guanosine-triphosphate and manumycin

Hepatotoxicity due to overdose of the analgesic and antipyretic acetaminophen (APAP) is a major cause of liver failure in adults. To better understand the contributions of different signaling pathways, the expression and role of Ras activation was evaluated after oral dosing of mice with APAP (400-500 mg/kg). Ras-guanosine triphosphate (GTP) is induced early and in an oxidative stress-dependent manner. The functional role of Ras activation was studied by a single intraperitoneal injection of the neutral sphingomyelinase and farnesyltransferase inhibitor (FTI) manumycin A (1 mg/kg), which lowers induction of Ras-GTP and serum amounts of alanine aminotransferase (ALT). APAP dosing decreases hepatic glutathione amounts, which are not affected by manumycin A treatment. However, APAP-induced activation of c-Jun N-terminal kinase, which plays an important role, is reduced by manumycin A. Also, APAP-induced mitochondrial reactive oxygen species are reduced by manumycin A at a later time point during liver injury. Importantly, the induction of genes involved in the inflammatory response (including iNos, gp91phox, and Fasl) and serum amounts of proinflammatory cytokines interferon-gamma (IFNgamma) and tumor necrosis factor alpha, which increase greatly with APAP challenge, are suppressed with manumycin A. The FTI activity of manumycin A is most likely involved in reducing APAP-induced liver injury, because a specific neutral sphingomyelinase inhibitor, GW4869 (1 mg/kg), did not show any hepatoprotective effect. Notably, a structurally distinct FTI, gliotoxin (1 mg/kg), also inhibits Ras activation and reduces serum amounts of ALT and IFN-gamma after APAP dosing. Finally, histological analysis confirmed the hepatoprotective effect of manumycin A and gliotoxin during APAP-induced liver damage.. This study identifies a key role for Ras activation and demonstrates the therapeutic efficacy of FTIs during APAP-induced liver injury.

Topics: Acetaminophen; Alanine Transaminase; Analgesics, Non-Narcotic; Animals; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Farnesyltranstransferase; Gliotoxin; Guanosine Triphosphate; Interferon-gamma; MAP Kinase Kinase 4; Mice; Mice, Inbred BALB C; Oxidative Stress; Polyenes; Polyunsaturated Alkamides; ras Proteins; Tumor Necrosis Factor-alpha

The majority of low molecular weight G proteins undergoes a series of post-translational modification steps, e.g., isoprenylation, at their C-terminal cysteine, which seem to be critical for the transport of the modified proteins to the membrane sites for interaction with their respective effector proteins. Using lovastatin, an inhibitor of mevalonic acid, and hence, isoprenoid biosynthesis, we demonstrated previously that protein isoprenylation is critical for physiological insulin secretion from normal rat islets. Herein, we used more selective synthetic inhibitors of protein prenylation to examine their effects on glucose- and calcium-mediated insulin secretion from betaTC3 cells. Both 3-allyl- and vinylfarnesols, which inhibit and/or modulate protein farnesyl transferases, significantly (80-95%) inhibited glucose- and KCl-stimulated insulin secretion from these cells. In a similar manner, the allyl and vinyl forms of geranylgeraniol, reagents targeted toward protein geranylation, attenuated insulin secretion elicited by glucose and KCl. Furthermore, manumycin A, a natural inhibitor of protein farnesylation, and geranylgeranyl transferase inhibitor-2147 (GGTI-2147), a peptidomimetic inhibitor of protein geranylgeranylation, also inhibited glucose- and KCl-induced insulin secretion to comparable degrees. Treatment of betaTC3 cells with either 3-vinylfarnesol or 3-vinyl geranylgeraniol resulted in accumulation of unprenylated proteins in the cytosolic fraction. These data further support our original formulation that inhibition of isoprenylation of small molecular weight G proteins might impede their interaction with their putative effectors, which may be required for physiological insulin secretion.

Topics: Alkyl and Aryl Transferases; Animals; Calcium; Cell Line; Cell Survival; Enzyme Inhibitors; Glucose; GTP-Binding Proteins; Guanosine Triphosphate; Imidazoles; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Leucine; Polyenes; Polyunsaturated Alkamides; Potassium Chloride; Protein Prenylation; Rats

To search for the intracellular signaling pathway critical for the secretion of matrix metalloproteinases (MMP), we studied the effects of dominant negative Ras (S17N Ras) and dominant negative MEK1 (MEK1AA) expression in v-crk-transformed 3Y1. Expression of either S17N Ras or MEK1AA dramatically suppressed the augmented secretion of MMP-2 and MMP-9 in v-crk-transfected 3Y1. Similarly, a Ras farnesyltransferase inhibitor, manumycin A, and a MEK1 inhibitor, U0126, suppressed MMP secretion in a dose-dependent manner, whereas a PI3 kinase inhibitor, wortmannin, could not. In addition, the suppression of MMP secretion by S17N Ras showed good correlation with the inhibition of in vitro invasiveness of the cells. In contrast, expression of dominant negative C3G did not suppress MMP secretion, although it substantially blocked the c-Jun N-terminal kinase activation. Taken together, the Ras-MEK1 pathway, but not the C3G-JNK pathway, seems to play a key role in the activation of MMP secretion and, hence, the invasiveness of v-crk-transformed cells.

Topics: Androstadienes; Animals; Butadienes; Cell Line, Transformed; Collagen; Drug Combinations; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Guanine Nucleotide-Releasing Factor 2; Guanosine Diphosphate; Guanosine Triphosphate; Immunoblotting; Laminin; MAP Kinase Kinase 1; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mitogen-Activated Protein Kinase Kinases; Nitriles; Oncogene Protein v-crk; Polyenes; Polyunsaturated Alkamides; Protein Serine-Threonine Kinases; Proteoglycans; ras Proteins; Rats; Retroviridae Proteins, Oncogenic; Signal Transduction; src Homology Domains; Wortmannin