QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.131961v1 325/1/256    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wu, D. Articles by Hu, Z. Search for Related Content PubMed PubMed Citation Articles by Wu, D. Articles by Hu, Z.

GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Rutaecarpine Induces Chloride Secretion across Rat Isolated Distal Colon

Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China (D.Z.W., Z.B.H.); and E-Institute of Traditional Chinese Medicine Internal Medicine, Shanghai Municipal Education Commission, Shanghai, China (D.Z.W.)

Received September 21, 2007; accepted January 9, 2008.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
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 E₂ 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 Ca²⁺ 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 Ba²⁺ ions and chromanol 293B. The evidence derived from present study suggests that Rut-stimulated Cl^– secretion is mediated by generation of endogenous prostaglandin E₂ 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.

Electrolyte transport of mammalian colonic epithelium involves both NaCl absorption and Cl^– secretion processes. Transport is regulated by a variety of neurotransmitters, hormones, and inflammatory mediators (Kunzelmann and Mall, 2002). NaCl is either absorbed electroneutrally by parallel luminal (apical) Na⁺-H⁺ and Cl^–- exchanges or electrogenically by luminal ENaC channels with Cl^– transport passively through the paracellular pathway located in the distal colon (Köttgen et al., 2003). Chloride secretion in the mammalian colonic crypt cells is a vectorial transport of Cl^– ions from the serosal (basolateral) to luminal compartment; this process requires the coordination of four distinct membrane events: 1) apical Cl^– passive diffusion via the cystic fibrosis transmembrane conductance regulator (CFTR) channel; 2) increase of K⁺ efflux through the basolateral Ba²⁺-sensitive channels; 3) activation of basolateral bumetanide-sensitive Na⁺-K⁺-2Cl^–-cotransporters; and 4) increase of Na⁺ efflux by the basolateral Na⁺-K⁺-ATPase pump (Greger, 2000). Cl^– secretion stimulated by both Na⁺-K⁺-2Cl^– cotransporters and Na⁺-K⁺-ATPase requires basolateral K⁺ channel for K⁺ recycling across the basolateral membrane. Two types of basolateral K⁺ channels were found to process Cl^– secretion via the apical membrane, namely, cAMP-activated KCNQ1 K⁺ channel and Ca²⁺-activated small-conductance Ca²⁺-activated K⁺ channel (Bleich et al., 1997; Warth, 2003).

Fructus evodiae in Chinese medicine is mainly recommended for treatment of gastrointestinal disorders, such as abdominal pain, acid regurgitation, nausea, diarrhea, and hernia (Yu et al., 2006). Thus, we hypothesize that its major active compound, Rut, may influence gastrointestinal function, especially electrolyte transport across the colonic epithelium. Therefore, the aim of the present study was to elucidate the effect of Rut on the ion transport process in rat distal colon epithelium and to determine the underlying mechanism(s). The information generated from this study demonstrates that Rut stimulates Cl^– secretion across rat colonic mucosa, and we suggest that this occurs by generation of endogenous prostaglandin E₂ (PGE₂) and also involves the stimulation of cAMP and protein kinase A (PKA) pathways.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals. Chemically pure Rut was obtained from the Laboratory of Chemistry (Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China). It was identified on the basis of chemical and spectroscopic evidence. Acetazolamide, amiloride, atropine sulfate, BAPTA-acetoxymethyl ester (AM), bisindolyimaleimide (BIS), bumetanide, charybdotoxin (ChTX), clotrimazole (CLT), 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), diphenylamine-2-carboxylate (DPC), forskolin, H-89, hexamethonium, indomethacin, 3-isobutyl-1-methylxanthine (IBMX), MDL-12,330A, nystatin, PGE₂, and thapsigargin were obtained from Sigma-Aldrich (St. Louis, MO). Chromanol 293B (293B) was purchased from Tocris Cookson Inc. (Ellisville, MO), glibenclamide and tetrodotoxin (TTX) were from BIOMOL Research Laboratories (Plymouth Meeting, PA), and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) was obtained from Calbiochem (San Diego, CA). TTX was dissolved in 0.25% acetic acid, and ChTX was dissolved in an aqueous stock solution containing 0.1% (w/v) bovine serum albumin. Atropine sulfate and hexamethonium were dissolved in aqueous stock solutions diluted in salt buffer just before use. All other drugs were dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration was less than 0.1% (v/v). For permeabilization studies, nystatin was used as a 100 mg · ml^–1 stock solution in DMSO [final concentration 0.1% (v/v)], and it was sonicated for 30 s just before use. If drugs were dissolved in a solvent rather than aqueous solution, the same volume of solvent was administered to the control tissue.

Solutions. The Parsons solution (Schultheiss et al., 2005) for tissue preparation and Ussing chamber experiments contained 107 mM NaCl, 4.5 mM KCl, 25 mM NaHCO₃, 1.8 mM Na₂HPO₄, 0.2 mM NaH₂PO₄, 1.2 mM CaCl₂, 1.0 mM MgSO₄, and 12 mM glucose. The solution was continuously aerated with carbogen (a mixture of 95% O₂ and 5% CO₂) to maintain the pH at 7.4. In ion substitution experiments, Cl^– was replaced with gluconate when Cl^–-free solution was used. When -free solution was used, was replaced by Cl^–, and 10 mM HEPES was added to solution. In Cl^–--free solution, both Cl^– and were replaced with gluconate and HEPES. Both -free and Cl^–--free solutions were gassed with 100% O₂. -free solution was also used in experiments with BaCl₂.

Animals and Tissue Preparation. Male Wistar rats (200–220 g) were obtained from Sino-British SIPPR/BK Lab Animal Ltd. (Shanghai, China). This research was approved by the university ethics committee. The animals were maintained on a 12-h light/dark cycle, and they were allowed free access to normal food and water until the day of the experiments. Animals were sacrificed rapidly by stunning and cervical dislocation. A 5-cm segment of the distal colon was removed without distention, and it was suspended in an ice-cold and oxygen-saturated Parsons solution. The colon was opened along the mesenteric border, and it was rinsed free of its fecal contents with Parsons solution. Thereafter, the colon was pinned with the mucosa facing down on a silicon-dissected plate, and then the longitudinal and circular muscles were carefully stripped away with fine forceps. Finally, the isolated mucosal sheet was cut into an appropriate size matched to Ussing chamber size.

Measurement of Electrophysiological Parameters. Freshly isolated rat colonic mucosal sheets were fixed to holding sliders, and then they were mounted in modified Ussing chambers (EasyMount chamber; Physiologic Instruments, San Diego, CA), with a window area of 0.5 cm². Two pieces of mucosal sheets were used from each animal. Tissues were bathed on both the mucosal and serosal sides with 5 ml of Parsons solution maintained at 37°C by heated water jackets; the pH was maintained at 7.4, and the solution was oxygenated with a 95% O₂, 5% CO₂ mixture. The tissues were voltage-clamped at 0 mV to monitor short-circuit current (I_SC) using a dual-voltage clamp amplifier (VCC MC2; Physiologic Instruments) connected via a PowerLab 8SP (ADInstruments Pty Ltd., Castle Hill, Australia) to a PC computer. The transepithelial electrical potential was measured by a pair of pipette-shaped voltage-sensing electrodes made of sintered silver-silver chloride wire (Physiologic Instruments) in agar bridges filled with a solution of 4% (w/v) agarose in 3 M KCl solution; the electrical current across mucosae was measured by a pair of pipette-shaped current-passing electrodes made of silver pellet (Physiologic Instruments) in agar bridges filled with a solution of 4% (w/v) agarose in 3 M KCl solution. The current deflection (I_SC) was caused by applying a 1-mV pulse for 0.5 s at 60-s intervals under short-circuit condition through the voltage-sensing electrodes. By this procedure, the transepithelial electrical resistance (R_te) was able to be calculated by Ohm's law (R_te = transepithelial electrical potential/I_SC). A positive I_SC is referred as a net flow of anions from the basolateral to the apical side, a net flow of cations from the apical to the basolateral side, or a combination.

Measurement of Apical Membrane Cl^– Current and Basolateral Membrane K⁺ Current. The apical membrane Cl^– current (I_Cl) was investigated according to Schultheiss et al. (2005). In brief, the basolateral membrane was depolarized with a solution containing high potassium concentration for 40 to 50 min. Thus, apical Cl^– current was measured in the presence of a serosal-to-mucosal Cl^– gradient with the following bath solution: apical, 107 mM potassium gluconate, 4.5 mM KCl, 25 mM NaHCO₃, 1.8 mM Na₂HPO₄, 0.2 mM NaH₂PO₄, 5.75 mM calcium gluconate, 1.0 mM MgSO₄, and 1 mM glucose; and basolateral, 111.5 mM KCl, 25 mM NaHCO₃, 1.8 mM Na₂HPO₄, 0.2 mM NaH₂PO₄, 1.25 mM CaCl₂, 1.0 mM MgSO₄, and 12 mM glucose. This procedure allows measurement of changes in the apical anion conductance, avoiding contaminations in the current response, e.g., by charybdotoxin-sensitive apical K⁺ channel (Schultheiss et al., 2005).

The basolateral membrane K⁺ current (I_K) was determined after permeabilization of apical membrane with nystatin (100 µg · ml^–1 at the mucosal side) in the presence of a mucosal-to-serosal K⁺ gradient (13.5 mM KCl at the mucosal and 4.5 mM KCl at the serosal side) established by the following bath solution: apical, 98 mM N-methyl-D-glucamine-Cl, 13.5 mM KCl, 25 mM choline , 1.8 mM Na₂HPO₄, 0.2 mM NaH₂PO₄, 1.25 mM CaCl₂, 1.0 mM MgSO₄, and 12 mM glucose; and basolateral, 107 mM potassium gluconate, 4.5 mM KCl, 25 mM NaHCO₃, 1.8 mM Na₂HPO₄, 0.2 mM NaH₂PO₄, 5.75 mM calcium gluconate, 1.0 mM MgSO₄, and 12 mM glucose. All solutions were adjusted to pH 7.4 at 37°C.

PGE₂ Measurement. After the isolated mucosal sheets were equalized in Ussing chamber with the normal Parsons solution at 37°C for 90 min, the tissues were treated with control (DMSO) and Rut (100 µM, basolateral) for 5 min. The total volumes of Parsons solutions in both serosal and mucosal chambers and tissues were removed, and then samples were quickly frozen in liquid nitrogen and stored at –80°C until PGE₂ measurement. For measurement of PGE₂ in incubates, 0.3 ml of each sample was assayed directly and without dilution; the PGE₂ level was calculated from a standard curve run in parallel with each assay, and the value was expressed as picograms per milliliter. For measurement of PGE₂ in tissue, tissue fragments (normal control mucosa and Rut-treated mucosa) were homogenized at 0 to 4°C in the presence of 10 µM indomethacin so as to prevent prostaglandin production during the procedure. Then, tissues were centrifuged at 600g. Supernatants of the tissue homogenates (500 µl) were used for PGE₂ determination using a competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and protein content was quantified by using Bradford (1976) method with bovine serum albumin as the standard. Determinations were performed in triplicate. The PGE₂ value was expressed as pg · mg^–1 of protein in the tissue sample.

Measurement of Intracellular cAMP Contents. Intracellular cAMP contents were measured with an enzyme immunoassay. The isolated mucosal sheets were equalized in Ussing chamber with the normal Parsons solution at 37°C for 90 min; the tissues were then exposed to DMSO, IBMX, Rut, Rut with IBMX, forskolin with IBMX, Rut with forskolin, and IBMX for 5 min; and finally, they were rapidly frozen in liquid nitrogen and stored at –80°C until homogenized in 0.5 ml of ice-cold 6% trichloroacetic acid using a glass homogenizer. The homogenate was centrifuged at 2000g for 10 min at 4°C. The supernatant was extracted three times with 3 volumes of diethyl ether before lyophilization. cAMP levels were assayed by a cAMP enzyme immunoassay kit (Cayman Chemical; or R&D Systems, Minneapolis, MN). The tissue residue was dissolved in 2 M NaOH, and protein content was determined by using Bradford (1976) method with bovine serum albumin as the standard. cAMP concentration was expressed as pmol · mg^–1 of protein.

PKA Activity Assay. The isolated mucosal sheets were equalized in Ussing chamber with the normal Parsons solution at 37°C for 90 min, and then the tissues were exposed to control (DMSO) and Rut (100 µM, basolateral) for 5 and 15 min. Finally, tissues were rapidly frozen in liquid nitrogen and stored at –80°C until PKA activity assay. Frozen colonic tissue was homogenized in 5 volumes of ice-cold (0–4°C) PKA extraction buffer (0.5 mM EDTA, 0.5 mM EGTA, 10 mM β-mercaptoethanol, 50 µM NaF, 5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 25 mM Tris-HCl, pH 7.4) containing protease inhibitor cocktails at 4°C, and then we centrifuged the lysate at 14,000g for 5 min at 4°C. Supernatant was used for the enzyme assay. The activity of PKA was detected by the nonradioactive PepTag test method with dye-labeled Kemptide as a substrate, according to the manufacturer's protocol (V5340; Promega, Madison, WI). The phosphorylated and nonphosphorylated samples were separated on a 0.8% agarose gel at 100 V for 18 min. The gel was photographed (Pharmacia Biotech Imaging System, Piscataway, NJ), and the PKA activity was quantitated by Beer's law through reading the absorbance at 570 nm on the microplate spectrophotometer (SpectraMax 190, Molecular Devices, Sunnyvale, CA). Protein concentrations in homogenate were determined by Bradford (1976) method with bovine serum albumin as a standard. The activity of PKA was expressed as pmol · min^–1 · µg^–1 of protein.

Data Analysis. Results are presented as the mean ± S.E.M. n was the number of tissue preparations. The difference between two different groups was analyzed by Student's paired or unpaired t test. The differences among multiple groups were analyzed by one-way analysis of variance. A P value less than 0.05 was considered to indicate statistical significance. The changes in I_SC (I_SC) were quantified by subtracting at the peak of a current response from its respective baseline values before drug administration. The IC₅₀ value was calculated from nonlinear regression analysis of dose-response data by GraphPad Prism software version 4.03 (GraphPad Software Inc., San Diego, CA).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Effect of Rut on I_SC. After a 90-min equilibration with normal Parsons solution, the transepithelial potential was –3.8 ± 0.1 mV, the basal I_SC was 34.5 ± 0.5 µA · cm^–2, and the transepithelial resistance was 111.1 ± 1.1 · cm² in 409 rat colonic preparations. When added to serosal (basolateral) side of the colonic tissue preparation, Rut (Fig. 1A) induced a rapid increase in I_SC that peaked approximately 2.5 min and subsequently returned to baseline value within approximately 10 min (Fig. 1B). Rut added to mucosal (apical) side of the preparation caused no alteration in I_SC (Fig. 1B). The concentration-dependent response to Rut showed that the amplitude of Rut-induced I_SC increased in a sigmoidal manner over the concentration range between 1.0 and 300 µM (Fig. 1C); the maximal I_SC response to Rut was 67.1 ± 7.9 µA · cm^–2, with an EC₅₀ value of 20.0 ± 7.0 µM (Fig. 1D). Application of 100 µM Rut to serosal side caused an increase in I_SC that was nearly to the maximal response, which was thus used to compare the responses between control and treated samples in the subsequent experiments. Initial experiments showed that second response to Rut was less than the first response, indicating that Rut had a desensitization effect. Therefore, only a single concentration of Rut was applied to one tissue, either in the absence or presence of inhibitors or antagonists. A similar desensitization phenomenon was observed from the alteration of positive inotropic and chronotropic actions induced by Rut in guinea pig isolated right atria (Kobayashi et al., 2001). The current response induced by 100 µM Rut was 63.3 ± 7.3 µA · cm^–2, and the corresponding tissue resistance was 105.7 ± 2.8 · cm² (n = 12), which was not different from its basal value (111.3 ± 2.6 · cm²).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Effect of Rut on ISC from rat colonic epithelium. A, chemical structure of Rut (mol. wt. 287.31). B, rat colonic mucosa were mounted in Ussing chambers, and application of Rut (100 µM) to basolateral rather than apical side induced a transient increase in ISC as shown in the original recording traces. Dashed line indicates zero current level. C, original recording traces of a concentration-dependent current response induced by Rut from 1.0 to 300 µM. Each concentration of Rut was added as a single dose to separate tissues. D, summary of concentration-response curve data collected from experiments as shown in C. Each point represented mean ± S.E.M., n = 7. The EC50 value was calculated to be 20.0 ± 7.0 µM.

Ionic Nature of Rut-Induced I_SC. To determine the ionic nature of Rut-induced I_SC, ion substitution experiments were performed as follows. When Cl^– was replaced with gluconate in bilateral bath solution, Rut-induced maximal I_SC was 15.4 ± 2.4 µA · cm^–2 (n = 8), or 23% of the control value (67.1 ± 7.9 µA · cm^–2; n = 7), whereas replacement of with HEPES did not influence Rut-induced I_SC. When using a Cl^–--free solution, Rut-induced maximal I_SC was 13.6 ± 1.8 µA · cm^–2 (n = 7), which was not significantly different from the response obtained in Cl^–-free solution (Fig. 2A). Pretreatment of tissues with the carbonic anhydrase inhibitor acetazolamide (100 µM, bilateral) did not influence I_SC response induced by Rut (Fig. 2, B and C). Rut-mediated peak response was strongly inhibited by pretreatment of tissues with a Na⁺-K⁺-2Cl^– cotransporter inhibitor bumetanide (100 µM, basolateral), but not by pretreatment of tissues with an epithelial Na⁺ channel inhibitor amiloride (100 µM, apical) (Table 1), suggesting that Rut-induced increase in I_SC was mainly carried out by Cl^– ions. Furthermore, the effect of Cl^– channel inhibitors on Rut-Induced I_SC was compared (Table 1) and showed that preincubation of mucosal membrane with DPC (100 µM) strongly inhibited basal I_SC by 19.8 ± 3.5 µA · cm^–2; subsequently, addition of Rut induced a response of I_SC by 5.6 ± 3.1 µA · cm^–2, which was 9.5% of control response (59.1 ± 3.8 µA · cm^–2; n = 9). In contrast, pretreatment of mucosal membrane with NPPB (100 µM) and glibenclamide (500 µM) did not influence basal I_SC, but it inhibited the subsequent Rut-induced I_SC by 50.3 ± 10.0 and 37.8 ± 6.2 µA · cm^–2, compared with their respective control responses. In contrast, the magnitude of Rut-induced I_SC was not influenced by prior application of the disulfonic stilbene DIDS (500 µM) to the mucosal side (63.1 ± 5.3 µA · cm^–2; n = 7) compared with the control value (66.0 ± 4.3 µA · cm^–2; n = 7). DPC, NPPB, and glibenclamide have been reported to block CFTR. DIDS is an inhibitor of Ca²⁺-activated Cl^– channel that is insensitive on CFTR. The combination of sensitivities to DPC, NPPB, and glibenclamide and insensitivity to DIDS suggests that the Cl^– secretion in response to Rut may be mediated via CFTR. Moreover, application of Rut to the basolateral rather than the apical surface activates apical CFTR Cl^– channel, suggesting that Rut-induced the opening of Cl^– channel can be explained via the activation of the intracellular second messengers, mostly cAMP.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Effect of ion substitution on rut-induced ISC. A, concentration-response curves for Rut-induced ISC in rat colonic epithelial preparations incubated in normal Parsons solution, solution, Cl–-free solution, and -Cl–-free solution, respectively. Each concentration of Rut was added as a single dose to separate tissues. Each point repressed as mean ± S.E.M., n = 7 to 8. B, original recordings were obtained from the tissues pretreatment with acetazolamide (100 µM, either side) or vehicle for 30 min before addition of Rut. C, summary data showed average ISC in response to Rut in the absence and presence of acetazolamide (ACTZ). Values are mean ± S.E.M., n = 6. n.s., not significantly different from control.

Involvement of Neurogenic Mechanisms in Rut-Induced Increase in I_SC. To define whether Rut-induced I_SC was involved in activation of submucosal neurons or increase of prostaglandin synthesis, Rut-induced I_SC was determined after pretreatment with neuronal blockers or indomethacin for 30 min. Figure 3B shows that preincubation of tissues with muscarinic receptor antagonist atropine (1 µM, serosal) did not change the basal I_SC or subsequent Rut-induced I_SC (61.4 ± 2.5 µA · cm^–2; n = 8) compared with the control value (64.8 ± 4.5 µA · cm^–2; n = 8). In addition, preincubating tissues with nicotinic receptor blocker hexamethonium (10 µM, serosal) also did not change the basal I_SC or subsequent Rut-induced I_SC. Whereas preincubation of basolateral membrane with neuronal blocker TTX (1 µM, serosal) decreased basal I_SC by 8.3 ± 3.1 µA · cm^–2 (from 36.3 ± 3.4 to 28.0 ± 6.0 µA · cm^–2), subsequent addition of Rut induced a response of I_SC by 11.3 ± 1.6 µA · cm^–2, which was 17.9% of control response (63.3 ± 4.2 µA · cm^–2; n = 8). Furthermore, pretreatment of tissues with the prostaglandin synthesis inhibitor indomethacin (1 µM, bilateral) slightly decreased basal I_SC by 4.5 ± 0.6 µA · cm^–2 (from 39.3 ± 3.2 to 34.8 ± 2.6 µA · cm^–2), and subsequent addition of Rut induced a response of I_SC by 19.6 ± 2.2 µA · cm^–2, which was 30.9% of control response (63.4 ± 3.9 µA · cm^–2; n = 8), indicating that Rut-induced Cl^– secretion was maintained by synthesis of prostaglandins in rat colonic mucosa. Most of the actions of prostaglandins were mediated by intracellular cAMP; therefore, Rut-induced I_SC response may be restored by the increase in intracellular cAMP levels in the presence of indomethacin. Figure 3C showed that Rut evoked an I_SC of 56.3 ± 4.6 µA · cm^–2 after intracellular cAMP level that was enhanced by the adenylate cyclase stimulator forskolin (2 µM, bilateral) in the presence of indomethacin (1 µM, bilateral), which was not statistically different from the control Rut-evoked I_SC response (63.8 ± 1.9 µA · cm^–2). Furthermore, determination of PGE₂ contents in bath solutions and tissues with a competitive enzyme immunoassay kit showed that Rut induced a significant increase in PGE₂ levels in both serosal incubate and colonic mucosa, indicating that Rut-induced Cl^– secretion is involved in prostaglandins release and synthesis in the colonic mucosa (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Effects of neuronal blockers and indomethacin on rut-induced increase in ISC. A, original recordings of Rut-induced ISC in rat colonic epithelial preparations pretreatment with indomethacin (Indo) (1 µM, bilateral) and TTX (1 µM, basolateral) for 30 min before addition of Rut to basolateral side. B, tissues were pretreated with atropine (Atrop) (1 µM, basolateral), hexamethonium (Hex) (10 µM, serosal), Indo (1 µM, bilateral), TTX (1 µM, basolateral), and their respective vehicles for 30 min before addition of Rut to basolateral side. Data are mean ± S.E.M., n = 5 to 10. n.s., not significantly different from respective vehicle. **, P < 0.01, significantly different from the respective vehicle. C, pretreatment with Indo (1 µM, bilateral) for 30 min, and Rut-induced ISC was obtained when the ISC had risen to a stable plateau level after the stimulation with forskolin (2 µM, bilateral) in the presence of indomethacin as shown in the original recordings. Dashed line indicates zero current level.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Effect of Rut on PGE2 synthesis in rat colonic mucosa. A, basolateral application of Rut for 5 min increased release of PGE2 into the serosal chamber, but it had no effect on PGE2 levels in the mucosal chamber. B, PGE2 levels in rat colonic mucosa were significantly increased after stimulation with Rut for 5 min. Each column represents mean ± S.E.M. (n = 6–9). n.s., not significantly different compared with control. *, P < 0.05 and **, P < 0.01, significantly different from the control.

Effect of Rut on Apical Cl^– Current and Basolateral K⁺ Current. To identify whether the increase of Cl^– secretion by Rut was involved in activation of the apical Cl^– channels, basolateral membrane was depolarized with high K⁺ solution in the presence of a basolateral-to-apical Cl^– gradient. Figure 5A shows that application of Rut induced a detectable outward current (5.8 ± 0.5 µA · cm^–2; n = 9), including a transient peak followed by a plateau phase, which under these conditions represented increased Cl^– efflux across apical membrane down their concentration gradient and showed activation of an apical Cl^– current. Figure 5B shows that Rut-induced I_Cl was not affected by pretreatment of tissue with DIDS (5.8 ± 0.7 µA · cm^–2; n = 5), but it was significantly inhibited by pretreatment of tissues with DPC (1.3 ± 0.2 µA · cm^–2; n = 6), glibenclamide (0.7 ± 0.2 µA · cm^–2; n = 5), and NPPB (0.8 ± 0.4 µA · cm^–2; n = 5). These data were similar to that obtained from I_SC experiments, indicating that Rut activates a clear and detectable cAMP-dependent Cl^– current in rat colonic mucosa.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Effect of Rut on apical ICl. A, after establishment of a basolateral-to-apical Cl– gradient in the high K+ solution by depolarizing the basolateral membrane, addition of Rut (100 µM, serosal) evoked a detectable outward current, consistent with a secretory Cl– flow from the basolateral-to-apical side. B, summary data showing the changes in ICl induced by Rut in the presence of NPPB (100 µM, mucosal), DPC (100 µM, mucosal), glibenclamide (Glib) (500 µM, mucosal), and DIDS (500 µM, mucosal). Each column represents mean ± S.E.M. (n = 5–9). n.s., not significantly different compared with vehicle (Rut). **, P < 0.01, significantly different from the control (Rut).

View larger version (19K): [in this window] [in a new window]   Fig. 6. Effect of Rut on basolateral K+ current. The K+ current across the basolateral membrane was measured after permeabilization of the apical membrane with nystatin in the presence of an apical-to-basolateral K+ gradient, with K+ as the sole of permeate ion. A, original recordings showing the basal and Rut-evoked outward K+ current in nystatin-permeabilized rat distal colon (100 µg · ml–1, mucosal). Dashed line indicates zero current level. B, effect of different K+ blockers on nystatin-evoked basal K+ current in rat distal colon. K+ blockers ChTX (100 nM), 293B (30 µM), CLT (30 µM), and BaCl2 (5 mM) were added to the serosal side for 30 min. C, Rut (100 µM) was added to the serosal bath for 30 min after presence of different K+ blockers in the same experiments shown as B. Each column shown as in B and C represents mean ± S.E.M. (n = 6–8). n.s., not significantly different from respective control. **, P < 0.01, significantly different from the respective control.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Interaction of Rut with cAMP-dependent secretagogues and the effect on ISC. A, original recording traces showed that Rut (100 µM, basolateral) induced an increase in ISC and subsequently increased to its maximal response by adenylate cyclase activator forskolin (5 µM, basolateral) and IBMX (100 µM, basolateral) in the presence of Rut or the effect of Rut on ISC after it reached to maximal response induced by forskolin and IBMX. After Rut or forskolin and IBMX, addition of bumetanide (100 µM, basolateral) inhibited cAMP-dependent ISC. B, average ISC in response to Rut in presence and absence of IBMX and forskolin (Fors), or the average ISC in response to IBMX and forskolin in presence and absence of Rut collected from experiments as shown A. Values are mean ± S.E.M., n = 6. **, P < 0.01, significantly different from the respective control. C, original recording traces showed that Rut (100 µM, basolateral) induced an increase in basolateral IK and subsequently increased to its maximal response by adenylate cyclase activator forskolin (5 µM, basolateral) and IBMX (100 µM, basolateral) in the presence of Rut or the effect of Rut on basolateral IK after it reached to maximal response induced by forskolin and IBMX. D, average basolateral IK in response to Rut in presence and absence of IBMX and forskolin, or average basolateral IK in response to IBMX and forskolin in presence and absence of Rut collected from experiments as shown C. Values are mean ± S.E.M., n = 6. **, P < 0.01, significantly different from the respective control.

Involvement of Ca²⁺- or cAMP-Dependent Signaling Pathway in Rut-Induced Increase in Cl^– Secretion. To identify the possible involvement of Ca²⁺- or cAMP-dependent signaling pathway in Rut-induced increase in Cl^– secretion, a pharmacological approach was taken. Preincubation with the adenylate cyclase inhibitor MDL-12,330A (50 µM, bilateral) or the cAMP-dependent PKA inhibitor H-89 (20 µM, bilateral) markedly inhibited the subsequent Rut-induced I_SC compared with their respective control values (Table 2), whereas preincubation with the Ca²⁺ chelator BAPTA-AM (10 µM, bilateral) or the protein kinase C inhibitor BIS (1.0 µM, bilateral) had no significant effect on the magnitude of the subsequent Rut-induced I_SC compared with their respective control values (Table 2). Pretreatment with a sarcoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (1 µM, bilateral) induced a transient increase in I_SC due to release of Ca²⁺ from internal calcium stores. When I_SC had returned to the basal level, addition of Rut to basolateral side induced a response that was not significantly different from that provided by same concentration of Rut without prior treatment with thapsigargin (Table 2). Furthermore, after the elimination of Ca²⁺ from serosal or mucosal side, Rut-induced response had no different effect from their respective control responses (serosal: control I_SC, 65.3 ± 4.1 µA · cm^–2, n = 6; Ca²⁺-free, 65.0 ± 5.3 µA · cm^–2, n = 6; and mucosal: control I_SC, 64.3 ± 3.4 µA · cm^–2, n = 6; Ca²⁺-free, 69.7 ± 4.3 µA · cm^–2, n = 6). These results suggest that Rut-increased Cl^– secretion was mainly mediated by a cAMP-dependent pathway. Intracellular Ca²⁺ seems to play a negligible role in I_SC response to Rut.

Rut-increased Cl^– secretion via a cAMP-dependent pathway was further characterized by measurements of intracellular cAMP level and PKA activity in rat colonic mucosa. As shown in Fig. 7A, the intracellular cAMP level under basal condition was 67.4 ± 14.3 pmol · mg^–1 protein (n = 7). After preincubation of Rut (100 µM), cAMP level was slightly enhanced to 123.6 ± 18.0 pmol · mg^–1 protein (n = 7; P < 0.05). The intracellular cAMP level in the presence of IBMX (100 µM, bilateral) was 139.3 ± 18.3 pmol · mg^–1 protein (n = 7), which was not markedly different from that induced by Rut. However, treatment of preparations with 100 µM Rut in the presence of IBMX increased cAMP level to 303.1 ± 43.3 pmol · mg^–1 protein (n = 7). There is a significant increase in cAMP level (1582.0 ± 362.3 pmol · mg^–1 protein) in the presence of both forskolin (1 µM) and IBMX (100 µM), which could not be further increased by Rut (1652.0 ± 326.7 pmol · mg^–1 protein).

View larger version (18K): [in this window] [in a new window]   Fig. 7. Effect of Rut on Intracellular cAMP content and PKA activity in rat colonic mucosa. A, cAMP content was accumulated after exposure to Rut (100 µM, basolateral), IBMX (100 µM, bilateral), Rut with IBMX, forskolin (Fors) (1 µM, bilateral) with IBMX, Rut with forskolin, and IBMX for 5 min. B, representative gel electrophoresis for PKA activity assay. Tissues were pretreated with either control alone or 100 µM Rut for 5 and 15 min. The positive and negative controls provided by the assay kit are shown in lanes 4 and 5. C, summary data showing the average PKA activities induced by Rut compared with control level. Each column represents mean ± S.E.M., n = 7 to 9. *, P < 0.05 and **, P < 0.01, significantly different from the control.

PKA activity is assayed using the PepTag nonradioactive cAMP-dependent protein kinase assay (V5340; Promega) that determines phosphorylated Kemptide, a synthetic substrate specific for PKA. As shown in Fig. 7B, phosphorylated peptide migrated toward the negative direction. Application of Rut (100 µM) to basolateral side for 5 and 10 min significantly increased PKA activity by about 21.1 and 39.5%, respectively, compared with control alone (Fig. 7C).

Rut-Induced I_SC Response Interacted with cAMP-Dependent Secretagogues. Experiments were performed to examine the effect of Rut on Cl^– secretion in presence or absence of cAMP-dependent secretagogues. Addition of Rut to the basolateral side induced an increase in I_SC by 63.3 ± 2.1 µA · cm^–2 (n = 6); subsequently, addition of cAMP-dependent secretagogues IBMX (100 µM) and forskolin (5 µM) in bilateral sides increased I_SC from 22.7 ± 2.3 to 228.7 ± 9.3 µA · cm^–2 after Rut-stimulated I_SC returned to the basal value (Fig. 8A). In contrast, when the maximal sustained I_SC response was elicited by application of 100 µM IBMX and 5 µM forskolin in bilateral sides, addition of Rut (100 µM, basolateral) inhibited I_SC from 321.7 ± 8.8 to 295.3 ± 10.6 µA · cm^–2 (n = 6) (Fig. 8B). The findings showed that pretreating tissues with Rut reduces a maximal IBMX and forskolin-stimulated I_SC response. In reverse, IBMX and forskolin-stimulated maximal I_SC response was inhibited by subsequent addition of Rut. Similar results were observed in the experiment on basolateral K⁺ current. To determine clear effects of Rut or IBMX and forskolin on basolateral K⁺ current, Rut or IBMX and forskolin were administered in the decaying phase of nystatin response (Fig. 8, C and D).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The present study showed that Rut stimulated a Cl^– secretion (an increase in I_SC) in a dose-dependent pattern in rat colonic mucosa. Evidence that Rut-stimulated I_SC was due to Cl^– secretion is based on 1) inhibition of I_SC by Na⁺-K⁺-2Cl^– cotransporter bumetanide; 2) inhibition of I_SC by cAMP-dependent Cl^– channel blockers DPC, NPPB, and glibenclamide; and 3) decrease in I_SC by substitution of Cl^– with gluconate ions in Parsons solution. Elimination of in Parsons solution or addition of the carbonic anhydrase inhibitor acetazolamide failed to influence Rut-driven I_SC. Furthermore, Rut-driven I_SC was not caused by activation of electrogenic Na⁺ absorption, because it was not affected by the epithelial Na⁺ channel inhibitor amiloride.

In mammalian colonic crypt cells, Cl^– secretion across apical membrane through Cl^– channels is predominantly CFTR, which is a major Cl^– secretory pathway that greatly contributes to regulating the amount of ions and water in the respiratory and colonic epithelia (Pilewski and Frizzell, 1999; Sheppard and Welsh, 1999). In this study, we find that Rut-induced I_SC is inhibited by CFTR inhibitors DPC, NPPB, and glibenclamide, whereas it is not inhibited by Ca²⁺-activated Cl^– channel inhibitor DIDS. Therefore, we hypothesize that apical Cl^– channels, including CFTR, may be the effective targets for Rut-mediated Cl^– secretion. Activation of apical Cl^– channels by directly activating CFTR at apical membrane or elevating intracellular cAMP content should be lead to the sustained electrogenic Cl^– secretion (Cuthbert et al., 1999). However, Rut mainly induced a transient increase in Cl^– secretion. Thus, it is doubtful that Rut-induced transient Cl^– secretion is a result of activation of apical CFTR Cl^– channel. So, it is necessary to identify whether Rut-induced Cl^– secretion is involved in the opening of apical Cl^– channels. To reach this end, we determine apical Cl^– current via depolarizing the basolateral membrane with high K⁺ in the presence of a basolateral-to-apical Cl^– gradient. The findings revealed that Rut induced a detectable sustained Cl^– current, which was also inhibited by CFTR inhibitors DPC, NPPB, and glibenclamide, but not by Ca²⁺-dependent Cl^– channel inhibitor DIDS. These observations further supported the hypothesis that the apical CFTR channel may be an effective target for Rut-mediated Cl^– secretion. The data derived from the present experiment indicate that Rut mostly induced a transient Cl^– secretion, whereas a detectable sustained response was only exhibited in the situation depolarized basolateral membrane with high K⁺. In general, cAMP-activated Cl^– secretion, including both transient and sustained phases, is dependent on the intracellular cAMP levels stimulated by secretagogues. In this study, we find that cAMP levels are less with Rut alone versus IBMX and forskolin (Fig. 7), indicating that Rut-generated cAMP levels could not sufficiently maintain a sustained Cl^– secretory response in contrast to the response obtained by IBMX and forskolin (Fig. 8).

It is essential that the activation of basolateral K⁺ channels maintains a membrane potential driving apical Cl^– secretion. Two types of K⁺ channels were reported to locate at the basolateral membrane of rat colon mucosa. One channel is cAMP-activated K⁺ channel that is predominantly Kv-LQT1 (Kunzelmann and Mall, 2002). Another channel is Ca²⁺-activated K⁺ channel that is mainly ChTX-sensitive small-conductance Ca²⁺-activated K⁺ channel (Warth et al., 1999; von Hahn et al., 2001). Under permeabilization of the apical membrane with nystatin and application of potassium gradient, Rut activated an outward K⁺ current, which was specifically diminished by a nonselective K⁺ channel blocker, Ba²⁺ ions; a KvLQT1 channel blocker, chromanol 293B; and CLT (a dual blocker of Ca²⁺-activated and cAMP-activated K⁺ channels), but not by a Ca²⁺-activated K⁺ channel blocker, ChTX. These observations indicate that cAMP-activated K⁺ channel is also a potential target for Rut in cAMP-generated Cl^– secretion.

Cl^– secretion in colonic mucosa involves the submucosal neural pathway. Pretreatment of tissues with neurotoxin tetrodotoxin inhibited both basal and Rut-stimulated I_SC, suggesting that Rut-stimulated Cl^– secretion was mediated via the activation of submucosal neural plexus. In contrast, pretreatment of tissues with muscarinic receptor antagonist atropine or nicotinic receptor blocker hexamethonium failed to alter Rut-stimulated I_SC, indicating that Rut-stimulated Cl^– secretion was not involved in activation of the acetylcholine-containing submucosal neurons. Further study revealed that the action of Rut to stimulate Cl^– secretion was mediated by cyclooxygenase-dependent metabolism of arachidonic acid, because indomethacin strongly inhibited Rut-induced I_SC. It is noteworthy that Rut-induced I_SC response is restored by the increase in intracellular cAMP level with an adenylate cyclase stimulator forskolin in the presence of indomethacin. The similar result was reported in rat colonic mucosa treated with baicalein (Ko et al., 2002). Determination of PGE₂ with enzyme-linked immunosorbent assay showed that a large part of Rut-mediated stimulation of Cl^– secretion was occurred through prostaglandins release and synthesis mostly via activation of E-prostanoid receptors. Subsequently, both the apical CFTR and basolateral cAMP-dependent K⁺ channels were activated (McNamara et al., 1999).

cAMP and Ca²⁺ are mostly the important intracellular second messengers in colonic mucosal Cl^– secretion (Cermak et al., 2002). We thus examined the roles of cAMP- and Ca²⁺-dependent signaling pathways underlying Rut-induced Cl^– secretion. Rut increased the intracellular cAMP concentration and PKA activity in colonic mucosa, suggesting that Rut may stimulate Cl^– secretion through the cAMP-PKA signal transduction pathway. The involvement of cAMP-PKA is further confirmed by the effectiveness of the adenylate cyclase inhibitor MDL-12,330A and the PKA inhibitor H-89 in reducing the responses to Rut (Table 2). In addition, Rut fails further to enhance cAMP level after the intracellular cAMP concentration is increased to the maximal level stimulated by IBMX and forskolin, which is consistent with the evidence derived from the experiments of I_SC and basolateral K⁺ current in which Rut decreases rather than increases I_SC and K⁺ current when a mixture of IBMX and forskolin elicited I_SC or K⁺ current reaches to the maximal response (Fig. 8). Similar alternations had been reported in T84 cells, in which genistein and baicalein inhibited I_SC response when cAMP-dependent channels were maximally activated by forskolin. This inhibitory effect was considered to be due to the blockage of basolateral K⁺ channels (Diener and Hug, 1996; Illek et al., 1996; Yue et al., 2004). It has been reported that the action of genistein on CFTR gating is involved in two binding sites in Hi-5 insect cells; genistein increased channel activity that reached maximum at the concentration of 35 µM through decreasing the close rate, whereas it decreased channel activity at higher concentrations through reducing open rate (Wang et al., 1998). Study of CFTR channel function within a bilayer demonstrated that genistein is able to modulate gramicidin channel function, which depends on the degree of hydrophobic mismatch between the bilayer-spanning channels and the host bilayer (Hwang et al., 2003). Therefore, similar to genistein, Rut processes dual effects of both stimulation and inhibition on the overall secretory response in the colonic mucosa.

Unlike cAMP, intracellular Ca²⁺ seems to play a negligible role in the I_SC response to Rut. Thus, pretreatment of tissue with a cell-permeant Ca²⁺ chelator, BAPTA-AM, that can chelate intracellular free Ca²⁺; a sarcoplasmic reticulum Ca²⁺-ATPase inhibitor, thapsigargin, that can lead to the depletion of Ca²⁺ stores; or with a PKC inhibitor, bisindolylmaleimide, did not significantly alter the I_SC response to Rut.

In contrast to the present experimental results, Rut has been reported to diminish prostaglandin production through inhibition of arachidonic acid release in RAW264.7 macrophages (Woo et al., 2001), and it directly inhibits COX-2 enzyme activity without altering COX-2 protein and mRNA levels in bone marrow-derived mast cells (Moon et al., 1999). It is suggested that many agents seem to exert the opposite pharmacological (excitation or inhibition) profiles acting on various tissues and cells under the different physiological and pathophysiological situations. For example, the flavonol quercetin and baicalein not only stimulated Cl^– secretion in rat colon mucosa under steady-state conditions (Cermak et al., 1998; Yue et al., 2004) but also inhibited Cl^– secretion increased by forskolin in T84 cells (Schuier et al., 2005). Moreover, Rut has been reported to cause vasorelaxation via inhibiting Ca²⁺ influx and Ca²⁺ release from intracellular stores in vascular smooth muscle cells and increasing Ca²⁺ influx in endothelial cells (Wang et al., 1996). Thus, it is not surprising that Rut stimulated Cl^– secretion by PGE₂ generation in rat colon mucosa under physiological conditions and it inhibited prostaglandin production under stimulation of inflammatory factors (Woo et al., 2001).

In summary, the evidence derived from the present study suggests that stimulation of Cl^– secretion by Rut was due to the production of endogenous PGE₂, which in turn activated both apical Cl^– channel, mostly CFTR, and basolateral K⁺ channel via a cAMP-PKA-dependent mechanism in rat colonic mucosa. Activation of both channel types would concert the secretion response from colonic crypt cells and then would help to lubricate the mucosal surface layer, to flush intestinal content, and to maintain host defense. In addition, Rut could be beneficial for treatment of colonic irritants or constipation associated with physiological and psychological stress.

	Acknowledgements

 
We acknowledge Drs. Jianye Yuan and Hailian Shi for excellent technical assistance.

	Footnotes

 
This work was supported by National Basic Research Program of China Grant 2006CB504704, the National Natural Science Foundation of China Grant 30171147, and E-Institutes of Traditional Chinese Medicine Internal Medicine, Shanghai Municipal Education Commission Grant E 03008, People's Republic of China.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.131961.

ABBREVIATIONS: Rut, rutaecarpine; COX, cyclooxygenase; CFTR, cystic fibrosis transmembrane conductance regulator; KvLQT1 (KCNQ1), voltage-dependent delayed activated K⁺ channel; PGE₂, prostaglandin E₂; PKA, protein kinase A; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; BIS, bisindolyimaleimide; ChTX, charybdotoxin; 293B, chromanol 293B; CLT, clotrimazole; DIDS, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid; DPC, diphenylamine-2-carboxylate; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide; IBMX, 3-isobutyl-1-methylxanthine; TTX, tetrodotoxin; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid; DMSO, dimethyl sulfoxide; I_SC, short-circuit current; I_Cl, Cl^– current; I_K, K⁺ current; Indo, indomethacin; MDL-12,330A, cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine.

Address correspondence to: Dr. ZhiBi Hu, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Rd., Zhangjiang Hi-tech Park, Shanghai 201203, People's Republic of China. E-mail: zhibihu{at}hotmail.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Bleich M, Briel M, Busch AE, Lang HJ, Gerlach U, Gögelein H, Greger R, and Kunzelmann K (1997) K_VLQT channels are inhibited by the K⁺ channel blocker 293B. Pflugers Arch 434: 499–501.[CrossRef][Medline]

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.[CrossRef][Medline]

Cermak R, Föllmer U, and Wolffram S (1998) Dietary flavonol quercetin induces chloride secretion in rat colon. Am J Physiol Gastrointest Liver Physiol 275: G1166–G1172.[Abstract/Free Full Text]

Cermak R, Kuhn G, and Wolffram S (2002) The flavonol quercetin activates basolateral K⁺ channels in rat distal colon epithelium. Br J Pharmacol 135: 1183–1190.[CrossRef][Medline]

Cuthbert AW, Hickman ME, Thorn P, and MacVinish LJ (1999) Activation of Ca²⁺- and cAMP-sensitive K⁺ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone. Am J Physiol Cell Physiol 277: C111–C120.[Abstract/Free Full Text]

Diener M and Hug F (1996) Modulation of Cl^– secretion in rat distal colon by genistein, a protein tyrosine kinase inhibitor. Eur J Pharmacol 299: 161–170.[CrossRef][Medline]

Fei XF, Wang BX, Li TJ, Tashiro S, Minami M, Xing DJ, and Ikejima T (2003) Evodiamine, a constituent of Evodiae Fructus, induces anti-proliferating effects in tumor cells. Cancer Sci 94: 92–98.[CrossRef][Medline]

Greger R (2000) Role of CFTR in the colon. Annu Rev Physiol 62: 467–491.[CrossRef][Medline]

Hu CP, Xiao L, Deng HW, and Li YJ (2002) The cardioprotection of rutaecarpine is mediated by endogenous calcitonin related-gene peptide through activation of vanilloid receptors in guinea-pig hearts. Planta Med 68: 705–709.[CrossRef][Medline]

Hwang TC, Koeppe RE 2nd, and Andersen OS (2003) Genistein can modulate channel function by a phosphorylation-independent mechanism: importance of hydrophobic mismatch and bilayer mechanics. Biochemistry 42: 13646–13658.[CrossRef][Medline]

Illek B, Fischer H, and Machen TE (1996) Alternate stimulation of apical CFTR by genistein in epithelia. Am J Physiol Cell Physiol 270: C265–C275.[Abstract/Free Full Text]

Iwata H, Tezuka Y, Kadota S, Hiratsuka A, and Watabe T (2005) Mechanism-based inactivation of human liver microsomal CYP3A4 by rutaecarpine and limonin from Evodia fruit extract. Drug Metab Pharmacokinet 20: 34–45.[CrossRef][Medline]

Jiang JK, Chiu JH, Yu IT, and Lin JK (2000) In vitro relaxation of rabbit and human internal anal sphincter by rutaecarpine, an alkaloid isolated from Evodia rutaecarpa. Life Sci 66: 2323–2335.[CrossRef][Medline]

Ko WH, Law VW, Yip WC, Yue GG, Lau CW, Chen ZY, and Huang Y (2002) Stimulation of chloride secretion by baicalein in isolated rat distal colon. Am J Physiol Gastrointest Liver Physiol 282: G508–G518.[Abstract/Free Full Text]

Kobayashi Y, Hoshikuma K, Nakano Y, Yokoo Y, and Kamiya T (2001) The positive inotropic and chronotropic effects of evodiamine and rutaecarpine, indoloquinazoline alkaloids isolated from the fruits of Evodia rutaecarpa, on the guinea-pig isolated right atria: possible involvement of vanilloid receptors. Planta Med 67: 244–248.[CrossRef][Medline]

Köttgen M, Löffler T, Jacobi C, Nitschke R, Pavenstädt H, Schreiber R, Frische S, Nielsen S, and Leipziger J (2003) P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport. J Clin Invest 111: 371–379.[CrossRef][Medline]

Kunzelmann K and Mall M (2002) Electrolyte transport in the mammalian colon: mechanisms and implications for disease. Physiol Rev 82: 245–289.[Abstract/Free Full Text]

Mall M, Gonska T, Thomas J, Schreiber R, Seydewitz HH, Kuehr J, Brandis M, and Kunzelmann K (2003) Modulation of Ca²⁺-activated Cl^– secretion by basolateral K⁺ channels in human normal and cystic fibrosis airway epithelia. Pediatr Res 53: 608–618.[CrossRef][Medline]

McNamara B, Winter DC, Cuffe JE, O'Sullivan GC, and Harvey BJ (1999) Basolateral K⁺ channel involvement in forskolin-activated chloride secretion in human colon. J Physiol 519: 251–260.[Abstract/Free Full Text]

Moon TC, Murakami M, Kudo I, Son KH, Kim HP, Kang SS, and Chang HW (1999) A new class of COX-2 inhibitor, rutaecarpine from Evodia rutaecarpa. Inflamm Res 48: 621–625.[CrossRef][Medline]

Pilewski JM and Frizzell RA (1999) Role of CFTR in airway disease. Physiol Rev 79: S215–S255.[Medline]

Schuier M, Sies H, Illek B, and Fischer H (2005) Cocoa-related flavonoids inhibit CFTR-mediated chloride transport across T84 human colon epithelia. J Nutr 135: 2320–2325.[Abstract/Free Full Text]

Schultheiss G, Lán Kocks S, and Diener M (2005) Stimulation of colonic anion secretion by monochloramine: action sites. Pflugers Arch 449: 553–563.[CrossRef][Medline]

Sheppard DN and Welsh MJ (1999) Structure and function of the CFTR chloride channel. Physiol Rev 79: S23–S45.[Medline]

Sheu JR, Hung WC, Wu CH, Lee YM, and Yen MH (2000) Antithrombotic effect of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, on platelet plug formation in in vivo experiments. Br J Haematol 110: 110–115.[CrossRef][Medline]

Tominaga K, Higuchi K, Hamasaki N, Hamaguchi M, Takashima T, Tanigawa T, Watanabe T, Fujiwara Y, Tezuka Y, Nagaoka T, et al. (2002) In vivo action of novel alkyl methyl quinolone alkaloids against Helicobacter pylori. J Antimicrob Chemother 50: 547–552.[Abstract/Free Full Text]

Ueng YF, Don MJ, Jan WC, Wang SY, Ho LK, and Chen CF (2006) Oxidative metabolism of the alkaloid rutaecarpine by human cytochrome P450. Drug Metab Dispos 34: 821–827.[Abstract/Free Full Text]

Ueng YF, Jan WC, Lin LC, Chen TL, Guengerich FP, and Chen CF (2002) The alkaloid rutaecarpine is a selective inhibitor of cytochrome P450 1A in mouse and human liver microsomes. Drug Metab Dispos 30: 349–353.[Abstract/Free Full Text]

von Hahn T, Thiele I, Zingaro L, Hamm K, Garcia-Alzamora M, Köttgen M, Bleich M, and Warth R (2001) Characterisation of the rat SK4/IK1 K⁺ channel. Cell Physiol Biochem 11: 219–230.[CrossRef][Medline]

Wang F, Zeltwanger S, Yang IC, Nairn AC, and Hwang TC (1998) Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating. Evidence for two binding sites with opposite effects. J Gen Physiol 111: 477–490.[Abstract/Free Full Text]

Wang GJ, Shan J, Pang PK, Yang MC, Chou CJ, and Chen CF (1996) The vasorelaxing action of rutaecarpine: direct paradoxical effects on intracellular calcium concentration of vascular smooth muscle and endothelial cells. J Pharmacol Exp Ther 276: 1016–1021.[Abstract/Free Full Text]

Wang GJ, Wu XC, Chen CF, Lin LC, Huang YT, Shan J, and Pang PK (1999) Vasorelaxing action of rutaecarpine: effects of rutaecarpine on calcium channel activities in vascular endothelial and smooth muscle cells. J Pharmacol Exp Ther 289: 1237–1244.[Abstract/Free Full Text]

Wang L, Hu CP, Deng PY, Shen SS, Zhu HQ, Ding JS, Tan GS, and Li YJ (2005) The protective effects of rutaecarpine on gastric mucosa injury in rats. Planta Med 71: 416–419.[CrossRef][Medline]

Warth R (2003) Potassium channels in epithelial transport. Pflugers Arch 446: 505–513.[CrossRef][Medline]

Warth R, Hamm K, Bleich M, Kunzelmann K, von Hahn T, Schreiber R, Ullrich E, Mengel M, Trautmann N, Kindle P, et al. (1999) Molecular and functional characterization of the small Ca²⁺-regulated K⁺ channel (rSK4) of colonic crypts. Pflugers Arch 438: 437–444.[CrossRef][Medline]

Woo HG, Lee CH, Noh MS, Lee JJ, Jung YS, Baik EJ, Moon CH, and Lee SH (2001) Rutaecarpine, a quinazolinocarboline alkaloid, inhibits prostaglandin production in RAW264.7 macrophages. Planta Med 67: 505–509.[CrossRef][Medline]

Wu SN, Lo YK, Chen H, Li HF, and Chiang HT (2001) Rutaecarpine-induced block of delayed rectifier K⁺ current in NG108-15 neuronal cells. Neuropharmacology 41: 834–843.[CrossRef][Medline]

Yi HH, Rang WQ, Deng PY, Hu CP, Liu GZ, Tan GS, Xu KP, and Li YJ (2004) Protective effects of rutaecarpine in cardiac anaphylactic injury is mediated by CGRP. Planta Med 70: 1135–1139.[CrossRef][Medline]

Yu J, Tan GS, Deng PY, Xu KP, Hu CP, and Li YJ (2005) Involvement of CGRP in the inhibitory effect of rutaecarpine on vasoconstriction induced by anaphylaxis in guinea pig. Regul Pept 125: 93–97.[CrossRef][Medline]

Yu X, Wu DZ, Yuan JY, Zhang RR, and Hu ZB (2006) Gastroprotective effect of fructus evodiae water extract on ethanol-induced gastric lesions in rats. Am J Chin Med 34: 1027–1035.[CrossRef][Medline]

Yue GG, Yip TW, Huang Y, and Ko WH (2004) Cellular mechanism for potentiation of Ca²⁺-mediated Cl^– secretion by the flavonoid baicalein in intestinal epithelia. J Biol Chem 279: 39310–39316.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.131961v1 325/1/256    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wu, D. Articles by Hu, Z. Search for Related Content PubMed PubMed Citation Articles by Wu, D. Articles by Hu, Z.