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)     

Tonoplast Action Potential of Characeae

The plasmalemma action potential was found to be indispensableto the production of the tonoplast action potential. In a solutionlacking Ca²⁺ and containing other divalent cations such as Ba²⁺,Mg²⁺ or Mn²⁺, the plasmalemma excited in Nitella but did notin Chara. In Nitella, however, both the tonoplast action potentialand EC-coupling were abolished due to depletion of Ca²⁺ fromthe external medium. Ca²⁺ ions injected into the cytoplasmiclayer caused a transient change in both plasmalemma and tonoplastpotentials. These results suggest that a transient rise in Ca²⁺concentration during excitation of the plasmalemma may triggerthe tonoplast action potential. (Received February 14, 1986; Accepted August 29, 1986)     

Dependence of Plasmalemma Conductance and Potential on Intracellular Free Ca2+ in Tonoplast-Removed Cells of a Brackish Water Characeae Lamprothamnium

In response to hypotonic treatment internodal cells of the brackishwater Characeae Lamprothamnium regulate turgor pressure by releasingK⁺ and Cl^–, accompanying membrane depolarization and atransient increase in membrane electrical conductance (Okazakiet al. 1984b). The hypothesis that a transient increase in cytoplasmicfree Ca²⁺ concentration ([Ca²⁺]_c) caused by hypotonic treatmenttriggers release of K⁺ and Cl^– from the cell (Okazakiand Tazawa 1986a, b, c) was tested using tonoplast-removed cells.These cells did not regulate turgor pressure. The plasmalemmaconductance remained almost constant for a change in the intracellularfree Ca²⁺ concentration ([Ca²⁺],) from 10^–6 to 10^–2mol?m^–3. The results suggest that some cytoplasmic Ca²⁺-sensitizingsoluble components, which work as mediators to activate K⁺ and/orCl^– channels in the plasmalemma and/or the tonoplast,were lost after desintegration of the tonoplast. The plasmalemmapotential was depolarized under high [Ca²⁺]_i. However, no membranedepolarization was observed upon hypotonic treatment. Sincemembrane depolarization has been suggsted to occur under normal[Ca²⁺]_c in intact cells (Okazaki and Tazawa 1986a, b), its absencesuggests that some cytoplasmic factors, which induce the membranedepolarization in a Ca²⁺-independent manner, are lost in tonoplast-removedcells. ¹ Present address: Department of Biology, Osaka Medical College,Sawaragi-cho 2-41, Takatsuki, Osaka 569, Japan. (Received October 22, 1986; Accepted March 31, 1987)     

Intracellular Ca2+ Concentration and Cytoplasmic Streaming in Vallisneria Mesophyll Cells

Intracellular Ca²⁺ concentration regulating the cytoplasmicstreaming in Vallisneria mesophyll cells was estimated. Theleaf segment was cut open at the middle of the mesophyll celllayers and the exposed mesophyll cells were treated with testsolutions of various Ca²⁺ concentrations in the dark. This allowedA23187 [GenBank] , a calcium ionophore, to exert its full effect on thecell membrane. The streaming was induced or maintained in solutions which containedCa²⁺ at lower than 10^–6M. However, Ca²⁺ at concentrationshigher than 10^–5M had a definite, inhibitory effect. Theinduction and cessation of streaming could be repeated by alternatelychanging the solutions. (Received March 14, 1986; Accepted May 15, 1986)     

NAD Kinase in Corn: Regulation by Far Red Light is Mediated by Ca2+ and Calmodulin

Far red light irradiation of intact corn seedlings (Zea maysL.) has neither an effect on the cellular distribution nor onthe Ca²⁺, calmodulin-dependence of the NAD kinase (EC 2.7.1.23 [EC] ).The enzyme is located in the outer mitochondrial membrane andits activity is totally dependent on the presence of both Ca²⁺and calmodulin, independently of the illumination. In intactmitochondria and the presence of calmodulin the enzyme activityincreases linearly from 100 nM to 1 mM. At 100 µM Ca²⁺halfmaximal activation occurs at about 10 nM calmodulin. After solubilizationand purification by calmodulin-Sepharose chromatography theCa²⁺dependence of the enzyme changes. The activation reachesa plateau at about 100 µM Ca²⁺ and half maximal activationoccurs at about 6 µM Ca²⁺. On the other hand irradiationof intact corn seedlings as well as an increase of the cellularCa²⁺ concentration leads to an increase of NADP and a correspondingdecrease of NAD. Based on these data we suggest that the lighteffect on the NAD kinase activity is mediated by Ca²⁺ and calmodulin. (Received May 31, 1986; Accepted July 14, 1986)     

Inhibition of Cl- Channel Activation in Chara corallina Membrane by Lanthanum Ion

We examined a role of Ca²⁺ in the activation of the two majorion channels, i.e., Cl^– and K⁺ channels at the excitationof the characean plasmalemma. The current-voltage relation (I-Vcurve) of the Chara membrane was compared under the ramp voltageclamp condition before and after external application of 20µMof La³⁺ (a Ca²⁺ channel blocker). The transient inward currentcomponent, which is carried mainly by the efflux of Cl^–,disappeared almost completely in about 30 min with La³⁺ treatment.On the other hand, no effect was observed on the late largeoutward current, which is mainly carried by the efflux of K⁺in a large depolarization region (less negative than –50mV). These results suggest that the Cl^– channel in theChara plasmalemma is activated by Ca²⁺ influx, while the K⁺channel is simply activated by depolarization. (Received April 7, 1986; Accepted June 6, 1986)     

Changes of Some Membrane-Associated Enzyme Activities Degradation of Membrane Phospholipids in Cucumber Roots Due to Ca2+ Starvation

Deprivation of Ca²⁺ from a complete culture medium affectedthe enzyme activities associated with five membrane fractionsof cucmber roots obtained by discontinuous sucrose density gradientcentrifugation. The total activity of K⁺-ATPase, Cyt. c oxidaseand NADPH-Cyt. c reductase of Ca²⁺-deficient roots, starvedfor only 4 days, had decreased to 14, 38 and 60% of the activityof the control roots. In general, loss of enzyme activitieswas accompanied by a shift of activity distribution from theheavier density fractions to lighter ones. The amounts of Ca²⁺ associated with membranes from Ca²⁺-starvedroots decreased to 50–60% of those of the control roots.Both phospholipid and neutral lipid contents in the membranesdecreased markedly while the protein content was not changedby Ca²⁺ deficiency. Phospholipid analysis indicated a drasticdrop in the percent composition of phosphatidylinositol butan increase of phosphatidic acid. Also, phospholipase D activityincreased remarkably during Ca²⁺ starvation, paralleling theappearance of Ca²⁺-deficiency symptoms. Thus, the major effects of Ca²⁺ deficiency appear to be to stimulatephospholipase D activity and a reduction in membrane bound Ca²⁺.These effect may be involved in disorganization of the membranestructure and the changes of enzyme activities associated withthe altered membranes. ¹Rubber Research Institute of Sri Lanka, Dartonfield, Agalwatta,Sri Lanka. (Received July 15, 1985; Accepted November 21, 1985)     

Changes of Polysomes in Cucumber Plant due to Ca2+ Deficiency

The amounts of monosomes plus polysomes in cucumber plant decreaseddue to Ca²⁺ deficiency. The decrease was more prominent in themembrane-bound forms than free forms. Polysomes in immatureleaves and the proportion of large polysomes in roots decreasedeven at the early stage of Ca²⁺ deficiency. (Received November 28, 1986; Accepted May 22, 1987)     

Studies on factors in sweet potato root which induce spore germination of Ceratocystis fimbriata

Spore germination of Ceratocystis fimbriata was studied in termsof host-parasite specificity. The sweet potato, coffee and cacaostrains of Ceratocystis fimbriata germinated well in a fractionof sweet potato root water extract which had been passed througha column of cation exchange resin. The results showed that germinationof these strains was independent of exogenous cations. On theother hand, the prune, oak, taro and almond strains requiredfor germination both the absorbed and unabsorbed fractions ofsweet potato root water extract which were separated from eachother with a cation exchange resin column. Divalent cationssuch as Ca²⁺ Mg²⁺, Mn²⁺ and Zn²⁺ were identified as the activeprinciples in the absorbed fraction and Ca²⁺ showed the highestinductive activity for spore germination in the presence ofthe unabsorbed fraction. The active principle(s) in the unabsorbedfraction has not yet been identified. There was no relationshipbetween the Ca²⁺ and Mg²⁺ contents of the spores and the requirementof exogenous Ca²⁺ for germination. Ca²⁺ appeared to functionas a trigger of spore germination, not as a normal nutrient.These results suggest that the divalent cations such as Ca²⁺and Mg²⁺ in sweet potato contribute to the establishment ofhost-parasite specificity of this system. (Received August 10, 1977; )     

The Ca2+-Induced Phase Transformation in Soybean Root Microsomal Membranes is a Consequence of Phospholipase Activation

Microsomes from soybean (Glycine max L.) roots contain a Ca²⁺-activatedphospholipase D which hydrolyzes phosphatidylethanolamine andphosphatidylcholine to phosphatidic acid. The phosphatidic acidbinds Ca²⁺ in the medium and undergoes a liquid crystal to gelphase transformation (measured by wide-angle x-ray diffraction).The gel phase formation in the microsomes half-saturates at1 to 10 mM Ca²⁺. The upper limit for the transition temperaturefor the microsomes is –10 to 10°C in the native state.After treatment with Ca²⁺, the transition temperature increasesto 35 to 50°C. Under the same experimental conditions, incubationwith 10 mM Ca²⁺ at 4°C causes an increase in phosphatidicacid content from 8 mole % to 49% with a concomitant decreasein phosphatidylethanolamine and phosphatidylcholine from about22% and 41%, respectively, to 14% each. These results suggest that Ca²⁺ effects in systems where Ca²⁺plays a multifunctional role be interpreted with caution. ³Present address: Department of Physiology, Yale UniversitySchool of Medicine, New Haven, CT 06510, U.S.A. (Received June 15, 1985; Accepted June 13, 1986)     

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)     

Relations Between Indoleacetic Acid, Calcium Ions and Ethylene in the Regulation of Growth and Cell Wall Composition in Picea abies

Effects of indoleacetic acid, calcium ions and ethylene on thegrowth of and deposition of different cell wall fractions inthe hypocotyl of Norway spruce (Picea abies (L.) Karst.) seedlingswere investigated. Indoleacetic acid progressively stimulated cellulose depositionas the amount of added Ca²⁺ increased. In contrast, indoleaceticacid promoted lignification and the deposition of non-cellulosicpolysaccharides only in the absence of added Ca²⁺ . When Ca²⁺was added, the indoleacetic acid effect disappeared. Similarly,indoleacetic acid promoted non-cellulosic polysaccharide depositiononly in the absence of ethylene. At increasing ethylene levelsthe effect of indoleacetic acid on non-cellulosic polysaccharidedeposition disappeared and indoleacetic acid instead promotedcellulose deposition. The response to indoleacetic acid depended on the Ca²⁺ concentrationand on the rate of ethylene production. The relationship betweenindoleacetic acid and Ca²⁺ seemed complex, but clearly indoleaceticacid could partially overcome a Ca²⁺ deficiency. The resultssuggest that ethylene may be a factor of particular importancefor the type of polysaccharide deposition during cell wall formation. Key words: Calcium, cell wall, conifers, ethylene, indoleacetic acid     

Bidirectional Ca2+ coupling of mitochondria with the endoplasmic reticulum and regulation of multimodal Ca2+ entries in rat brown adipocytes

How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca²⁺ entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca²⁺ or Mg²⁺ and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) produced by thapsigargin was abolished by removing extracellular Ca²⁺, depressed by depleting extracellular Na⁺, and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [Ca²⁺]_i, in which the first phase was accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin but not by ruthenium red, cyclosporine A, U-73122, a Ca²⁺-free EGTA solution, and an Na⁺-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca²⁺ and fully blocked by oligomycin but not by thapsigargin or an Na⁺-deficient solution, was accompanied by a rise in cytoplasmic Mg²⁺ concentration, and was summated with a high pH-induced rise in [Ca²⁺]_i, whereas the extracellular Ca²⁺-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca²⁺ but not by thapsigargin, depressed by decreasing Na⁺, and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca²⁺]_i in a Ca²⁺-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca²⁺ release, activating Ca²⁺ release from the ER and store-operated Ca²⁺ entry, and directly elicits a novel plasmalemmal Ca²⁺ entry, whereas Ca²⁺ release from the ER activates Ca²⁺ accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. plasmalemmal calcium entry; calcium release; mitochondrial depolarization; FCCP     

Mg2+ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca2+ concentrations

To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca²⁺-dependent [³H]ryanodine binding, a biochemical measure of Ca²⁺-induced Ca²⁺ release (CICR), was determined using SR vesicle fractions isolated from rabbit and rat cardiac muscles. In the absence of an adenine nucleotide or caffeine, the rat SR showed a complicated Ca²⁺ dependence, instead of the well-documented biphasic dependence of the rabbit SR. In the rat SR, [³H]ryanodine binding initially increased as [Ca²⁺] increased, with a plateau in the range of 10–100 µM Ca²⁺, and thereafter further increased to an apparent peak around 1 mM Ca²⁺, followed by a decrease. In the presence of these modulators, this complicated dependence prevailed, irrespective of the source. Addition of 0.3–1 mM Mg²⁺ unexpectedly increased the binding two- to threefold and enhanced the affinity for [³H]ryanodine at 10–100 µM Ca²⁺, resulting in the well-known biphasic dependence. In other words, the partial suppression of RyR2 is relieved by Mg²⁺. Ca²⁺ could be a substitute for Mg²⁺. Mg²⁺ also amplifies the responses of RyR2 to inhibitory and stimulatory modulators. This stimulating effect of Mg²⁺ on RyR2 is entirely new, and is referred to as the third effect, in addition to the well-known dual inhibitory effects. This effect is critical to describe the role of RyR2 in excitation-contraction coupling of cardiac muscle, in view of the intracellular Mg²⁺ concentration. [³H]ryanodine binding; CICR; stimulation by physiological Mg²⁺, excitation-contraction coupling in the heart     

Na+/Ca2+ exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function

The role of theNa⁺/Ca²⁺exchanger in intracellular Ca²⁺regulation was investigated in freshly dissociated catfish retinalhorizontal cells (HC).Ca²⁺-permeable glutamate receptorsand L-type Ca²⁺ channels as wellas inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitiveintracellular Ca²⁺ stores regulateintracellular Ca²⁺ in these cells.We used the Ca²⁺-sensitive dyefluo 3 to measure changes in intracellularCa²⁺ concentration([Ca²⁺]_i)under conditions in whichNa⁺/Ca²⁺exchange was altered. In addition, the role of theNa⁺/Ca²⁺exchanger in the refilling of the caffeine-sensitiveCa²⁺ store followingcaffeine-stimulated Ca²⁺ releasewas assessed. Brief applications of caffeine (1-10 s) producedrapid and transient changes in[Ca²⁺]_i.Repeated applications of caffeine produced smallerCa²⁺ transients until no furtherCa²⁺ was released. Store refillingoccurred within 1-2 min and required extracellularCa²⁺. Ouabain-induced increases inintracellular Na⁺ concentration([Na⁺]_i)increased both basal free[Ca²⁺]_iand caffeine-stimulated Ca²⁺release. Reduction of external Na⁺concentration([Na⁺]_o)further and reversibly increased[Ca²⁺]_iin ouabain-treated HC. This effect was not abolished by the Ca²⁺ channel blocker nifedipine,suggesting that increases in[Na⁺]_ipromote net extracellular Ca²⁺influx through aNa⁺/Ca²⁺exchanger. Moreover, when[Na⁺]_owas replaced by Li⁺, caffeine didnot stimulate release of Ca²⁺ fromthe caffeine-sensitive store afterCa²⁺ depletion. TheNa⁺/Ca²⁺exchanger inhibitor 2',4'-dimethylbenzamil significantlyreduced the caffeine-evoked Ca²⁺response 1 and 2 min after store depletion.

     

Calmodulin-Like Activity Associated with Chromatin from Pea Buds

NAD kinase and cyclic AMP phosphodiesterase were activated bya factor prepared from pea chromatin. About 62% of the originalamount of the factor in the purified chromatin was recoveredin the reassociated chromatin. The NAD kinase- and cyclic AMP phosphodiesterase-activatingfactor was released from the chromatin by heat treatment withethylene glycol-bis(ß-aminoethyl ether)- N,N,N',N'-tetraaceticacid (EGTA) then adsorbed on an affinity gel of phenothiazineagarosederivatives in the presence of excess Ca²⁺ over EGTA, afterwhich it was eluted by a flush of EGTA. Activation of NAD kinaseand cyclic AMP phosphodiesterase by this factor depended onthe presence of Ca²⁺. The NAD kinase-activating factor and chromatin were coelutedwhen soluble chromatin was applied to a Bio-Gel A50 column.When chromatin was chromatographed on the same column afterdigestion by DNase I, the factor was eluted in association withthe digested products of the chromatin. The activation propertiesof this factor indicate that a calmodulin-like activity existsin association with pea chromatin. The activation curves of cyclic AMP phosphodiesterase with thepea bud factor and with bovine brain calmodulin were compared.The amount of the factor in the chromatin fraction that correspondedto authentic calmodulin was calculated as 5.7 µg per mgDNA. (Received August 6, 1982; Accepted February 17, 1983)     

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     

Ion Efflux during a Single Action Potential of Nitella axilliformis in Medium Lacking Ca2-

Ion efflux during excitation of Nitella axilliformis was measuredconductometrically. In medium lacking Ca²⁺ but with 0.1 mM MgCl₂,the duration of the action potential and the total efflux weremuch larger than those in APW, while the efflux rate, givenas the total efflux divided by the duration, was about halfof that in APW. (Received September 4, 1986; Accepted November 25, 1986)     

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells

Cytoplasmic Ca²⁺concentration ([Ca²⁺]_i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca²⁺ concentration ([Ca²⁺]_o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca²⁺]_i due to Ca²⁺influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca²⁺-sensing receptor.[Ca²⁺]_o-induced[Ca²⁺]_i increase was inhibited byCa²⁺ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca²⁺]_i increasewas potentiated by depletion of intracellular Ca²⁺ storesand depressed by inactivation of the Na⁺/Ca²⁺exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca²⁺]_i increase in L6-C5 cells, was notmodified by high [Ca²⁺]_o. On the contrary,AVP potentiated the [Ca²⁺]_i increase in thepresence of elevated [Ca²⁺]_o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca²⁺]_o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.