Edited by Ma JY
Published online Dec 15, 2001. doi: 10.3748/wjg.v7.i6.852
Revised: June 1, 2001
Accepted: June 11, 2001
Published online: December 15, 2001
AIM: To study the distribution of nitric oxide synthase (NOS) in rat stomach myenteric plexus.
METHODS: The distribution of NOS in gastric wall was studied in quantity and location by the NADPH-diaphorase (NDP) histochemical staining method and whole mount preparation technique.
RESULTS: NOS was distributed in whole stomach wall, most of them were located in myenteric plexus, and distributed in submucosal plexus. The shape of NOS positive neurons was basically similar, most of them being round and oval in shape. But their density, size and staining intensity varied greatly in the different parts of stomach. The density was 62 ± 38 cells/mm2 (antrum), 43 ± 32 cells/mm2 (body), and 32 ± 28 cells/mm2 (fundus), respectively. The size and staining intensity of NOS positive neurons in the fundus were basically the same, the neurons being large and dark stained, while they were obviously different in antrum. In the body of the stomach, the NOS positive neurons were in an intermediate state from fundus to antrum. There were some beadlike structures which were strung together by NOS positive varicosities in nerve fibers, some were closely adherent to the outer walls of blood vessels.
CONCLUSION: Nitric oxide might be involved in the modulation of motility, secretion and blood circulation of the stomach, and the significant difference of NOS positive neurons in different parts of stomach myenteric plexus may be related to the physiologic function of stomach.
- Citation: Peng X, Feng JB, Yan H, Zhao Y, Wang SL. Distribution of nitric oxide synthase in stomach myenteric plexus of rats. World J Gastroenterol 2001; 7(6): 852-854
- URL: https://www.wjgnet.com/1007-9327/full/v7/i6/852.htm
- DOI: https://dx.doi.org/10.3748/wjg.v7.i6.852
Recent pharmacological and physiological studies indicated that nitric oxide (NO) is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves in the mammalian gastrointestinal tract[1-15]. It may play a very important role in the neuronal regulation of gut. Previous studies have revealed the distribution, morphological features and projections of nitric oxide synthase (NOS) activity neurons and fibers in the enteric nervous system in intestine[16-25]. However, there have been much fewer studies of NOS activity in stomach wall since it is very difficult to make whole mount preparations of stomach. If NO is a transmitter of NANC inhibitory nerves, it should be present in neurons innervating the muscularis in stomach. What are the regularity of distribution of NOS in rat gastric wall, and the pat tern of innervation of these neurons To answer these questions, we examined the distribution and morphological feature of NOS positive neurons in the stomach wall using improved whole mount preparation technique.
Adult male or female Wistar strain rats (210 g-250 g in body weight) were provided by the Center of Laboratory Animals of the Third Military Medical University. The rats were fasted overnight prior to the experiment, and anaesthetized with sodium pentobarbitone (50 mg•kg¯¹, ip). Stomach was rinsed with 0.01 mol•L¯¹ phosphate buffered saline (PBS, pH7.4) and poured into gastric cavity with 40 g.L-1 paraformaldehyde (until 3-4 times of normal stomach volume), put in the same fixed liquid for 4 h-6 h (4 °C), and soaked in 300 g•L¯¹ sucrose liquid for 24 h (4 °C). Thereafter, wash away with PBS, wrap stomach up with bandage, rub gently for 2-3 min, stuff tightly with cotton, then demarcate trace along greater curvature of stomach toward lesser curvature with an extremely thin piece of bamboo, and strip slightly. Finally, trim the longitudinal muscular layer and dip in PBS for staining.
Whole mount preparations were rinsed with PBS three times, and incubated in a solution of 0.1 mol•L¯¹ PBS (pH8.0) containing 0.5 g•L¯¹β-NADPH, 0.2 g•L¯¹ nitroblue tetrazolium (NBT), 2.0 g•L¯¹ L-malic acid, 3 g•L¯¹ Triton X-100, hatched at 37 °C for 45 min, and rinsed three times with PBS, then mounted on glass slide, paved nicely (drug ratio less than 1:1.1), dehydrated through graded alcohol, cleared in xylene, and coverslipped with DPX. Incubative solution without adding β-NADPH served as negative control.
All results were expressed as the mean ± SD, data were analyzed by Student t test. P values < 0.05 were considered statistically significant.
Our study showed that NOS was widely distributed in rat stomach wall, most of them being localized in myenteric plexus, and distributed in submucosal plexus, gastric mucosal epithelium and gastric gland as well. In the myenteric plexus, NOS positive neurons were clearly identified by their sharply defined dark blue cytoplasmic stain with almost no background, the nuclei appeared as colorless “Holes” (Figure 1). The shape of neurons was basically similar, most of them varied from round, oval to fusiform, while their density, size and staining intensity varied greatly in the different parts of stomach. The density was 62 ± 38 cells/mm2, 43 ± 32 cells/mm2 and 32 ± 28 cells/mm2, respectively in antrum, body and fundus. Two subtypes of NOS positive neurons could be distinguished on the basis of size, staining intensity and progress in number. In fundus, about 75% neurons were large, dark stained and had some long progresses (Figure 2). Neurons of the second subtype were slightly smaller, with some short processes and mainly located in antrum (approximately 65%) (Figure 3). In the body of stomach, the NOS positive neurons were in an intermediate state from fundus to antrum. Moreover, some beadlike structure was strung together by NOS positive varicosities in nerve fibers, and some were colsely adherent to the outer walls of blood vessels (Figure 4).
Since the early 1960s, it has been known that discrete populations of nerve cells can be labeled by an obscure, but simple, histochemical reaction involving the reduction of tetrazolium salts to form dark formazan products. This reaction is catalyzed by an unidentified enzyme contained in these cells and requires NADPH. The enzyme, called NADPH diaphorase, has been identified as being identic al to NOS[26-39]. It has been demonstrated that NADPH diaphorase reacticity and NOS immunoreactivity coexist in enteric neurons. The simple histochemical procedure to visualize NADPH diaphorase may be useful to localize NOS in other tissues in view of the currently limited availability of antisera to NOS[40-50]. Therefore, the NADPH-d is now wildly used as a marker for neuronal structures in the CNS and ENS which contain the enzyme NOS.
The result showed that NOS was widely distributed in the gastric wall. Briefly, many NOS positive neurons were present in the myenteric plexus but relatively few in the submucous plexus. The majority of the NOS positive neurons in stomach could be classified as either Dogiel type 1 or type 2 neurons, although they were not as typical as the Dogiel neurons in small intestine, suggesting that NOS is both in the circular muscle motor neurons and in some interneurons. Some NOS positive nerve fibers formed a dense network in the circular muscle and in myenteric ganglia, and few nerve fibers in the mucosa. Based on this evidence, it is clear that the distribution of NOS in the enteric nervous system is compatible with a transmitter role of NO in stomach.
The significant difference of NOS positive neurons in different parts of the stomach myenteric may be related to the physiologic function of stomach. The density of NOS positive neurons was scarce in fundus, it is advantageous to keep tonic contraction at fundus and maintain foundation tension, but the activity of NOS was more powerful than that in antram. It benefits gastric lumen relaxation for food store after diet. Teng et al found that involvement of NO in the reflex relaxation of the stomach to accommodate food or fluid, and the NO released from fundus plays a principal role in adjusting, gastric lumen volume and press. We also found that the density of NOS in pyloric part was intensive, it is possibly related to stomach peristalsis since there were not only NOS neurons, but also other types, especially cholinergic neurons. They released transmitters to coordinate each other, adjust stomach smooth muscle to shrink, thus producing peristalsis as a mechanical pump pushing food to duodenum. Meanwhile, the results show that staining intensity of NOS neurons was different in pylorus, hinting that some NOS neurons may be in a reserving condition, once required NOS will be activated and NO sythesized to regulate stomach movement and the tension of pyloric sphincter. But the detailed mechanism is far from clear. We also found that there were many NOS positive varicosities, and bead-like structure strung together in nerve fibers. Some of them were closely adherent to the outer walls of blood vessels and smooth muscle fibers. This finding has provided morphological evidence of NO involved in the modulation of motility and blood circulation of gastrointestinal tract.
|1.||Qu XW, Wang H, Rozenfeld RA, Huang W, Hsueh W. Type I nitric oxide synthase (NOS) is the predominant NOS in rat small intestine. Regulation by platelet-activating factor. Biochim Biophys Acta. 1999;1451:211-217. [PubMed]|
|2.||Belai A, Burnstock G. Distribution and colocalization of nitric oxide synthase and calretinin in myenteric neurons of developing, aging, and Crohn's disease human small intestine. Dig Dis Sci. 1999;44:1579-1587. [PubMed] [DOI]|
|3.||Brehmer A, Stach W. Morphological classification of NADPHd-positive and -negative myenteric neurons in the porcine small intestine. Cell Tissue Res. 1997;287:127-134. [PubMed] [DOI]|
|4.||Toma H, Nakamura K, Emson PC, Kawabuchi M. Immunohistochemical distribution of c-Kit-positive cells and nitric oxide synthase-positive nerves in the guinea-pig small intestine. J Auton Nerv Syst. 1999;75:93-99. [PubMed] [DOI]|
|5.||Brehmer A, Stach W, Krammer HJ, Neuhuber W. Distribution, morphology and projections of nitrergic and non-nitrergic submucosal neurons in the pig small intestine. Histochem Cell Biol. 1998;109:87-94. [PubMed] [DOI]|
|6.||Teng B, Murthy KS, Kuemmerle JF, Grider JR, Sase K, Michel T, Makhlouf GM. Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells. Am J Physiol. 1998;275:G342-G351. [PubMed]|
|7.||Taguchi T, Guo R, Masumoto K, Nada O, Nakao M, Yanai K, Suita S. Chronological change of distribution in nitric oxide and peptidergic neurons after rat small intestinal transplantation. J Pediatr Surg. 1999;34:341-345. [PubMed] [DOI]|
|8.||Gao Y, Xu XP, Hu HZ, Yang JZ. Cultivation of human liver cell lines with microcarriers acting as biological materials of bioartificial liver. World J Gastroenterol. 1999;5:221-224. [PubMed]|
|9.||Li L, Wu PH, Mo YX, Lin HG, Zheng L, Li JQ, Lu LX, Ruan CM, Chen L. CT arterial portography and CT hepatic arteriography in detection of micro liver cancer. World J Gastroenterol. 1999;5:225-227. [PubMed]|
|10.||Lin QY, Du JP, Zhang MY, Yao YG, Li L, Cheng NS, Yan LN, Xiao LJ. Effect of apolipoprotein E gene Hha I restricting fragment length polymorphism on serum lipids in cholecystolithiasis. World J Gastroenterol. 1999;5:228-230. [PubMed]|
|11.||Jiang Y, Liu AH, Zhao KS. Studies on the flow and distribution of leukocytes in mesentery microcirculation of rats. World J Gastroenterol. 1999;5:231-234. [PubMed]|
|12.||Zhou GH, Luo GA, Cao YC, Zhu MS. Study on the quality of recombinant proteins using matrix-assisted laser desorption ionization time of flight mass spectrometry. World J Gastroenterol. 1999;5:235-240. [PubMed]|
|13.||Zhong QP. Pathogenic effects of Opolysaccharide from Shigella flexneri strain. World J Gastroenterol. 1999;5:245-248. [PubMed]|
|14.||Cui J, Yang DH, Bi XJ, Fan ZR. Methylation status of c-fms oncogene in HCC and its relationship with clinical pathology. World J Gastroenterol. 2001;7:136-139. [PubMed]|
|15.||Ran XZ, Su YP, Wei YJ, Ai GP, Cheng TM, Lin Y. Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation. World J Gastroenterol. 2001;7:140-142. [PubMed]|
|16.||Ma LS, Pan BR. Strengthen international academic exchange and promote development of gastroenterology. World J Gastroenterol. 1998;4:1. [PubMed]|
|17.||Wu XN. Current concept of Spleen-Stomach theory and Spleen deficiency syndrome in TCM. World J Gastroenterol. 1998;4:2-6. [PubMed]|
|18.||Gong Y, Liu KD, Zhou G, Xue Q, Chen SL, Tang ZY. Tumor radioimmunoimaging of chimeric antibody in nude mice with hepatoma xenograft. World J Gastroenterol. 1998;4:7-9. [PubMed]|
|19.||Tao HQ, Lin YZ, Wang RN. Significance of vascular endothelial growth factor messenger RNA expression in gastric cancer. World J Gastroenterol. 1998;4:10-13. [PubMed]|
|20.||Guo BY, Zhang SY, Mukaida N, Harada A, Kuno K, Wang JB, Sun SH, Matsushima K. CCR5 gene expression in fulminant hepatitis and DTH in mice. World J Gastroenterol. 1998;4:14-18. [PubMed]|
|21.||Chen XQ, Zhang WD, Song YG, Zhou DY. Induction of apoptosis of lymphocytes in rat mucosal immune system. World J Gastroenterol. 1998;4:19-23. [PubMed]|
|22.||Zhang XC, Gao RF, Li BQ, Ma LS, Mei LX, Wu YZ, Liu FQ, Liao ZL. Clinical and experimental study of therapeutic effect of Weixibaonizhuan pills on gastric precancerous lesions. World J Gastroenterol. 1998;4:24-27. [PubMed]|
|23.||Deng ZL, Ma Y. Aflatoxin sufferer and p53 gene mutation in hepatocellular carcinoma. World J Gastroenterol. 1998;4:28-29. [PubMed]|
|24.||Zhang L, Li SN, Wang XN. CEA and AFP expression in human hepatoma cells transfected with antisense IGF-I gene. World J Gastroenterol. 1998;4:30-32. [PubMed]|
|25.||Fan J, Ten GJ, He SC, Guo JH, Yang DP, Wang GY. Arterial chemoembolization for hepatocellular carcinoma. World J Gastroenterol. 1998;4:33-37. [PubMed]|
|26.||Zhu HZ, Zhang XL, Chen YS. Expression of glutathione S-transferase placental mRNA in hepatic preneoplastic lesions in rats. World J Gastroenterol. 1998;4:38-40. [PubMed]|
|27.||Li J, Wang Y, Li QX, Wang YM, Xu JJ, Dong ZW. Cloning of 3 h11 mAb variable region gene and expression of 3 h11 human-mouse chimeric light Chain. World J Gastroenterol. 1998;4:41-44. [PubMed]|
|28.||Wang ZQ, He J, Chen W, Chen Y, Zhou TS, Lin YC. Relationship between different sources of drinking water, water quality improvement and gastric cancer mortality in Changle County-A retrospective-cohort study in high incidence area. World J Gastroenterol. 1998;4:45-47. [PubMed]|
|29.||Zhang QX, Dou YL, Shi XY, Ding Y. Expression of somatostatin mRNA in various differentiated types of gastric carcinoma. World J Gastroenterol. 1998;4:48-51. [PubMed]|
|30.||Yi YF, Huang YR. Arylsulfatase, betagalactosidase and lysozyme in gastric cancer cells and its relationship to invasion. World J Gastroenterol. 1998;4:52-54. [PubMed]|
|31.||Cao GH, Yan SM, Yuan ZK, Wu L, Liu YF. A study of the relationship between trace element Mo and gastric cancer. World J Gastroenterol. 1998;4:55-56. [PubMed]|
|32.||Qiao GB, Han CL, Jiang RC, Sun CS, Wang Y, Wang YJ. Overexpression of p53 and its risk factors in esophageal cancer in urban areas of Xi'an. World J Gastroenterol. 1998;4:57-60. [PubMed]|
|33.||Zhang SL, Han XB, Yue YF. Relationship between HBV viremia level of pregnant women and intrauterine infection: neated PCR for detection of HBV DNA. World J Gastroenterol. 1998;4:61-63. [PubMed]|
|34.||Zhang LF, Peng WW, Yao JL, Tang YH. Immunohistochemical detection of HCV infection in patients with hepatocellular carcinoma and other liver diseases. World J Gastroenterol. 1998;4:64-65. [PubMed]|
|35.||Li XN, Benjamin I, Alexander B. A new rat model of portal hypertension induced by intraportal injection of microspheres. World J Gastroenterol. 1998;4:66-69. [PubMed]|
|36.||Tong WB, Zhang CY, Feng BF, Tao QM. Establishment of a nonradioactive assay for 2'-5' oligoadenylate synthetase and its application in chronic hepatitis C patients receiving interferon-alpha. World J Gastroenterol. 1998;4:70-73. [PubMed]|
|37.||Wang YJ, Li MD, Wang YM, Ding J, Nie QH. Simplified isolation and spheroidal aggregate culture of rat hepatocytes. World J Gastroenterol. 1998;4:74-76. [PubMed]|
|38.||Sun WB, Han BL, Peng ZM, Li K, Ji Q, Chen J, Wang HZ, Ma RL. Effect of aging on cytoskeleton system of Kupffer cell and its phagocytic capacity. World J Gastroenterol. 1998;4:77-79. [PubMed]|
|39.||Xu HB, Zhang YJ, Wei WJ, Li WM, Tu XQ. Pancreatic tumor: DSA diagnosis and treatment. World J Gastroenterol. 1998;4:80-81. [PubMed]|
|40.||Wei HS, Lu HM, Li DG, Zhan YT, Wang ZR, Huang X, Cheng JL, Xu QF. The regulatory role of AT1 receptor on activated HSCs in hepatic fibrogenesis: effects of RAS inhibitors on hepatic fibrosis induced by CCl (4). World J Gastroenterol. 2000;6:824-828. [PubMed]|
|41.||Zhao YL, Meng ZD, Xu ZY, Guo JJ, Chai SA, Duo CG, Wang XY, Yao JF, Liu HB, Qi SX. H2 strain attenuated live hepatitis A vaccines: protective efficacy in a hepatitis A outbreak. World J Gastroenterol. 2000;6:829-832. [PubMed]|
|42.||Yan J, Dennin RH. A high frequency of GBV-C/HGV coinfection in hepatitis C patients in Germany. World J Gastroenterol. 2000;6:833-841. [PubMed]|
|43.||Pan QS, Fang ZP, Huang FJ. Identification, localization and morphology of APUD cells in gastroenteropancreatic system of stomach-containing teleosts. World J Gastroenterol. 2000;6:842-847. [PubMed]|
|44.||Gao HJ, Yu LZ, Bai JF, Peng YS, Sun G, Zhao HL, Miu K, L XZ, Zhang XY, Zhao ZQ. Multiple genetic alterations and behavior of cellular biology in gastric cancer and other gastric mucosal lesions: H. pylori infection, histological types and staging. World J Gastroenterol. 2000;6:848-854. [PubMed]|
|45.||Chen JP, Lin C, Xu CP, Zhang XY, Wu M. The therapeutic effects of recombinant adenovirus RA538 on human gastric carcinoma cells in vitro and in vivo. World J Gastroenterol. 2000;6:855-860. [PubMed]|
|46.||Wu HG, Zhou LB, Shi DR, Liu SM, Liu HR, Zhang BM, Chen HP, Zhang LS. Morphological study on colonic pathology in ulcerative colitis treated by moxibustion. World J Gastroenterol. 2000;6:861-865. [PubMed]|
|47.||Chen MY, Huang ZQ, Chen LZ, Gao YB, Peng RY, Wang DW. Detection of hepatitis C virus NS5 protein and genome in Chinese carcinoma of the extrahepatic bile duct and its significance. World J Gastroenterol. 2000;6:800-804. [PubMed]|
|48.||Yan FM, Chen AS, Hao F, Zhao XP, Gu CH, Zhao LB, Yang DL, Hao LJ. Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis C. World J Gastroenterol. 2000;6:805-811. [PubMed]|
|49.||Yin ZZ, Jin HL, Yin XZ, Li TZ, Quan JS, Jin ZN. Effect of Boschniakia rossica on expression of GST-P, p53 and p21 (ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats. World J Gastroenterol. 2000;6:812-818. [PubMed]|
|50.||Huang GC, Zhang JS, Zhang YE. Effects of retinoic acid on proliferation, phenotype and expression of cyclin-dependent kinase inhibitors in TGF-beta1-stimulated rat hepatic stellate cells. World J Gastroenterol. 2000;6:819-823. [PubMed]|