Effects of Calcium Ion Concentration on Cel Wall Synthesis

Cuttings of 6-week-old Norway spruce (Picea abies (L.) Karst.)seedlings were placed in liquid media containing various concentrationsof Ca²⁺. Cytoplasmic concentrations of Ca²⁺ were manipulatedusing the ionophore A 23 187. The effects of Ca²⁺ concentrationson the deposition of total cell wall material as well as onthe deposition of cellulose, lignin, and non-cellulosic polysaccharidesin the hypocotyls were investigated. At low concentrations ofCa²⁺ wall deposition was reduced, mainly as a result of theinhibition of lignin and non-cellulosic polysaccharide deposition.High concentrations of Ca²⁺ stimulated non-cellulosic polysaccharideand lignin deposition, whereas cellulose deposition was almosttotally inhibited. Key words: Conifers, calcium, cell wall, lignin, cellulose     

Salinity Effects on the Activity of Plasma Membrane H+and Ca2+Transporters in Bean Leaf Mesophyll: Masking Role of the Cell Wall

Net fluxes of H⁺and Ca²⁺were measured in the mesophyll tissueof broad bean (Vicia faba L.) leaves and in protoplasts derivedfrom these cells. NaCl at 90 m M enhanced H⁺extrusion in bothprotoplasts and tissue, but in different ways. Proton extrusionwas inhibited by vanadate, suggesting the involvement of theplasma membrane H⁺-ATPase in cell responses to salinity. Therewas virtually no effect of NaCl on the net Ca²⁺flux in protoplasts,while in the tissue a large transient Ca²⁺efflux followed thesalt treatment. Salt-induced Ca²⁺efflux was essentially independentof external Ca²⁺concentrations in the range 0.1 to 10 m M. Also,Ca²⁺flux responses were ‘saturated’ above 50 m MNaCl. It is suggested that almost all the measured Ca²⁺fluxoriginates from Na⁺/Ca²⁺and H⁺/Ca²⁺ion exchange in the cellwall. This conclusion was supported by the results of modellingcation exchange in the cell wall. Copyright 2000 Annals of BotanyCompany Salinity, membrane transporters, wall ion exchange, proton, calcium, Vicia faba     

Calcium Antagonist TMB-8 Inhibits Cell Wall Formation and Growth in Pea

The effects on auxin-stimulated growth and cell wall formationof 8-(N, N-diethylamino)-octyl-3, 4, 5-trimethoxybenzoate.HCI(TMB-8), an intracellular Ca²⁺ antagonist, were investigatedin abraded stem segments from aetiolated seedlings of Pisumsativum L. cv. Alaska. Incubation of segments at pH 6.0 with200 mmol m^–3 TMB-8 resulted in a 50% inhibition of auxin-stimulatedgrowth. Added Ca²⁺ did not restore normal auxin-stimulated growth,presumably because of its well-known stiffening effect on thecell wall. In segments incubated at a pH (7–2) which preventedelongation, auxin promoted the incorporation of [³H]glucoseinto the cell wall relative to total uptake of label. TMB-8abolished about 60% of the total incorporation of label intocell walls in the presence of auxin, but was not effective inthe absence of auxin. Exogenous CaCl₂ reversed the inhibitoryeffect of TMB-8 on relative cell wall incorporation in a parabolicmanner, with a 50% reversal at about 100 mmol m^–3 andcomplete reversal at 1.0 mol m^–3 Ca²⁺. Other ions tested(Mg²⁺, Mn²⁺, Cu²⁺, Zn²⁺) were without substantial effect atconcentrations of 0.5 mol m^–3. Both apparent uptake ofCa²⁺ and consequent reversal of TMB-8 inhibition of cell wallincorporation were blocked by the Ca²⁺ channel blockers verapamiland La³⁺. The data provide further evidence that auxin-stimulatedgrowth is dependent upon continued cell wall incorporation,and suggest that a Ca²⁺ messenger system may be involved inthe promotory actions of auxin on cell wall synthesis and long-termgrowth. Key words: Auxin, calcium, cell wall synthesis     

Germination of Monocolpate Angiosperm Pollen: Effects of Inhibitory Factors and the Ca2+-Channel Blocker, Nifedipine

Hydration of pollen of Narcissus pseudonarcissus was retardedand germination blocked in media with supra-optimal concentrationsof osmoticum. Activation of the grains, expressed in circulatorymovement in the vegetative cell, was not blocked. Wall developmentwas disrupted, and pectic material and callose were depositedthroughout. In the absence of calcium many grains burst on hydration.The survivors showed evidence of activation, but few tubes wereformed. In medium with supra-optimal Ca²⁺, activation proceeded,but where tube tips were produced they became occluded withcallose, which eventually formed a general lining to the intine.Nifedipine, a Ca²⁺-blocker, did not prevent activation at 10^–4M, but reduced callose deposition and inhibited polarized movementin the vegetative cell. Prominences formed at the germinationsites were mostly low and rounded. During recovery in normalmedium, tube tips with normal callose linings were formed. Colchicine,a microtubule inhibitor, had no effect on activation or germination.Cytochalasin D, an actin inhibitor, prevented activation ofthe vegetative cell, but did not arrest all wall deposition.Movement began soon after transfer to normal medium, and somegrains produced adventitious tube tips. While Ca²⁺ appears notto be essential for activation, these results may be interpretedas indicating links in the normal course of germination betweenthe initial Ca²⁺ influx at the potential germination sites and:(a) polarization of movement in the vegetative cell, probablyrelated to re-orientation of the actin cytoskeleton; and (b)patterned deposition of callose, which appears to have an importantmorphogenetic role. Narcissus pseudonarcissus, pollen activation, pollen germination, osmotic effects, actin cytoskeleton, nifedipine, cytochalasin D, colchicine, role of Ca²⁺ flux     

Effects of Calcium on NAD(H)-Glutamate Dehydrogenase from Turnip (Brassica rapa L.) Mitochondria

The effect of Ca²⁺ and ammonia on mitochondrial NADH-glutamatedehydrogenase (GDH: EC 1.4.1.2 [EC] ) isolated from turnip root (Brassicarapa L.) activity was examined. Increasing the ammonia [(NH₄)₂SO₄]concentration led to significant substrate inhibition whichcould be reversed by micromolar levels of Ca²⁺. The sensitivityof the enzyme to ammonia inhibition and its reversal by Ca²⁺was affected by proteolysis. After treatment with various proteases,lower concentrations of Ca²⁺ were capable of fully activatingthe enzyme or overcoming the inhibitory effects of high ammonium,compared to non-treated enzyme. However, the protease-treatedenzyme was still sensitive to ethylene glycol-bis(ß-aminoethylether) N,N,N',N'-tetraacetate (EGTA). In contrast, NADH-GDHactivity was inhibited approx. 30% by organic mercurials (200µm), but the residual activity was not affected by thesubsequent additions of EGTA. NADH-GDH activity could also bestimulated by additions of high concentrations of NaCl (300mM) in the absence of added Ca²⁺. These results suggest thathydrophobic and -SH groups may be involved in the regulationof mitochondrial NADH-GDH activity by Ca²⁺. ² Present address: CSIRO Division of Horticulture, Urrbrae,S.A. 5064, Australia (Received April 18, 1990; Accepted July 23, 1990)     

Time Course of Cell Biological Events Evoked in Legume Root Hairs by Rhizobium Nod Factors: State of the Art

In many common legumes, when host-specific nodule bacteria meettheir legume root they attach to it and enter through root hairs.The bacteria can intrude these cells because they instigatein the hairs the formation of an inward growing tube, the infectionthread, which consists of wall material. Prior to infectionthread formation, the bacteria exploit the cell machinery forwall deposition by inducing the hairs to form a curl, in whichthe dividing bacteria become entrapped. In most species, Nodfactor alone (a lipochito-oligosaccharide excreted by bacteria)induces root hair deformation, though without curling, thusmost aspects of the initial effects of Nod factor can be elucidatedby studying root hair deformation. In this review we discussthe cellular events that host-specific Nod factors induce intheir host legume root hairs. The first event, detectable onlya few seconds after Nod factor application, is a Ca²⁺influxat the root hair tip, followed by a transient depolarizationof the plasma membrane potential, causing an increase in cytosolic[Ca²⁺] at the root hair tip. Also within minutes, Nod factorschange the cell organization by acting on the actin cytoskeleton,enhancing tip cell wall deposition so that root hairs becomelonger than normal for their species. Since the remodellingof the actin cytoskeleton precedes the second calcium event,Ca²⁺spiking, which is observed in the perinuclear area, we proposethat the initial cytoskeleton events taking place at the hairtip are related to Ca²⁺influx in the hair tip and that Ca²⁺spikingserves later events involving gene expression. Copyright 2001Annals of Botany Company Review, Nod factor, tip growth, root hair, Rhizobium, legume, cytoskeleton, calcium, symbiosis     

Aluminium Effects on Pollen Germination and Tube Growth of Chamelaucium uncinatum. A Comparison with Other Ca2+Antagonists

An interaction between aluminium (Al) and calcium (Ca) may bea cause of Al toxicity in plants. The pollen tube is a suitablesystem to test the interaction between Al and Ca since Ca ionsplay a pivotal role in pollen germination and tube growth. Weinvestigated how Al and other known blockers of Ca²⁺-permeablechannels (trivalent cations, ruthenium red, verapamil and nifedipine)influence pollen of an Australian native species Geraldton waxflower(Chamelaucium uncinatum). Pollen germination was inhibited bymicromolar concentrations of trivalent cations (La³⁺>Al³⁺>Gd³⁺)and ruthenium red, but it was relatively insensitive to a micromolarconcentration of verapamil. Exposure of the growing pollen tubesto micromolar concentrations of Al³⁺and La³⁺, and a millimolarconcentration of Ca²⁺chelator ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA) led to rapid tip bursting.In contrast, exposure to Gd³⁺, nifedipine, ruthenium red, verapamiland the organic trivalent cation tris (ethylenediamine)cobalt(TEC³⁺) caused only inhibition of pollen tube growth. The Al³⁺-relatedpollen tube bursting was reduced significantly by increasingeither solution pH from 4.5 to 6 or activity of Ca²⁺from 0.25to 5 m M. In contrast, La³⁺-related pollen tube bursting wasinsensitive to changes in Ca²⁺activity. The results are discussedin terms of Al interactions with cell wall Ca²⁺and the plasmamembrane Ca²⁺-permeable channels. Copyright 1999 Annals of BotanyCompany Aluminium toxicity, Ca²⁺-channel blockers, cell wall, Chamelaucium uncinatum, pollen germination, pollen tube growth.     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

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.

     

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

The plasmalemmal Na⁺-K⁺-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca²⁺. However, the ability of PTX to directly increase cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Na⁺ pump channels and to initiate Ca²⁺ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca²⁺]_i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca²⁺]_i that was dependent on extracellular Ca²⁺. The increase in [Ca²⁺]_i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC₅₀ < 1 µM. The elevation in [Ca²⁺]_i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca²⁺]_i occurs via the Na⁺ pump. Subsequent to the rise in [Ca²⁺]_i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca²⁺ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca²⁺ overload. necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2     

Isolation and characterization of adenosine triphosphatase from Salicornia pacifica var. utahensis

Membrane-bound ATPase associated with plasma membrane and solubleATPase associated with the cytoplasm were prepared from shootsof Salicornia pacifica var. utahensis by sucrose density gradientcentrifugation. The isolated ATPases were tolerant to high concentrationsof NaCl. The Km for membrane-bound ATPase was 1.75 mM and forsoluble ATPase, it was 1.4 mM. The relative effectiveness ofdivalent cations for stimulation of membrane-bound ATPase wasMg²⁺>Fe²⁺>Mn²⁺>Co²⁺>Cu²⁺. Soluble ATPase activitywas stimulated by Ba²⁺>Ca²⁺>Mg²⁺ and was inhibited byCu²⁺, Zn²⁺, Co²⁺ and Fe²⁺. The compounds N,N¹-dicyclohexylcarbodiimide,NaF and ADP, did inhibit the ATPases but ouabain, triphenyltinhydroxide, sodium azide, indoleacetic acid and abscisic aciddid not inhibit the ATPases from s. pacifica var. utahensis. ¹ Present address: Department of Biology, Kong-Ju National College,Kong-Ju, Korea. (Received April 1, 1980; )     

Effects of Calcium and Abscisic Acid on Volume Changes of Guard Cell Protoplasts of Commelina

Calcium ions contracted guard cell protoplasts (GCP) of Commelinacommunis L., being particularly effective within the concentrationrange of 0 to 0.2 mol m^–3. Abscisic acid (ABA) in thepresence of EGTA, which chelates free Ca²⁺ in the medium, contractedGCP to a similar extent to Ca²⁺ alone or Ca²⁺ and ABA together.Similarly, ABA in the absence of free Ca²⁺ (i.e. an ABA/EGTAtreatment) inhibited K⁺-induced swelling of contracted GCP,as did Ca²⁺ alone or ABA and Ca²⁺ together. Lanthanum, a Ca²⁺channel blocker, prevented the contraction of GCP by Ca²⁺ buthad no effect if ABA was also present with Ca²⁺. The inhibitionof swelling of GCP by Ca²⁺ was also prevented by the presenceof lanthanum or verapamil (another Ca²⁺ channel blocker). These results indicate that Ca²⁺ and ABA can act independentlyof each other in contracting swollen GCP and in preventing K⁺-inducedswelling of contracted GCP of C. communis. If swelling and contractionof GCP are equivalent to stomatal opening and closure, respectively,the results do not support the hypothesis that ABA opens Ca²⁺channels in the plasma membrane of guard cells allowing Ca²⁺to enter the cells and, as a second messenger, to set in motionclosing processes. Key words: Abscisic acid, calcium, guard cell protoplasts, stomata     

Ca(2+)-dependent activation of c-jun NH(2)-terminal kinase in primary rabbit proximal tubule epithelial cells

Previous work from this laboratorydemonstrated that arachidonic acid activates c-junNH₂-terminal kinase (JNK) through oxidative intermediatesin a Ca²⁺-independent manner (Cui X and Douglas JG.Arachidonic acid activates c-jun N-terminal kinase throughNADPH oxidase in rabbit proximal tubular epithelial cells. ProcNatl Acad Sci USA 94: 3771-3776, 1997.). We now report thatJNK can also be activated via a Ca²⁺-dependent mechanism byagents that increase the cytosolic Ca²⁺ concentration(Ca²⁺ ionophore A₂₃₁₈₇, Ca²⁺-ATPaseinhibitor thapsigargin) or deplete intracellular Ca²⁺stores [intracellular Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(BAPTA)-AM]. The activation of JNK by BAPTA-AM occurs despite adecrease in cytosolic Ca²⁺ concentration as detected by theindicator dye fura 2, but appears to be related to Ca²⁺metabolism, because modification of BAPTA with two methyl groups increases not only the chelation affinity for Ca²⁺, butalso the potency for JNK activation. BAPTA-AM stimulates Ca²⁺ influx across the plasma membrane, and the resultinglocal Ca²⁺ increases are probably involved in activation ofJNK because Ca²⁺ influx inhibitors (SKF-96365, nifedipine)and lowering of the free extracellular Ca²⁺ concentrationwith EGTA reduce the BAPTA-induced JNK activation.

     

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding

Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K⁺ (Kv) channel expression andcytosolic free Ca²⁺ concentration([Ca²⁺]_cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca²⁺ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K⁺ currents, membrane hyperpolarization, and arise in [Ca²⁺]_cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca²⁺]_cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca²⁺]_cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca²⁺ influx during restitution.

     

An Excel-based model of Ca2+ diffusion and fura 2 measurements in a spherical cell

We wrote a program that runs as a Microsoft Excel spreadsheet to calculate the diffusion of Ca²⁺ in a spherical cell in the presence of a fixed Ca²⁺ buffer and two diffusible Ca²⁺ buffers, one of which is considered to be a fluorescent Ca²⁺ indicator. We modeled Ca²⁺ diffusion during and after Ca²⁺ influx across the plasma membrane with parameters chosen to approximate amphibian sympathetic neurons, mammalian adrenal chromaffin cells, and rat dorsal root ganglion neurons. In each of these cell types, the model predicts that spatially averaged intracellular Ca²⁺ activity ([Ca²⁺]_avg) rises to a high peak and starts to decline promptly on the termination of Ca²⁺ influx. We compared [Ca²⁺]_avg with predictions of ratiometric Ca²⁺ measurements analyzed in two ways. Method 1 sums the fluorescence at each of the two excitation or emission wavelengths over the N compartments of the model, calculates the ratio of the summed signals, and converts this ratio to Ca²⁺ ([Ca²⁺]_avg,M1). Method 2 sums the measured number of moles of Ca²⁺ in each of the N compartments and divides by the volume of the cell ([Ca²⁺]_avg,M2). [Ca²⁺]_avg,M1 peaks well after the termination of Ca²⁺ influx at a value substantially less than [Ca²⁺]_avg because the summed signals do not reflect the averaged free Ca²⁺ if the signals come from compartments containing gradients in free Ca²⁺ spanning nonlinear regions of the relationship between free Ca²⁺ and the fluorescence signals. In contrast, [Ca²⁺]_avg,M2 follows [Ca²⁺]_avg closely. intracellular calcium; kinetic model; diffusion coefficient; fura 2ff; furaptra     

Effect of Calcium on the Structure and Template Activity of Pea Chromatin

The absorption spectrum of native pea chromatin solubilizedunder minimal shearing conditions changed with increasing Ca²⁺concentration; the ratio of maximum to minimum absorption decreasedand the maximum absorption peak shifted to a longer wavelength.The concentration of Ca²⁺ to cause half complete sedimentationof chromatin was much lower for the solubilized native chromatin(more condensed and larger in size) than for the sonicated chromatin(less condensed and smaller in size). Solubilized native chromatinshowed a two-step melting profile in the absence of Ca²⁺. In the presence of Ca²⁺ the two T_ms disappeared and a new higherT_m appeared. Template activity of solubilized native chromatinincreased 3-fold upon dispersion and fragmentation by sonication.Addition of a small amount of ethylene glycol-bis (ß-aminoethylether)-N, N'-tetraacetic acid (EGTA) promoted the template activityof solubilized native chromatin, but not that of sonicated ordenatured DNA. The effect of EGTA was reversed by Ca²⁺. Thechromatin reconstituted in the presence of EGTA showed a lowerT_m than the chromatin reconstituted in the presence of Ca²⁺.The relationship between chromatin structure and its templateactivity is discussed in relation to Ca²⁺. (Received August 12, 1985; Accepted December 7, 1985)     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells

The possibility that protein kinase C (PKC) could control theactivity of L-type Ca²⁺ channelsin A7r5 vascular smooth muscle-derived cells in the absence of agoniststimulation was investigated using the patch-clamp technique.Consistent with the possibility that L-typeCa²⁺ channels are maximallyphosphorylated by PKC under these conditions, we show that1) activation of PKC with thephorbol ester phorbol 12,13-dibutyrate was ineffective in modulatingwhole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine orchelerythrine inhibited channel activity,3) inhibition of proteinphosphatases by intracellular dialysis of okadaic acid did not affectwhole cell currents, and 4) theinhibitory effect of staurosporine was absent in the presence ofokadaic acid. The inhibition ofCa²⁺ currents by PKC inhibitorswas due to a decrease in channel availability and long open events,whereas the voltage dependence of the open probability and thesingle-channel conductance were not affected. The evidence suggeststhat in resting, nonstimulated A7r5 cells there is a high level of PKCactivity that modulates the gating of L-typeCa²⁺ channels.

     

Medium Acidification by Auxin- and Fusicoccin-Treated Peeled Stem Segments from Etiolated Seedlings of Pisum sativum

Several reports have suggested that peeling off the epidermisfrom dicotyledonous stems also removes the capacity of thesesegments to show an auxin-induced elongation response. Thiscould be due either to the removal of the auxin-responsive tissue,or to the loss of H⁺ into the medium when the impermeable cuticleis removed. Medium acidification by peeled etiolated segmentsof Pisum sativum L. cv. Alaska, and the effects of indoleaceticacid (IAA) and fusicoccin (FC) on this process, were thus investigatedin an attempt to resolve this problem. It was found that mediumacidification occurred even with control segments, and thatonly in the presence of the divalent cations Ca²⁺ or Mg²⁺ didIAA give any enhancement of acidification. Under these conditions,the main effect of IAA was a reduction in the lag time betweenIAA addition and an observed acidification response. Additionof these ions is not, however, required to obtain normal auxin-inducedgrowth. Similarly, peeled segments of mature non-elongatingtissue also acidified the medium. In contrast, FC gave someenhancement of medium acidification even in the absence of Ca²⁺and Mg²⁺. With both IAA and FC, lag times before observableacidification occurred were reduced (from about 40–50min to 20 min and 2/emdash 4 min respectively) in the presenceof Ca²⁺ or Mg²⁺. These ions appeared to exert similar effects.IAA, when added once a stabilized pH had been attained, hadlittle effect on pH, whereas FC caused a further drop of about0.5 unit. It thus appears that auxin has little appreciableeffect on the acidification of the medium caused by peeled segments,either of non-elongating or of growing stems, and we concludethat peeling must therefore remove the auxin-responsive tissuein pea stem.