ATP-Dependent Ca2+ Transport in Tonoplast Vesicles from Apple Fruit

Protoplasts and vacuoles were isolated from immature apple fruit(Malus pumila Mill. cv. Golden Delicious). ATP-stimulated Ca²⁺uptake was identified in both protoplast vesicles and tonoplastvesicles. The apparent K_m for Ca²⁺ of the tonoplast transportsystem was 43.4 µM. The pH optima were 7.2 and 6.7 forCa²⁺ transport by protoplast and tonoplast vesicles, respectively.Ca²⁺ transport in tonoplast vesicles was strongly inhibitedby the calmodulin antagonists fluphenazine and N-(6-aminohexyl)-5-chloro-l-naphthalensulfonamidehydrochloride (W-7), while N-aminohexyl)-l-naphthalensulfonamidehydrochloride (W-5) was relatively ineffective. Addition ofexogenous calmodulin stimulated transport by 35%. Ca²⁺ uptakewas inhibited by vanadate, but not by the ionophores carbonylcyanidem-chlorophenyl hydrazone (CCCP) or valinomycin. The resultsindicate that apple tonoplasts have a Ca²⁺ transport systemthat is driven by the direct hydrolysis of ATP, and may be calmodulindependent. ¹Present address: Morioka Branch, Fruit Tree Research Station,Ministry of Agriculture, Forestry and Fisheries, Shimokuriyagawa,Morioka 020-01, Japan. To whom reprint requests should be addressed. (Received October 18, 1985; Accepted January 29, 1986)     

Control of Glutamate Dehydrogenase from Green Tobacco Callus Mitochondria by Ca2$ and pH

Glutamate dehydrogenase (GDH) (EC 1.4.1.3 [EC] .) purified from greentobacco callus mitochondria was activated markedly by Ca^2$ inthe amination reaction. This activation was detectable evenat concentrations below 5 µM Ca^2$. Saturation curves for the three substrates of the aminationreaction showed normal Michaelis-Menten kinetics in the presenceof 1 mM of Ca^2$, but pronounced substrate inhibition occurredwithout Ca^2$. The effect of Ca^2$ was chiefly on the maximalvelocity. The saturation curve for NH₄Cl in the presence of Ca^2$ was modulatedby a change in pH. The apparent K_m value for NH₄Cl markedlydecreased whereas that for -ketoglutarate increased slightlywhen the pH was raised from 7.3 to 9.0. In contrast, the K_mfor NADH was little affected by raising the pH. The characteristicof GDH which increases its affinity for NH₄Cl when the pH israised may be compatible with the detoxification of ammonia. ¹ Present address: Mochida Pharmaceutical Co., Ltd. (Received August 24, 1981; Accepted November 28, 1981)     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

Characterization, Functional Reconstitution and Activation by Fusicoccin of a Ca2+-ATPase from Corydalis sempervirens Pers. Cell Suspension Cultures

Cell suspension cultures of Corydalis sempervirens have provenideal for the study of fusicoccin action [Schulz et al. (1990)Planta 183: 83] and express the fusicoccin-binding protein aswell as a plasma membrane H⁺-ATPase which is activated by thefungal toxin. Microsomal vesicles prepared from these cellsaccumulate Ca²⁺ in the presence of Mg-ATP. The protonophorecar-bonylcyanide m-chlorophenylhydrazone did not inhibit theMg-ATP dependent Ca²⁺-transport into the vesicles. This processis thus due to the activity of at least one primary active,ATP-driven, Ca²⁺-pump. The enzyme was characterized in detail.It has a pH optimum of 7.2, an apparent K_m of 0.3 mu (ATP),12pm (Ca²⁺), accepts ATP>ITP GTP>CTP UTP, and is strongly(Kⁱ, app 0.75 µmM) inhibited by erythrosine B but lessso (Kⁱ, app 95 µM) by or-thovanadate. These characteristicsare typical for the plasma membrane Ca²⁺-ATPase characterizedfrom differentiated tissues [Graf and Weiler (1990) Physiol.Plant. 75: 634]. Fusicoccin activates the erythrosine-sensitiveCa²⁺-pump by lowering its K_m for ATP, when added to living cellsprior to tissue homogenization. Thus, fusicoccin appears toactivate at least two ion-translocating ATPases in one and thesame tissue, suggesting that the toxin's mechanism of actionis complex and not restricted to activation of the H⁺-ATPase.FC has no effect when administered to microsomes. The microsomalenzyme was solubilized and reconstituted into asolec-tin liposomesin functional form. The reconstituted, erythrosine sensitiveCa²⁺-ATPase was insensitive to fusicoccin. Thus, componentsessential for toxin action are either lost or inactivated duringsubcellular fractionation. It is likely that FC action requiressoluble components. (Received April 22, 1991; Accepted July 24, 1991)     

Measurement of Changes in Cytosolic Ca2+ in Arabidopsis Guard Cells and Mesophyll Cells in Response to Blue Light

Phototropins (phot1 and phot2) are blue light (BL) receptorsthat mediate responses including phototropism, chloroplast movementand stomatal opening, and increased cytosolic Ca²⁺. BL absorbedby phototropins activates plasma membrane H⁺-ATPase in guardcells, resulting in membrane hyperpolarization, and drives K⁺uptake and stomatal opening. However, it is unclear whetherthe phototropin-mediated Ca²⁺ increase activates the H⁺-ATPase.Here, we determined cytosolic Ca²⁺ concentrations in guard cellprotoplasts (GCPs) from Arabidopsis transformed with aequorin.Cytosolic Ca²⁺ increased rapidly in response to BL in GCPs fromboth the wild type and phot1 phot2 double mutants, but was mostlysuppressed by an inhibitor of photosynthetic electron flow (DCMU).With depleted external K⁺, we observed another slower Ca²⁺ increase,which was phototropin- dependent. Fusicoccin, a H⁺-ATPase activator,mimicked the effect of BL. The slow Ca²⁺ increase thus appearsto result from membrane hyperpolarization. The slow Ca²⁺ increasewas suppressed by external K⁺ and was restored by blockers ofinward-rectifying K⁺ channels, CsCl and tetraethylammonium,suggesting the preferential uptake of K⁺ over Ca²⁺. Such efficientK⁺ uptake in response to BL was not found in mesophyll cells.Both the fast and the slow Ca²⁺ increases were inhibited byCa²⁺ channel blockers (CoCl₂ and LaCl₃) and a chelating agent(EGTA). These results indicate that the phototropin-mediatedCa²⁺ increase was not observed prior to H⁺-ATPase activationin guard cells and that Ca²⁺ entered guard cells via Ca²⁺ channelsthrough photosynthesis and phototropin-mediated membrane hyperpolarization.     

Plasma Membrane H+-ATPase in Guard-Cell Protoplasts from Vicia faba L.

The activity of plasma membrane H⁺-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO₃^–and vanadate. In the presence of oligomycin, ammonium molybdateand NO₃^–, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca²⁺, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theK_m was 0.5 mM for ATP. The activity required Mg²⁺ but was notaffected by K⁺, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)^–1.h^–1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H⁺-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987)     

Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes

Decoding of fast cytosolic Ca²⁺ concentration ([Ca²⁺]_i) transients by mitochondria was studied in permeabilized cat ventricular myocytes. Mitochondrial [Ca²⁺] ([Ca²⁺]_m) was measured with fluo-3 trapped inside mitochondria after removal of cytosolic indicator by plasma membrane permeabilization with digitonin. Elevation of extramitochondrial [Ca²⁺] ([Ca²⁺]_em) to >0.5 µM resulted in a [Ca²⁺]_em-dependent increase in the rate of mitochondrial Ca²⁺ accumulation ([Ca²⁺]_em resulting in half-maximal rate of Ca²⁺ accumulation = 4.4 µM) via Ca²⁺ uniporter. Ca²⁺ uptake was sensitive to the Ca²⁺ uniporter blocker ruthenium red and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and depended on inorganic phosphate concentration. The rates of [Ca²⁺]_m increase and recovery were dependent on the extramitochondrial [Na⁺] ([Na⁺]_em) due to Ca²⁺ extrusion via mitochondrial Na⁺/Ca²⁺ exchanger. The maximal rate of Ca²⁺ extrusion was observed with [Na⁺]_em in the range of 20–40 mM. Rapid switching (0.25–1 Hz) of [Ca²⁺]_em between 0 and 100 µM simulated rapid beat-to-beat changes in [Ca²⁺]_i (with [Ca²⁺]_i transient duration of 100–500 ms). No [Ca²⁺]_m oscillations were observed, either under conditions of maximal rate of Ca²⁺ uptake (100 µM [Ca²⁺]_em, 0 [Na⁺]_em) or with maximal rate of Ca²⁺ removal (0 [Ca²⁺]_em, 40 mM [Na⁺]_em). The slow frequency-dependent increase of [Ca²⁺]_m argues against a rapid transmission of Ca²⁺ signals between cytosol and mitochondria on a beat-to-beat basis in the heart. [Ca²⁺]_m changes elicited by continuous or pulsatile exposure to elevated [Ca²⁺]_em showed no difference in mitochondrial Ca²⁺ uptake. Thus in cardiac myocytes fast [Ca²⁺]_i transients are integrated by mitochondrial Ca²⁺ transport systems, resulting in a frequency-dependent net mitochondrial Ca²⁺ accumulation. mitochondrial Ca²⁺; excitation-contraction coupling; cardiomyocytes     

Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration

Polyamines are essential for cell migrationduring early mucosal restitution after wounding in the gastrointestinaltract. Activity of voltage-gated K⁺ channels (Kv) controlsmembrane potential (E_m) that regulates cytoplasmicfree Ca²⁺ concentration([Ca²⁺]_cyt) by governing thedriving force for Ca²⁺ influx. This study determinedwhether polyamines are required for the stimulation of cell migrationby altering K⁺ channel gene expression,E_m, and[Ca²⁺]_cyt in intestinal epithelialcells (IEC-6). The specific inhibitor of polyamine synthesis,-difluoromethylornithine (DFMO, 5 mM), depleted cellularpolyamines (putrescine, spermidine, and spermine), selectivelyinhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression,and resulted in membrane depolarization. Because IEC-6 cells did notexpress voltage-gated Ca²⁺ channels, the depolarizedE_m in DFMO-treated cells decreased [Ca²⁺]_cyt as a result of reduceddriving force for Ca²⁺ influx through capacitativeCa²⁺ entry. Migration was reduced by 80% in thepolyamine-deficient cells. Exogenous spermidine not only reversed theeffects of DFMO on Kv1.1 channel expression, E_m,and [Ca²⁺]_cyt but also restoredcell migration to normal. Removal of extracellular Ca²⁺ orblockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cellmigration by exogenous spermidine in polyamine-deficient cells. Theseresults suggest that polyamine-dependent intestinal epithelial cellmigration may be due partially to an increase of Kv1.1 channelexpression. The subsequent membrane hyperpolarization raises[Ca²⁺]_cyt by increasing the drivingforce (the electrochemical gradient) for Ca²⁺ influx andthus stimulates cell migration.

     

Repression of the K+ Uptake and Cation-Stimulated ATPase Activity Associated with the Plasma Membrane-Enriched Fraction of Cucumber Roots Due to Ca2+ Starvation

The uptake of K⁺ by cucumber plants decreased markedly duringCa²⁺ starvation. A plasma membrane-enriched fraction, judgedfrom the distribution of marker enzymes, was prepared from controland Ca²⁺-starved roots. The Mg²⁺- and K⁺-Mg²⁺-ATPase activitiesassociated with the plasma membrane-enriched fraction of controlroots were maxima at pH 6.5. Various monovalent cations andpotassium salts of monovalent anions stimulated Mg²⁺-ATPaseactivity. Vanadate, DES and DCCD inhibited K⁺- Mg²⁺-ATPase activity.Of the divalent cations and phosphate esters tested, Mg²⁺ andATP were most effective for the stimulation of ATPase by K⁺,whereas Ca²⁺ was ineffective in replacing Mg²⁺. Mg²⁺- and K⁺-Mg²⁺-ATPase activities associated with the plasmamembrane enriched fraction of Ca²⁺-starved roots were much lowerthan those of control roots. K_m values of K⁺-Mg²⁺-ATPase forATP were comparable for control and Ca²⁺-starved roots. The K⁺-stimulated activity of Mg²⁺-ATPase in Ca²⁺-starved rootswas approximately one fourth that of the control, whereas therate of stimulation was only slightly lower in Ca²⁺-starvedroots. (Received May 9, 1984; Accepted September 17, 1984)     

Repression of Proton extrusion from Intact Cucumber Roots and the Proton Transport Rate of Microsomal Membrane Vesicles of the Roots due to Ca2+ Starvation

Proton extrusion from cucumber roots decreased markedly duringCa²⁺ starvation in the presence of KC1. Vesicles with ATP-dependentproton transport activity were prepared from the microsomalmembrane fraction of control and Ca²⁺-starved roots. The protontransport rate of the vesicles from Ca²⁺-starved roots was repressedto less than half of the vesicles prepared from the controlroots. K⁺-Mg²⁺-ATPase activity associated with the vesiclesprepared from Ca²⁺-starved roots was approximately one-thirdof the activity associated with those prepared from controlroots. K_m values of the proton transport rate and K⁺-Mg²⁺-ATPasefor ATP were much higher in vesicles prepared from Ca²⁺-starvedroots. The repression of proton extrusion linked with K⁺ uptake inthe Ca²⁺-starved roots could be largely caused by the reducedproton pumping activity associated with microsomal membranesin the roots. (Received May 25, 1987; Accepted October 14, 1987)     

Stimulatory Effect of Chloride Ions on NADP Malic Enzyme from Leaves of two C4 Species of Cyperus

NADP malic enzyme (EC 1.1.1.40 [EC] ) from leaves of two C₄ speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl^–. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na₂SO₄ and stimulated by relatively high concentration ofTris-HCl (50–100 mM, pH 7–8), the activation ofthe enzyme by NaCl appears to be due to Cl^–. Variationsin the concentration of Mg²⁺ affected the K_A (the concentrationof activator giving half-maximal activation) for Cl^–,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg²⁺ from 0.5 mM to 7 mM. The K_m for Mg²⁺ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of V_max was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC₄ species, was able to utilize Mn²⁺. The K_m for Mn²⁺ was 5mM, a value similar to that for Mg²⁺. The addition of 91 mMNaCl markedly decreased the K_m for Mn²⁺ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Cl^–than other C₄ species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989)     

ATP-Promoted Sorbitol Transport into Vacuoles Isolated from Apple Fruit

Sorbitol was transported actively into vacuoles isolated fromapple (Malus pumilla Mill, var domestica Schneid.) fruit flesh.The uptake was stimulated up to twofold by the addition of ATP,and the ATP dependent uptake showed a saturation curve as tothe substrate concentration. The optimum uptake of sorbitolwas pursued in the acidic range of pH 5 to 6. The K_m value forthe ATP dependent sorbitol uptake was about 5 mM. Sorbitol uptake was clearly inhibited by PCMB and uncouplers(CCCP and DCCD), and to a lesser extent by orthovanadate, butonly slightly by oligomycin. K⁺ stimulated sorbitol uptake.Sorbitol was converted to other sugars (glucose) only very slowlywhen transported across the tonoplast. This suggests that sorbitolis transported into vacuoles by a carrier mediated transportsystem coupled with H⁺- ATPase, localized on the tonoplast.Sucrose uptake into the vacuoles was also enhanced by ATP. (Received May 31, 1986; Accepted March 2, 1987)     

Characterization of Hexose Transporter for Facilitated Diffusion of the Tonoplast Vesicles from Pear Fruit

Tonoplast vesicles were prepared from the flesh tissue of maturepear fruit. Sugar uptakes into the vesicles determined by twodifferent methods, the membrane and the gel filtration methods,were quite similar. The uptake was highest for glucose and subsequently,in order, for fructose, sucrose and sorbitol. It was not stimulatedby addition of ATP, although the vesicles could create a protongradient. However, the uptakes were significantly inhibitedby p-chloromercuribenzene sulphonate (PCMBS, SH-reagent andinhibitor of sugar transporter). Further, the PCMBS-sensitiveuptakes of glucose and fructose saturated with their increasedconcentrations. Thus, these PCMBS-sensitive uptakes are mediatedby the transporter of facilitated diffusion. The uptakes ofglucose or fructose each had two K_m values. K_m values for glucosewere 0.35 and 18 mM, and those for fructose were 1.6 and 25raM. The uptake of 0.2 mM glucose was inhibited by 2 mM fructoseand that of 2 mM fructose was inhibited by 2 mM glucose, butneither was inhibited by sucrose or sorbitol. O-methyl-glucose(OMG) also inhibited both the glucose and fructose uptakes.Therefore, the same transporter may mediate both glucose andfructose uptakes at lower concentrations; this hexose transportsystem differed from the sucrose and sorbitol transport systems. ¹Research Fellow of the Japan Society for the Promotion of Science. ²Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan.     

Role of Coccoliths in the Utilization of Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi: a Survey Using Intact Cells and Protoplasts

The utilization of inorganic carbon and role of the coccolithswere investigated in intact cells and protoplasts of a marineunicellular calcareous alga, Emiliania huxleyi. Protoplastswith high photosynthetic activity were obtained by artificialdecalcification with 50 mM MES-NaOH (pH5.5). (1) The kineticsof the photosynthetic evolution of O₂ at various concentrationsof externally added NaHCO₃ were the same for intact cells andprotoplasts, indicating that the kinetic properties with respectto dissolved inorganic carbon (DIC) were not affected by thepresence or absence of the coccoliths on the cell surface. Double-reciprocalplots and plots of the concentration of substrate divided byvelocity (s/v) against the concentration of substrate (s) werebiphasic in the case of both intact cells and protoplasts. TheCO₂-utilization reaction was, therefore, considered to involvetwo processes with different values of K_m and V_max. From thekinetic analyses, K_m and V_max [µmoles O₂ (ml PCV)^–1h^–1] were deduced to be 92 µM and 76.3 for a "low-K_m"reaction and 4.1 mM and 252 for a "high-K_m" reaction, respectively.(2) In short-term (40-min) experiments, time courses of thetotal uptake of ¹⁴C-DIC and the incorporation of ¹⁴C into acid-stableproducts of photosynthesis and the internal pool of DIC, determinedas acid-labile compounds, under CO₂-limiting conditions (80µM) were very similar for intact cells and protoplasts.However, incorporation of ¹⁴C into CaCO₃ apparently occurredmore slowly in protoplasts than in intact cells. (3) In longterm (24-h) experiments, patterns of incorporation of ¹⁴C werealmost same for intact cells and protoplasts, with the exceptionthat the amount of ¹⁴C incorporated into CaCO₃ was much smallerin the former than the latter. The production of Ca¹⁴CO₃ increasedduring the course of 10 h after a 4-h lag. However, after 10h the level of Ca¹⁴CCO₃ started to decrease. The decrease wasaccompanied by an increase in ¹⁴C in the products of photosynthesis,suggesting that CaCO₃ was reutilized for the photosyntheticfixation of CO₂ and, therefore, that the coccoliths functionas sites of storage of DIC. However, the internal level of DICremained at the same level even after the supply of externalDIC has been almost completely depleted. (Received July 25, 1995; Accepted December 11, 1995)     

Activation of K+ channels induces apoptosis in vascular smooth muscle cells

Intracellular K⁺ playsan important role in controlling the cytoplasmic ion homeostasis formaintaining cell volume and inhibiting apoptotic enzymes in thecytosol and nucleus. Cytoplasmic K⁺ concentration is mainlyregulated by K⁺ uptake viaNa⁺-K⁺-ATPase and K⁺ efflux throughK⁺ channels in the plasma membrane. Carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), a protonophorethat dissipates the H⁺ gradient across the inner membraneof mitochondria, induces apoptosis in many cell types. In ratand human pulmonary artery smooth muscle cells (PASMC), FCCP opened thelarge-conductance, voltage- and Ca²⁺-sensitiveK⁺ (maxi-K) channels, increased K⁺ currentsthrough maxi-K channels [I_K(Ca)], and inducedapoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM)decreased I_K(Ca) by blocking the sarcolemmalmaxi-K channels and inhibited the FCCP-induced apoptosis inPASMC cultured in media containing serum and growth factors.Furthermore, inhibition of K⁺ efflux by raisingextracellular K⁺ concentration from 5 to 40 mM alsoattenuated PASMC apoptosis induced by FCCP and theK⁺ ionophore valinomycin. These results suggest thatFCCP-mediated apoptosis in PASMC is partially due to anincrease of maxi-K channel activity. The resultant K⁺ lossthrough opened maxi-K channels may serve as a trigger for cellshrinkage and caspase activation, which are major characteristics ofapoptosis in pulmonary vascular smooth muscle cells.

     

Bafilomycin A1 is a non-competitive inhibitor of the tonoplast H+-ATPase of maize coleoptiles

When microsomal membranes from maize (Zea mays L. cv. Clipper)coleoptiles were separated by isopyc-nic centrifugation on acontinuous 10–45% sucrose gradient, bafilomycin A1-inhibitedATPase activity co-localized with the activities of the tonoplastmarker-enzymes, nitrate-Inhibited ATPase and K⁺-dependent pyrophosphatase.Thus, bafilomycin A1 is a specific inhibitor of the vacuolarH⁺-ATPase of maize coleoptiles. Inhibition of the vacuolar H⁺-ATPaseby bafilomycin A1 was strictly dependent upon the concentrationof the enzyme present in the assay medium, suggesting a stoichiometricassociation between bafilomycin A1 and the vacuolar H⁺-ATPase.In tonoplast-enriched preparations, half-maximal inhibitionwas obtained at 43 pmol bafilomycin A1 mg^–1 protein. BafilomycinA1 inhibited the vacuolar H⁺-ATPase in a simple non-competitivemanner: increasing bafilomycin A1 concentrations reduced theV_max, of the H+ -ATPase, but had no effect on its K_m towardsATP. Key words: Bafilomycin A1, coleoptile, H⁺-ATPase (vacuolar), maize, Zea mays L     

Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification

The mechanism involved inN-methyl-D-glucamine(NMDA)-induced Ca²⁺-dependentintracellular acidosis is not clear. In this study, we investigated indetail several possible mechanisms using cultured rat cerebellargranule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].When 100 µM NMDA or 40 mM KCl was added, a marked increase in theintracellular Ca²⁺ concentration([Ca²⁺]_i)and a decrease in the intracellular pH were seen. Acidosis wascompletely prevented by the use ofCa²⁺-free medium or1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca²⁺. The following fourmechanisms that could conceivably have been involved were excluded:1)Ca²⁺ displacement of intracellularH⁺ from common binding sites;2) activation of an acid loader or inhibition of acid extruders; 3)overproduction of CO₂ or lactate; and 4) collapse of the mitochondrialmembrane potential due to Ca²⁺uptake, resulting in inhibition of cytosolicH⁺ uptake. However,NMDA/KCl-induced acidosis was largely prevented by glycolyticinhibitors (iodoacetate or deoxyglucose in glucose-free medium) or byinhibitors of the Ca²⁺-ATPase(i.e.,Ca²⁺/H⁺exchanger), including La³⁺,orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca²⁺-ATPaseis involved in NMDA-induced intracellular acidosis in granule cells. Wealso provide new evidence that NMDA-evoked intracellular acidosisprobably serves as a negative feedback signal, probably with theacidification itself inhibiting the NMDA-induced[Ca²⁺]_i increase.

     

Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation

Pulmonary vasoconstriction and vascularmedial hypertrophy greatly contribute to the elevated pulmonaryvascular resistance in patients with pulmonary hypertension. A rise incytosolic free Ca²⁺ ([Ca²⁺]_cyt)in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (E_m) regulates[Ca²⁺]_cyt by governing Ca²⁺influx through voltage-dependent Ca²⁺ channels. Thusintracellular Ca²⁺ may serve as a shared signaltransduction element that leads to pulmonary vasoconstriction andvascular remodeling. In PASMC, activity of voltage-gated K⁺(Kv) channels regulates resting E_m. In thisstudy, we investigated whether changes of Kv currents[I_K(V)], E_m, and[Ca²⁺]_cyt affect cell growth by comparingthese parameters in proliferating and growth-arrested PASMC. Serumdeprivation induced growth arrest of PASMC, whereas chelation ofextracellular Ca²⁺ abolished PASMC growth. Resting[Ca²⁺]_cyt was significantly higher, andresting E_m was more depolarized, inproliferating PASMC than in growth-arrested cells. Consistently, wholecell I_K(V) was significantly attenuated in PASMCduring proliferation. Furthermore, E_mdepolarization significantly increased resting[Ca²⁺]_cyt and augmented agonist-mediatedrises in [Ca²⁺]_cyt in the absence ofextracellular Ca²⁺. These results demonstrate that reducedI_K(V), depolarized E_m, and elevated [Ca²⁺]_cyt may play a criticalrole in stimulating PASMC proliferation. Pulmonary vascular medialhypertrophy in patients with pulmonary hypertension may be partlycaused by a membrane depolarization-mediated increase in[Ca²⁺]_cyt in PASMC.

     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)     

Potassium-Ammonium Uptake Interactions in Tobacco Seedlings

Short-term (< 12 h) uptake experiments were conducted with6–7-week-old tobacco (Nicotiana tabacum L. cv. Ky 14)seedlings to determine absorption interactions between K⁺ andNH₄⁺. At equal solution concentrations (0.5 mol m^–3) netK⁺ uptake was inhibited 30–35% by NH₄⁺ and NH₄⁺ uptakewas decreased 9–24%. Removal of NH₄⁺ resulted in completerecovery in K⁺ uptake rate, but NH⁴⁺ uptake rate did not recoverwhen K⁺ was removed. In both cases, inhibition of the uptakerate of one cation saturated as the concentration of the othercation was increased up to 0.5 mol m^–3. The relative effectof K⁺-NH₄⁺ interactions was not altered when Cl- was replacedwith SO₄^2–, but the magnitudes of the uptake rates wereless in the absence of Cl-. The V_max for NH₄⁺ uptake was reducedfrom 128 to 105 µmol g^–1 dry wt. h^–1 in thepresence of 0.5 mol m^–3 K⁺ and the K_m for NH₄⁺ doubledfrom 12 to 27 mmol m^–3 in the presence of K⁺. The resultsof these K⁺-NH₄⁺ experiments are interpreted as mixed-noncompetitiveinteractions. However, an enhanced efflux of K⁺ coupled to NH₄⁺influx via an antiporter cannot be ruled out as contributingto the decrease in net K⁺ uptake. Key words: Nicotiana tabacum, K⁺, NH₄⁺, Uptake interactions