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)     

Patch-Clamp Study on Ion Channels in the Tonoplast of Nitellopsis obtusa

Cytoplasmic drops covered with the tonoplast were prepared frominternodal cells of Nitellopsis grown in fresh water. Applyingthe patch-clamp technique and the microinjection technique tosuch drops, we characterized the ion channels in the tonoplast.Both in cell-free patches and in the cytoplasmic-drop-attachedpatches, the tonoplast K⁺ channel was identified. The permeabilityratio between Na⁺ and K⁺ was calculated to be 0.2. This channelwould provide a molecular basis for the Na⁺/K⁺ exchange at thetonoplast. In cell-free patches, the K⁺channel was not activatedby Ca²⁺. However, in the case of attached patches, microinjectionof Ca²⁺ into a drop activated the K⁺ channel with a lag of afew seconds, suggesting that some cytoplasmic factor(s) maymediate the activation of the K⁺ channel by Ca²⁺. The conductanceof this channel was not changed by cytoplasmic Ca²⁺, but theprobability of opening increased markedly. In addition to theK⁺ channel, a second type of channel was also identified incell-free patches. This channel may be the Cl^– channel. ³ Present address: Department of Insect Physiology and Behavior,National Institute of Sericultural and Entomological Science,Tsukuba, Ibaraki, 305 Japan (Received August 6, 1990; Accepted December 6, 1990)     

A Soluble Chloroplast Protease Processes the Euglena Polyprotein Precursor to the Light Harvesting Chlorophyll a/b Binding Protein of Photosystem II

The Euglena light harvesting chlorophyll a/b binding proteinof photosystem II (LHCPII) is synthesized as a polyprotein precursorscomposed of 8 LHCPIIs covalently joined by a decapeptide. Asoluble chloroplast protease releases LHCPII from the polyprotein.The polyprotein processing peptidase has a pH optima between8.0 and 9.0. It is inhibited by Zn²⁺, Cu²⁺, phenylmethylsulfonylfluoride and E64 suggesting it is a novel thiol protease. ¹ Present address: Department of Food Sciences, Ishikawa AgriculturalCollege, Nonoichi, Ishikawa, 921 Japan.     

褐飞虱和桃蚜粗提液中酚氧化酶原激活系统的激活特点

采用研磨法制备褐飞虱Nilaparvata lugens (Stål)和桃蚜Myzus persicae (Sulzer)粗提液,用于研究几种化学因子对粗提液中酚氧化酶原激活系统(proPO-AS)的激活特点。结果表明, 在0.1～100 mmol/L的Ca²⁺浓度范围内,虱液和蚜液的酚氧化酶(PO)活性随Ca²⁺浓度升高而增强,并在30 mmol/L处PO活性达到最高,在此限之上PO活性反而下降。当昆布多糖浓度从10^-4 mg/mL升至10 mg/mL时,虱液和蚜液的PO活性也随之升高,但再提高昆布多糖浓度却未见PO活性的峰顶出现。在6种葡聚糖对PO的激活作用中,昆布多糖和酵母聚糖能显著增强PO活性,但curdlan对虱液和蚜液的PO无明显激活作用,而甘露聚糖、右旋糖苷和纤维素则降低PO活性。这些结果显示β-1,3-葡聚糖能有效激活proPO-AS。Ca²⁺和丝氨酸蛋白酶抑制剂PMSF加样顺序的不同也会影响PO活性。     

Effect of intracellular pH on acetylcholine-induced Ca2+ waves in mouse pancreatic acinar cells

We have used fluo3-loaded mouse pancreatic acinar cells to investigate the relationshipbetween Ca²⁺ mobilization andintracellular pH (pH_i). TheCa²⁺-mobilizing agonist ACh (500 nM) induced a Ca²⁺ release in theluminal cell pole followed by spreading of the Ca²⁺ signal toward the basolateralside with a mean speed of 16.1 ± 0.3 µm/s. In the presence of anacidic pH_i, achieved by blockade of theNa⁺/H⁺exchanger or by incubation of the cells in aNa⁺-free buffer, a slowerspreading of ACh-evoked Ca²⁺ waveswas observed (7.2 ± 0.6 µm/s and 7.5 ± 0.3 µm/s,respectively). The effects of cytosolic acidification on thepropagation rate of ACh-evokedCa²⁺ waves were largely reversibleand were not dependent on the presence of extracellularCa²⁺. A reduction in the spreadingspeed of Ca²⁺ waves could also beobserved by inhibition of the vacuolarH⁺-ATPase with bafilomycinA₁ (11.1 ± 0.6 µm/s), whichdid not lead to cytosolic acidification. In contrast, inhibition of theendoplasmic reticulum Ca²⁺-ATPaseby 2,5-di-tert-butylhydroquinone ledto faster spreading of the ACh-evokedCa²⁺ signals (25.6 ± 1.8 µm/s), which was also reduced by cytosolic acidification or treatmentof the cells with bafilomycin A₁.Cytosolic alkalinization had no effect on the spreading speed of theCa²⁺ signals. The data suggestthat the propagation rate of ACh-induced Ca²⁺ waves is decreased byinhibition of Ca²⁺ release fromintracellular stores due to cytosolic acidification or toCa²⁺ pool alkalinizationand/or to a decrease in the proton gradient directed from theinositol 1,4,5-trisphosphate-sensitiveCa²⁺ pool to the cytosol.

     

Effects of elevated physiological temperatures on sarcoplasmic reticulum function in mechanically skinned muscle fibers of the rat

Properties of the sarcoplasmic reticulum (SR) with respect to Ca²⁺ loading and release were measured in mechanically skinned fiber preparations from isolated extensor digitorum longus (EDL) muscles of the rat that were either kept at room temperature (23°C) or exposed to temperatures in the upper physiological range for mammalian skeletal muscle (30 min at 40 or 43°C). The ability of the SR to accumulate Ca²⁺ was significantly reduced by a factor of 1.9–2.1 after the temperature treatments due to a marked increase in SR Ca²⁺ leak, which persisted for at least 3 h after treatment. Results with blockers of Ca²⁺ release channels (ruthenium red) and SR Ca²⁺ pumps [2,5-di(tert-butyl)-1,4-hydroquinone] indicate that the increased Ca²⁺ leak was not through the SR Ca²⁺ release channel or the SR Ca²⁺ pump, although it is possible that the leak pathway was via oligomerized Ca²⁺ pump molecules. No significant change in the maximum SR Ca²⁺-ATPase activity was observed after the temperature treatment, although there was a tendency for a decrease in the SR Ca²⁺-ATPase. The observed changes in SR properties were fully prevented by the superoxide (O₂^–) scavenger Tiron (20 mM), indicating that the production of O₂^– at elevated temperatures is responsible for the increase in SR Ca²⁺ leak. Results show that physiologically relevant elevated temperatures 1) induce lasting changes in SR properties with respect to Ca²⁺ handling that contribute to a marked increase in the SR Ca²⁺ leak and, consequently, to the reduction in the average coupling ratio between Ca²⁺ transport and SR Ca²⁺-ATPase and muscle performance, and 2) that these changes are mediated by temperature-induced O₂^– production. skeletal muscle; calcium ion leak; superoxide; skinned fibers     

Native Cadmium-Metallothionein from the Yeast Saccharomyces cerevisiae: Its Primary Structure and Function in Heavy-Metal Resistance

A Cd-resistant strain of yeast (Saccharomyces cerevisiae, strain30IN) accumulated Cd with the concomitant synthesis of a Cd-bindingprotein of low molecular weight when grown in Cd²⁺-containingmedium. Analysis of the amino acid composition, N-terminal sequenceand immunological properties of the protein revealed its structuralhomology to Cu-metallothionein (Cu-MT) in S. cerevisiae 2186,a Cu-resistant strain of yeast (Winge et al. 1985). The synthesisof MT in Cu-resistant strains of yeast is known to be underthe strict control of Cu²⁺ ions, while that in 301N was inducedboth by Cd²⁺ and Cu²⁺ ions. When 301N was precultured for 48h in the presence of 1 mM CuSO₄, its resistance to Cu²⁺ andthe synthesis of MT in response to Cu²⁺ were enhanced whileanalogous responses to Cd²⁺ were conversely reduced. These resultssuggest that the synthesis of MT is controlled by Cd²⁺ and Cu²⁺in a counteractive manner in strain 301N and, therefore, theregulation of the synthesis of MT plays a role in the adaptationof this strain to conditions when either metal is present. (Received November 1, 1990; Accepted February 22, 1991)     

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.

     

Protective Effect of External Ca2+ on Elongation and the Intracellular Concentration of K+ in Intact Mung Bean Roots under High NaCl Stress

The effects of Ca²⁺ in the external medium on intact mung beanroots under high NaCl stress were investigated. With increasingexternal concentrations of NaCl, mung bean roots showed suppressionof elongation and a decrease in the intracellular concentrationof K⁺. Addition of Ca²⁺ to the external medium alleviated theinhibition of root elongation under the high NaCl stress andmaintained a high intracellular concentration of K⁺ in the elongatingregion of the roots. This counter effect of Ca²⁺ against theNaCl stress on roots was correlated with the ratio of [Ca²⁺]/[Na⁺]²in the external medium. A value above 5.0 ? 10^–4 mM^–1resulted in almost complete recovery of root elongation undervarious high concentrations of NaCl. Root elongation for 24h under NaCl stress was correlated with the extent to whichthe intracellular concentration of K⁺ was in excess of 10 mM.Maintenance of an adequate concentration of K⁺ in root cellsis essential for root elongation under salt stress. These findingsindicate that Ca²⁺ prevents the leakage of intracellular K⁺and thereby supports the elongation of roots under salt stress. (Received November 13, 1989; Accepted June 5, 1990)     

Control of Ca2+ wave propagation in mouse pancreatic acinar cells

We haveinvestigated control mechanisms involved in the propagation ofagonist-induced Ca²⁺ waves inisolated mouse pancreatic acinar cells. Using a confocal laser-scanningmicroscope, we were able to show that maximal stimulation of cells withacetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initialCa²⁺ release of comparable amountswith both agonists at the luminal cell pole. SubsequentCa²⁺ spreading to the basolateralmembrane was faster with ACh (17.3 ± 5.4 µm/s) than with bombesin(8.0 ± 2.2 µm/s). The speed of bombesin-inducedCa²⁺ waves could be increased upto the speed of ACh-induced Ca²⁺waves by inhibition of protein kinase C (PKC). Activation of PKCsignificantly decreased the speed of ACh-inducedCa²⁺ waves but had only littleeffect on bombesin-evoked Ca²⁺waves. Within 3 s after stimulation, production of inositol1,4,5-trisphosphate [Ins(1,4,5)P₃]was higher in the presence of ACh compared with bombesin, whereasbombesin induced higher levels of diacylglycerol (DAG) than ACh. Thesedata suggest that the slower propagation speed of bombesin-inducedCa²⁺ waves is due to higheractivation of PKC in the presence of bombesin compared with ACh. Thehigher increase in bombesin- compared with ACh-induced DAG productionis probably due to activation of phospholipase D (PLD). Inhibition ofthe PLD-dependent DAG production by preincubation with 0.3% butanolled to an acceleration of the bombesin-induced Ca²⁺ wave. In further experiments,we could show that ruthenium red (100 µM), an inhibitor ofCa²⁺-inducedCa²⁺ release in skeletal muscle,also decreased the speed of ACh-induced Ca²⁺ waves. The effect ofruthenium red was not additive to the effect of PKC activation. Fromthe data, we conclude that, following Ins(1,4,5)P₃-inducedCa²⁺ release in the luminal cellpole, secondary Ca²⁺ release fromstores, which are located in series between the luminal and the basalplasma membrane, modifies Ca²⁺spreading toward the basolateral cell side byCa²⁺-inducedCa²⁺ release. Activation of PKCleads to a reduction in Ca²⁺release from these stores and therefore could explain the slower propagation of Ca²⁺ waves in thepresence of bombesin compared with ACh.

     

Effect of extracellular polymeric substances (EPS) on the attachment of activated sludge

Extracellular polymeric substances (EPS) were removed by mechanical (high-speed centrifugation) and chemical (EDTA treatment) methods. The number of attached microorganisms decreased from 33.0 2 10⁷ CFU/cm² to 17.5 2 10⁷ CFU/cm² and 12.5 2 10⁷ CFU/cm², respectively. When the activated sludge was treated with polysaccharide-oxidizing agent (sodium meta periodate) and protease (pronase E), the number of attached microorganisms decreased to 41% and 43.5%, respectively. Transmission electron microphotographs showed that polysaccharide-oxidizing agent and protease treatment caused the removal of filamentous extracellular structures.     

Ca2+ mediates the effect of inhibition of Na+-K+-ATPase on the basolateral K+ channels in the rat CCD

We investigated the effect ofinhibiting Na⁺-K⁺-ATPase on the basolateral18-pS K⁺ channel in the cortical collecting duct (CCD) ofthe rat kidney. Inhibiting Na⁺-K⁺-ATPase withstrophanthidin decreased the activity of the 18-pS K⁺channel and increased the intracellular Ca²⁺ to 420 nM.Removal of extracellular Ca²⁺ abolished the effect ofstrophanthidin. When intracellular Ca²⁺ was raised with 5 µM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the18-pS K⁺ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism ofCa²⁺-induced inhibition, the effect of 400 nMCa²⁺ on channel activity was studied in the presence ofcalphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62,inhibitors of calmodulin-dependent kinase II. Addition of calphostin Cor KN-93 or KN-62 failed to block the inhibitory effect of highconcentrations of Ca²⁺. This suggested that the inhibitoryeffect of high concentrations of Ca²⁺ was not mediated byprotein kinase C or calmodulin-dependent kinase II pathways. To examinethe possibility that the inhibitory effect of high concentrations ofCa²⁺ was mediated by the interaction of nitric oxide withsuperoxide, we investigated the effect of 400 nM Ca²⁺ onchannel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) orN-nitro-L-arginine methyl ester.Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonicacid or N-nitro-L-arginine methylester completely abolished the inhibitory effect of 400 nMCa²⁺ on channel activity. Moreover, application of4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effectof strophanthidin. We conclude that the effect of inhibitingNa⁺-K⁺-ATPase is mediated by intracellularCa²⁺ and the inhibitory effect of high concentrations ofCa²⁺ is the result of interaction of nitric oxide with superoxide.

     

Hormone-induced rise in cytosolic Ca2+ in axolotl hepatocytes: properties of the Ca2+ influx channel

Calcium entry in nonexcitable cells occurs throughCa²⁺-selective channels activatedsecondarily to store depletion and/or through receptor- orsecond messenger-operated channels. In amphibian liver, hormones thatstimulate the production of adenosine 3',5'-cyclic monophosphate (cAMP) also regulate the opening of an ion gate in theplasma membrane, which allows a noncapacitative inflow ofCa²⁺. To characterize thisCa²⁺ channel, we studied theeffects of inhibitors of voltage-dependent Ca²⁺ channels and of nonselectivecation channels on 8-bromoadenosine 3',5'-cyclicmonophosphate (8-BrcAMP)-dependentCa²⁺ entry in single axolotlhepatocytes. Ca²⁺ entry provokedby 8-BrcAMP in the presence of physiologicalCa²⁺ followed first-order kinetics(apparent Michaelis constant = 43 µM at the cellsurface). Maximal values of cytosolicCa²⁺ (increment ~300%) werereached within 15 s, and the effect was transient (half time of 56 s).We report a strong inhibition of cAMP-dependentCa²⁺ entry by nifedipine[half-maximal inhibitory concentration(IC₅₀) = 0.8 µM], byverapamil (IC₅₀ = 22 µM), andby SK&F-96365 (IC₅₀ = 1.8 µM).Depolarizing concentrations of K⁺were without effect. Gadolinium and the anti-inflammatory compound niflumate, both inhibitors of nonselective cation channels, suppressed Ca²⁺ influx. This "profile"indicates a novel mechanism ofCa²⁺ entry in nonexcitable cells.

     

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)     

Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms

We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Because the sarcoplasmic reticulum (SR) and Na⁺/Ca²⁺ exchanger (NCX) play crucial roles in regulating [Ca²⁺]_i and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca²⁺ overload by maintaining Ca²⁺ homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca²⁺ transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca²⁺ transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca²⁺]_i levels and attenuated the depression of the Ca²⁺ transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca²⁺ release channels and/or ryanodine receptors (RyRs) and SR Ca²⁺ pump ATPase (SERCA2) and SR Ca²⁺ release and uptake. In addition, a delayed decay rate time constant of Ca²⁺ transients and cell shortening of Ca²⁺ transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca²⁺ homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R. intracellular Ca²⁺ concentration; Ca²⁺ transients; Ca²⁺ transporters; myofilament Ca²⁺ sensitivity     

Mitochondrial transport in processes of cortical neurons is independent of intracellular calcium

Mitochondria show extensive movement along neuronal processes, but the mechanisms and function of this movement are not clearly understood. We have used high-resolution confocal microscopy to simultaneously monitor movement of mitochondria and changes in intracellular [Ca²⁺] ([Ca²⁺]_i) in rat cortical neurons. A significant percentage (27%) of the total mitochondria in cortical neuronal processes showed movement over distances of >2 µM. The average velocity was 0.52 µm/s. The velocity, direction, and pattern of mitochondrial movement were not affected by transient increases in [Ca²⁺]_i associated with spontaneous firing of action potentials. Stimulation of Ca²⁺ transients with forskolin (10 µM) or bicuculline (10 µM), or sustained elevations of [Ca²⁺]_i evoked by glutamate (10 µM) also had no effect on mitochondrial transit. Neither removal of extracellular Ca²⁺, depletion of intracellular Ca²⁺ stores with thapsigargin, or inhibition of synaptic activity with TTX (1 µM) or a cocktail of CNQX (10 µM) and MK801 (10 µM) affected mitochondrial movement. These results indicate that movement of mitochondria along processes is a fundamental activity in neurons that occurs independently of physiological changes in [Ca²⁺]_i associated with action potential firing, synaptic activity, or release of Ca²⁺ from intracellular stores. calcium transient; dendrites     

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)     

Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger

To assess the role of Ca²⁺in regulation of theNa⁺/H⁺exchanger (NHE1), we used CCL-39 fibroblasts overexpressing theNa⁺/Ca²⁺exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca²⁺ rise andlong-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant withthe calmodulin (CaM)-binding domain deleted (amino acids 637-656),suggesting that the effect of NCX1 transfection involves Ca²⁺-CaM binding. Expression ofNCX1 only slightly inhibited platelet-derived growth factor BB-inducedalkalinization and did not affect hyperosmolarity- or phorbol12-myristate 13-acetate-induced alkalinization. Downregulation ofprotein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely inNCX1-transfected cells, suggesting that the thrombin effect is mediatedexclusively via Ca²⁺ and PKC. Onthe other hand, deletion mutant study revealed that PKC-dependentregulation occurs through a small cytoplasmic segment (amino aids566-595). These data suggest that a mechanism involving directCa²⁺-CaM binding lasts for arelatively long period after agonist stimulation, despite apparentshort-lived Ca²⁺ mobilization, andfurther support our previous conclusion that Ca²⁺- and PKC-dependent mechanismsare mediated through distinct segments of the NHE1 cytoplasmic domain.

     

Further study on the role of HSP70 on Ca2+ homeostasis in rat ventricular myocytes subjected to simulated ischemia

We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca²⁺ at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), and Na⁺/Ca²⁺ exchanger (NCX) handled Ca²⁺ in rat ventricular myocytes preconditioned with a -opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca²⁺ transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca²⁺ homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca²⁺ fluxes via NCX by the use of ⁴⁵Ca²⁺ in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca²⁺ from the sarcoplasmic reticulum via RyR and in the removal of Ca²⁺ from the cytoplasm by reuptake of Ca²⁺ to the SR via SERCA and by extrusion of Ca²⁺ out of the cell via NCX. UP also reduced mitochondrial Ca²⁺ accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca²⁺ homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca²⁺ overload, improved recovery in contractile function, and cardioprotection. intracellular Ca²⁺, -opioid receptor; Na⁺/Ca²⁺ exchanger; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase     

Elementary events of agonist-induced Ca2+ release in vascular endothelial cells

The subcellular spatial and temporal organization ofagonist-induced Ca²⁺ signals wasinvestigated in single cultured vascular endothelial cells.Extracellular application of ATP initiated a rapid increase ofintracellular Ca²⁺ concentration([Ca²⁺]_i)in peripheral cytoplasmic processes from where activation propagated asa[Ca²⁺]_iwave toward the central regions of the cell. The average propagation velocity of the[Ca²⁺]_iwave in the peripheral processes was 20-60 µm/s, whereas in thecentral region the wave propagated at <10 µm/s. The time course ofthe recovery of[Ca²⁺]_idepended on the cell geometry. In the peripheral processes (i.e.,regions with a high surface-to-volume ratio)[Ca²⁺]_ideclined monotonically, whereas in the central region[Ca²⁺]_idecreased in an oscillatory fashion. Propagating[Ca²⁺]_iwaves were preceded by small, highly localized[Ca²⁺]_itransients originating from 1- to 3-µm-wide regions. The average amplitude of these elementary events ofCa²⁺ release was 23 nM, and theunderlying flux of Ca²⁺ amountedto ~1-2 × 10¹⁸mol/s or ~0.3 pA, consistent with aCa²⁺ flux through a single orsmall number of endoplasmic reticulum Ca²⁺-release channels.