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Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells in vitro. The used approach is based on the following considerations a) use low concentrations of DAF-2 (0.1 µM) in order to reduce the contribution of DAF-2 auto-fluorescence to the measured total fluorescence, and b) subtract the DAF-2 auto-fluorescence from the measured total fluorescence. The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated. Here we focus in addition to the previous publication (Leikert et al., FEBS Lett 2001, 506:131-134) on aspects of validation procedures as well as limitations and pitfalls of this method.

A pathological increase of the permeability of the mitochondrial membranes may culminate in the irreversible rupture of the mitochondrial outer membrane. Such a permeability transition is lethal because it results in the release of death-inducing molecules from mitochondria and/or metabolic failure. Current methods to assess this outer membrane damage are mostly indirect or scarcely representative of the overall mitochondrial population. Here we present an analytical and preparative approach using free flow electrophoresis to directly distinguish rat liver mitochondria that have undergone the permeability transition from unaffected organelles or from organelles that are damaged to a minor degree. Mitochondrial populations, which considerably differ in outer membrane integrity or cytochrome c content, were separated by this means. We further show that the relative abundance of each population depends on the dose of the permeability transition inducer and the duration of the treatment time. Finally, we have employed this approach to investigate the impairment of mitochondria that were isolated from livers subjected to ischemia/reperfusion damage.

Atrial natriuretic peptide (ANP)-preconditioned livers are protected from ischemia-reperfusion injury. ANP-treated organs show increased expression of heme oxygenase (HO)-1. Because HO-1 liberates bound iron, the aim of our study was to determine whether ANP affects iron regulatory protein (IRP) activity and, thus, the levels of ferritin. Rat livers were perfused with Krebs-Henseleit buffer [[+ or -]ANP, 8-bromo-cGMP (8-Br-cGMP), and tin protoporphyrin, 20 min], stored in University of Wisconsin solution (4[degrees]C, 24 h), and reperfused (120 min). IRP activity was assessed by gel-shift assays, and ferritin, IRP phosphorylation, and PKC localization were assessed by Western blot. Control livers displayed decreased IRP activity at the end of ischemia but no change in ferritin content during ischemia and reperfusion. ANP-pretreated livers showed reduced IRP activity, an effect mimicked by 8-Br-cGMP. Ferritin levels were increased in ANP-pretreated organs. Simultaneous perfusion of livers with ANP and tin protoporphyrin did not reduce ANP-induced action, arguing against a role for HO-1 in changes in IRP activity. ANP and 8-Br-cGMP decreased membrane localization of PKC-[alpha] and PKC-[member of], but this modulation of PKC seems unrelated to inhibition of IRP binding. This work shows the cGMP-mediated attenuation of IRP binding activity by ANP, which results in increased hepatic ferritin levels. This change in IRPs is independent of ANP-induced HO-1 and reduced PKC activation. cGMP; ischemia-reperfusion injury; heat-shock proteins; hepatoprotection; hormonal preconditioning

In liver resection and transplantation ischemiareperfusion injury (IRI) is one of the main causes of organ dysor nonfunction. The aim of the present study was to determine whether [alpha]-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA ([+ or -] wortmannin), followed by ischemia (1 h, 37[degrees]C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-KB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 [micro]mol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-[kappa]B and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway. adenosine 5'-triphosphate; transcription factors; p38 mitogen-activated protein kinase; wortmannin; phosphatidylinositol 3-kinase

Garlic is proposed to have immunomodulatory and anti-inflammatory properties. This paper shows that garlic powder extracts (GPE) and single garlic metabolites modulate lipopolysaccharide (LPS)-induced cytokine levels in human whole blood. GPE-altered cytokine levels in human blood sample supernatants reduced nuclear factor (NF)-[kappa]B activity in human cells exposed to these samples. Pretreatment with GPE (100 mg/L) reduced LPS-induced production of proinflammatory cytokines interleukin (IL)-1 [beta] from 15.7 [+ or -] 5.1 to 6.2 [+ or -] 1.2 [micro]g/L and tumor necrosis factor (TNF)-[alpha] from 8.8 [+ or -] 2.4 to 3.9 [+ or -] 0.8 [micro]g/L, respectively, whereas the expression of the anti-inflammatory cytokine IL-10 was unchanged. The garlic metabolite diallydisulfide (1-100 [micro]mol/L) also significantly reduced IL-1 [beta] and TNF-[alpha]. Interestingly, exposure of human embryonic kidney cell line (HEK293) cells to GPE-treated blood sample supernatants (10 or 100 mg/L) reduced NF-[kappa]B activity compared with cells exposed to untreated blood supernatants as measured by a NF-[kappa]B-driven luciferase reporter gene assay. Blood samples treated with extract obtained from unfertilized garlic (100 mg/L) reduced NF-[kappa]B activity by 25%, whereas blood samples treated with sulfur-fertilized garlic extracts (100 mg/L) lowered NF-[kappa]B activity by 41%. In summary, garlic may indeed promote an anti-inflammatory environment by cytokine modulation in human blood that leads to an overall inhibition of NF-[kappa]B activity in the surrounding tissue. KEY WORDS: * garlic * cytokines * nuclear factor-[kappa]B

Liver injury is known to occur when reactive oxygen species is generated by activated Kupffer cells following liver preservation, shock or endotoxemia. However, no pharmacological interventions for protecting liver cells from the action of Kupffer cells have yet been found. To test the idea that cell-induced oxidant injury can be prevented by atrial natriuretic peptide (ANP), the mechanism of cytoprotection by ANP is investigated in male Sprague-Dawley rats. Results suggest that ANP signaling could provide the basis for a new pharmacological approach for protecting liver cells.

Liver cancers, including hepatocellular carcinoma (HCC), are the second leading cause of cancer death worldwide, and novel therapeutic strategies are still highly needed. Recently, the endolysosomal cation channel TRPML1 (also known as MCOLN1) has gained focus in cancer research because it represents an interesting novel target. We utilized the recently developed isoform-selective TRPML1 activator ML1-SA1 and the CRISPR/Cas9 system to generate tools for overactivation and loss-of-function studies on TRPML1 in HCC. After verification of our tools, we investigated the role of TRPML1 in HCC by studying proliferation, apoptosis and proteomic alterations. Furthermore, we analyzed mitochondrial function in detail by performing confocal and transmission electron microscopy combined with Seahorse™ and Oroboros® functional analysis. We report that TRPML1 overactivation mediated by a novel, isoform-selective small-molecule activator induces apoptosis by impairing mitochondrial function in a Ca2+-dependent manner. Additionally, TRPML1 loss-of-function deregulates mitochondrial renewal, which leads to proliferation impairment. Thus, our study reveals a novel role for TRPML1 as regulator of mitochondrial function and its modulators as promising molecules for novel therapeutic options in HCC therapy. [ABSTRACT FROM AUTHOR]