Biomedical subjects

Y Katagiri

Publications and source records attributed to Y Katagiri.

At least 37 records · Page 2Linked to original sources

PubMed2000

HPLC analysis of white lupin isoflavonoids.

An investigation of the HPLC analytical conditions for simple isoflavones, prenylated isoflavones and some of their glucosyl derivatives resulted in reasonable separation and total elution in 35 min when using a reversed-phase C18 Lichrospher column and a gradient elution system of MeCN-THF-H2O. This method was successfully applied to quantify the changes in isoflavonoid constituents in white lupin (Lupinus albus L.) tissues: (a) young legumes (pods and seeds) during maturation, and (b) soaked, germinating seeds. In developing legumes, genistein and 2'-hydroxygenistein, as well as their prenylated derivatives, were present in the pods as the major components, together with minor amounts of glucosides, whereas only minute amounts of isoflavonoids were detectable in the ripening seeds. When soaked with water, mature lupin seeds which normally contain trace amounts of isoflavonoids, started rapidly to biosynthesize simple isoflavones and accumulate large amounts of genistein 7-O-glucoside and its 6"-O-malonyl derivative. These dynamic changes are discussed in relation to the role of isoflavonoids in the lupin defense system.

Chromatography, High Pressure Liquid↗

PubMed1999

Nerve growth factor (NGF)-induced calcium influx and intracellular calcium mobilization in 3T3 cells expressing NGF receptors.

The neurotrophins have been implicated in the acute regulation of synaptic plasticity. Neurotrophin-stimulated presynaptic calcium uptake appears to play a key role in this process. To understand the mechanism of neurotrophin-stimulated calcium uptake, the regulation of calcium uptake and intracellular mobilization by nerve growth factor (NGF) was investigated using NIH 3T3 cells stably transfected with either the high affinity NGF receptor p140(trk) (3T3-Trk) or the low affinity NGF receptor p75(NGFR) (3T3-p75). In 3T3-Trk cells, NGF increased both calcium uptake and intracellular calcium mobilization. In 3T3-p75 cells, NGF increased calcium uptake but not intracellular calcium mobilization. K-252a alone increased intracellular calcium in 3T3-Trk cells but not in 3T3-p75 cells. Nifedipine, an inhibitor of calcium uptake through L-type calcium channels, inhibited the action of NGF on both 3T3-Trk cells and 3T3-p75 cells, indicating that both p140(trk) and p75(NGFR) receptors are linked to nifedipine-sensitive L-type calcium channels. These studies show that either NGF receptor will support increases in intracellular calcium but that p140(trk) does so by increasing both uptake and mobilization, whereas p75(NGFR) does so by increasing uptake only.

3T3 Cells↗

PubMed1999

Urinary excretion of the vitronectin receptor (integrin alpha V beta 3) in patients with Fabry disease.

A renal disorder is one of the important manifestations of Fabry disease, but the details of the pathogenesis have not been clarified yet. We examined the possibility that the vitronectin receptor (VNR, integrin alpha V beta 3), one of the integrins, is involved in the progression of the renal injury in Fabry disease. We measured the urinary excretion of beta 3 originating from VNR in Fabry patients by immunoblotting analysis and enzyme-linked immunosorbent assay (ELISA). Immunofluorescent microscopic analyses for VNR and globotriaosylceramide were performed on urinary sediments from Fabry patients. Furthermore, beta 3 and vitronectin in kidney tissues were analyzed immunohistochemically. Immunoblotting analysis and ELISA showed that the urinary excretion of beta 3 originating from VNR was significantly increased in the Fabry group compared with both the pathological and healthy control groups. Immunofluorescent microscopy revealed the expression of VNR and accumulation of globotriaosylceramide in urinary sediments from the Fabry patients. Increased expression of beta 3 was observed in glomerular epithelial cells, and in Bowman's capsular epithelial layer and tubular cells, and the amount of vitronectin was moderately increased in the kidney tissues from the Fabry patients. The urinary excretion of VNR was increased, and the expression of VNR was observed in Fabry kidney tissues. The expression of VNR may be involved in the progression of the renal injury in this disease.

Adolescent↗

PubMed1999

Distribution of the cfiA gene among Bacteroides fragilis strains in Japan and relatedness of cfiA to imipenem resistance.

The cfiA gene, encoding an imipenem-hydrolyzing metallo-beta-lactamase produced by Bacteroides fragilis, and insertion-like elements were detected by PCR amplification with B. fragilis strains isolated in Japan. The cfiA gene was found in 1.9 and 4.1% of the imipenem-susceptible B. fragilis isolates collected from 1987 to 1988 and from 1992 to 1994, respectively. Insertion-like elements adjacent to the cfiA gene were found in all nine metallo-beta-lactamase-producing imipenem-resistant strains tested but not in nine cfiA-positive strains with no detectable metallo-beta-lactamase activity.

Bacterial Proteins↗

PubMed1999

Two cases of cutaneous apocrine ductal carcinoma of the axilla. Case report and review of the literature.

We report 2 cases of cutaneous apocrine ductal carcinoma (CADC) of the axilla in a 64- and a 54-year-old male. Histological examination revealed 2 solid, ductal and glandular tumors with decapitation secretion. Tumor cells showed cellular and nuclear atypism, and infiltrative growth of tumor cell nests was also observed. Although there were no characteristic features of extramammary Paget's disease on the overlying skin, case 1 exhibited a typical Paget's phenomenon. We concluded that the Paget's phenomenon of case 1 was a result of upward extension of the tumor in the dermis. The neoplastic cells of both cases were immunohistochemically positive for gross cystic disease fluid protein, lysozyme, CD15 and carcinoembryonic antigen but negative for S-100 protein. Based on these findings, we concluded that these tumors were cutaneous apocrine ductal carcinomas. There was no evidence of tumor remnants in the axilla, and the patients have shown no signs of local recurrence or metastasis. We also reviewed the literature and summarize here the clinical features of CADC.

Adenocarcinoma↗

PubMed1998

Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity.

Amino Acid Sequence↗

PubMed1998

Nerve growth factor treatment prevents the increase in superoxide produced by epidermal growth factor in PC12 cells.

Stimulation of pheochromocytoma (PC12) cells with the mitogen epidermal growth factor (EGF) produced a rapid and robust accumulation of intracellular reactive oxygen species (ROS), an accumulation which, in other systems, has been shown to be essential for mitogenesis. Brief pretreatment of the cells with nerve growth factor (NGF) suppressed the EGF-mediated ROS increase. EGF failed to produce elevations in ROS in a PC12 variant stably expressing a dominant-negative p21(ras) construct (PC12-N17) or in cells pretreated with the MEK inhibitor PD098059. NGF failed to suppress the increase in ROS in the PC12 variant nnr5, which lacks p140(trk) receptors. The suppression of the increase in ROS by NGF was restored in nnr5 cells stably expressing p140(trk) (nnr5-trk), but NGF failed to prevent the increase in ROS in nnr cells expressing mutant p140(trk) receptors that lack binding sites for Shc and phospholipase Cgamma. Among several inhibitors of superoxide-generating enzymes, only the lipoxygenase inhibitor, nordihydroguaiaretic acid reduced EGF-mediated ROS accumulation. The inhibitory action of NGF on ROS production was mimicked by the nitric oxide donor, sodium nitroprusside, and was blocked by an inhibitor of nitric-oxide synthetase, L-nitroarginine methyl ester. These results suggest a novel mechanism for the rapid interruption of mitogenic signaling by the neurotrophin NGF.

Animals↗

PubMed1998

Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant.

Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [Hamasaki-Katagiri, N., Tabor, C. W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35-43].

Amino Acid Sequence↗

PubMed1998

Transcriptional down-regulation of epidermal growth factor receptors by nerve growth factor treatment of PC12 cells.

Treatment of PC12 cells with nerve growth factor leads to a decrease in the number of epidermal growth factor receptors on the cell membrane. The mRNA for the epidermal growth factor receptor decreases in a comparable fashion. This decrease appears due to a decrease in the transcription of the epidermal growth factor receptor gene because first, there is no difference in the stability of the epidermal growth factor receptor mRNA, second, newly transcribed epidermal growth factor receptor mRNA is decreased in nerve growth factor-differentiated cells, and third, constructs containing the promoter region of the epidermal growth factor receptor gene are transcribed much less readily in nerve growth factor-differentiated cells than in untreated cells. The decreases in mRNA are not seen in the p140(trk)-deficient variant PC12nnr5 cells nor in cells containing either dominant-negative Ras or dominant-negative Src. Treatment with nerve growth factor also increases the cellular content of GCF2, a putative transcription factor inhibitory for the transcription of the epidermal growth factor receptor gene. The increase in GCF2, like the decrease in the epidermal growth factor receptor mRNA, is not seen in PC12nnr5 cells nor in cells expressing either dominant-negative Ras or dominant-negative Src. The results suggest that nerve growth factor-induced down-regulation of the epidermal growth factor receptor is under transcriptional control, is p140(trk)-, Ras-, and Src-dependent, and may involve transcriptional repression by GCF2.

Animals↗

PubMed1998

Development of porous apatite ceramic for local delivery of chemotherapeutic agents.

An experimental study was conducted on a drug delivery system (DDS), using porous apatite ceramics (PAC): hydroxyapatite block (HAb) [Ca10(PO4)6(OH)2] having a porosity of 35-48% and pore size range of 50-300 microm, and beta-tricalcium phosphate block (TCP) [Ca3(PO4)2] having a porosity of 75-80% and pore size range of 100-400 microm, for sustained release of a chemotherapeutic agent. Methotrexate (MTX) was loaded in the pores of PAC blocks by centrifuging the blocks in MTX solution. Impregnation of MTX in PAC blocks (1 cm3) was confirmed by a magnetic resonance imaging (MRI) study using Gadolinium-DTPA enhancement. The MRI showed high signal intensity in the PAC, which was confirmed by dye loading into the pores. To estimate the MTX-releasing capability of the PAC, the blocks were stored in 3 mL of phosphate-buffered saline (PBS) at 37 degrees C and the PBS was replaced every 48 h. The amount of MTX released was assayed by high-performance liquid chromatography. This study showed that MTX-impregnated PAC (0.63-2.25 mg/block) released the drug in a steady manner and maintained its concentration (0.1-1.0 microg/mL) up to 12 days. This concentration is high enough to be effective against tumor cells. Chemotherapeutic agent-impregnated PAC, prepared by simple centrifugation, could be a valuable form of local chemotherapy when used as a strut graft to repair bone defects. This new DDS material could also be used as an adjuvant to extended curettage and provide a means to reduce the recurrence of tumors without risk of systemic toxicity.

Antimetabolites, Antineoplastic↗

PubMed1998

Involvement of tyrosine phosphorylation in inhibition of fMLP-induced PLD activation by N-acetyl-L-cysteine in differentiated HL60 cells.

N-acetyl-L-cysteine (NAC), which is known as a multipotential agent; an antioxidant, a thiol reagent, or a tyrosine kinase inhibitor, inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced phospholipase D (PLD) activation in HL60 cells in a concentration-dependent manner (IC50 = 2 mM). Its inhibitory mechanism was examined in this study to gain insight into the regulation of PLD activity. NAC had no direct effect on membrane PLD activity in an in vitro assay system. fMLP-induced formation of inositol phosphates via phospholipase C (PLC) was not affected by the drug, suggesting that the receptor-G protein coupling was not inhibited. H2O2, which is known to induce PLD activation in several types of cells, failed to activate PLD in HL60 cells. Pretreatment of 3-amino-1,2,4-triazole (ATZ), a catalase inhibitor, did not enhance fMLP-induced PLD activation. NAC inhibited fMLP-induced tyrosine phosphorylation of several protein bands (42, 44, 64, and 138 kDa) in a concentration-dependent manner. The temporal and concentration-dependent inhibitory profiles for tyrosine phosphorylation of 64- and 138-kDa proteins were well correlated with PLD activation. However, thiol reagents, 1 mM 2,3-dimercapto-l-propanol (2,3-DMP), 1 mM dithiothreitol (DTT), and 2 mM cysteine also did not suppress protein tyrosine phosphorylation or PBut formation by fMLP. Wortmannin, a selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, inhibited these two tyrosine phosphorylation bands. These results suggest that NAC inhibits fMLP-induced PLD activation through blockage of protein tyrosine phosphorylation, which is located at the downstream of PI-3 kinase.

Acetylcysteine↗

PubMed1998

Influence of intermittent ischemia on thioacetamide-induced rat liver cirrhosis.

We studied the influence of intermittent ischemic injury on thioacetamide-induced liver cirrhosis in rats. Wistar rats were divided into group A, intermittent ischemic injury to liver cirrhosis, and group B, continuous ischemic injury to liver cirrhosis. Total ischemic time was 60 min in both groups. In group A, ischemic injury consisted of a repetition 4 times of 15 min ischemia and 5 min reperfusion. The ATP level of the liver was measured before ischemia, before reperfusion, and 60 min after reperfusion. Bile was collected to determine bile flow rate. The ATP level in the liver tissue 60 min after reperfusion was significantly (p < 0.05) higher in group A than in group B. The ATP level immediately before reperfusion was also significantly (p < 0.05) higher in group A than in group B. The survival rate 1 week after ischemic injury and bile flow rate 60 min after reperfusion were significantly (p < 0.01) higher in group A compared with those in group B. The energy level was much higher in intermittent ischemic injury than in continuous ischemic injury immediately before reperfusion and after reperfusion. Survival rate and bile flow rate were higher in intermittent ischemic injury than in continuous ischemic injury. Therefore it suggests that the viability of the liver was maintained better in intermittent ischemic injury than in continuous ischemic injury.

Adenosine Triphosphate↗

PubMed1998

Dual effects of anti-inflammatory 2-arylpropionic acid derivatives on a major isoform of human liver 3alpha-hydroxysteroid dehydrogenase.

Nonsteroidal anti-inflammatory drugs have been shown to be potent inhibitors of mammalian 3alpha-hydroxysteroid dehydrogenase. Here, we report that the drugs of the 2-arylpropionic acid class act as both activators and inhibitors for a predominant isoform of the human liver enzyme which is involved in the metabolism of steroid hormones, bile acids, drug ketones and xenobiotic aromatic hydrocarbons. Flurbiprofen, fenoprofen, ibuprofen, naproxen, ketoprofen and suprofen stimulated the activity of the human enzyme (1.5-2.4-fold) at low concentrations of less than 20-100 microM, whereas at higher concentrations they inhibited the activity. Comparison of the effects of the structurally related compounds with the drugs revealed that the essential structure required as the activator molecule is 2-phenylpropionic acid with a hydrophobic substituent on the aromatic ring. In addition, an R-enantiomer of ibuprofen showed higher activation (3-fold) than its S-enantiomer. Kinetic analysis with respect to NADP+ concentration indicated that R- and S-ibuprofens are nonessential activators showing binding constants of 23 and 34 microM, respectively. Neither enantiomers activated, but rather inhibited the enzyme, with Met replacing Arg-276 which has been shown to interact with the 2'-phosphate of NADP+. The inhibitions of the mutant enzyme by R- and S-ibuprofens were competitive with respect to the substrate, giving Ki values of 95 and 18 microM, respectively. The results suggest that the human liver 3alpha-hydroxysteroid dehydrogenase isoform possesses the two distinct sites, activator and inhibitor sites, to which anti-inflammatory 2-arylpropionates stereoselectively bind.

Alcohol Oxidoreductases↗

PubMed1998

Activation of human liver 3alpha-hydroxysteroid dehydrogenase by clofibrate derivatives.

The NADP+-dependent dehydrogenase activity of a predominant isoenzyme of human liver 3alpha-hydroxysteroid dehydrogenase was activated by antihyperlipidemic drugs, such as bezafibrate and clinofibrate, and by clofibric acid and fenofibric acid (active metabolites of clofibrate and fenofibrate, respectively). The optimal pH of the activation by the drugs was about 7.5, and the concentrations giving maximum stimulation (1.8- to 2.4-fold) were 100, 50, 400 and 50 microM for bezafibrate, clinofibrate, clofibric acid and fenofibric acid, respectively. Clofibrate and fenofibrate acted as weak inhibitors, and the clofibric acid derivatives that lack the chloro group, methyl group on the alpha-carbon or carboxyl group greatly decreased the stimulatory effects. The activation by the drugs increased both Km and kcat (turnover number) values for the coenzyme and substrates. Kinetic analysis with respect to NADP+ showed that bezafibrate, clinofibrate, clofibric acid and fenofibric acid were nonessential activators showing dissociation constants of 32, 6, 103 and 11 microM, respectively. The combined activators experiments with one of the above drugs and sulfobromophthalein, a known activator specific for this enzyme, and comparison of their effects on the activities of mutant enzymes (with Met replacing Lys-270 or Arg-276) indicated that sulfobromophthalein and the drugs bind to an identical site on the enzyme. These results suggest that the long-term therapy with the antihyperlipidemic drugs influences the metabolism of steroid hormones, bile acids and several ketone-containing drugs mediated by the enzyme.

Alcohol Oxidoreductases↗

PubMed1998

Introduction of an osteopontin gene confers the increase in B1 cell population and the production of anti-DNA autoantibodies.

Osteopontin (OPN) is an Arg-Gly-Asp-containing phosphoprotein that is secreted by activated T cells. The concentration of serum OPN protein is elevated in autoimmune-prone MRL-lpr mice as well as in patients with systemic lupus erythematosus. Previously, it was shown that OPN induces the polyclonal activation of B cells, resulting in the augmented production of immunoglobulin, indicating that OPN plays some role in the development of autoimmune disease. However, the link between OPN and development of autoimmune disease remains unclear. To analyze the role of OPN in immune system and autoimmune diseases, we have generated two kinds of transgenic mice: one carries the immunoglobulin (Ig) enhancer/SV40 promoter and the other carries the cytomegalovirus enhancer/chicken beta-actin (CAG) promoter. In both groups of transgenic mice, the B1 cell population in peritoneal cavity was markedly increased and titer of IgM and IgG3 antibodies in the serum was considerably higher than that in wild-type mice. Most important, the titer of the IgM class of anti-double-stranded DNA antibody was significantly elevated in transgenic mice. These results strongly suggest that OPN may have an important role in the propagation and differentiation of B1 cells and production of autoantibodies.

Animals↗

PubMed1997

Differential regulation of the transcriptional activity of the orphan nuclear receptor NGFI-B by membrane depolarization and nerve growth factor.

The immediate-early gene NGFI-B (also called nur77) encodes an orphan nuclear receptor that activates transcription through a unique response element (NBRE). NGFI-B is rapidly induced and modified via phosphorylation by a variety of stimuli that induce cells to differentiate or to proliferate. We have shown that the in vitro phosphorylation of Ser350 located within the "A-box," a motif necessary for DNA binding by NGFI-B, results in a decrease in the binding of NGFI-B to its response element (Hirata, Y., Kiuchi, K., Chen, H.-C., Milbrandt, J., and Guroff, G. (1993) J. Biol. Chem. 268, 24808-24812). We show here that nerve growth factor (NGF)-induced changes in the in vivo phosphorylation of Ser350 accompany transcriptional deactivation of NGFI-B in PC12 cells, that membrane depolarization and NGF treatment cause differential phosphorylation of NGFI-B, and that the transcriptional activation caused by exogenous expression of NGFI-B or membrane depolarization can be inhibited by NGF treatment. In addition, the mutation of Ser350 to Ala abolished the inhibitory effect of NGF on the transcriptional activation of NGFI-B in PC12 cells. These data could provide new insights into the regulation of transcriptional activity required for some neurons to switch from activity-dependent survival to neurotrophin-dependent survival during development.

Animals↗

PubMed1997

Expression of human p140trk receptors in p140trk-deficient, PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis.

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140trk. These biological effects of NGF depend upon the signal-mediating function of p140trk substrates which are likely to differ from cell to cell. To define p140trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140trk cDNA sequence. Two stably transfected clones, one expressing p140trk with higher affinity (PC12EN-trk3; Kd 57.4 pM, Bmax 9.7 pmole/mg) and one expressing p140trk with a lower affinity (PC12EN-trk1; Kd 392.4 pM, Bmax 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140trk receptors show slow NGF-dissociation kinetics, are resistant to trypsin or Triton X-100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140trk receptors. NGF stimulates p140trk tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70S6k, SNT, and phospholipase C gamma, demonstrating that the major NGF-stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [3H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140trk receptor substrates.

Animals↗

PubMed1997

Effect of wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) on N-formyl-methionyl-leucyl-phenylalanine-induced phospholipase D activation in differentiated HL60 cells: possible involvement of phosphatidylinositol 3-kinase in phospholipase D activation.

Phospholipase D (PLD) plays an important role in neutrophil activation. However, despite various proposed mechanisms, its detailed regulatory mechanism is not fully understood. The functional coupling between phosphatidylinositol 3-kinase (PI 3-kinase) and PLD was investigated in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated human promyelocytic leukemia HL60 cells, using wortmannin, a fungal metabolite that is known as a selective inhibitor for phosphatidylinositol 3-kinase. Treatment of cells with this drug inhibited the formation of both phosphatidylinositol 3,4,5-trisphosphate (PIP3), a product of PI 3-kinase, and phosphatidylbutanol (PBut), the specific product of transphosphatidylation due to PLD in the presence of butanol, with similar concentration dependence (IC50 = 30-70 nM). Another PI 3-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also inhibited PBut formation in a concentration-dependent manner. However, wortmannin failed to inhibit phorbol 12-myristate 13-acetate-induced PLD activation in whole cells and membrane PLD activity in an in vitro assay system, indicating that inhibition of fMLP-induced PLD activation by wortmannin was not due to its direct effect on PLD activity. These results suggest that a major part of inhibition of PLD activation by wortmannin might be mediated through its effect on PI 3-kinase.

Androstadienes↗