herbimycin and Hypoxia

Muscle contractions and insulin stimulate glucose transport into muscle by separate pathways. The contraction-mediated increase in glucose transport is mediated by two mechanisms, one involves the activation of 5'-AMP-activated protein kinase (AMPK) and the other involves the activation of calcium/calmodulin-dependent protein kinase II (CAMKII). The steps leading from the activation of AMPK and CAMKII to the translocation of GLUT4 to the cell surface have not been identified. Studies with the use of the tyrosine kinase inhibitor genistein suggest that one or more tyrosine kinases could be involved in contraction-stimulated glucose transport. The purpose of the present study was to determine the involvement of tyrosine kinases in contraction-stimulated glucose transport in rat soleus and epitrochlearis muscles. Contraction-stimulated glucose transport was completely prevented by pretreatment with genistein (100 microM) and the related compound butein (100 microM). However, the structurally distinct tyrosine kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyridine and herbimycin did not reduce contraction-stimulated glucose transport. Furthermore, genistein and butein inhibited glucose transport even when muscles were exposed to these compounds after being stimulated to contract. Muscle contractions did not result in increases in tyrosine phosphorylation of proteins such as proline-rich tyrosine kinase and SRC. These results provide evidence that tyrosine kinases do not mediate contraction-stimulated glucose transport and that the inhibitory effects of genistein on glucose transport result from direct inhibition of the glucose transporters at the cell surface.

Topics: Animals; Benzoquinones; Biological Transport; Chalcone; Chalcones; Deoxyglucose; Electric Stimulation; Focal Adhesion Kinase 2; Genistein; Glucose; Glucose Transporter Type 4; Hypoxia; In Vitro Techniques; Insulin; Lactams, Macrocyclic; Male; Muscle Contraction; Muscle, Skeletal; Phosphorylation; Protein-Tyrosine Kinases; Pyrimidines; Quinones; Rats; Rats, Wistar; Rifabutin; src-Family Kinases

Hypoxia is classically considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, considered a feature of apoptosis, in DNA damage and cell death in chemical hypoxic injury to renal tubular epithelial cells (LLC-PK1 cells). Tyrosine phosphorylation has been implicated to be involved in cell signaling pathway leading to cell growth, proliferation, and apoptotic death. However, a role of tyrosine phosphorylation as a signal transduction pathway involved in DNA damage and cell death has not been previously examined in hypoxic injury in any tissue. In the present study, we have demonstrated that chemical hypoxia with a combination of antimycin A, a mitochondrial respiration inhibitor, and substrate deprivation resulted in rapid increase in protein tyrosine kinases activity and protein tyrosine phosphorylation prior to any evidence of cell death in LLC-PK1 cells. The inhibitors of protein tyrosine kinases, genistein, lavendustin A, tyrphostin, and herbimycin A provided a marked protection against chemical hypoxia-induced DNA damage (as measured by alkaline unwinding assay) and cell death (as measured by trypan blue exclusion assay). In a separate study, we confirmed the ability of the inhibitors, lavendustin A and herbimycin A to prevent chemical hypoxia-induced increase in protein tyrosine kinases activity and protein tyrosine phosphorylation. In addition, the inhibitors used did not affect ATP depletion induced by antimycin A, suggesting that the inhibitors do not alter cellular uptake of antimycin A. Taken together, our data provide a strong evidence that tyrosine phosphorylation plays as important role in DNA damage and cell death in chemical hypoxic injury to renal tubular epithelial cells.

Topics: Animals; Benzoquinones; Cell Death; DNA Damage; Enzyme Inhibitors; Hypoxia; Lactams, Macrocyclic; LLC-PK1 Cells; Phenols; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; Swine; Tyrosine

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen, which also enhances vascular permeability. Because this angiogenic factor has been suggested to play a role in brain tumor biology, we have begun to investigate the regulation of VEGF expression in cultures of rat type I astrocytes. In this report, we have focused on the influence of hypoxia on VEGF expression. Under standard in vitro conditions (21% O2) VEGF expression in astrocytes in barely detectable by northern analysis. However, after exposure to 0.2% O2 for as little as 3 h VEGF mRNA levels are markedly increased reaching a maximum by approximately 8 h of exposure. Treatment of astrocytes with CoCl2 or desferrioxamine results in a similar induction of VEGF, suggesting that the oxygen sensor regulating VEGF expression in astrocytes is a heme-containing molecule. Although acute treatment with TPA (6 h) induces VEGF expression, chronic exposure to TPA (24 h) to deplete PKC activity does not reduce the hypoxia-induced VEGF expression. These data indicate that VEGF induction in astrocytes can proceed through PKC-dependent and -independent pathways. Furthermore, chronic exposure to TPA or treatment with herbimycin A results in the enhancement of the hypoxia-mediated increase in VEGF mRNA levels. These results suggest that PKC and herbimycin-sensitive tyrosine kinase may serve as negative regulators of the hypoxia-activated signal transduction pathway that leads to the induction of VEGF expression. However, treatment of astrocytes with the nonspecific kinase inhibitors H7 and H8 reduced the level of VEGF induction by hypoxia, indicating that some type of kinase activity is required in this signaling pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Astrocytes; Base Sequence; Benzoquinones; Blotting, Northern; Cells, Cultured; Deferoxamine; Endothelial Growth Factors; Endothelium, Vascular; Hemeproteins; Hypoxia; Isoquinolines; Lactams, Macrocyclic; Lymphokines; Molecular Sequence Data; Oxygen; Piperazines; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; Siderophores; Signal Transduction; Tetradecanoylphorbol Acetate; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors