The Mechanism Underlying The Inhibitory Effect Of Vasopressin On The Contraction Of Circular Muscle Strips From Colon In Male Rats

Arginine vasopressin (AVP) is a non peptide secreted from neurohypophysis. It is traditionally recognized as a vasoconstrictor and antidiuretic hormone. Arginine vasopressin receptors are the members of G-protein coupled receptors on plasma membrane. They are divided into three subtypes, including V1 receptors (V1Rs), V2 receptors (V2Rs) and V3 receptors (V3RS). Exogenous AVP agonists induce vasoconstriction, so they are widely used as alternative non-adrenergic vasopressors for hemodynamic support of patients with critical illness or to treat the gastrointestinal (GI) hemorrhage. However, the effect of AVP might impair tissue perfusion and induce the genesis of ischemic tissue injury and impairment of smooth muscle contractility.It has been demonstrated that AVP receptors were widely expressed in the human GI tract, the effect of AVP on GI motility seemed to be dose dependent and species different. Although the effect of AVP at physiological level might not influence colonic motility of the rat and human, the effect at high level has not been clearly understood.Nitric oxide (NO) is the major relaxant of GI muscle. NO is synthesized by the activation of NO synthase (NOS) in the myenteric plexus. Released NO plays an important physiological role in various parts of the GI tract. NO regulates the muscle tone of the sphincter in the lower esophagus, pylorus, sphincter of Oddi, and anus. NO also regulates the accommodation reflex of the fundus and the peristaltic reflex of the intestine. Previous studies have shown that NOS inhibitors delay gastric emptying and colonic transit. The reduction of NOS expression, associated with impaired local production of NO, may be responsible for motility disorders in the GI tract.we hypothesized that exogenous AVP might inhibit colonic contraction in rats via activation of NO synthase (NOS) and increase of NO production. The aim of this study is to investigate the effect of AVP on the contraction of circular muscle strips motility and involved mechanism in rats.MATERIALS AND METHODSExperimental animalsIn this study, only male Wistar rats (280-320 g) were used. Before the experiments, the rats were fasted for 12 h with free access to tap water.Muscle strips preparationAfter each rat was sacrificed, a segment of proximal colon (1 cm from cecum) was cut and opened along the mesenteric border. After rinsed with Krebs solution, the rectangular sheet from the proximal colon was pinned flat (mucosa up) in a silica gel dish which was filled with Krebs solution and bubbled with 95% O2 and 5% CO2. The mucosa was carefully removed by fine forceps. Muscle strips (8×3 mm) were cut parallel to the circular fibers and were designated as circular muscle strips (CM).Recording of the contraction of muscle stripsCircular muscle strips was suspended in a chamber containing 5 mL of oxygenated (95% O2 and 5% CO2) Krebs solution (37℃). One end of CM was tied to a hook at the bottom of the chamber. The other end was connected to an isometric force transducer. The tension of CM was recorded by a polygraph. Before any chemical administration, the CM with 1 g preload was equilibrated in Krebs solution for at least 60 min. Each CM was exposed to AVP only once. The tension of CM was recorded continuously for 30 min after AVP administration. In some experiments, the strips were pretreated with one of the six specific inhibitors for 10-30 min before AVP application. These inhibitors include [deamino-Penl, O-Me-Tyr2, Arg8]-Vasopressin (V-1880, the antagonist of V,Rs), Pyrrolidine dithiocarbamate (PDTC, the inhibitor of NF-κB), N (G)-nitro-L-arginine methyl ester (L-NAME, the inhibitor of NOS), S-methylisothioure (SMT, the specific inhibitor of iNOS, tetrodotoxin (TTX, the inhibitor of the voltage dependent Na+ channel on neuron membrane) and N-Propyl-L-Arginine (NPLA, a specific inhibitor for nNOS).ImmunohistochemistryFour-micron-thick sections were prepared from 4% paraformaldehyde fixed, paraffin-embedded colonic segments that were removed from the proximal colon of male rats. Sections were de-waxed and hydrated. After antigen retrieval, the endogenous peroxidase was quenched for 10 min using 3% hydrogen peroxide in a Two-Step IHC Detection Reagent. Following three rinses in phosphate-buffered saline (PBS) and treated by 5% bovine serum for 1 h, the sections were incubated with rabbit anti-iNOS antibody (1:200) or goat anti-V1R receptor polyclonal antibody (1:100) overnight at 4℃. After washing with PBS, the sections were incubated with biotinylated secondary antibodies for 30 min at 20℃. The sections were then washed and treated with horse radish peroxidase (HRP) labeled streptavidin-complex for 30 min at 20℃. After three rinses, the peroxidase was revealed by a 3,3'-diaminobenzidine tetrahydrochloride substrate kit. At the final step, the sections were counterstained with hematoxylin. The sections of negative control were incubated with PBS instead of primary antibody.Immunofluorescence stainingV1 receptors and neuronal nuclei (NeuN) were co-located by dualimmunoflurescence staining on paraffin sections of the proximal colon. After the endogenous immunoglobulins was blocked by bovine serum, the sections were incubated in anti-V1Rs polyclonal antibody (1:100) and the anti-NeuN monoclonal antibody (1:100) overnight at 4℃. After washed with PBS, the sections were incubated for 1 h at 20℃with tetramethyl-rhodamine isothiocyanate (TRITC) (rhodamine)-conjugated rabbit anti-goat IgG (1:50) and fluorescein isothiocyanate (FITC) (fluorescein)-conjugated rabbit anti-mouse IgG(1:50).In order to co-locate V1Rs and iNOS in the proximal colon, paraffin sections from the proximal colon were incubated in anti-V1Rs polyclonal antibody (1:100) and anti-iNOS polyclonal antibody (1:200) overnight at 4℃. After washed with PBS, the sections were incubated at 20℃for 1 h with FITC (fluorescein)-conjugated donkey anti-goat IgG (1:50) and TRITC (rhodamine)-conjugated donkey anti-rabbit IgG (1:50).The immunopositive cells on the sections were detected by a fluorescence microscope.Extraction of nuclear and cytoplasmic proteinAfter incubating with AVP or vehicle for 5 min, the segments of proximal colon without mucosa were homogenized in cytoplasmic extraction reagent A (CERA). After the vortex and centrifugation at 4℃, the supernatant containing cytoplasmic proteins was collected. The sediment was dissolved in phenylmethanesulfonyl fluoride buffer (0.05 ml), diluted by same volume of nuclear extraction reagent B, and rotated for 30 min at 4℃. After centrifuged at 21 130 g for 10 min at 4℃, the supernatant was collected as nuclear extracts.Western blotThe amount of protein in the cytoplasmic or nuclear extracts was quantified by Protein Quantitative Analysis kit. The supernatant was electrophoresed and transferred to nitrocellulose membrane. The membrane was incubated in blocking buffer [5% non-fat dry milk in tween/tris-buffered salt solution (TTBS)] for 1 h at 20℃, washed in TTBS, and incubated overnight with one of the three primary antibodies, including rabbit anti-iNOS antibody (1:500), rabbit anti-NF-κB (P65) antibody (1:500), and rabbit anti-I-kB antibody (1:800). After multiple washes, the membranes were incubated at 20℃for 1 h with secondary antibodies (1:20000) conjugated with HRP. The immunopositive proteins on the membrane were detected by ECL plus.Measurement of NO content in proximal colonAfter incubating with AVP (10-8 mol/L) or vehicle for 5 min, the segments of proximal colon without mucosa layer were homogenized. After centrifuging at 94 g for 5 min at 4℃, supernatant (50μL) was mixed with an equal volume of Griess reagents I and II at room temperature. The amount of nitrite production was measured at the absorbance of 560 nm by a Universal Microplate Spectrophotometer. Data analysisBoth the tonic and phasic contraction of the muscle strips were simultaneously recorded during the experiments. In order to facilitate the quantification of the muscle contractions, the recording trace was integrated with an interval of 10 s. The mean tension in 1 min was calculated by dividing the summarized integration value with the corresponding period (60 s). The mean tension of the muscle strips before AVP treatment was defined as reference value. The average tension for a period after each chemical treatment was normalized to a standardized ratio (R) where the reference value for each experiment was equal to one. The R value was taken as the change in muscle contraction due to each AVP treatment.The Western blot band was quantified by Scion Image software. The band was expressed as relative protein amounts compared to c-jun (nuclear protein) orβ-actin (cytoplamic protein).All the values in these experiments were presented as mean±SEM. Significant differences between several treatment groups and one control group were determined by one way ANOVA on ranks followed by Dunn's test, and that between two groups was determined by student's t-test. P< 0.05 was considered to be a significant difference.Key Results(1) Effects of exogenous AVP on the contraction of CM of proximal colon in male rats. Arginine Vasopressin (10-8 mol/L) inhibited the contraction of CM. The spontaneous contraction of the strips decreased immediately after AVP administration, reached the lowest level at 3-5 min, and returned to normal at 12 min. Pretreatment with V-1880 (10-7 mol/L), a potent V1Rs antagonist, abolished this effect. The inhibitory effect of AVP on muscle contraction was in a dose-dependent manner. Higher concentration of AVP (10-10～10-6 mol/L) significantly decreased the muscle contraction, whereas, the effect was not observed in lower level (10-12 and 10-11 mol/L).(2) Effect of NOS inhibitors on AVP-induced inhibition on CM contraction. Both L-NAME (10-4 mol/L) and NPLA (10-7 mol/L) increased the tension of CM while SMT (10-3 mol/L) did not exert any effect. Pretreatment with L-NAME (10-4 mol/L) significantly attenuated the inhibitory effect of AVP (10-8 mol/L) on the contraction of the muscle strips. Similar results were found in the groups pretreated with SMT or NPLA. The inhibitory effect of L-NAME was greater than that of NPLA at 3 min following AVP administration.(3) Involvement of enteric nervous system. Tetrodotoxin (TTX) itself increased the CM contraction. Pretreatment of TTX (10-5 mol/L) significantly reversed the inhibitory effect of AVP on CM contraction.(4) NO content and iNOS expression in colon following AVP treatment. Arginine vasopressin (10-8 mol/L) significantly increased the production of NO and iNOS expression in proximal colonic segments. With the pretreatment of SMT, the increase of NO content following AVP (10-8 mol/L) administration was significantly attenuated. Pretreatment of PDTC (10-3 mol/L), the inhibitor of NF-κB, significantly reversed this increase in iNOS expression following AVP administration. Same dose of PDTC attenuated the inhibitory effect of AVP (10-8 mol/L) on CM contraction.Effect of AVP administration on the expression of cytoplasmic I-κB and nuclear NF-κB (P65) in proximal colon. Arginine vasopressin (10-8 mol/L) significantly decreased the amount of cytoplasmic I-κB and increased the nuclear P65 in colon. Pretreatment of PDTC (10-3 mol/L) partly reversed these changes.(5) Localization of V1Rs in colon.V1 receptors-positive cells were located in myenteric plexus of proximal colonic sections. A double staining was performed for V1Rs and NeuN and the result indicated that both V1Rs and NeuN were expressed in myenteric plexus. Inducible NOS was detected in the myenteric plexus and V1Rs and iNOS were also co-located in the cells in myenteric plexus.Conclusions:In this study, we found that exogenous AVP inhibited the contraction of CM in rat colon through production of NO via activation of two pathways, including NF-κB-iNOS and nNOS, in myenteric plexus.