Tumor necrosis factor (TNF) has been shown to induce the phosphorylation of a 27 kDa protein in a time‐ and concentration‐dependent manner in HeLa D98/AH2, ME 180 and bovine aortic endothelial cells. This phosphorylation could be reproduced by the calcium ionophore, A23187. However, this phosphorylation was not observed in L929 cells, for which TNF is highly cytotoxic, suggesting that it might play a role in actions of TNF other than the induction of cell death.

Tumor necrosis factor-alpha has been shown to rapidly increase the phosphorylation of three 28 kDa proteins in bovine aortic endothelial cells but not in L929 cells. Tumor necrosis factor-alpha induces the necrosis of the latter cells but not of the former. Arsenite enhanced the phosphorylation of the same 28kDa proteins as tumor necrosis factor-alpha in the endothelial cells. As stress proteins often play a protective role, we suggest that the phosphorylation of these proteins in endothelial cells may be responsible for the resistance of these cells to tumor necrosis factor-alpha.

L929 cells were incubated with tumor necrosis factor-alpha (TNF-alpha) in the presence or absence of various inhibitors of arachidonic acid metabolism. The addition of either hydrocortisone or nordihydroguaiaretic acid (NDGA) decreased the cytotoxic effect of TNF-alpha but exogenously added arachidonate or linoleate, indomethacin and eicosatetraynoic acid (ETYA) were without effect. While it was found that TNF-alpha stimulated arachidonic acid release, no metabolites of this fatty acid could be evidenced. Cytotoxicity of TNF-alpha could also be decreased by the addition of either cholera or pertussis toxin. These results suggest that a GTP-binding protein is involved in the cytotoxic action of TNF-alpha. Arachidonic acid, released possibly by a phospholipase A2, might also play a role, but probably not via its conversion to known metabolites.

No abstract

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI-ATP. On sodium dodecylsulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approximately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively. This purified myosin exhibited high ATPase activity in the presence of K + + EDTA or Ca2+ and very little activity in the presence of M8'. (K+ + EDTA)-ATPase activity showed one pH optimum at 8.0, while Ca2+-ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (KC + EDTA)-stimulated enzyme activity was specific for ATP whereas Ca2+-stimulated activity showed low specificity for nucleoside triphosphates.Actin and myosin may provide the force required for all cytoplasmic and cell movements in a variety of non-muscle cells [I] and secretion is among the numerous functions possibly attributable to actomyosin. Recently, both actin and myosin were localized by immunofluorescence microscopy beneath the luminal border of rat pancreatic acinar cells [2] and tropomyosin was extracted from calf pancreas [3]. G-actin is also found to form a 1 : 1 complex with deoxyribonuclease I in rat pancreatic juice [4]. In addition, cytochalasin B, a compound with destabilizes actin-microfilaments, inhibits the secretory response of the rat pancreas to secretagogues [5 -71, suggesting the contribution of actin to intracellular movements of zymogen granules. Pancreatic myosin has not yet been isolated, to the best of our knowledge. As different cell types contain distinct species of contractile proteins [l], it was felt of interest to separate myosin from actin in the rat pancreas and to further characterize pancreatic myosin by chromatographic, electrophoretic, and ATPase properties. MATERIALS AND METHODS Preparation of Actomyosin from the Rat PancreasAbout 7 g of rat pancreas were homogenized in 3 vol. of a KCl buffer containing 15 mM Tris/HCl pH 7.5, 2 mM EDTA, 0.2 mM dithiothreitol, 0.6 M KCl and aprotinin (500 kallikrein inhibitor units Trasylol Bayer/ml) as protease inhibitor. After magnetic stirring for 1 h at 4"C, the homogenate was centrifuged for 45 min at 70000 x g and the resulting opalescent supernatant was submitted to a second centrifugation for 2 h at 150000 x g. The clear supernatant beneath the surface lipid layer (15 ml) was carefully removed with a pipette and applied to a 2.6 x 90 cm column of Abbreviatiom. Taps, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; Mops, 4-morpholinepropanesulfonic acid.Sepharose CL 4B equilibrated with the KC1 buffer lacking Trasylol, and eluted with the same buffer. Preparation of Myosin from the Rat PancreasThe only difference with the preparation of actomyosin was that the Sepharose column was eluted with the discontinuous buffer system of Pollard et al. [8]. The support was preequilibrated with two column volumes of KCl buffer pH ...