Effect of EGTA on Nyctinastic Closure Mediated by Phytochrome in Albizzia lophantha

The role of extracellular calcium on nyctinastic closure ofAlbizzia lophantha leaflets has been studied by testing theeffect of ethyleneglycol-bis-(ß-aminoethylether)-N,N,N',N'-tetraaceticacid (EGTA) and its reversibility by calcium. EGTA (1 and 5mM) causes an inhibition of nyctinastic closure and at a concentrationof 1 mM EGTA it decreases the difference between the effectof red light (R) and far-red light (FR) irradiation on leafletclosure. A simultaneous or subsequent supply of CaCl₂ (5 or10 mM) reverses EGTA (5 mM) inhibition on closure as well ascausing an additional promotion of closure. We suggest that external calcium could play a dual role in nyctinasticclosure. Phytochrome control of leaflet closure probably needsexternal Ca²⁺ and, in addition, Ca²⁺ could regulate the closuremechanism by controlling ionic fluxes through the plasma membranein pulvinular motor cells. (Received June 9, 1989; Accepted November 27, 1989)     

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.

     

The concentration of selected cations in normal and crown-gall tumors obtained from potato discs

Crown-gall tumor tissue obtained from potato discs inoculatedwith virulent strains of Agrobacterium tumefadens containedhigher concentrations of K⁺, Mg²⁺ and Ca²⁺ than the correspondingnormal tissue. These tumors also contained higher concentrationsof these cations than normal tissue inoculated with an avirulentstrain of Agrobacterium tumefadens, or than normal tissue adjacentto the crown-gall tumors on the same potato disc. The concentrationof these cations remained significantly higher than controltissue regardless of the tumor age. 2,4-Dinitrophenol and myo-inositol,while affecting the concentration of Ca²⁺ in these tissues,had no effect on the Mg²⁺ and K⁺ concentrations. These resultssuggest increased concentrations of certain cations may be aspecific property of crown-gall tumors. (Received August 16, 1978; )     

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; )     

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.     

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)     

Injection of a Ca2+-Chelating Agent into the Cytoplasm Retards the Progress of Turgor Regulation upon Hypotonic Treatment in the Alga, Lamprothamnium

The turgor regulation induced by hypotonic treatment (hypotonicturgor regulation) in the brackish-water alga Lamprothamniumsuccinctum is accompanied by a transient increase in the electricalconductance of the membrane, membrane depolarization and a transientincrease in the cytoplasmic concentration of free Ca²⁺ ([Ca²⁺([Ca²⁺]_c) (Okazaki and Tazawa 1990). In the present study, weloaded a Ca²⁺-chelating agent, EGTA, into the cytoplasm by mechanicalinjection in order to suppress the increase in [Ca²⁺]_c thatoccurs during the hypotonic turgor regulation. The rate of thecytoplasmic streaming was taken as an indirect indicator of[Ca²⁺]_c, since cytoplasmic streaming has been shown to be inhibitedby high [Ca²⁺]_c in Lamprothamnium cells. The lag time for theinhibition of the cytoplasmic streaming upon hypotonic treatmentwas significantly prolonged in EGTA-loaded cells as comparedto that in intact cells. This result indicates that the loadedcytoplasmic EGTA functioned as a buffer of Ca²⁺ to retard theincrease in [Ca²⁺]_c. It took a longer time for the membraneconductance to reach the peak value in EGTA-loaded cells thanin intact cells. Membrane depolarization was affected to aninsignificant extent by the cytoplasmic EGTA. The regulationof turgor pressure itself was partially inhibited. These resultsstrongly support the idea that the net efflux of ions that occursduring the recovery from hy-potonically induced changes in turgorpressure is controlled by [Ca²⁺]_c. (Received August 22, 1990; Accepted December 6, 1990)     

Effects of cations on the sugar receptor-site of the blowfly, Phormia

1) Ca^{+ +} (1 to 10 mM) lowered the binding affinity of sugarreceptor-site for sucrose in the labellar sugar receptor ofthe blowfly, Phormia regina, without changing the maximum-responseamplitude. It also elevated the values of the Hill coefficient(n_H) in some degrees. 2) Other divalent cations such as Mg^{+ +}, Ba^{+ +} or Cd^{+ +} alsoshowed almost the same property as above. The sequence of theeffect is as follows: Ba^{+ +}, Mg^{+ +} x Ca^{+ +} x Cd^{+ +}. Trivalentcation, La^{+ + +} (1 mM), changed the value of n_H from 1 (La^{++ +}-free) to 2. 3) On the contrary, the action of monovalent cations such asK⁺ or Na⁺, of which ionic strength was made the same as thatof the divalents hardly suppressed the response. 4) The results obtained do not support the hypothesis, at leaston the sugar receptor of the fly, that the receptor potentialis attributable to a change of the surface potential (zeta potential)as is proposed for the frog sugar receptor.     

Modulation of Ca2+-gated cardiac muscle Ca2+-release channel (ryanodine receptor) by mono- and divalent ions

The effects of mono- and divalent ions onCa²⁺-gated cardiac muscleCa²⁺-release channel (ryanodinereceptor) activity were examined in [³H]ryanodine-bindingmeasurements. Ca²⁺ bound with thehighest apparent affinity to Ca²⁺activation sites in choline chloride medium, followed by KCl, CsCl,NaCl, and LiCl media. The apparentCa²⁺ binding affinities ofCa²⁺ inactivation sites were lowerin choline chloride and CsCl media than in LiCl, NaCl, and KCl media.Sr²⁺ activated the ryanodinereceptor with a lower efficacy thanCa²⁺. Competition studiesindicated that Li⁺,K⁺,Mg²⁺, andBa²⁺ compete withCa²⁺ forCa²⁺ activation sites. In 0.125 MKCl medium, the Ca²⁺ dependence of[³H]ryanodine bindingwas modified by 5 mM Mg²⁺ and 5 mM,-methyleneadenosine 5'-triphosphate (a nonhydrolyzable ATPanalog). The addition of 5 mM glutathione was without appreciable effect. Substitution of Clby 2-(N-morpholino)ethanesulfonic acid ion caused anincrease in the apparent Ca²⁺affinity of the Ca²⁺ inactivationsites, whereas an increase in KCl concentration had the oppositeeffect. These results suggest that cardiac muscle ryanodine receptoractivity may be regulated by 1)competitive binding of mono- and divalent cations toCa²⁺ activation sites,2) binding of monovalent cations toCa²⁺ inactivation sites, and3) binding of anions to anionregulatory sites.

     

Patch-Clamp Study on a Ca2+-Regulated K+ Channel in the Tonoplast of the Brackish Characeae Lamprothamnium succinctum

Cytoplasmic drops were prepared from internodal cells of thebrackish Characeae Lamprothamnium succinctum. Applying the patch-clamptechnique to single drops covered with tonoplast, we demonstratedthe presence of Ca²⁺-regulated K⁺ channels in the tonoplast.In a cell-attached mode, the selectivity of such channels forK⁺ was about 50 times that for Na⁺. This channel showed a tendencyto rectify in an outward direction. In the negative region ofthe pipette voltage, the conductance of this channel was 50pS, while it was 100 pS in the positive voltage region. Whenthe pipette voltage was increased above 50 mV, two conductancelevels were found in the cell-attached mode as well as in theexcised patch (cytoplasmic-side-out patch), which was obtainedby pulling the patch pipette from the cytoplasmic drop underconditions of low levels of Ca²⁺. Using the excised patch, wecontrolled the level of Ca²⁺ on the cytoplasmic side of thechannels. At a low level of Ca²⁺ (pCa=8) on the cytoplasmicside, the open frequency was very low and the opening time wasshort. An increase in Ca²⁺ on the cytoplasmic side (pCa = 5)increased both the frequency and the duration of opening. However,the conductance of the channels did not change. This regulationby Ca²⁺ of the K⁺ channels was reversible, that is, additionof EGTA on the cytoplasmic side inactivated the channels. Thepresent study demonstrates a direct action of Ca²⁺ on the K⁺channels. The physiological role of the K⁺ channel in the regulationof turgor in Lamprothamnium is discussed. (Received January 9, 1989; Accepted March 8, 1989)     

Negative Phototropism of Vaucheria terrestris Regulated by Calcium II. Inhibition by Ca2+-Channel Blockers and Mimesis by A23187

As reported in a previous article [Kataoka (1988a) Plant CellPhysiol. 29: 1323], growing apices of the xanthophycean coenocyticalga, Vaucheria terrestris, bends away from a unilateral bluelight (BL) source, if they are simultaneously irradiated withstrong background BL in a solution containing 1–4 mM Ca²⁺.Since the negative bending is a function of the product of theexternal Ca²⁺ concentration and the fluence rate of backgroundBL, a BL-induced Ca²⁺-influx at the apex was hypothesized tobe the cause of the phototropic inversion. The present reportprovides strong evidence for this hypothesis. Addition of theCa²⁺ channel blockers, La³⁺, verapamil, nifedipine and nitrendipineto media containing 4 mM Ca²⁺ completely inhibited the phototropicinversion. By contrast, 1 µM A23187 [GenBank] (plus 4 mM Ca²⁺) notonly enhanced the phototropic inversion under background BL,but also mimicked the background BL; i.e. it caused negativebending under safe red light. Inhibition of phototropic inversionby La³⁺ is also observed under conditions where the algae areirradiated with unilateral BL for 1 week. A BL-dependent Ca²⁺influx and a consequential elevation of the cytoplasmic Ca²⁺-levelin the apical growth region must be involved in the early stepsof phototropic response. A BL-controlled opening of L-type Ca²⁺channels is also suggested. ¹A part of this study was reported at the 3rd Phycological Congressat Melbourne 1988 (Kataoka 1988b) and XXII Yamada Conferenceon Plant Water Relations and Growth Under Stress at Osaka 1989(Kataoka 1989) ²Dedicated to Prof. em. Dr. Noburo Kamiya on the occasion ofhis 77th birthday (Received May 23, 1990; Accepted July 13, 1990)     

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; )     

Involvement of Water Channels in the Hydration-Induced Calcium Release in Nitella flexilis

An internodal cell of Nitella flexilis treated with 10 mM KC1was vacuole-perfused with an isotonic solution containing ethyleneglycol-bis-(rß-aminoethylether)N,N,N,N-tetraaceticacid (EGTA) and its content including cytoplasm was squeezedout into a vessel and covered with silicone oil. When the hypotonicsolution was added into the cytoplasmic drop which had beenmixed with aequorin, a significant increase in the light emissionfrom aequorin was detected with the photomultiplier, indicatinga release of Ca²⁺ from some cell organdies storing Ca²⁺. Thisincrease in the light emission was strongly inhibited by treatingcells with 0.1 mM HgCl₂ which is known to inhibit water channelsin the plasma membrane. The inhibition was completely recoveredby washing HgCl₂ with 2-mer-captoethanol. This suggests thatwater channels may exist in the membrane of Ca²⁺ stores andplay an essential role in the hydration-induced Ca²⁺ release. (Received February 12, 1998; Accepted May 21, 1998)     

Passive Interactions of Ca2+ and other Multivalent Cations with the Membranes of Isolated Corn Mitochondria

The presence of Ca²⁺ in a hypo-osmotic reaction medium reducessuccinate: cytochrome c reductase activity and the release ofouter membrane-specific antimycin A-insensitive NADH: cytochromec reductase. The action of Ca²⁺ is non-competitive and approximately30 mmol m^–3 Ca²⁺ affords half-maximal (I₅₀) protection.The effect of a range of inorganic and organic multivalent cationson succinate: cytochrome c reductase activity suggests thatthe action of Ca²⁺ is non-specific and probably involves Ca²⁺binding to outer membrane component(s) which may be proteins. Valinomycin- or gramicidin-induced passive swelling of isolatedcorn mitochondria in isotonic K.C1 is also non-competitivelyinhibited by up to 50% with Ca²⁺. Half-maximal inhibition (I₅₀)occurs at 0-35 mol m^–3 Ca²⁺ for valinomycin and 1-0 molm^–3 Ca²⁺ for gramicidin. Other divalent cations, Mg²⁺,Sr²⁺ and Ba²⁺, seem to inhibit similarly while the trivalentcations La³⁺ and Ho³⁺ show a maximum inhibition of up to 85%,with an I₅₀ of 0.1 mol m^–3 for valinomycin. It is suggestedthat non-specific cation binding may reduce membrane fluiditythereby slowing down the rate of ionophore penetration throughthe inner membrane. Key words: Calcium, Mitochondria, Membranes     

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)     

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.

     

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)     

Role of aspartate residues in Ca(2+) affinity and permeation of the distal ECaC1

The Ca²⁺ affinity andpermeation of the epithelial Ca²⁺ channel (ECaC1) wereinvestigated after expression in Xenopus oocytes. ECaC1displayed anomalous mole-fraction effects. Extracellular Ca²⁺ and Mg²⁺ reversibly inhibited ECaC1 wholecell Li⁺ currents: IC₅₀ = 2.2 ± 0.4 µM (n = 9) and 235 ± 35 µM (n = 10), respectively. These values compare well with theCa²⁺ affinity of the L-type voltage-gated Ca²⁺(Ca_V1.2) channel measured under the same conditions,suggesting that high-affinity Ca²⁺ binding is awell-conserved feature of epithelial and voltage-gated Ca²⁺channels. Neutralization of D550 and E535 in the pore region had nosignificant effect on Ca²⁺ and Mg²⁺ affinities.In contrast, neutralization of D542 significantly decreasedCa²⁺ affinity (IC₅₀ = 1.1 ± 0.2 mM,n = 6) and Mg²⁺ affinity(IC₅₀ > 25 ± 3 mM, n = 4).Despite a 1,000-fold decrease in Ca²⁺ affinity in D542N,Ca²⁺ permeation properties and theCa²⁺-to-Ba²⁺ conductance ratio remainedcomparable to values for wild-type ECaC1. Together, our observationssuggest that D542 plays a critical role in Ca²⁺ affinitybut not in Ca²⁺ permeation in ECaC1.

     

Enhanced response to caffeine and 4-chloro-m-cresol in malignant hyperthermia-susceptible muscle is related in part to chronically elevated resting [Ca2+]i

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. We have measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) using double-barreled, Ca²⁺-selective microelectrodes in myoballs prepared from skeletal muscle of MH-susceptible (MHS) and MH-nonsusceptible (MHN) swine. Resting [Ca²⁺]_i was approximately twofold in MHS compared with MHN quiescent myoballs (232 ± 35 vs. 112 ± 11 nM). Treatment of myoballs with caffeine or 4-chloro-m-cresol (4-CmC) produced an elevation in [Ca²⁺]_i in both groups; however, the concentration required to cause a rise in [Ca²⁺]_i elevation was four times lower in MHS than in MHN skeletal muscle cells. Incubation of MHS cells with the fast-complexing Ca²⁺ buffer BAPTA reduced [Ca²⁺]_i, raised the concentration of caffeine and 4-CmC required to cause an elevation of [Ca²⁺]_i, and reduced the amount of Ca²⁺ release associated with exposure to any given concentration of caffeine or 4-CmC to MHN levels. These results suggest that the differences in the response of MHS skeletal myoballs to caffeine and 4-CmC may be mediated at least in part by the chronic high resting [Ca²⁺]_i levels in these cells. calcium homeostasis; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid     

Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes

This study examines whether fluid pressure (FP) modulates the L-type Ca²⁺ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (16 dyn/cm²) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca²⁺ current (I_Ca) and cytosolic Ca²⁺ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed I_Ca (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of I_Ca. The level of I_Ca suppression by FP depended on the level and duration of pressure. The Ba²⁺ current through the Ca²⁺ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca²⁺ channel during FP stimulation. The cytosolic Ca²⁺ transients and the basal Ca²⁺ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the I_Ca and on the Ca²⁺ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca²⁺ buffer, eliminated the FP-induced acceleration of I_Ca inactivation and reduced the inhibitory effect of the FP on I_Ca by 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca²⁺ release, eliminated the accelerating effect of FP on the I_Ca inactivation, and they reduced the inhibitory effect of FP on the I_Ca. These results suggest that the fluid pressure indirectly suppresses the Ca²⁺ channel by enhancing the Ca²⁺-induced intracellular Ca²⁺ release in rat ventricular myocytes. L-type Ca²⁺ current; fluid pressure; ventricular myocytes; cytosolic Ca²⁺ transient