Interrelationships between Potassium, Calcium, and Magnesium Nutrition of Zea mays L.

The uptake of potassium, calcium, and magnesium ions by maizeand the interrelationships among the cations have been investigatedat 48 K: Ca: Mg ratios in culture solutions. Calcium was foundto stimulate K⁺ and Mg⁺⁺ uptake at certain cation ratios butinhibit it at others. Potassium did the same for Ca⁺⁺ uptake,and Mg⁺⁺ for Ca⁺⁺ and K⁺. The uptake of Mg⁺⁺ was generally enhancedby K⁺. The sum of the cations in the plants expressed in meqwas fairly constant for treatments of the same K⁺ concentrationat the low to moderate levels of K⁺, but at considerably higher(> 24 meq l^–1) K⁺ levels the constancy was not dependenton K⁺ concentration. Potassium depressed, but Mg⁺⁺ stimulatedphosphorus accumulation. Calcium stimulated phosphate absorptionat certain cation ratios but had no effect at others. The plantyield increased with increasing K⁺ up to 24 meq l^–1 ofK⁺ after which the yield tended to fall with further increasein K⁺. The yield was also increased by Ca⁺⁺. Magnesium increasedthe yield at certain cation ratios and either depressed it orwas without effect at others.     

Effect of Calcium on Cell Wall Structure, Protein Phosphorylation and Protein Profile in Senescing Apples

The effect of Ca⁺⁺ on various parameters of apple fruit senescencewas investigated. Distinct and specific changes in polypeptideand phosphoprotein patterns were observed in Ca⁺⁺ treated ascompared to control fruits. A 70 kDa salt-extracted polypeptidebecame apparent in control fruits after 8 months of cold storagewhich was not apparent in Ca⁺⁺-treated fruits until 12 months.The soluble protein profile of Ca⁺⁺-treated fruits showed anaccumulation of a 30 kDa polypeptide while the control fruitsaccumulated a 60 kDa polypeptide. Autoradiographs of phosphorylatedpolypeptides revealed a 60 kDa membrane polypeptide becomingphosphorylated in the Ca⁺⁺-treated and not in the control fruitprotein fractions. Transmission electron micrographs of thecell showed Ca⁺⁺ to be effective in maintaining the cell wallstructure, particularly the middle lamella. Furthermore, increasein fruit Ca⁺⁺ reduced CO₂ and C₂H₂ evolution and altered chlorophyllcontent, ascorbic acid level and hydraulic permeability. ¹Scientific Paper No: 7930, College of Agriculture and HomeEconomics Research Center, Washington State University, Pullman,Washington, Project 0321. ²Supported by grants from the National Science Foundation CB-8502215and Washington State Tree Fruit Research Commission to BWP. (Received September 3, 1987; Accepted March 3, 1988)     

Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals

In luteal cells, prostaglandin (PG)F_2a mobilizes intracellular calcium concentration ([Ca]_i), generates reactive oxygen species (ROS), depletes ascorbic acid (AA) levels, inhibits steroidogenesis, and ultimately induces cell death. We investigated the hypothesis that [Ca]_i mobilization stimulates ROS, which results in depletion of cellular AA in rat luteal cells. We used a self-referencing AA-selective electrode that noninvasively measures AA flux at the extended boundary layer of single cells and fluorescence microscopy with fura 2 and dichlorofluorescein diacetate (DCF-DA) to measure [Ca]_i and ROS, respectively. Menadione, a generator of intracellular superoxide radical (), PGF_2a, and calcium ionophore were shown to increase [Ca]_i and stimulate intracellular ROS. With calcium ionophore and PGF_2a, but not menadione, the generation of ROS was dependent on extracellular calcium influx. In unstimulated cells there was a net efflux of AA of 121.5 ± 20.3 fmol · cm^–1 · s^–1 (mean ± SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 ± 4.9 fmol · cm^–1 · s^–1; n = 5, P < 0.05). PGF_2a and menadione stimulated AA efflux, but calcium ionophore had no significant effect. These data suggest two AA regulatory mechanisms: Under basal conditions, AA efflux is calcium dependent and may represent recycling and maintenance of an antioxidant AA gradient at the plasma membrane. Under luteolytic hormone and/or oxidative stress, AA efflux is stimulated that is independent of extracellular calcium influx or generation of ROS. Although site-specific mobilization of calcium pools and ROS cannot be ruled out, the release of AA by PGF_2a-stimulated luteal cells may occur through other signaling pathways. luteolysis; apoptosis; self-referencing microelectrode     

Extracellular calcium dependence of contracture and modulation by serotonin in buccal muscle E1 of Aplysia

Serotonin [5-hydroxytryptamine (5-HT)] enhances acetyl choline (ACh)-elicited contractures of Aplysia buccal muscles E1 and I5. The possible role of external calcium in regulating the magnitude of ACh contracture in the presence and absence of 5-HT was investigated. Superfusion of E1 with zero calcium medium caused ACh contractures to fail within one to two minutes. Recovery of ACh contracture upon restoring normal medium occurred within two to four minutes. In the absence of 5-HT, ACh contracture decreased proportionally to external [Ca⁺⁺] in the concentration range of 0–10 mM; however, the amount of enhancement of of ACh contracture following 5-HT treatment did not decrease with external [Ca⁺⁺] as long as [Ca⁺⁺] was above a threshold concentration that varied from preparation to preparation. For most preparations, the enhancement of ACh contracture by 5-HT was dependent on the presence of external calcium during 5-HT treatment. Calcium influx into muscles E1 and I5 increased approximately two and a half fold in the presence of 10^?6 M 5-HT. A model in which 5-HT brings about calcium “loading” of an ACh releasable intracellular storage site is discussed.     

Uncoupling Properties of the Herbicide N-n-Pentyl-N-Methyl-N'-(3,4-Dichlorophenyl)-Urea

The herbicide D₅ (N-n-pentyl-N-methyl-N'-(3, 4-dichlorophenyl)-ureacan uncouple oxidative phosphorylation in isolated plant mitochondria.This paper confirms that D₅ is an uncoupler that catalyzes thecollapse of the transmembrane potential gradient by inducinga movement of protons across the membrane. However, D₅ is notitself capable of transporting protons. D₅ gives complete uncouplingat 40 µM, a lower concentration than that required foruncoupling by the n-butyl homologue ‘neburon’. Analysisof the shape of the state 4 stimulation curve suggests thatD₅ might act as a dimer in the membrane. Attempts to demonstrate binding of D₅ to a membrane target gaveambiguous results, binding is not evident at 10 °C and 25°C but might occur at 15 °C and 20 °C. The calculatedherbicide concentration in the membrane (40 µM of which4–0 µM is as the dimer) is high and similar to thatof the major phospholipids. The calculated partition coefficientbetween medium and membrane (3.8 x 103) is in agreement withthe lipophilicity of the substituted urea herbicides. In the presence of a substrate, D₃ blocks both influx and effluxcalcium movement through the mitochondrial membrane but in theabsence of substrate, D₅ induces binding of calcium. Bindingrequires Mg⁺⁺ but not K⁺ or phosphate and leads to a releaseof H⁺. Ruthenium Red causes a partial inhibition of bindingbut no other reagent or ionophore tested had any effect. Sincebinding does not occur in turnip mitochondria which are unableto transport Ca⁺⁺ it is concluded that the effect is not directlylinked to the uncoupling action. The mechanism of action of D₅ is discussed and it is concludedthat D₅ probably acts as a dimer and perturbs membrane structure.The site of action is probably the lipid components of the membrane. Key words: Plant mitochondria, Herbicide, Substituted ureas, Calcium, Uncoupling     

Effect of intracellular pH on depolarization-evoked calcium influx in human sperm

Human sperm are endowed with putative voltage-dependent calcium channels (VDCC) that produce measurable increases in intracellular calcium concentration ([Ca²⁺]_i) in response to membrane depolarization with potassium. These channels are blocked by nickel, inactivate in 1–2 min in calcium-deprived medium, and are remarkably stimulated by NH₄Cl, suggesting a role for intracellular pH (pH_i). In a previous work, we showed that calcium permeability through these channels increases approximately onefold during in vitro "capacitation," a calcium-dependent process that sperm require to fertilize eggs. In this work, we have determined the pH_i dependence of sperm VDCC. Simultaneous depolarization and pH_i alkalinization with NH₄Cl induced an [Ca²⁺]_i increase that depended on the amount of NH₄Cl added. VDCC stimulation as a function of pH_i showed a sigmoid curve in the 6.6–7.2 pH_i range, with a half-maximum stimulation at pH 7.00. At higher pH_i (7.3), a further stimulation occurred. Calcium release from internal stores did not contribute to the stimulating effect of pH_i because the [Ca²⁺]_i increase induced by progesterone, which opens a calcium permeability pathway that does not involve gating of VDCC, was unaffected by ammonium. The ratio of pH_i-stimulated-to-nonstimulated calcium influx was nearly constant at different test depolarization values. Likewise, depolarization-induced calcium influx in pH_i-stimulated and nonstimulated cells was equally blocked by nickel. In our capacitating conditions pH_i increased 0.11 pH units, suggesting that the calcium influx stimulation observed during sperm capacitation might be partially caused by pH_i alkalinization. Additionally, a calcium permeability pathway triggered exclusively by pH_i alkalinization was detected. mammalian sperm; capacitation; intracellular calcium     

Long-term Respiratory Cost of Maintenance and Growth of Field-grown Young Hinoki Cypress (Chamaecyparis obtusa)

Respiration rate of the entire above-ground parts of field-grown8-year-old hinoki cypress [Chamaecyparis obtusa(Sieb. et Zucc.)Endl.] was measured at monthly intervals over a 5-year period,to evaluate the trend in proportion of maintenance and growthcomponents of respiration with stand development. Representativesample trees were selected for respiration measurements. Theannual respiration rates of individual sample trees were combinedand partitioned into maintenance and growth components by regressingspecific respiration rate on mean relative growth rate. Maintenanceand growth respiration coefficients generated in this way were5.2 mol CO₂kg^-1year^-1and 39 mol  CO₂kg^-1, which are equivalentto 14.3 mg C kg^-1C h^-1(at mean annual air temperature of 15.1°C) and 0.94 kg C kg^-1C. Considering the maintenance andgrowth respiration coefficients, and phytomass and phytomassincrement of individual trees in the stand, the maintenanceand growth respiration rates of the stand were estimated. Theproportion of the maintenance respiration increased, whereasthat of the growth respiration decreased with stand development,due to decreasing relative growth rate.Copyright 1997 Annalsof Botany Company Chamaecyparis obtusa; growth respiration coefficient; hinoki; maintenance respiration coefficient; stand respiration     

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes

Spontaneous Ca²⁺ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 µM), but the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 µM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca²⁺ release events were also observed in 20% of cells treated with IP₃R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 µM; 0 Ca²⁺)-evoked Ca²⁺ transients but increased caffeine (10 mM; 0 Ca²⁺) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 µM; 0 Ca²⁺) were also enhanced by 2-APB. Ca²⁺ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 µM), which sensitizes the IP₃R to IP₃. In cells voltage clamped at –40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca²⁺ store content in smooth muscle is limited by tonic release of Ca²⁺ via an IP₃-dependent pathway. Blockade of IP₃Rs elevates sarcoplasmic reticulum store content, promoting Ca²⁺ sparks and STOC activity. calcium ion release; calcium ion transients; smooth muscle     

Palytoxin disrupts cardiac excitation-contraction coupling through interactions with P-type ion pumps

Palytoxin is a coral toxin that seriously impairs heart function, but its effects on excitation-contraction (E-C) coupling have remained elusive. Therefore, we studied the effects of palytoxin on mechanisms involved in atrial E-C coupling. In field-stimulated cat atrial myocytes, palytoxin caused elevation of diastolic intracellular Ca²⁺ concentration ([Ca²⁺]_i), a decrease in [Ca²⁺]_i transient amplitude, Ca²⁺ alternans followed by [Ca²⁺]_i waves, and failures of Ca²⁺ release. The decrease in [Ca²⁺]_i transient amplitude occurred despite high sarcoplasmic reticulum (SR) Ca²⁺ load. In voltage-clamped myocytes, palytoxin induced a current with a linear current-voltage relationship (reversal potential 5 mV) that was blocked by ouabain. Whole cell Ca²⁺ current and ryanodine receptor Ca²⁺ release channel function remained unaffected by the toxin. However, palytoxin significantly reduced Ca²⁺ pumping of isolated SR vesicles. In current-clamped myocytes stimulated at 1 Hz, palytoxin induced a depolarization of the resting membrane potential that was accompanied by delayed afterdepolarizations. No major changes of action potential configuration were observed. The results demonstrate that palytoxin interferes with the function of the sarcolemmal Na⁺-K⁺ pump and the SR Ca²⁺ pump. The suggested mode of palytoxin toxicity in the atrium involves the conversion of Na⁺-K⁺ pumps into nonselective cation channels as a primary event followed by depolarization, Na⁺ accumulation, and Ca²⁺ overload, which, in turn, causes arrhythmogenic [Ca²⁺]_i waves and delayed afterdepolarizations. atrial myocytes; intracellular calcium     

Effects of External Calcium Deprivation on Single Muscle Fibers

Deprivation of external calcium causes sudden potentiation of the twitch response of single muscle fibers. The potentiation was 64 ± 8%. Potentiation is simultaneous with membrane depolarization occurring after Ca⁺⁺ removal. This depolarization amounted to 9 ± 2 mv. Ca⁺⁺ removal also alters the action potential. 3 min after calcium withdrawal, action potential amplitude fell by 36 ± 3 mv; maximum rates of rise and fall of the spike decreased by 55 ± 5 and 63 ± 5% respectively. Changes in shape of the A. P. differ from those seen with other potentiators of the twitch response, such as Zn⁺⁺. After short exposure to calcium-free media, potassium-induced contractures show potentiation of peak tension. The S-shaped curve relating potassium contracture tension to log [K]_o shifts to the left after such treatment. Calcium deprivation also increased the rate of relaxation of the contractures. This effect depends on the duration of calcium deprivation, and is probably related to the effect of calcium lack on the membrane. The change in relaxation occurred immediately after calcium deprivation, and was reversed by sudden readmission of calcium. Relaxation of twitch and tetanus responses also were affected by Ca lack, but not as rapidly as potassium contractures. The results suggest that external calcium is not directly involved in the process responsible for tension development, supporting the view that this process is mediated by translocation of intracellular calcium. The relaxation process, however, appears to be rapidly affected by deprivation of external calcium.     

Gibberellic-Acid-Promoted Transport of Assimilates in Stems of Phaseolus vulgaris. Effects on Assimilate Accumulation by the Sink

Under conditions of apoplastic unloading from the sieve element-companioncell (se-cc) complexes in fully-elongated stems of Phaseolusvulgaris plants, gjbberellic acid (GA₃ stimulated in vitro uptakeof [¹⁴C]sucrose by the stem tissues. The GA₃, response dependedupon the incubate containing calcium ions and being bufferedat pH 6. The GA₃ action could be accounted for by a reductionin the Michaelis-Menten constant of the uptake process. Promotedtransport by GA₃ in the decapitated stems resulted in all thetissues accumulating higher levels of [¹⁴C]photosynthates. Comparisonof this response with that for in vitro uptake of [¹⁴C]sucroseindicated that GA₃ stimulation of the sucrose uptake processcontributed significantly to the accumulation of photosynthatesby the pith alone. The bulk of enhanced photosynthate accumulationby the remaining stem tissues can be accounted for by a GA,-inducedelevation of the apoplast sucrose concentration. In terms ofonset and change in rate, the time-course kinetics of GA₃ stimulationof [¹⁴C]photosynthate transport and of in vitro [¹⁴CJsucroseuptake were found to be similar. It is proposed that GA₃ promotionof photosynthate accumulation by the pith tissues is a minorcontributing factor to GA₃ regulation of phloem translocation Phaseolus vulgaris L., french bean, stem, assimilate transport, gibberellic acid, rink accumulation     

The Effect of Calcium on the Fluidity and Phase Properties of Microsomal Membranes Isolated from Postclimacteric Golden Delicious Apples

Calcium and magnesium have been found to stabilize and preservethe ethylene-synthesizing capacity of postclimacteric GoldenDelicious apple slices. In order to assess if this reflectsan effect at the membrane level, we have used three spin labelsto assay changes in the physical properties of isolated microsomalmembranes treated with calcium. A surface spin label, 18NP (an18 carbon alkane with a terminal quaternary amine attached toa nitroxyl-containing pyrollidine ring), reported a 20.8% increasein rotational correlation time (_c) at 25?C and a 25.3% increasein activation energy (E_a) calculated from linear Arrhenius plotsof _c for microsomal membranes treated with 50 mM CaCl₂. Regionsdeeper within the bilayer were probed using two fatty acid spinlabels—I(l2,3), stearic acid bearing a paramagnetic nitroxidegroup on carbon 5, and I(1,14), stearic acid bearing a paramagneticnitroxide group on carbon 16. The calcium effect was less pronounceddeeper within the lipid bilayer, there being only a 10.6% increasein _c and a 6% increase in the value of an order parameter (S)calculated from spectra recorded at 25?C for microsomes treatedwith 50 mM CaCl₂ and labelled with I(1,14) and I(12,3) respectively.There was no significant change in E_a for I(1,14)-labelled membranestreated with 50 mM CaCl₂. Similar trends were observed for membranestreated with 5 mM CaCl₂, although the degree of change was lessand differences were not always significant. Wide-angle X-raydiffraction revealed that untreated microsomal membranes frompostclimacteric apples were exclusively liquid-crystalline at25?C, as were microsomes treated with 50 mM CaCl₂. The transitiontemperature, defined as the highest temperature at which gelphase lipid could be detected, was below –25?C for bothtreated and untreated membranes. It is apparent, therefore, that Ca^2$ rigidifies and stabilizesmembranes, particularly at their surfaces. Inasmuch as partsof the ethylene biosynthetic pathway appear to be membrane-associated,this rigidification may, in part, act to preserve ethylene production. (Received August 3, 1981; Accepted November 4, 1981)     

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

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

Upregulation of Ca2+ removal in human skeletal muscle: a possible role for Ca2+-dependent priming of mitochondrial ATP synthesis

In muscle, ATP is required for the powerstroke of the myosin head, the detachment of actin and myosin filaments, and the reuptake of Ca²⁺ into the sarcoplasmic reticulum. During contraction-relaxation, large amounts of ATP are consumed at the sites of action of the myosin-ATPase and sarcoplasmic reticulum Ca²⁺-ATPase. The present study addresses the consequences of a reduction in mitochondrial ATP production capacity on sarcoplasmic Ca²⁺ handling. To this end, myotubes were cultured from patient quadriceps with a biochemically defined decrease in the maximal rate of mitochondrial ATP production and were loaded with indo 1 for imaging of sarcoplasmic Ca²⁺ changes in real time by confocal microscopy. Myotubes were field-stimulated with 10-ms pulses of 16 V to evoke transient rises in sarcoplasmic Ca²⁺ concentration ([Ca²⁺]_S). Three single pulses, two pulse trains (1 Hz), and one single pulse were applied in succession to mimic changing workloads. Control myotubes displayed [Ca²⁺]_S transients with an amplitude that was independent of the strength of the stimulus. Intriguingly, the rate of sarcoplasmic Ca²⁺ removal (CRR) was significantly upregulated during the second and subsequent transients. In myotubes with a reduced mitochondrial ATP production capacity, the amplitude of the [Ca²⁺]_S transients was markedly increased at higher stimulus intensities. Moreover, upregulation of the CRR was significantly decreased compared with control. Taken together, these results are in good agreement with a tight coupling between mitochondrial ATP production and sarcoplasmic Ca²⁺ handling. Moreover, they support the existence of a relatively long-lasting mitochondrial memory for sarcoplasmic [Ca²⁺] rises. This memory, which manifested itself as an increase in CRR upon recurrent stimulation, was impaired in patient myotubes with a reduced mitochondrial ATP production capacity. sarcoplasmic Ca²⁺ removal; video-rate imaging; indo 1; electrical stimulation; mitochondrial memory     

The effects of valinomycin on respiration and volume changes in plant mitochondria

The effects of valinomycin on the respiration and volume changeshave been studied with isolated mitochondria from bean hypocotyl(Phaseolus vulgaris L.) and cauliflower bud (Brassica oleraceaL.). In the presence of 10 mM K salts of chloride, acetate,phosphate, and sulfate respiration is stimulated by valinomycinconcomitant with osmotic swelling. When swelling declines respirationwith organic acid substrates also declines. In the presenceof the K salts of acetate and PO₄ but not Cl^– the terminationof respiration leads to contraction. The contraction in K-PO₄is inhibited by addition to the external medium of between 65to 100 mM K-PO₄. The results are interpreted to suggest thatvalinomycin in the presence of KCl facilitated the movementof K down an electrical gradient, with the Cl anion followingand osmotic swelling resulting. However, in a medium containingacetate or PO₄ the anions are actively transported against anelectrical gradient at the expense of metabolic energy. Valinomycinfacilitates the influx of K⁺ with the actively transported anionand swelling follows. When respiration terminates the activelytransported anions move passively back down their electrochemicalgradient and osmotic contraction follows. ¹ Present address: Department of Biology, Fort Lewis College,Durango, Colorado 81301, U.S.A. (Received July 21, 1972; )     

Effects of nitrogen on Pinus palustris foliar respiratory responses to elevated atmospheric CO2 concentration

Indirect effects of atmospheric CO₂ concentration [CO₂], onlongleaf pine (Pinus palustris Mill.) foliage respiration werestudied by growing trees in a factorial arrangement of low andhigh [CO₂] (369 and 729µmol CO₂ mol^–1) and low andhigh N (40 and 400 kg ha^–1 yr^–1). Direct effectsof [CO₂] on leaf respiration were tested by measuring respirationrates of foliage from all treatments at two CO₂ levels (360and 720µmol CO₂mol^–1) at the time of measurement.Elevated CO₂ did not directly or indirectly affect leaf respirationwhen expressed on a leaf area or mass basis, but a significantincrease in respiration per unit leaf N was observed in treesgrown in elevated [CO₂] (indirect response to elevated [CO₂]).The lack of a [CO₂] effect on respiration, when analysed onan area or mass basis, may have resulted from combined effectsof [CO₂] on factors that increase respiration (e.g. greateravailability of non-structural carbohydrates stimulating growthand carbon export from leaves) and on factors that decreaserespiration (e.g. lower N concentration leading to lower constructioncosts and maintenance requirements). Thus, [CO₂] affected factorsthat influence respiration, but in opposing ways. Key words: Pinus palustris, elevated CO₂, nitrogen, foliar, respiration     

Calcium-stimulated respiration and active calcium transport in the isolated chick chorioallantoic membrane

Summary Calcium markedly stimulates the respiration of the isolated chick chorioallantoic membrane. This stimulation of oxygen uptake appears to be closely associated with the membrane's active transcellular calcium transport mechanism. In the presence of 1mm Ca⁺⁺ the rate of uptake increases from 9.3±0.15 to 13.0±0.2 liters O₂/cm²/hr, an increase of about 40%. The calcium-stimulated respiration is specific for the ectodermal layer of cells, the known location of the calcium transport mechanism, and only occurs when the calcium transport mechanism is operative. Sr⁺⁺ and Mn⁺⁺ are transported by the tissue at a lower rate than Ca⁺⁺ and cause a smaller stimulation of oxygen consumption. Mg⁺⁺ and La³⁺ have no effect on tissue respiration. In the presence of Ca⁺⁺, the organic mercurialp-chloromercuribenzene sulfonate (PCMBS) inhibits calcium transport and specifically decreases the oxygen uptake of the ectoderm to a rate identical to that obtained in a calcium-free medium. Stripping the inner shell membrane away from the chorioallantoic membrane mimics these effects. The specificity and locus of action of these two inhibitors suggest that a vital component of the active transcellular calcium transport mechanism resides on or near the outer surface of the plasma membrane of the ectodermal cells and that sulfhydryl groups are important to the normal function of this component.     

Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells

Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]_mt). Stimulation of mitochondrial Ca²⁺ uptake by exposure to different cytoplasmic Ca²⁺ concentrations (1, 2, and 5 µM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]_mt was sensitive to the NOS antagonist L-N⁵-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca²⁺ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl--cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca²⁺ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis. nitric oxide; nitric oxide synthase; calcium; endothelium; mitochondria     

EGF stimulates proliferation of mouse embryonic stem cells: involvement of Ca2+ influx and p44/42 MAPKs

We examined the effect of EGF on the proliferation of mouse embryonic stem (ES) cells and their related signal pathways. EGF increased [³H]thymidine and 5-bromo-2'-deoxyuridine incorporation in a time- and dose-dependent manner. EGF stimulated the phosphorylation of EGF receptor (EGFR). Inhibition of EGFR tyrosine kinase with AG-1478 or herbimycin A, inhibition of PLC with neomycin or U-73122, inhibition of PKC with bisindolylmaleimide I or staurosporine, and inhibition of L-type Ca²⁺ channels with nifedipine or methoxyverapamil prevented EGF-induced [³H]thymidine incorporation. PKC-, -_I, -, -, and - were translocated to the membrane and intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased in response to EGF. Moreover, inhibition of EGFR tyrosine kinase, PLC, and PKC completely prevented EGF-induced increases in [Ca²⁺]_i. EGF also increased inositol phosphate levels, which were blocked by EGFR tyrosine kinase inhibitors. Furthermore, EGF rapidly increased formation of H₂O₂, and pretreatment with antioxidant (N-acetyl-L-cysteine) inhibited EGF-induced increase of [Ca²⁺]_i. In addition, we observed that p44/42 MAPK phosphorylation by EGF and inhibition of EGFR tyrosine kinase, PLC, PKC, or Ca²⁺ channels blocked EGF-induced phosphorylation of p44/42 MAPKs. Inhibition of p44/42 MAPKs with PD-98059 (MEK inhibitor) attenuated EGF-induced increase of [³H]thymidine incorporation. Finally, inhibition of EGFR tyrosine kinase, PKC, Ca²⁺ channels, or p44/42 MAPKs attenuated EGF-stimulated cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, and CDK4, respectively. In conclusion, EGF partially stimulates proliferation of mouse ES cells via PLC/PKC, Ca²⁺ influx, and p44/42 MAPK signal pathways through EGFR tyrosine kinase phosphorylation. calcium; epidermal growth factor; mitogen-activated protein kinases; protein kinase C     

Elevated resting [Ca(2+)](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca²⁺ microelectrodes in myotubes expressing wild-type RyR1 (_WTRyR1) or RyR1 with one of four common MH mutations (_MHRyR1), we measured resting intracellular Ca²⁺ concentration ([Ca²⁺]_i). Changes in resting [Ca²⁺]_i produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the _MHRyR1s had a 2.0- to 3.7-fold higher resting [Ca²⁺]_i than those expressing _WTRyR1. Exposure of myotubes expressing _MHRyR1s to ryanodine (500 µM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 µM) had no effects on their resting [Ca²⁺]_i. However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca²⁺]_i was significantly reduced (P < 0.01). Interestingly, the percent decrease in resting [Ca²⁺]_i in myotubes expressing _MHRyR1s was significantly greater than that for _WTRyR1. From these data, we propose that the high resting myoplasmic [Ca²⁺]_i in _MHRyR1 expressing myotubes is due in part to a related structural conformation of _MHRyR1s that favors "passive" calcium leak from the sarcoplasmic reticulum. ryanodine; FLA 365; bastadin 5; resting intracellular calcium concentration; sarcoplasmic reticulum