Extracellular GTP Causes Membrane-Potential Oscillations through the Parallel Activation of Mg2+ and Na+ Currents in Paramecium tetraurelia

Paramecium tetraurelia responds to extracellular GTP (≥ 10 nm) with repeated episodes of prolonged backward swimming. These backward swimming events cause repulsion from the stimulus and are the behavioral consequence of an oscillating membrane depolarization. Ion substitution experiments showed that either Mg²⁺ or Na⁺ could support these responses in wild-type cells, with increasing concentrations of either cation increasing the extent of backward swimming. Applying GTP to cells under voltage clamp elicited oscillating inward currents with a periodicity similar to that of the membrane-potential and behavioral responses. These currents were also Mg²⁺- and Na⁺-dependent, suggesting that GTP acts through Mg²⁺-specific (I _Mg) and Na⁺-specific (I _Na) conductances that have been described previously in Paramecium. This suggestion is strengthened by the finding that Mg²⁺ failed to support normal behavioral or electrophysiological responses to GTP in a mutant that specifically lacks I _Mg (``eccentric'), while Na<sup>+</sup> failed to support GTP responses in ``fast-2,' a mutant that specifically lacks I _Na. Both mutants responded normally to GTP if the alternative cation was provided. As I _Mg and I _Na are both Ca²⁺-dependent currents, the characteristic GTP behavior could result from oscillations in intracellular Ca²⁺ concentration. Indeed, applying GTP to cells in the absence of either Mg²⁺ or Na⁺ revealed a minor inward current with a periodicity similar to that of the depolarizations. This current persisted when known voltage-dependent Ca²⁺ currents were blocked pharmacologically or genetically, which implies that it may represent the activation of a novel purinergic-receptor–coupled Ca²⁺ conductance. Received: 28 October 1996/Revised: 24 December 1996     

Feedback inhibition of sodium uptake in K+-depolarized toad urinary bladders

Ouabain-blocked toad urinary bladders were maintained in Na⁺-free mucosal solutions, and a depolarizing solution of high K⁺ activity containing only 5 mM Na⁺ on the serosal side. Exposure to mucosal sodium (20 mM activity) evoked a transient amiloride-blockable inward current, which decayed to near zero within one hour. The apical sodium conductance increased in the initial phase of the current decay and decreased in the second phase. The conductance decrease required Ca²⁺ to be present on the serosal side and was more rapid when the mucosal Na⁺ activity was higher. At 20 mM mucosal Na⁺ and 3 mM serosal Ca²⁺ the initial (maximal) rate of inhibition amounted to 20% in 10 min. The conductance decrease could be accelerated by raising the serosal Ca²⁺ activity to 10 mM. The inhibition reversed on lowering the serosal Ca²⁺ to 3 μM and, in addition, the mucosal Na⁺ to zero. Exposure of the mucosal surface to the ionophore nystatin abolished the Ca²⁺ sensitivity of the transcellular conductance, showing that the Ca²⁺-sensitive conductance resides in the apical membrane. The data imply that in the K⁺-depolarized epithelia, cellular Ca²⁺, taken up from the serosal medium by means of a Na⁺-Ca²⁺ antiport, cause feedback inhibition by blockage of apical Na⁺ channels. However, the rate of inhibition is small, such that this regulatory mechanism will have little effect at 1 mM serosal Ca²⁺ and less than 20 mM cellular Na⁺.     

Zinc mediated effects on leaf CO2 diffusion conductances and net photosynthesis in Phaseolus vulgaris L.

Supra-optimal levels of zinc in primary leaves of Phaseolus vulgaris increased the CO₂ compensation point and inhibited net photosynthesis. Leaf morphology was modified: mesophyll intercellular area, stomatal slit length and interstomatal distance were reduced, but stomatal density increased. Internal and stomatal conductances to CO₂ diffusion decreased. These changes are discussed in relation to the observed effects on leaf gas exchange and to the previously reported inhibition of different photosynthetic and photorespiratory enzymes.     

Uptake and distribution of abscisic acid in Commelina leaf epidermis

Closure of stomata by abscisic acid (ABA) was studied by floating leaf epidermal strips of Commelina communis L. in PIPES buffer (pH 6.8) containing a range of KCl concentrations. Control apertures were greatest at high concentrations of the salt, and the effects of ABA, in terms of closure, were most pronounced below 100 mol m^-3 KCl. Stomata opened on strips floated on buffer plus 50 mol m^-3 KCl and closed within 10 min when transferred to the same medium plus 0.1 mol m^-3 ABA. [2-¹⁴C]ABA was used to study uptake and distribution of the hormone by the epidermal strips. It was calculated that no more than 6 fmol ABA were present per stomatal complex at the time of closure, although uptake continued thereafter. Microautoradiography indicated that radioactivity from [2-¹⁴C]ABA accumulated in the stomatal complex at or near the guard cells within 20 min. TLC was used to examine the state of the label after 1 h incubation. Efflux of label from preincubated tissue appeared to occur in three phases (t_1/2=7.2 s, 4.0 min, 35.2 min). Efflux was correlated with stomatal re-opening. The results confirm that ABA can accumulate in the epidermis of C. communis.Abbreviation ABA Abscisic acid     

Light effects on leaf development and photosynthetic capacity of Hydrocotyle bonariensis Lam.

Rates of net CO₂ uptake were examined in developing leaves of Hydrocotyle bonariensis. Leaves that developed under high photosynthetically active radiation (48 mol m^-2 day^-1 PAR) were smaller, thicker, and reached maximum size sooner than did leaves that developed under low PAR (4.8 mol m^-2 day^-1). Maximum net CO₂ uptake rates were reached after 5 to 6 days expansion for both the low and the high PAR leaves. Leaves grown at high PAR had higher maximum photosynthetic rates and a higher PAR required for light saturation but showed a more rapid decline in rate with age than did low PAR leaves. To assess the basis for the difference observed in photosynthetic rates, CO₂ diffusion conductances and the mesophyll surface available for CO₂ absorption were examined for mature leaves. Stomatal conductance was the largest conductance in all treatments and did not vary appreciably with growth PAR. Mesophyll conductance progressively increased with growth PAR (up to 48 mol m^-2 day^-1) as did the mesophyll surface area per unit leaf area, but the cellular conductance exhibited most of its increase at low PAR (up to 4.8 mol m^-2 day^-1).     

Uptake and metabolism of carbohydrates by epidermal tissue

Isolated epidermis of Commelina communis L. and Tulipa gesneriana L. assimilated ¹⁴CO₂ into malic acid and its metabolites but not into sugars or their phosphates; epidermis could not reduce CO₂ by photosynthesis and therefore must be heterotrophic (Raschke and Dittrich, 1977). If, however, isolated epidermis of Commelina communis was placed on prelabelled mesophyll (obtained by an exposure to ¹⁴CO₂ for 10 min), radioactive sugars appeared in the epidermis, most likely by transfer from the mesophyll. Of the radioactivity in the epidermis, 60% was in sucrose, glucose, fructose, 3-phosphoglyceric acid and sugar phosphates. During a 10-min exposure to ¹⁴CO₂, epidermis in situ incorporated 16 times more radioactivity than isolated epidermal strips. Isolated epidermis of Commelina communis and Tulipa gesneriana took up ¹⁴C-labelled glucose-1-phosphate (without dephosphorylation), glucose, sucrose and maltose. These substances were transformed into other sugars and, simultaneously, into malic acid. Carbons-1 through-3 of malic acid in guard cells can thus be derived from sugars. Radioactivity appeared also in the hydrolysate of the ethanol-insoluble residue and in compounds of the tricarboxylic-acid cycle, including their transamination products. The hydrolysate contained glucose as the only radioactive compound. Radioactivity in the hydrolysate was therefore considered an indication of starch. Starch formation in the epidermis began within 5 min of exposure to glucose-1-phosphate. Autoradiograms of epidermal sections were blackened above the guard cells. Formation of starch from radioactive sugars therefore occurred predominantly in these cells. Epidermis of tulip consistently incorporated more ¹⁴C into malic and aspartic acids than that of Commelina communis (e.g. after a 4-h exposure to [¹⁴C]glucose in the dark, epidermis, with open stomata, of tulip contained 31% of its radioactivity in malate and aspartate, that of Commelina communis only 2%). The results of our experiments allow a merger of the old observations on the involvement of starch metabolism in stomatal movement with the more recent recognition of ion transfer and acid metabolism as causes of stomatal opening and closing.Abbreviation G-1-P glucose-1-phosphate     

Electrophysiological properties of onion guard cells

Intracellular electrical recordings in onion (Allium cepa L.) guard cells show that they maintain a membrane potential difference (MPD), inside negative. The MPD of intact cells averaged -72±29 mV (n=45); MPD of cells partially digested with a cellulolytic enzyme, -39±7 mV (n=65). Evidence indicates that the guard cells have two electrically distinct compartments, presumably delimited by the plasmalemma and tonoplast. Epidermal cells in partially digested preparations also showed MPD that could be either positive (+15±7 mV; n=23) or negative (-15 ±8 mV; n=13). Guard cells exposed to light-dark cycles hyperpolarized in the light and depolarized in the dark. The largest observed voltage changes reached 52 mV during hyperpolarizations and 60 mV during depolarizations. The light responses saturated with roughly exponential kinetics, with the depolarizations exhibiting a slower second phase that might be related to the contracting movements of the guard cells. Initial rates of the responses averaged about 14 mV min^-1 in the dark and about 8 mV min^-1 in the light. The results can be interpreted as electrical correlates of fluctuations in intracellular potassium concentration, as light-induced changes in membrane permeability, or as the photoactivation of an electrogenic proton pump. The last possibility seems to be the simplest interpretation of the data that also provides us with a mechanism driving the ion fluxes associated with stomatal function.     

cAMP-Dependent protein kinase inhibits the chloride conductance in apical membrane vesicles of hunman placenta

Summary The role of adenosine 3,5-monophosphate (cAMP) dependent protein kinase (PK-A) on the Cl^– conductance has been studied in the apical membrane vesicles purified from the chorionic villi of human placenta. In order to phosphorylate the cytosolic side of the membranes, vesicles have been hypotonically lysed, loaded with 100nm catalytic subunit of PK-A purified from human placenta and 1mm of the phosphatase resistant adenosine 5-thiotriphosphate (ATP-gamma-S) and resealed. Cl^– conductance has been measured by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) at 23°C with membrane potential clamped at 0 mV. The actual volume of the resealed vesicles was measured in each experiment by trapping an impermeable radioactive molecule ([¹⁴C]-sucrose) and included in each Cl^– flux calculation. In 19 independent experiments, the mean Cl^– conductance in placental membranes in the absence of phosphorylation was 3.67±3.18 whereas with the addition of PK-A and ATP-gamma-S it was 1.97±1.75 nmol·sec^–1·(mg protein)^–1 (mean±sd). PK-A dependent phosphorylation reduced the Cl^– conductance in 14/19 experiments. The same protocol applied to the apical membranes of bovine trachea, where PK-A is known to activate the Cl^– channels, confirmed that the PK-A dependent phosphorylation increased the Cl^– conductance in 11/13 experiments, from 1.01±0.61 to 1.85±0.99 nmol·sec^–1·(mg protein)^–1(mean±sd). These studies indicate that the PK-A dependent phosphorylation inhibits one or more Cl^– channel(s) of the apical membranes of human placenta.