Artificial Pond Water Research Articles

Effect of light upon membrane potential, conductance, and ion fluxes inRiccia fluitans

The membrane potentialEm, slope conductancegm, and fluxes of86Rb+/K+ and36Cl− have been measured on cells of the aquatic liverwortRiccia fluitans in artificial pond water of pH 4 to 8 and 0.1 to 10mm K0+, in the dark and 1 mW/cm2 of white light. In the darkEm reflects a passive diffusion potential according to the Goldman equation with relative ionic permeabilities,PH/PK/PNa=10∶1∶0.02. In the light,Em of −200 to −240 mV exceeds the most negative ion diffusion potential, i.e.EK, by more than 100mV, is more sensitive to H0+ than K0+, and is reduced to the dark level by uncouplers of phosphorylation. At 1mm K0+,gm is 33 and 50 μS/cm2 in the light and dark, respectively.gm is sensitive to H0+ only in the light, and more sensitive toK0+ in the dark. Current-voltage relationships are given. Light increases the influx at the plasmalemma of86Rb+/K+ and36Cl− less than would be expected from the increase ofEm. It is concluded that the electrogenic pump operates in the dark as a constant current source which is shunted by the diffusive channels whereas in the lightEm approaches the H+-dependent electromotive force of the electrogenic pump.

The electrical potential difference and resistance of Chara vulgaris

A study has been made of the electrical properties of the large central internodal cell of Chara vulgaris. The mean electrical potential difference (± standard deviation) measured in the light between the vacuole and the external, artificial pond water was −165 (±10) mV. This was much larger than the Nernst potentials for potassium, sodium, magnesium, and calcium and it was concluded that (a) if these ions were in flux equilibrium, then all of them were actively pumped out of the cell; and (b) no diffusion potential based on these ions alone could account for the large, electrical potential difference. The combined electrical resistance of the tonoplast and plasmalemma in the light was about 20 kΩ cm2. In the dark the electrical potential fell to a value which could have resulted from the diffusion of the above-mentioned cations while the resistance tripled in size. These observations are discussed in terms of Spanswick's model (Spanswick, R. M. 1974. Can. J. Bot. 52: 1029–1034).

Ionic regulation in the freshwater mussel,Ligumia subrostrata (Say)

The freshwater mussel,Ligumia subrostrata, maintains an ionic and osmotic steady state in an artificial pond water. Blood osmolality is 50 mOsm/l with Na, Ca, HCO3 and Cl accounting for 90% of the total solute. Steady state unidirectional influx of Na and Cl from pond water are 1.1±0.2 and 1.5±0.4 μEq/g dry tissue·hr, respectively. The transepithelial potential,in vivo, is −15±5 mV in pond water but both Na and Cl are actively transported, independently, by non0electrogenic mechanisms. Exposure of the mussels to distilled water results in a loss of blood Na and Cl and stimulates influx 2–5 times control levels when returned to pond water.

Cytoplasmic streaming and ionic regulation inNitella flexilis having artificial cell saps of low ionic strength

The cell sap of the internode ofNitella flexilis was replaced with the isotonic artificial pond water of high Ca2+-concentration (0.1 mM KCl, 0.1 mM NaCl, 10 mM CaCl2 and 275 mM mannitol) and changes in osmotic value and concentrations of K+, Na+ and Cl− of the cells were followed. When the operated cells were incubated in the artificial pond water containing 0.1 mM each of KCl, NaCl, CaCl2, they survived for only a short period of time (<10 hr). The cells did not absorb ions from the artificial pond water and showed a conspicuous decrease in the rate of cytoplasmic streaming. In such cell the concentration of K+ in the protoplasm decreased significantly. In order to reverse normal concentration gradients of K+ and Na+ across the protoplasmic layer, the cells of low vacuolar ionic concentrations were incubated in the artificial cell sap (90 mM KCl, 40 mM NaCl, 15 mM CaCl2, 10 mM MgCl2). It was found that the cells rapidly absorbed much K+, Na+ and Cl− and survived for a longer period (1–2 days). During this period the rate of cytoplasmic streaming was nearly normal. Furthermore, the cell lost much mannitol, indicating an enormous increase in permeability to it. Since both absorption of ions and leakage of mannitol at 1 C occurred at nearly the same rates as at 22 C, the processes are assumed to be passive.

Electrical responses of isolated protoplasm from Nitella.

Isolated protoplasmic droplets of the alga Nitella were investigated with microelectrodes in artificial vacuolar sap. The following observations were made: 1. Two types of preparations could be distinguished differing in size and in time of adaptation to artificial pond water but only slightly in their electrical behaviour. 2. The droplets proved to be electrically excitable in the sense that short current pulses elicited electrical responses which outlasted the stimuli. 3. The responses resembled nerve action potentials in shape and duration but they were graded and could be elicited as well in positive as in negative direction. Moreover, spontaneous changes of the normal resting potential (a few millivolts inside negative) did not influence their amplitudes. 4. In most cases the amplitudes of the responses grew with time and saturated after about 90 min. Before saturation the relation between stimulus strength and amplitude of responses was almost linear but became slightly S-shaped after saturation. The saturation value of the responses caused by 100 mus pulses of 1 muA/mm2 was taken as a standard response. In 32 experiments the standard response varied considerable between 2 and 90 mV and was 13 mV on the average. The observations suggest that quite different mechanisms are responsible for the transients of the Nitella droplets and the all-or-none responses of nerve fibres.

Active chloride transport across the skin of the earthworm, Lumbricus terrestris L

1. 1. Earthworms acclimated to an artificial pond water actively transport Cl across the integument. The Cl transport system displays saturation kinetics: V max = 1.2 μ-equiv/10 g-hr, K s = 0.3 mM/1. 2. 2. Salt depletion stimulates Cl uptake across the skin. Influx of Cl is elevated with no change in efflux. 3. 3. Net uptake of Cl occurs from KC1 or choline chloride independent of the cation. 4. 4. Chloride influx is abolished by injection of acetazolamide (12 μg/g body weight) suggesting a C1/HCO 3 exchange mechanism. Sodium influx is not affected by acetazolamide.

Dielectric measurements of Nitellopsis obtusa cells with intracellular electrodes.

The paper deals with the measurement of the passive electrical properties ofNitellopsis obtusa cells by means of intracellular electrodes. TheNitellopsis cell is placed into a special sample holder and surrounded by isotonic solution of artificial pond water and saccharose. A platinum wire is inserted into the vacuole without destroying the membranes or disturbing the cytoplasmic motion. The electrode arrangement consists of a coaxial cylinder condenser with guard-rings. The guard-rings guarantees a well-defined and strictly cylinder-symmetric field with a direction of current lines independent of frequency. A special three-terminal bridge with inductively coupled ratio arms was constructed to measure between 20 Hz and 500 kHz the conductance and the capacitance of the sample with an accuracy better than 1 % without falsification by additional guard-ring currents. The measured values are shown as circular arcs with slightly depressed centres in the complex conductance plane as well as in the complex capacitance plane. The results were analysed using the theory of heterogeneous dielectrics. In accordance with the structure of the cell it is shown that a model with frequency-independent electrical properties is sufficient to explain the data.

A transcellular water flux induced by light in Nitella

When Nitella cells in artificial pond water (APW) are illuminated in an electroosmometer, water appears to move into the closed chamber of the instrument. This has been termed the "basic flow" of the instrument. A part of this basic flow is thermal expansion but an appreciable part is a water flow through the living cell. This transcellular water flow is increased by illumination of any part of the cell and is mostly removed by 10−3 mM N-dichlorophenyl-N′,N′-dimethylurea (DCMU). It is readily altered by conditioners which alter the membrane properties at one end of the cell compared with the other: carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) at 5 × 10−3 mM, dinitrophenol (DNP), 10−1 mM ouabain, and particularly pH. Asymmetrical changes in the hydraulic permeability of the membrane are indicated as the most probable cause of this water flow.An electric current flows between the two ends of the cell when the cell is differentially illuminated, particularly at the beginning or end of the light change. The current is thought to indicate the presence of some electrogenic pumps on the plasma membranes.

The effect of some growth-regulating compounds upon electroosmotic measurements, transcellular water flow, and Na, K, and Cl influx in Nitella flexilis

By using a small, constant, applied current on a cell of Nitella in artificial pond water, it was possible to compare the effect of different growth-regulating compounds on the apparent electroosmotic water coupling in the cell wall – membrane complex. Indoleacetic acid (IAA) at first enhanced the electro-osmotic effect and then ceased to alter it, but began to change the asymmetric transcellular water flow. Gibberellic acid (GA), when added to IAA, increased this effect, while abscissic acid (ABA) added to IAA decreased it. Some compounds like naphthaleneacetic acid (NAA), alar-83, skatol, or ABA alone, had no effect on the electroosmotic (eo.) coefficient; but NAA, skatol, ABA (alone) changed the asymmetric water flow (basic flow) and some ion influxes. Light-degraded IAA acted in a manner very similar to skatol, which suggests that IAA is quickly converted to skatol in the cell in the presence of light. With light–dark transitions or with IAA, the electroosmotic coefficient changed in a manner parallel to passive ion (Na+ and K+) influx, but was not related to basic flow or Cl− influx changes.Under controlled conditions of low constant current applied to a test cell, it has been shown that a change in apparent eo. coefficient upon auxin addition is largely a reflection of change in the true electroosmotic properties of the cell membrane at the positive end of the cell.

The influence of small applied electrical currents on Na, K, and Cl fluxes in Nitella translucens

The changes of influx and efflux of Na, K, and Cl were studied in Nitella in artificial pond water (APW) under varying current densities below 1.3 μamp cm−2. The electric current seemed to act only on the passive ion influx and K efflux. Cl influx was insensitive to applied current. Na influx normally carried about 1/5 of the current, K much less unless the K concentration of the APW was increased. The relative percentage of each ion carrying the current at the positive end of the cell was readily altered by changes in K concentration or pH. It is presumed that H ion is also involved. In our experiments, low current densities below 0.7 μamp cm−2 gave no detectable effect on K, Na, or Cl effluxes at either end of the cell. Thus in electroosmotic experiments, Na influx at the positive end of the cell is an important flux, while K efflux is important at the negative end.Microinjection of tracer into the vacuole of Nitella is a feasible technique. Once isotopes have penetrated the cell membrane, they seem to move to some extent independently of each other.

Changes of Ionic and Electrical Properties of Charaaustraliswhen the External Ca is replaced by Mg

The calcium in the normal artificial pond water (A.P.W.) for Chara australis was replaced by magnesium and the resulting changes in resting potential, action potential, and ion permeabilities were measured. The effect of lowering the pH of the A.P.W. from 6.5 to 4.5 in the presence of either Ca or Mg was also observed. It was concluded that ionic permeability changes induced by varying the pH are Ca or Mg mediated and the ionic permeability changes occuring during an action potential can be mediated by Ca but not by Mg. © 1970 Oxford University Press.

The Effects of pH on the Ionic and Electrical Properties of the Internodal Cells ofChara australis

The effects of pH on the resting and action potentials and on the fluxes of potassium, sodium, and chloride across the membranes of internodal cells of Chara australis have been investigated. Experiments were carried out in an artificial pond water (A.P.W.) of standard composition: CaCl2, 01 mM; KCl, 0.1 mM; NaCl, 1.0 mM. The resting potential decreased as the pH was lowered from 6.5, being depolarized by about 75 mV at pH 4.5. Measurements of the ion fluxes as a function of pH suggested that this depolarization was caused by an increase in the permeability to sodium and a decrease in permeability to potassium at pH 4.5. Action potentials of constant peak value can be elicited for some time at pH 4.5, but after 20 min or so the cell becomes refractory. All these effects on resting and action potentials are fully reversible. We briefly speculate about the mechanism of these pH effects.

The influence of H+ on the membrane potential and ion fluxes of Nitella.

The resting membrane potential of the Nitella cell is relatively insensitive to [K](o), but behaves like a hydrogen electrode. K(+) and Cl(-) effluxes from the cell were measured continuously, while the membrane potential was changed either by means of a negative feedback circuit or by external pH changes. The experiments indicate that P(K) and P(Cl) are independent of pH but are a function of membrane potential. Slope ion conductances, G(K), G(Cl), and G(Na) were calculated from efflux measurements, and their sum was found to be negligible compared to membrane conductance. The possibility that a boundary potential change might be responsible for the membrane potential change was considered but was ruled out by the fact that the peak of the action potential remained at a constant level regardless of pH changes in the external solution. The conductance for H(+) was estimated by measuring the membrane current change during an external pH change while the membrane potential was clamped at K(+) equilibrium potential. In the range of external pH 5 to 6, H(+) chord conductance was substantially equal to the membrane conductance. However, the [H](i) measured by various methods was not such as would be predicted from the [H](o) and the membrane potential using the Nernst equation. In artificial pond water containing DNP, the resting membrane potential decreased; this suggested that some energy-consuming mechanism maintains the membrane potential at the resting level. It is probable that there is a H(+) extrusion mechanism in the Nitella cell, because the potential difference between the resting potential and the H(+) equilibrium potential is always maintained notwithstanding a continuous H(+) inward current which should result from the potential difference.

REPETITIVE ACTION POTENTIALS IN NITELLA INTERNODES

Typical spontaneous action potentials can be elicited in 10–100 mM NaCl or LiCl solution. The period of repetition is 0.5–2 seconds and the action potential generally consists of a rapid spike alone. Similar spontaneous action potentials are also demonstrated by adding either 1 mM EDTA (pH 6.6) or 2 mM ATP (pH 6.6) to the artificial pond water. In these cases, however, the period of repetition is much longer and the action potential is of a normal shape, a rapid spike being followed by a slow transient depolarization. The period of repetition and the size of the action potential decrease with the elevation of the vacuolar potential level. The cause of the spontaneous firing is supposed to be the removal of Ca++ from the outer surface of the Nitella membrane.

The Laboratory Population Ecology of Kerona Pediculus (O.F.M.) Epizoic on Hydra SPP.

In attempt to determine the factors regulating the distribution and abundance of Kerona pediculus, populations of the hypotrich and hydras were raised in the laboratory in artificial pond water. Factors governing the survival of hypotrichs on hydras were examined by varying the feeding conditions of hydras and the relative numbers available to Kerona. Ciliate populations did not maintain themselves on starved hydras. Availability of undetermined food substances from the hydras seems to limit the numbers of Kerona per hydra. The rate of increase of the hypotrichs varied inversely with density per hydra over a wide range. The densities ranged from below normal (3) to extremely crowded (96). Kerona densities increased in abnormal ways when hydras were infested with Hydramoeba hydroxena, or small histiophagic ciliates and flagellates. Only the amoebic infestation increased the absolute numbers of hypotrichs. Under certain culture conditions in the laboratory, the growth—rate of hydras was greater in the presence of Kerona than in its absence.

Ca Fluxes and Membrane Potentials inNitella translucens

The concentrations of Ca have been measured in the flowing cytoplasm and the vacuole of the single cells of Nitella translucens, the cells being immersed in an artificial pond water (composition: NaCl, 10 mM; KC1, o-i mM; CaCL, o r mM). In the flowing cytoplasm the total concentration is 8 mM and in the vacuole 12 mM. Measurements of the electrical potential differences across the plasmalemma and tonoplast membranes show that the cytoplasm is at a potential of —134 mV with respect to the bathing medium and — 24 mV with respect to the vacuole. An attempt has been made to measure the tracer fluxes of Ca and it is shown that the cells are not in flux equilibrium. The influx is 0-046 ijlix moles cm-2 sec-1; the efflux was too small to measure with any degree of accuracy. The observed potential differences across both membranes are compared with the Nernst potentials for Ca. This analysis shows that Ca is not in electrochemical equilibrium across either membrane and that the driving forces on Ca are directed from the bathing medium and the vacuole into the cytoplasm. It is suggested that there is no necessity for a metabolically driven Ca pump at the plasmalemma because the low cytoplasmic Ca content could be due to the low permeability of the plasmalemma; the Goldman flux equation gives a value of Pea — 4'3 X to 8 cm sec-1. A Ca pump at the tonoplast appears to be necessary to explain the steep electrochemical potential gradient from the vacuole to the cytoplasm. The efflux of Ca from the isolated cell wall has been measured. From these measurements it was possible to estimate the concentration of indiffusible anions in the Donnan Free Space; the value obtained was 0 74 equiv. I.-1.

Electrical Potentials and Na, K, and Cl Concentrations in the Vacuole and Cytoplasm ofNitella translucens

The potential differences across the tonoplast and plasmalemma membranes have been measured in the single cells of Nitella translucens, the cells being immersed in an artificial pond water (composition: NaCl 1.0 mM., KC1 0.1 mM., CaCl2, 0.1 mM.). The potential of the cytoplasm is –138 m V with respect to the bathing medium and –18 mV with respect to the vacuole. The concentrations of Na, K, and Cl have been measured in the two cell fractions. The concentrations in the flowing cytoplasm are: Na 14 mM., K 119 mM., and Cl 65 mM.; the vacuolar concentrations are: Na 65 mM., K 75 mM.,and Cl 160 mM. The observed potential differences across the two membranes are compared with the Nernst potentials for all three ions. This analysis shows that all three ions are actively transported at the plasmalemma: Na is pumped outwards while K and Cl are pumped inwards. At the tonoplast Na is pumped into the vacuole while K and Cl are close to electrochemical equilibrium. The inhibitor, ouabain, has no effect on the cell resting potential.

Osmotic and ionic regulation in Ambystoma tigrinum

1. 1. Larval and adult A. tigrinum maintain NaCl balance in artificial pond water even when starved. 2. 2. Sodium is lost in the urine and across the body surface at a mean rate of 1·3 μg-equiv/10 g per hr. Urinary loss represents 60–90 per cent of the total efflux in larvae and 55 per cent of the total in adults. 3. 3. sodium loss is balanced by uptake across the body surface against a large gradient of electrochemical potential. Chloride is also actively transported inward. 4. 4. Osmotic influx of water occurs at the rate of 26 per cent of body weight per day in larvae, 32 per cent of body weight per day in adults. The osmotic steady state is preserved by excretion of an equivalent volume of dilute urine. 5. 5. Sodium and chloride concentrations were lower in adult than in larval urine.

Accumulation of strontium-90 and calcium-45 by fresh water fishes.

Our previous studies suggested that fresh water fishes do not discriminate against strontium relative to calcium when the isotopes are taken up from water in which they swim (1, 2). Unlike fresh water fishes. Boroughs et al. (3) found that marine Tilapia discriminate against strontium relative to calcium. Discrimination against strontium also appears to be well documented in small laboratory mammals and in man (4-6). However, Wasserman et al. (7) found that strontium discrimination in rats is not affected by dietary calcium levels. The present results, using double tracer technics, indicate that fresh water fishes display only a small degree of discrimination against strontium relative to calcium and essentially substantiate our previous data. Methods and materials. Adult fresh water fishes, obtained from commercial sources, were of mixed sexes for zebra fish (Dunio) and white cloud mountain fish (Tanicthys) but guppies (Lebistes) were sexually mature males. Fish were placed in artificial pond water (8) containing both strontium-90 and calcium-45. The basic solution, adjusted to pH 7.0, contained 50 mg/liter of each of the following analytical reagent grade salts: sodium nitrate, potassium sulfate and magnesium sulfate. Solutions were modified by additions of calcium chloride, strontium chloride and sodium chloride as shown in results. Addition of strontium-90 and calcium-45 contributed approximately 0.25 mM/liter calcium(2). Small amouilts of these ions in the hasic solution are not included in the calculation. After 5 da57s in various solutions, fish were sacrificed and prepared for radioactive assay as previously described (9). Samples were held for 21 days to attain secular equilibrium Of stroritiuni-90 and its yttrium-90 daughter nuclide. Total beta activity (strontium-90 plus calcium-45) was determined with windowless gas flow counter.