Contribution of mucosal chloride to chloride in toad bladder epithelial cells

Summary Epithelial cells were scraped from the bladders of toads of the speciesBufo marinus obtained from the Dominican Republic. These epithelial cells exchanged their chloride virtually completely with³⁶Cl in the medium within 60 min. Of this chloride, about 93% came from the serosal medium. The approximately 20 mmole/kg dry wt of chloride which equilibrates with³⁶Cl in the mucosal medium was still present when choline replaced sodium in the medium in the presence of amiloride (10^–4 M) and was almost all readily removed by rapid washing of the mucosal surface immediately prior to analysis. These observations suggest that little chloride of mucosal origin is truly intracellular. This conclusion is supported by the fact that after vasopressin the increased cellular chloride was not of mucosal origin.     

Studies on halotolerance in a moderately halophilic bacterium. Effect of betaine on salt resistance of the respiratory system

The role of betaine as a factor influencing the salt resistance of the respiratory system in resting cells of the moderately halophilic halotolerant bacterium Ba(1) was studied. Betaine accelerated succinate oxidation in cells obtained from low-salt medium, and stimulation of the respiratory rate was stronger the higher the sodium chloride concentration in the assay medium. The stimulatory effect also depended on the ratio of betaine concentration to the amount of bacteria present. Accumulation of labelled betaine by the bacterial cells was demonstrated; like the respiratory stimulation, it was favourably influenced by an increase in the sodium chloride concentration of the medium. In cells harvested from a high-salt medium and washed with 2.0m-sodium chloride, betaine caused no increase in the respiratory rate, nor was the already high salt resistance of the respiratory system further improved by the addition of betaine. When, however, these cells lost their salt resistance as a result of washing in the absence of sodium chloride, betaine was able to restore it to its original level. In contrast with respiration in low-salt-grown bacteria, that in high-salt-grown cells was not affected by betaine, even after they were washed in the absence of sodium chloride, when the sodium chloride concentration was optimum.     

Extrusion of Sodium Ions by Barley Roots: III. The Effect of High Salinity on Long-distance Sodium Ion Transport

The quantity of sodium transported to the shoots of intact barleyplants was stimulated by 0·5 mM ouabain when the sodiumchloride level of the bathing medium was below 100 mM. At sodiumchloride concentrations of 100 mM or more this ouabain-stimulatedsodium transport was not observed. Equiosmotic mannitol, equimolarpotassium chloride or equivalent calcium chloride solutionsdid-not affect the ouabain-stimulated sodium transport froma basic medium containing 10 mM sodium chloride. It is suggestedthat under the present experimental conditions the increasedsodium uptake by the root cells at sodium chloride concentrationsof 100 mM or more masks the extrusion process.     

Starvation and Encystment of a Soil Amoeba Hartmannella castellanii*

SYNOPSIS. The behavior of the amoeba H. castellanii was investigated in various carbon and nitrogen deficient media with a view to developing a satisfactory replacement medium for the study of encystment and excystment. Media which had been devised for other soil amoebae did not cause H. castellanii to encyst. In these media there was an efflux of material from the cells which was independent of osmolarity but which was minimized by the addition of magnesium. Maximal encystment occurred in a medium containing magnesium chloride alone. The cysts produced in the magnesium chloride replacement medium are viable and readily excyst when resuspended in the growth medium. The cysts contain cellulose, which is not present in the vegetative amoebae, and differ from the amoebae in their greater resistance to induced lysis and mechanical injury.     

Effects of impermeant medium ions on the composition of rabbit renal cortical slices

When incubated in isosmotic oxygenated medium in which chloride was completely replaced by gluconate, rabbit renal cortical slices lost chloride with sodium, potassium and water before reaching a new steady-state composition after 15-30 min. When corrected for extracellular space, there was an electroneutral loss of alkali metal cations (Na + K) with chloride, accompanied by isosmotic loss of water from the cells. The losses of chloride and water were independent of medium pH over the range of 6.4-8.2, and were the same with potassium rather than sodium as the dominant medium cation. Incubation in isosmotic sodium chloride medium restored tissue composition of slices transferred from gluconate medium. This recovery was not dependent specifically upon medium chloride, for slice water content also recovered when nitrate rather than chloride was substituted for medium gluconate. With sodium completely replaced by n-methyl d-glucamine (nmdG+), cells in slices lost far more sodium and potassium than chloride before reaching a new steady-state composition after some 30 min. However, the loss of water was as predicted from the total losses of measured inorganic ions. With sodium and chloride completely replaced by nmdG+ and gluconate, there was a greater loss of water than found with unilateral substitutions. Again, the combined loss of diffusible inorganic cations exceeded the loss of chloride but the water loss was that expected for isosmotic loss accompanying the measured losses of ions. These results reveal that both gluconate and nmdG+ behave as impermeant ions in this tissue preparation. It is suggested that, in the absence of medium sodium, sodium-hydrogen exchange is inhibited. Retained hydrogen ions are buffered on charged cellular non-diffusible solutes and the associated hydroxyl (or bicarbonate) ions are lost from the cells accompanied by the inorganic univalent cations lost in excess of chloride in nmdG+ medium.     

Chloride and water secretion and absorption by the gills of the eel

Summary Perfusion experiments with the heart-gill preparation of the common eel, Anguilla vulgaris, are presented. Various concentrations of perfusion medium were used with eels from both sea water and fresh water. The external medium was sea water in the case of the sea water eels and fresh water in the other case.Perfusate was collected over a period of hours in each experiment and the exchange of chloride and water was studied.In the early experiments it was shown that normal permeability of the gills to these substances is small. The development of an extremely precise chloride method made it possible to evaluate the magnitudes of the exchanges in accurate quantitative terms.Experiments with fresh water as external medium showed that there is a small but perfectly significant change in chloride concentration in the perfusion medium as it passes through the gills so that it becomes more dilute. Reasons are adduced from which it is concluded that the dilution is due to the diffusion of water into the internal medium through the surface of the gills. This branchial water intake is calculated to be something like 40 cc. per kilogram of eel per day. This figure is in substantial agreement with Smith's measurements of the rate of urine formation in fresh water fishes.It was found that when the external medium is sea water, the chloride concentration of the internal medium decreases at a considerably faster rate than in the case where fresh water is present outside the gills. Experiments in which the external medium was analysed showed that chloride was being secreted into the concentrated sea water. It was found possible to measure volume changes in the external medium as well as the changes of chloride concentration in both mediums. These experiments demonstrated beyond doubt that a concentrated chloride solution is secreted by the gills in opposition to a large concentration gradient.Calculations made from several different bases showed that the measurements of volume and of chloride concentration consistently agree in yielding similar values for the volume and concentration of the chloride solution secreted by the gills of the sea water eel.It is shown that the magnitude of the concentration changes effected are related to the concentration of the internal medium used so that a small increase in the concentration of the perfusion medium results in a large increase in the amount of chloride concentration change occurring in the perfusion. Below a certain concentration of the internal medium the gills no longer give evidence of doing concentration work, and may even become permeable as evidenced by the internal medium increasing in concentration as it passes through the gills.Similarities between the activities of the gill of the sea water eel and of the normal mammalian kidney are pointed out. A rough calculation indicates that the work performed by the two organs is of the same order when computed in terms of grams of tissue involved.Experiments in which urea analyses were made showed that the gills of the eel are permeable to urea and that the amount of urea excreted through the gills as determined by direct measurement agrees quite well with Smith's figures for extfa-renal urea excretion.Possible objections to some of the conclusions are brought forth and their untenability demonstrated.Fellow of the National Research Council of America.     

Stimulation of tyrosinase activity of cultured melanoma cells by lysosomotropic agents

The tyrosinase (EC 1.14.18.1) activity of cell-free extracts (TyH) of B16 melanoma cells cultured in the presence of 5 to 10 mM ammonium chloride was considerably higher than that of cells from control cultures. This increase in TyH in the presence of ammonium chloride seemed to be due to de novo synthesis of the enzyme, because it was inhibited by 1 microgram/ml of cycloheximide. In the presence of the latter, however, ammonium chloride did increase the tyrosinase activity of living cells in culture (TyC) resulting in about threefold increase in the TyC/TyH ratio, a measure of the extent of tyrosinase reaction exerted by the enzyme present in living cells. This higher TyC/TyH ratio induced by ammonium chloride was also observed in the absence of cycloheximide. Similar increases in TyH, TyC, and TyC/TyH occurred in the presence of methylamine or ethylamine instead of ammonium chloride, but not in the presence of tetraethylammonium chloride, and also in culture medium of higher pH. The apparently similar effects of lysosomotropic bases and medium of higher pH on the TyC/TyH ratio suggest that there are some mechanisms that control the intramelanosomal pH lower than the cytoplasmic pH.     

Anion transport in red blood cells and arginine specific reagents. (1). Effect of chloride and sulfate ions on phenylglyoxal sensitive sites in the red blood cell membrane

Inhibition of anion transport by the arginine specific reagents phenylglyoxal and 1,2 cyclohexandione depends on the pH and anion concentration in the medium. At pH 8.0, chloride ions protect the transport system against inhibition by PG and 1,2 CHD, while sulfate ions do not protect (1). In the present paper it is shown that at pH 6.5 and 7 both sulfate ions and chloride ions protect the transport system. The protection increases with increasing concentration of the two substrate ions.     

Regeneration fragmentierter,nackter Protoplasten in Hefezellen

Summary The organism used wasSaccharomyces ellipsoideus. Exposure of yeast cells to a hypertonic strontium chloride generally does not establish typical plasmolysed cells. Rather, during a short period of exposure to plain hypertonic strontium chloride solution or to a hypertonic medium supplemented with high content of strontium chloride, the cell walls ruptured and subsequently the formation of spherical osmotically fragile bodies observed. Being associated with large droplet of protoplast together with the plasmolysed cells in the medium, abundantly small and numerous particles imperfectly for reliable counts to be made appear.About other kinds of salts, the protoplast of the yeast cell used separated from the wall smoothly when plasmolysed with sodium chloride or potassium chloride. But calcium chloride increasingly produced the cell rupture. However subsequent successive growth of small particles of protoplast could not be observed in the medium containing calcium chloride. Despite similar results using manganese chloride or magnesium chloride, it seems to cause the damage of the sub cellular structure during prolonged incubation. Similar result was obtained, when the cells were treated in polyethyleneglycol solution.After incubation into liquid strontium medium containing 2 mol strontium chloride, subcellular particles were isolated by the peculiar method (Fig. 6) and were incubated further. Such particles multiplicated, they being designated as K-fragment, and enlarged in size, and became K-body. The size of K-body was threefold as large as that of K-fragment. After further prolonged incubation K-body also enlarged in size, calling it K-cell. The protoplasm of K-cell was less well organized, but the early stage of the formation of daughter K-cell as revealed by microscopical observation beared resemblance to the budding found in mother yeast cell.The findings considered in conjunction with our cytochemical studies of the action of solutions of RNase or DNase, or the stain with some basic dyes, have led us to the conclusion that macromolecular fragment and nuclear substance present in the K-fragment, K-body and K-cell, respectively. Regeneration will result in the most miniature feature of the yeas cell.

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Herrn Prof. Dr. Karl Höfler zu seinem 70. Geburtstag in Verehrung gewidmet.     

Effect of osmolarity on potassium transport in isolated cerebral microvessels

Potassium transport in microvessels isolated from rat brain by a technique involving density gradient centrifugation was studied in HEPES buffer solutions of varying osmolarity from 200 to 420 mosmols, containing different concentration of sodium chloride, choline chloride, or sodium nitrate. The flux of 86Rb (as a tracer for K) into and out of the endothelial cells was estimated. Potassium influx was very sensitive to the osmolarity of the medium. Ouabain-insensitive K-component was reduced in hypotonic medium and was increased in medium made hypertonic with sodium chloride or mannitol. Choline chloride replacement caused a large reduction in K influx. Potassium influx was significant decrease when nitrate is substituted for chloride ion in isotonic and hypertonic media, whereas a slight decrease was found in hypotonic medium. The decrease of K influx in the ion-replacement medium is due to a decrement of the ouabain-insensitive component. Potassium efflux was unchanged in hypotonic medium but was somewhat reduced in hypertonic medium. The marked effect of medium osmolarity on K fluxes suggests that these fluxes may be responsible for the volume regulatory K movements. The possible mechanism of changes of K flux under anisotonic media is also discussed.     

The Effect of Osmotic Value of the Medium on the Growth of the Young Sunflower Embryos Cultured in Vitro

The present paper deals with the effect of osmotic value of the medium on the growth of the young sunflower embryos grown in vitro. Based upon the results of a series of experiments carried out during 1963 the following conclusions are reached: 1. The sunflower embryos grown in vitro require different concentrations of sucrose, varying with the age at which the embryos are excised. Generally speaking, the optimum concentration of sucrose for the embryos 1,000–1,100μ in length is about 17.5%, for 2,000–2,500 μ, about 16% and for 5,000–5,500 μ, about 12.5%. 2. The sucrose may be partially replaced by sodium chloride (0.2%–0.4%) or mannitol (1.125%–5.5%) as an osmotically active substance, but 0.8% sodium chloride is apparently toxic to the young embryos. 3. Embryos cultured in hypotonic medium usually germinate precociously, those in approximately isotonic medium continue their embryonic growth, while those in hypertonic medium become more or less checked in their growth.     

Effect of reduction of culture medium sodium, using different sodium chloride substitutes, on the proliferation of normal and Rous sarcoma virus-infected chicken fibroblasts

We have substituted choline chloride, tetramethylammonium chloride, sucrose, or glucose for culture medium sodium chloride. When culture medium sodium is reduced below physiological levels (143 mM) by replacement of graded concentrations of sodium chloride with equivalent concentrations of choline chloride, normal fibroblasts approach proliferative inactivity in the presence of 90 mM Na, while their Rous sarcoma virus (RSV)-infected counterparts proliferate actively; both normal and neoplastic cells die with further sodium reduction. When culture medium NaC; is replaced with tetramethylammonium chloride, however, both normal and RSV-infected fibroblasts alike approach proliferative inactivity in the presence of 110 mM Na and both die off in the presence of 90 mM Na. When culture medium NaCl is replaced with sucrose or glucose yet another set of results is obtained: both normal and RSV-infected fibroblasts proliferate at reduced, although significant, rates in the presence of 42 mM Na. It is clear from our experimental results that the effects of reduction of culture medium sodium on cell proliferation differ markedly with the use of different sodium chloride substitutes. Caution must be exercised, therefore, in drawing inferences concerning the role of sodium in mitogenesis from experimental studies based on the tactic of reduction of external sodium.     

Spheroplasts of plague microbe strains from the Transcaucasian uplands and their capacity for pesticin synthesis]

Spherical formations of the plague microbe strains from the Transcaucasian Upland, I plague microbe strain of the sandwort variety and I strain of the marmot variety were obtained under the effect of lithium chloride. They had the remains of the cell wall, were viable, sensitive to osmotic shock, preserved sensitivity to the specific bacteriophage and pesticins. All this was evident of isolation of the spheroplasts of the plague microbe. The spheroplasts showed a capacity for pesticin production. The pesticin synthesis by the spheroplasts of the plague causative agent from the Transcaucasian Upland increased with an increase in the content of lithium chloride in the medium. The largest inhibiiton zones were observed, when 0.7-0.8 per cent of lithium chloride were present in the medium. In the spheroplasts of the plague causative agent from the Mountain Altai (the marmot variety) the pesticin synthesis was inhibited with an increase in the content of lithium chloride in the medium. The activity spectrum of the pesticins of the spheroplasts of the plague causative agent from the Transcaucasian Upland and the spheroplasts of the strains of the marmot and sandwort varieties was broader than that of the rod-like forms of these strains. The indicator properties were found in the strains of the plague microbe of the marmot and sandwort varieties with respect to the pesticins of the spheroplasts of the sel-like producing organisms and organisms from the Transcaucasian Upland.     

N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) as a photoaffinity probe for identifying membrane components containing the modifier site of the human red blood cell anion exchange system

Exposure of cells to intense light with the photoactivatable reagent, N- (4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), present in the external medium results in irreversible inhibition of chloride or sulfate exchange. This irreversible inhibition seems to result from covalent reaction with the same sites to which NAP-taurine binds reversibly in the dark. As shown in the preceding paper, high chloride concentrations decrease the reversible inhibition by NAP-taurine in the dark, in a manner suggesting that NAP-taurine and chloride compete for the modifier site of the anion transport system. In a similar fashion, high chloride concentrations in the medium during exposure to light cause a decrease in both the irreversible binding of NAP-taurine to the membrane and the inhibition of chloride exchange. Most of the chloride- sensitive irreversibly bound NAP-taurine is found in the 95,000 dalton polypeptide known as band 3 and, after pronase treatment of intact cells, in the 65,000 dalton fragment of this protein produced by proteolytic cleavage. After chymotrypsin treatment of ghosts, the NAP- taurine is localized in the 17,000 dalton transmembrane portion of this fragment. Although the possible involvement of minor labeled proteins cannot be rigorously excluded, the modifier site labeled by external NAP-taurine appears, therefore, to be located in the same portion of the 95,000 dalton polypeptide as is the transport site.     

Sulfide Production from Cysteine by Desulfovibrio desulfuricans

Two rumen nitrate-reducing isolates of Desulfovibrio desulfuricans were found to hydrolyze cysteine with the production of sulfide and pyruvate. When cultured on agar medium containing yeast extract with nitrate as the primary electron acceptor and ferrous chloride as the indicator, blackening of colonies occurred. The blackening of colonies appeared sooner and was more intense when either cysteine or sulfate was added to the culture medium with nitrate present.     

The formation of choline O-sulphate by Pseudomonas C12B and other Pseudomonas species

Pseudomonas C(12)B and other Pseudomonas species released larger amounts of a (35)S-labelled metabolite into the medium when cultured on growth-limiting concentrations of Na(2)SO(4) as opposed to growth in SO(4) (2-)-sufficient media. The metabolite was found at all stages of the culture cycle of Pseudomonas C(12)B and maximum quantities occurred in stationary-phase culture supernatants. The metabolite was not detected when the bacterium was cultured on growth-limiting concentrations of potassium phosphate. The amount of the metabolite present in the medium greatly exceeded that which could be extracted from intact cells and, except for choline chloride, it was independent of the carbon source used for growth. If choline chloride was present in high concentration, then larger amounts of the metabolite were found in the culture medium. The metabolite was not detected extracellularly or intracellularly when the bacterium was grown in SO(4) (2-)-deficient media containing 5mm-l-cysteine. The same metabolite was also synthesized in vitro only when Pseudomonas C(12)B extracts were incubated with choline chloride, ATP, MgCl(2) and Na(2) (35)SO(4). The metabolite-forming system was not subject to repression by Na(2)SO(4) and was completely inhibited by 0.5mm-l-cysteine and activated by Na(2)SO(4) (up to 1.0mm). The metabolite was identified as choline O-sulphate by electrophoresis, chromatography and isotope-dilution analysis. Another (35)S-labelled metabolite was also detected in culture supernatants, but was not identified.     

Further Investigations on the Nature of the Heat Resistance of Thermophilic Bacteria

When cells of Bacillus stearothermophilus, strain NCA 1503, were grown in tryptone starch broth and subsequently transferred to tris buffer, a fraction of the cells: rapidly died in ttie buffer. This fraction increased with increasing content of calcium chloride in the growth medium. The' addition of sodium, potassium or magnesium chloride to the growth medium had no such effect. The rapid dying of the cells in tris buffer was associated with a leakage of organic material and calcium ions from the cells. The results obtained are probably caused by a damage to the osmotic barrier of the cells during their contact with the buffer. Observations: made during the present investigation and a previous one (Ljunger 1970) indicate that the heat resistance of thermophilic bacteria depends on the maintenance of a high intracellular concentration of free calcium ions.     

Catabolism of newly synthesized decorin by explant cultures of bovine ligament.

The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.     

Release of Citric Acid into the Medium by Aluminum-Tolerant Carrot Cells

One physiological characteristic of an Al-tolerant cell line(TA-1) selected from a cultured carrot cell line (SO-1) wasthe release of more citric acid into the medium than the parentalSO-1 line. Aluminum chloride was added to the media at a concentration,at which SO-1 as well as TA-1 could grow normally without inhibition.The amounts of citric acid and the soluble Al present in themedium were determined during the growth period. Much citricacid was released from TA-1 cells into the medium in the firsthalf of the culture period. At the time of maximum growth, theamount of citric acid in the medium of TA-1 cells was twiceas much as in the medium of SO-1 cells. The precipitates ofAl compound(s), which were formed in the medium by the additionof AlCl₃ as the Al source, became soluble as culture proceeded,depending on the amount of citric acid present in the medium. (Received September 3, 1983; Accepted May 9, 1984)     

The change from symmetry to asymmetry of a sodium transport system in red cell membranes

A study has been made with human red cells of sodium movements that are sensitive to the drug furosemide. The aim was to see if furosemide-sensitive movements that are symmetrical (exchange) became asymmetrical (net transport) on replacement of chloride with nitrate as the major external anion. Cells were incubated for 4 h at 37 degrees C with 140 mM sodium, and chloride or nitrate as the principal anion. Under a variety of conditions (presence and absence of ouabain or furosemide, or both) the cell sodium concentration was always higher when chloride was replaced with nitrate. The cells became leakier to sodium. Tracer studies indicated that, in contrast to the results in chloride medium, the decrease in sodium influx was greater than the fall in efflux when furosemide was added to cells in nitrate medium. The results confirm that the sensitivity of sodium efflux to furosemide depended on chloride. However, influx showed a different sensitivity in that furosemide still inhibited in cells incubated in nitrate medium. The stimulation of sodium influx with nitrate medium was independent of external potassium (10-50 mM) and the furosemide-sensitive influx was also constant. It is concluded that symmetrical transmembrane sodium movements with cells in chloride medium became downhill asymmetrical in nitrate medium, giving a net gain of cell sodium that was insensitive to ouabain and sensitive to furosemide. The drug thus partly retarded the gain of cell sodium that otherwise occurred in the somewhat leaky cells.