Robust anti-obesity and metabolic effects of a dual GLP-1/glucagon receptor peptide agonist in rodents and non-human primates.

AIMS: To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. MATERIALS AND METHODS: MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. RESULTS: MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. CONCLUSIONS: Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

Stiffer double-stranded DNA in two-dimensional confinement due to bending anisotropy.

Using analytical approach and Monte Carlo (MC) simulations, we study the elastic behavior of the intrinsically twisted elastic ribbons with bending anisotropy, such as double-stranded DNA (dsDNA), in two-dimensional (2D) confinement. We show that, due to the bending anisotropy, the persistence length of dsDNA in 2D conformations is always greater than three-dimensional (3D) conformations. This result is in consistence with the measured values for DNA persistence length in 2D and 3D in equal biological conditions. We also show that in two dimensions, an anisotropic, intrinsically twisted polymer exhibits an implicit twist-bend coupling, which leads to the transient curvature increasing with a half helical turn periodicity along the bent polymer.

Extreme bendability of DNA double helix due to bending asymmetry.

Experimental data of the DNA cyclization (J-factor) at short length scales exceed the theoretical expectation based on the wormlike chain (WLC) model by several orders of magnitude. Here, we propose that asymmetric bending rigidity of the double helix in the groove direction can be responsible for extreme bendability of DNA at short length scales and it also facilitates DNA loop formation at these lengths. To account for the bending asymmetry, we consider the asymmetric elastic rod (AER) model which has been introduced and parametrized in an earlier study [B. Eslami-Mossallam and M. R. Ejtehadi, Phys. Rev. E 80, 011919 (2009)]. Exploiting a coarse grained representation of the DNA molecule at base pair (bp) level and using the Monte Carlo simulation method in combination with the umbrella sampling technique, we calculate the loop formation probability of DNA in the AER model. We show that the DNA molecule has a larger J-factor compared to the WLC model which is in excellent agreement with recent experimental data.

Hepatitis C virus infection among multi-transfused patients and personnel in haemodialysis units in central Islamic Republic of Iran.

A cross-sectional study was made of the prevalence of HCV and associated risk factors in 382 multi-transfused patients and haemodialysis staff in Yadz province in 2006. Of those tested for anti-HCV antibodies, 50.6% of patients with inherited bleeding disorders, 11.8% with thalassaemia and 5.0% undergoing haemodialysis were seropositive. First transfusion before 1996 (when blood donor screening started) was the common risk factor associated with HCV infection. Only 1/52 haemodialysis staff members was HCV infected (an intravenous drug user). Infection control measures were poor in all centres. In patients with inherited bleeding disorders genotype 1 (65.0%) was the predominant followed by genotype 3 (35.0%). The results provide evidence that blood donor screening and use of virus-inactivated factor concentrates have lowered the risk of HCV infection among multi-transfused patients.

A role for phosphatidylinositol 3-kinase in platelet aggregation in response to low, but not high, concentrations of PAF or thrombin.

In this study we show that platelet activating factor (PAF) activates PI 3-kinase over a rapid time course that correlates closely with the aggregation response. Tyrosine kinases are involved in this response, since there is increased PI 3-kinase activity associated with tyrosine-phosphorylated proteins. PI 3-kinase inhibitors were used to probe the dependence of PAF-induced aggregation on PI 3-kinase. Both wortmannin and LY-294002 inhibited PAF-induced aggregation that correlated with PI 3-kinase inhibition only when using lower concentrations of PAF giving reversible aggregation (primary phase). Similar results were obtained with human platelets using thrombin or thrombin receptor activating peptide. The same pattern of response was observed when activation of GPIIbIIIa was assessed by flow cytometry, i.e., PI 3-kinase inhibitors blocked integrin activation only when lower concentrations of agonist were used. We suggest that PI 3-kinase is important for reversible (primary) aggregation of platelets in response to PAF or thrombin, perhaps by contributing to the 'inside-out' activation of the platelet integrin GPIIbIIIa, only when submaximal concentrations of agonists are used. The lack of effect of PI 3-kinase inhibitors, when high concentrations of agonist are used, suggests that PI 3-kinase-independent pathways contribute to aggregation under these conditions.

High-performance liquid chromatography determination of residue levels on chicken carcasses treated with cetylpyridinium chloride.

Cetylpyridinium chloride (CPC) has been found to be effective in reducing contamination of chicken carcasses from a variety of microorganisms, including Escherichia coli O157:H7, Salmonella typhimurium, Campylobacter jejuni, Aeromonas hydrophila, Listeria monocytogenes, and Staphylococcus aureus. A procedure has been developed to determine residue levels on chicken carcasses after CPC treatment. For the analysis, chicken carcasses were extracted with 95% ethanol. The CPC concentration in the extract was measured by high-performance liquid chromatography (HPLC) with ultraviolet detection using dodecylpyridinium chloride (DPC) as an internal standard. The method was validated in the concentration range of 3-200 microg/ml CPC in ethanolic extract. This assay is rapid, precise, and accurate.

Is tyrosine kinase activation involved in basophil histamine release in asthma due to western red cedar?

Occupational asthma due to western red cedar is associated with histamine release from basophils and mast cells on exposure to plicatic acid (PA), but the mechanisms underlying this response remain unclear. Specific kinase inhibitors were used to study the role of tyrosine and serine/threonine kinases in PA-induced histamine release from human basophils. Pretreatment with the tyrosine kinase inhibitor methyl 2,5-dihydroxy-cinnamate (MDHC) attenuated histamine release from basophils triggered by anti-IgE (29.8% inhibition; n = 15; P < 0.01) or grass pollen (48% inhibition; n = 6; P < 0.01). Inhibition was concentration-dependent and could be reversed by washing the cells in buffer, while the inactive stereoisomer of MDHC did not affect histamine release. In contrast, the protein kinase C inhibitor staurosporine did not affect histamine release by either anti-IgE or grass pollen. Pretreatment with MDHC partially inhibited PA-induced histamine release from basophils of 6/9 patients with red cedar asthma (25.4% vs 33.8%; P = NS). Staurosporine gave a similar level of inhibition of PA-induced histamine release (25.3% vs 33.8%; P = NS). Thus, signal transduction of the human basophil Fc epsilon RI appears to depend upon tyrosine kinase activation, but not on protein kinase C (serine/threonine kinase) activation. The lack of specific effect on plicatic acid-induced histamine release in basophils obtained from patients with occupational asthma due to western red cedar suggests that tyrosine kinases are not as important in this disease as in atopic asthma, and is consistent with the view that histamine release in red cedar asthma is largely IgE-independent.

The antineoplastic ether lipid, s-phosphonate, selectively induces apoptosis in human leukemic cells and exhibits antiangiogenic and apoptotic activity on the chorioallantoic membrane of the chick embryo.

INTRODUCTION: We investigated the cytotoxic and antiangiogenic activity of the ether lipid, 2'-(trimethylammonio)ethyl 4-(hexadecyloxy)-3(S)-methoxybutane-phosphonate (termed s-phosphonate). METHOD: Cytotoxicity was determined using an XTT bioassay. Apoptosis was measured by either DNA fragmentation or immunolabelling techniques. Angiogenesis was measured using the in vivo chorioallantoic membrane (CAM) of the chick embryo. RESULTS: S-phosphonate was selectively cytotoxic towards the human leukemic cell lines, HL-60 and AML-14, whereas leukemic K-562 cells and the murine mast cell line, MC-9, were resistant to this agent at concentrations as high as 50 microM. This selectivity resulted from the induction of apoptosis (or programmed cell death) by s-phosphonate in HL-60 and AML-14 cells but not in resistant K-562 or MC-9 cells. S-phosphonate induced localized antiangiogenic effects and membrane thinning in the CAM. This concentration-dependent antiangiogenic effect was associated with apoptosis in the CAM as measured by DNA fragmentation in extracted CAM tissue. The localized areas of membrane thinning and antiangiogenesis on the CAM caused by s-phosphonate were also the only areas of the membrane in which apoptosis occurred. CONCLUSION: We conclude that s-phosphonate selectively induces apoptosis in human leukemic cells and exhibits antiangiogenic and apoptotic activity on the CAM.

Markers of airway smooth muscle cell phenotype.

Airway smooth muscle plays a principal role in the pathogenesis of asthma. Primary cultures are being used to investigate airway myocyte proliferation and cellular pathways regulating contraction. Airway smooth muscle cells (SMC) modulate from a contractile to a noncontractile phenotype in culture, but no systematic study of the concomitant changes in expression of cytocontractile and cytoskeletal proteins has been reported. We measured temporal changes in protein marker expression of canine tracheal SMC in primary culture, using specific antibodies and cDNA probes. Immunoblot analysis revealed that when cells became proliferative after 5 days of culture, the content of smooth muscle myosin heavy chain (sm-MHC), calponin, sm-alpha-actin, and desmin diminished by > 75%; myosin light chain kinase, h-caldesmon, and beta-tropomyosin had also decreased significantly (P < 0.05). Northern blots revealed that mRNA levels for sm-MHC and sm-alpha-actin were also significantly reduced in proliferative SMC. Conversely, immunoblotting demonstrated the content of non-muscle myosin heavy chain, l-caldesmon, vimentin, alpha/beta-protein kinase C (PKC), and CD44 homing cellular adhesion molecule (HCAM) increased one- to sixfold as cells became proliferative. The content of sm-MHC and sm-alpha-actin protein increased after confluence, suggesting that cultured airway SMC are capable of phenotypic plasticity. Marker protein contents were also compared, by immunoblot assay, between SMC dissociated from trachealis or pulmonary arterial media. Cytocontractile protein content was higher in the trachea, which shortens faster than the pulmonary artery. The identification of these markers provides tools for assessing the phenotype of airway SMC in culture and the airways of asthmatic patients.

Selective inhibition of phosphatidylinositol 3-kinase by phosphatidic acid and related lipids.

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is necessary for stimulation of cell division and inhibition of apoptosis in several cell types. We report that a synthetic phosphonolipid, 4-(hexadecyloxy)-3-(S)-methoxybutyl phosphonic acid (PoA), as well as the naturally occurring lipids, phosphatidic acid and lyso-phosphatidic acid, are potent and specific inhibitors of PI 3-kinase. The IC50's for inhibition using phosphatidylinositol as substrate ranged from 10-20 microM. PoA is also the putative primary intracellular metabolite following phospholipase D hydrolysis of the anti-tumour ether lipid, 2'-(trimethylammonio) ethyl-4-(hexadecyloxy)-3-(S)-methoxybutanephosphonate. These results suggests that inhibition of PI 3-kinase following metabolic degradation of ether lipids by phospholipase D may contribute to the cytotoxicity of these compounds. The sensitivity of PI 3-kinase to PA and lyso-PA could imply cross-talk between the phospholipase D and PI 3-kinase signal transduction pathways in vivo.

Effects of IZP-94005 (contignasterol) on antigen-induced bronchial responsiveness in ovalbumin-sensitized guinea-pigs.

1. We have investigated the novel naturally occurring marine compound, IZP-94005 (contignasterol), as a potential anti-asthma agent, using both in vivo and in vitro models of allergen-induced bronchoconstriction and airway smooth muscle contraction. 2. Tracheal rings from ovalbumin (OA)-sensitized guinea-pigs were treated with various concentrations of IZP-94005 for 20 min prior to challenge with ovalbumin. IZP-94005 (3-30 microM) inhibited responses of sensitized tracheal rings stimulated with OA in a concentration-dependent manner, with an IC50 of 10 microM. 3. IZP-94005 (10 microM) had no effect on carbachol-induced contractions of sensitized guinea-pig tracheal rings, although it did inhibit histamine-induced responses of OA sensitized guinea-pig tracheal rings. 4. The effects of IZP-94005 in vivo were examined using OA-sensitized guinea-pigs which were tracheotomized under anaesthesia and placed in a body plethysmograph. Measurements of lung resistance and compliance were performed by isovolumetric analysis of volume and trans-pulmonary pressure. 5. IZP-94005 (50 and 200 micrograms kg-1), by inhalation 20 min prior to OA challenge caused significant inhibition of the increase in lung resistance induced by OA in sensitized guinea-pigs, compared to vehicle-treated animals. Nedocromil sodium (20 mg kg-1), with a similar protocol, also inhibited OA-induced responses in this model. 6. We therefore suggest that IZP-94005 is a good candidate for further investigation as a possible antiasthma agent.

A comparison of the lytic properties of two ether-linked lipids, one with and one without antineoplastic activity.

We have synthesized two ether lipids: 2'-(trimethylammonio)ethyl 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (compound 1) with antineoplastic activity and a maltosyl derivative (compound 2) without antineoplastic activity. We have compared the antineoplastic activity of these two compounds against WEHI-3B cells with their ability to disrupt the membranes of erythrocytes or neutrophils. Since ether lipids are highly hydrophobic molecules, it is possible that they may exert their cytotoxic action by inducing the nonspecific perturbation of cellular membranes, causing lysis and cell death. Membrane disruption was monitored by the lysis of cells or the change in erythrocyte membrane microviscocity and compared with the effect of detergents (known nonspecific lytic agents). Both compounds 1 and 2 caused the lysis of erythrocytes and neutrophils. The rate of lysis of erythrocytes was comparable to the rate of change of erythrocyte membrane microviscosity caused by both compounds 1 and 2. Both compounds caused the lysis of erythrocytes via a noncolloid osmotic mechanism that displayed features of the lysis caused by detergents at high concentrations.

Involvement of immunologic mechanisms in a guinea pig model of western red cedar asthma.

Western red cedar asthma is the most common form of occupational asthma in the Pacific Northwest. Plicatic acid (PA) is the chemical component of Western red cedar that causes asthma. The role of immunologic processes involved in the PA-induced asthmatic reaction has not been established. To characterize the mechanisms of PA-induced asthmatic reaction, guinea pigs were sensitized to PA through biweekly injection of PA-ovalbumin conjugate with aluminum hydroxide as an adjuvant for a period of 6 months. Specific IgG1 antibodies to PA were detected in the blood 3 months after sensitization of animals. The level of specific IgG1 antibodies to ovalbumin after 6 months was about two times the level of specific IgG1 to PA. At 6 months, tracheal tissue from PA-ovalbumin-sensitized guinea pigs contracted after exposure to either PA or ovalbumin in vitro. The degree of contraction induced by PA was two to three times less than the contraction induced by ovalbumin. PA caused histamine, prostaglandin D2, and leukotriene D4 release from both lung mast cells and blood basophils. The amount of histamine and eicosanoids released by PA was also two to three times less than the amount of mediators released by ovalbumin. When the trachea of normal guinea pigs was passively sensitized with serum from PA-ovalbumin-sensitized guinea pigs, it contracted in response to PA or ovalbumin in an organ bath. When the serum of PA-ovalbumin-sensitized guinea pigs was depleted of immunoglobulins and then used for passive sensitization of normal trachea, no contraction was observed when challenged with PA, suggesting that IgG1 antibodies mediate the tracheal reaction to PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Isosteric phosphonate analogs of ET-16-OMe. Synthesis and biological evaluation of the enantiomers of 2'-(trimethylammonio)ethyl 4-(hexadecyloxy)-3-methoxybutanephosphonate and 2'-(trimethylammonio)ethyl 4-(hexadecylthio)-3-methoxybutanephosphonate.

The enantiomers of two isosteric phosphonate analogs of the ether-linked antitumor agent 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OMe) were synthesized and evaluated for their cytotoxicity against various mouse leukemic cell lines in vitro and in vivo. The key step in the synthesis of the alkyloxy and alkylthio analogs (1 and 2, respectively) is the opening of an epoxide [hexadecyl 2-oxiranylmethyl ether (4) or hexadecyl 2-oxiranylmethyl thioether (8)] by LiCH2P(O)(OMe)2 using BF3.Et2O in tetrahydrofuran at low temperature. The cytotoxic activities of the hexadecyloxy and hexadecylthio phosphonate analogs of ET-18-OMe (1 and 2) against the murine leukemias WEHI-3B,L1210, and P388 were similar, indicating that substitution of a sulfur atom for oxygen in the long-chain ether does not result in a significant difference in cytotoxicity. The IC50 values of 1 and 2 were in the range of 1-5 microM. Alkyloxy phosphonate 1 was highly effective in inhibiting the growth of WEHI-3B and P388 tumors implanted in BALB/C mice. The alkyloxy and alkylthio phosphonates 1 and 2 prolonged the survival of CD1 mice bearing L1210 tumors. The antitumor activities of the phosphonate analogs of ET-18-OMe in these in vitro and in vivo studies were independent of chirality, consistent with previous studies with the enantiomers of 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine.

Inhibition of crystal-induced neutrophil activation by a protein tyrosine kinase inhibitor.

The objective of this work was to investigate the role of tyrosine kinase in monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystal-induced neutrophil activation using the tyrosine kinase inhibitor lavendustin C (LVC). Human neutrophils pretreated with LVC at concentrations between 10 and 150 microM or control neutrophils were stimulated by plasma-coated CPPD or uncoated MSUM, and chemiluminescence, superoxide generation, intracellular calcium concentration, and degranulation (myeloperoxidase and lysozyme release) were monitored with time. LVC strongly inhibited chemiluminescence, superoxide anion generation, myeloperoxidase and lysozyme release, and calcium mobilization. After 1-min crystal-neutrophil incubations, neutrophil cytosolic fractions showed extensive inhibition of tyrosine kinase activity by LVC. We conclude that the inhibition of neutrophil responses to crystal stimulation, by the protein tyrosine kinase inhibitor LVC, provides evidence that supports the involvement of tyrosine kinases in crystal-induced neutrophil activation.

Effect of phospholipase C inhibitor U-73122 on antigen-induced airway smooth muscle contraction in guinea pigs.

The importance of phospholipase C (PLC) in airway smooth muscle contraction was studied, using an inhibitor of PLC, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dione (U-73122). Tracheas from ovalbumin (OA)-sensitized guinea pigs contracted rapidly after exposure to low concentrations of antigen (OA). However, tracheas treated with U-73122 for 10 min prior to the addition of antigen, demonstrated a 3 log rightward shift in the OA dose-response curve with an IC50 of 7 microM. The analogue of U-73122, 1-[6[[17 beta-3-methoxyestra-1,3,5 trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73433), was approximately 5-fold less active in inhibiting smooth muscle contraction. In addition to the inhibition of antigen-induced smooth muscle contraction, U-73122 inhibited carbachol- and leukotriene D4-induced smooth muscle contraction. Furthermore, U-73122 inhibited in a dose-dependent manner antigen-induced histamine release from guinea pig tracheal tissue. The inhibition of smooth muscle contraction by U-73122 correlated well with the inhibition of polyphosphoinositide mediates smooth muscle contractile responses to muscarinic agonists and leukotrienes as well as antigenic-induced contraction.

Immunologic studies of the mechanisms of occupational asthma caused by western red cedar.

BACKGROUND: Occupational asthma caused by western red cedar (Thuja plicata) is a common problem in sawmill industries. The objective of this study was to examine the cellular and immunologic mechanisms of western red cedar asthma (WRCA) more closely. METHODS: Bronchial biopsy specimens, bronchoalveolar lavage (BAL) mast cells and peripheral blood basophils from patients with WRCA, patients with atopic asthma, and nonatopic control subjects were challenged in vitro with plicatic acid (PA), PA-human serum albumin conjugate (PA-HSA), grass pollen, or calcium ionophore. RESULTS: PA (100 micrograms/ml) released histamine from the basophils of 9 of 11 patients with WRCA, 1 of 7 patients with atopic asthma, and 2 of 7 normal subjects. PA triggered histamine release from 10 of 11 bronchial biopsy specimens and 8 of 8 BAL samples from patients with WRCA. Interestingly, PA released histamine from BAL cells and bronchial biopsy specimens from 3 of 7 normal subjects but in none of the patients with atopic asthma. PA-HSA-induced histamine release from basophils and biopsy specimens was confined to patients with WRCA. PA-specific IgE was not detectable in serum from most patients with WRCA, and their serum did not transfer PA sensitivity to human lung fragments or lactate-stripped basophils. After pretreatment with anti-IgE in the absence of calcium, basophils from 14 subjects with WRCA still responded to PA (mean 64% to 67% of pretreatment response), whereas responses to grass pollen or anti-IgE were abolished. CONCLUSIONS: This study confirms that PA releases histamine from bronchial mast cells of most patients with WRCA but not from those of patients with atopic asthma. The PA response of some normal subjects suggests that PA may have both specific and nonspecific actions on mast cells and basophils, whereas the serologic studies indicate histamine release in WRCA cannot simply be attributed to PA-specific IgE.

Forskolin inhibits platelet-activating factor binding to platelet receptors independently of adenylyl cyclase activation.

The effect of forskolin on platelet-activating factor (PAF) receptor was investigated. Rabbit platelets treated with forskolin showed approximately a 9-fold increase in cAMP levels over the control. After treatment of platelets with forskolin prior to PAF binding, a 30-40% (P < 0.005) decrease in PAF binding was observed. The decrease in PAF binding caused by forskolin was concomitant with a decrease in the physiological responses of platelets induced by PAF. However, this forskolin-induced decrease in PAF binding was not a consequence of cAMP formation as the addition of a cAMP analog could not mimic the action of forskolin. Additionally, the inactive analog of forskolin, dideoxyforskolin, which does not activate adenylyl cyclase, also reduced PAF binding to its receptor. Reduction of PAF binding by forskolin and dideoxyforskolin was also observed with isolated platelet membranes. To understand the mechanism of forskolin induced changes in PAF binding, the involvement of a G-protein in this process was investigated. Cells treated with GTP gamma S showed approximately a 25% reduction in PAF binding. Addition of forskolin to the GTP gamma S treated cells resulted in a further reduction in PAF binding, suggesting the action of forskolin was independent of G-protein activation. The data suggests that the action of forskolin was independent of adenylyl cyclase or G-protein involvement. It is speculated that the action of forskolin on PAF binding was due to a direct effect of this molecule and its analog on the PAF receptor itself or to components of the post-receptor signalling for PAF.

1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine inhibits diacylglycerol kinase in WEHI-3B cells.

The effects of 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-16-OCH3-GPC) and its metabolite 1-O-hexadecyl-2-O-methyl-sn-glycerol (AMG) on the activity of diacylglycerol kinase (DGK) in WEHI-3B cells were investigated. Treatment of WEHI-3B cells with 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 5 min leads to the activation of cytosolic DGK without significant effect on microsomal DGK. When these cells were first exposed to 50 microM ET-16-OCH3-GPC for 30 min prior to activation with TPA, the activity of DGK was inhibited by about 70%, as measured by the ability of enzyme to form [32P]phosphatidic acid ([32P]PA). Addition of either ET-16-OCH3-GPC or AMG to the preparation of enzyme in vitro also inhibited 1,2-dioleoyl-sn-glycerol (DG) phosphorylation in the presence of [gamma-32P]ATP. The IC50 value for inhibition of cytosolic DGK by ET-16-OCH3-GPC and AMG were about 8.5 and 15 microM, respectively. ET-16-OCH3-GPC also inhibited the ability of guanosine 5'-O-(3-thiophosphate) (GTP-gamma S) to activate DGK in vitro. The potency of ET-16-OCH3-GPC at 10 microM in inhibiting DGK was greater than that of sphingosine at 50 microM, but less than that of R59022 (a specific DGK inhibitor) at 10 microM.(ABSTRACT TRUNCATED AT 250 WORDS)