PubMed · 9246147
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G Todd. 1997. Asthma guidelines.. https://pubmed.ncbi.nlm.nih.gov/9246147/
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Tissue factor is the prime initiator of blood coagulation. Expression of tissue factor in tumor endothelial cells leads to thrombus formation, occlusion of vessels and development of hemorrhagic infarctions in the tumor tissue, often followed by regression of the tumor. Tumor cells produce endogenous vascular endothelial growth factor (VEGF), which sensitizes endothelial cells for systemically administered tumor necrosis factor alpha (TNF alpha) and synergistically enhances the TNF-induced expression of tissue factor. We have analyzed the pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells (HUVECs) after combined stimulation with TNF and VEGF. By using specific low molecular weight inhibitors, we demonstrated that protein kinase C (PKC), p44/42 and p38 mitogen-activated protein (MAP) kinases, and stress-activated protein kinase (JNK) are essentially involved in the induction of tissue factor. In contrast, the application of wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, led to strongly enhanced expression of tissue factor in TNF- and VEGF-treated cells, implicating a negative regulatory role for PI3-kinase. In vivo, the application of wortmannin promoted the formation of TNF-induced hemorrhages and intratumoral necroses in murine meth A tumors. The co-injection of wortmannin lowered the effective dose of applied TNF. Therefore, it is conceivable that the treatment of TNF-sensitive tumors with a combination of TNF and wortmannin will ensure the selective damage of the tumor endothelium and minimize the risk of systemic toxicity of TNF. TNF-treatment in combination with specific inhibition of PI3-kinase is a novel concept in anti-cancer therapy.
Wortmannin reportedly induces the formation of enlarged cytoplasmic endosomes. Such vesicles were observed in a definite time window after wortmannin treatment (250 nM) in HEK-293 cells stably expressing a B2R (B2 receptor)--green fluorescent protein conjugate and other cell types. The alternative PI3K (phosphoinositide 3-kinase) inhibitor LY 294002 (100 microM) and a dominant-negative form of the enzyme (p85alpha DeltaiSH2) induce a more modest vesicle enlargement. PI3K inhibition by drugs did not affect agonist-induced [3H]arachidonate release. The wortmannin-induced formation of giant endosomes also involves Rab5 activity, since a dominant-negative form of this GTPase (Rab5 S34N) partially inhibits the wortmannin effect and a constitutively active form of Rab5 (Rab5 Q79L) induces the formation of enlarged endosomes. Moreover, agonist stimulation targeted B2R-green fluorescent protein towards the periphery of the giant vesicles and led to partial receptor degradation only in wortmannin-treated cells. Receptor degradation was decreased by protease inhibitors and by bafilomycin A1, a drug that inhibits lysosome function. Accumulation of fluorescent material inside the enlarged endosomes was observed in cells treated with bafilomycin A1, wortmannin and an agonist. [3H]Bradykinin binding was decreased in HEK-293 cells treated with both wortmannin and the agonist, but not with either separately. Furthermore, a wortmannin-induced functional down-regulation of B2R was observed in rabbit jugular veins after repeated agonist stimulation (contractility assay). This is the first report of a G-protein-coupled receptor down-regulation induced by an alteration of its usual routing in the cell. These results suggest that both PI3K and Rab5 influence B2R intracellular trafficking.
We evaluated the possible autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from normal hamsters. We measured 14CO2 and 3H2O production from d-[U-14C]glucose and d-[5-3H]glucose, respectively, in islets incubated with 0.6, 3.3, 8.3, and 16.7 mM glucose alone or with 5 or 15 mU/ml insulin, anti-insulin guinea pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured (radioimmunoassay) in islets incubated with 3.3 or 16.7 mM glucose with or without 75, 150, and 300 nM wortmannin. Insulin significantly enhanced 14CO2 and 3H2O production with 3.3 mM glucose but not with 0.6, 8.3, or 16.7 mM glucose. Addition of anti-insulin serum to the medium with 8.3 and 16.7 mM glucose decreased 14CO2 and 3H2O production significantly. A similar decrease was obtained in islets incubated with 8.3 and 16.7 mM glucose and wortmannin or nifedipine. This latter effect was reversed by adding 15 mU/ml insulin to the medium. Glucose metabolism was almost abolished when islets were incubated in a Ca2+-deprived medium, but this effect was not reversed by insulin. No changes were found in 14CO2 and 3H2O production by islets incubated with 3.3 mM glucose and anti-insulin serum, wortmannin, or nifedipine in the media. Addition of wortmannin significantly decreased insulin release induced by 16.7 mM glucose in a dose-dependent manner. Our results suggest that insulin exerts a physiological autocrine stimulatory effect on glucose metabolism in intact islets as well as on glucose-induced insulin release. Such an effect, however, depends on the glucose concentration in the incubation medium.