Development of a UPLC-MS/MS method for the determination of narciclasine and 7-deoxynarciclasine in mouse blood and its application in pharmacokinetics.

In this study, we used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to measure the concentration of narciclasine and 7-deoxynarciclasine in mouse blood after intravenous (i.v.) and oral administration (p.o.), and we used this method to investigate their pharmacokinetics profiles in mice. Chromatographic separation of the analytes was achieved using a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm) with a mobile phase consisting of acetonitrile-water (0.1% formic acid) by gradient elution. Electrospray ionization (ESI positive-ion mode)-tandem mass spectrometry in multiple reaction monitoring (MRM) mode was employed for quantitative analysis of the analytes in mouse blood samples. Twelve mice were administered narciclasine and 7-deoxynarciclasine (2 mg/kg) intravenously (iv), while the other twelve mice were administered narciclasine and 7-deoxynarciclasine (10 mg/kg) orally. The mouse blood was withdrawn from the caudal vein to be processed, after which the blood was analyzed by UPLC-MS/MS, and the corresponding data were fitted using the Drug and Statistics (DAS) software. Standard curves of narciclasine and 7-deoxynarciclasine were generated over the concentration range of 5-5000 ng/mL. The intra-day accuracy of narciclasine and 7-deoxynarciclasine was 90-105%, and the corresponding inter-day accuracy was 87-108%. The intra-day precision was less than 13%, while the inter-day precision was less than 14%. Matrix effects were also observed (between 94% and 104%), and the recovery calculated was higher than 70%. The developed and validated UPLC-MS/MS method was then successfully applied in determining the mouse pharmacokinetics of narciclasine and 7-deoxynarciclasine. From this, thebioavailabilityofnarciclasine and 7-deoxynarciclasinewasdetermined to be 10.3%and35.4%, respectively.

18F-Labeled dihydromethidine: positron emission tomography radiotracer for imaging of reactive oxygen species in intact brain.

Dihydromethidine (DHM) labeled with 18F at the para position of the peripheral benzene ring was designed as a positron emission tomography (PET) radiotracer for non-invasive imaging of reactive oxygen species (ROS). This compound readily crosses the blood-brain barrier and is oxidized by ROS, and the oxidation product is retained intracellularly. PET imaging of ROS-producing rat brain microinfused with sodium nitroprusside identified specific brain regions with high ROS concentrations. This tracer should be useful for studies of the pathophysiological roles of ROS, and in the diagnosis of neurodegenerative diseases.

Lycorine: A prospective natural lead for anticancer drug discovery.

Nature is the most abundant source for novel drug discovery. Lycorine is a natural alkaloid with immense therapeutic potential. Lycorine is active in a very low concentration and with high specificity against a number of cancers both in vivo and in vitro and against various drug-resistant cancer cells. This review summarized the therapeutic effect and the anticancer mechanisms of lycorine. At the same time, we have discussed the pharmacology and comparative structure-activity relationship for the anticancer activity of this compound. The researches outlined in this paper serve as a foundation to explain lycorine as an important lead compound for new generation anticancer drug design and provide the principle for the development of biological strategies to utilize lycorine in the treatment of cancers.

Lipid-modified cell-penetrating peptide-based self-assembly micelles for co-delivery of narciclasine and siULK1 in hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer, and one therapeutic approach is to target both the AMPK and autophagy pathways in order to synergistically promote programmed cell death. Here, a series of amphiphilic, lipid-modified cell-penetrating peptides were synthesized and allowed to self-assemble into micelles loaded with the AMPK activator narciclasine (Narc) and short interfering RNA targeting the unc-51-like kinase 1 (siULK1). The size of these micelles, their efficiency of transfection into cells, and their ability to release drug or siRNA cargo in vitro were pH-sensitive, such that drug release was facilitated in the acidic microenvironment of the tumor. Transfecting the micelles into HCC cells significantly inhibited protective autophagy within tumor cells, and delivering the micelles into mice carrying HCC xenografts induced apoptosis, slowed tumor growth, and inhibited autophagy. Our results indicate that co-delivering Narc and siULK1 in biocompatible micelles can safely inhibit tumor growth and protective autophagy, justifying further studies into this promising therapeutic approach against HCC. STATEMENT OF SIGNIFICANCE: We have focused on the targeted therapy of HCC via synergistically inhibiting the autophagy and inducing apoptosis. The lipid-modified cell-penetrating peptide can not only aggregate into micelles to load natural product narciclasine and ULK1 siRNA simultaneously, but also facilitate uptake and endosome escape with a pH-sensitive manner in HepG2 cells. HepG2 cell treated with siULK1-M-Narc has increased apoptotic levels and declined autophagy via the targeted regulation of AMPK-ULK1 signaling axis. The in vivo studies have confirmed that siULK1-M-Narc efficiently reduce the growth of tumor on HCC xenograft models with good safety. Thus, we suppose the lipid-modified cell-penetrating peptide has good application prospects in the targeted combinational therapy of HCC.

Evaluation of a novel radiotracer for positron emission tomography imaging of reactive oxygen species in the central nervous system.

INTRODUCTION: Few, if any, radiotracers are available for the in vivo imaging of reactive oxygen species (ROS) in the central nervous system. ROS play a critical role in normal cell processes such as signaling and homeostasis but overproduction of ROS is implicated in several disorders. We describe here the radiosynthesis and initial ex vivo and in vivo evaluation of [11C]hydromethidine ([11C]HM) as a radiotracer to image ROS using positron emission tomography (PET). METHODS: [11C]HM and its deuterated isotopologue [11C](4) were produced using [11C]methyl triflate in a one-pot, two-step reaction and purified by high performance liquid chromatography. Ex vivo biodistribution studies were performed after tail vein injections of both radiotracers. To demonstrate sensitivity of uptake to ROS, [11C]HM was administered to rats treated systemically with lipopolysaccharide (LPS). In addition, ex vivo autoradiography and in vivo PET imaging were performed using [11C]HM on rats which had been microinjected with sodium nitroprusside (SNP) to induce ROS. RESULTS: [11C]HM and [11C](4) radiosyntheses were reliable and produced the radiotracers at high specific activities and radiochemical purities. Both radiotracers demonstrated good brain uptake and fast washout of radioactivity, but [11C](4) washout was faster. Pretreatment with LPS resulted in a significant increase in brain retention of radioactivity. Ex vivo autoradiography and PET imaging of rats unilaterally treated with microinjections of SNP demonstrated increased retention of radioactivity in the treated side of the brain. CONCLUSIONS: [11C]HM has the attributes of a radiotracer for PET imaging of ROS in the brain including good brain penetration and increased retention of radioactivity in animal models of oxidative stress.

LC-MS/MS method for the determination of haemanthamine in rat plasma, bile and urine and its application to a pilot pharmacokinetic study.

Evidence gathered in various studies points to the fact that haemanthamine, an isoquinoline alkaloid, has multiple medicinally interesting characteristics, including antitumor, antileukemic, antioxidant, antiviral, anticonvulsant and antimalarial activity. This work presents, for the first time, a universal LC-MS/MS method for analysis of haemanthamine in plasma, bile and urine which has been verified in a pilot pharmacokinetic experiment on rats. Chromatographic separation was performed on a pentafluorophenyl core-shell column in gradient elution mode with a mobile phase consisting of acetonitrile-methanol-ammonium formate buffer. A sample preparation based on liquid-liquid extraction with methyl tert-butyl ether was employed with ambelline used as an internal standard. Quantification was performed using LC-MS-ESI(+) in Selected Reaction Monitoring mode. The method was validated according to the European Medicines Agency guideline in a concentration range of 0.1-10 µmol/L in plasma, bile and urine. The concentration-time profiles of haemanthamine in plasma, bile and urine after a single i.v. bolus of 10 mg/kg have been described for the first time. The presented study addresses the lack of information on haemanthamine pharmacokinetics and also introduces a new universal method of haemanthamine analysis in complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd.

Rapid and sensitive HPLC-MS/MS method for quantitative determination of lycorine from the plasma of rats.

A simple, rapid and sensitive high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method was developed and validated for determining lycorine in rat plasma. Plasma samples were prepared by a simple protein precipitation with methanol containing dextrorphan as internal standard. The chromatographic separation was performed on a Kromasil 60-5CN column (3 µm, 2.1 mm × 150 mm) with the mobile phase of methanol/water (containing 0.1% formic acid) (40:60, v/v) at a flow rate of 0.2 mL/min, the total analytical runtime was 5 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with Electronic Spray Ion by selected reaction monitoring (SRM) of the transitions at m/z 288.1→147.1 for lycorine and m/z 258.1→157.2 for dextrorphan, respectively. The calibration curve was linear over the range of 1-1000 ng/mL with the lower limit of quantification of 1 ng/mL for lycorine. The intra- and inter-day precision (R.S.D.%) were less than 8.5% and accuracy (R.E.%) was within ±7.0%. Lycorine was sufficiently stable under all relevant analytical conditions. This method was successfully applied to the pharmacokinetic study of lycorine in rats after intraperitoneal administration with different doses of 5, 10 and 20 mg/kg.

An LC-MS/MS method for the simultaneous determination of lycorine and galanthamine in rat plasma and its application to pharmacokinetic study of Lycoris radiata extract in rats.

A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of lycorine and galanthamine, two major constituents in Lycoris radiata extract, in rat plasma. Liquid-liquid extraction with ethyl ether was carried out using diphenhydramine as the internal standard. The two bioactive alkaloids were separated on a Zorbax SB-C18 reserved-phase column (150 mm × 4.6 mm, i.d., 5 µm) by gradient elution using a mobile phase consisting of methanol with 0.1% formic acid (A) and water with 0.1% formic acid (B) at a flow rate of 0.6 mL/min. All analytes showed good linearity over a wide concentration range (r (2)>0.99) and the lower limit of quantification was 3.00 ng/mL for each analyte. The average extraction recovery of the analytes from rat plasma was more than 82.15%, and the intra-day and inter-day accuracy and precision of the assay were less than 12.6%. The validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of two Amaryllidaceous alkaloids in rat plasma after an oral administration of Lycoris radiata extract.

Study on pharmacokinetic and tissue distribution of lycorine in mice plasma and tissues by liquid chromatography-mass spectrometry.

A fast and simple liquid chromatography-mass spectrometry method for the determination of lycorine in mice plasma and tissues was developed and well used in the pharmacokinetic and tissue distribution study of lycorine after tail vein injection and intraperitoneal administration. Biological samples were processed with ethyl acetate by liquid-liquid extraction, and evodiamine was used as the internal standard. Chromatographic separation was performed on an Amethyst C18 column (4.6 × 150 mm) with a mobile phase consisting of methanol and water. Quantification was performed by selected ion monitoring with m/z 288 [M+H](+) for lycorine and m/z 304 [M+H](+) for the internal standard. Good linearity was observed over the concentration ranges. Limits of quantification were low up to 10.0 ng/mL in plasma samples, 9.0 ng/g for lung, 12.0 ng/g for heart, 18.0 ng/g for spleen and 6.5 ng/g for other tested tissues. The intraday accuracy and precision in plasma and tissues ranged from -7.4% to 9.1%. Recoveries in plasma and tissue were more than 80%. The method was rapid, accurate and fully validated. It was successfully applied to the investigation of the pharmacokinetics and tissue distribution of lycorine in mice.

Phenanthriplatin, a monofunctional DNA-binding platinum anticancer drug candidate with unusual potency and cellular activity profile.

Monofunctional platinum(II) complexes of general formula cis-[Pt(NH(3))(2)(N-heterocycle)Cl]Cl bind DNA at a single site, inducing little distortion in the double helix. Despite this behavior, these compounds display significant antitumor properties, with a different spectrum of activity than that of classic bifunctional cross-linking agents like cisplatin. To discover the most potent monofunctional platinum(II) compounds, the N-heterocycle was systematically varied to generate a small library of new compounds, with guidance from the X-ray structure of RNA polymerase II (Pol II) stalled at a monofunctional pyriplatin-DNA adduct. In pyriplatin, the N-heterocycle is pyridine. The most effective complex evaluated was phenanthriplatin, cis-[Pt(NH(3))(2)(phenanthridine)Cl]NO(3), which exhibits significantly greater activity than the Food and Drug Administration-approved drugs cisplatin and oxaliplatin. Studies of phenanthriplatin in the National Cancer Institute 60-cell tumor panel screen revealed a spectrum of activity distinct from that of these clinically validated anticancer agents. The cellular uptake of phenanthriplatin is substantially greater than that of cisplatin and pyriplatin because of the hydrophobicity of the phenanthridine ligand. Phenanthriplatin binds more effectively to 5'-deoxyguanosine monophosphate than to N-acetyl methionine, whereas pyriplatin reacts equally well with both reagents. This chemistry supports DNA as a viable cellular target for phenanthriplatin and suggests that it may avoid cytoplasmic platinum scavengers with sulfur-donor ligands that convey drug resistance. With the use of globally platinated Gaussia luciferase vectors, we determined that phenanthriplatin inhibits transcription in live mammalian cells as effectively as cisplatin, despite its inability to form DNA cross-links.

Comparison of the D1 dopamine full agonists, dihydrexidine and doxanthrine, in the 6-OHDA rat model of Parkinson's disease.

RATIONALE: Preclinical evidence indicates that D1 dopamine receptor full agonists have potential as therapeutic agents for a variety of neurological conditions. Dihydrexidine (DHX) was the first high potency selective D1 dopamine receptor full agonist and has been studied as a possible treatment for Parkinson's disease (PD). Recently, we discovered doxanthrine (DOX), an oxygen bioisostere of DHX that has even greater selectivity for the D1 dopamine receptor. OBJECTIVES: Using the unilateral 6-hydroxydopamine-lesioned rat model of PD, DOX and DHX were compared at several doses (0.625, 1.25, 2.5, or 5.0 mg/kg) for their ability to elicit contralateral rotation by either intraperitoneal injection or oral gavage. RESULTS: After intraperitoneal administration, both DOX and DHX showed robust contralateral rotation at doses of 2.5 and 5.0 mg/kg compared to vehicle. In addition, after intraperitoneal administration at doses of 2.5 and 5.0 mg/kg, DHX had a significantly longer duration of action than DOX (p < 0.05). Areas under the curves (AUC) for DOX and DHX were not significantly different, however, indicating that DOX and DHX have similar potency after intraperitoneal administration. By contrast, after oral administration, 2.5 and 5.0 mg/kg of DOX produced significant contralateral rotations (p < 0.05), whereas DHX showed no significant activity after oral administration of any dose. CONCLUSION: These results demonstrate that although DHX and DOX have similar activity after intraperitoneal administration, DOX demonstrated greater activity after oral administration compared to DHX. Despite its catechol functionality, DOX may possess sufficient oral availability for development as a human therapeutic agent.

Dose-dependent emetic effects of the Amaryllidaceous alkaloid lycorine in beagle dogs.

Ingestions of plant material from Amaryllidaceae, especially the bulbs of daffodils, are known to be toxic, representing a persistent cause of poisoning in human and animals. Empiric data from case reports suggested, that the alkaloid lycorine could be the toxic constituent of the multi-component mixture responsible for symptoms like nausea and emesis. Systematic studies of the in vivo effects of the amaryllidaceaeous-type alkaloids are not available. Therefore, in an open, prospective, randomized and controlled trial we studied the dose-effect relationship of lycorine-induced nausea and emesis and the toxicokinetics of lycorine in beagle dogs. Subcutaneously administered lycorine-induced nausea and emesis starting at 0.5 mg/kg body weight reaching statistical significance at 1.0 mg/kg. The maximum emetic dose of lycorine (ED(100)) was 2 mg/kg body weight. There was a correlation between dose and nausea score as well as between dose and number of the induced emetic events. Nausea and emesis were short-lasting and occurred not later than 2.5 h post dose. Lycorine showed linear plasma kinetics with a mean elimination half-life of 0.67 and 0.3 h after single s.c. and i.v. administration, compatible with the clinical course of nausea and emesis. The mean oral bioavailability was calculated to be about 40%. Biochemical and haematological parameters of safety showed no pathological signs. The results provide evidence that lycorine can be considered as a main, if not the crucial constituent responsible for nausea and emesis in human and animals in poisoning due to ingestion of plant material of the Amaryllidaceae.

Characterization of in vivo pharmacokinetic properties of the dopamine D1 receptor agonist DAR-0100A in nonhuman primates using PET with [11C] NNC112 and [11C] raclopride.

DAR-0100A, the active enantiomer of dihydrexidine, is a potent dopamine D1 agonist under investigation for treatment of cognitive impairment and negative symptoms of schizophrenia. We measured the dose-occupancy relationship for DAR-0100A at D1 receptors using positron emission tomography (PET) imaging in baboons with [(11)C] NNC112 and its binding to D2 with [(11)C] raclopride. Two baboons were scanned with [(11)C] NNC112 at baseline and after three different doses of DAR-0100A. Two baboons were scanned with [(11)C] raclopride at baseline and after one dose of DAR-0100A. Occupancy (ΔBP(ND)) was computed in the striatum and cortex. A clear relationship was observed between plasma concentration of DAR-0100A and ΔBP(ND). ΔBP(ND) was larger in the striatum than in the cortex, consistent with reports showing that 25% of [(11)C] NNC112 BP(ND) in the cortex is attributed to 5-HT(2A). Plasma EC(50) estimates ranged from 150 to 550 ng/mL according to the constraints on the model. There was no detectable effect of DAR-0100A on [(11)C] raclopride BP(ND). These data suggest that at doses likely to be administered to patients, occupancy will not be detectable with [(11)C] NNC112 PET and binding of DAR-0100A to D2 will be negligible. This is the first demonstration with PET of a significant occupancy by a full D1 agonist in vivo.

Study of aldehyde oxidase-catalyzed metabolic pathway of phenanthridine using MCR-ALS method.

Evaluation of metabolic pathways is one of the challenging areas in biological and pharmaceutical sciences. Phenanthridine oxidation to phenanthridinone is used commonly to study aldehyde oxidase activity. This reaction could pass through phenanthridine N-oxide intermediate. In the present study, the application of multivariate curve resolution, optimized by alternating least squares (MCR-ALS) to investigate this metabolic pathway has been described. The results obtained from MCR-ALS analysis along with those obtained from the use of potassium ferrocyanide method indicated that phenanthridine is directly oxidized to phenanthridinone by rat liver aldehyde oxidase without passing through phenanthridine N-oxide intermediate. It was also found that the later compound is not metabolized by this enzyme.

Two color RNA intercalating probe for cell imaging applications.

Here we report on a phenanthridine derivative which has a covalently linked fluorescein molecule in order to increase the light absorption and hence fluorescence signal intensity when bound to duplex RNA. Steady-state fluorescence shows that the energy transfer efficiency from the fluorescein to the phenanthridine fluorophore is approximately 77%, which results in the probe being over 5x brighter than other phenanthridine derivatives when bound to RNA. Due to the relatively long lifetime (approximately 20 ns) of the probe, time-resolved fluorescence is used to increase the signal to background ratio in cell growth medium from 7 (steady-state value) to over 40. Moreover, fluorescence images of cells containing the probe show that the fluorescein signal is readily apparent along with that of the intercalated fluorophore, allowing this probe to be used as a dual color probe which simultaneously reports the probes' location and that of RNA.

Metabolism of isometamidium in hepatocytes isolated from control and inducer-treated rats.

Little is known about the metabolism and mechanism of action of the trypanocide, isometamidium (ISM), the major drug used for prophylaxis of trypanosomiasis. We have investigated its metabolism and distribution in isolated rat hepatocytes using liquid chromatography-mass spectrometry and confocal laser scanning microscopy (CLSM). Two putative metabolites were formed, which were proposed to be a mono-acetyl derivative and an oxidized metabolite (SII). This is the first demonstration of the hepatic metabolism of ISM, as previous in vivo studies were hampered by dose-limiting toxicity and insensitive analytical methods. The intrinsic fluorescence of the drug enabled its intracellular uptake to be followed by CLSM. It is taken up rapidly into the nucleolus, nuclear membrane and endoplasmic reticulum within 5 min, and retained in the nucleus for at least 24 h. Persistent binding of ISM to cellular macromolecules may contribute to its prophylactic effect in vivo. Pretreatment of rats with 3-methylcholanthrene, phenobarbitone (PB) or the widely used pyrethroid pesticide, deltamethrin, resulted in an increase in metabolism of ISM to the proposed SII after 1 h incubation with hepatocytes. 3-methylcholanthrene was the most potent inducer, causing a maximal 19.5-fold induction of SII formation after exposure of hepatocytes to ISM for 1 h compared with formation by control hepatocytes. In comparison, at the 1 h timepoint deltamethrin pre-treatment caused a 10.2-fold induction, and PB only 8.2 fold.

Evaluation of isometamidium levels in the serum of sheep and goats after prophylactic treatment against trypanosomosis.

Isometamidium chloride has been used for the control of trypanosomosis in animals for over 36 years, but recently there have been reports of prophylaxis failure under natural conditions. In this study, use of the drug for prophylactic purpose against trypanosomosis in small ruminants was investigated. Forty-two sheep and 44 goats were divided into four treatment groups. Groups 1 and 2 were treated with isometamidium chloride (Samorin, Rhone Merieux, Lyon, France) at 3-month intervals while groups 3 and 4 were used as controls. All the animals were exposed to natural tsetse challenge and monitored for serum isometamidium levels and anti-trypanosome antibodies. Seven days after drug administration, isometamidium levels were significantly higher in goats 13.7+/-0.07 ng/ml than in sheep 6.2+/-0.06 ng/ml. However, the elimination half-life in the sheep was 14.2+/-0.92 days and was significantly higher (P> 0.05) than that of the goats 12+/-0.5 days. This study established that isometamidium metabolism differs between sheep and goats and this difference may have important implications in high tsetse challenge areas.

Sanguinarine and chelerythrine: assessment of safety on pigs in ninety days feeding experiment.

Sanguinaria canadesis, Chelidonium majus and Macleya cordata have been used for centuries as alternative medicines. Currently the extracts from these medicinal plants are components of veterinary and human phytopreparations, and of oral-hygiene agents. Sanguinarine and chelerythrine (SA/CHE) are biologically active components of these extracts. They display distinct antibacterial and anti-inflammatory properties, but, on the other hand, they have been reported as having adverse effects - genotoxicity and hepatotoxicity. This paper is aimed at evaluation of the effects of daily administration of the extract from Macleya cordata (2 mg and 100 mg in 1 kg feed, sanguinarine:chelerythrine 3:1) in the diet on the health status of swine. After 90-day administration, alkaloids were retained to a different extent in tissues. The highest SA/CHE retention was detected in the gingiva (0.55 microg/g) and liver (0.15 microg/g), no SA/CHE were detected in muscles. Plasma SA levels attained 0.11 microg/ml. Treated animals did not display any results of hematological, biochemical or histological assay different from controls. A (32)P-postlabeling assay proved that no DNA-adducts with SA/CHE were detected in pig livers. We did not observe any symptom linked to epidemic dropsy syndrome often attributed to sanguinarine. In conclusion, an average daily oral dose of alkaloids up to 5 mg per 1 kg animal body weight proved to be safe.

Endothelin-1 limits vascular smooth muscle beta-adrenergic receptor sensitivity by a PKC-dependent pathway.

Endothelin-1 reduces the chronotropic and inotropic effects of the beta-adrenoceptor agonist isoproterenol in rabbit isolated atria. Vascular interactions between endothelin-1 and isoproterenol have not been reported. Rings of the rabbit aorta without endothelium were mounted on myographs to measure isometric tension. Vessels were precontracted to similar levels with phenylephrine (30 micromol/L) or endothelin-1 (30 nmol/L). Relaxation to isoproterenol and forskolin were obtained. Vascular sensitivity (pD2) to isoproterenol was not different in the presence of endothelin-1 (7.6 +/- 0.3; n = 13) and phenylephrine (7.5 +/- 0.3; n = 11). The maximal relaxation (Emax) however, was doubled (P < 0.05) by endothelin-1 (42 +/- 5%), as compared with phenylephrine (23 +/- 4%). In the presence of endothelin-1, chelerythrine (protein kinase C inhibitor; 10 micromol/L) increased (P < 0.05) vascular sensitivity to isoproterenol (8.6 +/- 0.4, n = 7), but had no influence on the Emax. In contrast, in the presence of phenylephrine, pD2 was unaffected by chelerythrine, whereas the Emax to isoproterenol was increased (P < 0.05; 50 +/- 4%, n = 8). Vascular sensitivity and Emax to forskolin were similar in the presence of endothelin-1 and phenylephrine. In conclusion, endothelin-1 reduces vascular sensitivity to isoproterenol in a PKC-dependent pathway. The permissive effect of endothelin-1 appears to directly target the beta-adrenoceptor/G protein complex upstream of adenylate cyclase.

Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells.

Rat multidrug resistant protein 2 (Mrp2; Abcc2), an ATP-driven pump located on the canalicular domain of hepatocytes, exports glutathione S-conjugates (GS-X) and GSH among its wide variety of substrates. Previous studies have shown that chelerythrine (CHEL), a quaternary benzophenanthridine cation, reacts with GSH to form a reversible adduct under physiological conditions. Here we report that CHEL can strongly stimulate GSH efflux by Mrp2, when it is constitutively expressed in polarized canine kidney cells, thereby leading to the depletion of cellular GSH. Transepithelial transport experiments indicate that Mrp2 transports GSH and CHEL with a 1:1 stoichiometry, which can be readily inhibited by GS-bimane, a GS-X substrate for Mrp2. Moreover, CHEL can block Mrp2-mediated leukotriene C4 uptake by membrane vesicles with an IC50 approximately 100 microM in the presence of GSH, but not S-methyl GSH or ophthalmic acid. Thus the thiol group of GSH is required for inhibition of Mrp2 in the presence of CHEL. Our results suggest that CHEL stimulates GSH efflux by forming a reversible GS-CHEL adduct, which is transported by Mrp2 and dissociates extracellularly.