Osmotic and Ionic Regulation of North American Zebra Mussels (Dreissena polymorpha)

SYNOPSIS. Unlike other freshwater bivalves that survive formonths in deionized water, Dreissena polymorpha requires minimalconcentrations of Na, K, Mg, and Cl in the bathing medium forlong-term survival. Although ion transport rates are higherin D. polymorpha compared to other freshwater bivalves, theytend to have lower blood solute concentrations. D. polymorphahas an unusually "leaky" epithelium with a high paracellularpermeability to solutes. Thus, even with high transport rates,it may not be possible for zebra mussels to retain higher bloodsolutes because of the extensive passive loss of ions. Undera hyperosmotic stress, D. polymorpha will rapidly osmoconform(about 12 hr) due primarily to the diffusion of solutes andpartially to the osmotic loss of water. D. polymorpha is notcapable of surviving an imbalance of Na/K in the external medium.In the absence of K the cells will tend to lose volume to achieveisosmotic balance with the blood, but the animals usually diewithin a few days. If D. polymorpha is exposed to excess K inthe environment (1 mM), they will accumulate K in the blood.If the K enters the cells, cellular volume would expand dueto increase in osmolyte concentration, yet, if K remains inthe blood, there will be an electrochemical imbalance. In eithercase, the animal cannot survive much longer than a day. WhenNa and K are present in the medium in a balanced combinationapproximated by artificial seawater (ASW), D. polymorpha willsurvive an acute transfer to 100 mosm ASW indefinitely (months).Our preliminary studies have shown that D. polymorpha will toleratestep-wise acclimation to solutions >250 mosm provided thechanges in salinity do not exceed 50–100 mosm. Freshwaterbivalves, unlike the marine bivalves, have limited free aminoacids in their body fluids and must rely on inorganic ions forosmotic regulation. The free amino acids serve as an importantosmolyte buffer for volume regulation when an animal experiencesan environment of changing salinity. The inability of Dreissena,and perhaps other freshwater bivalves, to tolerate hyperosmoticallyinduced dehydration may be due, in part, to the inability toaccumulate or retain sufficient intracellular K to facilitateregulatory volume adjustments.     

Ammonia Uptake in the Alkalophilic Cyanobacterium Spirulina platensis

Ammonia uptake was studied in the alkalophilic cyanobacteriumSpirulina platensis. In continuous cultures under optimal growthconditions ammonia supported optimal growth (doubling time of9.3 h), causing a reduction of glutamine synthetase activityto 25% of that found in cultures grown on NO₃. Long term (20min) ammonia uptake assays were performed to study the dependencyon metabolism: 1) Ammonia uptake proceeded at the same ratesin the light and in the dark, the pH dependency pattern correlatingwith light-dependent O₂ evolution and dark O₂ consumption. 2)The uptake of ammonia was pH dependent with an optimum at pH9.3. 3) The uptake was totally dependent upon the activity ofglutamine synthetase and was completely inhibited by methoininesulfoximine. To study the mechanism by which NH₄⁺/NH₃ enters the cells, shortterm experiments (up to 1 min) were performed at pH 7.0 andpH 10.0: At pH 7.0 the uptake was slow and at a constant rate.At pH 10.0, the uptake did not saturate even at 1 mM ammoniaand the kinetics were biphasic, consisting of a fast componentlasting less than 5 seconds and of a subsequent slower component.The fast phase was insensitive to methionine sulfoximine, whereasthe slower phase was completely inhibited by this compound.We suggest that under optimal (alkaline) pH the entry of ammoniainto Spirulina cells is likely to be a pH driven diffusion process,continuously supported by its intracellular assimilation. ¹Contribution number 35 of the Microalgal Biotechnology Laboratory. (Received September 19, 1988; Accepted January 16, 1989)     

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     

Ammonia Induces Starch Degradation in Chlorella Cells

When ammonia was added to cells of Chlorella which had fixed¹⁴CO₂ photo synthetically, ¹⁴C which had been incorporated intostarch was greatly decreased. A similar effect was observedwhen potassium nitrate and sodium nitrite were added. The ammonia-induceddecrease in ¹⁴C-starch was observed in all species of Chlorellatested. With cells of C. vulgaris 11h, most of the radioactivityin starch was recovered in sucrose, indicating that ammoniainduces the conversion of starch into sucrose. The percent of¹⁴C recovered in sucrose differed from species to species andpractically no recovery in sucrose was observed in C. pyrenoidosa.In most species tested, the enhancing effects of blue lightand ammonia on O₂ uptake as well as the ammonia effect on starchdegradation were greater in cells which had been starved inphosphate medium in the dark than in non-starved cells. In contrast,the enhancing effect of ammonia on dark CO₂ fixation was muchgreater in non-starved cells. C. pyrenoidosa was unique in thatblue light did not show any effect on its O₂ uptake. (Received August 15, 1984; Accepted November 16, 1984)     

THE KINETICS OF PENETRATION : XII. HYDROGEN SULFIDE

The rate of entrance of H₂S into cells of Valonia macrophysa has been studied and it has been shown that at any given time up to 5 minutes the rate of entrance of total sulfide (H₂S + S^-) into the sap is proportional to the concentration of molecular H₂S in the external solution. This is in marked contrast with the entrance of ammonia, where Osterhout has shown that the rate of entrance of total ammonia (NH₃ + NR₄ ⁺) does not increase in a linear way with the increase in the external concentration of NH₃, but falls off. The strong base guanidine also acts thus. It has been shown that the rate of entrance of H₂S is best explained by assuming that it enters by diffusion of molecular H₂S through the non-aqueous protoplasmic surface. It has been pointed out that the simple diffusion requires that the rate of entrance might be expected to be monomolecular. Possible causes of the failure of H₂S to follow this relationship have been discussed.     

THE ACCUMULATION OF ELECTROLYTES : I. THE ENTRANCE OF AMMONIA INTO VALONIA MACROPHYSA

When 0.005 M NH₄Cl is added to sea water containing cells of Valonia macrophysa ammonia soon appears in the sap and may reach a concentration inside over 40 times as great as outside. It appears to enter as undissociated NH₃ (or NH₄OH) and tends to reach a pseudoequilibrium in which the activity of undissociated NH₃ (or NH₄OH) is the same inside and outside. When ammonia first enters, the pH value of the sap rapidly rises but it soon reaches a maximum and subsequently falls off. At the same time there is an increase of halide in the sap which, however, does not run a parallel course to the ammonia accumulation, but it comes to a new equilibrium value and remains constant. The increase in NH₃ in the sap is accompanied by a decrease in the concentration of K. As NH₃ enters the specific gravity of the sap decreases and the cells rise to the surface and continue to grow as floating organisms. The growth of the cells is increased.     

Metabolism of urea in Chlorella ellipsoidea

The effect of cyanide on ammonia and urea metabolism was studiedwith intact cells of Chlorella ellipsoidea Gerneck, a greenalga which apparently lacks urease. Ammonia uptake was inhibited more readily by cyanide than wasurea uptake. Urea uptake was stimulated by lower concentrationsof cyanide. The addition of cyanide caused the formation ofammonia from some cellular nitrogenous compounds. In the presenceof exogenously added urea, the molar ratio of ammonia accumulatedin the medium to urea taken up exceeded 2.0 as the cyanide concentrationincreased. However, the molar ratio of ammonia actually producedfrom urea nitrogen to urea taken up was less than 1.35 at anyconcentration of cyanide tested. In the presence of higher concentrationsof cyanide, the rate of incorporation of ¹⁵N into amino acidsfrom ¹⁵N-urea was higher than that from ¹⁵N-ammonium sulfate. The results suggest that Chlorella ellipsoidea possesses a pathwaythrough which urea nitrogen is assimilated directly withouta preliminary breakdown to ammonia. (Received October 18, 1976; )     

The Membrane Potential of Nitella translucens

The effects of changing the external concentrations of Na, K,Ca, and Cl on the potentials of the cytoplasm and the vacuolewith respect to the bathing medium of the internodal cells ofNitella translucens have been investigated. The potential differencebetween the vacuole and the cytoplasm is practically unaffectedby the concentration changes. The observed changes of potentialdifference are therefore attributed to the boundary separatingthe cytoplasm from the medium; this boundary is possibly a plasmalemma–cellwall complex. The difference of potential between the cell walland the medium has also been measured and, in the presence ofCa, shown to be markedly sensitive only to the external Ca concentration.The results are divided into two sections: (a) for cells pretreatedin 5 mM NaCl, the subsequent experiments being carried out inCa-free media, and (b) for cells initially immersed in a standardartificial pond water containing the chlorides of Na, K, Ca.With the pretreated cells the external Na/K ratio was variedwith the total NaCl+KCl concentration kept constant at 1.1 mM.The results suggest that over a limited range of concentrationsthe cytoplasm-medium potential difference can be described byan equation similar in form to a Goldman equation but containingonly terms for Na and K, the average value of the permeabilityratio (= P_Na/P_K) being 0.27. In the presence of Ca the effectsof Na and K on the cytoplasm-medium potential difference aregreatly reduced, while the effect of Ca is relatively large.The results cannot be fitted to any form of Goldman equationcontaining terms for the major ions. The possibility of a contributionto the plasmalemma potential from electrogenic pumps is brieflydiscussed. Measurements of the Na and K content of the cytoplasmand the vacuole have been made for the pretreated cells. TheNa concentration in the cytoplasm is 37 mM and in the vacuole73 mM; the K concentration is 93 mM in the cytoplasm and 67mM in the vacuole. The Nernst potentials for both ions are comparedwith the cytoplasm-medium and cytoplasm-vacuole potential differences.This analysis shows that Na is actively transported from thecytoplasm into the medium as well as into the Vacuole; K ispumped into the cytoplasm from the medium but appears to beclose to electrochemical equilibrium across the tonoplast. ThisConfirms previously published work.     

THE PENETRATION OF CO2 INTO LIVING PROTOPLASM

The experiments indicate that little or no CO₂ enters normal cells of Valonia except in the form of undissociated molecules. Whenever the interior of a cell is more acid than the surrounding medium (excess base being the same in both) we may expect that at equilibrium the internal concentration of total CO₂ will be less than the external.     

The Assimilation of Nitrogen from Ammonium Salts and Nitrate by Fungi

1. The assimilation of inorganic nitrogen by Scopulariopsis brevicaulisand some physiologically similar species has been studied. Theirfailure to assimilate completely from ammonium sulphate hasbeen shown to be due to the fall in pH of the medium inducedby the initial uptake of ammonia.
2. Complete assimilation ofammonia takes place in the presenceof the neutral salts ofeach of thirteen organic acids investigated.The organic acidsact primarily through their buffering effectwhich preventsor slows down the fall in pH. They are not specificallyrequiredfor ammonia assimilation by these fungi and can beeffectivelyreplaced by certain inorganic buffers.
3. The influence of severalexternal factors on the rate of assimilationof ammonia, nitrate,and nitrite has been studied in S. brevicaulis.In correspondingconditions the mycelium assimilates ammoniamore rapidly thannitrate over a wide range of conditions.
4. Ammonia, even invery low concentration, completely suppressesnitrate assimilationwhen both sources of nitrogen are presenttogether. Nitrite,however, is assimilated simultaneously withammonia. It is thereforeconcluded that ammonia blocks the reductionof nitrate to nitriteby the fungus.
5. The suppression of nitrate assimilation inthe presence of ammoniais common to many mould fungi besidesS. brevicaulis, and isbelieved to have adaptive significancein natural habitats.
6. The nitrate-reducing and assimilatingsystem is formed, evenwhen S. brevicaulis is grown in completeabsence of nitrate(ammonia medium with organic acid). It comesinto action rapidlywhen the inhibiting effect of ammonia isremoved. Similarly,nitrate-grown mycelium is capable of assimilatingammonia atmaximal rate without any adaptive lag.

     

Metabolic Effects on Ion Fluxes in Tolypella intricata

The uptake of C1 ions by cells of Tolypella intricata is greatlyincreased by light, and must be an active process. K ions inthe cells (at 90–110 mM concentration) are in approximateelectrochemical equilibrium with the external solution, butthe K influx is affected (directly or indirectly) by cellularmetabolism. The K influx is increased by light, and the increaseis greater in the presence of C1 than when C1 is removed fromthe solution. K uptake is inhibited by chemicals which alsoinhibit the C1 pump.It is suggested that light increases thepermeability of the plasmalemma to K, but there must also belinks between K and C1 uptake. The possible nature of theselinks is discussed.The internal Na concentration (3–10mM) is considerably below the expected equilibrium concentration,but the Na influx is also very low (and is not increased bylight). The permeability of the plasmalemma to Na is thus verylow, and there can be little active extrusion of Na under normalconditions.     

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.     

The Assimilation of Ammonia by Nitrogen-starved Cells of Chlorella vulgaris: Part I. The Correlation of Assimilation with Respiration

The addition of ammonium sulphate to a suspension of nitrogen-starvedChlorella cells is followed immediately by the rapid assimilationof ammonia and a large increase of the respiration rate. Theassimilation of ammonia and the high rate of respiration continueuntil either all the ammonia has been assimilated or some carbonreserve within the cells has been exhausted. Which happens firstdepends on the amount of ammonia added and the quantity of cellspresent. The respiration which accompanies, ammonia assimilationis sensitive to cyanide and it has a respiratory quotient of0•75 compared with 1•2–1•3 for normal reapiration.The addition of glucose to nitrogen-starved cells when ammoniais being assimilated does not increase either the rate of respirationor the rate of assimilation. The rates of reapiration and ammoniaassimilation by normal cells are markedly increased by the additionof glucose. Light has little effect on the rate of ammonia assimilationby nitrogen-starved cells, but doubles the assimilation rateof normal cells.     

The Effect of Molybdenum upon the Nitrogen Metabolism of Anabaena cylindrica: II. A More Detailed Study of the Action of Molybdenum in Nitrate Assimilation

Molybdenum-deficiency in Anabaena cylindrica results in a decreaseof all the organic nitrogen fractions determined analyticallywith the exception of amide which occurs at approximately thesame concentration irrespective of the molybdenum status. Molybdenum-rich,nitrogen-starved cells assimilate nitrate to protein rapidlyin the dark; similarly treated molybdenum-deficient cells rapidlyreduce nitrate to ammonia and amide, but the further synthesisto peptides and proteins proceeds very slowly. The rate of endogenousrespiration is higher in deficient than in normal cells, butthe rate of glycolysis is lower. No period of adaptation isrequired, after the addition of molybdenum to deficient cells,before normal nitrogen metabolism commences. Glucose, acetate,fumarate, succinate, and citrate are respired by normal anddeficient cells in the dark. Acetate has no effect on nitratereduction, glucose stimulates the reduction for a short period,while fumarate (and probably succinate) and, somewhat less efficiently,citrate, stimulate reduction in both normal and deficient cellsand enable the deficient cells to assimilate nitrate completelyto protein. It would seem that, in the absence of molybdenum,there is an insufficient supply of hydrogen and/or energy donorsto permit complete nitrate assimilation.     

Ionic Currents across the Plasmalemma of Chara inflata Cells: II. EFFECTS OF EXTERNAL Na+, Ca2+ AND Cl- ON K+ AND Cl CURRENTS

The electrical conductance of the plasmalemma of cells of Charainflata, due to the diffusion of ions, consists predominantlyof K⁺, Cl^– and leak components. When the membrane electricalpotential difference is stepped in a negative direction witha voltage-clamp, the resulting inward current has componentsI_K, I_Cl and I_L (leak). During such voltage-clamp steps I_K isinactivated, and Ic activated with voltage-dependent half-times.Increases in the external NaCl concentration reduce the magnitudeof I_K and increase the magnitude of I_c, but reduce the half-timeof inactivation or activation. The NaCl-induced changes in I_kand I_Cl and their kinetics were more pronounced at pH₀ =6.5than at pH₀ =9.5. When the concentration of external CaCl₂ wasincreased, I_k, I_Cl and the half-time of inactivation, (T_1/2),of I_k were all reduced. The half-time of activation of I_Cl wasincreased. The NaCI-induced changes could result from increases in bothexternal ion concentration and osmotic pressure. Previous experimentshave shown that an increase in external osmotic pressure alonealters the properties of the conductances. In this paper weattempt to separate the purely ionic effects from the osmoticones. Key words: Chara inflata, ionic effects, K⁺ and Cl^– currents     

The Interrelation between the Effects of 2,4-Dinitrophenol and of Substrate Concentration on the Rate of Exogenous Respiration

1. The specific activity of the CO₂ evolved by sunflower hypocotylsegments supplied with labelled glucose or glutamic acid isrelated to the external concentration by a rectangular hyperbolaover a concentration range of approx. 2–60 mM. The overallrespiration rate over this range is approximately 15 per cent.above that of a buffer control, and rises only slightly withincreasing external concentration. 2. The curve for total exogenous substrate respired is alsoclose to a rectangular hyperbola over this concentration range. 3. 2,4-dinitrophenol (DNP) reduces the slope of the hyperbolafor specific activity, but not its maximum value. Increasingthe glucose concentration while maintaining the DNP concentrationconstant reduces the degree of inhibition. 4. The effect of DNP on absolute amount of exogenous substraterespired depends on the external concentration of the substrate.At lower concentrations, absolute exogenous respiration is inhibited:at higher concentrations, it is stimulated. 5. The results have been analysed with the object of determiningwhether the respiratory sites lie in the ‘sugar free space’of the cell. Kinetic analysis indicates (a) that exogenous substratesdo not diffuse to these sites through an appreciable diffusionbarrier; (b) that there is no evidence for an active transportstep; and (c) that the data accord well with the view that exogenoussubstrates diffuse freely to the respiratory sites where theycompete on equal terms with endogenous substrates.     

Effect of ammonia on nitrogen fixation by the blue-green alga Anabaena cylindrica

Ammonia at a concentration of 1 ? 10^–3M completely inhibitednitrogenase activity, as measured by acetylene reduction, inthe blue-green alga Anabaena cylindrica. Free ammonia was undetectablein cells grown either on N₂ or ammonia within the limits ofprecision of the method used. Glutamic acid formed a major aminoacid pool in N₂-grown cells, and basic amino acids, i.e. lysine,histidine and arginine were abundant in ammonia-grown cells.A 10-fold increase in the amounts of labile amino compound(s)was observed when N₂-grown cells were exposed to ammonia. When cells were incubated under anaerobic conditions, the acetylene-reducingactivity increased 2-fold or more; ammonia had no effect. Oxygenwas required for ammonia to inhibit acetylene reduction. Modes of inhibition by ammonia on acetylene reduction were comparedwith those by chloramphenicol, puromycin, cycloheximide, DCMUand CCCP. On the basis of these comparisons we concluded thatammonia not only acts as a suppressor of nitrogenase synthesisbut also inhibits acetylene-reducing activity by lowering thesupply of reductant and/or of energy for the nitrogenase system. ¹This work was supported by grant No. 38814 from the Ministryof Education. (Received July 30, 1973; )     

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.     

TRANSPORT OF UNCONTAMINATED AND MOLLUSCICIDE-CONTAINING FOOD IN THE DIGESTIVE TRACT OF THE SLUG DEROCERAS RETICULATUM (MULLER)

By X-ray analysis of food transport in the alimentary tractof Deroceras reticulatum it was shown that even ten hours afteringestion of a thorium sulfate-containing bait, this materialcan be detected in the crop. After 2.5 h, some parts of labelledfood passes down from the anterior to the posterior part ofthe gut. After 13 h, thorium sulfate-containing material canbe observed only in the gut. After 19 h, no more labelled materialis present in the alimentary tract of the animals. After addition of Cloethocarb, the animals feed on only smallamounts of food. The labelled material only enters the anteriorpart of the gut. After ten hours, the food does not move anymore and does not leave the crop even 19 h after feeding. After molluscicide application, the crop epithelium is moreinfolded than in control animals and the cells are elongated.After 30 h, cells protrude into the lumen of the digestive tract. (Received 11 May 1992; accepted 26 June 1992)     

Differential Effects of Methionine Sulfoximine on Light and Dark Uptake of Nitrate by Anabaena cylindrica

Methionine sulfoximine (MSX), a glutamine synthetase inhibitor,suppressed the inhibitory effect of ammonia on nitrate uptakeby Anabaena cells in both the light and dark. In the light,MSX did not inhibit nitrate uptake in the absence of ammonia,but under dark conditions, MSX above 2 µM inhibited nitrateuptake. Nitrite uptake, which is not affected by ammonia ineither the light or the dark, was inhibited by MSX in the darkbut not in the light. (Received October 3, 1984; Accepted April 22, 1985)