The occurrence of an unusual tubular organelle in surface epithelial cells of the mouse ascending colon after injection of diazo-oxo-norleucine.код для вставкиСкачать
THE ANATOMICAL RECORD 196:413-420 (1980) The Occurrence of an Unusual Tubular Organelle in Surface Epithelial Cells of the Mouse Ascending Colon after Injection of Diazo-0x0-Norleucine JOHN E. MICHAELS Department ofdnatomy, Uniuersity of Cincinnati College of Medicine, Cincinnati. Ohio 45267 ABSTRACT Tubular structures were observed in surface epithelial cells of mice that had been injected with high dosages of diazo-0x0-norleucine(DON), a glutamine antagonist. The tubules often occurred in bundles which contained a variable number of tubules, often as many as one hundred being present. Within the bundles, the tubules were oriented either randomly or parallel t o one another. They measured 25 to 35 nm in diameter with angular or circular profiles and were as long as 1to 2 pm. In the center of each tubule, a smaller tubule-like component was evident that measured 5 to 7 nm in diameter. With the exception of endoplasmic reticulum, often with attached ribosomes, organelles were excluded from the bundles. Since the tubules and the endoplasmic reticulum occasionally were observed to be continuous, it is suggested that the tubules may originate from this organelle. There have been many reports of modified endoplasmic reticulum containing formed elements, some of which are tubular. Such modifications have occurred in a variety of plant and animal cells derived from normal, pathological, or drug treated specimens. A tubular organelle, which may be a modified form of endoplasmic reticulum, was observed in surface epithelial cells of murine ascending colon after injection of high dosages of the drug diazo-0x0-norleucine (DON). The current report will be limited to a description of this structure. DON is a glutamine antagonist that interferes with glycosylation of forming glycoproteins, including those that comprise the cell coat (Greene and Pratt, '77). The compoundbinds irreversibly (Rando, '75) t o transamidases, such as those responsible for the glutamine dependent conversion of fructose to glucosamine, a precursor of two carbohydrates of cell coat glycoproteins, N-acetylglycosamine and N-acetylgalactosamine. Both of these carbohydrates apparently are added to peptides within rough endoplasmic reticulum (Whur et al., '69). Absence of the compounds would prevent further addition of sugars to forming glycoproteins. DON also inhibits purine synthesis (Livingston et al., '70). The effects of DON on the surface columnar epithelial cells of the ascending colon were of interest, since these cells have a n abundant cell coat composed of glycoproteins on their apical surface, and they rapidly incorporate carbohydrate precursors of glycoproteins (Bennett et al., '74). Moreover, the mode ofmigrationof cell coat glycoproteins from the Golgi apparatus to the cell surface has been established for these cells (Michaels and Leblond, '76). MATERIALS AND METHODS Thirteen adult male CD-1 outbred albino mice were injected intraperitoneally with diazo-0x0-norleucinein a saline solution as follows. One pair of mice was given a single dosage of 10 mglkg body weight 18 hours before sacrifice. Four pairs of mice received a total dosage of 5, 10, 20, or 100 mg/kg body weight; these mice were given two injections of equal quantity a t a 24 hour interval and sacrificed 24 hours later. Three additional mice received a total dosage of 150 mgkg body weight; these mice were given three injections of equal quantity a t 24 hour intervals and sacrificed 24 hours later. Two control animals received three injections at 24 hour intervals of a volume of saline equivalent to that injected into the animals dosed with 150 mgkg of DON, 0.5 ml. At all times, mice were given feed and water ad libitum. 000-3276X/80/1964-0413$02.400 1980 ALAN R. LISS, INC. Received April 3, 1979; accepted July 30, 1979 413 414 JOHN E. MICHAELS In order to prepare the tissue for electron microscopy, the ascending colon of each anesthetized mouse was incised about 1 cm from the cecum and fixative was injected into the lumen of the intestine 2 cm distally. A 2 cm segment of jejunum was fixed in a similar manner. The fixative consisted of 2.0% glutaraldehyde and 0.5% formaldehyde (prepared from paraformaldehyde) buffered with 0.1 M sodium cacodylate, pH 7.2. After about two minutes of continuous intraluminal injection of fixative a t room temperature, each segment of intestine was dissected, cut into small pieces with a razor blade, and additionally fixed by immersion a t room temperature for about 30 minutes and then at 4°C for two to three hours. The tissue was washed in 0.2 M sodium cacodylate buffer (pH 7.2) for one hour and postfixed for one and a half hours with 1.5%osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.2) a t 4°C. Dehydration was initiated with cold alcohols and specimens were brought to room temperature in 100% alcohol. Tissue was immersed in two rinses of propylene oxide for a total of 20 minutes, then infiltrated in a mixture of propylene oxide and Epon 812 (Luft, '611, and embedded in Epon. Sections were cut with a diamond knife on a Reichert OMU-3 Ultramicrotome mounted on uncoated grids and stained with uranyl acetate (Watson, '58) and lead citrate (Reynolds, '63). A Philips EM 301 electron microscope operated a t 60 or 80 kV was used to observe the specimens. OBSERVATIONS In the basal half of normal surface epithelial cells of the mouse ascending colon (Fig. 11, mitochondria are the most predominant organelle and profiles of rough endoplasmic reticulum are frequent. At the lateral surfaces, processes from adjacent cells interdigitate (Fig. 1). After injection of DON, long tubular structures, composed of unit membrane, appeared in many of the surfaae epithelial cells (Fig. 2) as well as mid-crypt cells that had characteristics of surface cells. The tubular profiles measured 25-35 nm in diameter and as long as 2-3 pm (Figs. $7) before passing out of the plane of section. The tubules occurred singly, but more often in bundles containing as few as three or four (Fig. 2, upper left corner) to as many as one hundred or more tubules (Fig. 3 ) . The bundles of tubules were most abundant and largest in mice that received the highest dosages of DON (150 mg/kg body weight) and occurred, to a lesser extent, in animals that had been injected with an intermediate dosage (100 mg/kg body weight); but they were absent in mice receiving lower dosages. The tubules, singular or in bundles, were not found in goblet cells nor in argentaffin cells. Many absorptive cells from the jejunum of mice that had received the highest dosage of DON were observed for the presence of the peculiar tubular structures, but the modified organelle was never apparent in these cells. Since only stomach, jejunum, and ascending colon were fixed and observed, it is not known whether cells of other organs from animals injected with DON exhibited the tubular organelle. The tubules were not uniform in orientation; in each bundle they were oriented either randomly (Fig. 3 ) or more or less parallel to one another for 1-2 pm (Fig. 4). Most often, a single bundle of tubules was observed in a cell (Fig. 2). The bundles frequently occurred basal to or near the nucleus, but they were not limited to this region. Some bundles occurred above the nucleus and others near the lateral cell membrane. In sections that included the subnuclear region of the cells, bundles were observed in many adjacent cells (Fig. 2). When this region was not present in the section, the bundles were less numerous. Therefore, the overall frequency of the structures in sections of cells varied from 1&15% t o 80-9W0, depending on the region sectioned. It is possible that serial sectioning would have revealed the structures in every absorptive cell. The bundles were approximately circular in cross section (Fig. 2) and measured as large as 1-2 pm in diameter. In cells that contained more than one bundle, the orientation of each bundle of tubules was independent of other bundles in the cell. Most large organelles were excluded from the bundles. However, profiles of rough endoplasmic reticulum were located near and within the bundles (Figs. 2-6) and lipid droplets occasionally appeared to be associated with the bundles (Figs. 3, 5). The distance between many tubules in each of the bundles of parallel tubules varied between 10 to 20 nm (Fig. 4). In some instances, connections appeared to occur between one or more tubules and smooth or rough endoplasmic reticulum (Figs. 4-6). Yet, the tubules themselves always remained agranular. In cross section (Figs. 6,7),the tubulesvaried in shape, some appearing triangular, others polygonal or round. In the center of each tubule, a very small circular structure ( 5 7 nm in diameter) was evident (Figs. 6,7),composed of a n electron opaque wall and an electron lucent core, therehy appearing tubular. The wall of the central tubule was approximately 1.5 nm thick. In every tubule that was sectioned transversely, the inner 7 nm tubule was evident. Once the central tubule was recognized in transverse sections, close observation of TUBULAR ORGANELLE IN CELLS OF ASCENDING COLON 415 Fig. 1. In this eleetmn miciugraph of normal epithelial cells of the ascending colon, the region basal ta the nucleus is illustrated in longitudinal section. Mitochondria (M)are numerous and profiles of rough endoplasmic reticulum (R) are frequent. At the lateral surfaces (LS) processes from adjacent cells interdigitate. x 17,750. The following electron micrographswere obtained from sectionsof surface epithelial cells of murine ascending colons. The mice had received dosages of DON that totalled either 100 or 150 mgikg body weight. Fig. 2. This low magnification micrograph shows cells sectioned transversely at a level just basal to the nucleus. In most cells, bundles (B) of tubule-like structures are evident. Many of the tubules are sectioned transversely; a few longitudinally sectioned tubules (LT) also appear. Profiles of rough endoplasmic reticulum (R) lie close to or within the bundles of tubules. Other organelles are not observed within the bundles of tubules. x 17.750. Fig. 3. In this cell, a large bundle of irregularly arranged tubules is located at the basal end of the nucleus. Numerous tubules are evident, some of which are sectioned transversely (T),and a few profiles of rough endoplasrnic reticulum are present. Two lipid droplets occur in the bundle (LD). Two microtubules (arrows) are smaller in diameter than the tubules. x 37,500. Fig. 4. A portion of a bundle of parallel tubules, sectioned longitudinally, is illustrated. Many of the tubules maintain a rather uniform distance from each other. A connection between a profile of rough endoplasmic reticulum and a tubule is evident (large arrow). The central tubule may be observed in many of the parallel tubules (small arrow). x 102.000. 418 JOHN E. MICHAELS TUBULAR ORGANELLE IN CELLS OF ASCENDING COLON tubules that were sectioned longitudinally revealed faint images of the central tubules also oriented longitudinally (Fig. 4). DISCUSSION The tubular structures, containing a central tubule-like component as described in this report, a r e apparently unique. A few generalizations concerning their characteristics may be suggested. The most common location of the bundles of tubules, basal to and near the nucleus, implies that this region had the most suitable environment for formation of the structure, although neither smooth nor rough endoplasmic reticulum was especially abundant a t this site. This region was also the most prevalent location of another very different and infrequent modification of endoplasmic reticulum observed in the same cell type in normal mice (Michaels, '75). (This modification consisted of stacks of multiple parallel cisternae. The cisternae were separated by a regular spacing in which a single layer of small vesicle was found). Few organelles were observed between the tubules of a single bundle with the exception of the many profiles of rough endoplasmic reticulum, indicating that the bundles, regardless of the orientation of the tubules, were fairly cohesive. The profiles of endoplasmic reticulum within the bundles had occasional sites where connections to the tubules were evident, suggesting that the tubules may be formed from endoplasmic reticulum. At the subnuclear location, the tubules were oriented either randomly or parallel to each other. In either case, the tubules themselves maintained the same diameter and the 7 nm central tubule was always present. A superficial resemblance between the tubules and microtubules was apparent, especially in the instances of parallel tubules (Fig. 4). However, the diameter of microtubules is somewhat smaller (Fig. 3); microtubules are not formed from unit membrane; continuity between microtubules and endoplasmic reticulum is not observed; microtubules tend not to occur in irregularly oriented bundles (Fig. 3); and their profiles in transverse sections are more uniformly round t h a n those of the 419 tubules. Moreover, a central tubule is lacking in microtubules. Previously tubular elements (e.g., Uzman et al., '71; Valeri et al., '71; Baringer and Swoveland, '72; Quan et al., '74; Hruban et al., '76) or fibers (Baic et al., '73; Wang, '74) have been observed within endoplasmic reticulum, occurring in different cell types of diverse organisms. Several types of tubules have been described, many of which conform to two broad categories, but only on the basis of their morphological appearance. In one group, multiple tubules in endoplasmic reticulum have been described that were highly curved and branched and had diameters of 2530nm. Examples of this type of intracisternal tubules were found in 1)tumor cells or cells of animals that had been infected with a virus (Uzman et al., '71, who also reviewed earlier work; Baringer and Swoveland, '72; Hruban et al., '76), and 2) in human 1 . ~ phoid cells treated with 5-brom0-2-deoxpr1dine (Grimley et al., '73). In some cases, the tubules apparently were formed as invaginations of the endoplasmic reticulum and therefore consisted of unit membrane (Baringer and Swoveland, '72; Hruban et al., '76). In a second category, the intracisternal tubules were multiple, straight, and most had diameters of 20-45 nm. Examples of these occurred in pars intermedia of toad hypophysis (Valeri et al., '71), in a thyroid tumor of a dog (Deutschlander, '72), and in a variety of plant cells, each instance of which differed from the others (Jansen, '68; Hepler and Newcomb, '69; Schnepf and Diechgraber, '72; Quan et al., '74; Heywood, '72; Cresti et al., '74). In addition, other arrangements of intracisternal tubules have been described, such as triangular prism-shaped inclusions in rough endoplasmic reticulum of malpighian tubules of the mosquito, Culex tarsalis (Houk, '77). Recently, tubular structures with a diameter similar to the tubules reported here and containing a central 7 nm core were observed in Langerhans cells (Kobayashi and Hoshino, '78). However, long profiles were not observed and the cores were not tubular. The authors suggested that the cored tubules were distinct from Langerhans cell granules. Fig. 5. This oblique section through a bundle of tubules includes profiles of rough endoplasmic reticulum. A few tubules (arrowheads) appear to connect with the membrane that forms the endoplasmic reticulum. x 62,500. Fig. 6. In this micrograph, a bundle of tubules has been sectioned transversely. The profiles measure about 30 nm in diameter and reveal circular, triangular, and irregular shapes. Each tubule displays the central tubule-like element (small arrow) measuring about 7 nm in diameter. Many tubules maintain a fairly uniform distance from one another. Toward the center of the bundle, the membrane of a profile of agranular endoplasmic reticulum is continuous with two tubules (arrowhead). x 62,500. Fig. 7. At higher magnification, the tubules may be observed to be composed of unit membrane (arrows).The central tubular element is depicted more clearly in this micrograph. x 100,000, 420 JOHN E. MICHAELS The tubules possibly derived from endoplasmic reticulum and those containing central tubules in the mice treated with DON, are clearly different from the endoplasmic reticulum that contains intracisternal tubules described by others. The relationship of the tubules to DON, whether they arise as a primary response to the drug o r a secondary toxic reaction to DON or its metabolic products, can only be speculated upon. The tubules did not stain positively for the presence of glycoproteins with periodic acid-chromic acid silver methenamine technique (not illustrated), but this was also the case for other endoplasmic reticulum as well. Whatever the relationship to DON, it is worth noting that another gastrointestinal cell type which contains a significant amount of glycoproteins, the parietal cells, exhibited an abnormal structure-i.e., concentric saccules (Michaels,’79)-after injection of high dosages of DON. The difference in response of the jejunum and the ascending colon epithelial cells to DON or its metabolites is interesting. These two cell types have different functions, but morphological similarity, including a well developed cell coat composed of glycoproteins. Yet, the bundles were never observed in epithelial cells of the jejunum. It is also of interest that malignant tumors occur much more frequently in the colon and they may be accompanied by alterations of surface glycoproteins (Gold and Freedman, ’65).Further analysis of the effects of DON on these cells would seem to be warranted. Gold, P., and S.O. Freedman (1965) Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J. Exp. Med., 121:43%462. Greene, R.M., and R.M. Pratt (1977)Inhibition of diazo-oxonorleucine (DON)of rat palatal glycoprotein synthesis and epithelial cell adhesion invitro. Exp. Cell Res., 105:27-37. Grimley, P.M., D.W. Barry, and Z. SchafT(1973)Induction of tubular structures in the endoplasmic reticulum of human lymphoid cells by treatment with 5-bromo-2’-deoxyuridine. J. Natl. Canc. Instit., 51:1751-1760. Hepler, P.K., and E.H. Newcomb (1964) Microtubules and fibrils in the cytoplasm of coleus cells undergoing secondary wall deposition. J. Cell Biol., 20t529-533. Heywood, P. (1972) Structure and origin of flagellar hairs in Vucuoluria uirescens. J. Ultrastruct. Res., 39:60%623. Houk, E.J. (1977) Endoplasmic reticulum inclusions in the Malpighian tubules of the mosquito Culex tarsalis. J. Ultrastruct. Res., 60:6%70. Hruban, Z., T.-W. Wong, M. Itabashi, and E.S. Lyon (1976) Glomiform and fibrillar cytoplasmic inclusions. Virchows Archiv b Cell. Pathol., 20.9-102. Jensen, W.A. (1968) Cotton embryogenesis embryogenesis: The tube-containing endoplasmic reticulum. J. Ultrastruct. Res., 22:2=302. Kobayashi, M., and T. Hoshino (1978) Occurrence of cored tubule in the Birbeck granule-containing cells of mice. J. Electron Microsc., 27,199-205, Livingston, R.B., J.M. Venditti, D.A. Cooney, andS.K. Carter (1970) Glutamine antagonists in chemotherapy. Adv. Pharmacol. Chemother., 8:57- 120. Luft, J.H. (1961) Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9:40%414. Michaels, J.E. (1975) An unusual modification of endoplasmic reticulum in epithelial cells of the mouse ascending colon. Anat. Rec., 183:27-38. Michaels, J.E. (1979) Formation of concentric saccules in murine parietal cells after injection of diazo-oxo-norleucine. Anat. Rec., 193:775-790. Michaels, J.E., and C.P. Leblond (1976) Transport of glycoprotein from Golgi apparatus to cell surface by means of “carrier” vesicles, as shown by radioautography of mouse colonic epithelium after injection of 3H-fucose.J. Microsc. Biol. Cell., 25:243-248. Quan, S.G., E.Y. Chi, and S.M. Caplin (1974) Tubular ACKNOWLEDGMENTS structures in endoplasmic reticulum cultured in broccoli. It is a pleasure to thank Mrs. Julia T. Hung J. Ultrastruc. Res., 48t9Z-101. for her excellent and dedicated technical assis- Rando, R.R. (1975)On the mechanism of action of enzymes which act a s irreversible enzyme inhibitions. Biochem. tance. Pharmacol., 24: 1153- 1160. This work was supported initially by a grant Reynolds, E.S. (1963)Theuseof lead citrate a t high pH as a n from the University of Cincinnati Research electron opaque stain in electron microscopy.J. Cell Biol., 13r405-421. Council and subsequently by a grant from the Schnepf, E., and G. Deichgraber (1972)Tubular inclusions in National Institutes of Health, GM-24604. the endoplasmic reticulum of the gland hairs of Onionis repens. L. (Fabaceac)J. Microscopie, 14:361-364. LITERATURE CITED Uzman, B.G., H. Saito, and M. Kasae (1971) Tubular arrays in endoplasmic reticulum in human tumor cells. Lab. InBaic, D., B.E. Frye, and B.G. Ladewski (1973) Intracisternal vest., 24:492-498. fibers in the liver of starved frogs. J. Ultrastruct. Res., 43;478-482. Valeri, V., R.P. Goncalves, A.R. Cruz, and E.M. Laicine Baringer, J.R., and P. Swoveland (1972)Tubular aggregates (1971)Tubular structure within the granular endoplasmic in endoplasmic reticulum: Evidence against their viral reticulum of the pars intermedia of toad hypophysis. J. Ultrastruct. Res., 35:197-200. nature. J. Ultrastruct. Res., 41;270-276. Bennett, G., C.P. Leblond, and A. Haddad (1974) Migration Wang, N.S. 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