Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: II. Inhibition of secretion of thyroglobulin in rat thyroid follicular cells as visualized by radioautography after 3H-fucose injection

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 thyroid follicular cells of control animals, at this time interval, 57% of the total label was associated with colloid and secretory vesicles in the apical cytoplasm while 27% was localized in the Golgi apparatus and neighboring vesicles. In experimental animals, the proportion of label in colloid and apical vesicles was reduced by more than 69% after colchicine and more than 83% after vinblastine treatment. The proportion of label in the Golgi region, on the other hand, increased by more than 125% after colchicine and more than 179% after vinblastine treatment. Within the Golgi region, the great majority of the label was associated with secretory vesicles which accumulated adjacent to the trans face of the Golgi stacks. It is concluded that the drugs do not interfere with passage of newly synthesized thyroglobulin from the Golgi saccules to nearby secretory vesicles, but do inhibit intracellular migration of these vesicles to the cell apex. In most cells the number of vesicles in the apical cytoplasm diminished, but this was not always the case, suggesting that exocytosis may also be partially inhibited. The loss of microtubules in drug-treated cells suggests that the microtubules may be necessary for intracellular transport of thyroglobulin.     

The formation of glandular secretion in larval Austramphilina elongata (Amphilinidea)

The ultrastructure of three types of gland cells of embryos and free-swimming larvae of Austramphilina elongata is described. Type I gland cells contain large, more or less round electron-dense granules which are formed by numerous Golgi complexes. Type II gland cells contain thread-like, membrane-bound secretory granules with longitudinally arranged microtubules inside the granules; secretory droplets are produced by Golgi complexes and the microtubules apparently condense in the cytoplasm or in the droplets. Type III gland cells contain irregular-ovoid membrane-bound granules with coiled up microtubules which have an electron-dense core; the granules are formed by secretionderived from Golgi complexes and the microtubules aggregate around and migrate into the secretion; microtubules are at first hollow and the early secretory granules have a central electron-dense region.     

The fine structural effect of sialectomy on the taste bud cells in the rat.

Taste buds in the rat and other mammals share a secretory activity with their transduction function as taste receptor. The present work shows the effect of bilateral removal of the main salivary glands on taste bud cells' components related to secretion in the vallate papilla of the rat. In the sialectomized rats remarkable changes were evidence in the dark and intermediate types of taste bud cells, which are known to be the secretory components. Such changes involve hypertrophy of either the protein synthetizing machinery, the smooth endoplasmic reticulum or the Golgi complex. Lucent and coated vesicles associated to Golgi cisternae increased in number but the amount of dense-core vesicles (secretory vesicles) at the apical cytoplasm of cells decreased. Images of exocytosis of secretory products were observed. The hypertrophy of Golgi complex components was clearly detected with the OsO4 impregnation method for light and electron microscopy. Alteration in the acid phosphatase activity of taste bud cells was not observed in the sialectomized rats. These findings suggest that sialectomy stimulates the entire secretory cycle of dark and intermediate taste bud cells. The light taste bud cells, which are not engaged in secretion, are hardly affected by the treatment. Although taste buds in mammals are neuro-dependent structures, present evidence indicates that they are also sensitive to non-neural influences.     

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.     

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.     

Effect of monoamines on the taste buds in the mouse

Summary Mouse taste buds were investigated following administration of monoamines and their precursors by fluorescence and electron microscopy. The appearance of fluorescent cells within the taste bud and the ultrastructural changes of vesicles in the gustatory cells were due to the treatment of 5-hydroxytryptophan. Small dense-cored vesicles (30–60 nm in diameter) appeared throughout the cytoplasm and accumulated especially at the presynaptic membranes of afferent synapses. Large dense-cored vesicles (80–100 nm) increased twice in number, and electron densities of their cores became more dense as compared with untreated mice. Fluorescent cells appeared in the taste bud of l-DOPA treated mice, whereas no ultrastructural changes were observed. These results suggest that the gustatory cells of the taste bud are capable of taking up and storing monoamines, which might act as neurotransmitters from the gustatory cells to the nerves.     

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.     

Ultrastructure of palatal taste buds in the perihatching chick.

Palatal taste buds of perihatching chicks were examined by electron microscopy. Four intragemmal cell types were characterized. 1) Light: with voluminous, electron-lucent cytoplasm containing scattered free ribosomes, rough and smooth endoplasmic reticulum, plump mitochondria, sparse perinuclear filaments, occasional Golgi bodies, and numerous clear and dense-cored vesicles. Clear vesicles sometimes aggregate in a presynaptic-like configuration apposed to an axonal profile. These cells contained large, spherical, uniformly granular nuclei with one nucleolus. 2) Dark: with dense cytoplasm containing filamentous bundles surrounding the nucleus, occasional clear vesicles, centrioles, rough endoplasmic reticulum, and compact mitochrondria. The apical cytoplasm noticeably lacks dense secretory granules. Irregular to lobulated nuclei are densely granular, and contain scattered clumps of chromatin, adhering especially to the inner leaflet of the nuclear membrane, and at least one nucleolus. Cytoplasmic extensions of dark cells envelop other intragemmal cell types and nerve fibers. Light and dark cells project microvilli into the taste pore. 3) Intermediate: contain gradations of features of light and dark cells. 4) Basal: darker than the other intragemmal cell types and confined to the ventral bud region. Putative afferent synapses in relation to light cells, and axo-axonal contacts are described. While the appearance of axo-axonal contacts may be a transient developmental event, other bud features are consonant with observations in adult chickens and suggest that the peripheral gustatory apparatus is mature at hatching in this precocial avian species.     

Secretory organelles in ECL cells of the rat stomach: an immunohistochemical and electron-microscopic study

ECL cells are numerous in the rat stomach. They produce and store histamine and chromogranin-A (CGA)-derived peptides such as pancreastatin and respond to gastrin with secretion of these products. Numerous electron-lucent vesicles of varying size and a few small, dense-cored granules are found in the cytoplasm. Using confocal and electron microscopy, we examined these organelles and their metamorphosis as they underwent intracellular transport from the Golgi area to the cell periphery. ECL-cell histamine was found to occur in both cytosol and secretory vesicles. Histidine decarboxylase, the histamine-forming enzyme, was in the cytosol, while pancreastatin (and possibly other peptide products) was confined to the dense cores of granules and secretory vesicles. Dense-cored granules and small, clear microvesicles were more numerous in the Golgi area than in the docking zone, i.e. close to the plasma membrane. Secretory vesicles were numerous in both Golgi area and docking zone, where they were sometimes seen to be attached to the plasma membrane. Upon acute gastrin stimulation, histamine was mobilized and the compartment size (volume density) of secretory vesicles in the docking zone was decreased, while the compartment size of microvesicles was increased. Based on these findings, we propose the following life cycle of secretory organelles in ECL cells: small, electron-lucent microvesicles (pro-granules) bud off the trans Golgi network, carrying proteins and secretory peptide precursors (such as CGA and an anticipated prohormone). They are transformed into dense-cored granules (approximate profile diameter 100 nm) while still in the trans Golgi area. Pro-granules and granules accumulate histamine, which leads to their metamorphosis into dense-cored secretory vesicles. In the Golgi area the secretory vesicles have an approximate profile diameter of 150 nm. By the time they reach their destination in the docking zone, their profile diameter is between 200 and 500 nm. Exocytosis is coupled with endocytosis (membrane retrieval), and microvesicles in the docking zone are likely to represent membrane retrieval vesicles (endocytotic vesicles).     

The cytoarchitecture of gustatory receptors from the rabbit foliate papillae

Summary The apical region of the taste bud, delimited by the stratum disjunctum of the papillary epithelium, is divided into a distal taste pore, a channel, and a proximal chamber. In addition to fuzzy coated microvilli, the chamber contains an amorphous dense material histochemically defined as a neutral mucopolysaccharide.Abounding in the apical cytoplasm of the taste cell is a randomly oriented filamentous component 60–70 Å in diameter extending into each microvillus. Likewise in this area membrane-bounded electron-dense bodies are found whose content is considered to be synthesized by the rough endoplasmic reticulum and subsequently concentrated and packaged by the Golgi complex. These bodies are thought to contain the neutral mucopolysaccharide, which finally reaches the chamber. Other components of the taste cell cytoplasm include vesicles, mitochondria, ribosomes, and nucleus. Two centrioles, each possessing a pair of rootlets, have been observed in the apical cytoplasm. Adjacent taste cells are attached apically by a zonula occludens followed by a zonula adhaerens. The synaptic clefts between taste cells and nerve fibers are acetylcholinesterase positive but are negative for butylcholinesterase and adenosine triophosphatase. A lineage of taste cells is postulated.This work was supported in part by In House Independent Research Grant No. 6.11.30.01, 3A013001A91C; by National Defense Education Act Group IV Fellowship, awarded to the author; and in part by Grant No. GM-0877, awarded to Dr. Everett Anderson, University of Massachusetts, by the National Institutes of Health. I am grateful to Drs. Everett AnderSon, James N. Dumont, Gunter F. Bahr, and Joe L. GRiffin for their expert guidance and support in the preparation of this paper.     

Morphometric and ultrastructural features of the mare oviduct epithelium during oestrus

Morphometric, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) investigations have displayed regional differences in the mare oviductal epithelium. The entire mucosa of the oviduct was lined with a pseudostratified epithelium, which consisted of two distinct cell types, ciliated and non-ciliated. Ciliated cells were predominant in the three different segments of the oviduct and their percentage increased from fimbriae to ampulla and significantly decreased in the isthmus. SEM revealed in the infundibulum finger-like mucosal folds, some of them interconnected, in the ampulla numerous and elaborated branched folds of the mucosa, whereas the isthmus displayed a narrow lumen, short and non-branched mucosal folds. In the ampulla and isthmus the majority of non-ciliated cells showed apical blebs provided or not of short microvilli. TEM displayed different ultrastructural features of ciliated and non-ciliated cells along the oviduct. Isthmus ciliated cells presented a more electron-dense cytoplasm than in infundibulum and ampulla cells and its cilia were enclosed in an amorphous matrix. The non-ciliated cells of infundibulum did not contain secretory granules but some apical endocytic vesicles and microvilli coated by a well developed glycocalyx. Non-ciliated cells of ampulla and isthmus contained secretory granules. Apical protrusions of ampulla displayed two types of secretory granules as well as occasional electron-lucent vesicles. Isthmus non-ciliated cells showed either electron-lucent or electron-dense cytoplasm and not all contained apical protrusions. The electron-dense non-ciliated cells displayed microvilli coated with a well developed glycocalyx. Three types of granules were observed in the isthmus non-ciliated cells. The regional differences observed along the epithelium lining the mare oviduct suggest that the epithelium of the each segment is involved in the production of a distinctive microenvironment with a unique biochemical milieu related to its functional role.     

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.     

Ultrastructure of mouse incisor ameloblasts after vascular perfusion with colchicine

Summary In order to revalue the effects of colchicine on incisor secretory ameloblasts, entire mice were perfused with Krebs solution supplemented with a buffer and amino acids, through the right common carotid artery. The normal ultrastructure of the cells was maintained for 2 h with the perfusate alone. When colchicine (0.3–3.0 g/ml) was added to the perfusate, it induced ultrastructural changes, such as the loss of cytoplasmic microtubules, the loss of secretory granules in Tomes' process, the abnormal accumulation and secretion of secretory granules, disarranged Golgi apparatus and the fragmentation of rough endoplasmic reticulum. Vesicles (150–400 nm in diameter) resembling immature secretory granules also accumulated, the degree of accumulation depending on the duration of colchicine treatment. The accumulation of secretory granules and these vesicles suggests that the intracellular transport system was affected by colchicine but that the production of secretory granules was continuous throughout the experimental period. The present perfusion system has enabled us to treat ameloblasts with an agent that is a useful experimental tool for elucidating cell functions, despite being lethal to animals in vivo.     

Fine structure of the dorsal lingual epithelium of the lizard, Gekko japonicus (Lacertilia, Gekkonidae)

Three different types of lingual papilla were observed by scanning electron microscopy on the dorsal lingual epithelium of the lizard Gekko japonicus. Dome-shaped lingual papillae were located at the apex. Flat, fan-shaped lingual papillae were seen in the widest area of the lingual body. Long, scale-like lingual papillae were arranged on the latero-posterior dorsal surface. At higher magnification, microvilli and microridges were seen to be widely distributed over the surface of the papillae. By light microscopy, the epithelium of the dome-shaped papillae was composed of single, columnar epithelial cells filled with secretory granules. The tip of the epithelium of the fan-shaped and scale-like papillae was composed of stratified squamous epithelial cells without granules. The major part of the epithelium of these two types of papilla, except the tip area, was also composed of single, columnar epithelial cells with secretory granules. By transmission electron microscopy, a nucleus without a defined shape was seen to be located in the basal part of each of the single, columnar epithelial cells. Rough-surfaced endoplasmic reticulum and Golgi apparatus were well developed around the nucleus. The other, major part of the cytoplasm was filled with the spherical secretory granules, a large number of which had very electron-dense cores and moderately electron-dense peripheral regions. In the stratified squamous epithelium, a nucleus, which tended to be condensed on the free-surface side, was located in the center of each cell. Mitochondria, endoplasmic reticulum, and vesicles were observed in the cytoplasm.     

Immunoelectron-microscopic demonstration of histamine depletion in the gastric enterochromaffin-like cells of rats treated with α-fluoromethylhistidine

We conducted an immunoelectron-microscopic study for histamine (HA) in the enterochromaffin-like (ECL) cells of normal rats and rats given alpha-fluoromethylhistidine (alpha-FMH, 3 mg/kg per hour) via osmotic minipumps over a period of 24 h. The indirect immunoperoxidase procedure utilized a mouse monoclonal antibody (mAb), AHA-2, which is produced against glutaraldehyde-conjugated HA. alpha-FMH is a potent and irreversible inhibitor of the HA-forming enzyme histidine decarboxylase and is known to reduce tissue HA concentrations in several tissues. The present study clearly demonstrated that HA immunoreactivity, which was found to a high degree in the cores of the granules and secretory vesicles and in the cytoplasm of ECL cells of control rats, was completely abolished from the corresponding compartments in the cells of alpha-FMH-treated rats. Furthermore, treatment with alpha-FMH drastically lowered the number of secretory vesicles and was associated with larger cores in the granules of the ECL cells. These results seem to support the idea of a HA-pathway mechanism, emphasizing that the granules in normal ECL cells take up HA from the cytosol during its transport from the Golgi zone to the more peripheral portion of the cell and condense it in their cores, thus forming mature secretory vesicles. However, the present study showed that not only the secretory vesicles but also almost all the granules seen in ECL cells were already loaded with HA in their cores, suggesting that the newborn granules very rapidly take up HA from the cytosol. Also suggested was the fact that HA depletion impairs the maturation of the granules into secretory vesicles.     

Ultrastructural and immunocytochemical studies of neuromuscular junctions in oviduct of Locusta migratoria

The ultrastructure of nerve endings in the oviduct visceral muscles of Locusta migratoria was studied by electron microscopy and by immunogold labeling for two kinds of neuromodulators, the pentapeptide proctolin and FMRFamide-related peptides. Nerve endings contained electron-lucent round vesicles and two kinds of granules (round and avoid), and formed two types of synapses or release sites with the muscle. The morphologically distinct nerve endings were classified into three different categories based on the composition of synaptic vesicles and granules. Type-I nerve endings were dominated by electron-lucent round vesicles and contained only a few round electron-dense granules. Type-II nerve endings contained mostly electron-dense round granules and electron-lucent round vesicles. A few electron-dense ovoid granules were also present. Electron-dense ovoid granules dominated the type-III nerve endings, which usually contained less electron-lucent vesicles than either type-I or II nerve endings. Both proctolin and FMRFamide-like immunoreactivity was associated with electron-dense round granules. However, FMRFamide-like immunoreactivity was only found in the type-II nerve endings, while proctolin immunoreactivity was found within type-I nerve endings as well as in some type-II nerve endings. Immunological results therefore allow us to further divide type-II nerve endings into type-IIa (immunonegative for proctolin) and type-IIb (immunopositive for proctolin). Type-III nerve endings show no immunolabeling to either proctolin or FMRFamide.     

Effect of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells. Immunocytochemical observations

We studied the effects of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells using the direct immunoenzyme technique. Livers were perfusion-fixed 0.5, 1, and 2 h after injection of colchicine. Vibratome sections of the fixed liver were stained using peroxidase-conjugated Fab' of anti-albumin or anti-fibrinogen. By light microscopy, reaction deposits showing albumin and fibrinogen were observed in the cytoplasmic granules of hepatocytes. Such stained granules decreased 30 min after injection, but later increased gradually and crowded in the cytoplasm. The Golgi complex stained for the proteins decreased after 30 min but increased in the juxtanuclear region after 60 min. The analysis of serial sections showed that colchicine severely disturbed the spatial relationship between the Golgi apparatus and the bile canaliculus. We obtained similar results by electron microscopy; a positive reaction for albumin and fibrinogen was observed in a small number of the cytoplasmic granules after 30 min. After 1 h of treatment, most of the Golgi complexes were fragmented and lost their stacked cisternae. However, they reappeared accompanied with vacuolated cisternae and secretory granules, which were partially stained for albumin and fibrinogen. After 2 h, the secretory granules positive for both proteins accumulated further. Some of them lined a long the plasma membrane, and others made a cluster in the cytoplasm. The profiles showing exocytosis were very rarely seen. These results showed that in the first 30 min, colchicine primarily disturbs partially the Golgi assembly but does not affect the post Golgi secretory pathway much. Later, the drug affects both the post Golgi pathway and the Golgi assembly, and it causes a marked accumulation of secretory granules.     

Induction of apoptosis by colchicine in taste bud and epithelial cells of the mouse circumvallate papillae

Apoptotic cells in the taste buds and epithelia of mouse circumvallate papillae after colchicine treatment were examined by the methods of in situ DNA nick-end labeling, immunocytochemistry, and electron microscopy. After colchicine treatment, numerous positive cells appeared in the taste buds by DNA nick-end labeling, and some epithelial cells in the basal and suprabasal layers in and around the circumvallate papillae also revealed positive staining. Condensed and fragmented nuclei with a high density were occasionally found in the taste bud cells and in the basal and suprabasal layer epithelial cells by electron-microscopic observation. An immunocytochemical reaction for tubulin revealed weak staining in taste bud cells, because of the depolymerization of microtubules, and a decrease of the microtubules in the taste bud cells was observed by electron microscopy. These results indicate that colchicine treatment of mice induces the apoptosis of taste bud and epithelial cells in the circumvallate papillae and dorsal epithelial cells around the circumvallate papillae.     

Morphometric analysis of epithelial cells of frog urinary bladder, II. Effect of ADH, calcium ionophore (A23187) and verapamil on isolated dissociated cells

Isolated frog urinary bladder epithelial cells, upon dissociation lose their polarity and develop microridges and occasional microvilli in a global fashion. These cells, when exposed only to isotonic Ringer's solution manifest a membrane conformation with smooth discontinuous microridges, a cytoplasm with numerous free ribosomes, rough ER, thin Golgi cisternae, mitochondria, small vacuoles, electron-dense granules, few microtubules, and numerous microfilaments and intermediate filaments with an apparent random distribution, the dissociated cells, when treated with ADH or calcium ionophore (A23187), have the appearance of numerous elongated microvilli over the entire cell surface. The cytoplasm, under these conditions, is occupied by large vacuoles with a distribution of long profiles of aggrephores and associated vesicles. The peripheral cytoplasm as well as the cavities of the elongated microvilli of these cells contain large concentrations of microfilaments often showing a strong axial orientation to the long axis of the microvilli. Many of these filamentous elements appear in contact with the apical membrane of these microvilli with an alignment with the external glycocalyx. There is an indication that these morphocytological changes as revealed by SEM and TEM studies, correlated with a redistribution and realignment of microfilaments and possibly microtubules as detected by fluorescent microscopy using immunofluorescent antibody labeling for actin and tubulin. Cells treated with verapamil, a calcium antagonist, presented dwarf and stout microvilli with little detectable alterations in the cytoplasmic compositions from that of non-hormonal treated cells. Verapamil prevented ADH induction of microvilli, with the membrane, under these conditions, appearing as compact microridges. The results indicate that calcium ionophore, like ADH, produces intense formation of microvilli in dissociated cells, mobilization and realignment of microfilaments, microtubules, increase in the density of vesicles, aggrephores and possibly secretory granules, whereas the calcium antagonist, verapamil, opposes these actions. The results suggests a prominent role of calcium in the morphological changes induced by ADH.