Renata Rybová

Ionic relations of the alga Hydrodictyon reticulatum. The effects of light conditions and inhibitors

Summary Flows of sodium, potassium and chloride ions across the plasmalemma of the alga Hydrodictyon reticulatum were studied and their light dependence examined. Both the inflows and the outflows of the ions were found to be light-dependent in the sense of being considerably reduced in the dark. In the case of potassium the two flows appear to be influenced approximately to the same degree, since the potassium content is not significantly changed in the dark. It seems that a special mechanism is responsible for changes of the content of sodium, the highly significant decrease of which during prolonged dark periods may be shown to be due to a release of sodium bound by intracellular structures. Most of the intracellular sodium is found to be in a non-exchangeable form under normal day-and-night pattern of illumination and part of this sodium fraction is released reversibly during 48 hours in the dark, irreversibly after still more prolonged dark periods. The outflow of chloride ions in the dark seems to be less reduced than the inflow, for a significantly lower content of chloride ions was found in the dark. Coupling of individual flows with photosynthetic processes was examined with specific inhibitors. Chloride flows were found to be practically abolished with DCMU, an inhibitor of the second photosynthetic system; the flows of sodium and potassium, on the other hand, were markedly reduced with CCCP, an inhibitor of photophosphorylation. The most important conclusion drawn from these experiments seems to be that not only the unidirectional flows proceeding against a gradient of electrochemical potential are inhibited by the inhibitors, the flows in the opposite direction and in the direction of simple physico-chemical forces being inhibited in the same sense and often to the same degree. Hence the passive permeability of the plasmalemma of Hydrodictyon reticulatum appears to be very low, the flows of the ions taking place almost exclusively through metabolically-coupled active pumps in both directions. When these mechanisms are impaired by inhibition, both net flows and tracer exchange are almost completely prevented, the plasmalemma becoming virtually impermeable. In addition to that, the sodium transport in Hydrodictyon reticulatum is once more found to be insensitive to ouabain like in some of the algal cells and unlike in those of other algae.

Nonpolarized frog bladder preparation

SummaryIn a frog bladder preparation with the mucosal surface covered with liquid paraffin the content of sodium chloride and of water in the epithelial cells is reduced, while the polarity and the magnitude of the membrane potential across the non-mucosal parts of the membranes are preserved. The transport of ions between the cells and their surroundings appears to be mediated solely by the non-mucosal membranes of the epithelial cells, and for this reason the tissue in this condition was called a nonpolarized preparation. Oxytocin (50 mU/ml) brings about an extrusion of sodium and potassium chlorides and of water from the epithelial cells of the nonpolarized preparation. Since sodium ions leave the cells against their electrochemical potential gradient, a conclusion about a direct stimulation of the sodium pump by oxytocin (possibly due to a reduction of its internal resistance) appears to be warranted.

Effects of electron acceptors on anion uptake in Hydrodictyon reticulatum

Summary The artificial electron acceptors, potassium ferricyanide and tetrahiafulvalene cation radical, are shown to inhibit uptakes of chloride, nitrate and sulphate anions in the green fresh-water alga Hydrodictyon reticulatum . The same is true about the phosphate anion in an alkaline medium, where its divalent form prevails. The unidirectional efflux of these anions was not inhibited. TTF + , the more potent of the two inhibitors, appears to be partly located in the plasma membrane. An electron-anion antiport (a redox pump) is suggested as the mechanism of the active uptake of most of the physiologically important anions in the alga. Notable exceptions to this rule are represented by bicarbonate, the carbon source for photosynthesis at air levels of CO 2 , and by phosphate (probably univalent) in a medium buffered to pH6.

The plasmalemma redox system of a fresh-water alga and membrane electrical parameters

In order to learn more details about the plasmalemma redox system in the alga Hydrodictyon reticulatum the electrophysiological characteristics of the plasma membrane as influenced by artificial electron acceptors were estimated. Ferricyanide anion as well as TTF+ depolarized the membrane potential, the effect being more marked in the dark than in the light. In the presence of ferricyanide the membrane resistance in the light was decreased by about 29% on the average, in the dark it remained unchanged. On the other hand, TTF+ brought about a large increase in membrane resistance, notwithstanding the light conditions. The results are discussed in view of the impairing influence of both electron acceptors on the active inflow of chloride ions (Rybová, R. et al. (1990) Bot. Acta 103, 404-407). The electrogenic outflow of electrons appears to make the largest contribution to the membrane depolarization. The inhibition of a pumping mechanism coupling the active uptake of Cl- in some way to the transmembrane electron flow is envisaged as a plausible explanation for membrane resistance increase by TTF+.

Adaptive mechanisms of inorganic carbon transport and metabolism inHydrodictyon reticulatum

Abstract A marked difference between the half-saturation constants in the alga Hydrodictyon reticulatum (L.) Lagerh. living at a low CO 2 concentration ( K 1/2(CO 2 ) = 9.1 μM) and of cells pre-treated for 24 h in a CO 2 -rich media ( K 1/2(CO 2 ) = 36.1 μM) points clearly to the adaptivity of the mechanism which supplies the photosynthetic apparatus with CO 2 . Light-induced pH increases, which reflect the photosynthetic utilization of bicarbonate, were impaired by the inhibitors of carbonic anhydrase and by specific anion channel blockers. The results are consistent with the conclusion that in H. reticulatum the dissolved inorganic carbon substrate enters the plasmalemma mainly in the form of HCO 3 − which is then decomposed to CO 2 and OH − in a reaction catalyzed by carbonic anhydrase. Furthermore, the uptake of bicarbonate appears to be dependent on the activity of H + -ATPase as judged from the inhibitory actions of diethylstilbestrol and dicylocarboheximide on light alkanization. During the first 5 s of photosynthesis a relatively high incorporation of radioactive H 14 CO − 3 into C 4 compounds took place under conditions of limited carbon source.

Sulphate ion influx and efflux inhydrodictyon reticulatum

Sulphate ion is accumulated in the cells of the algaHydrodidyon reticulatum to a concentration approximately 30 mmol 1-1. The rate of SO42- uptake is increased markedly in the light, however, the effect is observable only after the cells have been kept in the light for about one hour. Uptake of sulphate is strongly inhibited by phosphorylation uncouplers and also by DIDS. The two-phase sulphate uptake kinetics, reported earlier was confirmed in experiments in which competition with phosphate anion was tested. Phosphate competes with sulphate only in the range of higher substrate concentrations (from 0.2 mmol 1-1) and does not affect the system which works at low substrate concentrations. Efflux of SO42- is very slow; the reason of the flux hindrance is not yet known.

Sodium-potassium pump and cell volume regulation in frog bladder

Abstract In the so-called nonpolarized preparation of frog bladder the mucosal membranes are blocked by liquid paraffin and the intracellular ionic composition is governed solely by the non-mucosal parts of the cell membranes. Results obtained with this preparation support the original idea of a coupled Na + -K + pump operating in ion-transporting epithelial layers and implied in the frog skin model by Ussing and Koefoed-Johnsen. Under the physiological conditions of a normal Na + gradient the operation of the pump leads to a one-to-one exchange of Na + for K + and is inhibited equally by ouabain and by the absence of K + in the adjacent medium. Along with the coupled pump a mechanism regulating the cell volume is present which is insensitive to treatment with ouabain and to K + -free media but is sensitive to inhibition with 2,4-dinitrophenol.

Independent stimulation of sodium entry and sodium extrusion in frog urinary bladder by aldosterone

Summary10−6M d-aldosterone was found to increase the sodium content in a conventional preparation (frog Ringer solution on both sides) of the urinary bladder of female frogsRana temporaria, whereas the same preparation of male frogs showed a highly significant decrease of the sodium content, which did not occur in preparations inhibited by 5·10−4M ouabain. In female frog bladders, nevertheless, the extrusion of sodium ions across the nonmucosal surfaces of epithelial cells is also stimulated by aldosterone, as shown by an increase in the transcellular sodium transport, an independent stimulation of the sodium pump by aldosterone being also involved, as demonstrated by a decrease of the sodium content in a nonpolarized preparation of female frog bladders (vesicles filled with liquid paraffin). The conclusion is drawn that aldosterone in sodium-transporting epithelial layers stimulates independently the entry of sodium ions across the mucosal membranes of the epithelial cells and their active extrusion across the nomucosal surfaces of the cells. In the nonpolarized preparation of male frog bladders no further decrease in the sodium content is observed with aldosterone, the minimum content of sodium in the transport pool being probably achieved here already by the prolonged incubation of the untreated preparation.

Ion transport in the Alga Hydrodictyon reticulatum under conditions of carbonic anhydrase inhibition

Summary The effect of a specific inhibitor of carbonic anhydrase, hydrochlorothiazide, on the content and fluxes of the principal ions in the alga Hydrodictyon reticulatum was investigated using chemical analysis and radioisotopes. Pronounced changes in the intracellular content of Na + , K + and Cl − ions after a 24-hour application of the inhibitor are correlated with the transmembrane flows of labelled ions. A diminished uptake of both protons and bicarbonate ions observed in the inhibited cells indicates changes in the intensity of photosynthesis and points to the possible role of carbonic anhydrase in the process.

Salicylhydroxamic-acid-stimulated chloride transport in Hydrodictyon reticulatum

Summary SHAM was found to stimulate the chloride uptake assumed to be operated by the redox pump. Nevertheless, peroxidase-like enzymes do not seem to be involved in the anion transport since catalase does not abolish this effect; on the contrary, its presence increases the 36Cl uptake additively to SHAM. More NAD(P)H appears to be available for chloride transfer into the cells in the presence of SHAM and/or catalase.