adapter proteins TANK, NAP1, and/or SINTBAD. Taken together, our data suggest alternative splicing as a novel regulatory mechanism suitable to shift the balance between different functions of IKKe.Key words: IFN . Innate immunity . NF-kB pathway . Protein/protein interactions . Signal transductionSupporting Information available online IntroductionViral infections are recognized by the innate immune system, which is essential for the subsequent initiation of adaptive immunity. Invading viruses are sensed by pattern-recognition receptors (PRR) recognizing pathogen-associated molecular patterns such as singleor double-stranded RNA. These PRR comprise TLR with endosomal/lysosomal localization like TLR3 and cytoplasmic receptors such as the retinoic acid-inducible protein I and melanoma differentiation-associated gene 5. Activation of these PRR engages intracellular signaling cascades leading to the secretion of type I IFN, which are important anti-viral cytokines ultimately facilitating viral clearance [1,2]. The signal transduction pathways leading to type I IFN expression involve activation of the serine/threonine kinases TANK-binding kinase 1 (TBK-1), also known as NF-kB activating kinase NAK [3], and inhibitor of kB kinase e (IKKe), also known as IKKi [4]. After virus infection, both kinases are activated and phosphorylate the transcription factor IFN regulatory factor 3 (IRF3), leading to homo-dimerization, translocation into the nucleus, and activation of promoters containing the IRF3 binding site termed IFN-stimulated response element [5,6]. For IRF3 activation after triggering of different PRR, the three related scaffold proteins NAP1, TANK, and SINTBAD are essential [7][8][9], whereas the use of a distinct scaffold protein depends on the respective stimulus activating the TBK1/IKKe pathway [10]. Ultimately, the formation of a multisubunit complex c...

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is upregulated during hepatic fibrogenesis and considered to promote fibrosis in the injured liver by inhibition of matrix metalloproteases (MMP) and degradation of extracellular matrix. Moreover, TIMP-1 displays anti-apoptotic properties, in patients with hepatocellular carcinoma (HCC) TIMP-1 serum levels are elevated and high TIMP-1 expression levels in HCC are associated with a poor prognosis. Therefore, TIMP-1 could functionally link fibrogenesis and carcinogenesis in the liver. The aim of our study was to characterize the role of TIMP-1 in hepatic fibrogenesis and carcinogenesis. Experimental hepatic fibrogenesis as well as diethylnitrosamine (DEN) -induced hepatocarcinogenesis were studied in TIMP-1-deficient mice and wild type littermates. Hepatic TIMP-1 expression was upregulated following induction of liver fibrosis by bile duct ligation (BDL) or by carbon tetrachloride (CCl4). Unexpectedly, in comparison to wild type littermates, TIMP-1-deficient mice were not protected from liver fibrosis induced by BDL or CCl4. TIMP-1 expression was significantly higher in HCC nodules than in surrounding liver tissue. However, experimental hepatic carcinogenesis was similar in TIMP-1-deficient mice and wild type littermates following DEN-treatment or combined treatment with DEN and CCl4. Therefore we concluded that TIMP-1 is not essential for hepatic fibrogenesis and carcinogenesis in mice.