Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway

We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [(3)H]oleic acid and [(3)H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [(3)H]oleic acid uptake (V(0)) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (V(max) 854 +/- 46 pmol mg protein(-1) min(-1), K(m) 100 +/- 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [(3)H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [(3)H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with alpha-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells.     

Inhibitory effect of Cordyceps sinensis and Cordyceps militaris on human glomerular mesangial cell proliferation induced by native LDL

Native LDL, in low concentrations, promotes proliferation of cultured human glomerular mesangial cells. LDL stimulated [(3)H]-thymidine incorporation into DNA of human glomerular mesangial cells. Increased concentrations of LDL led to increased [(3)H]-thymidine incorporation. When LDL concentrations were 5, 10 and 50 microg ml(-1), [(3)H]-thymidine incorporation was 919.5+/-216, 1106+/-132, and 1200+/-210, respectively. When Cordyceps sinensis 100, 200, 300, 400 microg ml(-1) plus LDL 10 microg ml(-1) were added, [(3)H]-thymidine incorporation was 99+/-19 and 53+/-8, respectively, P<0.01 compared with controls. With Cordyceps militaris at similar concentrations plus LDL 10 microg ml(-1), [(3)H]-thymidine incorporation was respectively 192+/-75, 168+/-66, 145+/-53 and 72+/-16, P<0.01 compared with controls. The data suggest that LDL may play a critical role in mediating mesangial cell hypertrophy or proliferation involved in the development of glomerulosclerosis. Cordyceps sinensis and Cordyceps militaris inhibited, to a certain degree, proliferation of cultured human glomerular mesangial cell induced by LDL.     

Protection against diethylnitrosoamine-induced hepatocarcinogenesis by an indigenous medicine comprised of Nigella sativa, Hemidesmus indicus and Smilax glabra: a preliminary study

BACKGROUND: A decoction comprised of Nigella sativa seeds, Hemidesmus indicus root and Smilax glabra rhizome is used to treat cancer patients in Sri Lanka. However, the anti-carcinogenic properties of this decoction have not been experimentally confirmed. The purpose of this study was to determine whether the above decoction could protect against chemically induce hepatocarcinogenesis. METHODS: The effects of this decoction on diethylnitrosamine (DEN) induced hepatocarcinogenesis were examined in male Wistar rats using the medium term bioassay system of Ito, based on a 2-step model of hepatocarcinogenesis. Rats were randomly divided into 6 groups of 10 each. Groups 1 to 4 were injected with DEN (200 mg/kg) to initiate carcinogenesis. Twenty-four hours later groups 1 and 2 were administered the decoction at 4 g/kg body weight/day (dose 1) and 6 g/kg body weight/day (dose 2), respectively. Group 3 and group 4 were given distilled water instead of the decoction and a suspension of garlic powder (20 g/kg body weight/day) in distilled water (positive control), respectively. Group 5 and 6 were injected with normal saline and twenty-four hours later group 5 was given distilled water (normal control) while group 6 was given decoction dose 2 (decoction control). Oral feeding continued for two weeks after which all rats were subjected to 2/3 partial hepatectomy to promote carcinogenesis. Oral feeding continued for eight more weeks. At the end of the 10th week, rats were sacrificed and samples of livers taken for immunohistochemical studies.Carcinogenic potential was scored by comparing the number, area and staining intensity of glutathione S-transferase placental form (GST-P) positive foci and the number of cells/cm2 of the positive foci in the livers of the six groups of rats. RESULTS: The number and area of DEN-mediated GST-P positive foci, number of cells/cm2 of foci and staining intensity of the foci were significantly (P > 0.001) reduced by the decoction and garlic in the order dose 2 = garlic >dose 1. CONCLUSION: Overall results indicate that the decoction comprised of N. sativa, S. glabra and H. indicus has the potential to protect rat liver against DEN induced hepatocarcinogenesis     

Selective stimulation by tri-iodothyronine of myocardial deoxyribonucleic acid polymerase-α in neonatal rats

Three forms of DNA polymerase (alpha, beta and gamma) were separated from isolated rat myocardial cells on the basis of template, pH and ionic requirements, sensitivity to N-ethylmaleimide and position on sucrose gradients. Tri-iodothyronine administration (20mug/100g intraperitoneally) to 3-week-old rats resulted in selective stimulation of DNA polymerase-alpha (198+/-7.1 versus 102+/-5.8pmol of [(3)H]dTMP/30min per mg of protein in untreated controls, P<0.01), with no change in polymerases-beta and -gamma. [(3)H]Thymidine incorporation into myocardial DNA was also enhanced in tri-iodothyronine-treated neonatal rats (132+/-11.2 versus 53+/-4.1c.p.m./mug of DNA in controls, P<0.001). Increased incorporation was associated with an expansion of deoxyribonucleoside 5'-triphosphate pools, especially that of dTTP (24+/-1.6 versus 10+/-1.1pmol/mg of DNA, P<0.01). Neither DNA polymerase activities nor [(3)H]thymidine incorporation were changed in 6-month-old rats in response to tri-iodothyronine. Unstimulated adult myocardial cells had DNA polymerase activities comparable with those in 3-week-old animals, but significantly lower [(3)H]-thymidine incorporation and deoxyribonucleoside triphosphate concentrations. Enhancement of both DNA polymerase-alpha activity and [(3)H]thymidine incorporation in tri-iodothyronine-treated young rats was prevented by concomitant administration of either vinblastine (1mug/g) or daunomycin (2mug/g); actinomycin D (0.1mug/g) or cycloheximide (8mug/g), on the other hand, prevented the increase in [(3)H]thymidine incorporation, but not DNA polymerase-alpha activation. These results demonstrate an age-dependent stimulation of myocardial DNA replication by tri-iodothyronine and suggest an inter-relationship between DNA synthesis and subsequent entry into mitosis.     

In vitro keratinocyte antiproliferant effect of Centella asiatica extract and triterpenoid saponins.

Psoriasis is a hyperproliferative skin disorder estimated to be present in 1-3% of most populations. Conventional therapy using corticosteroids, Vitamin D analogs and cytotoxic agents eg psoralens is associated with low success rate and many side effects. Traditional plant remedies may provide leads for new treatments. A rapid-throughput, in vitro bioassay has been utilised to examine plants for inhibitory effects on the growth of SVK-14 keratinocytes. Centella asiatica, a reputed anti-psoriatic herb, has been compared against the psoralen-containing seeds of Psoralea corylifolia and the synthetic anti-psoriatic agent dithranol (anthralin). Aqueous extracts of Psoralea corylifolia and Centella asiatica inhibited keratinocyte replication with IC50 values of 18.4 +/- 0.6 microg/ml and 209.9 +/- 9.8 mg/ml respectively prior to treatment with polyvinylpolypyrrolidone (PVPP) and 36.3 +/- 3.3 mg/ml and 238.0 +/- 2.5 mg/ml respectively after PVPP treatment of the extracts. The effect produced by C. asiatica is thus unlikely to be due to phenolic compounds. It may, however, be due to its two constituent triterpenoid glycosides madecassoside and asiaticoside which had IC50 values of 8.6 +/- 0.6 microM respectively. These values were comparable to their concentrations in the crude extract and to the IC50 of dithranol (5.1 +/- 0.4 microM). These results suggest that the potential use of C. asiatica extracts as a topical anti-psoriatic agent is worthy of further investigation.     

Hepatocellular bioactivation and cytotoxicity of the synthetic endoperoxide antimalarial arteflene

Arteflene is a synthetic endoperoxide antimalarial. Its peroxide bridge undergoes iron(II)-mediated reduction in vitro which yields a carbon-centered cyclohexyl radical and a mixture of cis- and trans-alpha,beta-unsaturated ketones (enones). The enones are biliary metabolites in rats and therefore surrogate markers of bioactivation. Arteflene is reported to be more cytotoxic to primary rat hepatocytes than some non-endoperoxide antimalarials. Hepatic metabolism of arteflene was investigated in recirculating isolated perfused rat livers, and the drug's metabolism and cytotoxicity were compared using hepatocytes from male rats. Both preparations metabolized [(14)C]arteflene to cis- and trans-[(14)C]enone, 8-hydroxyarteflene glucuronide and an unassigned isomeric glucuronide. During a 2 h liver perfusion, the cis- and trans-enones recovered in bile represented 8.1 +/- 3.4 and 11.3 +/- 4.6% (mean +/- S.D., N=6), respectively, of the [(14)C]arteflene (52 microM) added to the perfusate. After a 3 h incubation of [(14)C]arteflene (10 microM) with hepatocytes in suspension, the cis- and trans-enones comprised, respectively, 14.8 +/- 7.1 and 2.1 +/- 1.0% (N = 4) of the recovered radioactivity; the corresponding data for cultured hepatocytes being 18.6 +/- 6.9 and 3.3 +/- 2.2%. Arteflene was significantly (P < 0.05) toxic to isolated hepatocytes with reference to extramitochondrial reductase activity (tetrazolium reduction) but not enzyme leakage when the cells were exposed to drug concentrations > or =50 microM for 24 h. Cellular glutathione was depleted under these conditions. Therefore arteflene was acutely cytotoxic, though only at relatively high concentrations, when it was metabolized via a pathway which generates carbon-centered radicals.     

Evaluation of cytotoxic compounds in different organs of the sea bream Sarpa salpa as related to phytoplankton consumption: an in vitro study in human liver cell lines HepG2 and WRL68

The present study was aimed to assess the cytotoxic effects of not-yet identified compounds present in organ extracts of Sarpa salpa, collected in autumn, the period with a peak in health problems. In addition, we studied the cytotoxicity of extracts of epiphytes found in the stomach content of S. salpa collected in summer and of epiphytes collected from the sea in the Sfax area at the end of spring. We tested these fractions in two human hepatic cell lines: HepG2 and WRL68. We observed a significant loss of viable cells when HepG2 cells were exposed for 72?h to acetone extracts of livers of S. salpa at a concentration of 2.5?mg/ml protein. Proteins extracted from brain or muscle did not significantly induce cell death at the studied concentrations (??10?mg/ml). There was a significant loss of viable cells when treated with liver extract of S. salpa dissolved in DMSO. Extracts of epiphytes collected in late spring showed a cytotoxic effect in a concentration-dependent manner. Moreover, we observed a significantly decreased cell viability of HepG2 at a dilution (1/40) of epiphyte extracts from stomach contents of two fish we had collected. The cytotoxic effect of the observed epiphyte extracts confirms the transfer of toxins originating from toxic dinoflagellates which live in epiphyte on the Posidonia oceanica leaves to fish organs by grazing. Hence, the liver of this fish can cause a threat to human health and consumption should for this reason be dissuaded.     

Production of glycyrrhizin in callus cultures of licorice

Licorice plants, Glycyrrhiza glabra, G. uralensis, and G. inflata, were investigated for callus induction using Murashige and Skoog (MS) medium combined with auxins and cytokinins. After 4 weeks of culture, 33-100% of leaf or stem explants formed calli. Maximum of shoot induction from callus cultures was achieved by G. inflata stem explants cultured on MS medium supplemented with 1 mg/l alpha-naphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzyladenine (BA) (67%) which also gave maximum shoot formation per explant (two shoots per explant). These results indicated that all three Glycyrrhiza species regenerated shoots from callus cultures on MS medium combined with NAA and BA or only thidiazuron (TDZ; 0.1 and 0.5 mg/l). Glycyrrhizin contents of G. uralensis calli induced using MS medium in combination with NAA and BA [(27.60 +/- 8.47) microg/g DW] or TDZ alone [(36.52 +/- 2.45) microg/ g DW] were higher than those found in other combinations.     

Mechanism of nicotinic acid transport in human liver cells: experiments with HepG2 cells and primary hepatocytes

This study reports on the functional expression of a specific, high-affinity carrier-mediated mechanism for the transport of niacin (nicotinic acid) in human liver cells. Both human-derived liver HepG2 cells and human primary hepatocytes were used as models in these investigations. The initial rate of transport of nicotinic acid into HepG2 cells was found to be acidic pH, temperature, and energy dependent; it was, however, Na(+) independent in nature. Evidence for the existence of a carrier-mediated system that is specific for [(3)H]nicotinic acid transport was found and included the following: 1) saturability as a function of concentration with an apparent K(m) of 0.73 +/- 0.16 microM and V(max) of 25.02 +/- 1.45 pmol.mg protein(-1).3 min(-1), 2) cis-inhibition by unlabeled nicotinic acid and nicotinamide but not by unrelated organic anions (lactate, acetate, butyrate, succinate, citrate, and valproate), and 3) trans-stimulation of [(3)H]nicotinic acid efflux by unlabeled nicotinic acid. Transport of the vitamin into human primary hepatocytes occurs similarly via an acidic pH-dependent and specific carrier-mediated process. Inhibitors of the Ca(2+)-calmodulin-mediated pathway (but not modulators of the PKC-, PKA-, and protein tyrosine kinase-mediated pathways) inhibited nicotinic acid transport into both HepG2 cells and human primary hepatocytes. Maintenance of HepG2 cells (for 48 h) in growth medium oversupplemented with nicotinic acid (or nicotinamide) did not affect the subsequent transport of [(3)H]nicotinic acid into HepG2 cells. These results show, for the first time, the existence of a specific and regulated membrane carrier-mediated system for nicotinic acid transport in human liver cells.     

Mechanism for antiplatelet effect of onion: AA release inhibition, thromboxane A(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade

Antiplatelet actions of aqueous extract of onion were investigated in rat and human platelet. IC(50)values of onion extract for collagen-, thrombin-, arachidonic acid (AA)-induced aggregations and collagen-induced thromboxane A(2)(TXA(2)) formation were 0.17 +/- 0. 01, 0.23 + 0.03, 0.34 +/- 0.02 and 0.12 +/- 0.01 g/ml, respectively. [(3)H]-AA release induced by collagen (10 microg/ml) in rat platelet was decreased by onion compared to control (22.1 +/- 2.13 and 5.2 +/- 0.82% of total [(3)H]-AA incorporated, respectively). In fura-2 loaded platelets, the elevation of intracellular Ca(2+)concentration stimulated by collagen was inhibited by onion. Onion had no cytotoxic effect in platelet. Onion significantly inhibited TXA(2)synthase activity without influence on COX activity. Platelet aggregation induced by U46619, a stable TXA(2)mimetic, was inhibited by onion, indicating its antagonism for TXA(2)/PGH(2)receptor. These results suggest that the mechanism for antiplatelet effect of onion may, at least partly, involve AA release diminution, TXA(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

In vivo and in vitro evidence for ACh-stimulated L-arginine uptake

Whereas L-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y(+) transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), the effects of ACh (9.25 and 37 microg/min), Iso (25-50 and 200 microg/min), and sodium nitroprusside (SNP) (1-2 and 8 microg/min) on forearm plasma flow (FPF), L-[(3)H]arginine uptake, and L-[(3)H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on L-[(3)H]arginine uptake were studied in BAEC. L-Arginine uptake was inversely related to FPF (r = -0.50; P < 0.005). At a similar FPF (ACh 56.82 +/- 9.25, Iso 58.49 +/- 5.56, SNP 57.92 +/- 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of L-[(3)H]arginine (54,655 +/- 8,018 dpm/min), compared with that observed with either Iso (40,517.23 +/- 6,841 dpm/min; P = 0.01) or SNP (36,816 +/- 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced L-[(3)H]citrulline release compared with Iso and SNP (P = 0.046). Similarly, in BAEC, ACh significantly increased L-[(3)H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 x 10(7) +/- 1.83 x 10(7) vs. control 6.67 x 10(7) +/- 1.16 x 10(7) vs. Iso 7.35 x 10(7) +/- 1.63 x 10(7) vs. SNP 6.01 x 10(7) +/- 1.11 x 10(7) fmol.min(-1).mg(-1) at 300 micromol/l L-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.     

Interaction of ACTH synthetic fragments with rat adrenal cortex membranes.

Synthetic peptide, corresponding to the amino acid sequence 11-24 of human adrenocorticotropic hormone (ACTH), was labeled with tritium (specific activity of 22 Ci/mmol). [(3)H]ACTH (11-24) was found to bind to rat adrenal cortex membranes with high affinity and specificity (K(d) = 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) have been synthesized and their ability to inhibit the specific binding of [(3)H]ACTH (11-24) to adrenocortical membranes has been investigated. Unlabeled fragment ACTH 15-18 (KKRR) was found to replace in a concentration-dependent manner [(3)H]ACTH (11-24) in the receptor-ligand complex (K(i) = 2.3 +/- 0.2 nM). ACTH (15-18) was labeled with tritium (specific activity of 20 Ci/mmol). [(3)H]ACTH (15-18) was found to bind to rat adrenal cortex membranes with high affinity (K(d) = 2.1 +/- 0.1 nM). The specific binding of [(3)H]ACTH (15-18) was inhibited by unlabeled ACTH (11-24) (K(i) = 2.2 +/- 0.1 nM). ACTH (15-18) at the concentration range of 1-1000 nM did not affect the adenylate cyclase activity in adrenocortical membranes.     

A permanent fish cell line (EPC) for genotoxicity testing of marine sediments with the comet assay.

Genotoxicity and cytotoxicity were evaluated in an in vitro system with a permanent cell line Epithelioma papulosum cyprini (EPC) derived from a skin tumour of carp (Cyprinus carpio L.). EPC cells were exposed to different concentrations of organic sediment extracts from the North Sea for 24h. After incubation the cells were analysed for viability and DNA strand breaks with the comet assay or single cell gel electrophoresis (SCGE). The results confirm the sensitivity of this assay. Out of 10 marine sediment samples from the North Sea, 9 showed a dose-dependent genotoxic effect. The EC(50) of sediment extracts ranged from 7 to 307 mg sediment dry weight/ml assay volume. Hepatic microsomal enzymes from dab (Limanda limanda L.) was proposed for enzymatic activation of benzo[a]pyrene (BAP) or sediment extracts, respectively. The suitability of this in vitro test system for assessing genotoxic and cytotoxic effects of marine sediment extracts on EPC cells could be demonstrated.     

Phytoecdysteroids of Silene guntensis and their in vitro cytotoxic and antioxidant activity

Phytoecdysteroids from aerial parts of Silene guntensis B. Fedtsch were investigated and three phytoecdysteroids were isolated: 2,3-diacetate-22-benzoate-20-hydroxyecdysone (1), 2-deoxy-20-hydroxyecdysone (2), and 20-hydroxyecdysone (3). Their chemical structures were elucidated by DEPT, COSY, 1H and 13C NMR spectroscopy. The isolated compounds 1-3 and crude extracts were evaluated for their antiproliferative and antioxidant activities. They exhibited substantial inhibition of cell growth against human cervix adenocarcinoma (HeLa), hepatocellular carcinoma (HepG-2), and breast adenocarcinoma (MCF-7) cells. The chloroform extract showed potent cytotoxic effects [IC50 values (26.58 +/- 1.88) microg/mL, (20.99 +/- 1.64) microg/mL, and (18.89 +/- 2.36) microg/mL, respectively]. The new compound 1 was mildly cytotoxic compared to extracts [(127.97 +/- 11.34), (106.76 +/- 7.81), and (203.10 +/- 19.56) microg/mL, respectively]. Water and n-butanol extracts exhibited good antioxidant activities [IC50 values of (68.90 +/- 6.45) microg/mL and (69.12 +/- 5.85) microg/mL, respectively].     

Development of a microtitre-based spectrophotometric method to monitor Babesia divergens growth in vitro

Babesia divergens multiplication cycle involves erythrocyte invasion, intracellular division, and erythrocyte lysis with the simultaneous liberation of hemoglobin. We have decided to set up a spectrophotometric protocol based on hemoglobin concentration in the culture supernatants to monitor B. divergens in vitro growth. After the selection of 405 nm as the most appropriate endpoint hemoglobin wavelength in our conditions (hemoglobin concentration in the supernatant), cultures were standardized [1 x 10(9) red blood cell (RBC)/ml, 1-2.5 x 10(5) infected red blood cell (iRBC)/ml] to allow their monitoring over 3 days. The protocol was then compared to the most commonly used growth measurement methods: parasitemia counting and [(3)H]hypoxanthine incorporation. An excellent correlation was demonstrated between A(405) of the culture supernatant and parasitemia of the iRBC, whatever the RBC concentration used in the medium. This correlation was also evidenced between A(405) and [(3)H]hypoxanthine incorporation for [(3)H]hypoxanthine concentrations lower than 4 microCi/ml. Our assays also highlighted the inhibitory effect of [(3)H]hypoxanthine on B. divergens growth even when used at low concentrations (0.8 microCi/ml) and for a short incorporation duration (24 h). This effect was confirmed by both A(405) and parasitemia counting. In conclusion, A(405) measurement of B. divergens culture supernatant represents a simple, rapid, safe, and reliable way to measure the in vitro growth of this parasite. Generation times of three different B. divergens strains were then determined by the protocol described here and varied between 8 h 36 min and 13 h 8 min.     

A novel cytotoxic aryltetraline lactone from Bupleurum marginatum (Apiaceae)

A detailed phytochemical study of the aerial parts of Bupleurum marginatum Wall. ex DC revealed a novel aryltetraline lactone lignan identified as 9-(benzo[d][1,3]dioxol-5-yl)-6,7,8-trimethoxy-3a,4,9,9a-tetrahydronaphtho[2,3-c]furan-1(3H)-one (marginatoxin) along with nine known compounds and characterization of five other compounds either by GLC/MS or LC/MS techniques. Chemical structures of the isolated compounds were unambiguously elucidated by both 1D, 2D NMR and mass spectrometry techniques.The in vitro cytotoxic activity of both methanol and dichloromethane extracts as well as the isolated compounds was assessed in two human cancer cell lines HepG2 and HeLa using the MTT assay. The new aryltetraline lactone lignan exhibited a potent cytotoxic activity with IC₅₀ values of 12.14 and 16.90 μM after 24 h treatment for HepG2 and HeLa cells, respectively.     

Biological half-life and organ distribution of [3H]8-arginine vasopressin following administration of vasopressin receptor antagonist OPC-31260

The effects of the antidiuretic (V(2)) non-peptide receptor antagonist OPC-31260 on the plasma vasopressin level and the biological half-life and organ distribution of radiochemically pure, biologically active [(3)H]8-arginine vasopressin [spec. act.: 15.9 mCi/mmol (588 GBq/mmol)] were studied in Wistar rats. The plasma vasopressin level increased significantly throughout the whole experimental period (24 h). There was no change in the fast phase of the curves of total radioactivity disappearance from the plasma after the administration of [(3)H]arginine vasopressin (control: 1.51+/-0.17 min, OPC-31260-treated: 1.42+/-0.12 min, n=10). The fast phase of the disappearance curves of intact [(3)H]arginine vasopressin did not change either following the administration of OPC-31260 in a dose of 30 mg/kg p.o. (control: 1.06+/-0.19 min, OPC-31260-treated: 1.00+/-0.15 min, n=6). The slow phase of the biological half-life, which is characteristic for the examined compound, proved to be significantly longer (total radioactivity control: 9.29+/-0.61 min, OPC-31260-treated: 12.33+/-0.42 min, P<0.05, n=10; [(3)H]arginine vasopressin radioactivity: control: 5.96+/-0.58 min, OPC-31260-treated: 8.90+/-0.37 min, P<0.05, n=6). In the control rats, the radioactivity was accumulated to the greatest extent in the neurohypophysis, adenohypophysis and kidney. Following OPC-31260 administration, significantly more radioactive compounds accumulated in the kidney (control: 0.30+/-0.052 total radioactivity %/100 mg organ weight, OPC-31260-treated: 0.50+/-0.133 total radioactivity %/100 mg organ weight, P<0.05, n=10) and neurohypophysis (control: 0.37+/-0.053 total radioactivity %/100 mg organ weight, OPC-31260-treated: 0.52+/-0.076 total radioactivity %/100 mg organ weight, P<0.05, n=10). Our results permit the conclusion that the antidiuretic antagonist OPC-31260 not only blocks the V(2) receptors, but also increases the biological half-life of vasopressin. The longer biological half-life of vasopressin following OPC-31260 administration may play a role in the elevation of the plasma vasopressin level.     

Alterations in 5-HT(1A) receptors and adenylyl cyclase response by trazodone in regions of rat brain

The in vivo effect of trazodone on the density of [(3)H]5-HT binding sites and 5-HT(1A) receptors and adenylyl cyclase (AC) response was studied in regions of rat brain. The chronic administration of trazodone (10 mg/Kg body wt, 40 days) resulted in a significant downregulation of [(3)H]5-HT binding sites and 5-HT(1A) receptors in cortex and hippocampus. Trazodone significantly (p < 0.0001) decreased the density of [(3)H]5-HT binding sites in cortex (42.6 +/- 3.6 fmol/mg protein, 65%) and hippocampus (12.6 +/- 1.6 fmol/mg protein, 87%) when compared to control values of 121.9 +/- 5.4 and 99.3 +/- 7.5 fmol/mg protein in these regions, respectively. Similarly there was a significant (p < 0.0001) decrease in the density of 5-HT(1A) receptors in both cortex (7.2 +/- 0.5 fmol/mg protein, 70%) and hippocampus (6.3 +/- 1.2 fmol/mg protein, 79%) when compared to control values of 24.2 +/- 2.1 and 30.6 +/- 3.7 fmol/mg protein, in these regions respectively. However, the affinity of [(3)H]5-HT to 5-HT binding sites (1.83 +/- 0.26 nM, p < 0.0001) and [(3)H]8-OH-DPAT to 5-HT(1A) receptors (0.60 +/- 0.06 nM, p < 0.05) was significantly decreased only in cortex when compared to the control K(d) values of 0.88 +/- 0.04 nM and 0.47 +/- 0.02 nM in these regions, respectively.The basal AC activity did not alter in treated rats, where as, the inhibition of forskolin-stimulated AC activity by 5-HT (10 microM) was significantly (p < 0.0001) decreased both in cortex (43%) and hippocampus (40%) when compared to control levels. In conclusion, chronic treatment with trazodone results in downregulation of 5-HT(1A) receptors in cortex and hippocampus along with concomitant increased AC response, suggesting the involvement of 5-HT(1A) receptor-mediated AC response in the mechanism of action of trazodone.     

Decreased [3H]nitrendipine binding in the brainstem of deoxycorticosterone-NaCl hypertensive rats

Binding studies with the 1,4-dihydropyridine calcium channel antagonist [3H]nitrendipine [( 3H]NTD) were performed in uninephrectomized, deoxycorticosterone (DOCA)-NaCl hypertensive rats and vehicle treated normotensive control littermates. After 6 weeks of treatment, hypertensive (199 mmHg, systolic arterial pressure) DOCA rats showed significantly increased heart, left ventricle, and kidney weight in contrast to normotensive (135 mmHg) controls. [3H]NTD binding in the brainstem was significantly reduced (51 +/- 5 fmol/mg protein) in DOCA-NaCl rats, as compared to controls (116 +/- 24 fmol/mg protein). However, no significant differences were found in the [3H]NTD dissociation constants for DOCA-NaCl (0.43 +/- 0.03 nM) or control rats (0.62 +/- 0.06 nM). Cerebral cortical and left ventricular tissue showed no significant alterations in receptor binding density or affinity. Specific [3H]NTD binding was not significantly altered in other selected brain regions or the atria. These data suggest that alterations in the dihydropyridine binding sites associated with calcium channels in the brainstem may be involved in the etiology of DOCA-NaCl-induced hypertension.