Nitrate Transport into Chara corallina Cells using as an Analogue for Nitrate: III. INTERACTIONS BETWEEN CHLORIDE AND NITRATE TRANSPORT PROCESSES

The effects of Cl^– and pretreatment on ³⁶Cl^–influx and influx into Characorallina cells were examined. Both treatments reduced ³⁶Cl^–influx into C. corallina cells in the acid pH range (4.5–7.0). pretreatment stimulated influx into C. corallina cells, but Cl^– pretreatment hadno effect. There was a direct inhibitory effect of CI on influx into C. corallina cells, but no apparenteffect on net NO uptake. The time course of ³⁶Cl accumulation into cytoplasmic and vacuolarcompartments during incubation of the cells with showed that significant radioactivity appeared in the vacuolarsap after 30 min. There was a linear increase in the percentageof total ³⁶CI which crossed the tonoplast (_c-v). There was noeffect of Cl^– or pretreatment on accumulation of radioactivity in the vacuole. Thin layer chromatography ofthe vacuolar sap showed that after 2 h only one component waspresent which had an R_F which was similar to ³⁶CI^–. Therate of accumulation of ³⁶C1^– in the vacuole could beused to estimate rates of reduction. Key words: Chloride, Chlorate, Chara, Nitrate     

Nitrate Transport into Chara corallina Cells using 36C1O3 as an Analogue for Nitrate: II. COMPARISON WITH 14C METHYLAMINE FLUXES AT DIFFERENT pH0 AND NH+4sol;NO3 INTERACTIONS

³⁶C1O₃/NO₃ influx into Chara cells was found to be sensitiveto pH_o and a maximum was found at pH_o = 4.5. By contrast ¹⁴Cmethylamine influx into Chara showed a maximum at pH_o = 8.5,and at this pH_o influx rates were about 150 times higher thanrates of ³⁶C1O₃/NO₃ influx. However, at pH_o = 4.5, ³⁶C1O₃/NO₃influx rates were, in some cases, comparable with rates of ¹⁴Cmethylamine influx. ³⁶C1O₃/NO₃ influx into Chara cells was stimulatedby Rb ⁺, K⁺, Na ⁺, and NH₄⁺, but not by Cs⁺ or Li ⁺. NO₃ andCl reduced ¹⁴C methylamine influx into Chara by 30%. NH₄⁺ causedvery considerable inhibition of ¹⁴C methylamine influx intoChara, but had no effect on ³⁶C1O₃/NO₃ influx in the presenceof K ⁺. Net NO³ uptake into Chara was completely prevented byNH₄⁺ even at relatively low NH₄⁺ concentrations (25 mmol m ^–3).This latter effect was reversed by diethylstilbestrol (DES).Evidence is presented for the stimulation of NO₃ efflux by NH⁴⁺as the mechanism responsible for the immediate effects of NH₄⁺on net NO₃ uptake into Chara cells. Key words: Chara, ¹⁴C methylamine, ³⁶ClO^–₃, pH     

Potassium Transport Across the Membranes of Chara: I. THE RELATIONSHIP BETWEEN RADIOACTIVE TRACER INFLUX AND ELECTRICAL CONDUCTANCE

Smith, J. R., Smith, F. A. and Walker, N. A. 1987. Potassiumtransport across the membrane of Chara. I. The relationshipbetween radioactive tracer influx and electrical conductance.—J.exp. Bot. 38:731–751. The ⁴²K influx () and the electrical conductance (G_m) were measured simultaneously for the ‘membrane’of internodal cells of Chara australis as a function of theexternal [KCl] (K?. In bathing solutions of pH = 5?0, progressively increased from 20?5to 430?60 nmol m^–2 s^–1 and G_m increased from 0?36?0?02to 3?8?0?8 S m^–2 when K? was increased from 0?1 to 10mol m^–3. The resting membrane potential difference (p.d.)was approximately -135 mV for low K? and approached the expectedNernst equilibrium p.d. for K⁺ ions when K? > 1?0 mol m^–3.Measurements of ³⁶Cl influx suggested that the ⁴²K influx waspredominantly electrogenic. The equivalent Goldman permeabilityto K⁺ ions (P_k) was approximately 20–30 nm s^–1 anddid not vary significantly with increasing K?. The equivalentconductance attributable to the electrogenic transport of K⁺ ions was calculated from assuming passive, independent diffusionof K⁺ ions and the ratio was found to be typically close to one. It was also found that themagnitudes of and G_m measuredsimultaneously for each individual cell were also well correlatedfor K? 1?0 mol m^–3, and that the slope of the line ofbest fit was close to one. For each K? it was found that theconductance not attributable to K⁺ translocation and presumablyassociated primarily with the transport of protons or theirequivalents was typically 0?2–0?5 Sm^–2. For K? >1?0 mol m^–3 the results indicated that the transport ofK⁺ ions was essentially independent, i.e. there was no evidencefor flux interactions. The results also indicated that the equivalentconductance derived from the measured ⁴²K influx could usefullyindicate the fraction of the electrical conductance attributableto the translocation of K⁺ ions. Key words: Potassium, conductance, influx     

Physiological Control of Chloride Transport in Chara corallina: I. EFFECTS OF LOW TEMPERATURE, CELL TURGOR PRESSURE, AND ANIONS

The rate of Cl⁻ transport at the plasma membrane of the freshwater alga Chara corallina is investigated with respect to possible in vivo controls acting in addition to the two well established ones of cytoplasmic Cl⁻ and cytoplasmic pH. In contrast with results from many other plant tissues, halides appear to be the only anions capable of inhibiting Cl⁻ transport, either from the outside or inside surfaces of the plasma membrane. Cell turgor pressure was also investigated. It was found that neither the influx of Cl⁻ nor that of K⁺ or HCO₂⁻ is sensitive to turgor. Internal osmotic pressure is also insensitive to turgor, a situation contrasting with that in closely related brackish water charophytes.     

The Transport and Metabolism of Urea in Chara australis: I. PASSIVE DIFFUSION, SPECIFIC TRANSPORT AND METABOLISM OF UREA, THIOUREA AND METHYLUREA

Most of the urea entering Chara australis cells is rapidly metabolizedto produce CO₂, which diffuses out of the cells into the surroundingmedium. A simple and convenient apparatus to measure both the¹⁴C-urea retained by cells and the ¹⁴CO₂ released into the mediumwas developed and used in a study of urea transport in Chara.The permeability coefficient for urea in the Chara plasmalemmawas estimated from the slope of an uptake versus concentrationfunction as 85 nm s^-1. Computer modelling of urea uptake andmetabolism suggests that this could be a 20% underestimate ofthe true value.The corresponding permeability coefficients forthiourea and N-methyl-urea were estimated in the same way as34 and 35 nm s^-1, respectively. These permeabilities are muchgreater than expected on the basis of either/water partitioncoefficients for the solutes and are consistent with the diffusionof urea and its similarly-sized analogues through aqueous poresin the plasmalemma.At external concentrations of urea less than20 mmol m^-3, the bulk of the uptake is effected by a specifictransport mechanism with an apparent K_m for urea of less than1.0 mmol m^-3. This transport system operates most rapidly withexternal pH in the range 6.5–7.5 and is influenced bythe nitrogen status of the cell.Evidence is produced here suggestingthat the specific transport of urea may be an active process. Key words: Chara, urea uptake, metabolism, diffusion, specific transport     

Ionic Currents across the Plasmalemma of Chara inflata Cells: I. OSMOTIC EFFECTS OF SORBITOL ON K+, CI- AND LEAK CURRENTS

This paper describes experiments designed to investigate theeffects of increases in external osmotic pressure on the electrophysiologicalbehaviour of the plasmalemma in cells of the brackish-wateralga, Chara inflata. The electrical conductance of the plasmalemmaof these cells of, due to the diffusion of ions, consists mainlyof K⁺, Cl^– and leak components. The addition of sorbitolat concentrations in the range 40–280 mol m^–3 tothe external solution bathing the cells, progressively and reversiblydepolarized the cell membrane and increased the total membraneconductance, chiefly due to increases in each of the separateionic conductances. At concentrations higher than about 280mol m^–3 when the cells became plasmolysed, the effectsof sorbitol on the electrical properties of the cell ceasedto be fully reversible. When the membrane electrical potentialdifference is stepped in a negative direction with a voltage-clamp,the resulting inward current has voltage-dependent componentsconsisting of an inactivating K⁺ current, an activating Cl^–current and a constant leak current. The addition of sorbitolto the external solution modified these currents by increasingtheir magnitude, by increasing the half-time of the inactivationof the K⁺ current, and by decreasing the half-time of activationof the Cl^– current. Key words: Chara inflata, osmotic effects, K⁺ and Cl^– currents     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : I. Antagonistic Effect of Progesterone on Estrogen-Induced Proliferation and Differentiation of Tubular Gland Cells

Daily administration of estrogen to immature female chicks results in marked oviduct growth and appearance of characteristic tubular gland cells which contain lysozyme. Although a rapid increase in total DNA and RNA content begins within 24 hr, cell specific protein, lysozyme, is first detectable after 3 days of estrogen. Progesterone administered concomitantly with estrogen antagonizes the estrogen-induced tissue growth as well as appearance of tubular gland cells and their specific products, lysozyme and ovalbumin. When the initiation of progesterone administration is delayed for progressively longer periods (days) during estrogen treatment, proportionally greater growth occurs with more lysozyme and tubular gland cells after 5 days of total treatment. Progesterone does not inhibit the estrogen-stimulated increase in uptake of α-aminoisobutyric acid and water by oviduct occurring within 24 hr or the estrogen-induced increase in total lipid, phospholipid, and phosphoprotein content of serum. The above results of progesterone antagonism can best be explained by the hypothesis that progesterone inhibits the initial proliferation of cells which become tubular gland cells but does not antagonize the subsequent cytodifferentiation leading to the synthesis of lysozyme and ovalbumin once such cell proliferation has occurred.     

Mechanism of nitrate uptake into Chara corallina cells: lack of evidence for obligatory coupling to proton pump and a new NO3−/NO3− exchange model

Abstract. Nitrate uptake into Chara corallina cells at different external pH (pH_o) after different NO₃⁻ pretreatment conditions has been investigated. Following NO₃⁻ pretreatment (0.2 mol m⁻³ NO₃⁻) there was little effect of pH_o on subsequent net NO₃⁻ uptake into Chara cells. After N deprivation (2 mmol m⁻³ NO₃⁻) there was a pronounced effect of pH_o on nitrate uptake, the maximum rate occurring at pH_o 4.7. There was no consistent relationship between OH⁻ efflux and NO₃⁻ uptake in short term experiments (< 1 h). NO₃⁻ efflux was also sensitive to pH_o, the maximum rate occurring at ∼ pH_o 5.0. An inhibitor of the proton pump, DES, immediately stimulated NO₃⁻ uptake into cells pretreated with NO₃⁻ and prevented the time-dependent decrease in NO₃⁻, uptake into Chara cells that had been previously treated with low N (2 mmol m⁻³ NO₃⁻). NO₃⁻ efflux was found to be very sensitive to DES with K_i= 0.7 mmol m⁻³. At the optimum pH_o for NO₃⁻ uptake the effect of DES on membrane potential (ψ_m) were slight, and only apparent after 30 min. The results are interpreted in context of current models relating NO₃⁻ uptake and H⁺ pump activity. A new model for NO₃⁻ uptake is proposed which involves NO₃⁻/NO₃⁻ exchange at steady state.     

Nitrate Uptake into Barley (Hordeum vulgare) Plants : A New Approach Using ClO(3) as an Analog for NO(3)

Evidence is presented that chlorate is an extremely good analog for nitrate during nitrate uptake by intact barley (Hordeum vulgare cv. Fergus) roots. The depletion of ClO₃⁻ or NO₃⁻ from uptake media over 2 to 6 hours by seedlings was found to be dependent on combined NO₃⁻ plus ClO₃⁻ concentrations, and total anion uptake was equivalent at different NO₃⁻/ClO₃⁻ ratios. After loading barley seedlings with ³⁶ClO₃⁻ for 6 hours, kinetic parameters were derived from the analysis of efflux of [³⁶Cl] chlorate into unlabeled solution. On the basis of this analysis, the half times for exchange for the cytoplasmic and vacuolar phases were 17 minutes and 20 hours, respectively.     

The Incorporation of 14C-Proline into the Proteins of Growing Cells: I. EVIDENCE OF SYNTHESIS IN DIFFERENT PROTEINS AND CELLULAR COMPONENTS

¹⁴C-proline was supplied to aerated potato disks, in which celldivision was occurring, and also to rapidly growing potato carrotexplants. It was absorbed and incorporated into all the subcellularprotein fractions examined, including the electrophoreticallydistinguishable fractions of the soluble protein of the potatodisks and explants. The ¹⁴C-proline was partially convertedto ¹⁴C- hydroxyproline in all the protein fractions, exceptfor one of the soluble protein fractions of potato explantsand the soluble proteins of one set of potato disks. Most ofthe ¹⁴C-proline and ¹⁴C-hydroxyproline contained in the tissuewas found in the soluble protein and also in the cellular fragmentsobtained by centrifugation at 500 g. The relative importanceof the soluble protein in the incorporation of ¹⁴C-proline andits conversion to ¹⁴C-hydroxyproline was greatest over a shortperiod of a few hours of contact with the ¹⁴C-proline supplied.Over a longer period (70 hours) the cellular fragments (500g) had become the most important and contained over 40 per cent.of the total ¹⁴C, and more than 60 per cent. of the ¹⁴C-hydroxyproline,in the protein of the tissues. In the soluble fraction of potatoexplants, seven protein bands were distinguishable on electrophoresis.A different but characteristic value of the ratio ¹⁴C-hydroxyprolineto ¹⁴C-proline was associated with each protein band, exceptfor the one region where ¹⁴C-hydroxyproline did not occur. Thebasic proteins (i.e. those moving towards the cathode) werethe most active in the incorporation of ¹⁴C-proline and itsconversion to ¹⁴C-hydroxyproline. The rather general distributionof the ¹⁴C-hydroxyproline is noted and the possible siginificanceof the basic proteins and the proteins associated with the cellularfragments (500 g) is considered in relation to the growth, celldivision, and cell wall formations which occurs in the rapidlygrowing tissue cultures.     

Studies on the Movement of Solutes between the Sieve Tubes and Surrounding Tissues3: I. INTERFERENCE BETWEEN SOLUTES AND RATE OF TRANSLOCATION MEASUREMENTS

Certain aspects of the secretion of solutes into, and removalfrom, the sieve tubes of isolated stem segments and rooted cuttingsof Salix viminalis have been studied. Sieve-tube sap was obtainedeither as honeydew from whole individuals or via the severedstylets of the aphid Tuberolachnus salignus (Gmelin). It was shown that interference occurred between the chemicallyunrelated solutes, sucrose and the cations potassium and rubidium.On raising the potassium concentration in the sieve-tube sapby passing a solution of this ion through the xylem, the sucroseconcentration declined. When the sucrose concentration fellover a period of days due to respiratory loss of carbohydratesfrom an isolated stem segment, a concomitant rise in eitherthe potassium or rubidium level in the sap occurred. When a solution of sodium was passed through the xylem, theconcentration of this ion in the sieve-tube sap rose, whilstthat of potassium fell at first, but later rose higher thanits initial value, indicating that both antagonism and synergycan occur between these ions. On introducing both these cationsinto the xylem simultaneously, more sodium than potassium wastaken up by the segment, though the increase in the sodium concentrationin the sieve-tube sap was less than that of the potassium. Perfusingthe xylem with a calcium solution had no effect upon the concentrationof potassium in the sieve tube. It has been shown that the rate of translocation of a solutealong the sieve tube, as measured by the two colony technique,depends upon the rate of removal of this solute from the sievetube. The amount of such lateral loss from the sieve tube isrelated to the potential gradient for a solute between the sievetube and surrounding cells.     

RELATIONSHIP BETWEEN RNA SYNTHESIS, CELL DIVISION, AND MORPHOLOGY OF MAMMALIAN CELLS : I. Puromycin Aminonucleoside As An Inhibitor of RNA Synthesis and Division in HeLa Cells

Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P³² and H³-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division within the period studied (3 generation times). The cell content of RNA returned to normal when the inhibitor was removed.     

The Root Cap as a Test System for the Evaluation of Golgi Inhibitors: I. STRUCTURE AND DYNAMICS OF THE SECRETORY SYSTEM AND RESPONSE TO SOLVENTS

Maize (Zea mays) seedlings were grown under standard conditionsand the time for reformation of a slime droplet of standardsize was determined. The structure of the secretory cells wasexamined by light and electron microscopy. Osmium-zinc iodide(OZI) impregnation was used to provide contrast enhancementof the dictyosome forming-face cisternae and the endoplasmicreticulum (ER). The three-dimensional structure and relationshipsof these membrane systems were established from examinationof 300 nm thick sections. The presence of a marginal cisternalnetwork at the forming-face was confirmed, but no specific orientationof the ER to this face, or connection of ER to any part of thedictyosome could be demonstrated. Neither observation of reforming slime droplets, nor quantificationof secretory vesicle numbers gave any support to the suggestionthat slime formation is a phasic, or cyclic, process. Dilute solutions of solvents (1% ethanol, 1% and 0.1% DMSO)commonly used to solubilize potential inhibitors of Golgi activitywere found to delay significantly the time taken for reformationof a slime droplet of standard size. These solutions also hadslight effects on the numerical density of secretory vesicles. Cytochalasin D-induced accumulation of secretory vesicles wasused to determine rates of vesicle formation (0.39 dictyosome^–1min^–1) and hence turnover times for cisternal (6.5 min)and total dictyosome (26–39 min) membranes and the plasmamembrane (9.7 min). The volume contribution of the secretory vesicles to the slimedroplet was shown to be exceedingly small (0–1%), throwingdoubt on the value of using droplet reformation rates as anindicator of secretory activity. Key words: Maize, Root cap secretion, Dictyosome activity     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : I. Observations on the Specificity and Stability of Choline-14C and Glycerol-3H as Labels for Membrane Phospholipids

In order to determine the feasibility of using radioactive precursors as markers for membrane phospholipids in Acanthamoeba palestinensis, the characteristics of phospholipids labeled with choline-¹⁴C and glycerol-³H were examined. Choline-¹⁴C was found to be a specific label for phosphatidyl choline. There was a turnover of the radioactive moiety of phosphatidyl choline at a rate that varied with the concentration of nonradioactive choline added to the growth medium. Radioactivity was lost from labeled phosphatidyl choline into the acid-soluble intracellular pool and from the pool into the extracellular medium. This loss of radioactivity from cells leveled off and an equilibrium was reached between the label in the cells and in the medium. Radioactive choline was incorporated into phosphatidyl choline by cell-free microsomal suspensions. This incorporation leveled off with the attainment of an equilibrium between the choline-¹⁴C in the reaction mixture and the choline-¹⁴C moiety of phosphatidyl choline in the microsomal membranes. Therefore, a choline exchange reaction may occur in cell-free membranes, as well as living A. palestinensis. In contrast to choline-¹⁴C, the apparent turnover of glycerol-³H-labeled phospholipids was not affected by large concentrations of nonradioactive choline or glycerol in the medium. The radioactivity in lipids labeled with glycerol-³H consisted of 33% neutral lipids and 67% phospholipids. Phospholipids labeled with glycerol-³H turned over slowly, with a concomitant increase in the percentage of label in neutral lipids, indicating a conversion of phospholipids to neutral lipids. Because most (~96%) of the glycerol-³H recovered from microsomal membranes was in phospholipids, whereas only a minor component (~2%) of the glycerol-³H was in the phospholipids isolated from nonmembrane lipids, glycerol-³H was judged to be a specific marker for membrane phospholipids.