Calpain activation in plasma membrane bleb formation during tert-butyl hydroperoxide-induced rat hepatocyte injury.

BACKGROUND & AIMS: The mechanism of plasma membrane blebbing (dissociation of the lipid bilayer from the membrane cytoskeleton) in hepatocyte injury is not known. The aim of this study was to investigate the role of calpain, a calcium-dependent cytosolic protease, in bleb formation induced by oxidative stress. METHODS: Hepatocytes from Wistar rats were injured with tertbutyl hydroperoxide in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a specific calpain inhibitor, calpeptin (Z-Leu-nLeu-H). Bleb formation was examined by phase-contrast and transmission electron microscopies. Intracellular calcium concentration was measured using Fura-2. Western blot analyses were performed for cytoskeletal proteins (talin, alpha-actinin, and vinculin) and the intermediate (activated) and proactivated forms of calpain mu. RESULTS: tert-Butyl hydroperoxide induced a sustained increase in intracellular calciu, bleb formation, and, ultimately, hepatocyte death. Talin and alpha-actinin were degraded in a time-dependent manner, although no apparent changes of actin filament were observed. Before the cytoskeletal protein degradation, the intermediate form of calpain mu appeared as its proactivated form decreased. In addition, calpeptin or EGTA inhibited not only calpain mu activation but also cytoskeletal protein degradation and bleb formation. CONCLUSIONS: In tert-butyl hydroperoxide-treated hepatocytes, the activation of calpain promotes membrane blebbing via degradation of cytoskeletal proteins.

Involvement of protein phosphatase-1 in cytoskeletal organization of cultured endothelial cells.

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effects of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vein endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different according to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton.

Suppression by antithrombotic agents of pseudointimal hyperplasia of polytetrafluoroethylene graft implanted into venous system.

The lumen of the polytetrafluoroethylene graft (PTFE) implanted into a rabbit inferior vena cava (IVC) was markedly narrowed by 4 weeks after grafting. This was due to initial thrombosis on the luminal surface of the graft, which was followed by pseudointimal hyperplasia (PIH). To elucidate the role of the initial thrombosis in subsequent PIH, the effect of ticlopidine (T) and/or warfarin (W) on PIH was studied in an animal model. A PTFE tube graft was implanted into a rabbit IVC. Twenty eight rabbits were randomly assigned to the following experimental groups. Eight rabbits without antithrombotic agents were observed for 2 weeks (A, n = 4) and 4 weeks (B, n = 4) after grafting. T (100 mg/kg/day) and W (0.33 mg/kg/day) were orally administered for 4 weeks to group C (n = 4) and group D (n = 4), respectively. A combination of a half dose of T and W ((T + W)/2) was given for 2 weeks (E, n = 4) and 4 weeks (F, n = 4) after grafting. Four rabbits in group G received the combination of T and W for the first 2 weeks and were observed for an additional 2 weeks without medication. All the grafts were patent at time of harvest. The dry weight of the intraluminal deposit (DW) was determined as an indicator of PIH (A:35 +/- 1 mg/graft, B:40 +/- 8, C:22 +/- 3, D:22 +/- 3, E:14 +/- 2, F:15 +/- 3, G:23 +/- 2). Administration of T or W was equally effective in reducing DW and (T+W)/2 was more effective than a single agent.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane.

Platelets activated by various agonists produce vesicles (microparticles; MPs) from the plasma membrane. However, the mechanism of this MP formation remains to be elucidated. To investigate the possible involvement of protein phosphorylation and cytoskeletal reorganization in MP formation, the effects of various inhibitors on MP formation were investigated. Flow cytometry was employed to detect the amount of MP formation by using monoclonal antibodies against glycoprotein (GP) IIb-IIIa (NNKY 1-32) or GPIIb (Tab). The relationship between changes in cytoskeletal architecture and MP formation in the platelets activated by thrombin plus collagen was observed by scanning electron microscopy (SEM). MPs were observed in the vicinity of the terminals of pseudopods, suggesting that MPs may be related by budding of the pseudopods. Cytochalasin D (10 microM) inhibited MP formation from the activated platelets almost completely. Moreover, SEM of the cytochalasin D-treated platelets revealed the absence of shape change, pseudopod formation and MPs. These findings suggest that cytoskeletal reorganization is necessary for MP formation. Since cytoskeletal reorganization is considered to be regulated by a dynamic phosphorylation-dephosphorylation process, we investigated the effects of the protein phosphatase inhibitors, calyculin A (CLA) and okadaic acid (OA), on MP formation. Flow cytometry showed that these two inhibitors doubled MP formation in activated platelets. SEM of the platelets treated with CLA or OA demonstrated more prominent shape change and pseudopod formation in these platelets than in those without inhibitor. From these results, we conclude that cytoskeletal reorganization, which is controlled by phosphorylation, is involved in MP formation.