ucn-1028-c and tyrphostin-25

1. The aim of this study was to determine whether different signal transduction mechanisms underlie the Ca2+ sensitizing effects of guanosine 5'-O-(3-thiotriphosphate) (GTP(gamma)S) and receptor agonists on beta-escin-skinned smooth muscle of rabbit mesenteric artery. 2. In the homogenate of the beta-escin-skinned arterial strip, C3 exoenzyme of Clostridium botulinum catalyzed the [32P]-ADP-ribosylation of only one protein that had the same molecular mass as the protein detected in Western blots with anti-rho p21 antibody. Pretreatment of preparations with C3 resulted in great inhibition of GTP(gamma)S-induced Ca2+ sensitization, although the effect of GTP(gamma)S at higher concentrations (> or = 30 microM) was not completely blocked by this treatment. In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5'-triphosphate, of the Ca2+-induced contraction was not affected by C3 pretreatment. 3. The protein kinase C (PKC) inhibitors calphostin C and staurosporine completely eliminated the enhancement by phorbol ester 12,13-dibutyrate of the Ca2+-induced contraction. However, these PKC inhibitors had no effect on GTP(gamma)S- and receptor agonist-induced Ca2+ sensitization. 4. The tyrosine kinase inhibitors genistein and tyrphostin 25 caused an irreversible and complete block of the enhancement by GTP(gamma)S of the Ca2+-induced contraction without affecting this Ca2+ contraction. The inactive genistein analogue daidzein did not modify the effect of GTP(gamma)S. The Ca2+ sensitizing effects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors. 5. These results suggest that rho p21 predominantly mediates GTP(gamma)S-induced Ca2+ sensitization of beta-escin-skinned smooth muscle of rabbit mesenteric artery, while the Ca2+ sensitizing actions of heterotrimeric G protein-coupled receptor agonists do not involve this small G protein. However, it seems that tyrosine phosphorylation, but not PKC activation, plays an important role in both of the rho p21 protein- and heterotrimeric G protein-mediated Ca2+ sensitization mechanisms.

Topics: Adenosine Diphosphate; ADP Ribose Transferases; Animals; Botulinum Toxins; Calcium; Carcinogens; Dimerization; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genistein; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Histamine; In Vitro Techniques; Male; Mesenteric Arteries; Muscle Contraction; Muscle, Smooth; Naphthalenes; Phenylephrine; Phorbol 12,13-Dibutyrate; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Rabbits; rho GTP-Binding Proteins; Ribose; Staurosporine; Tyrosine; Tyrphostins; Vasoconstrictor Agents

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amides; Animals; Arachidonic Acid; Arachidonic Acids; Cyclic AMP; Dinoprostone; Endocannabinoids; Enzyme Activation; Ethanolamines; Genistein; GTP-Binding Proteins; Linoleic Acids; Macrophages; Mice; Naphthalenes; Neuroblastoma; Nitriles; Palmitic Acids; Phospholipases A; Polyunsaturated Alkamides; Second Messenger Systems; Staurosporine; Tumor Cells, Cultured; Tyrphostins; Virulence Factors, Bordetella

Exposing normal human breast epithelia (HBE) cells, which were growth arrested by a 3-day culture in EGF-deprived medium, to the microtubule disrupting agent, demecolcin (N-deacetyl-N-methyl-colchicine), for 2 hr significantly stimulated the initiation of DNA synthesis 22 hr later. The demecolcin-induced DNA synthesis was not accompanied by activation of the EGF receptor and it was inhibited by calphostin C, an inhibitor of protein kinase C (PKC), and U-73122, an inhibitor of phospholipase C (PLC). Contrary to this, the EGF-induced DNA synthesis was inhibited by tyrphostin A25, a specific inhibitor of the EGF receptor tyrosine kinase, and calphostin C. The results suggested that the involvement of PLC and PKC in the demecolcin-induced signal transduction pathway leads to the initiation of DNA synthesis.

Topics: Androstadienes; Breast; Cell Line; Cinnamates; Demecolcine; DNA; DNA Replication; Enzyme Inhibitors; Epidermal Growth Factor; Epithelium; ErbB Receptors; Estrenes; Female; Humans; Kinetics; Microtubules; Naphthalenes; Nitriles; Phosphorylation; Protein Kinase C; Pyrrolidinones; Type C Phospholipases; Tyrphostins; Wortmannin

Recent data suggest involvement of the Janus tyrosine kinase-2 (JAK2) in human GH-induced tyrosine phosphorylation of the GH receptor and the insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), leading to activation of the phosphatidylinositol 3-kinase and the acute insulin-like effects in primary rat adipocytes. To investigate the functional role of this kinase, we screened a number of tyrosine kinase inhibitors for their ability to inhibit three rapid effects of GH on primary adipocytes: increased lipogenesis, inhibition of noradrenaline-induced lipolysis, and promotion of JAK2 tyrosine phosphorylation. Only staurosporine was found to inhibit all three effects. The inhibition of lipogenesis and antilipolysis exhibited the same staurosporine dose dependency (IC50, approximately 40 nM) as inhibition of JAK2 and IRS-1 tyrosine phosphorylation as well as binding of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2. The unidentified cytosolic tyrosine-phosphorylated protein pp95, in contrast, was not affected, suggesting that it is not phosphorylated primarily by JAK2. Protein kinase C does not seem to be directly involved in the insulin-like effects, because the selective protein kinase C inhibitor calphostin C had no effect at levels up to 100 nM above which unspecific cellular effects occurred. Methyl-2,5-dihydroxy cinnamate inhibited GH-induced lipogenesis from [3-3H]glucose and nonstimulated lipogenesis from [2-14C]-pyruvate and [3H]acetate, but was without effect on GH-induced 2-deoxy-D-[1-3H]glucose uptake, JAK2 phosphorylation and antilipolysis, suggesting unspecific effects on mitochondrial metabolism rather than a direct effect on the GH-mediated signal. Tyrphostin 25 and herbimycin A had no effect on any of the parameters studied, except for a slight increase in JAK2 phosphorylation in response to tyrphostin 25. In summary, these data support the role for JAK2 in mediating the insulin-like effects of GH in adipocytes.

Topics: Acetates; Adipocytes; Animals; Benzoquinones; Cells, Cultured; Cinnamates; Deoxyglucose; Enzyme Inhibitors; Epididymis; Human Growth Hormone; Humans; Insulin; Kinetics; Lactams, Macrocyclic; Lipids; Lipolysis; Male; Naphthalenes; Nitriles; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Pyruvic Acid; Quinones; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Rifabutin; Staurosporine; Tyrphostins

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.

Topics: Cell Adhesion; Cell Line; Cell Movement; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Genistein; Glycoproteins; Humans; Insulin-Like Growth Factor I; Integrins; Isoflavones; Kinetics; Naphthalenes; Nitriles; Pancreatic Neoplasms; Phosphotyrosine; Polycyclic Compounds; Protein Kinase C; Receptor Protein-Tyrosine Kinases; Receptor, IGF Type 1; Receptors, Vitronectin; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine; Tyrphostins; Vitronectin