[Abstract unavailable

No abstract

Serum and certain growth factors have the ability to inhibit programmed cell death (apoptosis) and promote survival. The mechanism by which growth factors deliver an anti-apoptotic signal and the mechanism by which this survival signal is uncoupled from mitogenesis are not clear. We studied five downstream effectors of growth factor receptors-Ras, Raf, Src, phosphoinositide 3-kinase (PI 3-kinase), and Akt (PKB)-for their abilities to block apoptosis. Activated forms of Ras, Raf, and Src, although transforming, were not sufficient to deliver a survival signal upon serum withdrawal. In contrast, inhibition of PI 3-kinase accelerated apoptosis, and an activated form of the serine/threonine kinase Akt, a downstream effector of PI 3-kinase, blocked apoptosis. The ability of Akt to promote survival was dependent on and proportional to its kinase activity. In Ratla fibroblasts, activated Akt did not alter Bcl-2 or Bcl-X^ expression but inhibited Ced3/ICE-like activity. Thus, the PI 3-kinase/Akt (PKB) signaling pathway transduces a survival signal that ultimately blocks Ced3/ICE-like activity. These results suggest that uncoupling of survival and mitogenesis can be explained by differing abilities of distinct mitogens to efficiently induce the PI 3-kinase/Akt signaling pathway.

Digital-imaging microfluorimetry of the oxidation of hydroethidine (HEt) to ethidium can be used to monitor superoxide (O2-) production selectively within individual rat hippocampal pyramidal neurons in culture and in brain slices. Under assay conditions, oxidation was not accomplished by hydroxyl radical, singlet O2, H2O2, or nitrogen radicals. Neuronal O2- production varied with metabolic activity and age. O2- generation increased after treatment with AMPA, kainic acid, and NMDA, and the mitochondrial uncoupler carbonylcyanide p-(trifluoromethoxy)phenyl hydrazone, but usually not after depolarization (50 mM K+). O2- concentrations were sensitive to scavengers and nitric oxide. HEt oxidation was higher in Ca(2+)-containing versus Ca(2+)-free saline. However, Ca2+ ionophores did not increase oxidation greatly. H2O2 application produced a secondary increase in O2-. The major source of O2- under basal and stimulated conditions appeared to be the mitochondria. Consistent with this, ethidium staining in dendrites was punctate, colocalized with mitochondria, and blocked by CN-.

The potential toxic effects of high extracellular concentrations of fatty acids were tested in beta(INS-1)-cells cultured in the absence of serum, a condition known to alter cell survival in various systems. This may in part mimic the situation in type 1 or 2 diabetes where beta-cells are already insulted by various stressful conditions, such as cytokines and oxidative stress. Serum removal caused, over a 36-h period, oxidative stress and an early impairment of mitochondrial function, as revealed by increased superoxide production and markedly reduced mitochondrial membrane potential, but a lack of cytochrome c and apoptosis-inducing factor release in the cytosol. The fatty acids palmitate and oleate considerably accelerated the apoptosis process in serum-starved cells, as revealed by fluorescence-activated cell sorting analysis, morphological changes, chromatin condensation, DNA laddering, poly(ADP-ribose) polymerase cleavage, cytochrome c and apoptosis-inducing factor release, and increased levels of Bax and cytosolic caspase-2. The fatty acids also increased nitric oxide production, apparently independently of inducible nitric oxide synthase induction. Under the same experimental conditions, elevated glucose alone had only a marginal effect on beta-cell apoptosis. Together the results indicate that elevated concentrations of fatty acids are particularly efficient in accelerating the rate of apoptosis of already stressed beta(INS-1)-cells displaying altered mitochondrial function, and that the mitochondrial arm of the apoptosis process is involved in beta-cell lipotoxicity.