SANO, MASAHIRO

TOBE, TAKAYOSHI

Kyoto University Research Information Repository

Title Granule Release from Endocrine Cells in the Rat Stomach : AnElectron Microscopic StudyAuthor(s)SANO, MASAHIRO; TOBE, TAKAYOSHICitation日本外科宝函 (1977), 46(5): 576-584Issue Date1977-09-01URL http://hdl.handle.net/2433/208216RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityArch・ Jap Chir 46 (5), 576～584, Sept., 1977 Granule Release from Endocrine Cells in the Rat Stomach 一一AnElecto.ron Microscopic Study -MASAHIRO SANO Department of Surgery, Shizuoka City Hospital (Director : Toyoo HAsEGA wA) TAKAYOSHI TOBE The 2nd Department of Surgery, Kyoto University School of Medicine (Director : Prof. Dr. Y ORINORI H1KASA) Received for Publication May 12, 1977 Introduction Electron microscopic studies on the endocrine cells of the gastrointestinal tract under normal condition hav巴 beendone by many investigators in humans and in several species of animals including rats ・D 12l 16l17l They classified the endocrine cells into several types mainly according to the morphologic differences in their fine structures in an attempt to elucidate the relationships between cell types and hormones they produce. It is also known that gastrointestinal hormones including a polypeptide (gastrin) and/or a monoamine (serotonin) are released from these endocrine cells by various stimuli, che-micaP 1 l, nervous23l, and mechanical9l. The mechanisms for release of these hormones, how-ever, have not sufficiently been clarified. In this study the fine structures of the endocrine cells in the rat stomach after refeeding and electric vagal stimulation were examined under the electron microscope and, as com-pared with those of non-stimulated condition which had been already reported17l, the stimulation-induced morphologic changes in these cells, especially the secretory granules, were studied. Materials and Methods Male Wistar rats weighing between 200 to 300g were used as experimental animals after fasting for 24-48 hours. (1) Ten rats were sacrificed from 10 minut巴sto 2 hours after refeeding with an ordi-nary rat diet. One rat was sacrificed 30 minutes after administration of 50% glucose solu-tion into the stomach by the use of an oral tube. Each rat was laparotomized immediately after decapitation and the stomach removed. Key words : Endocrine cel, Emiocytosis Present address : Snrgical::> Department, Shizuoka City Hospital, 10-93, Otemachi, Shizuoka 420, Japan. GRANULE RELEASE FROM ENDOCRINE CELLS IN THE RAT STOMACH 577 (2) Two rats were anesthetized with sodium pentobarbital (Nembutal, 30 mg/kg., i.p.). After laparotomy the bilateral vagus nerves were dissected free and cut at the diaphrag-matic level. The peripheral ends of each nerve were placed on a bipolar platinum electrode connected to an electrical stimulator (Model ES 103, Sanei Sokki KK) delivering monop・hasic square wave stimuli of a duration of 5ms, an intensity of 4V and an impulse frequency of 5 Hz. The nerves were placed in paraffin oil to prevent drying during the experiment. Stimulation was continued for 30 minutes and then the stomach was removed. Tissues taken from the antral and fundus mucosae of the stomach in two groups were fixed in 4 % glutaraldehyde in 0.2M cacodylate buffer of pH7.3 at 4。cfor 2 hours, transfe-rred and washed in cold cacodylate buffer over 24 hours. Then, al samples were post fixed in a 1 % solution of osmium tetraxoide in the same buffer for 1 hour at 4°C, dehydrated in graded alcohols and propylene oxide, and then embedded in Epon 812. Each sample was cut on a Portor Blum MT I with a glass knife. Thick sections were stained with toluidine blue and examined under the light microscope. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined under a Hitachi HS7D electron micro-scope. Results The stomach removed after refeeding was dilated so that food was found in the lumen. Gastric juice also existed there for a brief period after refeeding. Gastrointestinal peristalsis was observed during electric vagal stimulattion. Light microscopic examination of the endocrine cells of the rat stomach under these conditions revealed no remarkable morphologic changes. The findidgs obtained by electron microscopic examination of various types of the endocrine cells in the rat stomach after stimulation consisted of the following. (1) G Cells The plasma membrane partly looking like an omega or a dimple was observed in some of the G cells after refeeding (Fig. 1). Enlargements of the rough endoplasmic reticulum in the cytoplasm, and the linear or round substances of high electron density in the Golgi complex, where a relatively large number of small corted vesicles existed, were also seen after refeeding (Fig. 2). After electric vagal stimulation G cells were found to have small vesicles either appea-ring to be in contact with the cell membrane or to have omega-shaped dimplings of the cel membrane (Figs. 3 and 4). The number and the electron density of the secretory granules in the cytoplasm of the G cel showed no remarkable changes after refeeding and electric vagal stimulation. Nor remarkable were the morphologic changes in the G cells after injection of a 50% glucose solution into the stomach. (2) EC Cells The EC cells showed finely granular contents within the membranous sacs and secretory granules with an eccentrically located small core in a relatively wide space within a 578 日外宝第46岱 第5号（昭和52年9月）2 ’‘ 3為. ~ ~ ‘ 炉句、’監• z‘ lMagnification approximately X 48000〕Fig. l. Part of a G cell after refeeding showing omega-shaped dimplings(thick arrows〕ofthe plasma membrane and microvesicles (thin arrows). Fig. 2. Golgi complex of a G cell after refeeding showing a relatively large number of progra-nules (thick arrows〕andcorted microvesicles (thin arrows). . Fig 3. and 4. Parts of G cels after electric vagr ーに ・、－ ,." . バーー ーー ι一目出叩 ‘’0°'cleswhich fuse with plasma membrane (arruw' '・ ；；』相← ζ弓，，，乙L5 GRANULE RELEASE FROM ENDOCRINE CELLS IN THE RAT STOMACH 579 ¥, ‘惨, • .. <r: ， ‘圃Fig. 5. and 6. Parts of EC cells after refeeding containing some secretory granules showing a fine granular or cored-vesicular structure (arrows). Fig. 7. An emiocytotic granule release 〔arrow〕 fromthe EC cells after electric vagal sti mulation. Fig. 8. Part of an EC cell after administration of 50% glucose solution by an oral tube showing various shaped small vesicles (arrows) near the plasma membrane. 580 日外宝第46巻 第5号（昭和52年9月）9 Jf. ‘闇 可官官 由』司、 ＇ I ~ .，仰 J E弘 ‘同’ ノJ r 占 ':li!7竺 や4ふ i与 s ぞ~ . '• 事 長• cf ，‘ ・a≪(, 6ろi<' 、主 向4";, '・ .，~ず,. 守王、 ιず司直時’d跡a除.I; . 司.J賞 、’a ・・，•v· ~~t:，ザ，，；； i'., 、・』可思d争.. L . v司帥．俳句多, ~ 日運営. ‘ －． j品。J_? _; ち ’偶・＇ .＇・..‘ー・． "' ’F ・’.’M ~12 ， 、， ’‘、． ?， ? ???? ，?i"1k a守主.ti:‘苛 4ぃ月、??? ??、ミFig. 9, and 10. Parts of D1 (Fig. 9) and D (Fig. 10) cells after electric vagal!stimulation indi・ cating an emiocytotic granule release (arrows〕．Fig. 11. and 12. Emiocytotic granule releases (arrows〕fromthe ECL cels after refeeding. GRANULE RELEASE FROM ENDOCRINE CELLS IN THE RAT STOMACH 581 membranous sac after refeeding (Figs. 5 and 6). Morphologic changes indicating an emiocytotic granule release or a contact with the cel membrane of the secretory granules were observed after electric vagal stimulation (Fig. 7). A relatively large number of microvesicles in the cytoplasm near the cell membrane were observed after injection of a 50% glucose solution into the stomach (Fig. 8.), although a definite figure showing an emiocytosis could not be found. There were no remarkable changes in the number and the distribution of the secretory granules in the cytoplasm of the EC cell after refeeding. However, the secretory granules seemed to be marginated or translocated to the cell membrane after electric vagal stimu-lation and injection of a 50% glucose solution into the stomach (Figs. 7 and 8). (3) Other Types of Endocrine Cells Morphologic changes as in the above two types of endocrine cells were observed in some of these endocrine cels after stimulation. Emiocytotic granule release from the D1 cells (Fig. 9) and the D cells (Fig. 10) occured after electric vagal stimulation, although they could not be found after refeeding. Some definite figures showing emiocytosis were frequently observed in the ECL cells after refeeding, especially after a brief period following refeeding (Figs. 11 and 12). Discussion The secretory mechanism of the endocrine cells in the gastrointestinal tract is not yet known in detail. As to the other types of endocrine cells involving the islet βcell and the endocrine cel of the adrenal grand, the following model for hormone secretion was proposed 13> 14>.The hormone is synthesized in the endoplasmic reticulum, transferred to the Golgi complex where the hormone is encapsulated and stored as secretory granules in the cytopl-asm. After specific stimulation, the secretory granules with their surrounding membranous sacs are translocated to the plasma membrane, where they are released by emiocytosis. After the release of the granules, the membranous sacs fuse with the plasma membrane and they are recaptured from the plasma membrane, and then they are returned as microvesi-des either to th巴 Golgicomplex or lysosomes where they are degrad巴d.The specific stimulus which results in the release of the hormone from the cells also initiates new formation of hormone in the endoplasmic reticulum. Morphologic evidence for this mode of secretion as to the endocrine cels of the gast-rointesinal tract was presented by FuJITA6> in the form of the D-like cells of the dog stomach observed after administration of O. lN hydrochloric acid into the stomach by oral catheteri-zation. On the other hand, some other investigators suggested that intracytoplasmic dissolution or diacrine secretion of the granule contents with an intact membranous sac occurred in the G cels of the cat的 afterrefeeding or of the human3>19> with hypergastrinemia. 582 日外宝第49巻第5号（昭和52年9月）We could not obtain morphologic evidence of diacrine secretion in the G cells after stimulation, because the electron density of most of the secretory granules in the G cells of the rat stomach was usually low with our method for electron microscopy. In the EC cells with usually high electron opaque secretory granules under normal condition, the secretory granules in the cytoplasm appearred to be finely granular or clear within the membranous sacs after stimulation, suggesting the decreased electron density of the granule cont巴nts.It remained unknown, however, whether these findings might serve as morphologic evidence of intracytoplasmic dissolution indicating different functional stages of diacrine secretion of granule contents including 5-HT (Serotonin) which is a monoamine produced in the EC cel, or merely an artificial product resulting from the fixation procedure for electron microscopy, because these findings were also observed in some of the EC cells under normal condition. On the other hand, the morphologic findings obtained time and again in several types of endocrine cells in the rat stomach after stimulation under various conditions, especially of the secretory granules, might be interpreted as morphologic evidence indicating various steps for emiocytotic granule release. This indicates that the same s巴cretorymechanism suggested by LACY13>14> in the other types of endocrine cells may be also present in the endocrine cells of the gastrointestinal tract as suggested by FuJITA6＞‘ The degranulation indicating a d巴finitedcfrease in the secretory granules in the cyto・plasm and the morphologic evidence showing final steps of emiocytosis were not always observed in most of the endocrine cells under stimulated conditions, especially physiological condition. Some other investigators10H8> failed to observe any morphologic change in the endocrine cells of the gastrointestinal tract after refeeding. The endocrine cells seem to control the rate of release of the stored hormone after stimulation. In our studies, emiocytotic granule release from the ECL cells and increased fromation of progranules in the G cells were often observed after refeeding. ECL cells in the rat stomach w巴reindentified as the cells containing a definite gastric acid stimulating mono-amine “histamine”and another unknown polypeptide which probably stimulates gastric secretion2>m. G cell has been known as the cell containing a hormone “gastrin”which also stimulates gastric secretion4> 17> It may b巴 reasonableto consider that refeeding, that is, a chemical, vagal or mecha-nical stimulus, is one of the specific physiologic stimuli which promote the release of the granules from these cells and, inevitably, the formation of the secetory granules in these cells. The cells seem to be responsible only for the specific stimuli. The emiocytotic granule release from the ECL cells observed after refeeding also indicates that emiocytosis occurs in the endocrine cells of the gastrointestinal tract under not only the non-physiological but also the physiological condition. In conclusion, the findings obtained in our mophologic studies seem to provide mor・ GRANULE RELEASE FROM ENDOCRINE CELLS IN THE RAT STOMACH 583 phologic evidence to indicate the secretion of the gastrointestinal hormones following refeed-ingB>11> and electric vagal stimulationm> I5H9>22> demonstrated by biochemical studies. The results also indicate that emiocytosis is at least one of the physiological mechanisms for secretion in the gastrointestinal endocrine cells, although the possibility that secretion by the mode of diacrine, especially the release of other granule contents than polypeptide, may occur under a certain condition is not completely precluded. Acknowledgments The authors wish to express their gratitude to Prof. YoR1NoR1 H1KASA for his useful advice and suggestions and to Miss Mm KANATO for her technical assistance. Part of this article was presented before the 61st General Meeting of Japanese Society of Gastroente-rology on April 1, 1975 in Kyoto. Reference自1) Becker, H. D., Reeder, D.D., and Thompson, J. C.: Direct measurement of vagal release of gastrin. Surg 75 : 101-106, 1974. 2) Bussolati, G. and Monga, G. : Histochemical and ultrastructural investigation on the endocrine cells of the stomach in hypersecreting (pylorusligated) rats. Virchows Arch Abt B Zellpath 13 55-67, 1973. 3) Creutzfeldt, W., Arnold, R., Creutzfeldt, C., Feurle, G., and Ketterer, H. : Gastrin and G cells in the antral mucosa of patients with pernicious anemia, acromegaly and hyperparathyroidism and in a Zollinger-Ellison tumors of the pancreas. Europ J Clin Invest 1 : 461 479, 1971. 4) Forssmann, W. G., Orci, L., Pictet, R., Renold, A. E., and Rouiller, C. : The endocrine cells in the epithlium of the gastrointestinal mucosa of the rats. J Cell Biol 40・692-715,1969. 5) Forssmann, W. G., and Orci, L. ・ Ultrastructure and secretory cycle of the gastrin producing cel. Z Zellforsch 101、419-432,1969. 6) Fujita, T. and Kobayashi, S. : Experimentally induced granule release in the endocrine cells of dog pyloric mucosa. Z Zellforsch 116 : 52-60, 1971. 7) Fyrii, B. : Reduction of antral and duodenal gastrin activity by electrical vagal stimulation. Acta Physiol Scand 71 : 334 340, 1967. 8) Fyrii, B.: Effect of feeding on antral and duodenal gastrin activity. Acta physiol Scand 74 : 166-172, 1968. 9) Grossman, M. I., Robertson, C.R., and Ivy, A. C .. Proof of a hormone mechanism for gastrin secretion-the humoral transmission of the distension stimulus. Am J Physiol 153 : 1-9, 1948. 10) Helander, H. F.: Ultrastructure of fundus glands of the mouse gastric mucosa. An electron micro-scopical study in fasted and refed animals with observation on ultrastructure after different fixation and embedding procedures. J Ultrastr Res Suppl., 4 : 1ー123,1962. 11) Korman, M. G., Soveny, C., and Hansky, J. : Effect of food on serum gastrin evaluated by radio-immunoassay. Gut 12 : 619-624, 1971. 12) Kurosumi, K., Shibasaki, S., Uchida, G., and Tanaka, Y. : Electron microscopic studies on the gastric mucosa of normal rats. Arch Hist Jap 15 : 587-624, 1958. 13) Lacy, P.E.: Beta cell secretion from the standpoint of a pathobiologist. Diabetes 19 895-905, 1970. 14) Lucy, P. E. : Endocrine secretory mechanisms. Am J Path 79 : 170 187, 1975. 15) Lanciault, G., Bonoma, C., and Brooks, F. P. : Vagal stimulation, gastrin release, and acid secretion in anesthetized dog. Am J Physiol 225 : 546-552, 1973. 16) Orci, L., Pictet, R., Forssmann, W. G., Renold, A. E., and Rouiller, C. : Structural evidence for glucagon producing cells in the intestinal mucosa of the rat. Diabetolatia 4 : 56-67, 1968. 17) Sano, M.『 Electronmicroscopic studies on endocrine cells in the gastric mucosa of rats. I. Endo・crine cells in the gastric mucosa of fasting rats. Arch Jap Chir 45 : 243-264, 1976. 584 日外宝第46巻第5号（昭和52年9月）18) Shibasaki, S.: Experimental cytological and electron microscope studies on the rat gastric mucosa. Arch Hist Jap 21 : 251-288, 1961. 19) Solcia, E., Capella, C., and Vassallo, G. : Endocrine cells of the stomach and pancreas in states of gastic hypersecretion. Rendic Gastroenterol 2 147-158, 1970. 20) Strauss, R. J., Demos, M. A., Strauss, R. D., and Stuckey, J.H. : Plasma 5・hydroxytryptamineand plasma 5-hydroxyindoleacetic acid levels after vagal stimulation. J Surg Res 12 : 334-340, 1972. 21) Tobe, T., Kimura, C., and Fujiwara, M. : Role of 5-hydroxytryptamine in the dumping syndrome after gastrectomy. Histochemical Study. Ann Surg 165 : 382 387, 1967. 22) Tobe, T., Izumi！《awa,F., Sano, M., and Tanaka, C. : Release Mechanisms of 5-HT in mammalian gastrointestinal tract一－especially,vagal release of 5・HT.Endocrine gut and pancreas. Ed. by Fujita, T., p. 371-380, Elsevier Scientific publ Comp., Amsterdam 1976. 23) Uvnis, B. Gastrin and vagus. Gastroenterology 56・812-815,1969. 和文抄録ラット胃内分泌細胞における頼粒放出について一電子顕微鏡的検索－静岡市立静岡病院外科（院長：長谷川豊男博士）佐 野 正 博京都大学医学部外科学教室第2講座（指導 ：日笠頼則教授）戸 音E 隆? ?給食刺激時，迷走神経電気刺激時におけるラット胃内分泌細胞の微細構造上の変化，ととlζ分泌願粒の形態学的変化について電子顕微鏡的IC検索した．刺激後の各種の内分泌細胞においては， 分泌頼粒の細胞膜周辺への局在傾向や，細胞膜との癒合， Q型の関口分泌像など，関口分泌における頼粒放出形式の種々の段階を示すと恩われる形態学的所見が観察された．以上の結果から，消化管内分泌細胞においても，非生理的あるいは生理的条件下をとわず，関口分泌による頼粒放出形式が存在するととが判明した．

ラット胃内分泌細胞における顆粒放出について : 電子顕微鏡的検索

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ラット胃内分泌細胞における顆粒放出について : 電子顕微鏡的検索

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