Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase.

Wortmannin is a fungal metabolite that so far has been shown to act as a selective inhibitor of phosphoinositide 3-kinase. It can therefore be used to investigate the convergence between two major cellular signalling systems: those involving G-protein-coupled receptors and those involving receptor tyrosine kinases. Importantly, wortmannin can enter intact cells, making whole-cell studies of the above signalling pathways possible.

Involvement of the beta gamma subunits of inhibitory GTP-binding protein in chemoattractant receptor-mediated potentiation of cyclic AMP formation in guinea pig neutrophils.

Cellular cyclic AMP formation in response to prostaglandin (PG) E1 was markedly potentiated by the chemoattractant formyl-Met-Leu-Phe (fMLP) in guinea pig neutrophils. This potentiation by fMLP was abolished by prior treatment of the cells with pertussis toxin, but not by the prevention of an fMLP-induced intracellular Ca2+ increase in the cells, indicating the direct involvement of the inhibitory GTP-binding protein (Gj), but not Ca2+, in the fMLP-induced potentiation of cyclic AMP formation. Cyclic AMP formation in the neutrophils was also unique in response to forskolin; the diterpene inhibited cyclic AMP formation stimulated by PGE1 plus fMLP at low concentrations, but it slightly stimulated the basal and fMLP-induced cyclic AMP formation at high concentrations. Such a forskolin-induced inhibition was also observed in the adenylyl cyclase of the cell membranes and detergent extract therefrom only when the cyclase was activated by GTP or its nonhydrolyzable analogue (GTP gamma S). The forskolin-inhibitable activity could be affinity-purified from the GTP gamma S-treated cell membranes with a forskolin-agarose column. The cyclase appeared to be purified as a complex with the GTP gamma S-bound alpha subunit of the stimulatory GTP-binding protein (Gs alpha), but not with the beta gamma subunits, as judged from immunoblot analysis with specific antisera. The GTP gamma S-bound Gs alpha-stimulated cyclase activity was further enhanced by beta gamma, and this enhancement was again inhibited by forskolin. These results suggest that the GTP-bound Gs alpha produced by PGE1 receptor stimulation and the beta gamma subunits released from Gj by fMLP receptor stimulation were acting synergistically in the cyclic AMP formation of intact neutrophils.

Involvement of the pertussis toxin-sensitive GTP-binding protein in regulation of expression and function of granulocyte complement receptor type 1 and type 3.

Human polymorphonuclear leukocytes (PMN) express receptors for complement (C) C3b and C3bi termed CR1 and CR3, respectively. The addition of PMA or fMLP to PMN enhances the capacity of these receptors to promote binding of C3b- and C3bi-coated erythrocytes. fMLP-dependent increase of the binding of these ligand-coated erythrocytes was completely abolished by prior exposure of the PMN to pertussis toxin (IAP). GTP-binding protein (Gi alpha) was ADP-ribosylated and dysfunctional by this treatment. On the other hand, PMA-dependent binding of these ligands, as well as control binding, was inhibited only slightly, if at all, by the IAP treatment. The levels of C receptor expression on cell surface were determined by flow cytometry using monoclonal antibody against CR1 and those against the alpha and beta chains of CR3 (CR3 is composed of alpha and beta chain). Upon exposure of PMN to the chemotactic factor or PMA, or upon incubation of the cells at 37 degrees C, the surface expression of CR1 and CR3 alpha was increased. IAP also blocked an fMLP-induced increase of CR1 and CR3 alpha, but did not block the temperature- or PMA-dependent increase of these receptors. Opsonized zymosan (SOZ), another ligand for CR3, also led to an increase of both CR1 and CR3 alpha. Neither PMA nor SOZ brought about an increase of the surface expression of CR3 beta, but fMLP caused a slight increase of CR3 beta in an IAP-sensitive manner. Based on the IAP-sensitivity of the receptor expression, therefore, it appears that at least two separate mechanisms are operative in the control of C receptors. In addition, the alpha and beta chains of CR3 are regulated independently. The present data offer evidence suggesting that C receptor functions are in part regulated through a GTP-binding protein via modulation of their surface expression.

Specific association of phosphatidylinositol 3-kinase with the protooncogene product Cbl in Fc gamma receptor signaling.

A tyrosine-phosphorylated protein with a molecular mass of 115 kDa was reported to be tightly associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, when the enzyme was essentially activated upon ligand engagement of Fc gamma receptors (Fc gamma R) leading to engulfment of IgG-coated erythrocytes by phagocytes [Ninomiya et al. (1994) J. Biol. Chem. 269, 22732-22737]. Here, the 115-kDa protein is identified as the product of human c-cbl protooncogene. Cross-linking of Fc gamma RII on the surface of THP-1 cells triggered (a) prominent tyrosine phosphorylation of Cbl, (b) activation of PI 3-kinase that was immunoprecipitated with the anti-Cbl or the anti-phosphotyrosine antibody, and (c) specific association of Cbl with p85. Thus, Cbl functions in phagocytes as a result of its association with PI 3-kinase in response to Fc gamma R ligation.

Association of phosphatidylinositol 3-kinase with the proto-oncogene product Cbl upon CD38 ligation by a specific monoclonal antibody in THP-1 cells.

We reported that ecto-NAD+ glycohydrolase activity induced upon differentiation of HL-60 cells with retinoic acid is localized on the extracellular domain of CD38 and that CD38 ligation by a specific monoclonal antibody, HB-7, is followed by rapid tyrosine phosphorylation of cellular proteins including a proto-oncogene product, Cbl. In the present study, we investigated intracellular signaling linked to the HB-7-induced Cbl phosphorylation in dibutyryl cAMP-treated THP-1 cells. The 85-kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3-kinase was immunoprecipitated with anti-Cbl antibody in a manner dependent on the tyrosine phosphorylation of Cbl. PI 3-kinase activity was also observed in the immunoprecipitated fractions containing tyrosine-phosphorylated Cbl. The phosphorylated form of Cbl, which had been separated from the CD38-stimulated cells, was capable of directly binding to a recombinant p85 fused to glutathione S-transferase. Thus, the direct association of tyrosine-phosphorylated Cbl with PI 3-kinase, possibly leading to the kinase activation, appeared to be involved in intracellular signaling caused by the CD38 ligation.

Noninvasive quantitative analysis of blood oxygenation in rat skeletal muscle.

Using the isolated perfused rat hindlimb and the fluorocarbon-transfused rat, we have examined the optical characteristics of the rat skeletal muscle in the near-infrared region. The total contribution of myoglobin and cytochromes to the overall absorbance change was less than 10%. Analyzing transmitted light at 700, 730, and 805 nm, we found linear relationships between the absorbance and the hemoglobin concentrations at hematocrit values from 15 to 50% in the inflowing perfusate. Based on the relationship, we determined the ratio of absorption coefficients at 700, 730, and 805 nm of oxy- and deoxy-hemoglobins of blood in the thigh muscle. The values in thigh muscle were significantly smaller than those in hemoglobin solutions for deoxygenated blood. On the other hand, the values in thigh muscle were larger than those in hemoglobin solutions for oxygenated blood. Solving simultaneous equations by the use of these absorption coefficients, we calculated the changes in the contents of oxy-, deoxy-, and total hemoglobins in the anesthetized rat hindlimb under various conditions. The oxygen saturation of blood determined by our optical method in the thigh muscle was very close to that in the vena cava measured directly with a gas analyzer.

Potentiation of chemotactic peptide-induced superoxide generation by CD38 ligation in human myeloid cell lines.

CD38 is a type II transmembrane glycoprotein possessing an NAD+ glycohydrolase activity in its extracellular domain. We previously reported that the ligation of CD38 by a monoclonal antibody (mAb), HB-7, induces the tyrosine phosphorylation of cellular proteins including p120(c-cbl) in differentiated human myeloid cell lines and that the phosphorylated p120(c-cbl) is capable of binding to phosphatidylinositol (PI) 3-kinase. In the present study, we found that the agonistic anti-CD38 mAb markedly potentiates superoxide generation stimulated by chemotactic formyl-Met-Leu-Phe receptors in the CD38-producing cells. HB-7 neither generated superoxide by itself nor enhanced the cell response induced by phorbol 12-myristate acetate, indicating that the potentiating action of the anti-CD38 mAb is specific for the stimulation by the GTP-binding protein (G1)-coupled membrane receptors. The potentiation by HB-7 was abolished by prior treatment of the cells with a tyrosine kinase inhibitor, pertussis toxin, or a potent PI 3-kinase inhibitor, wortmannin. HB-7 also enhanced the product formation of PI 3-kinase in response to the chemotactic receptor stimulation, without significant changes in the receptor-stimulated accumulations of inositol-1,4,5-trisphosphate, arachidonate release, and intracellular Ca2+. These results indicate that the CD38-induced tyrosine phosphorylation has a cross-talk with the chemotactic receptor/G1-mediated signal transduction pathway resulting in the enhancement of superoxide generation, probably through the activation of PI 3-kinase.

Activation of c-Jun N-terminal kinase (JNK) by lysophosphatidic acid in Swiss 3T3 fibroblasts.

Lysophosphatidic acid (LPA) induced activation of c-Jun N-terminal kinase (JNK) in Swiss 3T3 fibroblasts. This activation reached the maximum at 20 min and required a high concentration of LPA with an EC50 value of approximately 3 microg/ml. LPA-induced activation of JNK was not suppressed by prior treatment of the cells with pertussis toxin, whereas it was completely blocked by suramin, a non-selective inhibitor of ligand-receptor interactions. The kinetics and concentration-dependency of LPA-induced JNK activation were in sharp contrast with those of LPA-induced extracellular signal-regulated kinase (ERK) activation, which reached the maximum within 3 min and occurred with an EC50 of 0.1 microg/ml. The ERK activation was susceptible to pertussis toxin, whereas it was not inhibited by suramin. These results indicate that the signal transduction pathways of LPA-induced JNK and ERK activations are distinct. Thus, this is the first report showing that LPA induces not only ERK activation but also JNK activation, which may be responsible for the induction of DNA synthesis in LPA-stimulated Swiss 3T3 fibroblasts.

Involvement of phosphoinositide 3-kinase in regulation of adhesive activity of highly metastatic hepatoma cells.

We have established hepatoma clones from benzopyrene-treated liver cells, one of which (G-5) shows extensive metastasis to the lung when injected subcutaneously into mice [Tanigaki, Y. et al. (1995) Invasion Metastasis 15, 70-80]. In the present study, we performed in vitro assays suitable for examination of the adhesive and invasive properties of the highly metastatic cells. G-5 cells efficiently entered the pores of fibronectin-coated filters. Treatment of the cells with an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), wortmannin, significantly impaired the invasive activity. A structurally unrelated inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also prevented invasion. Both inhibitors suppressed cell adhesion to fibronectin-coated dishes. G-5 cells were next transfected with a mutant regulatory subunit (Deltap85) of PI 3-kinase, which was expected to impair the function of PI 3-kinase. The transfectants showed suppressed adhesion to the dishes and did not efficiently migrate into the filters. The lower adhesive ability of the transfected cells was not further affected by inhibitors of PI 3-kinase. Thus, PI 3-kinase activity contributes significantly to the adhesive and invasive properties of G-5 cells.

Quantitative analysis of hemoglobin oxygenation state of rat brain in situ by near-infrared spectrophotometry.

The light in the near-infrared region (700-900 nm) was illuminated on the rat head, and absorption spectra were measured with the transmitted light under various conditions. The absorbance changes less than 780 nm were attributable to hemoglobin in the brain tissue, whereas those greater than 780 nm were associated with both hemoglobin and cytochrome oxidase. The changes of oxy- and total (oxy- plus deoxy-) hemoglobin content in the rat head could be monitored quantitatively by expressions of delta A700--1.20 delta A730 and delta A700--1.52 delta A730, respectively. The oxyhemoglobin content in the tissue was decreased as the O2 tension in inspired gas was lowered. At 10% O2 approximately 50% of hemoglobin was deoxygenated. The total hemoglobin content was increased under anoxic conditions. Inhalation of 5% CO2 and intravenous injection of a Ca2+ blocker nicardipine increased the O2 saturation of hemoglobin in the brain. These conclusions were confirmed by measuring the difference absorption spectra in the near-infrared region.

Oxygen dependence of redox state of copper in cytochrome oxidase in vitro.

To obtain quantitative information about tissue oxygenation from near-infrared signals, the oxygen dependencies of the redox states of both heme a+a3 and copper in cytochrome oxidase of isolated mitochondria were determined at low oxygen concentrations (10(-6)-10(-9) M) using leghemoglobin as an oxygen indicator. The maximum absorbance changes caused by the aerobic-anaerobic transition measured at 830-760 nm of copper in state 3, state 4, and the uncoupled state were 10, 17, and 5% of those at 605-620 nm of heme a+a3, respectively. Thus the relative absorbance ratio of copper to heme a+a3 could be used as a sensitive indicator for the mitochondrial energy state. The oxygen concentrations required for the half-maximal reduction of heme a+a3 varied with the energy state and the respiratory rate and were 7.8 x 10(-8) and 1.6 x 10(-7) M in state 4 and state 3, respectively. In contrast, that of copper was 7.5 x 10(-8) M and was independent of both the energy state and the respiratory rate. The relationship between the percent oxidation of heme a+a3 and copper in the aerobic-anaerobic transition did not show a straight-line relationship. This was referred to as the difference in oxygen affinity between these two chromophores. The deviation from the straight line was larger in state 3 than in state 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Redox behavior of cytochrome oxidase in the rat brain measured by near-infrared spectroscopy.

Using near-infrared spectroscopy, we developed a new approach for measuring the redox state of cytochrome oxidase in the brain under normal blood-circulation conditions. Our algorithm does not require the absorption coefficient of cytochrome oxidase, which differs from study to study. We employed this method for evaluation of effects of changes in oxygen delivery on cerebral oxygenation in rats. When fractional inspired oxygen was decreased in a stepwise manner from 100 to <10%, at which point the concentration of oxygenated hemoglobin ([HbO2]) decreased by approximately 60%, cytochrome oxidase started to be reduced. Increases in arterial PO2 under hyperoxic conditions caused an increase in [HbO2], whereas further oxidation of cytochrome oxidase was not observed. The dissociation of the responses of hemogloblin and cytochrome oxidase was also clearly observed after the injection of epinephrine under severely hypoxic conditions; that is, cytochrome oxidase was reoxidized with increasing blood pressure, whereas hemoglobin oxygenation was not changed. These data indicated that oxygen-dependent redox changes in cytochrome oxidase occur only when oxygen delivery is extremely impaired. This is consistent with the in vitro data of our previous study.

Age-related changes in manganese superoxide dismutase activity in the cerebral cortex of senescence-accelerated prone and resistant mouse.

In this paper, we showed that the oxidative stress in brain of senescence-accelerated prone mouse 8 (SAMP8) at earlier stages was increased compared with that of senescence-accelerated resistant mouse 1 (SAMR1) irrespective of the breeding conditions. Furthermore, we found that manganese superoxide dismutase (Mn-SOD) activity in the cerebral cortex of 10-week-old SAMP8 was decreased by about 50% compared with that in age-matched SAMR1. These results indicate that the decrease of Mn-SOD activity may be involved in the increased oxidative stress in the brain of SAMP8 at younger stages. However, there was no difference in the expression of this protein between the two strains at 10 weeks of age, suggesting that Mn-SOD protein in SAMP8 was post-translationally modified to reduce its enzymatic activity.

Activation of PI 3-kinase by G protein betagamma subunits.

We have reported that fMLP-induced activation of pertussis toxin-sensitive GTP-binding proteins in THP-1 cells potentiates the insulin-induced accumulation of PtdIns(3,4,5)P3, a product of phosphoinositide 3-kinase (T. Okada et al., Biochem. J. 317, 475-480, 1996). The synergism in PtdIns(3,4,5)P3 accumulation was observed in Chinese hamster ovary cells expressing both insulin and fMLP receptors. In rat adipocytes, which represent the physiological target cells of insulin, receptor-mediated activation of GTP-binding protein by adenosine and prostaglandin E2 potentiated the insulin-induced PtdIns(3,4,5)P3 accumulation. In cell-free systems, the activity of the p85/p110beta subtype of phosphoinositide 3-kinase was, while that of p85/p110alpha was not, stimulated by the betagamma subunits of the GTP-binding proteins. We propose here a hypothesis that the p85/p110beta subtype is under the control of both the insulin receptors and the GTP-binding protein-coupled receptors in intact cell systems.

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.

Participation of a MEK-independent pathway in MAP kinase activation and modulation of cell growth in mouse hepatoma cell lines.

The mechanism of cell growth was investigated in GIT medium-supplemented in vitro assay using high and low metastatic mouse hepatoma cell sublines, G-5 and G-1, respectively. G-5 cells exhibited high growth rate compared to G-1 cells. The PI3-kinase inhibitor LY294002 and P70 S6 kinase inhibitor rapamycin partially blocked both G-1 and G-5 cell growth, suggesting that these two kinases are involved in hepatoma cell growth. In contrast, the MEK1 inhibitor PD98059 partially blocked G-5 cell growth but not G-1 cell growth. MAP kinases (MAPK) in both G-1 and G-5 cells were indistinguishably phosphorylated, yet MEK-dependent MAPK activation was observed only in G-5 cells. In G-1 cells, MAPK was phosphorylated in a manner not connected to MEK activation. Thus, the low degree of cell growth in G-1 cells was attributable to disruption of the MEK-dependent MAPK cascade. However, the molecular mechanism whereby MAPK phosphorylation does not parallel MAPK activation in G-1 cells remains unknown. Here, we suggest that there may be an as yet unidentified MAPK phosphorylation pathway in malignantly transformed cells, which may affect in vivo cell growth and metastatic capacities of cancers.

Relationship between time-resolved and non-time-resolved Beer-Lambert law in turbid media.

The time-resolved Beer-Lambert law proposed for oxygen monitoring using pulsed light was extended to the non-time-resolved case in a scattered medium such as living tissues with continuous illumination. The time-resolved Beer-Lambert law was valid for the phantom model and living tissues in the visible and near-infrared regions. The absolute concentration and oxygen saturation of haemoglobin in rat brain and thigh muscle could be determined. The temporal profile of rat brain was reproduced by Monte Carlo simulation. When the temporal profiles of rat brain under different oxygenation states were integrated with time, the absorbance difference was linearly related to changes in the absorption coefficient. When the simulated profiles were integrated, there was a linear relationship within the absorption coefficient which was predicted for fractional inspiratory oxygen concentration from 10 to 100% and, in the case beyond the range of the absorption coefficient, the deviation from linearity was slight. We concluded that an optical pathlength which is independent of changes in the absorption coefficient is a good approximation for near-infrared oxygen monitoring.

Near infrared quadruple wavelength spectrophotometry of the rat head.

A quadruple wavelength method to monitor the changes in concentration of oxygenated and deoxygenated hemoglobin and the redox state of cytochrome oxidase within a living tissue is presented. The expected advantages of this technique over the triple wavelength method are (i) that it can compensate for the light scattering change of tissue itself or for instabilities of light source and photomultiplier, (ii) that it can treat the optical properties of the red blood cell in a tissue in the same way as in an in vitro model system, and (iii) that it requires no estimation of the absorption coefficient of cytochrome oxidase in situ.

The oxygen dependency of the redox state of heme and copper in cytochrome oxidase in vitro.

The oxidation-reduction state of cytochrome oxidase in isolated mitochondria at low oxygen concentrations was measured by the use of leghemoglobin as an oxygen indicator. P50 a + a3 varied with energy state as well as the respiratory rate. In contrast to heme a + a3, copper was slower to reduce than heme a + a3. The P50Cu of 8 x 10(-8)M in State 4 and 7.4 x 10(-8)M in State 3 was independent of both the energy state and the respiratory rate.

The simultaneous measurements of tissue oxygen concentration and energy state by near-infrared and nuclear magnetic resonance spectroscopy.

The oxygenation and energy states of brain tissues were measured simultaneously by near-infrared photometry and nuclear magnetic resonance spectroscopy in situ. In both cat and dog, the critical hemoglobin oxygenation was 10%, below which the ratio of phosphocreatine (PCr) to inorganic phosphate (Pi) started to fall. The fall of PCr/Pi paralleled the reduction of copper in cytochrome aa3. The separation of the cytochrome aa3 signal from that of hemoglobin by our optical method was confirmed by the substitution of blood by fluorocarbon solution. The energy-oxygen diagram (PCr/Pi against hemoglobin oxygenation, HbO2) was the same in normal- and fluorocarbon substituted cats, but energy curve shifted to the right in the latter when PCr/Pi plotted against the inspired oxygen, FiO2.