Vanadate Sensitive ATPase and Phosphatase Activity in Guard Cell Protoplasts of Commelina

Flicker, M. D. and Willmer, C. M. 1986. Vanadate sensitive ATPaseand phosphatase activity in guard cell protoplasts of Commelina.—J.exp. Bot. 38: 642–648. Phosphatase activity was measured in extracts of guard cellprotoplasts of Commelina communis L. using the artificial substratep-nitrophenylphosphate. A pH optimum of 5.8 to 6.3 was determined.Ammonium molybdate (Ol mol m^–3) and sodium vanadate (1–0mol m^–3) gave almost complete inhibition of phosphataseactivity at pH 60. ATPase assays were, therefore, conductedin the presence of 0–2 mol m ^–3 molybdate and vanadatewas used as a specific inhibitor of plasmamembrane ATPase activity.Vanadate sensitive ATPase activity showed a pH optimum of 6.6and activity was stimulated by KC1. These properties are characteristicof plasmamembrane proton pumping ATPases in other systems andsuggest that proton extrusion in guard cells could be mediatedby a similar enzyme. The maximum ATPase activity is sufficientto account for all the proton flux observed during the stomatalopening response. Key words: ATPase, Commelina, guard cell protoplasts, phosphatase, vanadate     

A Comparison Between the ATPase and Proton Pumping Activities of Plasma Membranes Isolated from the Stele and Cortex of Zea mays Roots

Plasma membrane vesicles of high purity, determined by markerenzyme assays, were obtained by phase partitioning microsomalfractions from stelar and cortical tissues of Zea mays (cv.LG11) roots. ATP hydrolytic activities in both of the plasmamembrane fractions were inhibited by vanadate, SW26 and erythrosinB, but were insensitive to nitrate. Activity in both fractionsexhibited a marked pH optimum of 6·5 and displayed typicalMichaelis-Menten kinetics. A high substrate specificity wasapparent in both the stele and cortex plasma membrane fractions,while the lower fractions, after phase partitioning, showedlower specificity for nucleotide substrates. Specific activitiesof the stele (67·8 µmol Pi mg^–1 h^–1)and cortex (78·4 µmol Pi mg^–1 h–¹)plasma membrane H⁺ -ATPases were very similar. Proton pumping activities in microsomal membrane fractions fromstele and cortex were inhibited by nitrate and insensitive tovanadate. Homogenization of stele and cortex tissue in the presenceof 250 mol m^–3 KI resulted in microsomal fractions exhibitingvanadate-sensitive, nitrate-insensitive proton pumping activity,suggesting a plasma membrane origin for this activity. SW26was also an effective inhibitor of proton pumping activity,although results indicated an interaction between SW26 and thefluorescent probes quinacrine and acridine orange. The results are discussed in relation to models for the transportof ions into the stele and are consistent with a role for theH⁺ -ATPase activity in this process. Key words: ATPase, cortex, plasma membrane, stele, Zea mays     

Cytological studies on mitochondria-induced cytoplasmic transformation in yeasts

In Saccharomyces cerevisiae, protoplasts from respiratory-deficient(rho^–) cytoplasmic mutant cells were transformed intorespiratory-sufficient (rho⁺) cells by incubation with mitochondriaprepared from rho⁺ cells in the presence of polyethylene glycoland CaCl₂. Mitochondria prepared from different species, Hansenulawingei and Schizosaccharomyces pombe, also caused the transformationof S. cerevisiae rho^– protoplasts into the rho⁺ cellsas previously reported (14) The obtained transformants wereconfirmed to contain one nucleus and several mitochondrial DNAsby fluorescent staining of DNA. The transformants clearly restoredcytochromes a and b while untransformed recipient cells lackedthe cytochromes. In order to know the mechanism of the transformation,physiological measurement of endocytotic activity of protoplastsand cytological examination of mitochondria-protoplast aggregatesunder the transforming condition were performed. Protoplastshad significant endocytotic activity under this condition. Onthe other hand, fluorescence and electron microscopic observationsindicated that mitochondria forming aggregates with protoplastswere subsequently integrated into recipient protoplasts throughfusion rather than endocytosis. However, the possibility ofendocytosis could not be completely excluded when the low frequencyof the transformation (about 10^–6 to 10^–7) was takeninto account. This is discussed in this paper. A new convenientmethod for measuring endocytosis is also presented. (Received September 27, 1979; )     

Zinc deficiency enhanced NAD(P)H-dependent superoxider radical production in plasma membrane vesicles isolated from roots of bean plants

The production of superoxide radical (O₂^–) was studiedin plasma membrane vesicles isolated by aqueous polymer two-phasepartitioning from roots of zinc-sufficient and zinc-deficientbean (Phaseolus vulgaris L. cv. Prélude) plants. Thetwo populations of vesicles were highly enriched in plasma membraneand had similar composition as evidenced by the specific membranemarker enzymes. Vesicles from zinc-deficient roots showed higherrates of NAD(P)H oxidation compared to vesicles from zinc-sufficientplants. The NAD(P)H-dependent formation of O₂^– in plasmamembrane vesicles was also highly increased by zinc deficiency.For both activities, a higher response to zinc deficiency wasobserved when NADPH was used as electron source. Re-supply ofzinc to deficient plants for 24 h substantially decreased therates of NAD(P)H oxidation and 0₂^– production in isolatedvesicles. The NADPH-dependent O₂^– generation was stronglystimulated by FAD and showed a high pH optimum; it was scarcelyaffected by Triton X-100 or even inhibited in the presence ofFAD and was almost insensitive to Antimycin A. The results suggest the presence at the plasma membrane of beanroots of an O₂^– generating activity, preferentially utilizingNADPH, which is affected by the zinc nutritional status of theplant. This finding, together with previous observations oncytosolic and microsomal fractions prepared from zinc-deficientroots of different plants, is consistent with a role of zincin membrane stabilization by controlling the level of oxidizingO₂ species. Key words: NAD(P)H oxidase, superoxide radical, plasma membrane, zinc deficiency     

The Conductance of the Plasmalemma for CO2

Permeability coefficients (P_S values) for CO₂ of the plasmamembrane (PM) of the unicellular green algae Eremosphaera viridis,Dunaliella parva, and Dunaliella acidophila, and of mesophyllprotoplasts isolated from Valerianella locusta were determinedfrom ¹⁴CO₂ uptake experiments using the rapid separation ofcells by the silicone oil layer centrifugation technique. Theexperimental P_S values were compared with calculated numbersobtained by interpolation of Collander plots, which are basedon lipid solubility and molecular size, for D. parva cells,mesophyll protoplasts isolated from Spinacia oleracea, mesophyllcells and guard cells of Valerianella, and guard cell protoplastsisolated from Vicia faba. The conductivity of algal plasma membranes for CO₂ varies between0.1 and 9 ? 10^–6 m s^–1, whereas for the plasmalemmaof cells and protoplasts isolated from leaves of higher plantsvalues between 0.3 and 11 ? 10^–6 m s^–1 were measured.By assuming that these measurements are representative for plantsand algae in general, it is concluded that the CO₂ conductivityof algal PM is of the same order of magnitude as that of thehigher plant cell PM. P_s values of plasma membranes for CO₂are lower than those for SO₂, but are in the same order of magnitudeas those measured for H₂O. On the basis of these results itis concluded that theoretical values of about 3000 ? 10^–6m s^–1 believed to be representative for higher plant cells(Nobel, 1983) and which are frequently used for computer-basedmodels of photosynthesis, lack experimental confirmation andrepresent considerable overestimations. However, with severalsystems, including higher plant cells, the conductance of thePM for CO₂ was significantly higher in light than in darkness.This suggests that in light, additional mechanisms for CO₂ uptakesuch as facilitated diffusion or active uptake may operate inparallel with diffusional uptake. Key words: Conductivity, CO₂, permeability coefficient, photosynthesis, plasmalemma     

The Root Cap as a Test System for the Evaluation of Golgi Inhibitors: I. STRUCTURE AND DYNAMICS OF THE SECRETORY SYSTEM AND RESPONSE TO SOLVENTS

Maize (Zea mays) seedlings were grown under standard conditionsand the time for reformation of a slime droplet of standardsize was determined. The structure of the secretory cells wasexamined by light and electron microscopy. Osmium-zinc iodide(OZI) impregnation was used to provide contrast enhancementof the dictyosome forming-face cisternae and the endoplasmicreticulum (ER). The three-dimensional structure and relationshipsof these membrane systems were established from examinationof 300 nm thick sections. The presence of a marginal cisternalnetwork at the forming-face was confirmed, but no specific orientationof the ER to this face, or connection of ER to any part of thedictyosome could be demonstrated. Neither observation of reforming slime droplets, nor quantificationof secretory vesicle numbers gave any support to the suggestionthat slime formation is a phasic, or cyclic, process. Dilute solutions of solvents (1% ethanol, 1% and 0.1% DMSO)commonly used to solubilize potential inhibitors of Golgi activitywere found to delay significantly the time taken for reformationof a slime droplet of standard size. These solutions also hadslight effects on the numerical density of secretory vesicles. Cytochalasin D-induced accumulation of secretory vesicles wasused to determine rates of vesicle formation (0.39 dictyosome^–1min^–1) and hence turnover times for cisternal (6.5 min)and total dictyosome (26–39 min) membranes and the plasmamembrane (9.7 min). The volume contribution of the secretory vesicles to the slimedroplet was shown to be exceedingly small (0–1%), throwingdoubt on the value of using droplet reformation rates as anindicator of secretory activity. Key words: Maize, Root cap secretion, Dictyosome activity     

Interannual changes in the biomass of the Black Sea gelatinous zooplankton

The 15 year changes in the total gelatinous biomass consistof a general trend to increase, from 250 gm^–2 in 1980to 2500 g m^–2 in 1995, and fluctuations with a periodof –4 years performed against the background of this trendin the upper 150 m layer. Different species occupying distincttrophic niches form these peaks. When represented as the percentageof the total zooplankton biomass, Aurelia aurita and Mnemiopsisleidyi exhibit the contra-phase fluctuations where a sharp increasein M.leidyi is accompanied by a decrease in the A.aurita biornass.     

Rates of Photosynthesis in Characean Cells: II. PHOTOSYNTHETIC 14CO2 FIXATION AND 14C-BICARBONATE UPTAKE BY CHARACEAN CELLS

Photosynthetic ¹⁴C fixation by Characean cells in solutionsof high pH containing NaH¹⁴CO₃ gave a measure of the abilityof these cells to take up bicarbonate (H¹⁴CO₃^–). Whereascells of Nitella translucens from plants collected and thenstored in the laboratory absorbed bicarbonate at 1–1.5µµmoles cm^–2 sec^–1, rates of 3–8µµmoles cm^–2 sec^–1 were obtained withN. translucens cells from plants grown in the laboratory. Influxesof 5–6 µµmoles cm^–2 sec^–1 wereobtained with Chara australis, 3–8 µµmolescm^–2 sec^–1 with Nitellopsis obtusa, and 1–5µµmoles cm^–2 sec^–1 with Tolypella intricata.It is considered that these influxes represent the activityof a bicarbonate pump, which may be an electrogenic process. In solutions of lower pH, H¹⁴CO₃^– uptake would be maskedby rapid diffusion of ¹⁴CO₂ into the cells: the four Characeanspecies fixed ¹⁴CO₂ at maximum rates of 30–40 µµmolescm^–2 sec^–1 (at 21° C).     

Changes in the extracellular ion concentration in the main pulvinus of Mimosa pudica during rapid movement and recovery

A decrease in electric resistance and an increase in the extracellularCl^– concentration ([Cl^–]) in the main pulvinus ofMimosa occurred immediately after the action potential of themotor cells. The beginnings of both changes were almost coincidentalwith the beginning of the rapid movement. A remarkable increasein [Cl^–] was seen in the lower half of the pulvinus, butonly a slight increase in the upper half. Release of Cl^–,probably with K⁺; and other ions, from the motor cells impliesan ejection of liquid from the vacuole. When recovery of thepulvinus following rapid movement was fast in light, [Cl^–]decreased to its initial level within 20 min. When the recoverywas slow in darkness, [Cl^–] decreased at a slow rate andmaintained a higher level than its initial one for a long time.Photosynthetic inhibitors delayed recovery and the decreasein [Cl^–] even in light. These facts suggest that the re-entryof ions into motor cells during recovery partially requiresa photosynthetic energy supply. (Received February 12, 1980; )     

Activities of Ca2+ pump and low affinity Ca2+/H+ antiport in plasma membrane vesicles of corn roots

ATP-dependent Ca₂₊-uptake was investigated in sealed plasmamembrane vesicles isolated from corn roots (Zea mays L. cv.Hybrid-3352/Palma-Pioneer). In a chloride-containing medium,at high calcium concentrations, about 30% of the total Ca²⁺accumulation ({small tilde}4 nmol Ca²⁺ mg^–1 protein) wasshown to be protonophore-sensitive and corresponded to the fractionof Ca²⁺ not accumulated in a sulphate-containing medium. Furthermore,vesicles in the presence of nitrate, which stimulates H⁺ transport,or vesicles preloaded with H⁺, take up Ca²⁺ more rapidly, suggestingthat, at high calcium concentrations, there is a mechanism forCa²⁺ transport which depends on the magnitude of the protongradient across the membrane. The fraction of Ca²⁺ uptake shownto be sensitive to the protonophore CCCP increased by about150–200% as the Ca²⁺ concentration in the medium increasedfrom 50µM to 250µM. Under the same conditions, theCCCP-insensitive fraction of Ca²⁺ accumulated was reduced byabout 25–30% suggesting that different Ca²⁺ affinitiesexist in the two Ca²⁺ uptake processes. Although calmodulinstimulation was not observed, the sensitivity to Ca²⁺ and externalpH indicates that H⁺ gradient-independent Ca²⁺ accumulationreflects activity of the Ca²⁺–pump. These results indicatethat the plasma membrane of corn roots contain two distinctmechanisms of Ca²⁺ transport: a high Ca²⁺ affinity, proton gradient-independentCa²⁺ pump and a low Ca²⁺ affinity, proton gradient-dependentCa²⁺/H⁺ antiport, which have greatest activity at concentrationsof Ca²⁺ below and above 50+M, respectively. Key words: Ca²⁺/H+ antiport, Ca²⁺ pump, plasmalemma, roots, Zea mays L.     

X-Ray Microanalysis and Ultrastructural Studies of Cell Compartments of Dunaliella parva

Hajibagheri, M. A., Gilmour, D. J., Collins, J. C. and Flowers,T. J. 1986. X-ray microanalysis and ultrastructural studiesof cell compartments of Dunaliella parva. -J. exp. Bot. 37:1725–1732. Ultrastructural studies of the unicellular green alga Dunaliellaparva showed the presence of cytoplasmic vacuoles. X-ray microanalysiswas performed on sections of cells which had been freeze substitutedin acetone. It was found that the concentrations of both Naand Cl were much higher in the vacuoles than in the cytoplasm.When cells were grown in 0·4 kmol m^–3 NaCl theNa and Cl concentrations in the vacuoles were 349 and 283 molm ^–3 respectively, whilst cytoplasmic Na and Cl concentrationswere 37 and 26 mol m^–3. Corresponding values for cellsgrown in 1·5 kmol m^–3 NaCl were 392 mol m^–3Na and 325 mol m^–3 Cl in the vacuoles and 36 mol m^–3Na and 30 mol m^–3 Cl in the cytoplasm. Immediately afterexposure to an increase in external salinity Na and Q concentrationsincreased in both vacuoles and cytoplasm. The results are discussedwith reference to compartmental models for the ionic relationsof Dunaiiella. Key words: X-ray microanalysis, ultrastructural studies, Dunaliella parva     

Malate Accumulates in the Vacuole of Chara australis During Uptake of Ammonium From Chloride-free Solution

Internodal cells of Chara australis can accumulate ammoniumto high concentrations (10 to 70 mol m^–3) in their vacuoles.When Cl^– is included in the bathing solution, changesin the cellular concentrations of ammonium, K⁺, Cl^– andNa⁺ have been shown to meet the requirements for electroneutralityand to account for the changes in vacuolar osmotic pressureassociated with ammonium uptake. If accumulation occurs in theabsence of external Cl^–, however; changes in the inorganicions do not meet these criteria. Malate is found in the vacuolesof cells accumulating amine in the absence of external Cl^–and its presence (at 0·5 to 8·5 mol m^–3)allows us to account for electroneutrality and for changes inthe osmotic potential. Key words: Malate, Chara, electroneutrality, ammonium     

Plasmalemma ATPase activity modifications induced by traumatisms in Bidens pilosa

It has previously been shown that cotyledonary pricks inducedmodifications of ion levels (H⁺ and K⁺) in hypocotyl cells ofBidens pilosa. These modifications differed according to thelight quality: H ⁺ levels increased and K⁺ levels decreasedin white light (WL), whereas H⁺ levels decreased and K⁺ levelsincreased in blue light (BL). In this study, in order to determinethe mechanism responsible for these ionic modifications, plasmamembrane vesicles have been isolated and characterized fromhypocotyl cells. The effects of light quality and cotyledonarypricks on plasma-lemma ATPase activity (EC 3.6.1.3 [EC] ) were studied.Cotyledonary pricks induced, in WL, rapid (5 min) and transient(restoration in 60 min) inhibition of plasmalemma ATPase activity.Conversely, in BL, a rapid and transient stimulation was observed.These results suggest that, in Bidens pilosa, plasmalemma ATPaseis involved in 'short-term' ionic level modifications inducedby traumatisms. Key words: ATPase activity, short-term ionic regulation, growth inhibition     

Excitable Channels in Nitellopsis obtusa as Part of an Active Ion-Transport System

The present paper is a study on the rapid and the slow excitablechannels of Nitellopsis obtusa. The working hypothesis is thatduring the excitation of these cells a Ca²⁺-dependent activeion-transport system in the plasmalemma is activated (Gyenesand Bulychev, 1979; Gyenes, Bulychev, and Kurella, 1980) whichmay interact with a light-dependent active transport systemalso present in the plasmalemma. It is found that under conditionsof maximal light-induced current changes, registered in voltageclamp experiments, the amplitudes of both action current componentsare relatively small (10–15 µA cm²) and they increaseup to 100–150 µA cm^–2 during 15–30 minin the dark. Cells may also be excited chemically under conditionsof unchanged voltage across the plasmalemma. It is suggestedthat in the excitation process of Nitellopsis obtusa two typesof ion channels take part/emdash electrically excitable passiveand chemically excitable active channels-both incorporated inone proteolipid complex of a Ca²⁺-dependent ATPase of the plasmalemma.     

Effects of macronutrients upon buoyancy regulation by metalimnetic Oscillatoria agardhii in Deming Lake, Minnesota

Gas-vacuolate filaments of Oscillatoria agardhii form a metalimneticlayer in Oeming Lake, Minnesota. The environmental factors whichaffect buoyancy and the physiological processes which mediatechanges in buoyancy were determined. Buoyant filaments losttheir buoyancy in a few hours when incubated at light intensitiesabove those found in situ ({small tilde}15 µnol photonsm^–2 s^–1, or 1% of the surface value). The rate ofbuoyancy loss was accelerated by the addition of 10 µMphosphate at irradiances >200mol photons m^–2 s^–1.The effect of nutrient additions on buoyancy was also investigatedover a longer time period by incubating metalimnetic samplesin situ. The samples were deployed for 6 days at a depth wherethe irradiance was 8% of the surface value. As found in short-termexperiments, the addition of phosphate resulted in the largestdecrease in buoyancy. However, the addition of ammonia in additionto phosphate attenuated the buoyancy loss on day 2, and on day6 the filaments in these treatments were almost completely buoyant.The physiological status of the filaments in these treatmentswas assayed by analysis of elemental ratios of C, N and P, andby measurement of cellular chlorophyll, polysaccharide and protein.In addition, the cellular content of gas vesicles was determined.The construction of ballast balance sheets from these data indicatedthat changes in buoyancy were primarily due to differences inthe amount of polysaccharide ballast in the cells. However,in another set of in situ experiments, the increase in measuredballast molecules did not explain the observed loss of buoyancy.We hypothesized that another, undetected ballast-providing moleculehad accumulated in the cells.     

Effect of acidification on the location of H+-ATPase in cultured inner medullary collecting duct cells

In previousstudies, our laboratory has utilized a cell line derived from the ratinner medullary collecting duct (IMCD) as a model system for mammalianrenal epithelial cell acid secretion. We have provided evidence, from aphysiological perspective, that acute cellular acidification stimulatesapical exocytosis and elicits a rapid increase in proton secretion thatis mediated by an H⁺-ATPase. Thepurpose of these experiments was to examine the effect of acutecellular acidification on the distribution of the vacuolar H⁺-ATPase in IMCD cells in vitro.We utilized the 31-kDa subunit of theH⁺-ATPase as a marker of thecomplete enzyme. The distribution of this subunit of theH⁺-ATPase was evaluated byimmunohistochemical techniques (confocal and electron microscopy), andwe found that there is a redistribution of these pumps from vesicles tothe apical membrane. Immunoblot evaluation of isolated apical membranerevealed a 237 ± 34% (P < 0.05, n = 9) increase in the 31-kDa subunitpresent in the membrane fraction 20 min after the induction of cellularacidification. Thus our results demonstrate the presence of this pumpsubunit in the IMCD cell line in vitro and that cell acidificationregulates the shuttling of cytosolic vesicles containing the 31-kDasubunit into the apical membrane.     

Boron Nutrition of Cultured Tobacco BY-2 Cells: I. Requirement for and Intracellular Localization of Boron and Selection of Cells that Tolerate Low Levels of Boron

Cultured cells of tobacco (Nicotiana tabacum L. cv. Bright Yellow2) grown under the standard culture conditions (1 mg boron liter^–1medium as boric acid) contained boron at a concentration of2.26 mg boron kg^–1 oven-dried cells and the protoplastcontained 1.26% of the boron in the cells. The cells requiredboron for growth and the half-maximum growth rate was obtainedwith 0.056 mg of boron liter^–1 medium. Subculturing thecells in media with lower concentrations of boron allowed selectionof cells that can grow even in the presence of 1 µg boronliter^–1 medium. Cell walls of the selected cells seemedto be thicker than those of the control cells and Golgi bodieswere accompanied by more secretory vesicles than those in thecontrol cells. (Received May 25, 1992; Accepted September 10, 1992)     

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L

The sodium iodide symporter (NIS) mediates iodide (I^–) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I^–-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I^– induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I^– (35 µM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na⁺ dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I^– influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I^– influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I^– also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I^– uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I^– may be a useful tool by which to study the pathophysiology and pharmacology of NIS. thyroid; fluorescence microscopy; FRTL-5 cells     

Oxidative Damages and Repair in Euglena gracilis Exposed to Ozone II. Membrane Permeability and Uptake of Metabolites

Significant injuries to the plasma membrane were detected inEuglena gracilis cells during ozone exposure (240 µ1.liter¹,delivery rate of l µmol.min^–1), as assessed by measuringthe alterations of vitamin B₁₂ and acetate uptakes and the leakageof intracellular K⁺ (Rb⁺). A rapid decrease in the uptake ofvitamin B₁₂ and acetate was observed within 15 min of treatment,indicating that both transport systems are very sensitive toO₃. On the other hand, the leakage of intracellular K⁺ ions,as measured by the efflux of ⁸⁶Rb⁺ from prelabelled cells, couldonly be detected after 30 min of O₃ exposure. These resultssuggest that the initial metabolic symptoms of injury is atthe level of the two transport systems examined and that thealteration of the membrane permeability to K⁺ ions appears asa second step in the cascade of oxidative events at the plasmamembrane level. When Euglena cells were allowed to recover underautotrophic growth conditions following O₃ treatment, vitaminB₁₂ and ⁸⁶Rb⁺ (K⁺) ions uptakes returned gradually to controllevel within 5 h of the recovery period. Acetate uptake returnedto control level at a slower rate and needed 20 h for completerecovery. These results indicate that the cells were able toactively repair most of the initial oxidative damages inducedby O₃. The metabolic significance of the repair mechanism(s)is discussed. (Received December 25, 1989; Accepted July 23, 1990)     

Model of beta -cell mitochondrial calcium handling and electrical activity. II. Mitochondrial variables

In the preceding article[Am. J. Physiol. 274 (Cell Physiol. 43):C1158-C1173, 1998], we describe the development of a kinetic model for the interaction of mitochondrial Ca²⁺ handlingand electrical activity in the pancreatic -cell. Here we describefurther results of those simulations, focusing on mitochondrialvariables, the rate of respiration, and fluxes of metabolicintermediates as a function of D-glucose concentration. Oursimulations predict relatively smooth increases of O₂consumption, adenine nucleotide transport, oxidative phosphorylation,and ATP production by the tricarboxylic acid cycle asD-glucose concentrations are increased from basal to 20 mM.On the other hand, we find that the active fraction of pyruvatedehydrogenase saturates, due to increases in matrix Ca²⁺,near the onset of bursting electrical activity and that the NADH/NAD⁺ ratio in the mitochondria increases by roughly anorder of magnitude as glucose concentrations are increased. Themitochondrial ATP/ADP ratio increases by factor of <2 between theD-glucose threshold for bursting and continuousspiking. According to our simulations, relatively small changes inmitochondrial membrane potential (~1 mV) caused by uptake ofCa²⁺ are sufficient to alter the cytoplasmic ATP/ADP ratioand influence ATP-sensitive K⁺ channels in the plasmamembrane. In the simulations, these cyclic changes in the mitochondrialmembrane potential are due to synchronization of futile cycle ofCa²⁺ from the cytoplasm through mitochondria viaCa²⁺ uniporters and Na⁺/Ca²⁺exchange. Our simulations predict steady mitochondrial Ca²⁺concentrations on the order of 0.1 µM at low glucose concentrations that become oscillatory with an amplitude on the order of 0.5 µMduring bursting. Abrupt increases in mitochondrial Ca²⁺concentration >5 µM may occur during continuous electricalactivity.