Dark Metabolism of CO2 during Fibre Elongation of Two Cottons Differing in Fibre Lengths

A study has been made of the dark metabolism of CO₂ by elongatingfibres of Gossypium arboreum L. cv. LD 133 (a short staple type)and Gossypium hirsutum L. cv. LH 372 (a long staple type) atdifferent fibre ages. In both cultivars, phosphoenolpyruvatecarboxylasc, glutamate-oxalacetate transaminase and malate dehydrogenaseshow elevated activities during the period of rapid fibre growthand lowered activity with ageing. Malic enzyme activity increasesas extension growth levels off. Levels of K⁺ and malate riseduring rapid extension growth and fall as the rate of elongationdecreases. The results indicate that malate may act as an osmoticumand a counterion for K⁺ accumulation during rapid expansionof the fibres. Amounts of enzymes, K⁺ and malate are higherin the fibres of the long staple cultivar than the short staple.During the period of active elongation, K⁺/malate ratio is higherin the short staple cultivar. Key words: Gossypium hirsutum, CO₂ metabolism, Fibre extension     

A Histochemical Study of Nectaries of Hibiscus rosa-sinensis

Different histochemical and cytochemical methods were employedon nectaries of Hibiscus rosa-sinensis. Light microscopy revealedthe presence of oil and mucilage cells in the subglandular tissue.Electron microscopy showed intense activity of ATPase in thephloem subtending the nectary. When CaCl₂ or tannic acid areadded to the fixative, electron-dense globular deposits areencountered in close contact with the plasmalemma of the secretorycells. In this case the endoplasmic reticulum appears in alternatingelectron-dense areas. In young nectaries the application oftannic acid results in electron-opaque deposits at the cellplate of dividing cells. The prolonged incubation of nectariesin OsO₄ results in an obvious difference in staining betweennectary hairs and subglandular cells. Structures stained selectivelywith OsO₄ are the endoplasmic reticulum, nuclear envelope, plastids,and mitochondria. The cytochemical experiments support the viewthat in nectaries of Hibiscus rosa-sinensis, the pre-nectaroriginates from the phloem and it is symplastically carriedvia the plasmodesmata to the secretory cells of the hair fromwhere it is secreted. The principal element which is involvedboth in the pre-nectar transport and nectar secretion is theendoplasmic reticulum. Key words: Lipid staining, polysaccharides, tannic acid, calcium binding sites, ATPase activity, osmium impregnation     

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     

Translocation of Carbohydrate in Cotton: Movement to the Fruiting Bodies

The translocation of ¹⁴C-labelled assimilate was followed frommain stem leaves on the cotton plant (Gossypium hirsutum) tobolls on the lower sympodia. From the upper leaves the assimilatedescends the stem in well-defined strands in the phloem. Wheresympodia originate close to these strands some of the assimilateis distributed to the bolls on those sympodia. There is evidencethat the involucre, but not the green boll wall, contributesto boll nutrition by assimilation of external CO₂.     

Plant allocation to defensive compounds: interactions between elevated CO2 and nitrogen in transgenic cotton plants

Plant allocation to defensive compounds in response to growthin elevated atmospheric CO₂ in combination with two levels ofnitrogen was examined. The aim was to discover if allocationpatterns of transgenic plants containing genes for defensivechemicals which had not evolved in the species would respondas predicted by the Carbon Nutrient Balance (CNB) hypothesis.Cotton plants (Gossypium hirsutum L.) were sown inside 12 environmentalchambers. Six of them were maintained at an elevated CO₂ levelof 900 µmol mol^-1 and the other six at the current levelof     

Spatial and Temporal Aspects of Growth in the Primary Root of Cotton Seedlings: Effects of NaCl and CaCl2

Seedlings of cotton (Gossypium hirsutum L. cv. Acala SJ-2) weregrown in modified Hoagland nutrient solution with various combinationsof NaCl and CaCl₂. Marking experiments and numerical analysiswere conducted to characterize the spatial and temporal patternsof cotton root growth at varied Na/Ca ratios. At 1 mol m^–3Ca, 150 mol m^–3 NaCl reduced overall root elongation rateto 60% of the control, while increasing Ca to 10 mol m^–3at the same NaCl concentration restored the elongation rateto 80% of the control. Analysis of the spatial distributionof elongation revealed that the presence of 150 mol m^–3NaCl in the medium shortened the growth zone by about 2 mm fromthe approximate 10 mm in the control and also reduced the relativeelemental elongation rate (i.e. the longitudinal strain rate,defined as the derivatives of displacement velocity of a cellularparticle with respect to position on root axis). Supply of 10mol m^–3 Ca at the high salt condition restored partiallythe relative elemental elongation rate, but not the length ofthe growth zone. Compared to the control, the growth trajectoriesshowed that at 1 mol m^–3 CaCl2 it took more time for acellular particle to move through the growth zone at 150 molm^–3 NaCl, while at 10 mol m^–3 CaCl it took lesstime and there was no difference between the NaCl treatments Key words: Gossypium hirsutum, salinity stress, root growth kinematics     

The Location of the Transporting System for Inorganic Carbon and the Nature of the Form Translocated in Chlamydomonas reinhardtii

The permeability of the plasmalemma of Chlamydomonas reinhardtiicells was increased by treatment with poly-L-lysine or dimethylsulphoxideas indicated by 3-phosphoglyceric acid dependent O₂ evolution.These treatments decreased the ability of the cells to accumulateinorganic carbon internally and hence their photosynthetic affinityfor inorganic carbon in the medium. With saturating light andinorganic carbon, the photosynthetic rate was less affectedby the poly-L-lysine and dimethylsulphoxide treatments. Thusthe poly-L-lysine and dimethylsulphoxide did not alter the activityof the chloroplasts but rather made the intracellular inorganiccarbon pool more freely exchangeable with the medium. It isconcluded that the transporting system for inorganic carbonis located at the plasmalemma. Treatment with Diamox, an inhibitor of carbonic anhydrase, didnot affect photosynthetic rate and accumulation of inorganiccarbon when CO₂ was supplied but strongly inhibited both parameterswhen HCO^–₃ was supplied. In a mutant of Chlamydomonasreinhardtii lacking a cell wall, carbonic anhydrase leaks tothe medium and uptake of inorganic carbon is much faster whenCO₂ is supplied than when HCO^–₃ is supplied. These resultssuggest that CO₂ rather than HCO^–₃ is the inorganic carbonspecies that is actively translocated across the plasmalemma. Key words: Chlamydomonas, Inorganic carbon uptake     

Light-Induced H+ Accumulation in the Vacuole of Nitellopsis obtusa

The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H⁺-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.1–0.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pH_o). At pH_o 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pH_o valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.3–0.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pH_o 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pH_o 9.0 to pH_o 7.5results in a cell hyperpolarization by 60–80 mV and adecrease of the vacuolar pH by 0.4–0.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H⁺ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H⁺ in the cytoplasm. The H⁺ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H⁺ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H⁺ conductance and on the electrochemical gradientfor H⁺ at the plasmalemma.     

Changes of Cell Wall Composition and Polymer Size in Primary Roots of Cotton Seedlings Under High Salinity

The aim of this study was to investigate changes in cell wallchemical composition and polymer size in the root tip of intactcotton seedlings (Gossypium hirsutum L. cv. Acala SJ-2) grownin saline environments, in order to relate the interaction betweenhigh salinity and root growth to possible changes in cell wallmetabolism. Cotton seedlings were grown in modified Hoagland nutrient solutionwith various combinations of NaCl and CaCl₂. Cell walls werefractionated into four fractions (pectin, hemicellulose 1 and2, cellulose), and analysed for their total sugar content, neutralsugar composition and size of polysaccharides. At 1 mol m^–3Ca, 150 mol m^–3 NaCl resulted in a significant increasein the cell wall uronic acid content, but a reduction in cellulosecontent on a per unit dry weight basis. Supplemental Ca overcamethe inhibitory effect of high Na on cellulose content. The neutralsugar composition of the cell wall fractions showed no majorchanges caused by varied Na/Ca ratios. Determinations of polysaccharidepolymer size showed that high Na at 1 mol m^–3 Ca led toan increase in the amount of polysaccharides of intermediatemolecular size and a decrease in that of small size in the hemicellulose1 fraction, indicating a possible inhibition of polysaccharidedegradation by high Na. This change was not observed in the10 mol m^–3 Ca treatments. The results reveal a relationshipbetween the effects of high salinity on root growth and cellwall metabolism, particularly in regard to cellulose biosynthesis Key words: Gossypium hirsutum, salinity, root, cell wall     

Leaf Growth in Cotton (Gossypium hirsutum L.) 1. Growth in Area of Main-stem and Sympodial Leaves

A model is presented for growth of individual and successivemain-stem leaves of cotton, based on a series of indoor experimentsand data sets from the literature. Three variable parametersare used to describe individual leaf growth: relative growthrate of meristematic tissue (R¹), relative rate of approachof final area (R₂) and a ‘position parameter’ (t_0.5)which governs the transition from meristematic to extensiongrowth. Final area of a leaf does not occur in the model asa deterministic quantity but it is a result of the processesduring growth. The model generates successive mainstem leavesand sympodial leaves as an integrated system. Assimilate shortagesoccurring in the plant operate on R₁ leading to the characteristicchange of final leaf area along the mainstem. Gossypium hirsutumL., cotton, leaf growth, relative growth rate, meristematic tissue, extension growth, mathematical model     

Membrane Depolarization by Hexacyanoferrate (III), Hexabromoiridate (IV) and Hexachloroiridate (IV)

Three artificial electron acceptors of different E_o and charge,hexacyanoferrate (III) (K₃Fe(CN)₆), hexachloroiridate (IV) (K₂IrCl₆),and hexabromoiridate (IV) (K₂IrBr₆), were compared with respectto their rate of reduction by roots of Zea mays L., the concomitantproton secretion, and to the effect on plasmalemma depolarization. It has been shown that these plasma membrane impermeable electronacceptors were reduced by a plasmalemma reductase activity.At low concentrations proton secretion was slightly inhibited,at higher concentrations, however, the rate of proton secretionwas stimulated. The root cell plasmalemma showed a transientdepolarization after addition of all three electron acceptors.The depolarization was concentration-dependent for the iridatecomplexes but not for hexacyanoferrate (III). For both iridatecomplexes maximum depolarization was reached at 50 µmoldm^–3. A hypothetical model as an explanation of the redox dependentproton secretion will be given. Key words: Hexachloroiridate (IV), hexabromoiridate (IV), hexacyanoferrate (III), plasmalemma redox, membrane potential, Zea mays     

The Transport and Metabolism of Urea in Chara australis: I. PASSIVE DIFFUSION, SPECIFIC TRANSPORT AND METABOLISM OF UREA, THIOUREA AND METHYLUREA

Most of the urea entering Chara australis cells is rapidly metabolizedto produce CO₂, which diffuses out of the cells into the surroundingmedium. A simple and convenient apparatus to measure both the¹⁴C-urea retained by cells and the ¹⁴CO₂ released into the mediumwas developed and used in a study of urea transport in Chara.The permeability coefficient for urea in the Chara plasmalemmawas estimated from the slope of an uptake versus concentrationfunction as 85 nm s^-1. Computer modelling of urea uptake andmetabolism suggests that this could be a 20% underestimate ofthe true value.The corresponding permeability coefficients forthiourea and N-methyl-urea were estimated in the same way as34 and 35 nm s^-1, respectively. These permeabilities are muchgreater than expected on the basis of either/water partitioncoefficients for the solutes and are consistent with the diffusionof urea and its similarly-sized analogues through aqueous poresin the plasmalemma.At external concentrations of urea less than20 mmol m^-3, the bulk of the uptake is effected by a specifictransport mechanism with an apparent K_m for urea of less than1.0 mmol m^-3. This transport system operates most rapidly withexternal pH in the range 6.5–7.5 and is influenced bythe nitrogen status of the cell.Evidence is produced here suggestingthat the specific transport of urea may be an active process. Key words: Chara, urea uptake, metabolism, diffusion, specific transport     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species     

Photosynthetic Nitrogen Metabolism in High and Low CO2-adapted Scenedesmus: I. INORGANIC CARBON-DEPENDENT O2 EVOLUTION, NITRATE UTILIZATION AND NITROGEN RECYCLING

Larsson, M., Larsson, C.-M. and Guerrero, M. G. 1985. Photosyntheticnitrogen metabolism in high and low CO₂-adapted Scenedesmus.I. Inorganic carbon-dependent O2 evolution, nitrate utilizationand nitrogen recycling.—J. exp Bot. 36: 1373–1386 Scenedesmus obtusiusculus Chod. was grown on an inorganic mediumflushed with either air or air supplemented with 3% CO₂. Inair-grown cells, O₂ evolution dependent on low, but not high,HCO^–₃ concentrations was strongly inhibited by the carbonicanhydrase inhibitor acetazolamide. Cells grown with 3% CO² exhibitedlow rates of O2 evolution at low external inorganic C; however,after 30 min in air O2 evolution rates at low inorganic C approachedthose of air-grown cells. These results are compatible withthe view that Scenedesmus develops a ‘CO² concentratingmechanism’ in air, with carbonic anhydrase as an importantconstituent When 3% CO²-grown cells were subjected to air-level of CO²,just a transient decline in NO^–₃ utilization was observed,but in the presence of acetazolamide the rate of the processdecreased drastically in response to the decrease in the CO²level. In CO²-free air NO₃^– was taken up at high ratesbut in a deregulated manner, leading to release of NH₄⁺. A portionof the NO₃^– taken up in the absence of CO² was apparentlyassimilated Cellular nitrate reductase (NR) activity initially decreasedbut subsequently recovered after a transition from 3% CO² toair. In the presence of acetazolamide, a persistent decreasein NR activity was observed. Cellular glutamine synthetase (GS)activity increased after transition from 3% CO² to air, theactivity increase being unaffected by acetazolamide. NH₄⁺ releaseto the medium in the presence of L-methionine-D, L-sulphoximine(MSO) transiently increased in 3% CO²-grown cells in responseto a transfer to air. MSO-induced NH₄⁺ release was in fact higherin air-grown cells than in 3% CO²-grown cells. Glycollate wasinitially released after transition from 3% CO² to air, butthere was no difference in glycollate release between MSO-treatedand untreated cells. In air-adapted Scenedesmus, N recyclingseems to be of minor importance in comparison to primary N assimilation Key words: CO²-fixation, N recycling, nitrate uptake, Scenedesmus     

Evaluation of Arsenate- and Vanadate-Associated Changes of Electrical Membrane Potential and Phosphate Transport in Lemna gibba G1

Interference of arsenate and vanadate with phosphate uptakein Lemna gibba L. was studied by measuring voltage changes and(³²P)phosphate uptake. Arsenate proved to be competitive withthe high- and low-affinity phosphate uptake system. It inducedtransient membrane potential changes of up to 120 mV which weresimilar to those induced by phosphate and indicated a cotransportmechanism with at least 2H+/H₂As. The amplitude of the transient arsenate-induced membrane depolarization wasstrongly influenced by phosphate starvation. A permanent membranedepolarization to the diffusion potential was achieved within2 to 6 h in P-starved plants. Thus, arsenate is indeed a stronglycompetitive physiological analogue of phosphate in higher plants. Vanadate was easily transported into L. gibba as concluded fromtransient E_m changes of up to 110 mV. Vanadate interfered onlyslightly and non-specifically with the two phosphate transportmechanisms. Like phosphate, vanadate uptake seems to be an H+-cotransportmechanism, both with similar optima at pH 6.0. Unlike phosphateuptake, vanadate-linked membrane depolarization was not affectedby high intracellular phosphate concentrations. P-starvationdid not enhance the weak long-term effect on E_m. Hence, vanadate,in contrast to arsenate, is not regarded as a physiologicalphosphate analogue. The distinct and rapid vanadate-inducedand permanent membrane depolarization of Avena sativa, Triticumaestivum and Glycine max leaves was not seen in Lemna nor inleaves of Gossypium hirsutum and Nicotiana tabacum. Plasmalemma-enrichedpreparations of L. gibba revealed, however, a high vanadate-sensitiveATPase activity (87%). As a possible explanation for these differencesit is suggested that the latter plant species have cytosolicvanadate-detoxifying properties, i.e. they can reduce vanadateto vanadyl ions, in contrast to the former group of plant species. Key words: Arsenate, vanadate, H⁺/solute cotransport, membrane potential, phosphate competition     

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     

RNA Synthesis in Phaseolus Chloroplasts: I. RIBONUCLEIC ACID SYNTHESIS IN CHLOROPLAST PREPARATIONS FROM PHASEOLUS VULGARIS L. LEAVES AND SOLUBILIZATION OF THE RNA POLYMERASE

Chloroplast preparations from the young primary leaves of Phaseolusvulgaris L. cv. Canadian Wonder carry out the DNA-dependentincorporation of UTP into RNA at rates between 8 and 14 pmolUTP µg^–1 chlorophyll h^–1. It is estimatedthat 90% of the activity was localized in the chloroplasts.The incorporation proceeded for between 20 and 30 min at 35°C. The maximum rates of RNA synthesis were attained atpH 8.3, in the presence of 15 mM MgCl₂. Chloroplasts were alsoactive, to a lesser extent, with 1.5 mM MnCl₂. The simultaneouspresence of MnCl₂ and MgCl₂ resulted in inhibition of activity.Nuclear material prepared from young P. vulgaris leaves incorporatedUTP at a rate of about 12 pmol UTP µg^–1 DNA h^–1.On a chloroplast (Tritonsoluble) DNA basis chloroplast activitywas over 40-fold that of nuclei. Methods of solubilizing chloroplastRNA polymerase were explored. Yields of over 75% were achieved,but methods suitable for one species were not always successfulwhen applied to another. The highest yields of the P. vulgarisenzyme were obtained using EDTA and KCl. All methods resultedin solubilization of DNA. RNA synthesis by the soluble P. vulgarisenzyme proceeded for more than 40 min at 35 °C.     

Chloride Transport in Chara: I. KINETICS AND CURRENT-VOLTAGE CURVES FOR A PROBABLE PROTON SYMPORT

Chara cells show an inward positive electric current acrossthe plasmalemma when exposed to Cl^– under voltage-clampconditions. The rapid rise of this current suggests that itis directly associated with the inward transport of Cl^–.The dependence of the current on Cl^– concentration showssaturation, the data fitting the Michaelis-Menten equation withV_m up to 100 nmol m^–2 s^–1 (for Cl^–starvedcells) with K_M 10–20 µM, and with some allowancefor an unstirred layer of water adjacent to the membrane. Theeffects on the current of clamp potential, illumination, withdrawalof alkali metal cations, and addition of amine were also investigated.These results suggest that the mechanism is the symport of 2H⁺ with each Cl^–, and that the actions of light, externalK⁺, and amine in stimulating Cl^–, influx are indirect.     

Role of Carbonic Anhydrase and Identification of the Active Species of Inorganic Carbon Utilized for Photosynthesis in Chora corallina

Carbonic anhydrase (CA, EC. 4.2.1.1 [EC] ) activity in air-grown Characorallina was detected mainly in the intracellular fraction,most of which composed of chloroplasts and cytoplasmic gel,and not on the cell surface. Only minor levels of CA activity,on the basis of equivalent volumes, were detected in the cellsap and the cytoplasmic sol. The maximum rate of photosynthetic O₂ evolution by air-grownChara corallina at pH 6.0 was twice that at pH 7.6, while theapparent K_m for external inorganic carbon (C_i) at pH 7.6 wasabout three times that at pH 6.0. However, the apparent K_m(CO₂)was about three times larger at pH 6.0 than at pH 7.6. The K_m(C_i)-valueat pH 7.6 increased severalfold in the presence of acetazolamide(AZA), an inhibitor of CA, but no inhibition was observed atpH 6.0. The pH-dependence may be due to differences in the permeabilityof AZA at the given pH values. Fixation of ¹⁴CO₂ at 20 µMand of H¹⁴CO₃^– at 200 µM over the course of 5 swas very similar at pH 7.4. Addition of CA significantly suppressedthe photosynthetic ¹⁴CO₂-fixation but it stimulated the H¹⁴CO₃^–-fixation.This result indicates that free CO₂ is an active species ofC_i that is incorporated into the cell during photosynthesis. These results together suggest the following: (1) Free CO₂ isutilized for photosynthesis, (2) CA is mainly located insidethe cell and functions to increase the affinity for CO₂ in photosynthesisby facilitating the supply of CO₂ from the plasmalemma to thesite of CO₂-fixation. ³Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Chiba, 260 Japan. (Received December 9, 1988; Accepted March 22, 1989)     

Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore

The effects ofmaitotoxin (MTX) on plasmalemma permeability are similar to thosecaused by stimulation of P2Z/P2X₇ionotropic receptors, suggesting that1) MTX directly activatesP2Z/P2X₇ receptors or2) MTX andP2Z/P2X₇ receptor stimulationactivate a common cytolytic pore. To distinguish between these twopossibilities, the effect of MTX was examined in1) THP-1 monocytic cells before andafter treatment with lipopolysaccharide and interferon-, a maneuverknown to upregulate P2Z/P2X₇receptor, 2) wild-type HEK cells andHEK cells stably expressing theP2Z/P2X₇ receptor, and3) BW5147.3 lymphoma cells, a cellline that expresses functional P2Z/P2X₇ channels that are poorlylinked to pore formation. In control THP-1 monocytes, addition of MTXproduced a biphasic increase in the cytosolic freeCa²⁺ concentration([Ca²⁺]_i);the initial increase reflects MTX-inducedCa²⁺ influx, whereas the secondphase correlates in time with the appearance of large pores and theuptake of ethidium. MTX produced comparable increases in[Ca²⁺]_iand ethidium uptake in THP-1 monocytes overexpressing theP2Z/P2X₇ receptor. In bothwild-type HEK and HEK cells stably expressing theP2Z/P2X₇ receptor, MTX-inducedincreases in[Ca²⁺]_iand ethidium uptake were virtually identical. The response of BW5147.3cells to concentrations of MTX that produced large increases in[Ca²⁺]_ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- andBz-ATP-induced pores activate with similar kinetics and exhibit similarsize exclusion. Last, MTX-induced pore formation, but not channelactivation, is greatly attenuated by reducing the temperature to22°C, a characteristic shared by theP2Z/P2X₇-induced pore. Together,the results demonstrate that, although MTX activates channels that aredistinct from those activated byP2Z/P2X₇ receptor stimulation, thecytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.