Eduardo G. Lapetina

Nitric Oxide-induced S-Glutathionylation and Inactivation of Glyceraldehyde-3-phosphate Dehydrogenase

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) byS-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.

Superoxide Formation and Macrophage Resistance to Nitric Oxide-mediated Apoptosis

RAW 264.7 macrophages, when challenged with a combination of lipopolysaccharide (10 μg/ml) and interferon-γ (100 units/ml), respond with endogenous NO· formation, which ultimately results in apoptotic cell death. Apoptosis is detected morphologically by chromatin condensation. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO·-derived apoptosis was blocked by the NO·-synthase inhibitor NG-monomethyl-L-arginine. Repetitive treatment of RAW 264.7 macrophages with lipopolysaccharide/interferon-γ, followed by subculturing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/nitrate in response to agonist treatment but showed no apoptotic markers, i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO·, released from the NO-donors S-nitrosoglutathione or spermine-NO. Assessment of cytochrome c reduction established that RES cells released twice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreased apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage resistance toward NO·-mediated apoptosis is, at least in part, due to increased superoxide formation. Therefore, the balance between reactive nitrogen and reactive oxygen species regulates RAW 264.7 macrophage apoptosis.

Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells

Detachment-induced cell death (DICD) is considered to be one of the means by which intestinal epithelial cells (IEC) die of apoptosis as they reach the lumen and are shed. Caspases, a family of cysteine proteases, play a central role in initiating, amplifying, and executing apoptosis; however, the pattern of caspase activation in response to distinct apoptotic stimuli remains unknown. We investigated the kinetics of caspase activation during DICD in freshly isolated human IEC. DNA fragmentation is observed 90 min after detachment and is preceded by the sequential activation of preformed members of the CPP32 family of caspases. Activation of caspase 6 and cleavage of the endogenous caspase substrate poly(ADP-ribose) polymerase (EC 2.4.2.30 ) are detected within 15 min of detachment, 30-45 min before caspase 3 activation. Caspase 1 and caspase 10 are present as proenzymes, yet they remain inactive in response to this trigger of apoptosis. Human IEC are primed to rapidly undergo detachment-induced apoptosis involving the selective and sequential activation of preformed caspases. This study may enhance our understanding of physiological events occurring as IEC are shed. Their rapid apoptotic response to detachment may facilitate the high turnover of cells and ensure homeostasis in the intestinal epithelium.Detachment-induced cell death (DICD) is considered to be one of the means by which intestinal epithelial cells (IEC) die of apoptosis as they reach the lumen and are shed. Caspases, a family of cysteine proteases, play a central role in initiating, amplifying, and executing apoptosis; however, the pattern of caspase activation in response to distinct apoptotic stimuli remains unknown. We investigated the kinetics of caspase activation during DICD in freshly isolated human IEC. DNA fragmentation is observed 90 min after detachment and is preceded by the sequential activation of preformed members of the CPP32 family of caspases. Activation of caspase 6 and cleavage of the endogenous caspase substrate poly(ADP-ribose) polymerase (EC 2.4.2.30) are detected within 15 min of detachment, 30-45 min before caspase 3 activation. Caspase 1 and caspase 10 are present as proenzymes, yet they remain inactive in response to this trigger of apoptosis. Human IEC are primed to rapidly undergo detachment-induced apoptosis involving the selective and sequential activation of preformed caspases. This study may enhance our understanding of physiological events occurring as IEC are shed. Their rapid apoptotic response to detachment may facilitate the high turnover of cells and ensure homeostasis in the intestinal epithelium.

Rap1B and Rap2B Translocation to the Cytoskeleton by von Willebrand Factor Involves FcγII Receptor-mediated Protein Tyrosine Phosphorylation

Stimulation of human platelets with von Willebrand factor (vWF) induced the translocation of the small GTPases Rap1B and Rap2B to the cytoskeleton. This effect was specifically prevented by an anti-glycoprotein Ib monoclonal antibody or by the omission of stirring, but was not affected by the peptide RGDS, which antagonizes binding of adhesive proteins to platelet integrins. Association of Rap2B with the cytoskeleton was very rapid, while translocation of Rap1B occurred in a later phase of platelet activation and was totally inhibited by cytochalasin D. vWF also induced the rapid tyrosine phosphorylation of several proteins that was prevented by the tyrosine kinases inhibitor genistein and by cAMP-increasing agents. Under these conditions, also the association of Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the FcγII receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of selected substrates with molecular masses of about 75, 95, and 150 kDa. Three of these substrates were identified as the tyrosine kinase pp72 syk , the phospholipase Cγ2, and the inositol 5-phosphatase SHIP. Our results indicate that translocation of Rap1B and Rap2B to the cytoskeleton is regulated by tyrosine kinases and suggest a novel role for the FcγII receptor in the mechanism of platelet activation by vWF.

Cellular Adaptive Responses to Low Oxygen Tension: Apoptosis and Resistance

Oxygen plays such a critical role in the central nervous system that a specialized mechanism of oxygen delivery to neurons is required. Reduced oxygen tension, or hypoxia, may have severe detrimental effects on neuronal cells. Several studies suggest that hypoxia can induce cellular adaptive responses that overcome apoptotic signals in order to minimize hypoxic injury or damage. Adaptive responses of neuronal cells to hypoxia may involve activation of various ion channels, as well as induction of specific gene expression. For example, ATP sensitive K+ channels are activated by hypoxia in selective neuronal cells, and may play a role in cell survival during hypoxia/anoxia. Additionally, hypoxia-induced c-Jun, bFGF and NGF expression appear to be associated with prevention (or delay) of neuronal cell apoptosis. In this paper, these adaptive responses to hypoxia in neuronal cells are discussed to examine the possible role of hypoxia in pathophysiology of diseases.

INTERACTION OF THE LOW-MOLECULAR-WEIGHT GTP-BINDING PROTEIN RAP2 WITH THE PLATELET CYTOSKELETON IS MEDIATED BY DIRECT BINDING TO THE ACTIN FILAMENTS

The interaction of the low‐molecular‐weight GTP‐binding protein rap2 with the cytoskeleton from thrombin‐aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro‐promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin‐binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin‐activated platelets was subjected to actin depolymerization‐repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPγS. We also found that polyhistidine‐tagged rap2 immobilized on Ni2+‐Sepharose and loaded with either GDP or GTPγS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G‐protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPγS was able to bind to purified F‐actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein. J. Cell. Biochem. 75:675–685, 1999.

Integrin Expression and Macrophage Resistance to Apoptosis in Atherosclerosis

Development and progression of atherosclerosis is a complex pathologic event involving cells from both the arterial vessel wall and the immune system, including monocyte-derived macrophages. Monocytes are recruited to the atherosclerotic lesion via an elaborate process involving adhesion to and migration through the endothelial cell layer and into the arterial wall. Adhesion and migration of monocytes is accompanied by activation/differentiation to tissue macrophages. Once inside the atheroma, macrophages are exposed to many factors which further stimulate the cells. Cytokines, low density lipoproteins, and low oxygen levels (hypoxia; a product of tissue thickening in the absence of neovascularization [1]) are all factors which have profound effects on the state of differentiation, function, and survival of macrophages within the atheroma.