Differential binding of ceramide to MEKK1 in glomerular endothelial and mesangial cells.

Previously, we have shown that ceramide is able to directly bind to and activate c-Raf and to trigger the downstream classical mitogen-activated protein kinase (MAPK/ERK) cascade in glomerular mesangial cells [Proc. Natl. Acad. Sci. USA 93 (1996) 6959]. In this study, we show that ceramide acts differently in glomerular endothelial cells in that treatment of endothelial cells with exogenous ceramide leads to a potent activation of the stress-activated protein kinase (SAPK/JNK) cascade but not to an activation of the classical ERK cascade. A similar effect was observed with the inflammatory cytokines TNFalpha and IL-1beta, which activate a sphingomyelinase and thereby increase intracellular ceramide levels. The activation of JNKs as shown by c-Jun phosphorylation assays was paralleled by increased phosphorylation of the two JNK isoforms, p45 and p54. In addition, also the activator of JNKs, SEK1, was found to be increasingly phosphorylated by exogenous ceramide as well as by TNFalpha. In contrast, dihydroceramide had no effect on JNK or SEK1 phosphorylation. To see whether ceramide directly binds to MEKK1, which is the c-Raf analog in the SAPK cascade, a radioiodinated photoaffinity labeling analogue of ceramide, (N-[3-[[[2-(125I)iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl]oxy]-carbonyl] propanoyl]-D-erythro-sphingosine) ([125I]TID-ceramide) was used. Stimulation of endothelial cells with this [125I]TID-ceramide for 5 min followed by a short photolysis defined MEKK1 as a direct target of ceramide. With the same method, protein kinase C-alpha (PKC-alpha) was identified as a ceramide target. In contrast, no binding to c-Raf or the MEKK1 activator p65-PAK could be detected. A direct binding of ceramide to MEKK1 was also confirmed by affinity chromatography using a ceramide-coupled sepharose column. Furthermore, the ceramide-activated SAPK/JNK cascade is clearly involved in the mechanism of apoptosis, since in the presence of a JNK inhibitor, ceramide-induced DNA fragmentation is significantly reduced. In summary, we have shown that ceramide potently activates the SAPK cascade but not the ERK cascade in endothelial cells, which contrasts to mesangial cells where ceramide activates the ERK pathway and has only a minor effect on the SAPK cascade. Regarding the direct target of ceramide binding and action in endothelial cells, we identified MEKK1 as a further member of the growing family of ceramide-activated protein kinases.

Adrenal function during coronary artery bypass grafting.

OBJECTIVE: To assess adrenal function in patients undergoing coronary artery bypass grafting (CABG) by means of the low-dose (1 microg) ACTH test, and to correlate the adrenal function with clinical outcome. METHODS: During a 5-Month period we prospectively included 45 patients undergoing elective CABG with cardiopulmonary bypass and without symptoms of endocrine disease. Low-dose (1 microg) ACTH tests were performed on the day before surgery (day -1), immediately after the operation (day 0), on the two subsequent days in the intensive care unit (day 1 and day 2), and on the day of discharge from the hospital. A number of clinical, hemodynamic and laboratory parameters were monitored throughout. RESULTS: On day -1, 75% of the study patients had normal stimulated plasma cortisol concentrations. Eleven patients (25%) had an impaired adrenal response to 1 microg ACTH. The stimulated plasma cortisol concentrations in patients who had an inadequate adrenal response on day -1 remained significantly reduced on day 1 (756+/-205 vs 949+/-259 nmol/l, P=0.03) (mean+/-s.d.), day 2 (644 (580-793) vs 885 (713-1087), P=0.03) (median (interquartile range)), and on the day of discharge (698+/-201 vs 854+/-186, P=0.05). In patients with a normal adrenal response in the preoperative setting peak cortisol concentrations were reached on day 1, in patients with a blunted adrenal response they were reached on day 2. There were significant correlations between the stimulated plasma cortisol concentrations and the blood loss (r=-0.50, P=0.002) and Volume balance (r=0.41, P=0.015). CONCLUSIONS: Occult (partial) adrenal insufficiency is common in patients undergoing CABG who are otherwise asymptomatic as regards endocrine disease. The adrenal function in these patients differs both in the magnitude of cortisol response to ACTH and in the time course, with significantly delayed peak cortisol concentrations. Adequate regulation of Volume balance and the amount of blood loss seem to correlate with adequacy of adrenal function.

Cross-talk between nitric oxide and superoxide determines ceramide formation and apoptosis in glomerular cells.

BACKGROUND: The modulation of cell signaling by nitric oxide (NO) and superoxide (O(-)(2)) is associated with apoptotic cell death in inflammatory kidney diseases. Recently, we have shown that NO induces ceramide production in glomerular mesangial and endothelial cells and the ratio of NO and O(-)(2) determines whether cells live or die. METHODS: Glomerular endothelial and mesangial cells were labeled with [(14)C]serine, the precursor of all sphingolipids, then stimulated with reactive oxygen species- or reactive nitrogen species-generating substances and subjected to lipid extraction. Radioactive lipids were separated and analyzed by thin-layer chromatography. DNA fragmentation, as a characteristic feature of apoptosis, was measured by a nucleosome/DNA-ELISA, which quantitatively recorded the histone-associated DNA fragments. RESULTS: Exposure of glomerular endothelial and mesangial cells to either NO donors or superoxide-generating substances led to a delayed and sustained ceramide formation that paralleled the induction of apoptosis in both cell types. Coincubation of endothelial cells with NO and superoxide, which led to the generation of peroxynitrite, caused a synergistic enhancement of ceramide generation and apoptosis when compared to either stimulus alone. By contrast, in glomerular mesangial cells costimulation with superoxide neutralized not only NO-induced apoptosis but also NO-induced ceramide formation, although O(-)(2) alone triggered ceramide formation in mesangial cells and caused cell death. Moreover, SIN-1, a substance that simultaneously releases NO and O(-)(2) and thereby generates peroxynitrite, also stimulated a delayed ceramide formation in endothelial cells but not in mesangial cells. Furthermore, exposure of endothelial cells to glucose oxidase, which generates hydrogen peroxide, or to exogenous hydrogen peroxide, also showed a dose-dependent increase in ceramide formation and apoptosis, although to a lesser extent than did superoxide. CONCLUSIONS: These data suggest that ceramide represents an important mediator of reactive oxygen and nitrogen species-triggered cell responses, like apoptosis. There seem to be cell type-specific protective mechanisms that critically depend on a fine-tuned redox balance between reactive nitrogen and oxygen species to determine whether a cell undergoes apoptosis or survives when exposed to oxidative and/or nitrosative stress conditions.

Basic fibroblast growth factor selectively enhances TNF-alpha-induced apoptotic cell death in glomerular endothelial cells: effects on apoptotic signaling pathways.

Endothelial cell damage of glomeruli and kidney arterioles seems to play a pivotal role in several pathologic situations, such as Gram-negative sepsis, glomerulonephritis, and acute renal failure. Bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) have been identified as potent inducers of apoptotic cell death in bovine glomerular endothelial cells. Both agents elicited apoptotic DNA laddering within 12 to 24 h. Basic fibroblast growth factor (bFGF) was generally described as a protective factor for endothelial cells against radiation-, TNF-alpha-, and UV-light-induced programmed cell death. Therefore, whether bFGF also affects apoptosis of microvascular endothelial cells was questioned. Surprising was that simultaneous treatment of glomerular endothelial cells with bFGF and either LPS or TNF-alpha left LPS-induced death unaffected, whereas TNF-alpha-induced death induction was potentiated, amounting to 48.9+/-6.3% versus 22.4+/-4.3% DNA degradation with TNF-alpha alone. Comparably, acidic FGF also selectively potentiated TNF-alpha-induced apoptosis. In mechanistic terms, bFGF synergistically increased TNF-alpha-induced mitochondrial permeability transition, the release of cytochrome c from mitochondria to the cytosol, and upregulation of the proapoptotic protein Bak and significantly enhanced activation of caspase-8 protease activity. In contrast, stress-activated protein kinase and nuclear factor kappaB activation, which represent primary signals of TNF/TNF receptor interaction, downregulation of the antiapoptotic protein Bcl-x(L), and caspase-3-like protease activation, were unaffected. As bFGF did not affect LPS-induced apoptotic cell death, bFGF also left LPS-induced Bak upregulation and Bcl-x(L) downregulation unaffected. The results point to a selective bFGF-mediated enhancement of distinct proapoptotic pathways induced by TNF-alpha in glomerular endothelial cells.