Toad Bladder Epithelial Cells Research Articles

3H‐fucose incorporation into glycoproteins of toad bladder epithelial cells

Electron microscope autoradiography was used to detect the incorporation of 3H-fucose into glycoproteins of toad bladder epithelial cells. After short exposure to 3H-fucose, without a chase period, the Golgi regions of all four cell types were labeled. When exposure to 3H-fucose was followed by chase periods (1,3,4 and 6 hours) the apical and basal-lateral plasma membranes of granular cells were heavily labeled. Apical granules and the cytoplasm of granular cells were also labeled, suggesting that they both provide the means for glycoprotein transfer from the Golgi to the plasma membranes. The heaviest labeling in mitochondria-rich cells, after the 1- and 3-hour chase periods, was over the apical tubules, although the apical and basal-lateral plasma membranes were also heavily labeled. After 4- and 6-hour chases, the labeling of the apical tubules decreased, whereas the labeling of the plasma membranes increased, strongly suggesting that in these cells apical tubules play a major role in the transfer of glycoproteins from the Golgi to the plasma membrane. Our results demonstrate that the route of 3H-fucose incorporation into plasma membrane glycoproteins and the rate of glycoprotein synthesis and breakdown are not the same in the two major epithelial cell types in toad bladder.

Relationships between serosal medium potassium concentration and sodium transport in toad urinary bladder. III. Exchangeability of epithelial cellular potassium.

The exchangeability of toad bladder epithelial cell potassium has been investigated. An insignificant amount of cellular potassium exchanged with mucosal medium 42K. From the rate of uptake of 42K into the cells from the serosal medium at least two cellular potassium pools were identified. The more rapidly exchanging pool contained about one-quarter to one-third of the cellular potassium and exchanged with a half-time of about 30 min. It was from this pool that potassium was lost from cells exposed to ouabain or to a potassium-free medium. In addition, when 3.5 mM rubidium replaced 3.5 mM potassium in sodium Ringer's the epithelial cells lost in 60 min about one-quarter of their cellular potassium in exchange for rubidium. Inhibition of transepithelial sodium transport by amiloride, 10(-5) mM, seemed to depress the rate of potassium uptake into the more rapidly exchanging pool without affecting total cellular potassium content. However, stimulation of transepithelial sodium transport by vasopressin appeared not to affect the rate of potassium uptake. The rate of potassium uptake into this pool seemed much less than that required for a tight 1:1 coupling between transepithelial sodium transport and potassium uptake. The remaining cellular potassium exchanged at a much slower rate and even after 19 hours of incubation only 67% of cellular potassium was labelled. If this slower exchanging potassium represents a single pool, 99% of cellular potassium would be labelled only after incubation with 42K for 56 hours.

Differential covalent labeling of apical and basal-lateral membranes of the epithelium of the toad bladder.

The apical (luminal) plasma membrane of toad bladder epithelial cells has been labeled with (125I) diazo-diiodo sulfanilic acid (125I-DDISA) as demonstrated by electron-microscopic autoradiography. The silver grains (125I) were localized exclusively to the apical surface. At concentrations of DDISA of 10(-3) M or less, binding to the apical membrane had no significant effect on the fine structure of the epithelium. At concentrations of DDISA of 10(-6) M or less, the baseline short-circuit current (SCC), and the response to cyclic 3',5'-adenosine monophosphate (cAMP) plus theophylline were unimpaired. At 10(-5) M, baseline SCC was unchanged and the response to cyclic AMP plus theophylline was enhanced. At concentrations of 10(-4) M and greater baseline SCC was depressed and the response to the nucleotide inhibited. The basal-lateral epithelial plasma membranes were labeled by exposing the serosal side to pyridoxal phosphate and reducing the resultant Schiff base with sodium borotritide (3H-NaBH1). In electron-microscopic autoradiographs, the silver grains (3H) were found over the basal and lateral surfaces of the epithelium. At concentrations of pyridoxal phosphate of 10(-4) M and 3H-NaBH1 of 10(-3) M, there were no significant changes in the fine structure of the epithelium. Addition of pyridoxal phosphate (10(-4) M) and NaBH4 (10(-3) M) to the serosal side decreased the baseline SCC significantly but not the response to vasopressin. Covalent attachment of the 125I and the 3H was indicated by resistance to elution in the preparation of the sections for electron-microscopy and the reagent requirements for binding.

Estimation of the lifespan of amiloride binding sites in the membranes of toad bladder epithelial cells.

1. Sodium entry sites in the membranes of isolated epithelial cells prepared from bladders of toads (Bufo marinus) have been labelled with amiloride. The number of binding sites remained constant in suspensions for up to 100 hr. 2. In the presence of a protein synthesis inhibitor (cycloheximide, 0-5 mug/ml.) there was a decline in the density of binding sites was approximately exponential. Regression analysis gave a half-life of approximately 60 hr. 3. Aldosterone (5 X 10(-8) M) caused a significant (P less than 0-001) increase (50%) in the density of amiloride binding sites. Cells which had been treated with aldosterone had populations of binding sites which declined, in the presence of cycloheximide, at rates indistinguishable from those of untreated cells.

Selective inhibition of urea transport by oxidizing agents

Urea and water transport across the toad bladder epithelial cell appears to take place through independent vasopressin-stimulated pathways. Agents such as chromate, for example, when added to the luminal bathing medium, inhibit urea transport without inhibiting osmotic water flow, providing evidence for such independent pathways. In the present study, selective inhibition of urea transport is shown for permanganate and methylene blue, which like chromate, are oxidizing agents. Permanganate inhibits urea transport irreversibly, while methylene blue acts reversibly. Not all oxidizing agents are inhibitory; perchlorate, peroxide and ferricyanide have no effect on urea transport or water flow. Permanganate and chromate both act at a point beyond the generation of cyclic AMP, since they continue to inhibit urea transport in bladders treated with exogenous cyclic AMP, 8-bromoadenosine 3', 5'-cyclic monophosphate, and a combination of cyclic AMP and theophylline. These findings suggest that selective inhibition of urea transport can be brought about by oxidation of one or more components in its transport pathway, and that, in the case of chromate and permanganate, these components may be in the luminal membrane itself.

Effects of vasopressin and aldosterone on amiloride binding in toad bladder epithelial cells

Methods have been devised to measure the binding of [ 14 C]amiloride to isolated cells from bladders of toads, Bufo marinus . This agent blocks transepithelial sodium transport across bladders by preventing sodium entry to the transporting mechanism. A saturable binding component has been found with an affinity of 5.6 x 10 7 m -1 in the presence of 1.1 mM Na + , which corresponds to the affinity of amiloride when used as a transport inhibitor at the same sodium concentration. In freshly isolated cells the capacity of the binding sites is 3.6 x 10 5 sites/cell, but this value falls to about one third in aged suspensions. When cells are treated with vasopressin (100 mU/ml) somewhat less specific binding is measured at an amiloride concentration giving 50 % occupancy. The results are consistent with the view that vasopressin moves the binding curve to the right along the concentration axis, reducing the affinity of amiloride by a factor of approximately two, while leaving the total capacity unaffected. The affinity of amiloride when used as an inhibitor of transport is also found to be reduced by a factor of two by vasopressin, and complete inhibition of transport can still be achieved. d -Aldosterone in vitro increases the number of amiloride binding sites in isolated cells by approximately 115%, and results from transport studies indicate that there is no significant change in the affinity of amiloride after d -aldosterone treatment. Inhibitors of transcription and translation (actinomycin D and cycloheximide) prevent the increase in amiloride binding caused by d -aldosterone. In vivo the effects of d -aldosterone are more complex, but it is shown that the steroid increases the transport capacity of the tissue, when this is expressed as the number of amiloride binding sites per unit mass of tissue. The results are discussed in terms of the ways in which the two hormones may alter the entry of sodium into the epithelial cells, and so in turn affect transepithelial sodium transport.

Immunofluorescent Localization of Cyclic AMP in Toad Urinary Bladder: Possible Intercellular Transfer

By use of an immunofluorescent cytochemical staining technique, adenosine 3',5'-monophosphate (cyclic AMP) has been localized in toad bladder epithelial cells. Within 2 minutes after addition of vasopressin, staining intensity increases in both mitochondria-rich and granular cells. This finding, taken together with the precise anatomical relation between these two epithelial cell types and the observation that after separation of the two cell types vasopressin stimulates cyclic AMP accumulation in only mitochondria-rich cells, suggests that cyclic AMP may be transferred from mitochrondria-rich to granular cells as part of the response of the toad urinary bladder to vasopressin.

The effect of diuretics of Na+-K+-ATPase and c-AMP levels in toad bladder epithelial cells

Na+-K+-ATPase was inhibited by 1 times 10-4M ethacrynic acid and mercuderamide, and by 1 times 10-3M hydrochlorothiazide and furosemide. A modification of Gilman's (1970) protein displacement assay has been used to measure c-AMP levels in toad bladder epithelial cells. Vasopressin (50 mU/ml) caused c-AMP levels to rise from 4.27 to 9.27 pmol/mg protein. Ethacrynic acid had no effect on cellular c-AMP levels after 10 min exposure to the drug, but at 90 min caused a reduction of both basal and vasopressin stimulated levels. Furosemide caused an apparent rise in c-AMP levels, dilution ratio measurements indicated interference by this drug in the assay procedure, mecuderamide also caused substantial interference with the c-AMP assay. Hydrochlorothiazide had no effect on basal or hormone stimulated levels of c-AMP. It was concluded that the inhibition of sodium transport produced by ethacrynic acid in toad bladder is probably due to inhibition of adenylate cyclase, an effect not shared by other dieuretics.

Effect of hypotonicity on cyclic adenosine monophosphate formation and action in vasopressin target cells

We have shown previously that overhydration of toads renders their urinary bladders less responsive to the antidiuretic action of vasopressin (AVP). The present study investigates the relationship between osmotic swelling of vasopressin target cells and their sensitivity to AVP and dibutyryl cyclic adenosine monophosphate (db-cAMP). Conditions which engender osmotic swelling of toad bladder epithelial cells, such as immersing bladders on both surfaces in hypotonic Ringer's fluid or subjecting them to a net mucosal-to-serosal volume flux, markedly inhibited the effectiveness of db-cAMP in raising bladder permeability to water. This inhibitory phenomenon was seen both with serosal and mucosal applications of the nucleotide. Examination of isolated epithelial cells by phase contrast microscopy showed them to behave as osmometers, doubling their volume as the effective osmolality of the incubation medium was halved. AVP was found to increase the total content of cAMP about 3.5-fold both in the swollen and the normal cells, so that the actual concentration of cAMP may have diminished as the cell volume increased. Consistent with this suggestion was the observation that increasing exogenous db-cAMP abolished, in part, the inhibitory effects of hypotonicity. These observations indicate that homeostasis of body fluids in the toad depends in part upon the osmotic regulation of anti-diuretic homone action, and that intracellular cAMP may participate in coupling changes in cell volume to the altered state of responsiveness of the vasopressin target cell.

Extracellular space in some isolated tissues.

1. The spaces occupied by isotopically labelled inulin, polyethylene glycol, mol. wt. 4000 (PEG 4000), polyethylene glycol, mol. wt. 1000 (PEG 1000) and sucrose in metabolizing mammalian kidney and liver slices and in toad bladder epithelial cell preparations incubated in vitro have been examined.2. In slices of mammalian tissue, and in homogenized liver, it proved impossible to extract inulin completely from tissue which had been dried. However, inulin was recovered as completely from both dried and undried toad bladder epithelial cells scraped from hemibladders incubated in vitro.3. PEG 4000 occupied a space in all preparations similar to that from which inulin was extracted in dried tissue.4. PEG 1000 and sucrose entered cellular water in mammalian slices, but PEG 1000 occupied a similar space to inulin in toad bladder epithelial cell preparations.6. It is concluded that inulin enters cellular water in mammalian slices from which after drying of the slices it cannot be extracted. It thus rather fortuitously provides a measure of extracellular water under these conditions. In preparations of toad bladder epithelial cells inulin seems to be a satisfactory extracellular marker. PEG 4000, which did not appear to enter cellular water also allows a reasonable estimate of extracellular water. PEG 1000 is a suitable extracellular marker for toad bladder epithelial cell preparations but not for mammalian slices. Sucrose entered cellular water in both slices and toad bladder epithelial cells and is not a satisfactory extracellular marker in these tissues.

Iodination (125I) of the apical plasma membrane of toad bladder epithelium: Electron-microscopic autoradiography and physiological effects

The apical plasma membrane of toad bladder epithelial cells has been enzymatically iodinated, using lactoperoxidase, H2O2 (generated by a glucose-glucose oxidase system) and NaI. The site of labeling was demonstrated by electron-microscopic autoradiography; the silver grains (125I) were found exclusively overlying the luminal plasma membranes of the epithelium. The iodination reaction reached completion in less than 5 min. The dependence of the degree of iodination on NaI concentrations (range=6.3×10−8 to 6.3×10−2m) in the mucosal medium was determined. The results suggest that three classes of sites are iodinated within this concentration range. At concentrations of NaI of 6.3×10−6m or less, iodination of the apical membrane had no significant effect on either the fine structure of the epithelium or on electrophysiological properties. The baseline short-circuit current (SCC) remained steady and the response to vasopressin was unimpaired. At concentrations of 6.3×10−5m NaI and greater, the baseline SCC was depressed and the response to vasopressin was partially inhibited. The results indicate that125I may serve as a covalent marker (specific for tyrosine and histidine residues) of the apical plasma membrane of epithelia.

The effect of temperature and substrate concentration on the kinetics of [6- 3H]uridine incorporation into RNA of toad bladder epithelial cells: The role of aldosterone

1. The effects of temperature and uridine substrate concentration during incorporation of [6- 3H]uridine into RNA were studied in epithelial cells of the isolated urinary bladder of the toad, Bufo marinus, harvested after incubation. 2. Gross accumulation of newly synthesized RNA in the acid-insoluble fraction of whole epithelial cells was maximal at 33°C., even though net RNA synthesis, i.e. RNA specific activity, was significantly reduced compared to 24°C. Cold exposure at 8°C decreased accumulation of newly synthesized RNA when compared to warmer temperatures. 3. A uridine substrate concentration constant ( K′ m ), derived in a manner similar to the Michaelis-Menten constant ( K m ), was determined to be K′ m = 2.13 · 10 −5 mole/l at 24°C for non-hormonally treated urinary bladder. 4. The in vitro effect of the hormone, aldosterone, on RNA synthesis at 24°C was evaluated by quantitating the aldosterone-control difference of both RNA and RNA specific activity in paired samples of scraped epithelial cells. By minimizing dilution of the [6- 3H]uridine due to variations in the endogenous uridine RNA precursor pool size, through the technique of providing a constant excess of exogenous uridine with the radioisotope, aldosterone stimulation of RNA specific activity can be reproducibly demonstrated. 5. Aldosterone significantly increased RNA specific activity but not bulk RNA, per se, even after 20 h of hormone exposure. 6. The mean weight ratio of epithelial cell RNA/DNA determined in 116 different bladder parts derived from 30 toads, was 2.5 ± 0.32 S.E. 7. A 1.5-h exposure to aldosterone produced a significant increase (31.5 %) in the incorporation of [6- 3H]uridine into heterogenous RNA extracted from purified nuclei, but not in RNA extracted from either cytoplasm or whole cell. This primary nuclear response to aldosterone was followed by a significant increase in aldosterone-stimulated specific activity of whole cell RNA at 3 h (74 %) and 20 h (200 %).

Effect of ADH, aldosterone, ouabain, and amiloride on toad bladder epithelial cells.

Effect of ADH, aldosterone, ouabain, and amiloride on toad bladder epithelial cells.

The effect of aldosterone on the accumulation of adenosine 3':5'-cyclic monophosphate in toad bladder epithelial cells in response to vasopressin and theophylline.

Vasopressin and theophylline both increase the content of adenosine 3':5'-cyclic monophosphate (cAMP) in epithelial cells of the urinary bladder of toads (Bufo marinus). Incubation of the tissue with 0.2 muM aldosterone markedly increases this response to the two agents; incubation for a similar time without steroid reduces the response. The permeability responses (sodium transport and water flow) of the intact tissue to vasopressin, theophylline, and exogenous cAMP are also considerably greater in bladders incubated with aldosterone than without. The results are interpreted as indicating that the foregoing permissive effects of aldosterone on the permeability responses to vasopressin and theophylline are mediated by a steroid-dependent increase in the accumulation of cAMP in the pertinent epithelial cells, probably as a consequence of a diminution in the rate of degradation of the intracellular nucleotide.

Effects of aldosterone and vasopressin on electrolytes of toad bladder epithelial cells.

Effects of aldosterone and vasopressin on electrolytes of toad bladder epithelial cells.

Effects of vasopressin on the water and ionic composition of toad bladder epithelial cells

Isolated sheets of epithelial cells as well as epithelial cells scraped from paired hemibladders mounted in chambers both showed significant increases in water, sodium and chloride contents after exposure to vasopressin (100 mU/ml), without any change in potassium content. In the isolated cells these changes were prevented by amiloride (10(-5) M), suggesting that the gain of sodium after vasopressin occurs across the mucosal membrane. This hypothesis was confirmed in experiments in which it was found that, in hemibladders mounted in chambers and bathed on their mucosal surface by sodium Ringer's with(24)Na, the gains of chemical sodium and(24)Na after vasopressin were equivalent.

Identification of four types of steroid by their interaction with mineralocorticoid receptors in the toad bladder.

SUMMARY The effects of various steroids on sodium transport across the toad bladder were examined in vitro. Cortisone, alone, had no effect on sodium transport but was an antagonist of aldosterone. Cortisone was shown to bind to the two non-specific binding sites for steroids in the nucleus of the toad bladder epithelial cells when present at a concentration of 10−7m. At higher concentrations it interacted with the mineralocorticoid receptors and displaced aldosterone from these sites. Cortexolone had a weak stimulating effect on sodium transport and also antagonized the effect of aldosterone. Cortexolone, when present in excess, displaced aldosterone from its receptor sites. Four types of steroid could be differentiated by their interaction with the mineralocorticoid receptor sites and the subsequent biological response: (a) steroids that bind to the receptors and produce the full biological effect on sodium transport; (b) steroids that do not bind and therefore are incapable of producing a response; (c) steroids that bind to the receptor sites, have no effect alone but inhibit the action of aldosterone; (d) steroids that bind to the receptor sites, produce a partial response on sodium transport and subsequently antagonize the action of aldosterone. Multiple points of attachment are postulated for the steroid—receptor interaction.

Overestimation of DNA by ultraviolet measurement: A correction factor for toad bladder epithelial cells

Nondestructive ultraviolet light at 260 mμ may be used for measurement of DNA in toad bladder and other tissues rather than the diphenylamine method provided an ultraviolet correction factor is applied after the technique we have outlined. This factor can be obtained by calculating the slope of the regression line derived from the tissue DNA values measured in the same sample by the two methods described.

Incorporation of 3H-uridine into RNA of toad bladder epithelial cells : Method of determination

A quantitative method for the determination of 3H-uridine incorporation into PCA-insoluble RNA of epithelial cells harvested from the urinary bladder of the toad, Bufo marinus, has been developed. Information has been provided on 3H-uridine incorporation concerning: (1) the distribution and recovery of 3H-uridine (unquenched counts/minute) from fractions of whole bladder homogenates, (2) the effect of serial extraction of whole-bladder sediment with 0.3 n PCA on the removal of labile PCA-soluble 3H-uridine and (3) the measurement of 3H-uridine incorporation at 0–2°, 8°, room temperature and 24° into PCA-insoluble RNA extracted from whole bladder and from epithelial cells isolated from whole bladder. Evaluation of the method provided the following information: (1) there is no significant difference ( p 0.90) between the orcinol and ultraviolet methods for quantitating the nucleic acid content in PCA-insoluble RNA extracts of isolated epithelial cells, (2) one 0.3 n PCA washing of the sediment from either whole bladder or epithelial cells removes more than 99% of the unincorporated 3H-uridine but only 88% of the extractable UV-positive material, (3) approximately one third of the orcinol-positive, DNA-negative material appeared in the PCA-soluble fraction and two-thirds in the acid-insoluble fraction of the epithelial cell, (4) from simultaneous measurements in serially extracted PCA-soluble fractions of the epithelial cell, the ultraviolet-positive material exceeded the orcinol-positive material by a factor of 5 and (5) the reproducibility by replicate analysis of RNA extracted from PCA-insoluble fractions of toad bladder proved to be ± 7%.

Effects of sodium transport stimulating substances on enzyme activities in the toad bladder.

The activities of 10 enzymes of mitochondrial and extramitochondrial origin were studied in mucosal homogenates prepared 2 hours after addition of 1×10−8 M dexamethasone to the serosal medium bathing urinary bladders of the toad bufo marinus. The following increases in activity relative to the controls were found: Condensing enzyme +24%; TPN-isocitrate dehydrogenase +21%; glutamate dehydrogenase +23%; glutamate-oxaloacetate transaminase +16%; glyceraldehyde phosphate dehydrogenase +12%; aldolase +15%. The activities of lactate dehydrogenase, malic enzyme, hexokinase, and creatine kinase remained unchanged. Under the same conditions, neither 1×10−9 M dexamethasone, a concentration which has little effect on net sodium transport as measured by short circuit current (scc), nor 1×10−6 M progesterone, which lacks an effect on scc, showed any influence on the activity of the 10 enzymes studied. Vasopressin (25 milliunits per ml serosal medium) did not change the activities of condensing enzyme, isocitrate dehydrogenase, or glutamate dehydrogenase at a time of maximal response of scc (20–50 minutes after administration) while the activities of hexokinase and phosphorylaseb were increased by 9% and 20% at that time. 3′5′-cyclic AMP in combination with theophylline (11 mM) also failed to influence the activities of condensing enzyme, isocitrate dehydrogenase, or glutamate dehydrogenase at a time of maximal stimulation of scc. The results suggest that the toad bladder epithelial cells respond to dexamethasone with an activation of the citric acid cycle, as has been shown with aldosterone. This effect on intramitochondrial enzyme activity is not elicited by two other agents which stimulate sodium transport, namely vasopressin and 3′5′-cyclic-AMP. Therefore the increased activity does not occur as a direct consequence of increased metabolic activity in response to these substances, e.g. to an activation of glycogenolysis and glycolysis. The increased activity of the intramitochondrial enzymes appears to be an independent steroid effect.