31P-NMR saturation transfer measurements of exchange between Pi and ATP in the reactions catalysed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase in vitro

The rotational spectrum of yeast cells changed after pre-treatment of the cells with HgCl2 or Hg(NO3)2 and became indistinguishable from that of ultrasonically produced cell walls. The spectrum of the affected cells contained a peak which could only be explained by attributing a conductivity to the cell walls that was higher than that of the medium. Theoretical models of the rotational response are fully in accord with the experimental spectra. It is shown that the rotation method is capable of measuring even the low cell wall conductivity of yeast cells (which was found to be 33 microS/cm at 10 microS/cm medium conductivity). Knowledge of the spectra allowed a field frequency to be selected at which untreated cells showed no rotation, but at which cells affected by treatment with Hg(II) identified themselves by rotating in the same direction as the field. Calculation of the percentage of cells showing this co-field rotation gave an index (termed the co-field rotation value) of the proportion of the cells that were affected. Using this technique, effects of 25 nmol/l Hg(II) could be demonstrated. In media of low conductivity (10 microS/cm) the change in the rotational spectrum was usually 'all-or-none', whereas at 200 microS/cm a graded Hg(II)-mediated change became apparent. The co-field rotation method showed that the action of small quantities of Hg(II) was still increasing after 3 h of incubation and paralleled the Hg(II)-induced K+ release. A rapid reduction of the effects of Hg(II) was seen when 3-30 mM K+ (or Na+) or when 1 mM Ca2+ were present in the incubation medium, or as the pH was increased. At high incubation cell concentrations the toxic effect of Hg(II) was reduced, apparently due to binding by the cells.     

Binding of methylmercury(II) by HeLa S3 suspension-culture cells: Intracellular methylmercury levels and their effect on DNA replication and protein synthesis

HeLa S3 cells were exposed to varied concentrations of methylmercury over varied periods of time and its binding by the cells was studied using ²⁰³Hg-labeled methylmercuric chloride as radioactive marker. Also studied was the effect of cell-bound methylmercury on DNA replication and protein synthesis and on the growth rate of the cells. The results show that methylmercury binding is a rapid process, with much of the organomercurial bound within the the first 60 min of incubation, and that considerable quantities of organic mercury become affixed to the cells. The amounts of bound methylmercury, [CH₃Hg(II)]_bound, given in mol/cell, range from 2 × 10^?16 (at 1 h of incubation and at 1 μM CH₃Hg(II) in the medium) to almost 4 × 10^?14 (at 24 h of incubation and at 100 μM CH₃Hg(II) in the medium). A [CH₃Hg(II)]_bound value of about 30 × 10^?16 mol/cell appears to be the threshold below which cells display a normal growth pattern and below which metabolic events such as DNA replication or protein synthesis are affected only to a minor degree but above which major changes in cell metabolism and cell growth take place. Methylmercury binding by the cells is tight so that only 20% of the bound material is released from the cells over a 3-h incubation period when the cells are placed into fresh, methylmercury-free growth medium. Analysis of the binding data in terms of binding to identical and completely independent sites yields an association constant K of 7.92 × 10⁴ l/mol and for the maximum concentration of cellular binding sites the value 2.40 × 10^?14 mol/cell or 1.45 × 10¹⁰ sites/cell. Evidence is presented which shows that cellular sulfhydryl groups do not suffice to provide all the sites taken up by methylmercury and that binding, in all likelihood, involves basic nitrogen, too. The levels of cell-bound methylmercury are such that binding to HeLa DNA and HeLa chromatin, for instance, can readily take place. Methylmercury binding data obtained by using the technique of particle-induced X-ray emission (PIXE) are in good agreement with the data obtained via isotope dilution.     

The comparative influence of substituted phenols (especially chlorophenols) on yeast cells assayed by electro-rotation and other methods

The toxicity of 31 phenols was studied by electro-rotation of yeast cells. Control yeast cells show both anti-field and co-field rotation, depending upon the field frequency applied. After treatment with supra-threshold amounts of phenols the anti-field rotation is weakened or abolished and a stronger co-field rotation can be seen. The proportion of cells showing the co-field rotation was found to be a sensitive measure of toxicity. Doses of 2.2 mumol/l of pentachlorophenol, or of 0.3 mumol/l of pentabromophenol were detectable after 3 h incubation at pH 4.0. At a given pH, the toxicity of the chlorophenols correlated extremely well with their octanol:water partition coefficients (Pow). The complete set of phenols showed fair overall correlation with Pow, but less good correlation with their acidity constants (pKa). In particular the toxicity of a given phenol was less than predicted from its pKa if the incubation pH was higher than the pKa. Biochemical assays on 23 of the phenols showed that the rotational sensitivity runs closely parallel to the sensitivities of cell growth rate and of the plasmamembrane ATPase, but less closely to the inhibition of purine incorporation. It appears that the electro-rotation method provides a useful and rapid test for the presence of organic ecotoxins. The test enables us to distinguish differences between single cells, and is comparable in sensitivity to biochemical tests that use vesicles or homogenates derived from a cell population.     

Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti

Two-electrode voltage clamp (TEVC) methods were used to explore conductive transport pathways in principal cells, the dominant cell type in Malpighian tubules of the yellow fever mosquito. The basolateral membrane of principal cells had a voltage (V_bl) of -85.1 mV in 49 principal cells under control conditions. Measures of the input resistance R_pc together with membrane fractional resistance yielded estimates of the conductance of the basolateral membrane (g_bl = 1.48 μS) and the apical membrane (g_a = 3.13 μS). K⁺ channels blocked by barium accounted for 0.94 μS of g_bl. Estimates of transference numbers yielded the basolateral membrane Na⁺ conductance of 0.24 μS, leaving 0.30 μS (20%) of g_bl unaccounted. The secretagogue db-cAMP (0.1 mM), a known activator of the basolateral membrane Na⁺ conductance, significantly depolarized V_bl to -65.0 mV and significantly increased g_bl from 1.48 μS to 2.47 μS. The increase was blocked with amiloride (1 mM), a known blocker of epithelial Na⁺ transport. The inhibition of metabolism with di-nitrophenol significantly depolarized V_bl to -9.7 mV and significantly increased R_pc from 391.6 kΩ to 2612.5 kΩ. Similar results were obtained with cyanide, but it remains unclear whether the large increases in R_pc stem from the uncoupling of epithelial cells and/or the shutdown of conductive transport pathways in basolateral and apical membranes. Our results indicate that the apical membrane of principal cells is more than twice as conductive as the basolateral membrane. Partial ionic conductances suggest the rate-limiting step for transepithelial Na⁺ secretion at the basolateral membrane.     

Electrorotation of axolotl embryos

The frequency dependent dielectric properties of individual axolotl embryos (Ambystoma mexicanum) were investigated experimentally utilizing the technique of electrorotation. Individual axolotl embryos, immersed in low conductivity media, were subjected to a known frequency and fixed amplitude rotating AC electric field and the ensuing rotational motion of the embryo was monitored using a conventional optical microscope. None of the embryos in the pregastrulation or neurulation stages of development exhibited any rotational motion over the field frequency range (10 Hz-5 MHz). Over the same frequency range, the embryos in the gastrulation stage of development exhibited both co-field and counterfield rotation over different ranges of the applied field frequency. Typically, the counterfield rotation exhibited a peak in the rotation spectrum at similar 1 KHz while the co-field peak was located at similar 1-2 MHz. The rotational spectral data was analyzed using a multishelled spherical embryo model to determine the electrical character of embryos during the early development stages (Stages 5-16; i.e., 16 cell through open neural plate stages).     

Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K⁺ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba²⁺ (BaCl₂, 5 mmol/l) and the blocker of the Na⁺/K⁺ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B₄ receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 μmol/l). ATPγS (10 μmol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 μmol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPγS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.     

Distribution of mercury, methyl mercury and organic sulphur species in soil, soil solution and stream of a boreal forest catchment

Concentrations of methyl mercury, CH₃Hg (II), total mercury, Hg_tot = CH₃Hg (II) + Hg (II), and organic sulphur species were determined in soils, soil solutions and streams of a small (50 ha) boreal forest catchment in northern Sweden. The CH₃Hg (II)/Hg_tot ratio decreased from 1.2–17.2% in the peaty stream bank soils to 0.4–0.8% in mineral and peat soils 20 m away from the streams, indicating that conditions for net methylation of Hg (II) are most favourable in the riparian zone close to streams. Concentrations of CH₃Hg (II) bound in soil and in soil solution were significantly, positively correlated to the concentration of Hg_tot in soil solution. This, and the fact that the CH₃Hg (II)/Hg_tot ratio was higher in soil solution than in soil may indicate that Hg (II) in soil solution is more available for methylation processes than soil bound Hg (II). Reduced organic S functional groups (Org-S_RED) in soil, soil extract and in samples of organic substances from streams were quantified using S K-edge X-ray absorption near-edge structure (XANES) spectroscopy. Org-S_RED, likely representing RSH, RSSH, RSR and RSSR functionalities, made up 50 to 78% of total S in all samples examined. Inorganic sulphide [e.g. FeS₂ (s)] was only detected in one soil sample out of 10, and in none of the stream samples. Model calculations showed that under oxic conditions nearly 100% of Hg (II) and CH₃Hg (II) were complexed by thiol groups (RSH) in the soil, soil solution and in the stream water. Concentrations of free CH₃Hg⁺ and Hg²⁺ ions in soil solution and stream were on the order of 10^–18 and 10^–32M, respectively, at pH 5. For CH₃Hg (II), inorganic bi-sulphide complexes may contribute to an overall solubility at concentrations of inorganic sulphides higher than 10^–9M, whereas considerably higher concentrations of inorganic sulphides (lower redox-potential) are required to increase the solubility of Hg (II).     

The change in the electro-rotation of yeast cells effected by silver ions

Pre-treatment of brewer's yeast (Saccharomyces cerevisiae) cells with silver acetate or nitrate at concentrations of 20 nmol/l or higher caused a dramatic increase in the number of cells which rotated in the same direction as the field (‘Co-field rotation’). The change in rotation of single cells correlated very well with the chemically observed loss of potassium induced by Ag⁺. The sensitivity to Ag⁺ was lowered by increasing the cell concentration, and the extent of this change can be used to estimate the binding of Ag⁺ per cell and the limiting sensitivity of the method. The Ag⁺ concentration required to induce a response was found to be increased significantly in the presence of alkali ions (especially K⁺) during the Ag⁺ incubation. The Ag⁺ sensitivity was, therefore, observed to be a function of the type and strength of buffer used in the incubation. Under certain conditions, 1 mM Ca²⁺ increased the Ag⁺ sensitivity. These observations show that the presence or absence of ions that are so common that they are often overlooked may have interesting consequences for the bio-assay of heavy metals.     

Effects of 2-formylpyridine monothiosemicarbazonato copper II on red cell components

2-Formylpyridine monothiosemicarbazonato copper II (CuL⁺) is readily taken up by red cells and is initially bound to glutathione and hemoglobin. Glutathione was depleted within 5 hr of incubation, presumably by oxidation mediated by CuL⁺ and O₂ with concomittant generation of toxic oxygen species. Cupric ion was slowly transferred from CuL⁺ to hemoglobin within about 7 hr, and hemoglobin was oxidized until the major form prevailing after 10 hr was α₂β₂⁺. Little increase in hemolysis due to addition of CuL⁺ dissolved in the radical scavenger dimethyl sulfoxide was observed with prolonged incubation. Strong inhibition of red cell hexokinase by CuL⁺ was observed when the enzymes in red cell lysates and hemoglobin-free red cell lysates were examined. CuL⁺ was also an effective inhibitor of yeast hexokinase. However, the inhibitory effect of CuL ⁺ within the red cells was less pronounced. It is suggested that even though intracellular accumulation of CuL ⁺ creates an oxidizing environment and is potentially capable of inhibiting thiol enzymes such as hexokinase, protective effects are exerted in the red cell by the presence of hemoglobin, of radical scavengers, and of high levels of enzymes that detoxify toxic oxygen species. Address reprint requests to Dr. W.E. Antholine, Department of Radiology, or Dr. F. Taketa, Department of Bio     

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K_m for α-aminoisobutyric acid of 270 μM, a V_max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V_max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH₄⁺. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.     

Intracellular calcium chelator BAPTA protects cells against toxic calcium overload but also alters physiological calcium responses

The effect of the membrane-permeant calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N−'tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) on ionomycin-induced cellular calcium overload was studied in single differentiated NH15-CA2 neuroblastoma x glioma hybrid cells. To monitor [Ca²⁺]_i, we used the fluorescent indicator Fura-2. Preincubation of the cells with 3 μM BAPTA/AM reduced the number of cells showing deregulation of [Ca²⁺]_i during ionomycin-induced calcium influx. The calcium transients elicited by application of KCI were also severely affected by the chelator. These transients, although varying from cell to cell in shape, amplitude and duration, are well reproducible in individual cells. After incubation of cells for 1 h with 0.3–30 μM BAPTA/AM the time course of these cellular transients was markedly slowed. At 1 μM BAPTA/AM, the time constant of decline of [Ca²⁺]_i was increased by a factor of 4.1 ± 2.4 (n = 14) and the amplitude was reduced to about 50%. With 30 μM BAPTA/AM, the K⁺-induced calcium transients were almost completely inhibited. We conclude that intracellularly loaded calcium chelators may be used for the prevention of [Ca²⁺]_i-induced cell damage, however, at the expense of a disturbed calcium signalling.     

Interactions between selenium and methylmercury in rat brain

The interaction of selenium with methylmercury was investigated in brain of animals labeled with ⁷⁵SeO₃^2? and CH₃²⁰³Hg⁺. Brains were fractionated into subcellular components and the cytosol was further fractionated by chromatography on Sephadex G-150 and G-200. The main result of these studies was evidence suggesting a shift of ⁷⁵Se from the cytosol to the mitochondrial fraction in brain when CH₃Hg⁺ was given. Concurrent equimolar (10 μmoles/kg) selenite injections increased the uptake of Hg but did not alter ²⁰³Hg distribution in brain. Changing the dose of CH₃Hg⁺ from 1 to 38 μmoles/kg had little effect on Hg uptake (% of dose per g). Gel filtrations on Sephadex G-150 and G-200 revealed that ²⁰³Hg in cytosol followed a pattern more closely related to protein (A₂₈₀) than to ⁷⁵Se, although a considerable portion of both isotopes eluted with proteins in the void volume. Assays of whole brain homogenates revealed a slight reduction in glutathione peroxidase activity in CH₃Hg⁺-treated rats which was not seen when equimolar selenite was injected with the CH₃Hg⁺.     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.     

Further characterization of 17 β-hydroxysteroid dehydrogenase activity in cultured myometrial cells: Cofactor dependency and subcellular localization

To further characterize 17 β-hydroxysteroid dehydrogenase (17 β-SDH) from cultured ovine myometrial cells, an assay was established in whole cell homogenates and cell subfractions. Tritiated estradiol (E₂) was incubated in the presence of an excess of cofactor and estrone (E₁) formed purified by thin-layer chromatography. The enzyme activity was linear with time up to 2 hours and with protein concentration up to 0.7 mg/ml at the substrate concentration used (5 × 10^?9 M). The routine assay was for 30 min in the presence of 0.5 mg/ml of protein. Both NAD⁺ or NADP⁺ could sustain enzyme activity but NAD⁺ was twice as much efficient. Most of the enzyme activity was associated with the microsome and mitochondrial membranes. The addition of an excess (1000 μM) of NAD⁺ to the incubation medium prevented the progressive decline observed with time in a given subculture in the intact cell monolayer assay, supporting our previous hypothesis that this decline was due to cofactor depletion. In contrast, the slow and irreversible decline of enzyme activity observed in successive subcultures was not prevented by the addition of cofactor to the homogenate and thus reflects another phenomenon, probably a change in metabolism with age.     

Effect of divalent cations on the short-term NH 4 + inhibition of nitrogen fixation in Azotobacter chroococcum

Incubation of Azotobacter chroococcum in the presence of micromolar concentrations of MnCl₂, but not MgCl₂, prevented nitrogenase activity from NH ₄ ⁺ inhibition. Mg(II), at a 100-fold concentration with respect to Mn(II), counteracted the protective effect of Mn(II) on nitrogenase activity. When Mn(II) was added to cells that had been given NH₄Cl, stopping of NH ₄ ⁺ uptake and recovery of nitrogenase activity took place, and a raise of NH ₄ ⁺ concentration in medium developed. Furthermore, incubation of A. chroococcum cells with 20 M Mn(II) under air, but not under an argon: oxygen (79%:21%) gas mixture, resulted in NH ₄ ⁺ excretion to the external medium. The Mn(II)-mediated uncoupling of nitrogen fixation from ammonium assimilation leads us to conclude that Mn(II) may act as a physiological inhibitor of glutamine synthetase.Abbreviations Hepes N-2-Hydroxyethylpiperazine-N-ethanesulfonic acid - Mops 3-(N-Morpholino)propanesulfonic acid     

Different red cell populations in newborn, genetically low potassium sheep: relation to hematopoietic, immunologic and physiologic differentiation

Three red cell populations have been distinguished in genotypically low potassium (LK) newborn sheep by an improved electrical sizing method and were best approximated by a logarithmic normal distribution. Labeling studies with ⁵¹Cr and ⁵⁹Fe exclude transformation of the three red cell populations into each other. Population I, consisting of large red cells (mean volume 36 μm³), with a comparatively slow electrophoretic mobility is present at birth and disappears within three to four weeks from circulation. These cells possess a high potassium (HK) steady state concentration, a K⁺ pump influx activity at least 5-fold greater than observed in adult LK red cells, very low amounts of the L antigens generally associated with the LK property, and do not respond to the stimulatory action of the L antibody. The first population is gradually replaced by population II comprising small red cells (mean volume 28 μm³) of intermediate electrophoretic mobility and with a peak production around day 20 after birth. The potassium concentration, [K⁺]_c, in these cells appears to be lower than in the cells of population I but the L antigen content is increased. Formation of population III (mean volume 30 μm³ and comparatively fast electrophoretic mobility) follows closely that of population II and is preceded by a sharp increase in reticulocytosis. The red cells of population III exhibit parameters characteristic for adult LK cells: low [K⁺]_c and K⁺ pump activity, fully developed L antigen content, and an almost maximal response to the K⁺ pump stimulating effect of anti-L. In L and M antigen positive LK red cells of newborn sheep, the development of the M antigen parallels that of the L antigen. The data are consistent with the hypothesis that cellular replacement and not maturation is the major factor in controlling the HK-LK transition in newborn sheep.     

Peroxiredoxin II Regulates Effector and Secondary Memory CD8+ T Cell Responses

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H₂O₂ on an antigen-specific CD8⁺ T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8⁺ T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8⁺ T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8⁺ T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8⁺ T cell pool. During chronic viral infection, increased antigen-specific CD8⁺ T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8⁺ T cell expansion, secondary memory generation, and immunopathology.     

Mechanism of aldosterone-induced increase of K+ conductance in early distal renal tubule cells of the frog

Summary Isolated early distal tubule cells (EDC) of frog kidney were incubated for 20–28 hr in the presence of aldosterone and then whole-cell K⁺ currents were measured at constant intracellular pH by the whole-cell voltage-clamp technique. Aldosterone increased barium-inhibitable whole-cell K⁺ conductance (gK⁺) threefold. This effect was reduced by amiloride and totally abolished by ouabain. However, aldosterone could still raisegK⁺ in ouabain-treated cells in the presence of furosemide.We tested whether changes in intracellular pH (pH _i ) could be a signal for cells to regulategK⁺. After removal of aldosterone, the increase ingK⁺ was preserved by subsequent incubation for 8 hr at pH 7.6 but abolished at pH 6.6. In the complete absence of aldosterone, incubation of cells at pH 8.0 for 20–28 hr raised pH _i and doubledgK⁺.Using the patch-clamp technique, three types of K⁺-selective channels were identified, which had conductances of 24, 45 and 59 pS.Aldosterone had no effect on the conductance or open probability (P _o) of any of the three types of channels. However, the incidence of observing type II channels was increased from 4 to 22%. Type II channels were also found to be pH sensitive,P _o was increased by raising pH.These results indicate that prolonged aldosterone treatment raises pH _i and increasesgK⁺ by promoting insertion of K⁺ channels into the cell membrane. Channel insertion is itself triggered by raising both pH _i and increasing the activity of the Na⁺/K⁺ pump in early distal cells of frog kidney. Present address: Department of Physiology, The University of Leeds, Leeds, LS2 9NQ, England     

Influence of Ammonium on the Growth and Development of Suspension Cultures of Pauľs Scarlet Rose

In this work as in previous studies from this laboratory it was demonstrated that the presence of a trace amount of NH₄⁺ (72.8 μmol) stimulated the growth of Pau?s Scarlet Rose on a defined medium containing NO₃^? (1920 μmol) as the only other source of nitrogen. A kinetic analysis of several growth parameters showed that the rate of increase of dry weight, fresh weight, cell number, and cell volume were greater during early stages of growth (days 0–8) when NH₄⁺ was provided. During later stages (days 8–14) this relationship between the two cultures did not hold. The cells provided NH₄⁺ continued to increase in fresh weight and cell volume, but the cells which were not provided NH₄⁺ had a greater rate of dry weight and cell number increase. These differences led to 14-day-old cultures which were approximately equal in dry weight and cell number but differed by a factor of 2 in fresh weight. The presence of NH₄⁺ speeded up the development and growth of the cells.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.