5_HT_7受体对肠道运动的调节作用_邹百仓.pdf
Introduction ! In traditional medicine the powdered rhizome of ginger (Zingiber officinale Roscoe, Zingiberaceae) has been used for a long time to alleviate the symptoms of gastrointestinal diseases 13. Clin- ical studies have shown that ginger is effective in reducing motion sickness, postoperative nausea and vomiting, hyperemesis gravidarum, and gas- tric hypomotility 47. It has been hypothesized that the pharmacological activity of ginger lies in its pungent principles (gingerols and shogaols) and volatile oils (sesquiterpenes and monoter- penes) 8. The major chemical constituents of the nonvolatile fraction of ginger are the arylal- kanes 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol 3. Attempts were made to characterize the recep- tor(s) that might be responsible for the thera- peutic benefit of ginger on gastrointestinal func- tion. The mechanism of action of ginger is thought to lie in the ability of its active compo- nents to affect serotonin (5-HT) and muscarinic receptors in the gastrointestinal tract 911. It has been shown that ginger possesses antiemet- icpropertiesagainstchemotherapy-induced vomiting in laboratory animals 1214 as well as in humans 15. The antiemetic effect of gin- ger was attributed to an antagonist interaction with 5-HT3receptors 14. Interestingly, a con- tribution of the volatile oil to the 5-HT3recep- tor-mediated antiemetic effect of ginger has re- cently been suggested 16. However, ginger was less effective in reducing cisplatin-induced emesis than the selective 5-HT3receptor antag- onistgranisetron14.Furthermore,ginger failed to prevent apomorphine-induced emesis, ruling out an involvement of dopamine recep- tors 14. In addition, it has been suggested that the antiemetic effect of ginger is associated with an effect on gastric emptying; ginger and its ac- tive constituents 6-gingerol, 8-gingerol, 10- Abstract ! Theherbal drug ginger (Zingiber officinale Roscoe) may be effective for treating nausea, vomiting, and gastric hypomotility. In these conditions, cholinergic M3receptors and serotonergic 5-HT3 and 5-HT4receptors are involved. The major chemical constituents of ginger are 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol. We studied the interaction of 6-gingerol, 8-ginger- ol, 10-gingerol (racemates), and 6-shogaol with guinea pig M3receptors, guinea pig 5-HT3 receptors, and rat 5-HT4receptors. In whole seg- ments of guinea pig ileum (bioassay for contrac- tileM3receptors),6-gingerol,8-gingerol, 10-gingerol, and 6-shogaol slightly but signifi- cantly depressed the maximal carbachol response at an antagonist concentration of 10M. In the guinea pig myenteric plexus preparation (bioas- say for contractile 5-HT3receptors), 5-HT maxi- mal responses were depressed by 10-gingerol from 933% to 656% at an antagonist concen- tration of 3M and to 483% at an antagonist concentrationof5Mfollowingdesensitizationof 5-HT4receptors and blockade of 5-HT1and 5-HT2 receptors. 6-Shogaol (3M) induced depression to 613%. In rat esophageal tunica muscularis mucosae (bioassay for relaxant 5-HT4receptors), 6-gingerol, 8-gingerol, 10-gingerol, and 6- shogaol (26.3M) showed no agonist effects. The maximal 5-HT response remained unaffected in the presence of the compounds. It is concluded that the efficiency of ginger in reducing nausea and vomiting may be based on a weak inhibitory effect of gingerols and shogaols at M3and 5-HT3 receptors. 5-HT4receptors, which play a role in gastroduodenal motility, appear not to be in- volved in the action of these compounds. Effects of Ginger Constituents on the Gastrointestinal Tract: Role of Cholinergic M3 and Serotonergic 5-HT3and 5-HT4Receptors AuthorsHeinz H. Pertz1, Jochen Lehmann2,3, Ren Roth-Ehrang2, Sigurd Elz4 Affiliations 1Institut fr Pharmazie, Freie Universitt Berlin, Berlin (Dahlem), Germany 2Institut fr Pharmazie, Friedrich-Schiller-Universitt Jena, Jena, Germany 3College of Pharmacy, King Saud University, Riyadh, Saudi Arabia 4Institut fr Pharmazie, Universitt Regensburg, Regensburg, Germany Key words l ginger l Zingiber officinale Roscoe l Zingiberaceae l gastrointestinal diseases l cholinergic M3receptors l 5HT3receptors l 5HT4receptors receivedOctober 18, 2010 revisedJanuary 10, 2011 acceptedJanuary 14, 2011 Bibliography DOI http:/dx.doi.org/ 10.1055/s-0030-1270747 Published online February 8, 2011 Planta Med 2011; 77: 973978 Georg Thieme Verlag KG Stuttgart New York ISSN 00320943 Correspondence Prof. Dr. Heinz H. Pertz Institut fr Pharmazie Freie Universitt Berlin, Knigin-Luise-Strae 2+4 14195 Berlin (Dahlem) Germany Phone: +493083853135 Fax: +493083855576 hpertzzedat.fu-berlin.de 973 Pertz HH et al. Effects of GingerPlanta Med 2011; 77: 973978 Original Papers Downloaded by: IP-Proxy CONSORTIUM:360Counter (University of Florida), University of Florida. Copyrighted material. gingerol, and 6-shogaol have been reported to enhance gas- tric emptying of a charcoal meal in mice 17. This finding is in line with the ability of ginger extracts to reverse cisplatin-in- duced delay in gastric emptying in rats 18. The effect of gin- ger on gastric emptying was similar to that caused by metoclo- pramide, a 5-HT4receptor agonist with antagonist properties at 5-HT3receptors, and by ondansetron, a selective 5-HT3recep- tor antagonist 17,18. Important evidence for the participation of 5-HT4receptors in gastric emptying induced by gastrointes- tinal prokinetics has been provided 19,20. These findings un- equivocally rule out any relevant contribution of 5-HT3receptor blockade in the pharmacological action of gastroprokinetics such as metoclopramide 21. The aim of the present study was to evaluate the effectiveness of 6-, 8-, and 10-gingerol and 6-shogaol at cholinergic M3re- ceptors in whole segments of guinea pig ileum, at serotonergic 5- HT3receptors of the longitudinal muscle-myenteric plexus prep- aration of guinea pig ileum, and at 5-HT4receptors of rat esopha- geal tunica muscularis mucosae. Based on the pharmacological profile of 6-gingerol, 8-gingerol, 10-gingerol, and 6-sho- gaol, an antiemetic effect of ginger may be caused by the weak inhibitory properties of these compounds at the M3and 5-HT3 receptors. The prokinetic properties of ginger are not mediated by a specific interaction with 5-HT4receptors. Materials and Methods ! Chemicals 6-Gingerol, 8-gingerol, and 10-gingerol (racemates) were synthesized in-house according to previously described standard procedures 22. 6-Shogaol was obtained from 6-gingerol by dehydration. The purity of the compounds was 95% (analyzed by HPLC). The following drugs were obtained as gifts: methyser- gide hydrogen maleate (purity 99%) and tropisetron hydro- chloride (purity 99%) (Novartis). The following compounds were purchased: atropine sulfate (purity 97%) and cocaine hy- drochloride (purity 98.5%) (Merck); carbachol (purity 98%), corticosterone (purity 92%), 5-methoxytryptamine (purity 97%), pargyline hydrochloride (purity 98%), and SDZ-205557 hydrochloride (purity 99%) (Sigma-Aldrich); and choline chlo- ride (purity 99%) and 5-hydroxytryptamine creatinine sulfate (purity 99%) (Janssen). Animals Animal studies were approved by Landesamt fr Gesundheit und Soziales Berlin, Germany (ZH 12, T0035/00). All experimental procedures carried out in the present studies followed the guide- linesof the Animals (ScientificProcedures) Act 1986. Male Wistar rats (250300g) and guinea pigs (250450g) were housed in plastic cages in a special temperature-controlled room (222C, 50% humidity) on a 12:12-h light/dark cycle (light on at 07:00). They were allowed ad libitum access to food and water. Guinea pigs were stunned by a blow to the neck and bled. Rats were killed by decapitation following asphyxiation with CO2. Cholinergic M3receptor assay (guinea pig ileum) Whole segments of guinea pig ileum (1.5cm in length) were dis- sected and suspended in a water-jacketed 20-mL organ bath that contained Tyrode solution of the following composition (mM): NaCl 137, KCl 2.7, CaCl21.8, MgCl21.0, NaH2PO40.4, NaHCO3 11.0, and glucose 5.6. The solution was gassed with 95% O2/5% CO2and warmed to a constant temperature of 37C (pH 7.4). One end of the segment was fixed on the bottom of the organ bath and the other was attached to an isotonic lever for continu- ous measurement of length changes. Preload at the beginning of the experiment was 7.5mN. After a stabilization period of 20min, the segments were stimulated three times with carba- chol (1M) for a period of 45min until the magnitude of the con- tractile response became constant. Four cumulative concentra- tion-response curves were constructed at intervals of 15 20min: the first curve to carbachol and the second through fourth curves to carbachol in the presence of antagonist or ve- hicle. Antagonists were incubated for 10min. Previous studies by this group showed that successive concentration-response curves to carbachol in the absence of antagonist (control curves) were highly reproducible in guinea pig ileum (no differences in pEC50or Emaxvalues). Contractile responses were expressed as a percentage value of the maximal response to carbachol in the first curve. Serotonergic 5-HT3receptor assay (guinea pig myenteric plexus) Strips of longitudinal muscle with adhering myenteric plexus from guinea pig ileum (1.5cm in length) were prepared as previ- ouslydescribed 23. The strips were placedfor the measurement of isometric changes in tension in 20-mL organ baths filled with Tyrode solution that additionally contained choline (1M) for de novo synthesis of acetylcholine, 5-methoxytryptamine (10M) for desensitization of 5-HT4receptors, and methysergide (1M) for blockade of 5-HT1and 5-HT2receptors 24. The solution was continuously gassed with 95% O2/5% CO2and warmed to a constant temperature of 37C (pH 7.4). The resting tension was adjusted to 7.5mN at the beginning of each experiment. After a stabilization period of 30min, the tissues were stimulated three times with 5-HT (3M) for a period of 60min. Two noncumula- tive concentration-response curves were constructed at an inter- val of 60min on each tissue by adding increasing concentrations of 5-HT (130M) to the organ bath. Each concentration was left in contact with the tissue for 1530s followed by washout with Tyrode solution (100mL). The next higher concentration of 5-HT was administered 15min later. Antagonists or vehicle were added 30min before the second concentration-response curve and were retained in the bath fluid during construction of the second curve. Contractile responses were expressed as a percent- age value of the maximal response to 5-HT in the first curve. Two noncumulative concentration-response curves to 5-HT on the same tissue (control curves) were highly reproducible, with the pEC50of the first curve (5.570.02) being practically identical to that of the second curve (5.500.06; n=24). The Emaxof the sec- ond 5-HT curve was 971% relative to the Emaxof the first curve. Serotonergic 5-HT4receptor assay (rat esophageal tunica muscularis mucosae) The entire rat esophagus was excised and the outer striated muscle layer of the tunica propria was removed by microdissec- tion, leaving behind the inner smooth muscle tube of the tunica muscularis mucosae 25. The muscle tube was divided into a proximal (cervical), middle (thoracic), and distal (abdominal) segment. From each segment, 23 strips were cut longitudinally and placed in Tyrode solution (pH 7.4, 37C) for measurement of isometric changes in tension. The Tyrode solution additionally containedmethysergide (1M) toinhibit 5-HT1and 5-HT2recep- tors and cocaine (30M) and corticosterone (30M) to inhibit 974 Pertz HH et al. Effects of GingerPlanta Med 2011; 77: 973978 Original Papers Downloaded by: IP-Proxy CONSORTIUM:360Counter (University of Florida), University of Florida. Copyrighted material. neuronal and cellular uptake of 5-HT. The resting tensionwas ad- justed to 1.5mN at the beginning of each experiment. During a stabilization period of 30min, the tissues were exposed to pargy- line (100M) to inhibit monoamine oxidases. The tissues were allowed to equilibrate for a further 30min. After the stabilization and equilibration period, during which the tissues were washed with Tyrode solution (200mL) every 15min, the tissues were contracted with a submaximal concentration of carbachol (3M) until the magnitude of the contractile response became constant (usually after 40min). The relaxant response to 5-HTor arylalkanes was studied by constructing a cumulative concentra- tion-response curve. For determination of antagonist affinity, a second concentration-response curve to 5-HTwas performed fol- lowing a further carbachol-induced (3M) contraction. Antago- nists or vehicle were incubated for 60min. Effects were ex- pressed as a percentage value of the maximal relaxation evoked by 5-HT in the first curve. Two cumulative concentration-re- sponse curves to 5-HT on the same tissue (control curves) were highlyreproducible,withthepEC50ofthefirstcurve (8.020.07) being practically identical to that of the second curve (8.000.06; n=15). The Emaxof the second 5-HT curve was 1002% relative to the Emaxof the first curve. Data presentation and statistical evaluation Data are presented as the mean SEM for n animals. Agonist po- tencies and maximal responses are expressed as pEC50values (negative logarithm of the molar concentration of agonist pro- ducing 50% of the maximal response) and Emaxvalues, respec- tively. In cases where an antagonist caused a parallel shift of an agonist curve, a Schild plot of log (concentration ratio 1) versus log (antagonist concentration) was constructed, and a pA2was calculated from the x-intercept of this plot. A slope of the Schild plot of 1 indicated competitive antagonism. Where appropriate, differences between means were deter- mined by Students t-test (two-tailed), after checking the homo- geneity of the variances. P values of less than 0.05 were consid- ered to indicate a significant difference between the responses being compared. Results ! The effects of 6-gingerol, 8-gingerol, 10-gingerol, and 6- shogaol on cholinergic M3receptors are shown in l Fig.1AD. In contrast to the effect of the reference antagonist atropine, which behaved as a competitive M3receptor antagonist (pA2= 8.810.03, slope of the Schild plot=1.050.03; l Fig.1E), 6- gingerol, 8-gingerol, 10-gingerol, and 6-shogaol (130M) failed to evoke a rightward shift of the concentration-response curve to carbachol. However, the Emaxvalues of carbachol were significantly depressed at an antagonist concentration of 10M. Thus, ginger ingredients affected cholinergic M3receptors only at high concentrations and in a noncompetitive manner. 6-Gin- gerol, 8-gingerol, 10-gingerol, and 6-shogaol did not evoke a contractile response via stimulation of cholinergic M3receptors. The effects of 6-gingerol, 8-gingerol, 10-gingerol, and 6- shogaolonserotonergic5-HT3receptorsareshownin l Fig.2AD. The 5-HT3r
