Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucose injection

In the first paper of this series (Bennett et al., 1984), light-microscope radioautographic studies showed that colchicine or vinblastine inhibited intracellular migration of glycoproteins out of the Golgi region in a variety of cell types. In the present work, the effects of these drugs on migration of membrane glycoproteins have been examined at the ultrastructural level in duodenal villous columnar cells and hepatocytes. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In duodenal villous columnar cells, 3H-fucose labeling of the apical plasma membrane was reduced by 51% after colchicine and by 67% after vinblastine treatment; but there was little change in labeling of the lateral plasma membrane. Labeling of the Golgi apparatus increased. This suggests that labeled glycoproteins destined for the apical plasma membrane were inhibited from leaving the Golgi region, while migration to the lateral plasma membrane was not impaired. In hepatocytes, labeling of the sinusoidal plasma membrane was reduced by 83% after colchicine and by 85% after vinblastine treatment. Labeling of the lateral plasma membrane also decreased, although not so dramatically. Labeling of the Golgi apparatus and neighboring secretory vesicles increased. This indicates that the drugs inhibited migration of membrane glycoproteins from the Golgi region to the various portions of the plasma membrane. Accumulation of secretory vesicles at the sinusoidal front suggests that exocytosis may also have been partially inhibited. In both cell types, microtubules almost completely disappeared after drug treatment. Microtubules may, therefore, be necessary for intracellular transport of membrane glycoproteins, although the possibility of a direct action of these drugs on Golgi or plasma membranes must also be considered.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: I. Inhibition of glycoprotein migration in various rat cell types as shown by light microscope radioautography after injection of 3H-fucose

Previous studies have shown that colchicine and vinblastine inhibit secretion in many cell types by interrupting the normal intracellular migration of secretory products. In the present work, radioautography has been used to study the effects of these drugs on migration of membrane and secretory glycoproteins in a variety of cell types. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for light microscope radioautography. Examination of secretory cell types such as ameloblasts and thyroid follicular cells in control animals revealed reactions of approximately equal intensity over the Golgi region and over extracellular secretion products, while in drug-treated rats most of the reaction was confined to the Golgi region. In a variety of other cell types, including endocrine cells (e.g., hepatocytes) and cells generally considered as nonsecretory (e.g., intestinal columnar cells), reaction in control animals occurred both over the Golgi region and over various portions of the cell surface. In drug-treated animals, a strong Golgi reaction was present, but reaction over the cell surface was weak or absent. These results indicate that in many cell types, colchicine and vinblastine inhibit migration out of the Golgi region not only of secretory glycoproteins, but also of membrane glycoproteins destined for the plasma membrane.     

Synthesis and migration of 3H-fucose-labeled glycoproteins in the ciliary epithelium of the eye: effects of microtubule-disrupting drugs

3H-fucose was injected intravenously or intravitreously into albino rats. After time intervals of 10, 40, and 50 min, 1, 1.5, and 4 hr, 1, 3, and 7 days, and 1, 2, and 4 weeks after injection, the animals were sacrificed by intracardiac perfusion with glutaraldehyde. Samples of the ciliary body were prepared for light and electron microscope radioautography. Light microscope autoradiographs showed that the cells of both the inner and outer layers of ciliary epithelium actively incorporated 3H-fucose label in a reaction that peaked in intensity at 4 hr after injection, and then progressively declined. Electron microscope radioautographs revealed that, at early time intervals, most of the label was localized to the Golgi apparatus. With time, the plasma membrane of both cell types became increasingly labeled, and accounted for 60-70% of the total silver grains at 4 hr after injection. Adjacent to the basal cell surface of the inner layer cells, the fibers of the zonula became increasingly labeled from 1.5 hr onwards, providing strong evidence that these cells secrete glycoproteins to the zonula. When vinblastine was administered 30 min before 3H-fucose injection, followed by sacrifice 1.5 hr later, a much larger proportion of label remained localized to the Golgi apparatus than in controls, and the plasma membrane and zonula were much less labeled. These results suggest that, as documented in other cell types, microtubules may play a role in the intracellular transport of membrane and secretory glycoproteins in these cells.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.     

Effects of colchicine on the ultrastructure of mouse taste buds

Summary Effect of colchicine on the ultrastructure of taste bud cells was studied in the mouse. In untreated mice microtubules were abundant throughout the entire cytoplasm of type-III cells, but only in the apical cytoplasm of type-I cells. After 2 h of colchicine treatment, no microtubules were observed in any taste bud cells; dense secretory granules in the apical cytoplasm of type-I cells mostly disappeared, and instead, numerous phagosomes appeared. It is suggested that colchicine causes an interruption of the transport of the secretory granules in type-I cells from the Golgi apparatus to the membrane of the apical surface, from which release occurs. In type-III cells, after 4 or 5 h of treatment, dense-cored vesicles scattered throughout the cytoplasm tended to increase in number; they were often observed to accumulate in the vicinity of the Golgi apparatus. Five hours after treatment with 5-hydroxy-l-tryptophan (5-HTP) following colchicine pretreatment, monoamine specific fluorescent cells and vesicles with highly electron-dense cores of type-III cells were still present. On the other hand, 5 h after 5-HTP treatment alone both fluorescent cells and vesicles with highly electron-dense cores had already disappeared. These observations suggest that the treatment with colchicine interrupts the transport of densecored vesicles of type-III cells to synaptic areas, in which those vesicles are presumed to discharge the neurotransmitter substance.     

Membranes of mammary gland : XV. 5-Thio- -glucose decreases lactose content and inhibits secretory vesicle maturation in lactating rat mammary gland

Short-term administration of the glucose analog 5-thio- -glucose to primiparous lactating rats reduced mammary tissue lactose concentrations to half of control levels. Treatment with colchicine alone caused slight reductions in mammary tissue lactose content. These treatments did not alter the morphology or degree of development of rough endoplasmic reticulum or Golgi apparatus, but did cause alterations in secretory vesicles. In mammary tissue from untreated lactating animals, large, swollen secretory vesicles were abundant in apical regions of epithelial cells. After thioglucose administration secretory vesicles in the apical cytoplasm were smaller and were more densely packed with contents. While administration of colchicine alone caused accumulation of large numbers of nearly fully swollen vesicles, treatment with both colchicine and thioglucose induced accumulation of smaller, less fully developed secretory vesicles which contained morphologically recognizable casein micelles. Mammary tissue from late gestation rats was low in lactose; vesicles in this tissue resembled secretory vesicles in tissue from rats treated with thioglucose in that they were small and densely packed. These observations suggest that lactose, an osmoregulator in mammary gland, is transferred from Golgi apparatus to the apical cell surface within secretory vesicles. Lactose appears to be important for secretory vesicle maturation in mammary epithelial cells.     

Synthesis and migration of 3H-fucose-labeled glycoproteins in the retinal pigment epithelium of albino rats, as visualized by radioautography

3H-fucose was injected into the vitreous body of the eye(s) of 250-gm rats, which were then killed by means of an intracardiac perfusion with glutaraldehyde after intervals of 10 min, 1 and 4 hr, and 1 and 7 days. The eyes were removed and further fixed, and pieces of retina were processed for light and electron microscope radioautography. Light microscope radioautography showed that the pigment epithelial cells actively incorporated 3H-fucose label. The intensity of reaction peaked at 4 hr after injection of the label and then slowly declined. Quantitative electron microscope radioautography revealed that, at 10 min after 3H-fucose injection, over 70% of the label was localized to the Golgi apparatus, indicating that fucose residues are added to newly synthesized glycoproteins principally at this site. With time the proportion of label associated with the Golgi apparatus decreased, but that assigned to the infolded basal plasma membrane, the apical microvilli, and various apical lysosomes increased. These results indicate that in retinal pigment epithelial cells newly synthesized glycoproteins continuously migrate from the Golgi apparatus to lysosomes and to various regions of the plasma membrane. In this case, the membrane glycoproteins may play specific roles in receptor functions of the basal plasma membrane or phagocytic activities at the apical surface. Very little label migrated to Bruch's membrane, indicating either a very slow turnover or a paucity of fucose-containing glycoproteins at this site.     

Distribution and content of microtubules in relation to the transport of lipid. An ultrastructural quantitative study of the absorptive cell of the small intestine

To determine whether microtubules are linked to intracellular transport in absorptive cells of the proximal intestine, quantitative ultrastructural studies were carried out in which microtubule distribution and content were determined in cells from fasting and fed animals. Rats were given a 1-h meal of standard chow, and tissue was taken from the mid-jejunum before, 1/2 h, and 6 h after the meal. The microtubule content of apical, Golgi, and basal regions of cells was quantitated by point-counting stereology. The results show) that microtubules are localized in intracellular regions of enterocytes (apical and Golgi areas) previously shown to be associated with lipid transport, and that the microtubule content within apical and Golgi regions is significantly (P less than 0.01) reduced during transport of foodstuffs. To determine the effect of inhibition of microtubule assembly on transport, colchicine or vinblastine sulfate was administered to postabsorptive rats, and the lipid and microtubule content of enterocytes determined 1 and 3 h later. After treatment with these agents, lipid was found to accumulate in apical regions of the cells; this event was associated with a significant reduction in microtubule content. In conclusion, the regional distribution of microtubules in enterocytes, the decrease in assembled microtubules after a fat-containing meal, and the accumulation of lipid after the administration of antimicrotubule agents suggest that microtubules are related to lipid transport in enterocytes.     

Histochemical and radioautographic study of glycoprotein secretion in the epithelium lining the uterine tubes of mice

Summary Two-month-old female Swiss mice that had come into estrus were injected intravenously with L-³H-fucose and killed at 5, 15, 40 min, and 4 h after injection. Pieces of the isthmus and of the ampulla of the uterine tubes were processed for light-and electron-microscopic radioautography. Incorporation of ³H-fucose was more intense in the isthmian secretory cells than in the ciliated cells of the ampulla. Electron-microscopic radioautography of the isthmian secretory cells demonstrated that ³H-fucose was incorporated into newly synthesized glycoproteins in the Golgi apparatus from where labelled glycoproteins migrated mainly to secretory granules and apical microvilli. The histochemical technique using ruthenium red confirmed the presence of glycoproteins in the contents of the secretory granules released to the lumen of the uterine tubes as demonstrated by radioautography. Other glycoproteins are transported inside small vesicles and most likely are related to the renewal of the plasma membrane. The role of the secretory glycoproteins in various events of mammalian reproduction is discussed.     

Cytoplasmic organization and quantitation of microtubules in bovine mammary epithelial cells during lactation and involution

Summary Ultrastructural examination of milk secretory cells from lactating bovine mammary gland revealed presence of numerous microtubules in the apical and paranuclear cytoplasm, particularly in the vicinity of Golgi components. Most microtubules were oriented perpendicular to the apical plasma membrane and appeared to form a framework around Golgi dictyosomal elements and secretory vesicles. In comparison, non-secretory cells obtained from involuting glands displayed few microtubules and these were randomly located throughout the cytoplasm with no particular orientation.     

Distribution and orientation of microtubules in milk secreting epithelial cells of rat mammary gland

Summary Quantitative ultrastructural analysis of mid-lactation rat mammary gland demonstrated that cytoplasmic microtubules were present in nearly all secretory epithelial cells examined. Most microtubules were oriented perpendicular to the apical membrane and were found in the apical and medial portions of the cell cytoplasm. There was no statistical difference between the number of microtubules associated with vesicles and the number that were not. Most vesicles which were in contact with microtubules were small (50 to 150 nm), appeared electron lucent and were located in a supra-Golgi complex position. Many of these vesicles were seen to be aligned along the axis of longitudinally sectioned microtubules oriented perpendicular to the apical plasma membrane. As measured by a colchicine binding assay, the total tubulin content of mammary tissue from mid-lactation rats was about 107 g/100 mg wet weight. Approximately 19% of the total tubulin was in polymerized form. This study provides evidence that microtubules may be involved in guiding transport of small secretory vesicles to the apical regions of cells for exocytosis.     

Effects of colchicine on endocytosis and cellular inactivation of horseradish peroxidase in cultured chondrocytes

Horseradish peroxidase (HRP) was used as a marker to study the effects of microtubule-disruptive drugs on uptake and cellular inactivation of exogenous material in cultures of embryonic chick chondrocytes. HRP was ingested by fluid endocytosis, and intracellular enzyme activity subsequently diminished exponentially with time. Cytochemically, reaction product for HRP was found in vesicles often located close to the dictyosomes of the Golgi complex. Colchicine and vinblastine caused disappearance of cytoplasmic microtubules and disorganization of the Golgi complex with concomitant reduction in the cellular uptake of HRP to about half of that in the controls. Lumicolchicine, on the other hand, left cell fine structure and HRP uptake unaffected. These results indicate that microtubules are of considerable importance in the process of fluid endocytosis in cultured chondrocytes although the exact mechanism remains to be elucidated. The rate of intracellular inactivation of ingested HRP was not affected by colchicine or vinblastine. Double-labeling experiments with colloidal thorium dioxide and HRP likewise indicated that fusion of endocytic vesicles and lysosomes is not dependent on intact microtubules. The total specific activities of the three lysosomal enzymes examined were weakly or not at all changed by treatment of the cultures with colchicine or vinblastine. It therefore seems unlikely that microtubular organization plays an important role in the production or degradation of lysosomal enzymes in cultured chondrocytes.     

Microtubules and the organization of the Golgi complex

Electron microscopic and cytochemical studies indicate that microtubules play an important role in the organization of the Golgi complex in mammalian cells. During interphase microtubules form a radiating pattern in the cytoplasm, originating from the pericentriolar region (microtubule-organizing centre). The stacks of Golgi cisternae and the associated secretory vesicles and lysosomes are arranged in a circumscribed juxtanuclear area, usually centered around the centrioles, and show a defined orientation in relation to the rough endoplasmic reticulum. Exposure of cells to drugs such as colchicine, vinblastine and nocodazole leads to disassembly of microtubules and disorganization of the Golgi complex, most typically a dispersion of its stacks of cisternae throughout the cytoplasm. These alterations are accompanied by disturbances in the intracellular transport, processing and release of secretory products as well as inhibition of endocytosis. The observations suggest that microtubules are partly responsible for the maintenance and functioning of the Golgi complex, possibly by arranging its stacks of cisternae three-dimensionally within the cell and in relation to other organelles and ensuring a normal flow of material into and away from them. During mitosis, microtubules disassemble (prophase) and a mitotic spindle is built up (metaphase) to take care of the subsequent separation of the chromosomes (anaphase). The breaking up of the microtubular cytoskeleton is followed by vesiculation of the rough endoplasmic reticulum and partial atrophy, as well as dispersion of the stacks of Golgi cisternae. After completion of the nuclear division (telophase), the radiating microtubule pattern is re-established and the rough endoplasmic reticulum and the Golgi complex resume their normal interphase structure. This sequence of events is believed to fulfil the double function to provide tubulin units and space for construction of the mitotic spindle and to guarantee an approximately equal distribution of the rough endoplasmic reticulum and the Golgi complex on the two daughter cells.     

Localization of thyroglobulin antigenicity in rat thyroid sections using antibodies labled with peroxidase or (125)I-radioiodine

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling. Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin. It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.     

Effects of vinblastine and colchicine on the rat adrenal cortex: morphometric and cytochemical studies

The effects of administration of anti-microtubular drugs--vinblastine and colchicine--on the ultrastructure of the zona fasciculata cells of young rat adrenal were studied. Young male rats were injected with vinblastine and sacrificed 2 hr later or with colchicine and sacrificed 3 hr after drug administration. Animals injected with isotonic saline in same experimental conditions served as controls. Ultrastructural alterations provoked by both drugs, vinblastine or colchicine, were identical and were most prominent in the Golgi areas. They appeared enlarged and crowded with round, or slightly elongated light vesicles, acid phosphatase, and osmium negatives. The Golgi dictyosomes, although keeping their normal morphology, were less numerous and presented cisternae which were narrower and shorter than controls. Electron-dense vesicles, round or elongated, and acid phosphatase positive--lysosomes--were observed in great number in the Golgi areas, intermingled with light vesicles. The relative volume of light vesicles and lysosomes of the treated animals was significantly increased when compared with controls, but the relative volume of dictyosomes was significantly decreased. Also the numerical density of light vesicles and lysosomes of the injected rats was significantly increased when compared with controls. These alterations are highly suggestive of the Golgi involvement in the adrenal secretory process.     

Cytochemical localization of complex carbohydrates in the thyroid gland of normal and TSH-treated mice

Summary The silver methenamine method for the ultrastructural localization of carbohydrates and glycoproteins was applied to the thyroid glands of normal and TSH-treated mice. The majority of the cisternae of the rough endoplasmic reticulum showed a weak, but apparently positive reaction. These findings support the opinion that glycosylation of thyroglobulin occurs initially in the rough endoplasmic reticulum. By this method the Golgi apparatus was observed to display a staining gradient. The intermediate to inner saccules were intensely stained, whereas the outer saccules were not so heavily stained. This phenomenon indicates that the Golgi apparatus has a functional polarity for the addition of carbohydrates to thyroglobulin and other proteins. In the inner and/or the peripheral regions of the Golgi apparatus and in the apical cytoplasm, a large number of globules of various sizes, considered to be colloid droplets, lysosomes and apical secreting vesicles, showed a positive reaction. The luminal colloid was also positive with silver methenamine staining, with almost the same intensity as the globules and vesicles.This study was supported by a grant from the Japan Ministry of Education     

Translocation of dimeric IgA through neoplastic colon cells in vitro.

We studied the translocation of dimeric IgA across epithelium, using neoplastic human colon cells in culture as a source of epithelial cells, and immunoelectronmicroscopy with peroxidase-labeled antigens and antibodies. The cells had some of the ultrastructural characteristics of normal, mature epithelial cells, i.e., polarity, desmosomal junctions, and secretory component on their basal and lateral plasma membranes. Horseradish peroxidase-labeled dimeric IgA, exposed to the cells at 0 degrees C, bound selectively to secretory component on the cell surfaces. At 37 degrees C, the bound dimeric IgA was taken into the cells by endocytosis and transported apically through the cytoplasm in vesicles. After 30 min, IgA was discharged across the apical surface. Neither colchicine (10(-4) M) nor cytochalasin B (10(-5) M) interfered with binding or endocytosis of dimeric IgA, but colchicine inhibited intracellular transport of the IgA-containing vesicles. These experiments demonstrated that dimeric IgA can be transported through living intestinal epithelial cells in vitro. The transport includes 1) specific binding of IgA dimers to secretory component on plasma membranes, 2) endocytosis of IgA in vesicles, 3) transcytoplasmic transport of the IgA-containing vesicles by a process involving microtubules, and 4) discharge of IgA at the apical surfaces.     

The involvement of coated vesicles in the secretion of corticosterone by the zona fasciculata of the rat adrenal cortex

Following exposure of rats to unpredictable stress there was a marked increase in the number of ‘coated’ vesicles in contact with or close to the cell membrane of the zona fasciculata cells. The close correlation between the vesicle numbers and the plasma levels of corticosterone led to the hypothesis that the coated vesicles were intimately involved in the secretory process. The use of horseradish peroxidase as a tracer protein confirmed that the coated vesicles were not involved in pinocytosis and the inward movement of materials, this function being performed by a much larger uncoated vesicle. The presence of microtubules associated with the coated vesicles and radiating through the Golgi body region, the site of formation of the vesicles, suggested that they may be involved in the transport of the secretory product to the cell membrane. The use of microtubule inhibitors, colchicine and vinblastine, were found to significantly reduce the plasma steroid response to stress. On the basis of these findings a new secretory mechanism was postulated.     

Influence of antimicrotubular drugs on the Golgi apparatus of stomach secretory mucoid cells and small intestine absorptive cells

The effects of vinblastine and colchicine on the Golgi apparatus of stomach surface mucoid and absorptive intestinal cells were compared by cytochemical analysis. The two epithelial cells were chosen because of their different specific functions in the formation of secretory granules, the production of lysosomes and the intensity of membrane traffic in the cytoplasm. For the analysis, adult mice were injected with 1 mg/100 g b.w. of vinblastine and 1 mg/100 g b.w. of colchicine. For the demonstration of cis and trans cisternae of the Golgi apparatus, prolonged osmification, thiamine pyrophosphatase and acid phosphatase activity identification were applied. After treatment with vinblastine or colchicine, polarity of stacks in the Golgi apparatus of surface mucoid cells is preserved although the number of cisternae with thiamine pyrophosphatase or acid phosphatase activity decreases. However, the Golgi apparatus of intestinal absorptive cells completely disintegrates and only a few separated cis or trans cisternae can be identified. The main effect seems to be a reduction of vesicles which can be cytochemically identified as parts of the Golgi apparatus and an accumulation of vesicles which probably originate from budding ER. Communication between the ER and the Golgi apparatus seems to be interrupted.     

Immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment

Summary The purpose of this study was to investigate the effect of colchicine and vinblastine on the localization of alkaline phosphatase (AlPase) in rat duodenum in relation to structural changes. AlPase was localized on the membranes of rough endoplasmic reticulum, Golgi stacks, cytoplasmic vesicles, microvilli, on lateral plasma membranes, and in some lysosomes of the duodenal epithelial cells of rats treated with either lumicolchicine or 0.9% NaCl alone. Microvilli were most intensely stained, and AlPase-positive Golgi stacks were regularly distributed in the supranuclear regions. After colchicine treatment, microvilli were shortened and the staining intensity became weaker, whereas basal as well as lateral plasma membranes showed stronger staining. The AlPase-positive microvilli appeared not only on the luminal surfaces, but also on the baso-lateral plasma membranes and even on the surfaces of characteristic intracytoplasmic cysts. Golgi stacks became smaller and their distribution became less localized, and the staining intensity of the Golgi stacks became weaker. AlPase localization in rats treated with vinblastine was almost identical with that of rats treated with colchicine. Thus, colchicine and vinblastine appeared to have elicited a disorientation of intracellular transport of intestinal AlPase by inhibiting microtubule organization.