Cytochalasin B and water transport

Summary A morpho-functional study of the effects of cytochalasin B (CB) on Na and water transport was made in amphibian epithelia. The functional studies confirmed the dissociation of the natriferic and hydrosmotic effects of vasopressin in toad urinary bladders exposed to CB and showed in addition that the block of the hydrosmotic effect was reversible and could still be induced in epithelia maximally stimulated with the hormone. Scanning electron microscopy revealed that CB, per se, did not alter the apical surface of the bladders. An almost total loss of microvilli of granular cells was seen, however, if CB was associated with vasopressin and an osmotic gradient. The results suggest two points: a) the block of the hydrosmotic flow induced by CB is due to factors beyond the apical membrane; b) microfilaments may be important mechanochemical transducers in the chain of events leading to the hydrosmotic effect of vasopressin.Supported by the grants Nos 3.1300.73 and 3.043-0.76 of the Swiss National Science FoundationThe authors are grateful to Miss C. Brücher, SEM operator of the Department of Physics, Ciba-Geigy, for skillful collaboration, to Mr. R. Mira for the illustrations and to Mrs. A. Cergneux for secretarial assistance     

Mucosal surface morphology of the toad urinary bladder. Scanning electron microscope study of the natriferic and hydro-osmotic response to vasopressin

The mucosal cell surface of the toad urinary bladder was examined by scanning electron microscopy, and changes in the structure of the surface of the granular cell were correlated with specific physiological responses to vasopressin. Survey views of the mucosal surface demonstrated that there was no consistent repeating anatomical relationship between the granular cell and the mitochondria-rich cell that would support the concept of cooperativeness in the response to vasopressin. During base-line states of Na+-transport and water flux, the microvilli on the mucosal surface of the granular cell are arranged in a ridge-like network with occasional individual projections. When water flux is increased by exposing the tissue to vasopressin, in the presence of an osmotic gradient across the tissue the microvilli on the granular cell lose the ridge structure and appear, predominantly, as individual projection. Variability-of this appearance points out the necessity of examining large areas and many samples before the significance of any morphological change can be assessed. Blocking the simultaneously occurring natriferic response of the toad urinary bladder with 10(-2)M ouabain does not prevent these changes in the microvilli. When the hydro-osmotic response is blocked by eliminating the osmotic gradient, the granular cell shows no consistent change in mucosal surface morphology even when fixed at the height of the natriferic response. The mitochondria-rich and mucous cells did not show any change in morphology throughout these studies. We conclude that the changes in the mucosal surface morphology of the toad bladder seen after exposure to vasopressin are a result of the increased water flux that occurs when an osmotic gradient exists across the tissue, and are not related to the natriferic response or any specific alteration in the membrane properties.     

Alteration in surface substructure of frog urinary bladder by calcium ionophore, verapamil and antidiuretic hormone

The calcium antagonist verapamil and the calcium ionophore A23187 have been shown to inhibit the hydro-osmotic actions of antidiuretic hormone (ADH) presumably by different mechanisms. Presently, urinary bladders of the frog (Rana pipiens) were examined under SEM following exposure to calcium ionophore A23187, verapamil and ADH in the presence and absence of an osmotic gradient. Cells exposed to ADH show marked changes in surface substructure which is accompanied by an expansion of microridges, cell borders and the appearance of microvilli in the granular cells. The microvilli are pronounced and appear at the junction of microridges. In the presence of an osmotic gradient, ADH induces granular cell swelling and some cells show a blistering effect. Calcium ionophore, in the absence of an osmotic gradient, induced pronounced morphological changes in the granular cells, where the microvilli become prominently visible as 'finger-like' projections. This effect may be due to the action of calcium in promoting elongation of microtubules. Cells exposed to ionophore plus ADH are indistinguishable from ionophore alone. The most apparent effect of verapamil on surface substructure was on the elevation of the mitochondrial-rich cells above the surrounding granular cells. These cells show some degree of separation from the granular cells and are accentuated in tissues exposed to verapamil plus ADH. The present observations suggest that these agents, verapamil and calcium ionophore, have marked effects on cellular morphology. These actions are mediated through alterations in calcium movements and reflect the relative importance of cellular calcium in transepithelial water flow and the actions of antidiuretic hormone.     

The effects cytochalasin B on the surface morphology of the toad urinary bladder epithelium: a scanning electron microscopic study.

Cytochalasin B depresses the hydroosmotic response of the toad urinary bladder to vasopressin without affecting basal (bulk flow) permeability, diffusional permeability, or the hormone induced increase in short circuit current. Fine structural studies demonstrated that this macrolide fungal metabolite, in the presence of both an osmotic gradient and vasopressin, induces the formation of large intracellular vacuoles or 'lakes' in epitelial cells lining the bladder mucosa. Some surface changes (shortening and irregularity of microvilli, clumping of the glycocalyx, etc.) were reported by transmission electron microscopy. Scanning electron microscopy demonstrates that cytochalasin B drastically alters the mucosal surface morphology of the hormone stimulated bladder. Lesser changes were seen in the absence of vasopressin. In the presence of arginine vasopressin, excessive cellular swelling and possible rupturing, as well as surface membrane infolding and rippling, were seen in the cytochalasin treated tissues, The specific entity most affected by this treatment is the granular cell.     

Functional water channels and proton pumps are in separate populations of endocytic vesicles in toad bladder granular cells

Functional water channels are retrieved by endocytosis from the apical membrane of toad bladder granular cells in response to vasopressin [Shi, L.-B., & Verkman, A.S. (1989) J. Gen. Physiol. 94, 1101-1115]. To examine whether endocytic vesicles which contain the vasopressin-sensitive water channel fuse with acidic vesicles for entry into a lysosomal pathway, ATP-dependent acidification and osmotic water permeability were measured in endosomes from control bladders and bladders treated with vasopressin (VP) and/or phorbol myristate acetate (PMA). Endosomes were labeled with the fluid-phase markers 6-carboxyfluorescein or fluorescein-dextran. Osmotic water permeability (Pf) was measured by stopped-flow fluorescence quenching and proton ATPase activity by ATP-dependent, N-ethylmaleimide-inhibitable acidification. In a microsomal pellet, Pf was low (less than 0.002 cm/s, 20 degrees C) in labeled endocytic vesicles from control bladders but high (0.05-0.1 cm/s) in a subpopulation (50-70%) of vesicles from VP- and PMA-treated bladders. Following ATP addition, the average drop in pH was 0.1 (control), 0.3 (VP), and 0.2 (PMA) unit. Measurement of pH in individual endocytic vesicles by quantitative image analysis showed that less than 20% of vesicles from VP-treated bladders acidified by greater than 0.5 pH unit. To examine whether water channels and proton pumps were present in the same endocytic vesicles, the pH of endosomes with high and low water permeability was measured from the effect of ATP on the amplitude of the fluorescence quenching signal in response to an osmotic gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of the vasopressin V1 receptor to its hydrosmotic response

Toad urinary bladder epithelial cells grown in culture (primary) show a significant increase in water-soluble inositol phosphates when treated with 10(-8) M vasopressin (AVP), but not with (1-deamino-8-D-arginine)vasopressin (dDAVP), a V2-agonist. The increase in inositol phosphates was blocked by the V1-antagonist, d(CH2)5Tyr(Me)AVP, suggesting a V1-coupled phosphoinositide breakdown. The V1-antagonist had no effect on basal adenylate cyclase activity nor on that stimulated by AVP. However, the V1-antagonist was found to attenuate the hydrosmotic response of AVP, suggesting some role of the V1-receptor cascade in the water flow response. Mezerein (MZ), a non-phorbol activator of protein kinase C (PKC) increased osmotic water flow when added to the mucosal surface. The response was less in magnitude and occurred over a longer period (90 min) than that observed with AVP. In an attempt to emulate the V1-response, activation of PKC, and an increase in intracellular calcium, toad bladders were incubated with MZ and the calcium ionophore A23187 (IP). It was found that IP enhanced the water flow response to MZ at all times measured. Mz and IP were also found to enhance cAMP-mediated water flow, suggesting that apical membrane permeability may be regulated in part through V1-receptor stimulation and its respective second messengers. Collectively, these observations suggest that the V1 receptor may play a role not only as part of a negative feedback system, but also as an integral component of the enhanced water permeability that occurs at the apical membrane.     

The cellular specificity of the effect of vasopressin on toad urinary bladder

Summary Phase and electron micrographs of toad bladders were obtained following dilution of bathing media in the presence and absence of vasopressin. Dilution of the mucosal medium alone resulted in no morphologic changes. Subsequent addition of vasopressin produced an increase in the cell volume of the granular cells, manifested by some or all of the following changes: increased area of granular cell profiles as observed in sections, rounding of the cell nucleus, displacement of the two components of the nuclear envelope, loss of nuclear heterochromatin, sacculation of the endoplasmic reticulum and the Golgi apparatus, and reduction in the electron density of the cell cytoplasm. No such morphologic changes were noted in the other cell types comprising the mucosal epithelium — the mitochondria-rich, the goblet, and the basal cells. On the other hand, dilution of the serosal bathing medium in the absence of vasopressin caused a marked increase in the cell volume of all these cell types. The results demonstrate that the action of vasopressin to enhance bulk water flow across toad bladder is exerted specifically on the apical surface of the granular cells. It is suggested that the hormonal effect on sodium transport may also be limited to the granular cells. The route of osmotic water flow and the possible role of the other mucosal epithelial cells is discussed.     

Time course of vasopressin-induced formation of microvilli in granular cells of toad urinary bladder

Summary Vasopressin-induced transformation of ridges to microvilli on the surface of granular cells of toad urinary bladder occurs in conjunction with induced alterations in the water permeability of the luminal membrane. This study was designed to establish the relationship between the time course for induction of microvilli and the time course for induction of increased water permeability after vasopressin stimulation. Hemibladders were examined at 2.5, 5, 10, 20 and 30 min following exposure to 20 mU/ml of vasopressin and at 5, 10, 20, 30, 40, 50 and 60 min after washout of vasopressin. Within 2.5 min, vasopressin initiated complete transformation of ridges to microvilli on approximately 13% of the granular cells, while osmotic water flow (Jv) was 0.31±0.10 l·min^–1·cm^–2. Five minutes following vasopressin stimulation, microvilli were present on approximately 30% of granular cells andJv was 2.27±0.13 l·min^–1·cm^–2. At 10 minJv was maximum at 4.03±0.15 l·min^–1·cm^–2 and 50% of the granular cells were covered with microvilli. This percentage increased to 70% at 20 min and was maintained at 30 min, althoughJv decreased to 3.9±0.35 l·min^–1·cm^–2 at 30 min. Five minutes following vasopressin washout, ridges interspersed with microvilli reappeared asJv fell to 1.10±0.30 l·min^–1·cm^–2. At 10 min after vasopressin washout,Jv approached basal levels, but the reversal of microvilli to ridges remained incomplete. At 60 min after vasopressin washout, the granular cells had regained their original ridgelike surface structures. Thus, these studies establish a temporal relationship between the induction and reversibility of vasopressin-induced microvillous formation and alterations in the osmotic water permeability of the apical plasmalemma.     

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study

Summary The distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.     

Electron microscopic cytochemical localization of adenylate cyclase in amphibian urinary bladder epithelium: effects of antidiuretic hormone

A cytochemical technique for electron microscopic localization of adenylate cyclase was used to identify this enzyme in quiescent and hormone-stimulated toad urinary bladder epithelium. In the absence of vasopressin (antidiuretic hormone), adenylate cyclase was detected along the outer surface of the basolateral plasma membranes of granular cells, mitochondria-rich cells, and basal cells, the major cell types comprising the hormone-sensitive urinary epithelium. In the presence of antidiuretic hormone, the basolateral precipitates were markedly increased. The latter was true for both tissues incubated in the presence of an osmotic gradient and those stimulated in the absence of such a gradient. A significant mucosal reaction was never seen. Such data indicate that the hormone receptors for vasopressin are located along the basolateral membranes of all epithelial cells comprising the mucosal hormone-sensitive epithelium. All cells of the epithelium also demonstrate a vasopressin-sensitive adenylate cyclase. We discuss possible mechanisms that attempt to integrate the cytochemical data into an overall scheme for the physiological action of this hormone on amphibian urinary bladder.     

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study

The distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

Summary The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toadBufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to the serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Regulation of membrane permeability by vasopressin; activation of the water permeability pathway in toad urinary bladder by N-ethyl-maleimide

1. Vasopressin induces a rapid increase in water permeability and stimulates net sodium transport in responsive epithelia through the mediation of cAMP. 2. In amphibian urinary bladder, the increase in water permeability is dependent on an intact cytoskeleton and is associated with the exocytotic insertion of tubular vesicles containing particle aggregates (the putative water channels) into the apical membrane of the granular epithelial cells. 3. In the toad bladder, mucosal addition of NEM, 0.1 mM, elicits a slow and irreversible increase in transepithelial water flow, whilst decreasing net sodium transport. 4. The hydrosmotic response to mucosal NEM is inhibited by cellular acidification, by pretreatment with cytoskeleton-disruptive drugs, and by agents that increase cytosolic calcium. 5. Mucosal NEM potentiates the hydrosmotic response to a submaximal, but not a maximal, dose of vasopressin. 6. Mucosal NEM, like vasopressin, induces both vesicle fusion and the appearance of particle aggregates at the granular cell apical surface. 7. NEM, unlike vasopressin, does not increase cellular cAMP content. 8. Mucosal NEM appears to increase transcellular water flow by activating cellular processes normally triggered by vasopressin, at a step beyond cAMP.     

Scanning electron microscopic study of natural killer cell-mediated cytotoxicity

The interaction between human natural killer (NK) cells and NK-susceptible target cells, as well as the mechanism involved in target cell lysis, were studied with scanning electron microscopy (SEM). Low density human peripheral blood lymphocytes, highly enriched with large granular lymphocytes (LGL), were used as effector cells, and K562-cells were used as NK-susceptible target cells. The surface features of LGL/NK cells were examined under SEM. In the area of interaction, NK/target-cell conjugates showed microvilli and/or filipodia, and extensive areas of intercellular contact. In addition, the effector cells in some NK/target-cell conjugates were polarized toward the target cell. Changes in target cell surface features included loss of microvilli, large surface blebs and the appearance of small pore-like lesions on the cell membrane. Our findings show that target cell lysis occurred by apoptosis and plasma membrane lesions analogous to those seen during complement-mediated cytotoxicity.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toad Bufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to th serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Wheat germ agglutinin (WGA) reduces ADH-induced water flow and induces cell surface changes in epithelial cells of frog urinary bladder

The functional and structural changes induced by apical wheat germ agglutinin (WGA) 100 micrograms/ml exposure on frog urinary bladder have been investigated and the possible correlations between these effects discussed. Bladders, apically exposed to WGA for 30 min to 3 hr exhibit a marked reduction of their response to antidiuretic hormone (ADH) challenge and of their hydrosmotic reactivity. Structural changes triggered by WGA treatment are: 1. apical invaginations of the plasma membrane, interpreted as endocytotic in nature, taking into account the results of carbohydrate cytochemical detection and horseradish peroxidase (HRP) exposure: 2. cytoskeleton disorganization and microvilli collapse. These phenomena do not interfere with cortical granule traffic and are independent of ADH challenge: they occur in ADH-stimulated bladders as well as in bladders at rest. These findings could be interpreted as follows: binding of the divalent lectin WGA to its coat specific receptors would induce changes in the apical membrane structure which in turn could provoke disorganization and disruption of apical cytoskeletal elements associated with plasma membrane. Reduction of bladder response to ADH challenge could result from a reduced recycling of aggrephores, as they are associated with cytoskeletal elements in the subapical cytoplasm. Collapse of microvilli and endocytotic events also could result from apical cytoskeleton disruption, as microvilli are sustained by bundles of actin filaments interconnected with apical cytoskeletal filaments and as plasma membrane is associated with apical cytoskeleton. However, these two last events evidently occur in ADH-challenged or non-challenged bladders.     

Inhibition of ADH-enhanced transepithelial urea and water movement by prostaglandins

The effects of PGF_2α and PGE₂ on transepithelial urea flux and osmotic water flow were evaluated in toad bladders. Mucosal to serosal urea flux and osmotic water flow were not changed from basal values by the addition of either prostaglandin to the serosal bath. However, treatment with either PGF_2α or PGE₂ inhibited both urea flux and osmotic water flow in response to ADH stimulation in a concentration-dependent manner. The hydrosmotic response to ADH was more sensitive to prostaglandin inhibition than was urea flux. The inhibitory effect of the prostaglandins on ADH-enhanced urea flux was not dependent upon inhibition of the hydrosmotic response, since both PGF_2α and PGE₂ decreased urea flux in the absence of a trans-epithelial osmotic gradient. Prostaglandin E₂ was a more potent inhibitor than PGE_2α of both ADH-enhanced urea flux and osmotic water flow. The PGF_2α antagonism of osmotic water flow was apparently competitive, while antagonism of urea flux was apparently non-competitive. The results are consistent with the hypothesis of the existence of a “spare” population of prostaglandin receptors that modulate water flow, but the absence of a “spare” prostaglandin receptor population with respect to the modulation of urea flux.     

Regulation of luminal membrane water permeability by water flow in toad urinary bladder

Intramembranous particle aggregates (presumed sites for water flow) which appear in the luminal membrane consequent to ADH treatment are derived from cytoplasmic membrane structures (now termed "aggrephores") which fuse with the luminal membrane. We have previously shown that bladders stimulated in the absence of an osmotic gradient have about twice as many aggregates and about three times as many sites of aggrephore fusion as bladders stimulated with ADH in the presence of a 175 milliosmolal gradient. The present studies show that the frequency of fused aggrephores and luminal membrane aggregates can be modified as a consequence of alterations in transmembrane water flow initiated by changing the transbladder osmotic gradient during hormone stimulation. Bladders treated with ADH for 1 hr without a gradient and then for 1 hr with a gradient had approximately 1/3 as many aggregates and fusion sites as paired bladders treated for 2 hr without a gradient. Conversely, bladders treated with ADH for 1 hr with a gradient and then for 1 hr without a gradient had approximately 2x as many aggregates and fusion sites as bladders treated for 2 hr with a gradient. In other experiments we demonstrate that the time course of hormone washout is greatly accelerated if carried out in the presence of an osmotic gradient. In paired bladders that were first stimulated with ADH for 30 min in the absence of a gradient, aggregates and fusion sites as well as osmotic water permeability determined in fixed bladders, persisted at near maximum levels for 15 min of washout in the absence of a gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of colchicine and cytochalasin B on hypertonicity-induced changes in toad urinary bladder

Summary Coincident with an increase in the water permeability of toad urinary bladder induced by serosal hypertonicity, a transformation of the ridge-like surface structures of the granular cells into individual microvillous structures occurs. This study was initiated to establish whether the transformation is mediated by the cytoskeletal network and, thus, can be prevented by disruption of microtubulemicrofilament function with colchicine or cytochalasin B (CB). Scanning electron microscopy revealed the characteristic branching ridges on granular cells of control bladder incubated with colchicine or CB. In contrast, transformation of ridges to discrete microvilli was observed in experimental bladders exposed to serosal hypertonicity alone or in combination with either colchicine or CB. These results suggest that the mechanism underlying hypertonicity-induced surface changes which are associated with increased water permeability does not involve either microtubules or microfilaments.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothiocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.