Oxytocin (OT) is a nonapeptide. The wellknown physiological function of OT was to regulate uterine contractions and milk ejection reflex in humans and rats. In recent years, more and more studies indicated that both OT and OTR mRNA were expressed throughout the human gastrointestinal (GI) tract and might be involved in the regulation of GI motility.Cholecystokinin (CCK) is a classical GI hormone. It is reported that in vivo, systemic OT inhibited gastric emptying and intestinal transit, and decreasd the intragastric pressure. This effect was abolished by lorglumide, the CCK1 receptor antagonists. The OT concentration in plasma increased following OT administration. So it is possible that OT inhibited the gastric and intestinal motility via CCK but the mechanism was unknown. We hypothesize that OT might increase the relase of CCK in duodenum.In order to test this hypothesis, we investigated the effect of OT on the contraction of longitudinal muscle strips of duodenum. The possibility that CCK was involved in the OT effect on muscle contraction was tested. The expression of CCK mRNA was detected by RT-PCR analysis. The CCK protein was detected by ELISA. The tissue and cell that express OTR and CCK were located by immunohistochemistry and immuno fluorescence.MATERIALS AND METHODSAnimal and muscle strips preparationMale Wistar rats, weighing 280-300 g, were fasted overnight with free access to water before experiments. The muscle strips of duodenum were prepared as described elsewhere with some modification. Briefly, after rat was decapitation, a segment of duodenum (1 cm from pylorus) was removed immediately, opened along the mesenteric border and the resulting rectangular sheet was pinned flat (mucosa up) in a silica gel dish filled with oxygenated Krebs solution. Muscle strips (4 mm wide,10 mm long) parallel to the long axis of the longitudinal fibers were cut and used to record the tension of the longitudinal muscle fiber of duodenum The muscle strips were suspended in tissue chambers containing 5 ml Krebs solution (37℃) and bubbled continuously with 95% O2 and 5% CO2. Isometric contraction of the strips was monitored.RT-PCRWhole thick tissues of the duodenum (50-100 mg) were removed and stored at-80℃. The frozen specimens were homogenized with an electrokinetic homogenizer in Trizol (Invitrogen, Shanghai, China) for total RNA extraction. RNA reverse transcription was completed using the high-capacity cDNA reverse transcription kit (4368814; Applied Biosystems). Oligonucleotide primers were designed on the basis of the DNA sequence of rat CCK. A thermal cycle program according to the manufacturer’s protocol was conducted to amplify the homologus genes. The PCR-amplified fragment size of CCK was 350 bp. The products were separated by electrophoresis on a 1.5% agarose gel. The RT-PCR results were quantified using Scion Image software, background band was subtracted and the data were expressed as relative mRNA amounts compared toβ-actin (cytoplamic protein).Cells isolation and CCK ELISAMale Wistar rats, weighing 280-300g, were fasted overnight with free access to water before experiments. After anesthetized with amobarbital sodium (0.056 g/kg), a segment of duodenum (1 cm from pylorus) was removed immediately, opened along the mesenteric border and the resulting rectangular sheet was pinned flat (mucosa up) in a silica gel dish filled with oxygenated Krebs solution. The sheet was transfered to a germ free Krebs solution, the mucosa, submucous layer and circular muscle was teared with microinstrument under anatomical lens. The left sheet (longitudinal muscle-myenteric plexus, LMMP) was incubated for 50 min in papayotin solution (6mg/ml) at 37℃,and then incubated in collagenaseⅡsolution (1mg/ml) for 50 min. Cells was blowed for 30 times with suction-pipe, then centrifuged at 2,000 g at room temperature for 10 min, suspended in 1ml NBA. The culture medium was centrifuged at 1,000 g at room temperature for 10 min. The supernatants were stored at-20℃until assayed. CCK levels in the culture medium were measured by ELISA using a CCK ELISA commercial kit (GBD, C033-80).ImmunohistochemistryWhole thick tissue of duodenum was fixed in 4% paraformaldehyde, then rinsed, ehydrated, cleared, and immersed in wax. The tissue was then sectioned with 4-μm thickness. Sections were stained using a two-step method. After eparaffinaged, rehydrated, rinsed, antigens restoration,3% hydrogen peroxide was used to block the activity of endogenous peroxidase for 15 min, and then the sections were incubated with primary goat anti-OT receptor antibody (diluted 1:50 in PBS; Santa Cruz Biotechnology) and primary rabbit anti-CCK antibody (diluted 1:150 in PBS; Millipore) overninght in a humid chamber at 4℃. After rinsed 3 times in PBS, the sections were incubated with polymer peroxidase goat anti-rabbit serum (ZSGB-BIO, Beijing, China) for 30 minutes at room temperature. Then the sections were washed and treated with HRP-labeled streptavidin-complex (ZSGB-BIO) for 30 minutes at room temparture. After 3 rinses, peroxidase was revealed by a 3,3-diaminobenzidine tetrahydrochloride substrate kit (ZSGB-BIO). In negative controls, the sections were incubated with PBS instead of primary antibody.Tri-labeling ImmunofluorescenceThe procedure is similar to immunochemistry above with some modification. After prefusion in 5% for 1 h, the sections were incubated with the primary antibody mixture composed of primary goat anti-OT receptor antibody (diluted 1:50 in PBS; Santa Cruz Biotechnology), primary rabbit anti-CCK antibody (diluted 1:150 in PBS; Millipore) and primary mouse anti-NeuN antibody (diluted 1:50; Millipore) overninght in a humid chamber at 4℃. After rinsed 3 times in PBS, the sections were incubated with the secondary antibody mixture composed FITC-labeled donkey anti-goat (catalog no. sc-2024, Santa Cruz Biotechnology), RB-200-labeled donkey anti- rabbit IgG (catalog no. sc-2095, Santa Cruz Biotechnology), and CyTM5-labeled donkey anti-mouse (catalog no.715-175-150, Maojian Biotechnology limited company) for 1h at room temperature. In negative controls, the sections were incubated with PBS instead of primary antibody.Tri-labeling Immunocytochemistry.The cells isolation from LMMP was similar to that described above. The isolated cells were suspended in lml NBA, cultured at 37℃for 5 days. The culture medium was refreshed every day.Before the experiments, the cells were fixed using 4% (wt/vol) paraformaldehyde for 20 min, and then washed 3 times,5 min each time, with PBS. After preincubated in 5% donkey serum for 1 h, the cells were incubated with the primary antibody mixture composed of primary goat anti-OT receptor antibody (diluted 1:50 in PBS; Santa Cruz Biotechnology), primary rabbit anti-CCK antibody (diluted 1:150 in PBS; Millipore) and primary mouse anti-NeuN antibody (diluted 1:50; Millipore) overninght in a humid chamber at 4℃. After rinsed 3 times in PBS, the cells were incubated with the secondary antibody mixture composed FITC-labeled donkey anti-goat (catalog no. sc-2024, Santa Cruz Biotechnology), RB-200-labeled donkey anti-rabbit IgG (catalog no. sc-2095, Santa Cruz Biotechnology), and CyTM5-labeled donkey anti-mouse (catalog no.715-175-150, Maojian Biotechnology limited company) for lh at room temperature, In negative controls, the sections were incubated with PBS instead of primary antibody.RESULTSEffect of OT on the spontaneous contraction of the muscle strips.OT (10-5 M and 10-6 M) inhibited the spontaneous contraction of the muscle strips. The contraction of the muscle strips decreased immediately after OT administrated, reached the lowest level at the 30 s and returned to normal 1 min later. Thirty second following the two doses of OT administration (10-5M and 10-6 M), the R value of the muscle strip decreased from 1 (baseline) to 0.838±0.021 (P<0.05.n=6) and 0.759±0.842 (P<0.05, n=6). Lower dose of OT (10-7 M) did not significantly influence the contraction of the muscle strips. Effect of atosiban, TTX and lorglumide on the inhibitory effect of OT on duodenal muscle stripsPretreatment with atosiban (10-6 M), a competitive antagonist of oxytocin receptors, or TTX (10-5 M), the specific blocker of voltage-gated Na+ channels in the nerve fiber, or Lorglumide (3×10-6M), a potent CCK1 receptor antagonist, completely blocked the inhibitory effect of OT at 10-6 M on the contractions of the musle strips.Amount of of CCK mRNA on duodenum with OT treatmentThe amount of CCK mRNA expressed on duodenum did not change within 3 min following OT (10-7M-10-5M) administration.Amount of CCK expression in culture medium following OT treatmentThe amount of CCK expression in culture medium of the cells isolated from LMMP of duodenum increased significantly following OT (10-6 M) administration. Pretreatment with atosiban inhibited this effect of OT..Location of OTR and CCK in duodenumThe OTR immunoreactivity was expressed on the cell of enteric nerve plexus and smooth muscle of duodenum. No immunoreactive cell was detected in control sections where tissues were treated with PBS instead of primary antibody. The CCK immunoreactive cells were located at the enteric nerve plexus of duodenum. No immunoreactive cell was detective in control tissues in with PBS was treated instead of primary antibody.OTR and CCK were coexpressed on the neurons of myenteric nerve plexus of duodenum.CONCLUSIONAcute exposure of oxytosin inhibited the contraction of the longitudinal muscle strips in rat duodenum in rats through activation of OTR and the subsequent release of CCK from neurons in myenteric plexus.