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Results for “Wortmannin”

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At least 19 recordsLinked to original sources

PubMed1999

Wortmannin inhibits the growth of mammary tumors despite the existence of a novel wortmannin-insensitive phosphatidylinositol-3-kinase.

PURPOSE: Phosphatidylinositol (PtdIns) 3-kinase is an important mediator of many cellular functions. The study of PtdIns 3-kinase has been facilitated by the existence of the potent irreversible inhibitor of p110 PtdIns 3-kinase, wortmannin. The purpose of the study was to investigate the relationship between the cell growth inhibitory activity and antitumor activity of wortmannin and inhibition of PtdIns 3-kinase. METHODS: PtdIns 3-kinase activity was measured in cells and tumors and the effects of wortmannin investigated. RESULTS: Wortmannin inhibited the growth of murine C3H and human MCF-7 mammary tumors in vivo. However, the ability of wortmannin to inhibit C3H tumor growth was not related to inhibition of tumor PtdIns 3-kinase activity. The existence of wortmannin-insensitive PtdIns 3-kinase activity was demonstrated in C3H and MCF-7 cell culture lysates and solid tumors, and normal mouse tissue homogenates. In addition to being resistant to inhibition by wortmannin, MCF-7 cell lysate total PtdIns 3-kinase activity was also resistant to five additional known inhibitors of p110 PtdIns 3-kinase. Partial purification of wortmannin-insensitive PtdIns 3-kinase from MCF-7 cell lysate showed the activity to be independent of the PtdIns 3-kinase p85 regulatory subunit. CONCLUSION: The results of the current study demonstrate that wortmannin can inhibit the growth of murine and human mammary tumors despite the presence of novel wortmannin-insensitive PtdIns 3-kinases in these tissues suggesting that some other target is responsible for wortmannin's antitumor activity.

Androstadienes↗

PubMed1997

Wortmannin-sensitive and -insensitive steps in calcium-controlled exocytosis in pituitary gonadotrophs: evidence that myosin light chain kinase mediates calcium-dependent and wortmannin-sensitive gonadotropin secretion.

In cultured rat pituitary cells, increases in the cytosolic calcium concentration ([Ca2+]i) and LH release are induced by activation of GnRH receptors as well as by nonreceptor-mediated stimuli. Treatment of pituitary cells with the myosin light chain kinase (MLCK) inhibitor, wortmannin, attenuated GnRH-induced LH release. Wortmannin also reduced the LH responses to nonreceptor-mediated elevation of [Ca2+]i by ionomycin and activation of voltage-sensitive Ca2+ channels by Bay K 8644 or high K+, as well as Ca2+-induced LH release in permeabilized pituitary cells. The [Ca2+]i responses to these stimuli were unaltered in wortmannin-treated pituitary cells, indicating that this compound inhibits a Ca2+-dependent step in exocytosis without affecting Ca2+ signaling. In perifused pituitary cells, the GnRH-induced early spike phase of LH release was not affected by wortmannin, whereas the subsequent plateau phase was almost completely inhibited. No significant changes in GnRH-induced phospholipase D activity and diacylglycerol production were observed in wortmannin-treated pituitary cells during the sustained phase of agonist stimulation. Wortmannin also had no effect on LH responses to the protein kinase C activator, phorbol 12-myristate 13-acetate, further indicating that the attenuation of agonist-induced LH release is not related to inhibition of the diacylglycerol/protein kinase C pathway. In addition, agonist-induced LH release was attenuated by two other MLCK inhibitors, MS-347a and KT5926. These data suggest that MLCK mediates the downstream effects of Ca2+ on exocytosis, an action supported by the finding of wortmannin-sensitive phosphorylation of a 20-kDa protein in pituitary cells and alphaT3-1 gonadotrophs treated with GnRH, K+, and Bay K 8644. This protein was coprecipitated from pituitary extracts with a specific antibody to nonmuscle myosin IIB and comigrated with 20-kDa smooth muscle myosin light chain on SDS-PAGE. These results demonstrate that Ca2+ controls exocytosis through an initial wortmannin-insensitive step and a sustained wortmannin-sensitive step and suggest that the latter event in the cascade of cellular responses is dependent on phosphorylation of nonmuscle myosin IIB light chain by MLCK.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1989

Acute pathological effects on rats of orally administered wortmannin-containing preparations and purified wortmannin from Fusarium oxysporum.

The pathological effects in rats of orally administered wortmannin, a mycotoxin produced by Fusarium oxysporum, were studied. Weanling rats were fed a wortmannin-containing fungal culture for 4 or 5 days, or were given by intragastric gavage a single, lethal dose of extracts of a wortmannin-containing fungal culture or pure toxin. Haemoglobinuria, necrosis of lymphoid tissues and death occurred in rats fed the fungal culture. Administration by gavage of extracts of the wortmannin-containing culture and of purified wortmannin produced gastric and myocardial haemorrhage. Major microscopic lesions in gavaged rats were haemorrhage in the myocardium and gastric submucosa and necrosis of lymphocytes in the thymus, spleen and gut-associated lymphoid tissue. Necrosis of gastro-intestinal epithelium was not observed. Myocardial haemorrhage was severe, often transmural, and is an unusual lesion that may aid in the diagnosis of wortmannin toxicosis.

Administration, Oral↗

PubMed1995

Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase.

Wortmannin and its structural analogue demethoxyviridin (DMV) have been reported to be specific inhibitors of phosphatidylinositol 3-kinase activity. Here we report that these compounds are not as selective as assumed and demonstrate inhibition of bombesin-stimulated phospholipase A2 activity by both wortmannin and DMV with an IC50 (2 nM) which is slightly more potent than the inhibition of insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate generation in these cells (approximately 10nM). While it has not been possible to fully block in vitro phospholipase A2 activity with wortmannin, inhibition cannot be a consequence of inhibition of PI 3-kinase activity since bombesin fails to generate 3-phosphorylated lipids in the intact cell. Therefore, while wortmannin is indeed a PI 3-kinase inhibitor, it is not as specific as previously reported, and experimental conclusions based solely on its use should be treated with caution.

3T3 Cells↗

PubMed1992

Decreased immunological responses by wortmannin-containing rice culture of Fusarium oxysporum and by purified wortmannin in avian species.

Immunological assays were performed in young chicken and duck after they had been fed wortmannin-containing culture of Fusarium oxysporum or purified wortmannin for 2 weeks. The culture significantly decreased humoral response to sheep red blood cell, cell-mediated cutaneous hypersensitivity to phytohemagglutinin and phagocytic activity in isolated peritoneal exudate adherent cells, but only when the concentration was high enough to cause concurrent reduction in body weight gain and hematocrit. Increased dietary metabolizable energy and protein did not affect the toxicity of the culture. On the other hand, purified wortmannin (1 mg/kg diet) significantly inhibited the aforementioned immunological responses prior to the adverse effects on body growth and hematocrit. The data strongly indicate that wortmannin is an immunotoxic substance. The possibility that macrophage is the primary target cell type is discussed.

Androstadienes↗

PubMed1994

The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf.

Glycogen synthase kinase-3 (GSK3) is inactivated in vitro by p70 S6 kinase or MAP kinase-activated protein kinase-1 beta (MAPKAP kinase-1 beta; also known as Rsk-2). Here we show that GSK3 isoforms are inhibited by 40% within minutes after stimulation of the rat skeletal-muscle cell line L6 with insulin-like growth factor-1 (IGF-1) or insulin. GSK3 was similarly inhibited in rabbit skeletal muscle after an intravenous injection of insulin. Inhibition resulted from increased phosphorylation of GSK3, probably at a serine/threonine residue(s), because it was reversed by incubation with protein phosphatase-2A. Rapamycin blocked the activation of p70 S6 kinase by IGF-1 in L6 cells, but had no effect on the inhibition of GSK3 or the activation of MAPKAP kinase-1 beta. In contrast, wortmannin, a potent inhibitor of PtdIns 3-kinase, prevented the inactivation of GSK3 and the activation of MAPKAP kinase-1 beta and p70 S6 kinase by IGF-1 or insulin. Wortmannin also blocked the activation of p74raf-1. MAP kinase kinase and p42 MAP kinase, but not the formation of GTP-Ras by IGF-1. The results suggest that the stimulation of glycogen synthase by insulin/IGF-1 in skeletal muscle involves the MAP-KAP kinase-1-catalysed inhibition of GSK3, as well as the previously described activation of the glycogen-associated form of protein phosphatase-1.

Amino Acid Sequence↗

PubMed1996

Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

Adenosine Triphosphate↗

PubMed2000

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway.

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Inhibition by wortmannin of M-current in bullfrog sympathetic neurones.

1. The actions of wortmannin, an inhibitor of myosin light chain kinase (MLCK), on M-type potassium current of dissociated bullfrog sympathetic neurones have been examined. 2. The amplitude of M-current was measured by whole cell recordings from cells pretreated with wortmannin (0.01-10 microM) or the wortmannin vehicle, dimethylsulphoxide (0.0001-0.1 vol%), for 30 min. Internal (recording pipette) solutions having three different pCa values (6, 7 and 8) were used for the measurements. 3. Irrespective of the pCa, M-current was not detectable when the cells were pretreated with 10 microM wortmannin. Wortmannin, 3 microM, produced 85-95% inhibition of the M-current. Pretreatment with 10-30 nM wortmannin was without effect on M-current. 4. The M-current inhibition by wortmannin at concentrations of 0.1-1 microM depended on the pCa of the internal solution. Inhibition occurred only when the calcium-rich (pCa = 6) internal solution was used. 5. Pre-treatment of the cells with wortmannin (10 microM) did not affect rapidly-inactivating A-type or delayed rectifier-type potassium currents not did it alter inwardly rectifying sodium-potassium current (IH). 6. These observations show that M-current inhibition by wortmannin has two pharmacological profiles. One is calcium-dependent and occurs at lower concentrations (0.1-1 microM), and is attributed to inhibition of MLCK by wortmannin. At higher concentrations (3-10 microM), wortmannin has an additional, calcium-independent action, inhibiting the M-current by an unknown mechanism.

Androstadienes↗

PubMed1998

Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells.

Wortmannin, a specific inhibitor of PI-3 kinase, was recently found to be an effective radiosensitizer in cells of various human and murine cell lines. Another study indicated that wortmannin inhibited repair of DNA double-strand breaks (DSBs) in irradiated Chinese hamster ovary cells using the neutral elution assay. To further clarify the mechanism behind radiosensitization by wortmannin, we have studied DSB repair in gamma-irradiated normal human fibroblasts using pulsed-field gel electrophoresis. The rejoining of DSBs in irradiated cells was significantly inhibited when 20 microM or more of wortmannin was added to the cells. The colony formation assay in cultures treated with wortmannin showed that the radiosensitization occurred in a manner that was dependent on the drug concentration. However, significant sensitization was observed only with a concentration of wortmannin of 20 microM or higher, reflecting the results of DSB rejoining studies. No marked reduction in plating efficiencies was observed for cells treated with wortmannin alone. The studies of the levels of expression of DNA-dependent protein kinase (DNA-PK) indicated that, while there were no significant changes in expression of Ku protein, the expression of the DNA-PK catalytic subunit (DNA-PKcs) was reduced markedly in cultures treated with wortmannin using an antibody against the C-terminus region of DNA-PKcs. In addition, no reduction in the levels of expression of DNA-PKcs was observed in cells treated with wortmannin using an antibody which recognizes a mid-region of this large protein. These results together with those of related studies suggest that wortmannin radiosensitizes normal human cells by inhibiting DSB repair and that this inhibition is a consequence of an inactivation of kinase activity and/or a structural change caused by binding of wortmannin to the C-terminus region of DNA-PKcs.

Androstadienes↗

PubMed1996

Effects of a myosin light chain kinase inhibitor, wortmannin, on cytoplasmic Ca2+ levels, myosin light chain phosphorylation and force in vascular smooth muscle.

Biochemical studies have shown that wortmannin is an inhibitor of myosin light chain (MLC) kinase (Nakanishi et al. (1992) J. Biol. Chem. 267: 2157-2163). To investigate the role of MLC kinase in smooth muscle contractions, we examined the effects of wortmannin on isolated smooth muscles of the rat aorta. Wortmannin (1 microM) decreased MLC phosphorylation and the amplitude of contractions induced by high K+ (72.7 mM) to a level seen at rest. This occurred without a change in cytosolic Ca2+ levels ([Ca2+]i). In contrast, wortmannin only partially inhibited the sustained contractions induced by phenylephrine (1 microM) and prostaglandin F2 alpha (PGF2 alpha, 10 microM) without a change in the [Ca2+]i. On the other hand, wortmannin (1 or 10 microM) reduced the increase in MLC phosphorylation induced by phenylephrine and PGF2 alpha to a level seen at rest. In the absence of external Ca2+, caffeine (20 mM) induced a transient increase in [Ca2+]i and force with an increase in MLC phosphorylation. Wortmannin completely inhibited the increase in MLC phosphorylation and contraction induced by caffeine without affecting the increase in [Ca2+]i. In the absence of external Ca2+, phenylephrine induced a small transient increase in [Ca2+]i, MLC phosphorylation and generation of force. This was followed by a small sustained contraction without an increase in [Ca2+]i and MLC phosphorylation. Wortmannin (1 microM) inhibited the transient phase of the contraction and the increase in MLC phosphorylation without affecting the transient increase in [Ca2+]i nor the sustained contraction. Wortmannin inhibited the Ca2(+)-induced contraction in permeabilized rat mesenteric artery, although it did not inhibit the Ca(2+)-independent, ATP-induced contraction in the thiophosphorylated muscle. These results suggest that wortmannin inhibits MLC phosphorylation due to an increase in the entry of Ca2+ or through the release of Ca2+ from the sarcoplasmic reticulum. The results also suggest that the activation of receptors by norepinephrine and PGF2 alpha induces a contraction via a MLC phosphorylation-independent pathway or through a pathway which is dependent on the resting level of MLC phosphorylation. We conclude that wortmannin is a useful tool in studies of the physiological role of MLC kinase.

Androstadienes↗

PubMed1994

Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.

Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities.

3T3 Cells↗

PubMed1997

Lipid kinase and protein kinase activities of G-protein-coupled phosphoinositide 3-kinase gamma: structure-activity analysis and interactions with wortmannin.

Signalling via seven transmembrane helix receptors can lead to a massive increase in cellular PtdIns(3,4,5)P3, which is critical for the induction of various cell responses and is likely to be produced by a trimeric G-protein-sensitive phosphoinositide 3-kinase (PI3Kgamma). We show here that PI3Kgamma is a bifunctional lipid kinase and protein kinase, and that both activities are inhibited by wortmannin at concentrations equal to those affecting the p85/p110alpha heterodimeric PI3K (IC50 approx. 2 nM). The binding of wortmannin to PI3Kgamma, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. Truncation of more than the 98 N-terminal amino acid residues from PI3Kgamma produced proteins that were inactive in wortmannin binding and kinase assays. This suggests that regions apart from the core catalytic domain are important in catalysis and inhibitor interaction. The covalent reaction of wortmannin with PI3Kgamma was prevented by preincubation with phosphoinositides, ATP and its analogues adenine and 5'-(4-fluorosulphonylbenzoyl)adenine. Proteolytic analysis of wortmannin-prelabelled PI3Kgamma revealed candidate wortmannin-binding peptides around Lys-799. Replacement of Lys-799 by Arg through site-directed mutagenesis aborted the covalent reaction with wortmannin and the lipid kinase and protein kinase activities completely. The above illustrates that Lys-799 is crucial to the phosphate transfer reaction and wortmannin reactivity. Parallel inhibition of the PI3Kgamma-associated protein kinase and lipid kinase by wortmannin and by the Lys-799-->Arg mutation reveals that both activities are inherent in the PI3Kgamma polypeptide.

Adenosine Triphosphate↗

PubMed1996

Wortmannin is a potent inhibitor of DNA double strand break but not single strand break repair in Chinese hamster ovary cells.

Wortmannin, an inhibitor of p110 PI 3-kinase, also inhibits DNA-dependent protein kinase, which is known to mediate DNA double strand break repair. It was recently demonstrated that wortmannin sensitized cells to ionizing radiation (IR) (Price and Youmell, Cancer Res., 56, 246-250, 1996). Wortmannin was used to determine if the potentiation of IR-induced cytotoxicity in Chinese hamster ovary cells could be accounted for by an inhibition of DNA double strand break (DSB) repair. Wortmannin, at concentrations which were non-toxic per se (5 and 20 microM), increased IR cytotoxicity with dose enhancement factors at 10% survival of 2.7+/-0.28 (5 microM) and 5.3+/-0.86 (20 microM). The effects of wortmannin on DSB levels were assessed by neutral elution. The effects of wortmannin on the kinetics of DSB repair were evaluated over a 3 h time course. Wortmannin (50 microM) completely inhibited DSB repair over this period, without having any effect on DSB levels itself. The concentration-dependent effects of wortmannin on DSB levels showed that inhibition of DSB repair was significant at 1 microM, and near-maximal at 20 microM. In marked contrast, it exerted no effect on the kinetics of single strand break (SSB) repair as assessed by alkaline elution, even at concentrations as high as 50 microM. There was an excellent correlation between the concentration-dependence and exposure time of wortmannin required to enhance IR cytotoxicity and inhibit DSB repair. These data implicate inhibition of DNA-dependent protein kinase, and the consequent inhibition of DSB repair, as the mechanism whereby wortmannin potentiates the cytotoxicity of IR.

Androstadienes↗

PubMed1996

Wortmannin inhibits insulin secretion in pancreatic islets and beta-TC3 cells independent of its inhibition of phosphatidylinositol 3-kinase.

Glucose is the primary stimulus for insulin secretion by pancreatic beta-cells, and it triggers membrane depolarization and influx of extracellular Ca2+. Cholinergic agonists amplify insulin release by several pathways, including activation of phospholipase C, which hydrolyzes membrane polyphosphoinositides. A novel phospholipid, phosphatidylinositol 3,4,5- trisphosphate [PtdIns(3,4,5)P3], a product of phosphatidylinositol 3-kinase (PI 3-kinase), has recently been found in various cell types. We demonstrate by immunoblotting that PI 3-kinase is present in both cytosolic and membrane fractions of insulin-secreting beta-TC3 cells and in rat islets. The catalytic activity of PI 3-kinase in immunoprecipitates of islets and beta-TC3 cells was measured by the production of radioactive phosphatidylinositol 3-monophosphate from phosphatidylinositol (PtdIns) in the presence of [gamma-32P]ATP. Wortmannin, a fungal metabolite, dose dependently inhibited PI 3-kinase activity of both islets and beta-TC3 cells, with an IC50 of 1 nmol/l and a maximally effective concentration of 100 nmol/l, when it was added directly to the kinase assay. However, if intact islets were incubated with wortmannin and PI 3-kinase subsequently was determined in islet immunoprecipitates, approximately 50% inhibition of PI 3-kinase activity (but no inhibition of glucose- and carbachol-stimulated insulin secretion) from intact islets was obtained at wortmannin concentrations of 100 nmol/l. Wortmannin, at higher concentrations (1 and 10 micromol/l), inhibited glucose- and carbachol-induced insulin secretion of Intact rat islets by 58 and 92%, respectively. Wortmannin had no effect on the basal insulin release from rat islets. A similar dose curve of inhibition of glucose- and carbachol-induced insulin secretion by wortmannin was obtained when beta-TC3 cells were used. Cellular metabolism was, not changed by any wortmannin concentrations tested (0.01-10 micromol/l). Both basal cytosolic [Ca2+]i and carbamyl choline-induced increases of [Ca2]i were unaffected by wortmannin in the presence of 2.5 mmol/l Ca2+, while Ca2+ mobilization from intracellular stores was partially decreased by wortmannin. Together, these data suggest that wortmannin at concentrations that inhibit PI 3-kinase does not affect insulin secretion. PI 3-kinase is unlikely to have a major role in insulin secretion induced by glucose and carbachol.

Androstadienes↗

PubMed1997

The enhancement by wortmannin of protein kinase C-dependent activation of phospholipase D in vascular endothelial cells.

Phosphatidic acid generation by phospholipase D (PLD) activation has been implicated in agonist- and oxidant-mediated endothelial cell signal transduction. We examined the effect of wortmannin on PLD activation in pulmonary artery endothelial and smooth muscle cells in culture. Pretreatment of bovine pulmonary artery endothelial cells (BPAECs) with wortmannin potentiated TPA- (100 nM), ATP- (100 microM), and bradykinin- (1 microM) induced [32P]PEt formation, an index of PLD activation. However, wortmannin by itself had no effect on PLD activity. The potentiating effect of wortmannin on TPA-induced PLD activation was dose- (1-10 microM) and time-dependent (5-30 min) and was inhibited by bisindoylmalemide, an inhibitor of protein kinase C (PKC). Furthermore, down-regulation of PKC by prolonged treatment with TPA (100 nM, 18 h) attenuated the wortmannin effect. This effect of wortmannin was specific for TPA- or agonist-induced PLD activation as no potentiation of [32P]PEt formation was observed with H2O2 (1 mM) or ionomycin (1 microM). The effect of wortmannin was not due to activation of PKC alpha as determined by western blot analysis of PKC alpha in the cytosol and membrane fractions. Also, genistein, an inhibitor of tyrosine kinases, did not attenuate the wortmannin-mediated potentiation of PLD thereby suggesting non-involvement of protein tyrosine phosphorylation. These results indicate that wortmannin potentiates PKC-dependent stimulation of PLD in vascular endothelial cells.

Androstadienes↗

PubMed1996

Studies on the mechanism of phosphatidylinositol 3-kinase inhibition by wortmannin and related analogs.

Wortmannin, a fungal metabolite, was identified as a potent inhibitor (IC50 = 4.2 nM) of phosphatidylinositol 3-kinase (PI 3-kinase). Due to the importance of PI 3-kinase in several intracellular signaling pathways, structure-activities studies on wortmannin analogs were performed in an effort to understand the structural requirements necessary for PI 3-kinase inhibition. Since wortmannin is an irreversible inhibitor of PI 3-kinase, it was postulated that covalent attachment at the electrophilic C-21 site was a possible mode of action for PI 3-kinase inhibition. We have prepared various wortmannin analogs which address the possibility of this mechanism. Of particular interest are compounds which affect the C-21 position of wortaminnin either sterically or electronically. Our results support the conclusion that nucleophilic addition by the kinase onto the C-21 position of wortmannin is required for inhibition of PI 3-kinase by wortmannin analogs. Additionally, we have prepared several D-ring analogs of wortmannin, and their activities are reported herein. We conclude that the wortmannin D ring is an important recognition site since modifications have such a dramatic effect on inhibitor potency. Finally, the identification of 17beta-hydroxywortmannin represents the first reported subnanomolar inhibitor of PI 3-kinase. These studies, along with in vivo antitumor experiments, suggest that the mechanism of PI 3-kinase inhibition correlates to the associated toxicity observed with wortmannin-based inhibitors of PI 3-kinase.

Androstadienes↗

PubMed1996

Wortmannin-sensitive trafficking pathways in Chinese hamster ovary cells. Differential effects on endocytosis and lysosomal sorting.

Phosphatidylinositol (PI) 3'-kinases are a family of lipid kinases implicated in the regulation of cell growth by oncogene products and tyrosine kinase growth factor receptors. The catalytic subunit of the p85/p110 PI 3'-kinase is homologous to VPS-34, a phosphatidylinositol-specific lipid kinase involved in the sorting of newly synthesized hydrolases to the yeast vacuole. This suggests that PI 3'-kinases may play analogous roles in mammalian cells. We have measured a number of secretory and endocytic trafficking events in Chinese hamster ovary cells in the presence of wortmannin, a potent inhibitor of PI 3'-kinase. Wortmannin caused a 40-50% down-regulation of surface transferrin receptors, with a dose dependence identical to that required for maximal inhibition of the p85/p110 PI 3'-kinase in intact cells. The redistribution of transferrin receptors reflected a 60% increase in the internalization rate and a 35% decrease in the recycling rate. Experiments with fluorescent transferrin showed that entry of transferrin receptors into the recycling compartment and efflux of receptors out of the compartment were slowed by wortmannin. Wortmannin altered the morphology of the recycling compartment, which was more vesiculated than in untreated cells. Using Semliki Forest virus as a probe, we also found that delivery of the endocytosed virus to its lysosomal site of degradation was slowed by wortmannin, whereas endosomal acidification was unaffected. In contrast to these effects on endocytosis and recycling, wortmannin did not affect intracellular processing of newly synthesized viral spike proteins. Wortmannin did induce missorting of the lysosomal enzyme cathepsin D to the secretory pathway, but only at a dose 20-fold greater than that required to inhibit p85/p110 PI 3'-kinase activity or to redistribute transferrin receptors. Our data demonstrate the presence of wortmannin-sensitive enzymes at three distinct steps of the endocytic cycle in Chinese hamster ovary cells: internalization, transit from early endosomes to the recycling and degradative compartments, and transit from the recycling compartment back to the cell surface. The wortmannin-sensitive enzymes critical for endocytosis and recycling are distinct from those involved in sorting newly synthesized lysosomal enzymes.

Androstadienes↗