Astragaloside IV Promotes Adult Neurogenesis in Hippocampal Dentate Gyrus of Mouse through CXCL1/CXCR2 Signaling.

Astragaloside IV (ASI) has been reported to promote neural stem cells proliferation in vitro and CXCR2 expression on neutrophils. The present study was aimed to investigate the influence of ASI on adult neurogenesis in hippocampal dentate gyrus (DGs) of mouse and to discuss the possible underlying mechanisms. Total number of proliferative cells (BrdU⁺), pre-mature neurons (DCX⁺), early proliferative cells (BrdU⁺/DCX⁺), proliferative radial gila-like cells (BrdU⁺/GFAP⁺) and newly generated neurons (BrdU⁺/NeuN⁺) after ASI or vehicle administration for two weeks were counted, respectively. The results showed that BrdU⁺ cells and DCX⁺ cells were significantly increased in DGs of mice administered with ASI. The numbers of BrdU⁺/DCX⁺, BrdU⁺/GFAP⁺ cells and BrdU⁺/NeuN⁺ cells were also elevated in the ASI group. Correspondingly, ASI increased the protein expression of hippocampal DCX, GFAP and NeuN. Further study disclosed that ASI remarkably up-regulated the mRNA and protein expressions of CXCL1 as well as that of CXCR2 in the hippocampus. The promotive effect of ASI on DCX, GFAP and NeuN protein expression was abolished by SB225002, the inhibitor of CXCR2. Our results indicated that ASI modulated the homeostasis of the CXCL1/CXCR2 signaling pathway, which might be responsible for the increased neurogenesis within the hippocampal DGs of mice.

Combined transcriptome and metabolite profiling reveals that IiPLR1 plays an important role in lariciresinol accumulation in Isatis indigotica.

A lignan, lariciresinol, is an important efficacious compound for the antiviral effect of Isatis indigotica, a widely used herb for the treatment of colds, fever, and influenza. Although some rate-limiting steps of the lariciresinol biosynthetic pathway are well known, the specific roles of gene family members in I. indigotica in regulating lariciresinol production are poorly understood. In the present study, a correlation analysis between the RNA sequencing (RNA-Seq) expression profile and lignan content by using I. indigotica hairy roots treated with methyl jamonate (0.5 µM) at different time points as a source implicated that I. indigotica pinoresinol/lariciresinol reductase 1 (IiPLR1), but not IiPLR2 or IiPLR3, contributed greatly to lariciresinol accumulation. Gene silencing by RNA interference (RNAi) demonstrated that IiPLR1 indeed influenced lariciresinol biosynthesis, whereas suppression of IiPLR2 or IiPLR3 did not change lariciresinol abundance significantly. IiPLR1 was thus further characterized; IiPLR1 was constitutively expressed in roots, stems, leaves, and flowers of I. indigotica, with the highest expression in roots, and it responds to different stress treatments to various degrees. Recombinant IiPLR1 reduces both (±)-pinoresinol and (±)-lariciresinol efficiently, with comparative K cat/K m values. Furthermore, overexpression of IiPLR1 significantly enhanced lariciresinol accumulation in I. indigotica hairy roots, and the best line (ovx-2) produced 353.9 µg g(-1) lariciresinol, which was ~6.3-fold more than the wild type. This study sheds light on how to increase desired metabolites effectively by more accurate or appropriate genetic engineering strategies, and also provides an effective approach for the large-scale commercial production of pharmaceutically valuable lariciresinol by using hairy root culture systems as bioreactors.

Molecular diversity and hypoglycemic polypeptide-P content of Momordica charantia in different accessions and different seasons.

BACKGROUND: Momordica charantia (MC) has been used for treating diabetes mellitus from ancient times in Asia, Africa and South America. There are many MC accessions in local markets. Polypeptide-P as a main hypoglycemic component in MC was first studied in this experiment to illustrate the different contents in MC of different accessions and different harvesting times. RESULTS: Nineteen MC accessions collected from different regions were clustered into three groups using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers. Content of polypeptide-P in the tested MC accessions was detected by western blot (WB) method. The WB results revealed that polypeptide-P was detected in MC accessions harvested in June and July but not in September and October. Furthermore, Polypeptide-P content corresponded well with the MC accessions. CONCLUSION: Our results suggest that the MC accessions and the harvesting times or the weather during harvest play significant roles in high content of polypeptide-P.

[Identification of Dendrobii Caulis basing on ITS sequence].

Isolation of high-quality genomic DNA from dried and processed crude drug is the key for the DNA identification of Dendrobii Caulis. The DNA extract of Dendrobii Caulis was firstly compared using different method to isolate genomic DNA from dried and processed crude drug, including commercial DNA extracted kit and CTAB method. Using modified CTAB method (extracted from large samples), the genomic DNA was successfully isolated from Dendrobii Caulis, including Huangcao and Fengdou. The ITS regions were amplified using the purified DNA as template, and then cloned and sequenced. These ITS sequences were compared with data from Genbank database and our lab, 14 Dendrobium species and five similar species were identified from "Huangcao" and "Huangcao" slice, while six species and three similar species from "Fengdou" according to their sequence similarity. The study demonstrated that the dried Dendrobii Caulis could be identified using DNA molecular method, which could overcome deficiencies and limitations of traditional identification method and has a certain application prospects.

Early Growth Response Gene-1 Deficiency Interrupts TGFß1 Signaling Activation and Aggravates Neurodegeneration in Experimental Autoimmune Encephalomyelitis Mice.

Early growth response protein 1 (Egr-1) triggers the transcription of many genes involved in cell growth, differentiation, synaptic plasticity, and neurogenesis. However, its mechanism in neuronal survival and degeneration is still poorly understood. This study demonstrated that Egr-1 was down-regulated at mRNA and protein levels in the central nervous system (CNS) of experimental autoimmune encephalomyelitis (EAE) mice. Egr-1 knockout exacerbated EAE progression in mice, as shown by increased disease severity and incidence; it also aggravated neuronal apoptosis, which was associated with weakened activation of the BDNF/TGFß 1/MAPK/Akt signaling pathways in the CNS of EAE mice. Consistently, Egr-1 siRNA promoted apoptosis but mitigated the activation of BDNF/TGFß 1/MAPK/Akt signaling in SH-SY5Y cells. Our results revealed that Egr-1 is a crucial regulator of neuronal survival in EAE by regulating TGFß 1-mediated signaling activation, implicating the important role of Egr-1 in the pathogenesis of multiple sclerosis as a potential novel therapy target.

Antiangiogenic effects of GFP08, an agaran-type polysaccharide isolated from Grateloupia filicina.

An agaran-type polysaccharide, GFP08, isolated from Grateloupia filicina (C. Agardh) Lamouroux, was mainly composed of 1,3-linked ß-D-galactose partially sulfated at position O-2 and 1,4-linked α-L-galactose O-2, O-3-disulfate, α-L-galactose O-6-sulfate and 3,6-anhydro-α-L-galactose. Small quantities of xylose, 4,6-O-(1'-carboxyethylidene) and 6-O-methyl-ß-D-galactose were also present. In mice bearing sarcoma-180 cells, GFP08 decreased tumor weight in a dose-dependent manner. The antiangiogenic activity of GFP08 was evaluated using the chicken chorioallantoic membrane assay, and the results showed that GFP08 dose-dependently reduced new vessel formation. Meanwhile, GFP08 inhibited the differentiation of human umbilical vein endothelial cells (HUVECs) into capillary-like structures in vitro and reduced the number of migrated cells. However, there was no observed cytotoxicity of GFP08 toward HUVECs. Further study revealed that GFP08 decreased tissue factor (TF) expression without affecting the activities of matrix metalloproteinase-2 and -9. All those data indicated that GFP08 had an antitumor effect that might be associated in part with its antiangiogenic effect through down-regulating the expression of TF protein.

An efficient way from naringenin to carthamidine and isocarthamidine by Aspergillus niger.

Biotransformation of naringenin with Aspergillus niger CGMCC 3.4628 yielded two hydroxylation products which were identified unambiguously as 6-hydroxylnaringenin (carthamidin) and 8-hydroxylnaringenin (isocarthamidin) by ESI-MS and (1)H-NMR. Both products simultaneously arrived at high level after 48 h in the biotransformation process. The highest conversion efficiency of carthamidin was 0.38 mg/mg of naringenin and that of isocarthamidin was 0.43 mg/mg of naringenin. Antioxidant property assay using a thin layer chromatography-bioautographic-based DPPH scavenging method demonstrated that both hydroxylation metabolites exhibited much stronger activity than naringenin. The high efficiency and convenient procedure makes the biotransformation with A. niger described in current work a potential way to produce carthamidin and isocarthamidin.

[Expression of human estrogen receptor alpha and beta in Escherichia coli].

Estrogen participates in many life activities through combination with estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) in the body. In order to establish an in vitro estrogen-like compound screening model, the coding region of human ERalpha and ERbeta was separately constructed into pET32-ERalpha and pET43-ERbeta prokaryotic expression vector and water-soluble recombinant ERalpha and ERbeta proteins were expressed in Escherichia coli strain BL21. Western blotting revealed that both recombinant proteins have estrogen receptor binding sites. The proteins were purified using S-Tag affinity Purification Kit and digested with enterokinase to get the ERalpha and ERbeta proteins. About 0.90 mg of ERalpha and 0.65 mg of ERbeta were obtained at the concentration of 0.181 and 0.131 mg x mL(-1), respectively.

[Screening of endophytic fungi from Huperzia serrata for acetylcholinesterase inhibitory activity and its taxonomic identification].

OBJECTIVE: To screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata. METHOD: Endophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics. RESULT: Fungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium. CONCLUSION: Endophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.

Quantification of 1-(13) C-L-methionine in rat serum with hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry and its application in pharmacokinetic study.

A rapid, selective and sensitive hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry (MS/MS) method was developed to determine 1-(13) C-l-methionine in rat serum. Proteins in serum were precipitated using acetonitrile and the supernatant was separated after centrifugation. 1-(13) C-l-phenylalnine was used as the internal standard. HILIC-tandem mass spectrometry analysis was performed on a hydrophilic interaction silica column (TSK-GEL AMIDE-80) using a linear gradient elution system, acetonitrile-5 mm ammonium acetate containing 0.1% formic acid and multiple reaction monitoring mode for 1-(13) C-l-methionine and 1-(13) C-l-phenylalnine. The assay was validated with a linear range between 10 and 150 ng mL(-1) (r ≥ 0.99) and a lower limit of quantification of 10 ng mL(-1) , calculated with weighted (1/x(2) ) least squares linear regression. The RSD of intra-day precision was smaller than 3.6% and the inter-day RSD less than 6.5%, while the average recovery was 100.48% with an RSD of accuracy within 2.9%, determined from quality control samples. The HILIC-MS/MS method was fully validated and successfully applied to the in vivo pharmacokinetic study of stable-isotope 1-(13) C-l-methionine in rats.

Gene cloning and expression of a novel hypoglycaemic peptide from Momordica charantia.

BACKGROUND: Momordica charantia (MC) is used in many Asian countries as a traditional functional food and medicine. Polypeptide-P, a 166 amino acid (AA) polypeptide isolated from MC seeds, has been reported to show hypoglycaemic effects in patients with type I or type II diabetes. The AA sequence of this peptide has been determined, but its gene sequence has yet to be published. RESULTS: In this study a gene-cloning strategy was employed to obtain the polypeptide-P gene sequence using degenerate reverse transcription polymer chain reaction and genome-walking methods. A complete 498 bp sequence encoding the polypeptide-P protein was cloned from MC seeds. Subsequent assays of the bioactivity of the expressed recombinant protein revealed that it had significant hypoglycaemic activity in alloxan-induced diabetic mice. This result suggests that recombinant polypeptide-P has hypoglycaemic effects. CONCLUSION: This is the first report of cloning and expression of the MC polypeptide-P gene. The cloned gene could be helpful for exploring the mechanisms of polypeptide-P gene expression and regulation in MC. Furthermore, this gene could be used as a potential tool both for screening MC varieties with high hypoglycaemically active substance content and for breeding new varieties of MC with high economic value, which could in turn be beneficial to farmers.

[Regulation of Vitreoscilla hemoglobin on biosynthesis of astragaloside IV].

In the present study, the regulation of Vitreoscilla hemoglobin (VHb) on astragaloside IV biosynthesis was investigated. An intermediate expression vector consisting of the CaMV35S promoter fused to the vgb and nopaline synthase terminator was transferred into Astragalus membranaceus via Agrobacterium rhizogenes. The transgenic hairy roots were confirmed by PCR amplification and Southern blot hybridization. The expression of vgb in transgenic hairy roots was confirmed by RT-PCR. After 15 days cultivation, the dry weight and growth rate of transgenic hairy roots were higher than that of the non-transgenic hairy root. ELSD-HPLC analysis showed that astragaloside IV content of transgenic hairy roots was 5 to 6 times of non-transgenic hairy root control and 10 to 12 times of Radix Astragali from Shanxi Province. These results suggested that the expression of vgb promoted the growth of transgenic hairy roots, and increased the content of astragaloside IV.

Determination and biosynthesis of multiple salvianolic acids in hairy roots of Salvia miltiorrhiza.

Danshen (Salvia miltiorrhiza Bunge) hairy roots were obtained by infecting Danshen leaves with Agrobacterium rhizogenes 9402. Besides rosmarinic acid (RA) and salvianolic acid B (SAB), the hairy root could also produce salvianolic acid K (SAK), salvianolic acid L, ethyl salvianolic acid B (ESAB), methyl salvianolic acid B (MSAB), and a compound with a molecular weight of 538 (compound 538) identified by using LC-MS. Effects of methyl jasmonate (MeJA) and yeast elicitor (YE) on the accumulation of these compounds had been investigated. MeJA increased the accumulation of SAB, RA, SAK, and compound 538 from 4.21%, 2.48%, 0.29%, and 0.01% of dry weight to 7.11%, 3.38%, 0.68%, and 0.04%, respectively. YE stimulated the biosynthesis of RA from 2.83% to 5.71%, but depressed the synthesis of SAB, SAK and compound 538. It was indicated in all the results that these Danshen hairy roots could be used as alternative resources to produce salvianolic acids. Analysis of the content variation of these compounds after elicitation suggested that SAK and compound 538 might be the intermediates in the biosynthesis from RA to SAB in Danshen hairy roots.

[Evaluation on hepatotoxicity caused by Dioscorea bulbifera based on analysis of bile acids].

Metabolic profile of bile acids was used to evaluate hepatotoxicity of mice caused by ethanol extraction of Dioscorea bulbifera L. (ethanol extraction, ET) and diosbulbin B (DB), separately. Ultra-performance liquid chromatography coupled with quadrupole mass spectrometry (UPLC-MS) was applied to determine the contents of all kinds of endogenous bile acids including free bile acids, taurine conjugates and glycine conjugates. Obvious liver injuries could be observed in mice after administrated with ET and DB. Based on the analysis using principle components analysis (PCA), toxic groups could be distinguished from their control groups, which suggested that the variance of the contents of bile acids could evaluate hepatotoxicity caused by ET and DB. Meanwhile, ET and DB toxic groups were classified in the same trends comparing to control groups in the loading plot, and difference between the two toxic groups could also be observed. DB proved to be one of the toxic components in Dioscorea bulbifera L. Bile acids of tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), cholic acid (CA) and others proved to be important corresponds to ET and DB induced liver injury according to analysis of partial least square-discriminant analysis (PLS-DA) and the statistical analysis showed that there were significant differences between the control groups and toxic groups (P < 0.01). Furthermore, good correlation could be revealed between the foregoing bile acids and ALT, AST. It indicated that taurine conjugated bile acids as TUDCA, TCDCA, TCA and TDCA along with CA could be considered as sensitive biomarkers of ET and DB induced liver injury. This work can provide the base for the further research on the evaluation and mechanism of hepatotoxicity caused by Dioscorea bulbifera L.

Plantago asiatica L. seeds extract protects against cardiomyocyte injury in isoproterenol- induced cardiac hypertrophy by inhibiting excessive autophagy and apoptosis in mice.

BACKGROUND: Cardiac hypertrophy is the early stage of many heart diseases, such as coronary heart disease, hypertension, valvular dysfunction and cardiomyopathy. Cardiomyocyte autophagy and apoptosis play an important role in the process of cardiac hypertrophic response. Plantago asiatica L. seeds extract (PASE) is prepared from a traditional herbal medicine in Asia with tremendous pharmacological activities. However, whether PASE could relieve cardiac hypertrophy has not been elucidated. The present study is aimed to investigate the effect of PASE on cardiac hypertrophy and explore its potential underlying mechanism. METHODS: Cardiac hypertrophy was induced in C57BL/6 mice by subcutaneous injection of isoproterenol (ISO) for two weeks. Meanwhile, the mice were intraperitoneally injected with PASE at dosages of 20, 40 and 80 mg/kg/day. Cardiac hypertrophy was evaluated by echocardiographic examination, haematoxylin and eosin staining and quantitative real-time polymerase chain reaction. Expressions of proteins involved in autophagy and apoptosis such as Beclin1, p62, LC3II, Bax, Bcl-2 and Cleaved-caspase-3 were detected by western blot analysis. Western blot, transient transfection, acridine orange staining, TUNEL staining and autophagy inducer were used to observe the effect and explore the mechanism of PASE on cardiomyocyte and H9c2 cells with excessive autophagy and apoptosis induced by ISO. RESULTS: ISO induction for two weeks disturbed the myocardial contractility and cardiac function of left ventricles of mice. PASE treated mice showed significantly improved cardiac function indexes, including EF, FS, SV and CO, compared with the ISO group. Treatment with PASE also decreased the heart weight/body weight ratio and cardiomyocyte size, and downregulated the mRNA and protein expressions of hypertrophic markers ANP, BNP, and ß-MHC. Furthermore, the changes of autophagy and apoptosis markers, such as LC3II, Beclin1, p62, Bcl-2, Bax and Cleaved-caspase-3 induced by ISO were resumed by PASE treatment. Consistently, PASE demonstrated similar effects on ISO-induced H9c2 cells as it did in vivo. In addition, PASE could counteract the increased autophagy induced by the autophagy inducer, rapamycin. CONCLUSION: PASE attenuated ISO-induced cardiac hypertrophy in mice by inhibiting excessive autophagy and apoptosis in cardiomyocytes. The novel findings may pave the way for the clinical usage of PASE for the prevention of heart diseases related with cardiac hypertrophy.

Expression and characterization of Momordica Chanrantia anti-hyperglycaemic peptide in Escherichia coli.

A nucleic acid sequence MC, encoding Momordica Chanrantia anti-hyperglycaemic peptide MC6 (accession: AAX06814) synthesized according to Escherichia coli preferred codons, was cloned and expressed in E. coli. Recombinant protein pQE8-MC (about 3.5 kDa) was purified and analyzed by 20% SDS-PAGE and western blot. It revealed that the expressed pQE8-MC had good solubility in aqueous media. An HPLC assay was used to confirm the expression of pQE8-MC. Subsequent pharmacological activity assay revealed a significant hypoglycemic effect of low dose treatments of pQE8-MC on male kunming mice. Four hours after an intravenous tail injection, the blood sugar levels of mice treated with pQE8-MC saline solution A3 (1 mg/kg BW) decreased greatly (P < 0.01) relative to the levels of a control group. This suggests that pQE8-MC, expressed in bioengineered E. coli, has a similar hypoglycemic function to the natural protein MC6 from M. Chanrantia. These results reveal the possibility of using bio-engineered bacteria as an anti-diabetic agent.

[The utility of trnH-psbA gene for phylogenetic analysis of Huperziaceae and plant barcoding].

OBJECTIVE: The purpose of study was to discover the phylogenetic relations and plant barcoding of 17 plants from Huperziaceae. METHODS: Phylogenetic tree of chloroplast trnH-psbA gene of 17 plants from Huperziaceae was constructed by software. RESULTS: It showed that Huperziaceae could be divided into two genera Huperzia and Phlegmariurus and bootstrap value reached 91%. CONCLUSIONS: Holub and Qing' taxonomy was supported and 17 species in Huperziaceae were monophyletic groups and it suggested that trnH-psbA could be used as a DNA barcode to identify plants.

[Inhibitory effect on activated renin-angiotensin system by astragaloside IV in rats with pressure-overload induced cardiac hypertrophy].

OBJECTIVE: To investigate the effect of astragaloside IV (As IV) on the activation of rennin-angiotensin system in rats with pressure-overload induced cardiac hypertrophy. METHOD: Left ventricle hypertrophy was induced by abdominal aorta banding between bilateral renal aortas for 12 weeks. Rats were given astragaloside IV 1.0 mg x kg(-1) and 3.3 mg x kg(-1) for 12 weeks, respectively. After treatment, the left ventricular mass index (LVMI)was calculated by morphometry methods. Plasma and cardiac tissue angiotensin II, and plasma aldosterone were measured by ELISA method. Gene expressions of ACE, AT1 and AT2 in cardiac tissue were detected by real time PCR. Protein expressions of AT1 and AT2 in cardiac tissue were detected by Western blot. RESULT: Compared with model rats, LVMI was decreased by astragaloside IV treatment. Biochemical results indicated that the contents of angiotensin II in plasma and cardiac tissue as well as aldosterone in plasma were all increased in abdominal aorta banding rats comparing with sham-operated rats, then, decreased by astragaloside IV treatment. Gene expressions of cardiac ACE was downregulated by astragaloside IV, however, gene and protein expressions of cardiac AT2 were upregulated by astragaloside IV. Both elevated gene and protein expressions of AT1 were not attenuated by astragaloside IV. CONCLUSION: Excessive activated rennin-angiotensin system in rats with pressure-overload induced cardiac hypertrophy is inhibited by astragaloside IV treatment.

Child compound Endothelium corneum attenuates gastrointestinal dysmotility through regulating the homeostasis of brain-gut-microbiota axis in functional dyspepsia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Nowadays, there is no specific effective western medicine for functional dyspepsia (FD), especially in children. Clinically, child compound Endothelium corneum (CCEC) has shown to be effective for the therapy of FD, however, the underlying mechanism has not been elucidated yet. MATERIALS AND METHODS: FD was induced in rats by irregular diet plus dilute hydrochloric acid feeding. Gastric emptying and small intestinal transit were examined by intragastric gavage with Evans blue. Histopathology was assessed by H&E staining. Gastrointestinal hormones and brain gut peptides were measured by ELISA assay. mRNA expression level was quantified by real-time PCR. Protein expression level was detected by western blotting assay. Gut microbiota was analyzed by 16S rRNA miseq sequencing. RESULTS: CCEC significantly enhanced gastric emptying and small intestinal transit of FD rats, and prominently suppressed gastrointestinal microinflammation. At phylum level, CCEC prevented the decrease of Firmicutes and the increase of Bacteroidetes in gut of FD rats. In stomach of FD rats, MTL, CCK and VIP levels were significantly increased, which could be repressed by CCEC; however, the decreased GAS level could not be elevated by CCEC. In small intestine of FD rats, MTL and GAS levels were decreased, while VIP content was increased. These alterations could be effectively reversed by CCEC. NPY levels in serum, small intestine and hypothalamus of FD rats were significantly decreased, which could be rescued by CCEC. Moreover, the over-activated POMC/Stat3/Akt pathway in hypothalamus of FD rats could be suppressed by CCEC. CONCLUSION: CCEC enhanced gastrointestinal motility probably through rebalancing the homeostasis of brain-gut-microbiota axis in FD rats. The novel findings may provide insightful theoretical basis for its clinical employment.

Rutaecarpine induces chloride secretion across rat isolated distal colon.

The present study evaluated the effect of rutaecarpine (Rut) on Cl(-) secretion across rat distal colonic mucosa. Basolateral application of Rut elicited an increase in short-circuit current (I(SC)) response in a concentration-dependent manner. Evidence that Rut-stimulated I(SC) was due to Cl(-) secretion is based on 1) inhibition of current by bumetanide; 2) Cl(-) channel blockers diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and glibenclamide; and 3) removal of Cl(-) ions in bath solution. Determination of neurogenic blockers on Rut-induced I(SC) indicated that pretreatment of tissues with tetrodotoxin or indomethacin, but not atropine or hexamethonium, inhibited Rut-induced response. Treatment with Rut led to release and synthesis of prostaglandin E(2) in rat colonic mucosa. Rut-stimulated I(SC) was markedly reduced by pretreatment with MDL-12,330A [cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine] and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), but not with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, bisindolylmaleimide, and thapsigargin. Elimination of the extracellular Ca(2+) also did not alter Rut response. Rut treatment resulted in the increase in intracellular cAMP levels and the activation of protein kinase A. Depolarizing the basolateral membrane with high K(+) showed that Rut-stimulated apical Cl(-) current was largely prevented by cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors. Permeabilizing apical membrane with nystatin revealed that Rut-stimulated basolateral K(+) current was specifically inhibited by Ba(2+) ions and chromanol 293B. The evidence derived from present study suggests that Rut-stimulated Cl(-) secretion is mediated by generation of endogenous prostaglandin E(2) and that it also involves the stimulation of cAMP and protein kinase A pathways, which subsequently lead to the activation of apical Cl(-) channels, mostly the CFTR and basolateral cAMP-dependent K(+) channels.