The Permeability of Ammonia, Methylamine and Ethylamine in the Charophyte Chara corallina (C. australis)

Ritchie, R. J. 1987. The permeability of ammonia, methylamineand ethylamine in the charophyte Chara corallina (C. australis).—J.exp. Bot. 38: 67–76 The permeabilities of the amines, ammonia (NH₃), methylamine(CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the giant-celled charophyteChara corallina (C. australis) R.Br. have been measured andcompared. The permeabilities were corrected for uptake fluxesof the amine cations. Based on net uptake rates, the permeabilityof ammonia was 6?4?0?93 µm s^–1 (n = 38). The permeabilitiesof methylamine and ethylamine were measured in net and exchangeflux experiments. The permeabilities of methylamine were notsignificantly different in net and exchange experiments, norto that of ammonia (P_methylamine = 6?0?0?49 µm s^–1(n = 44)). In net flux experiments the apparent permeabilityof ethylamine was slightly greater than that of ammonia andmethylamine (P_{ethylamine, net} = 8?4?1?2 µm s^–1 (n= 40)) but the permeability of ethylamine based on exchangeflux data was significantly higher (P_{ethylamine, exchange} =14?1?2 µm s^–1 (n = 20)). Methylamine can be validlyused as an ammonium analogue in permeability studies in Chara. The plasmalemma of Chara has acid and alkaline bands; littlediffusion of uncharged amines would occur across the acid bands.The actual permeability of amines across the alkaline bandsis probably about twice the values quoted above on a whole cellbasis i.e. the permeability of ammonia across the permeablepart of the plasmalemma is probably about 12 µm s^–1. Key words: Chara, permeability, ammonia, methylamine     

Transport of Methylammonium and Ammonium Ions by Elodea densa

Excised leaves of Elodea densa rapidly absorb methylamine¹ fromdilute solutions (up to 2.0 mM). The influx isotherm is hyperbolic,with a K_? of approximately 160 µM. Influx is reduced followingtransfer of leaves from light to darkness, and at low temperature.Low concentrations of ammonia reduce the influx greatly, apparentlyby competition between NH⁺₄ and CH₃NH⁺₃, but K⁺ and Na⁺ havelittle effect, nor has removal of Cl^–. Influx is veryinsensitive to external pH over the range 5.0 to 9.0, with usuallya small increase between pH 9.0 and 10.0. When leaves are pretreatedin solutions containing nitrogenous compounds subsequent influxcan be decreased (by ammonia), unchanged (by methylamine) oreven increased (by arginine, proline and imidazol). Influx of methylamine and ammonia lowers influx of K⁺ (Rb⁺)and of Cl and increases efflux of K⁺ into solutions initiallyfree of K⁺. Fluxes of Ca⁺⁺ are not affected and there is netefflux of H⁺ into unbuffered solutions. The results show that uptake of methylamine and ammonia underthese conditions is primarily by transport (uniport) of CH3NHJand NHJ and that diffusion of CH₃NH⁺₃ and NH⁺₃ is insignificant.In Elodea, unlike some of the plants that have been previouslystudied, maintenance of charge-balance during transport of CH₃NH⁺₃and NH⁺₃ appears to involve accumulation of organic acid anions.     

Uptake of Methylamine by Ulva rigida: Transport of Cations and Diffusion of Free Base

Methylamine¹ is taken up rapidly by disks cut from fronds ofUlva rigida (‘Ulva lactuca’) and can be accumulatedat concentrations several hundred times greater than those inthe bathing medium. At pH 8.0 (the pH of sea-water) the relationshipbetween influx and concentration is normally linear up to 0.1–0.3mM, followed by a second, less steep linear phase, the slopeof which decreases or increases with decreasing or increasingexternal pH. When pH is greater than 9.0, however, net uptakesoon ceases. Methylamine influx is greatly reduced at low temperature,by low concentrations of ammonia and, depending on the lengthof storage of the material, by darkness. Influx is also greatlyreduced when disks are pretreated in solutions containing ammoniaand to a much lesser extent when they are pretreated in methylamine,imidazol or nitrate. Methylamine influx lowers intracellularK⁺, increases Cl^– and has no effect on Na⁺. We suggestthat the first linear phase of influx versus concentration reflectsthe operation of an amine cation porter that is rate-limitedby diffusion of CH₃NH₃⁺ through the external unstirred layer,and that the second phase is due to diffusion of CH₃NH₂ intothe tissue.     

Entry of Methylammonium and Ammonium Ions into Chara Internodal Cells

Low concentrations of ammonia and methylamine greatly affectmembrane transport by, and the electrical properties of, cellsof Chara corallina (=C. australis). In the presence of theseamines, influx of Cl^– and efflux of K⁺ increase and alarge depolarizing current flows through the cell membrane. Measurements with [¹⁴C]methylamine show that methylamine isabsorbed rapidly over a wide pH range, and that the absorptionisotherm is complex. Methylamine influx is not affected by presenceor absence of Cl^–, K⁺, or Na⁺, but is decreased by additionof . The depolarizing current is associated with a small increase in membrane conductance, except at highpH, and both these effects are reversible. The current showssaturation with increasing amine concentration; when methylamineis 10–12 times more concentrated than ammonia, it producesa current of the same magnitude. The results show that the amines enter the cells as cations( or CH₃) except where external pH is high, and that a specific, selective electrogenicporter is involved. There is no need to invoke active transport,as there is no evidence that internal amine concentrations exceedthe equilibrium (Nernst) concentrations.     

Comparison of the Effects of Ammonia and Methylamine on Chloride Transport and Intracellular pH in Chara corallina

Ammonium and methylammonium ions greatly increase the rate ofCl^– transport in Chara corallian. This effect is dependenton the pH of the bathing solution. The amine-stimulated Cl^–influx is small at pH 5·5, increases to a maximum atpH 6·5–7·5, and decreases again as the pHis raised to 8·5. Increased Cl^– influx is accompaniedby an increase in cytoplasmic pH, as calculated from the distributionof DMO. When the external pH lies between 5·5 and 7·3,cytoplasmic pH in the absence of amine is 7·65–7·70,with an increase of 0·15–0·25 in the presenceof amine. As external pH is increased above 7·3, cytoplasmicpH also increases, with progessively less effect of amine. Although the relationship between Cl^– influx and cytoplasmicpH is not simple, the results provide evidence in accord withthe hypothesis that Cl^– transport in Chara involves H⁺—Cl^–symport, or the equivalent OH^–—Cl^– antiport.The possible role of cytoplasmic pH as a factor involved inthe regulation of membrane transport in Chara is discussed.     

Uptake of Imidazole and its Effects on the Intracellular pH and Ionic Relations of Chara corallina

Ammonia (pK_a 9.25) and methylamine (pK_a, 10.65) increase cytoplasmicpH and stimulate Cl^– influx in Chara corallina, theseeffects being associated with influx of the amine cations ona specific porter. The weak base imidazole (pK_a 6.96) has similareffects but diffuses passively into the cell both as an unionizedbase and as a cation. When the external pH is greater than 6.0influx of the unionized species predominates. Imidazole accumulates to high concentrations in the vacuole,where it is protonated. Cytoplasmic pH and vacuolar pH riseby only 0.2–0.3 units, suggesting a large balancing protoninflux across the plasma membrane. Balance of electric chargeis partially maintained by net efflux of K⁺ and net influx ofCl^–. Calculation of vacuolar concentrations of imidazole(from (¹⁴C] imidazole uptake, assuming that there is no metabolism)plus K⁺ and Na⁺ indicates an excess of cations over inorganicanions (Cl^–). However, although the osmotic potentialof the cells increases, also indicating increased solute concentrations,the increase is less than that predicted by the calculated ionicconcentrations. This discrepancy remains to be resolved. Becausethe osmotic potential also increases when imidazole is absorbedfrom Cl^–-free solutions it is likely that maintenanceof charge-balance can also involve synthesis and vacuolar storageof organic or amino acids. Key words: Imidazole, potassium, intracellular pH, membrane transport, Chara     

Three distinct mechanisms of HCO3- secretion in rat distal colon

HCO₃^– secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO₃^– secretion have revealed evidence of lumen Cl^– dependence, suggesting a role for apical membrane Cl^–/HCO₃^– exchange, direct examination of HCO₃^– secretion in isolated crypt from rat distal colon did not identify Cl^–-dependent HCO₃^– secretion but did reveal cAMP-induced, Cl^–-independent HCO₃^– secretion. Studies were therefore initiated to determine the characteristics of HCO₃^– secretion in isolated colonic mucosa to identify HCO₃^– secretion in both surface and crypt cells. HCO₃^– secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO₃^– secretion (5.6 ± 0.03 µeq·h^–1·cm^–2) was abolished by removal of either lumen Cl^– or bath HCO₃^–; this Cl^–-dependent HCO₃^– secretion was also inhibited by 100 µM DIDS (0.5 ± 0.03 µeq·h^–1·cm^–2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl^– channel blocker. 8-Bromo-cAMP induced Cl^–-independent HCO₃^– secretion (and also inhibited Cl^–-dependent HCO₃^– secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl^–-independent, DIDS-insensitive, saturable HCO₃^– secretion that was not inhibited by NPPB. Three distinct HCO₃^– secretory mechanisms were identified: 1) Cl^–-dependent secretion associated with apical membrane Cl^–/HCO₃^– exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO₃^– exchange. chloride/bicarbonate exchange; short-chain fatty acid/bicarbonate exchange; anion channel; pH stat     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse     

Glucose 6-phosphate dehydrogenase from sweet potato: Effect of various ions and their ionic strength on enzyme activity

Activity of glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate:NADP oxidoreductase, EC 1.1.1.49 [EC] ) preparation from sweet potatoroot tissue was markedly altered in the presence of variousions. Cations or anions were effective in the following order:Na^$, K^$>Tris^$>NH₄^$>Mg^2$>Ca^2$, or Cl^–>NO₃^–,HPO₄^2–>SO₄^2–>HCO₃^–. Activity was inhibitedat high concentrations of Ca^2$, and HCO₃^–,. In an investigationon the dependence of the activity on pH, two activity peakswere clearly observed at low ionic strength. Ionic strength altered both the Km and Vmax for glucose 6-phosphate(G6P). A Lineweaver-Burk plot for the enzyme, with respect toG6P, showed a bimodal nature at low ionic strength; suggestingnegative cooperativity. Deviation from linearity of the plotwas less with an increase in the ionic strength. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, Tokyo 113. (Received September 18, 1971; )     

Chloride Transport in Chara: I. KINETICS AND CURRENT-VOLTAGE CURVES FOR A PROBABLE PROTON SYMPORT

Chara cells show an inward positive electric current acrossthe plasmalemma when exposed to Cl^– under voltage-clampconditions. The rapid rise of this current suggests that itis directly associated with the inward transport of Cl^–.The dependence of the current on Cl^– concentration showssaturation, the data fitting the Michaelis-Menten equation withV_m up to 100 nmol m^–2 s^–1 (for Cl^–starvedcells) with K_M 10–20 µM, and with some allowancefor an unstirred layer of water adjacent to the membrane. Theeffects on the current of clamp potential, illumination, withdrawalof alkali metal cations, and addition of amine were also investigated.These results suggest that the mechanism is the symport of 2H⁺ with each Cl^–, and that the actions of light, externalK⁺, and amine in stimulating Cl^–, influx are indirect.     

Plasmalemma Transport of HCO-3 and OH- in Chara corallina: Inhibitory Effect of Ammonium Sulphate

The influence of (NH₄)₂SO₄ on ¹⁴C assimilation and cyclosisin internodal cells of Chara corallina was investigated. Severeinhibition of ¹⁴C assimilation was found at pH values above7·0, this inhibition being correlated with the exogenouslevel of NH3 rather than NH⁺₄. Cyclosis was also affected athigher concentrations of (NH₄)₂SO₄. This effect was similarlycorrelated with exogenous levels of NH₃. ¹⁴C assimilation was inhibited non-competitively by (NH₄)₂SO₄,the apparent Km being increased from 0·55 to 1·5mM. The results suggest that the site(s) of inhibition is locatedat the plasmalemma, rather than at the chloroplasts. (Evidencein support of in vivo uncoupling of photophosphorylation, bylow concentrations of (NH₄)₂SO₄, was not obtained). Significant perturbation of the OH^– efflux pattern wasobserved as the level of (NH₄)₂SO₄ was increased. Induced migrationof efflux sites indicates that NH₃ may interfere with the cellularmechanism that controls OH^– transport. Using a cell-segmentisolating chamber it was shown that (NH₄)₂SO₄ inhibited OH^–efflux rather than HCO^–₃ transport. This inhibitory effectwas readily reversible. These data are discussed in terms of a possible relationshipbetween the observe NH₄)²SO₄ stimulation of ³⁶Cl^– influxand the effect of this compound on ¹⁴C assimilation.     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

Analogue Inhibition of the Active HCO-3 Transport Site in the Characean Plasma Membrane

Competitive inhibition of the HCO^–₃ transport site, atthe plasmalemma of Chara coraUina, by the CO^2–₃ ion isdemonstrated. This CO^2–₃ inhibition was used to demonstratethat HCO^–₃ ions enter the cell by facilitated ‘diffusion’when the HCO^–₃ transport system has been inactivated bytreatment with 10 mM K⁺. Use of CO^2–₃ as a HCO^–₃analogue is limited, however, because of the necessity to employsolutions of high pH. Inhibition was not observed in the presenceof a range of organic and inorganic acid anions. These resultsdemonstrate the stereo-specific nature of the HCO^–₃ bindingsite. A variety of amino compounds were found to inhibit H¹⁴CO^–₃influx. Inhibition appeared to be competitive, being completelyrelieved at higher substrate (HCO^–₃) concentrations. Asimple correlation was not found between the degree of inhibitionand the concentration of neutral base. A combination of thepresence of neutral base and experimental pH values of at least8·0 was required to produce the reactive species thatinhibited HCO^–₃ transport. This species is consideredto be the amino carbamate. These results are discussed withrespect to further HCO^–₃ analogue experiments.     

Influx and Efflux of Nitrate and Ammonium in Italian Ryegrass and White Clover Roots: Comparisons Between Effects of Darkness and Defoliation

Seedlings of Italian ryegrass (Lolium multiflorum Lam. cv. RVP)and clonal stolon cuttings of white clover (Trifolium repensL. cv. Blanca) were grown for 19 d in flowing solution culture,with N supplied as either 250 mmol m^–3 NO₃^– or NH₃⁺.Rates of net uptake, influx and translocation of NO₃^–and NH₄⁺ were then determined using ¹⁵N and ¹³N labelling techniques:between 3–5 h into the photoperiod following 8 h darknessfor white clover (CL), and for ryegrass plants that were eitherentire (IL) or with shoots excised 90 min prior to ¹³N influx(IC); and 75 min into the photoperiod following 37–39h darkness for ryegrass (ID). Rates of net uptake, influx andefflux of NH₄⁺ exceeded those of NO₃^– in IL and IC ryegrassplants: the opposite occurred in white clover (CL). The decreasein net uptake following defoliation of ryegrass was greaterfor NH₄⁺ (62%) than NO₃^– (40%). For NH₄⁺ this was associatedwith a large decrease in influx from 110 to 6.0µmol h^–1g^–1 root fr. wt; but for NO₃^–, influx only decreasedfrom 42 to 37 µmol h^–1 g^–1. Prolonged exposureto darkness (ID plants) also lowered net uptake of NO₃^–and NH₄⁺ by, respectively, 86% and 95% of IL levels. For NH₄⁺this was characterized by a large decrease in influx and a smalldecrease in efflux; whilst for NO₃^– the effect of a largedecrease in influx was reinforced by a smaller increase in efflux. The data were used to estimate the translocatory fluxes of NO₃^–(03–20µmol h^–1 g^–1) and NH₄⁺ (003–0.4µmolh^–1 g^–1), assimilation in the roots of NO₃^–(02–26µmol h^–1 g^–1) and NH⁺⁴ (05–89 µmolh^–1 g^–1), and the concentrations of NO₃^– (9–15mol m^–3) in the cytoplasmic compartment of the roots.The relevance of variable influx and efflux to models for theregulation of N uptake is discussed. Key words: Lolium multiflorum, Trifolium repens, influx, efflux, nitrate, ammonium, ¹³N     

The Mechanism of Photosynthetic Use of Bicarbonate by Hydrodictyon africanum

Hydrodictyon africanum can photosynthesize at high pH underconditions in which HCO₃^– rather than CO₂ is the carbonspecies entering the cell. A passive entry of HCO₃^– seemsunlikely; a metabolic HCO₃^– pump is proposed. It is possiblethat such a pump is related to a light-dependent reaction specificto the use of HCO₃^–. This reaction is dependent on photosystem2, but appears to be independent of ATP. These characteristicsare similar to those of active lightdependent Cl influx in H.africanum, and suggest a similar energy source for the two pumps.The HCO₃^– pump may be electrogenic.     

Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH

Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pH_i) and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl^–/Cl^– and Cl^–/HCO₃^– exchange are inhibited by acid pH_i or extracellular pH (pH_o). We have investigated the importance to pH sensitivity of the eight histidine (His) residues within the AE2 COOH-terminal transmembrane domain (TMD). Wild-type mouse AE2-mediated Cl^–/Cl^– exchange, measured as DIDS-sensitive ³⁶Cl^– efflux from Xenopus oocytes, was experimentally altered by varying pH_i at constant pH_o or varying pH_o. Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal ³⁶Cl^– efflux at pH_o 7.4 and acid shifted the pH_o vs. activity profile of wild-type AE2, suggesting that His residues might be involved in pH sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to Ala severely reduced transport and surface expression, other individual His mutants exhibited wild-type or near-wild-type levels of Cl^– transport activity with retention of pH_o sensitivity. In contrast to the effects of DEPC on wild-type AE2, pH_o sensitivity was significantly alkaline shifted for mutants H1144Y and H1145A and the triple mutants H846/H849/H1145A and H846/H849/H1160A. Although all functional mutants retained sensitivity to pH_i, pH_i sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pH_i and pH_o. pH regulation; histidine residues; Cl^–/HCO₃^– exchange     

Photosynthetic Carbon Sources of Stream Macrophytes

Rates of photosynthesis of four submerged stream macrophyteswere examined under varying pH and composition of inorganiccarbon species. Callitriche stagnatis and Sparganium simplexused only CO₂ for photosynthesis. Potamogeton crispus and P.pectinatus used HCO₃ in addition to CO₂, but with much lowerefficiency. The photosynthetic rates at air equilibrium anda total inorganic carbon concentration of 5.0 mM were 2–3times lower than maximum rates at CO2 saturation for the HCO₃users and 10–14 times lower for the CO₂ users. The CO₂compensation point of entire plants of Callitriche (2.5 µM)and Sparganium (6.0µM) was well below the equilibriumconcentration (15 µM). and the low saturation points (250–500µM) also pointed to efficient use of CO2. Callitricheand Sparganium compete successfully with HCO₃^– users inhardwater streams, which have a higher exchange and generationcapacity of CO2 than stagnant and more soft waters. Rates ofphotosynthesis of Potamogeton crispus and P. pectinatus decreasedat high pH. Depending on the two alternative hypotheses forHCO₃use, this decline can be explained by CO₃^––inhibition of HCO₃^– uptake or by increasing capacity tobuffer H+efflux from the plant. Habitats subject to high pH,e. g. small ponds with dense vegetation, may have a strong selectionfor efficient mechanisms of HCO₃ use. Key words: Photosynthesis, Macrophytes, Carbon-source     

Plasmalemma Transport of OH- in Chara corallina: II. FURTHER ANALYSIS OF THE DIFFUSION SYSTEM ASSOCIATED WITH- OH EFFLUX

The dynamic and steady state aspects of the pH and electricpotential () profiles, that develop in the experimental mediumin association with the photosynthetic assimilation of exogenousHCO^–₃ by internodal cells of Chara corallina, were investigated.A theoretical treatment is presented which explains the originof the phenomenon. This theory was tested by comparing thepH and values generated by a numerical analysis model (whichsimulated the experimental system) against experimental data.Verification of our model indicates that the steady state ionicfluxes, associated with HCO^–3 assimilation (HCO^–₃,OH^–, and CO^23–, are not significantly influencedby the electric potential gradients. The main driving forcecausing the observed fluxes is the diffusion gradient associatedwith the respective ion. By simultaneous measurement of and pH, at the centre of analkaline band, a direct correlation was established betweenlight-activation and dark-deactivation of the OH^– transportsystem and the light-mediated changes in at the cell surface.In addition, under steady state conditions, an almost perfectcorrelation was observed between alkaline band pH centres andthe negative electric potential maxima. These data offer strongsupport for the hypothesis that OH^–efflux, in this system,is an electrogenic process. Based on our present analysis, the profile along the cell indicatesthat, in terms of the spatial aspect of HCO3^– transport,the rate of HCO^–₃ influx varies quite dramatically alongthe length of an internodal cell. This aspect is discussed interms of the cellular integration of OH^– and HCO^–₃transport in this species.     

The regulation of ammonia uptake in Chara australis

The regulation of ammonia uptake was investigated in internodalcells of the freshwater alga Chara australis. Ammonia uptakewas estimated by monitoring (i) its depletion from the bathingsolution, (ii) the uptake of radiolabelled methylamine, an analogueof ammonia, and (iii) depletion of ammonia in the unstirredlayer with the microelectrode ion-flux estimation technique(MIFE). Distribution of methylamine (¹⁴CH₃NH₃⁺) between thevacuole and cytoplasm was estimated with efflux analysis. Whencells were bathed continuously in solutions containing ammoniaor methylamine, the uptake rates of both amines decreased over12 to 48 h despite the continuing existence of a large electrochemicalgradient favouring influx of the NH⁺₄ and CH₃NH⁺₄ cations. Treatmentwith 1.0 to 10.0 mM MSX, an inhibitor of glutamine synthetase,caused the internal ammonia concentration to rise and reducedthe subsequent uptake of ammonia and methylamine by up to 70%within 2 h. These results suggest that the permease facilitatingNH⁺₄/CH₃NH⁺₄ influx is under feedback or kinetic regulationfrom either internal ammonia or an intermediate of nitrogenassimilation. Treatment with metabolic inhibitors (CCCP, azide and DCMU) andsome weak acids (DMO and butyric acid) for 30 to 60 min inhibitedmethylamine uptake, but the changes in the electrical potentialdifference across the plasma membrane could not account forthe magnitude of inhibition. The rate of cytopiasmic streaming,which is an indicator of the cellular ATP concentration in Chara,was inhibited by many of these treatments. However, under certainconditions of external pH and concentration, butyric acid couldreversibly inhibit ammonia and methylamine uptake without affectingcytoplasmic streaming, demonstrating that a decrease in cytoplasmicATP concentration was not responsible for the inhibition. Theeffect of butyric acid was rapid, causing a 60% inhibition ofuptake in 15 min. We conclude that weak acids can inhibit theNH⁺₄/CH₃NH⁺₄ permease by acidifying the cytoplasm and suggestthat this may also explain the effects of the metabolic inhibitorson ammonia and methylamine uptake. Key words: Ammonia, methylamine, uptake, regulation, Chara     

Uptake of Methylammonium Ions by Hydrodictyon africanum

Methylamine influx into Hydrodictyon has been measured with[^l4C]methylamine. The influx increases with rising externalpH up to about pH 8. Between pH 8 and 9 influx remains quiteconstant, with a further increase above pH 9. Influx is light-dependent,temperature-sensitive, and is decreased by . During short-term influx (less than 4 h) metabolism of methylamineappears negligible. Prolonged influx results in CH₃ accumulation, and efflux of K⁺, Na⁺, and H⁺. There is no effecton Cl^– influx. Methylamine decreases the membrane electricp.d. by 60–120 mV at external concentrations of 0?2–1?0mM. The results indicate that, below pH 9, methylamine enters thecell almost entirely as CH₃. It is suggested that a passive electrogenic uniporter is involved, and thatby analogy uniport of may also be expected in Hydrodictyon. The results are discussed in relation to theevidence for uptake of CH₃ and by other plant cells.