Taurine Ameliorates Tunicamycin-Induced Liver Injury by Disrupting the Vicious Cycle between Oxidative Stress and Endoplasmic Reticulum Stress.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver dysfunction characterized by excess lipid accumulation; non-alcoholic steatohepatitis can transform into more severe conditions, such as cirrhosis and hepatocellular carcinoma. Although several pharmacologic approaches have been evaluated in clinical trials, there are no approved therapies for NAFLD. Previous studies have suggested that taurine supplementation alleviates fatty liver; however, the underlying mechanism remains obscure. In this study, we investigated the beneficial effects of taurine on fatty liver injury in vivo induced by tunicamycin, a chemical endoplasmic reticulum (ER) stressor. The mice were administered 2% taurine for 2 weeks prior to intraperitoneal tunicamycin injection; after 72 h of treatment, the mice were euthanized. Tunicamycin treatment significantly increased the levels of serum ALT and AST and hepatic triglycerides. Notably, these changes were alleviated by taurine supplementation. Taurine normalized the protein and/or mRNA levels involved in ER stress signaling (IRE1a, p-IRE1a, ATF6, XBP1, BiP, and CHOP) and lipid metabolism (CD36, MTTP, and ApoB), which were dysregulated by tunicamycin treatment. The stimulation of hepatic lipid export by taurine was evidenced by the recovery of blood VLDL levels. Furthermore, taurine supplementation prevented tunicamycin-induced lipid peroxidation and decreased glutathione (GSH) levels by correcting abnormal cysteine catabolism involved in the production of both taurine and GSH. Therefore, taurine supplementation can prevent tunicamycin-induced liver injury by counteracting oxidative and ER stress.

Preparation and in vivo evaluation of lecithin-based microparticles for topical delivery of minoxidil.

Minoxidil is widely used for treatment of androgenic alopecia. Commercial products containing minoxidil are usually in solution form. Repeated applications of minoxidil solution can lead to adverse effects such as skin irritation and horniness. The aims of this study were to prepare lecithin-based microparticle in minoxidil solution for enhancement of minoxidil topical delivery and skin protection and evaluate the ability of lecithin on in vitro delivery, in vivo hair growth, and skin trouble improvement compared to commercial minoxidil solution. In in vitro skin permeation study, minoxidil solution containing lecithin microparticle showed higher skin penetration rate and higher retention of drug inside the skin compared to minoxidil solution without lecithin. After topical application of minoxidil solutions with or without lecithin to C57BL/6 mice, minoxidil 5% solution containing lecithin microparticle showed hair re-growth as efficient as commercial product of minoxidil 5% solution. It also significantly improved skin troubles while commercial product presented horny substance and crust formation. Therefore, the lecithin-based microparticle in minoxidil 5% solution has good ability to promote hair growth without adverse effects.

3-Deoxysappanchalcone Promotes Proliferation of Human Hair Follicle Dermal Papilla Cells and Hair Growth in C57BL/6 Mice by Modulating WNT/ß-Catenin and STAT Signaling.

3-Deoxysappanchalcone (3-DSC) has been reported to possess anti-allergic, antiviral, anti-inflammatory and antioxidant activities. In the present study, we investigated the effects of 3-DSC on the proliferation of human hair follicle dermal papilla cells (HDPCs) and mouse hair growth in vivo. A real-time cell analyzer system, luciferase assay, Western blot and real-time polymerase chain reaction (PCR) were employed to measure the biochemical changes occurring in HDPCs in response to 3-DSC treatment. The effect of 3-DSC on hair growth in C57BL/6 mice was also examined. 3-DSC promoted the proliferation of HDPCs, similar to Tofacitinib, an inhibitor of janus-activated kinase (JAK). 3-DSC promoted phosphorylation of ß-catenin and transcriptional activation of the T-cell factor. In addition, 3-DSC potentiated interleukin-6 (IL-6)-induced phosphorylation and subsequent transactivation of signal transducer and activator of transcription-3 (STAT3), thereby increasing the expression of cyclin-dependent kinase-4 (Cdk4), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). On the contrary, 3-DSC attenuated STAT6 mRNA expression and IL4-induced STAT6 phosphorylation in HDPCs. Finally, we observed that topical application of 3-DSC promoted the anagen phase of hair growth in C57BL/6 mice. 3-DSC stimulates hair growth possibly by inducing proliferation of follicular dermal papilla cells via modulation of WNT/ß-catenin and STAT signaling.

Silymarin Prevents Restraint Stress-Induced Acute Liver Injury by Ameliorating Oxidative Stress and Reducing Inflammatory Response.

Silymarin is a flavonoid extracted from the milk thistle Silybum marianum. It has been reported to prevent liver injuries induced by various chemicals or toxins. Our recent study suggested that silymarin induces hepatic synthesis of glutathione by increasing cysteine availability, which may consequently contribute to increased antioxidant capacity of the liver. In the present study, we investigated the effects of silymarin on acute liver injury induced by restraint stress. Silymarin (100 mg/kg) was orally administered to BALB/c mice every 12 h (3 times in total). After the last dose, mice were subjected to restraint stress for 6 h, and serum levels of aspartate and alanine aminotransferases, and hepatic levels of lipid peroxidation were determined. Hepatic levels of sulfur-containing metabolites such as methionine, S-adenosylmethionine, cysteine, and glutathione were also measured. The level of pro-inflammatory mediators in both liver and serum was determined. To study the mechanism of the effects of silymarin, we assessed Jun N-terminal kinase (JNK) activation and apoptotic signaling. Restraint stress induced severe oxidative stress and increased mRNA levels of pro-inflammatory mediators; both effects of restraint stress were significantly inhibited by silymarin. Moreover, administration of silymarin significantly prevented acute liver injury induced by restraint stress by blocking JNK activation and subsequently apoptotic signaling. In conclusion, these results suggest that the inhibition of restraint stress-induced liver injury by silymarin is due at least in part to its anti-oxidant activity and its ability to suppress the inflammatory response.

Exacerbation of collagen antibody-induced arthritis in transgenic mice overexpressing peroxiredoxin 6.

OBJECTIVE: Peroxiredoxin 6 plays important and complex roles in the process of inflammation, but its role in the development of rheumatoid arthritis (RA) remains unclear. We undertook this study to investigate the roles and mechanisms of peroxiredoxin 6 in the development of collagen antibody-induced arthritis (CAIA) and antigen-induced arthritis (AIA) in peroxiredoxin 6-overexpressing transgenic mice, in peroxiredoxin 6-transfected RAW 264.7 cells, in macrophages isolated from peroxiredoxin 6-overexpressing transgenic mice, and in synoviocytes from arthritis patients. METHODS: CAIA and AIA were induced using standard methods. Peroxiredoxin 6-transfected RAW 264.7 cells, macrophages isolated from peroxiredoxin 6-overexpressing transgenic mice, and synoviocytes from arthritis patients were used to study proinflammatory responses and mechanisms. Clinical scores and histopathologic changes were determined in peroxiredoxin 6-overexpressing transgenic mice and wild-type (WT) mice with CAIA or AIA. Generation of nitric oxide (NO), expression of inducible NO synthase and cyclooxygenase 2, and activity of NF-κB and activator protein 1 (AP-1) were determined in cultured macrophages and synoviocytes as well as in joint tissue from mice by Western blotting, electrophoretic mobility shift assay, and immunohistochemical analysis. RESULTS: Development of CAIA and AIA and proinflammatory responses were more exacerbated in peroxiredoxin 6-overexpressing transgenic mice than in WT mice. Overexpression of peroxiredoxin 6 increased lipopolysaccharide-induced inflammatory responses in RAW 264.7 cells, in macrophages isolated from peroxiredoxin 6-overexpressing transgenic mice, and in synoviocytes from arthritis patients, and this was accompanied by up-regulation of the JNK pathway. Moreover, a JNK inhibitor completely blocked RA development and proinflammatory responses. CONCLUSION: Our findings suggest that overexpression of peroxiredoxin 6 might promote development of RA through NF-κB and AP-1 activity via the JNK pathway.

Sophoricoside isolated from Sophora japonica ameliorates contact dermatitis by inhibiting NF-κB signaling in B cells.

Sophoricoside (SOPH) is an isoflavone isolated from Sophora japonica (Leguminosae). In this study, the inhibitory effect of SOPH on contact dermatitis was investigated. At dosages of 3 and 10 mg/kg, SOPH ameliorated 2,4-dinitrochlorobenzene-induced acute and chronic contact dermatitis by 50-70%. As cellular targets, SOPH mainly affected the functions of B cells rather than T cells, macrophages and dendritic cells. As signaling targets, SOPH inhibited the phosphorylation and degradation of IκBα/ß and the nuclear translocation of NF-κB p65 in B cells, but not in dendritic cells and macrophages. SOPH did not affect the phosphorylation of ERK, p38, and JNK MAPKs, in B cells, dendritic cells, and macrophages. Taken together, these results suggest that SOPH ameliorates contact dermatitis by inhibiting mainly NF-κB signaling in B cells.

Obovatol enhances docetaxel-induced prostate and colon cancer cell death through inactivation of nuclear transcription factor-kappaB.

Nuclear transcription factor-kappaB (NF-kappaB) is constitutively activated in prostate and colon cancers and is related with the resistance of cancer cells against chemotherapeutics. Previously, we found that obovatol, an active compound isolated from Magnolia obovata, inhibited cancer cell growth through inhibition of NF-kappaB activity. We investigated here whether obovatol could sensitize cancer cells against docetaxel through inhibition of NF-kappaB activity in prostate cancer (LNCaP and PC-3) and colon cancer (SW620 and HCT116) cells. The combination treatment with each drug at one half the respective IC(50) dose (5 microM obovatol + 5 nM docetaxel) was more effective and significant (60%-70%) in the inhibition of cancer cell growth than single treatment by each drug (20%-40%); inhibition was exerted through a significant increase of apoptosis induction (60%-80%) by the combination treatment compared to the single treatment (10%-30%). Correlating well with the synergistic inhibition (combination indices are less than 1 in all cell types), the combination significantly inhibited NF-kappaB activities as well as expression of NF-kappaB target apoptotic cell death proteins, but decreased anti-apoptotic cell death proteins. Similar combination effects of obovatol with other chemotherapeutic agents (paclitaxel, cisplatin, and doxorubicin) on the inhibition of cell growth and NF-kappaB activity were also found. These results indicate that obovatol augments cell growth inhibition by chemotherapeutics through inactivation of NF-kappaB and suggest that obovatol may have therapeutic advantages in the combination treatment with other chemotherapeutics. [Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.09048FP].

Inhibition of NF-kappaB by ginsenoside Rg3 enhances the susceptibility of colon cancer cells to docetaxel.

Ginsenoside Rg3, the main constituent isolated from Panax ginseng, has been of interest for use as a cancer preventive or therapeutic agent. We investigated here whether Rg3 can inhibit the activity of NF-kappaB, a key transcriptional factor constitutively activated in colon cancer that confers cancer cell resistance to chemotherapeutic agents. To investigate whether RG3 can suppress activation of NF-kappaB, and thus inhibit cancer cell growth, we examined the susceptibility of colon cancer cells (SW620 and HCT116) to treatment with Rg3 (25, 50, 75, 100 microM) and RG3-induced activation of NF-kappaB. RG3 dose-dependently inhibited cancer cell growth through induction of apoptosis and decreased NF-kappaB activity. In a further study of compounds in colon cancer, we used half of the IC(50) dose, values in combined treatments of Rg3 (50 microM) with conventional agents - docetaxel (5 nM), paclitaxel (10 nM) cisplatin (10 microM) and doxorubicin (2 microM). Compared to treatment with Rg3 or chemotherapy alone, combined treatment was more effective (i.e., there were synergistic effects) in the inhibition of cancer cell growth and induction of apoptosis and these effects were accompanied by significant inhibition of NF-kappaB activity. NF-kappaB target gene expression of apoptotic cell death proteins (Bax, caspase-3, caspase-9) was significantly enhanced, but the expression of anti-apoptotic genes and cell proliferation marker genes (Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP), Cox-2, c-Fos, c-Jun and cyclin D1) was significantly inhibited by the combined treatment compared to Rg3 or docetaxel alone. These results indicate that ginsenoside Rg3 inhibits NF-kappaB, and enhances the susceptibility of colon cancer cells to docetaxel and other chemotherapeutics. Thus, ginsenoside Rg3 could be useful as an anti-cancer or adjuvant anti-cancer agent.

Protective effect of the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol on scopolamine-induced memory impairment and the inhibition of acetylcholinesterase activity.

Magnolol, honokiol, and obovatol are well-known bioactive constituents of the bark of Magnolia officinalis and have been used as traditional Chinese medicines for the treatment of neurosis, anxiety, and stroke. We recently isolated novel active compound (named 4-O-methylhonokiol) from the ethanol extract of Magnolia officinalis. The present study aimed to test two different doses of ethanol extracts of Magnolia officinalis (5 and 10 mg/kg/mouse, p.o., 1 week) and 4-O-methylhonokiol (0.75 and 1.5 mg/kg/mouse, p.o., 1 week) administered for 7 days on memory impairment induced by scopolamine (1 mg/kg body weight i.p.) in mice. Memory and learning were evaluated using the Morris water maze and the step-down avoidance test. Both the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol prevented memory impairment induced by scopolamine in a dose-dependent manner. The ethanol extract of Magnolia officinalis and 4-O-methylhonokiol also dose-dependently attenuated the scopolamine-induced increase of acetylcholinesterase (AChE) activity in the cortex and hippocampus of mice, and inhibited AChE activity in vitro with IC(50) (12 nM). This study, therefore, suggests that the ethanol extract of Magnolia officinalis and its major ingredient, 4-O-methylhonokiol, may be useful for the prevention of the development or progression of AD.

(-)-Epigallocatechin-3-gallate prevents lipopolysaccharide-induced elevation of beta-amyloid generation and memory deficiency.

Neuroinflammation has been known to play a role in the pathogenesis of AD. Our previous study showed that lipopolysaccharide (LPS) induced memory impairment through the accumulation of Abeta via the increase of beta- and gamma-secretase. In this study, we investigated the possible preventive effect of (-)-epigallocatechin-3-gallate (EGCG) on memory deficiency caused by LPS through the inhibition of Abeta(1-42) generation. Oral treatment with EGCG (1.5 and 3 mg/kg, for 3 weeks) into drinking water ameliorated LPS (1 microg/mouse, i.c.v.)-induced memory deficiency in a dose dependent manner. In addition, EGCG also dose-dependently inhibited LPS-induced elevation of Abeta level through attenuation of LPS-induced beta- and gamma-secretase activities and expression of its metabolic products; C99 and Abeta. Moreover, EGCG prevented LPS-induced neuronal cell death as well as the expression of inflammatory proteins, inducible nitric oxide synthetase and cyclooxygenase-2. This study therefore suggests that EGCG prevents LPS-mediated apoptotic cell death through the inhibition of the elevation of Abeta via the inhibition of beta- and gamma-secretases, and thus EGCG can be a useful agent against neuroinflammation-associated development or progression of AD.

Reduced anxiety in the mice expressing mutant (N141I) presenilin 2.

Alzheimer's disease (AD) is characterized by progressive cognitive impairment. The effect of presenilin 1 (PS1) and PS2 mutation on cognition has been well characterized in a variety of transgenic mice. However, noncognitive behaviors have not been considered in these mice. In the present study, we found that transgenic mice expressing mutant PS2 (N141I) displayed decreased anxiety behavior determined by the elevated plus maze test and the light dark box test. However, these mice showed biphasic ambulatory activity (hyperactivity followed by hypoactivity) in an open field test. Correlated well with the reduced anxiety, expression of GABA(A)alpha(1) receptor was higher whereas c-Fos was lower in the cortex, hippocampus, and amygdala of the mice expressing PS2 mutation than those of the wild-type PS2 or nontransgenic control mice. These data indicate that PS2 mutation causes reduction of anxiety, and this effect may be related to the change of the expression of GABA(A)alpha(1) receptor and c-Fos. These findings could be useful in the understanding and the treatment of AD patients.

Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation.

BACKGROUND: Alzheimer's disease (AD) is characterized by extensive loss of neurons in the brain of AD patients. Intracellular accumulation of beta-amyloid peptide (Abeta) has also shown to occur in AD. Neuro-inflammation has been known to play a role in the pathogenesis of AD. METHODS: In this study, we investigated neuro-inflammation and amyloidogenesis and memory impairment following the systemic inflammation generated by lipopolysaccharide (LPS) using immunohistochemistry, ELISA, behavioral tests and Western blotting. RESULTS: Intraperitoneal injection of LPS, (250 microg/kg) induced memory impairment determined by passive avoidance and water maze tests in mice. Repeated injection of LPS (250 microg/kg, 3 or 7 times) resulted in an accumulation of Abeta1-42 in the hippocampus and cerebralcortex of mice brains through increased beta- and gamma-secretase activities accompanied with the increased expression of amyloid precursor protein (APP), 99-residue carboxy-terminal fragment of APP (C99) and generation of Abeta1-42 as well as activation of astrocytes in vivo. 3 weeks of pretreatment of sulindac sulfide (3.75 and 7.5 mg/kg, orally), an anti-inflammatory agent, suppressed the LPS-induced amyloidogenesis, memory dysfunction as well as neuronal cell death in vivo. Sulindac sulfide (12.5-50 microM) also suppressed LPS (1 microg/ml)-induced amyloidogenesis in cultured neurons and astrocytes in vitro. CONCLUSION: This study suggests that neuro-inflammatory reaction could contribute to AD pathology, and anti-inflammatory agent could be useful for the prevention of AD.

Inhibitory effect of citral on NO production by suppression of iNOS expression and NF-kappa B activation in RAW264.7 cells.

Citral is a major compound of lemongrass (Cymbopogon citratus L.) that has many pharmacological activities such as anti-fungal and anti-bacterial effects. In this study, we investigated the anti-inflammatory effect of citral and defined its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Citral (3-12 microg/mL) significantly inhibited LPS-induced nitric oxide (NO) production in a concentration-dependent manner (IC50: 6.5 microg/mL). Furthermore, it was found that citral effectively inhibited the transcriptional activity and expression of iNOS, and potently suppressed the DNA binding activity and nuclear translocation of NF-kappa B as well as I kappa B phosphorylation in a concentration dependent manner. These results suggest that citral is anti-inflammatory, and its effects may be due to the inhibition of NO production through the suppression of NF-kappa B activation.

Growth inhibitory effects of obovatol through induction of apoptotic cell death in prostate and colon cancer by blocking of NF-kappaB.

Biphenolic components in Magnolia obovata including magnolol and honokiol have shown several pharmacological activities such as anti-tumor, anti-oxidant and anti-inflammatory effects. Previously in cultured macrophage Raw264.7 cells and fibroblast, we found that obovatol, an active compound isolated from M. obovata inhibited NF-kappaB activity which has been known to be a significant transcriptional factor to control of cancer cell growth. We investigated here whether obovatol could inhibit NF-kappaB activity, and thereby inhibit cancer cell growth in prostate (LNCaP and PC-3) and colon cancer (SW620 and HCT116) cells. Treatment of obovatol (10, 15, 20, 25 microM) inhibits cancer cell growth in the absence or the presence of tumor necrosis factor-alpha (TNF-alpha , 10 ng/ml) and tetradecanoyl phorbol acetate (TPA 10 or 50 nM) in a concentration-dependent manner through induction of apoptotic cell death. Cytotoxic activity was not observed in normal cells with up to 50 muM obovatol. It was also found that obovatol inhibited TNF-alpha and TPA-induced transcriptional and DNA binding activities of NF-kappaB. In further study, obovatol decreased translocation p65 and p50 into nucleus via decrease of phosphorylation of IkappaB. Correlated well with the induction of apoptosis, obovatol increased the expression of the apoptotic genes; Bax, caspase-3, caspase-9, whereas inhibited expression of anti-apoptotic genes; Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP) as well as the cell proliferation marker genes; Cox-2, c-Fos, c-Jun and cyclin D1. These results suggest that obovatol inhibits prostate and colon cancer cell growth via induction of apoptotic cell death, and that inhibition of NF-kappaB may be a significant as its action mechanism.

Berberine inhibits human neuroblastoma cell growth through induction of p53-dependent apoptosis.

Berberine, an alkaloid, has anti-tumor properties in some cancer cells, but action mechanisms are not clear yet. We here investigated the anticancer activity of berberine and possible mechanisms in human neuroblastoma SK-N-SH and SK-N-MC cells. The p53-expressing cells, SK-N-SH (IC50=37 microM) were more susceptible to berberine than the p53-deficient cells, SK-N-MC (IC50 > or =100 microM) without cytotoxic effect on the cortical neuronal cells. Berberine caused cell cycle arrest in G0/G1 phase and apoptotic cell death, and these effects were much greater in SK-N-SH cells than those in SK-N-MC cells. Berberine much greatly decreased G0/G1 phase-associated cyclin and cyclin-dependent kinase (cyclin D1, cyclin E, Cdk2, and Cdk4) expression, and increased apoptotic gene expression and activation of caspase-3 in SK-N-SH cells. Exploration of p53 siRNA or pifithrin-alpha (PFT-alpha), a p53 inhibitor, in the SK-N-SH cells resulted in increase of IC50 values for cell viability, and decreased apoptotic cell death, expression of p53 and activation of caspase-3. Therefore, these results showed that berberine causes p53-dependent apoptotic death of neuroblastoma cells, and suggested that berberine may be useful as an anticancer agent for neuroblastoma.

Inhibition of cell growth and induction of apoptosis via inactivation of NF-kappaB by a sulfurcompound isolated from garlic in human colon cancer cells.

Compounds such as S-allylmercaptocysteine, diallyl disulfide, and S-trityl-L-cysteine isolated from garlic have been known to be effective in chemoprevention. Nuclear transcription factor-kappaB (NF-kappaB) has been known to be an implicated factor in apoptotic cell death of several cancer cells. In this study, we investigated whether a sulfurcompound (named thiacremonone) isolated from garlic could modulate NF-kappaB activity and thereby induce apoptotic cell death of colon cancer cells. Treatment with different concentrations (30 - 150 microg/ml) of thiacremonone for various periods (0 - 48 h) inhibited colon cancer cell (SW620 and HCT116) growth followed by induction of apoptosis in a dose-dependent manner. We also found that thiacremonone modulated tumor necrosis factor-alpha (TNF-alpha) and tetradeanoyl phorbol acetate (TPA)-induced NF-kappaB transcriptional and DNA binding activity. Moreover, thiacremonone suppressed NF-kappaB target anti-apoptotic genes (Bcl-2, cIAP1/2, and XIAP) and inflammatory genes (iNOS and COX-2), whereas it induced apoptotic genes (Bax, cleaved caspse-3, and cleaved PARP) expression. These results suggest that a novel sulfurocompound from garlic inhibited colon cancer cell growth through induction of apoptotic cell death by modulating of NF-kappaB.

Decrease in NF-kappaB, AP-1 and SP-1 activities in neuronal cells expressing presenilin 2.

Decreases in activities of the NF-kappaB, AP-1 and SP-1 transcription factors, which could act as antiapoptotic factors, in the presenilin 2 transfected PC12 cells, either in nontreatment conditions or under apoptotic stimulation, were found in this study. Similar results were also found in mice brain cells carrying presenilin 2, especially in the mutant gene expressed ones. These findings suggested that presenilin 2 may be implicated in neuronal cell death by altering the antiapoptotic activity of the transcription factors.

Antiplatelet activity of J78 (2-Chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), an antithrombotic agent, is mediated by thromboxane (TX) A2 receptor blockade with TXA2 synthase inhibition and suppression of cytosolic Ca2+ mobilization.

We previously reported that J78 (2-chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), a newly synthesized 1,4-naphthoquinone derivative, exhibited a potent antithrombotic effect, which might be due to antiplatelet rather than anticoagulation activity. In the present study, possible anti-platelet mechanism of J78 was investigated. J78 concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 microg/ml), thrombin (0.05 U/ml), arachidonic acid (100 microM), and U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F(2); 1 microM), a thromboxane (TX) A(2) mimic, with IC(50) values of 0.32 +/- 0.01, 0.44 +/- 0.02, 0.50 +/- 0.04, and 0.36 +/- 0.02 microM, respectively. J78 also produced a shift to the right of the concentration-response curve of U46619, indicating an antagonistic effect on the TXA(2) receptor. J78 concentration-dependently inhibited collagen-induced arachidonic acid liberation. In addition, J78 potently suppressed TXA(2) formation by platelets that were exposed to arachidonic acid in a concentration-dependent manner but had no effect on the production of PGD(2), indicating an inhibitory effect on TXA(2) synthase. This was supported by a TXA(2) synthase activity assay that J78 concentration-dependently inhibited TXB(2) formation converted from PGH(2). Furthermore, J78 was also able to inhibit the [Ca(2+)](i) mobilization induced by collagen or thrombin at such a concentration that completely inhibited platelet aggregation. Taken together, these results suggest that the antiplatelet activity of J78 may be mediated by TXA(2) receptor blockade with TXA(2) synthase inhibition and suppression of cytosolic Ca(2+) mobilization.