BACKGROUND: Previous researches have proved that both atrial natriuretic polypeptide (ANP) and 5-hydroxytryptamine (5-HT) exist in the same endocrine granule of enterochromaffin cell (EC). However, whether ANP may promote or inhibit synthesis and secretion of 5-HT needs to be further studied. OBJECTIVE: To investigate the effect of ANP on synthesis and secretion of 5-HT in EC of rat gastric mucosa.DESIGN: Randomized controlled animals study.SETTING: Immunology Laboratory, Chengde Medical College. MATERIALS: This study was performed at the Immunology Laboratory, Chengde Medical College from October 2004 to July 2007. Forty adult male Wistar rats were provided by the Experimental Animal Center of Chengde Medical College. The experiment was in accordance with animal ethics standards. ANP, 5-HT antibody and serum were provided by Santa Cruz Biotechnology Company, USA.METHODS: Forty rats were randomly into endocrine and exocrine groups, and rats in the two groups were sub-grouped into control and experimental groups with 10 in each group. ANP (28 μg, 14 mg/L) was directly injected into the stomach to mimic ANP luminal secretion and ANP (14 μg, 14 mg/L) was directly injected into the sublingual vein to mimic ANP endocrinal secretion. In above condition, 5-HT immunoreactive positive cell was displayed by using immunohistochemistry technique, numerical density (Nv) of endocrine granule (SG) was counted by using electron microscopic morphometry, and 5-HT level in the serum was measured by using HPLC-ECD technique. And then, the results were compared to the control group. MAIN OUTCOME MEASURES: Effects of ANP on density of 5-HT immunoreactive positive cell, numerical density (Nv) of SG and 5-HT level in the serum. RESULTS: Effect of luminal and endocrine ANP on the 5-HT secretion: The density of immunoreactive positive cell and the numerical density (Nv) of SG were significantly increased by luminal and endocrine ANP (P＜0.05), while 5-HT level in serum was significantly reduced by luminal and endocrine ANP (P＜0.05). CONCLUSION: Luminal and endocrinal ANP can inhibit 5-HT release of gastric mucosa but may not change or enhancement its synthesis in rat.

BACKGROUND: Development of liver fibrosis accompanies many morphological and functional changes. The pathological alterations of dimethylnitrosamine (DMN)-induced liver fibrosis in rats are similar to those of human liver fibrosis.OBJECTIVE: To observe the dynamic changes in morphology and serum hyaluronic acid (HA), laminin (LN), and type Ⅳ collagen in rats with liver fibrosis induced by DMN.DESIGN: Randomized controlled animal trial.SETTING: Laboratory of Teaching and Research Section of Pathology, College of Medicine, Yanbian University.MATERIALS: Eighty 3-month-old male rats of clean grade with 175-200 g body mass were selected, which were provided by the animal center of College of Medicine, Yanbian University. Agents: Dimethylnitrosamine provided by Sigma company, α smooth muscle actin by Dako company, Sirius red by Aldrich chem company, serum hyaluronic acid,laminin and type Ⅳ collagen kit by Sino-American Biotechnology Company, rabbit-anti-rat I g by Dako, Denmak company. Devices: JEM-1200EX transmission electron microscope made in Japan; enzyme linked immuno analyzer made in Japan; and CMTAS multifunction true color pathological image analysis system developed by Beijing University of Aeronautics and Astronautics.METHODS: The experiment was conducted in the College of Medicine, Yanbian University from June 2004 to December 2005. The rats were divided into 2 groups by lot: model group (n =40): The rats were intraperioneally injected with 10 g/L DMN (10 μL/kg) once daily, 3 days a week for 4 weeks; control group (n =40): The matching normal saline was injected at the same period; the blood from the left ventricle was collected and frozen in refrigerator at -70 ℃ before the rats were killed at days 7, 14, 21, and 28 and the liver tissue was removed for electron and light microscope observation. ①The dynamic changes in the content of serum HA, LN and type Ⅳ collagen were measured by enzyme-linked immunoabsorbent assay (ELISA). ②The morphological changes and liver fibrosis grading were examined by HE staining,immunohistochemical Sirius-red staining (liver fibrosis degree was classified into 5 grades: grade 0: no fibrosis; grade 1:fibrosis in portal area; grade 2: fibrotic septa between portal tracts; grade 3: fibrosis septa and structure disturbance of hepatic lobule; grade 4: cirrhosis), meanwhile, the area-density percentage of collagen fibrosis was calculated. ③The hepatic stellate cells were detected with transmission electron microscope and immunohistochemical alpha smooth muscle actin (SMA) staining. ④The correlation between area-density percentage of collagen fibrosis during liver fibrosis formation and the serum levels of HA, LN and type Ⅳ collagen was analyzed.MAIN OUTCOME MEASURES: ①The changes in the serum levels of HA, type Ⅳ collagen and LN during liver fibrosis formation; ②The morphological changes and liver fibrosis grading and area-density percentage of collagen fibrosis; ③Transformation and distribution characteristics of hepatic stellate cells; ④The correlation between area-density percentage of collagen fibrosis and the serum levels of HA, LN and type Ⅳ collagen.RESULTS: Among the 80 rats, 34 of the experimental group were modeled successfully, which were involved in the result analysis with the 40 rats in the control group. ①The levels of serum HA, type Ⅳ collagen and LN of the model group were significantly higher compared with the control group from day 7 to 28 (P ＜ 0.05), especially that on the 28th day. ②In the model group, the portal area of the rats showed hemorrhagic necrosis at day 7 after injection of DMN; at day 14,hemorrhage, necrosis and thin fibrotic septa joining central areas of liver were found; at days 21 and 28, thick septa was found; The area-density percentage of collagen fibrosis of the model group was significantly higher compared with the control group at days 7, 14, 21 and 28 (P ＜ 0.05), especially that on the 28th day. There were significant differences in the liver pathologic grading between the two groups at each time point (P ＜ 0.01); the pathologic grading of the model group at day 7 differed from those at days 14 and 28 (P ＜ 0.01). ③The α-SMA positive cells and a transitional hepatic stellate cell were found under the electron microscopy; typical myofibroblast was observed in the model group at day 21 and 28 under the electron microscopy. ④The area-density percentage of collagen fibrosis was positively correlated with the content of serum HA, LN and type Ⅳ collagen (r=0.707, 0.675, 0.662, P＜ 0.01).CONCLUSTON: There are significantly progressional changes in morphological and serum levels of HA, type Ⅳ collagen and LN in different stages of DMN-induced liver fibrosis in rats, moreover, the area-density percentage of collagen fibrosis is positively correlated with the serum levels of HA, LN and type Ⅳ collagen at different stages.

This study sought to probe into the mechanism of spontaneous contraction of portal vein. The morphological and electrophysiological characteristics of the freshly isolated interstitial cells (ICs) of rabbit portal vein were investigated by using immunohistochemical and conventional whole-cell patch clamp techniques. The isolated interstitial cells exhibited stellate-shaped or spindle-shaped bodies with a variable number of thin processes projecting from cell bodies, and these cells were noted to be c-Kit immunopositive. Under conventional whole-cell patch clamp configuration, the membrane potential was held at -60 mV, the spontaneous rhythmic inward currents were recorded in ICs, and the frequencies of which were similar to those of spontaneous contraction of portal vein. The inward currents were insensitive to nicardipine (an L-type calcium channel blocker) but could be abolished by gadolinium (a non-selective cation channel blocker). The results suggested that the spontaneous rhythmic inward currents recorded in freshly isolated ICs may be pacemaker currents which elicit the spontaneous contraction of portal vein.
Action Potentials ; Animals ; Electrophysiology ; Female ; Interstitial Cells of Cajal ; physiology ; Male ; Muscle, Smooth, Vascular ; physiology ; Periodicity ; Portal Vein ; cytology ; physiology ; Rabbits ; Transient Receptor Potential Channels ; metabolism

BACKGROUND/AIMS: Interstitial cells play important roles in gastrointestinal (GI) neuro-smooth muscle transmission. The underlying mechanisms of colonic dysmotility have not been well illustrated. We established a partial colon obstruction (PCO) mouse model to investigate the changes of interstitial cells and the correlation with colonic motility. METHODS: Western blot technique was employed to observe the protein expressions of Kit, platelet-derived growth factor receptor-α (Pdgfra), Ca²⁺-activated Cl⁻ (Ano1) channels, and small conductance Ca²⁺- activated K⁺ (SK) channels. Colonic migrating motor complexes (CMMCs) and isometric force measurements were employed in control mice and PCO mice. RESULTS: PCO mice showed distended abdomen and feces excretion was significantly reduced. Anatomically, the colon above the obstructive silicone ring was obviously dilated. Kit and Ano1 proteins in the colonic smooth muscle layer of the PCO colons were significantly decreased, while the expression of Pdgfra and SK3 proteins were significantly increased. The effects of a nitric oxide synthase inhibitor (L-NAME) and an Ano1 channel inhibitor (NPPB) on CMMC and colonic spontaneous contractions were decreased in the proximal and distal colons of PCO mice. The SK agonist, CyPPA and antagonist, apamin in PCO mice showed more effect to the CMMCs and colonic smooth muscle contractions. CONCLUSIONS: Colonic transit disorder may be due to the downregulation of the Kit and Ano1 channels and the upregulation of SK3 channels in platelet-derived growth factor receptor-α positive (PDGFRα⁺) cells. The imbalance between interstitial cells of Cajal-Ano1 and PDGFRα-SK3 distribution might be a potential reason for the colonic dysmotility.
Abdomen ; Animals ; Apamin ; Blotting, Western ; Chloride Channels ; Colon ; Down-Regulation ; Feces ; Interstitial Cells of Cajal ; Mice ; Muscle, Smooth ; Myoelectric Complex, Migrating ; Nitric Oxide Synthase ; Platelet-Derived Growth Factor ; Silicon ; Silicones ; Small-Conductance Calcium-Activated Potassium Channels ; Up-Regulation

BACKGROUND/AIMS: Interstitial cells of Cajal (ICC) and their special calcium-activated chloride channel, anoctamin-1 (ANO1) play pivotal roles in regulating colonic transit. This study is designed to investigate the role of ICC and the ANO1 channel in colonic transit disorder in dextran sodium sulfate (DSS)-treated colitis mice. METHODS: Colonic transit experiment, colonic migrating motor complexes (CMMCs), smooth muscle spontaneous contractile experiments, intracellular electrical recordings, western blotting analysis, and quantitative polymerase chain reaction were applied in this study. RESULTS: The mRNA and protein expressions of c-KIT and ANO1 channels were significantly decreased in the colons of DSS-colitis mice. The colonic artificial fecal-pellet transit experiment in vitro was significantly delayed in DSS-colitis mice. The CMMCs and smooth muscle spontaneous contractions were significantly decreased by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an ANO1 channel blocker, and NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of nitric oxide synthase activity, in DSS-colitis mice compared with that of control mice. Intracellular electrical recordings showed that the amplitude of NPPB-induced hyperpolarization was more positive in DSS-colitis mice. The electric field stimulation-elicited nitric-dependent slow inhibitory junctional potentials were also more positive in DSS-colitis mice than those of control mice. CONCLUSION: The results suggest that colonic transit disorder is mediated via downregulation of the nitric oxide/ICC/ANO1 signalling pathway in DSS-colitis mice.
Animals ; Blotting, Western ; Chloride Channels ; Colitis ; Colon ; Dextrans ; Down-Regulation ; In Vitro Techniques ; Interstitial Cells of Cajal ; Mice ; Muscle, Smooth ; Myoelectric Complex, Migrating ; NG-Nitroarginine Methyl Ester ; Nitric Oxide Synthase ; Polymerase Chain Reaction ; RNA, Messenger ; Sodium

We explore the question of whether adenosine 5'-triphosphate (ATP) acts as an excitatory neurotransmitter in guinea-pig gastric smooth muscle. In an organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured in the presence of atropine and guanethidine. Under electrical field stimulation (EFS) at high frequencies (>20 Hz), NO-mediated relaxation during EFS was followed by a strong contraction after the cessation of EFS (a "rebound-contraction"). Exogenous ATP mimicked the rebound-contraction. A known P2Y-purinoceptor antagonist, reactive blue 2 (RB-2), blocked the rebound-contraction while selective desensitization of P2x-purinoceptor with alpha, beta-MeATP did not affect it. ATP and 2-MeSATP induced smooth muscle contraction, which was effectively blocked by RB-2 and suramin, a nonselective P2-purinoceptor antagonist. Particularly, in the presence of RB-2, exogenous ATP and 2-MeSATP inhibited spontaneous phasic contractions, suggestingthe existence of different populations of purinoceptors. Both the rebound-contraction and the agonist-induced contraction were not inhibited by indomethacin. The rank orders of agonists' potency were 2-MeSATP > ATP gtoreq UTP for contraction and alpha, beta-MeATP gtoreq beta, gamma-MeATP for inhibition of the phasic contraction, that accord with the commonly accepted rank order of the classical P2Y-purinoceptor subtypes. Electrical activities of smooth muscles were only slightly influenced by ATP and 2-MeSATP, whereas alpha, beta-MeATP attenuated slow waves with membrane hyperpolarization. From the above results, it is suggested that ATP acts as an excitatory neurotransmitter, which mediates the rebound-contraction via P2Y-purinoceptor in guinea-pig gastric antrum.
Adenosine Triphosphate* ; Adenosine* ; Atropine ; Baths ; Guanethidine ; Indomethacin ; Membranes ; Muscle, Smooth ; Neurotransmitter Agents* ; Pyloric Antrum* ; Receptors, Purinergic ; Relaxation ; Stomach ; Suramin ; Uridine Triphosphate