Role of extracellular signal-regulated kinase and phosphatidylinositol-3 kinase in chemoattractant and LPS delay of constitutive neutrophil apoptosis.

The present study examined the role of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3 kinase-stimulated Akt (PI-3K/Akt) in the regulation of constitutive human neutrophil apoptosis by bacterial lipopolysaccharide (LPS) and two chemoattractants, fMLP and leukotriene B(4) (LTB(4)). LPS and LTB(4) inhibited apoptosis, while fMLP had no effect. Inhibition of extracellular signal-regulated kinase (ERK) with PD098059 significantly inhibited the anti-apoptotic effect of both LPS and LTB(4), while inhibition of p38 kinase with SB203580 had no effect. Inhibition of PI-3K with wortmannin and LY294002 significantly attenuated the anti-apoptotic effect of LTB(4), but not LPS. LPS, fMLP, and LTB(4) stimulated similar levels of ERK and Akt activation. LTB(4) and LPS inhibited neutrophil apoptosis when added simultaneously with fMLP, and LTB(4) and LPS demonstrated an additive effect. We conclude that the ERK and/or PI-3K/Akt pathways are necessary, but not sufficient, for LPS and LTB(4) to delay apoptosis, but other anti-apoptotic pathways remain to be identified.

Hypokalemic nephropathy and nephrogenic diabetes insipidus due to excessive consumption of a soft drink.

Chronic diarrhea commonly causes hypokalemia. However, life-threatening hypokalemia due to chronic diarrhea has not been reported in the adult caused by chronic ingestion of a fructose beverage. We report an adult patient who had severe hypokalemia from chronic osmotic diarrhea as a result of drinking 4 to 6 liters of Big Red daily for several months. This resulted in sustained hypokalemia, complicated by hypokalemic nephropathy and subsequent nephrogenic diabetes insipidus. All abnormalities cleared with potassium replacement, salt restriction, modest thiazide treatment, and abstinence from Big Red.

Suppression by metallothionein of doxorubicin-induced cardiomyocyte apoptosis through inhibition of p38 mitogen-activated protein kinases.

Cardiomyopathy induced by doxorubicin (DOX) has long been a major impediment of clinical applications of this effective anticancer agent. Previous studies have shown that cardiac-specific metallothionein (MT)-overexpressing transgenic mice are highly resistant to DOX-induced cardiotoxicity. To investigate cellular and molecular mechanisms by which MT participates in this cytoprotection, transgenic mice containing high levels of cardiac MT and non-transgenic controls were treated intraperitoneally with DOX at a single dose of 15 mg/kg and sacrificed on the 4th day after treatment. Myocardial apoptosis was detected by a terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and confirmed by electron microscopy of immunogold staining of apoptotic nuclei. Dual staining of cardiac alpha-sarcomeric actin using an immunohistochemical method further identified apoptotic myocytes. Apoptosis was significantly inhibited in the transgenic myocardium. The anti-apoptotic effect of MT was further revealed in primary cultures of neonatal mouse cardiomyocytes. Furthermore, DOX activated p38 mitogen-activated protein kinase (MAPK), which was critically involved in the apoptotic process, as demonstrated by inhibition of DOX-induced apoptosis by a p38-specific inhibitor, SB203580. Both DOX-induced p38 MAPK activation and apoptosis were dramatically inhibited in the transgenic cardiomyocytes. The results thus demonstrate that DOX induces apoptosis in cardiomyocytes both in vivo and in vitro and MT suppresses this effect through at least in part inhibition of p38 MAPK activation.

Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways.

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.