herbimycin and tyrphostin-25

Selective inhibitors were used to study the role of tyrosine kinases in alpha(1A)-adrenoceptor-mediated responses in transfected PC12 cells. Ca(2+) responses to noradrenaline were measured using fura-2, and the effects of genistein, tyrphostin A25, and herbimycin A were examined. Neither genistein nor herbimycin A pretreatment altered noradrenaline-induced Ca(2+) responses, although tyrphostin A25 pretreatment caused some reduction. However, acute addition of genistein quickly reversed the apparent noradrenaline response, apparently, through a direct interaction with cytoplasmic fura-2. Both genistein and tyrphostin A25, at concentrations similar to those used to inhibit tyrosine kinases, markedly reduced fluorescence of fura-2 excited by both 340 and 380 nm, and genistein also reduced the 340/380 ratio. Tyrosine kinase inhibitors did not alter noradrenaline stimulated inositol phosphate formation in alpha(1A)-PC12 cells. These results suggest that tyrosine kinases are not involved in second messenger responses to alpha(1A)-adrenoceptors, but that tyrosine kinase inhibitors can interact directly with fura-2.

Topics: Animals; Benzoquinones; Calcium Signaling; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fura-2; Genistein; Inositol Phosphates; Lactams, Macrocyclic; Norepinephrine; PC12 Cells; Protein-Tyrosine Kinases; Quinones; Rats; Receptors, Adrenergic, alpha-1; Rifabutin; Spectrometry, Fluorescence; Transfection; Tyrphostins

We investigated the role of tyrosine kinases in the regulation of insulin release from a hamster beta-cell line, HIT T15, using selective tyrosine kinase inhibitors. Genistein increased the insulin release induced by glucose, but herbimycin A, tyrphostins and the erbstatin analogue failed to change the release. Lavendustin A at 0.1 nM-1 microM caused a concave-shaped inhibition of the insulin release stimulated by 7 mM glucose. The inhibitory effect of lavendustin A was overcome by higher concentrations of glucose. Lavendustin B, the negative control analogue, had no effect on the release. Lavendustin A at a nanomolar range progressively inhibited insulin release by high K+ (50 mM)-depolarization, whereas the inhibitor did not change the insulin release by Ca2+ ionophore (A23187). On the contrary, lavendustin A at 10 nM significantly increased insulin release when glucose-induced insulin release was enhanced by either 5 microM forskolin or 162 nM 12-O-tetradecanoylphorbol 13-acetate. Lavendustin A failed to influence the Ca(2+)-induced insulin release from HIT cells permeabilized with streptolysin-O. These findings suggest that tyrosine kinases may play versatile roles in the control of insulin release from the pancreatic beta-cell.

Topics: Animals; Benzoquinones; Cells, Cultured; Cricetinae; Genistein; Glucose; In Vitro Techniques; Insulin; Islets of Langerhans; Isoflavones; Lactams, Macrocyclic; Nitriles; Phenols; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tyrphostins

We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 microM and 0.1 microM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 microM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 microM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 mumol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity.

Topics: Animals; Benzoquinones; Bone Resorption; Caffeic Acids; Cells, Cultured; Chickens; Enzyme Inhibitors; Female; Femur; Genistein; Isoflavones; Lactams, Macrocyclic; Nitriles; Osteoclasts; Ovariectomy; Protein Biosynthesis; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Tyrphostins

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

Tyrosine phosphorylation is an important mechanism of cell regulation and has been recently implicated in defense strategies against a variety of pathogens. We have investigated the involvement of protein tyrosine kinase activity in the Leishmania attachment, invasion and survival within macrophages, as well as promastigote ability to trigger tyrosine phosphorylation, which could contribute to leishmanicidal activity. Treatment of murine macrophage monolayers with genistein, herbimycin A, tyrphostin 25 or staurosporine prior to infection decreased parasite invasion in a dose-dependent manner. Contrary, addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, phosphotyrosine and p-nitrophenyl phosphate to the interaction medium, significantly increased parasite binding and internalization, whereas phosphoserine and phosphothreonine had no effect. The phosphatase activity of intact promastigotes was greater than that of macrophages. Western blot analysis revealed tyrosine-phosphorylated bands from 198 to 28 kDa following macrophage challenge with promastigotes. Uninfected macrophages displayed no detectable tyrosine phosphorylated proteins, possibly indicating an inducible process, while in parasites it was constitutive, as seen by the presence of 42, 40 and 35 kDa phosphoproteins on the Leishmania lysates. Immunofluorescence and immunogold detection of phosphotyrosine residues in some promastigote-macrophage attachment areas, but not in the vicinity of ingested parasites, suggest that Leishmania-induced tyrosine phosphorylation is an early, local and short-lived event. Genistein treatment of Leishmania-infected cells significantly enhanced the parasite burden. This antagonist also diminished nitric oxide production in resting and interferon gamma/lipopolysaccharide-activated infected macrophages, which may account for the increased parasite survival. We propose that protein tyrosine kinase-linked pathways regulate the Leishmania promastigote invasion and the macrophage microbicidal activity.

Topics: Animals; Benzoquinones; Dose-Response Relationship, Drug; Fluorescent Antibody Technique, Indirect; Genistein; Immunohistochemistry; Isoflavones; Lactams, Macrocyclic; Leishmaniasis; Macrophage Activation; Macrophages, Peritoneal; Male; Mice; Microscopy, Electron; Nitriles; Organophosphorus Compounds; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Quinones; Rifabutin; Staurosporine; Tyrphostins; Vanadates