Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase

Light-mediated activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in intact spinach chloroplasts (Spinacia oleracea L.) is enhanced in the presence of 10⁻⁵ molar external free Ca²⁺. The most pronounced effect is observed during the first minutes of illumination. Ruthenium red, an inhibitor of light-induced Ca²⁺ influx, inhibits this Ca²⁺ stimulated activation. In isolated stromal preparations, the activation of fructose-1,6-bisphosphatase is already enhanced by 2 minutes of exposure to elevated Ca²⁺ concentrations in the presence of physiological concentrations of Mg²⁺ and fructose-1,6-bisphosphate. Maximal activation of the enzyme is achieved between 0.34 and 0.51 millimolar Ca²⁺. The Ca²⁺ mediated activation decreases with increasing fructose-1,6-bisphosphate concentration and with increasing pH. The data are consistent with the proposal that the illumination of chloroplasts leads to a transient increase of free stromal Ca²⁺. In dark-kept chloroplasts the steady-state concentration of free stromal Ca²⁺ is 2.4 to 6.3 micromolar as determined by null point titration. These observations support our previous proposal that light-induced Ca²⁺ influx into chloroplasts does not only influence the cytosolic concentration of free Ca²⁺ but also regulates enzymatic processes inside the chloroplast.     

Cytosolic Phosphofructokinase from Spinach Leaves : II. Affinity for Mg and Nucleoside Phosphates

Cytosolic ATP-phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was inhibited by submillimolar concentrations of free Mg²⁺. The free Mg²⁺ concentration required for 50% inhibition of PFK activity was 0.22 millimolar. Inhibition by free Mg²⁺ was independent of the MgATP²⁻ concentration. Inorganic phosphate (Pi) reduces the inhibition of PFK activity by Mg²⁺. Free ATP (ATP⁴⁻) also inhibits PFK activity. For free ATP the inhibition of PFK activity was dependent on the MgATP²⁻ concentration. Fifty percent inhibition of PFK activity requires 1.2 and 3.7 millimolar free ATP at 0.1 and 0.5 millimolar MgATP²⁻, respectively. It was proposed that free ATP competes for the MgATP²⁻ binding site, whereas free Mg²⁺ does not. Pi diminished the inhibitory effect of free ATP on PFK activity. Free ATP and Pi had substantial effects on the MgATP²⁻ requirement of cytosolic PFK. For half-maximum saturation of PFK activity 3 and 76 micromolar MgATP²⁻ was required at 0.007 and 0.8 millimolar free ATP in the absence of Pi. At 5 and 25 millimolar Pi, half-maximum saturation was achieved at 9 and 14 micromolar MgATP²⁻. PFK activity was inhibited by Ca²⁺. The inhibition by Ca²⁺ depends upon the total Mg²⁺ concentration. Fifty percent inhibition of PFK activity required 22 and 32 micromolar Ca²⁺ at 0.1 and 0.2 millimolar Mg²⁺, respectively. At physiological concentrations of about 0.5 millimolar free Mg²⁺, Ca²⁺ would have little effect on cytosolic PFK activity from spinach leaves. PFK is not absolutely specific for the nucleoside 5′-triphosphate substrate. Besides MgATP²⁻, MgUTP²⁻, MgCTP²⁻, and MgGTP²⁻ could be used as a substrate. All four free nucleotides inhibit PFK activity. The physiological consequences of the regulatory properties of cytosolic PFK from spinach leaves will be discussed. A model will be introduced, in an attempt to describe the complex interaction of PFK with substrates and the effectors Mg²⁺ and Pi.     

Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca²⁺ concentration. To better understand this process, we measured the cytosolic Ca²⁺ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca²⁺ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca²⁺-sensitive dyes that possess different affinities for Ca²⁺ allowed the quantitative determination of the cytosolic Ca²⁺ concentration in the steady state. Determining the cytosolic Ca²⁺ concentration as a function of the adapting light intensity shows that the Ca²⁺ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca²⁺ concentration reaches stable values higher than 10 μM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca²⁺ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca²⁺ concentration, we conclude that the Ca²⁺-buffering capacity is limited. The percentage of the Ca²⁺ influx that is buffered gradually decreases with increasing Ca²⁺ concentrations; at cytosolic Ca²⁺ concentration levels above 10 μM, buffering becomes minimal.     

High rates of calcium-free diffusion in the cytosol of living cells

Calcium (Ca²⁺) is a universal second messenger that participates in the regulation of innumerous physiological processes. The way in which local elevations of the cytosolic Ca²⁺ concentration spread in space and time is key for the versatility of the signals. Ca²⁺ diffusion in the cytosol is hindered by its interaction with proteins that act as buffers. Depending on the concentrations and the kinetics of the interactions, there is a large range of values at which Ca²⁺ diffusion can proceed. Having reliable estimates of this range, particularly of its highest end, which corresponds to the ions free diffusion, is key to understand how the signals propagate. In this work, we present the first experimental results with which the Ca²⁺-free diffusion coefficient is directly quantified in the cytosol of living cells. By means of fluorescence correlation spectroscopy experiments performed in Xenopus laevis oocytes and in cells of Saccharomyces cerevisiae, we show that the ions can freely diffuse in the cytosol at a higher rate than previously thought.     

Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores

Abstract: Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca²⁺ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca²⁺ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca²⁺ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca²⁺ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca²⁺ channel blocker Ni²⁺. Blocking the influx of extracellular Ca²⁺ had no effect on carbachol-mediated increases in cytosolic CaM (168 ± 18% of control values for carbachol treatment alone vs. 163 ± 28% for Ni²⁺ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca²⁺ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 ± 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca²⁺ by pretreatment with the cell-permeant Ca²⁺ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 ± 37% of control without BAPTA/AM vs. 136 ± 13% with BAPTA/AM). The effect of direct entry of extracellular Ca²⁺ into the cell by K⁺ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K⁺ elicited an immediate and persistent increase in intracellular free Ca²⁺ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca²⁺ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca²⁺, which releases Ca²⁺ from intracellular stores, induced an increase in cytosolic CaM (203 ± 30% of control). The mechanism for the CaM release may involve activation of the α isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K⁺ depolarization. These data demonstrate that release of Ca²⁺ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.     

Biophysical effects of the natural product euplotin C on the <Emphasis Type="Italic">Paramecium</Emphasis> membrane

The effect of euplotin C—a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus—on the voltage-dependent Ca²⁺ channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca²⁺ concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca²⁺ influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca²⁺ channel blockers, we demonstrate that euplotin C modulates Ca²⁺ channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca²⁺ channel functions through the reduction of cell membrane fluidity.     

Fluorescence anisotropy of labelled F-actin: Influence of Ca2+ on the flexibility of F-actin

We measured the fluorescence static anisotropy and the time-resolved fluorescence anisotropy decay of F-actin labelled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine at 20°C in solutions containing 100 mM KCl and free Ca²⁺ at various concentrations. The average fluorescence anisotropy and the fluorescence rotational correlation time of actin decreased in the presence of micromolar concentrations of free Ca²⁺. The change of the rotational correlation time of labelled actin could not be explained by a variation of the actin critical concentration. We concluded therefore that F-actin undergoes a conformational change induced by Ca²⁺ binding. The binding constant was 6 × 10⁶ M^?1.     

Plasma Membrane Ca-ATPase of Radish Seedlings : II. Regulation by Calmodulin

The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish (Raphanus sativus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca²⁺ concentration. Stimulation was marked at low, nonsaturating Ca²⁺ concentrations and decreased increasing Ca²⁺, so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca²⁺. The pattern of calmodulin stimulation of the plasma membrane Ca-ATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca²⁺, stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca-ATPase activity to comparable extents.     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

The determinants of contractility in the heart

The contraction-relaxation cycle of the heart represents the combined action of a variety of different components of the myocytes. For many years an ‘index’ of contractility has been sought as a means of describing and integrating the large amount of information available from the studies of heart muscle contraction. This review will undertake to show that dF/dt, recorded from the whole heart, and dT/dt, recorded in isometric studies of isolated heart muscle preparations, should not be considered as the ‘index’ of contractility. Examples will be presented in which an increasing dT/dt is paradoxically accompanied by a lower tension, while a decreasing dT/dt can occur concomitantly with an increased contractile tension. Arguments are further presented in support of the concept that Ca²⁺, in conjunction with troponin C, is the main determinant of cardiac contractility and that dT/dt reflects a dynamic equilibrium between free and troponin-bound Ca²⁺. Peak tension is thus the net result of overlapping events competing for Ca²⁺ during the latter part of contraction, that is, during Phase II of contraction as defined below. These suggestions are based upon the following considerations: (a) The Ca²⁺ pumps are active even during rest and serve to maintain low cytosolic Ca²⁺ levels, (b) As cytosolic Ca²⁺ concentration increases, Ca²⁺ pump activity also increases, (c) In addition, the Na⁺Ca²⁺ exchange is activated by elevated Ca²⁺ concentrations and serves to decrease cytosolic Ca²⁺ levels, (d) The net result is a decline in free Ca²⁺ concentration during Phase II and a reduction in the rate of cross-bridge formation until peak tension is reached. Thus, the Ca²⁺ handling elements of the myocyte serve as a finely tuned feedback device, regulating troponin C-Ca²⁺ interactions controlling the Ca²⁺ concentration of the cytosol and as a result, the actin and myosin interaction. Factors which influence the function of these elements will change the contractility of the heart.     

The role of the plasma-membrane Ca2+-ATPase in Ca2+ homeostasis in Sinapis alba root hairs

The regulation of cytosolic Ca²⁺ has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca²⁺-ATPase. For this purpose, erythrosin B was used to inhibit the Ca²⁺-ATPase, and the Ca²⁺ ionophore A₂₃₁₈₇ was applied to manipulate cytosolic free [Ca²⁺] which was then measured with Ca²⁺-selective microelectrodes. (i) At 0.01 M, A₂₃₁₈₇ had no effect on the membrane potential but enhanced the Ca²⁺ permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A₂₃₁₈₇ first caused a decrease in cytosolic Ca²⁺ by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca²⁺ spontaneously reversed and became an increase, a process which strongly depended on the external Ca²⁺ concentration, (iv) Upon removal of A₂₃₁₈₇, the cytosolic free [Ca²⁺] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca²⁺ is an activation of Ca²⁺ transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca²⁺ from the cytosol. The two observations that after the addition of A₂₃₁₈₇, (i) Ca²⁺ gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca²⁺-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH⁺/Ca²⁺-ATPase. Based on the finding that the Ca²⁺-ATPase inhibitor erythrosin B had no effect on cytosolic Ca²⁺, but caused a strong Ca²⁺ increase after the addion of A₂₃₁₈₇ we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca²⁺-pump. We further conclude that this ATPase is a major Ca²⁺ regulator in stress situations where the cytosolic Ca²⁺ has been shifted from its steady-state level, as may be the case during processes of signal transduction.Abbreviations and Symbols EB erythrosin B - E_m membrane potential - pCa negative logarithm of the Ca²⁺ concentration This work was supported by the Deutche Forschungsgemeinschaft (H.F.) and the Alexander-von-Humboldt-Foundation (A.T.).     

Regulation of rat brainstem tryptophan 5-monooxygenase. Calcium-dependent reversible activation by ATP and magnesium.

Tryptophan 5-monooxygenase in rat brainstem cytosol was activated about twofold by incubation with 0.5 mm ATP and 5 mm MgCl₂. The activation required micromolar concentrations of Ca²⁺ but was not dependent on either cyclic AMP or cyclic GMP. Rat brain cytosol was shown to possess an endogenous protein kinase which was markedly stimulated by the addition of Ca²⁺ using endogenous protein substrates. Following activation by ATP and Mg²⁺ in the presence of Ca²⁺, tryptophan 5-monooxygenase was reversibly deactivated to the original level by incubation at 30 °C after removal of Ca²⁺ by adding ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid and was then reactivated by incubation at 30 °C after subsequent addition of Ca²⁺ and ATP. The deactivation was markedly inhibited by the omission of Mg²⁺ or by the addition of NaF.     

Calcium-activated proteolytic activity in rat liver mitochondria

Soluble extracts from sonicated rat liver mitochondria and rat liver cytosol were each chromatographed on DEAE-cellulose columns, and the fractions assayed for Ca²⁺-activated proteolytic activity using ¹⁴C-casein as a substrate. The mitochondrial preparations were shown to be free of cytosolic and microsomal contamination by the lack of alcohol dehydrogenase activity, a cytosolic marker enzyme, and by a lack of cytochrome P-450 activity, a microsomal marker enzyme. Two peaks of Ca²⁺-activated neutral endoprotease activity were resolved from the mitochondrial fractions. One protease was half-maximally activated with 25 μM Ca²⁺, and the other by 750 μM Ca²⁺. Rat liver cytosol contained only a high Ca²⁺-requiring protease peak. This is the first demonstration of Ca²⁺-activated proteases in mitochondria.     

Stimulation of hepatic microsomal β-glucuronidase by calcium

Hydrolysis of 3-methylumbelliferyl glucuronide by liver microsomal β-glucuronidase is enhanced about 2-fold by micromolar concentrations of Ca²⁺; half-maximal stimulation occurs with 0.35 μM Ca²⁺. Dissociation of the enzyme from microsomal membranes by various treatments increases basal β-glucuronidase activity and markedly decreases the sensitivity of the enzyme to Ca²⁺. Under similar conditions, the soluble lysosomal form of the enzyme is insensitive to Ca²⁺. Ca²⁺ stimulation was unaltered by addition of calmodulin inhibitors or exogenous calmodulin. Thus, interaction of cytosolic Ca²⁺ with membrane bound β-glucuronidase may modulate glucuronidation in intact hepatocytes via a novel, calmodulin-independent mechanism.     

Formyl-Met-Leu-Phe induces calcium-dependent tyrosine phosphorylation of Rel-1 in neutrophils

Chemoattractant priming and activation of PMNs results in changes in cytosolic Ca²⁺ concentration, tyrosine kinase activity, and gene expression. We hypothesize that the initial signaling for the activation of a 105 kDa protein (Rel-1) requires Ca²⁺-dependent tyrosine phosphorylation. A rapid and time-dependent tyrosine phosphorylation of Rel-1 occurred following formyl-Met-Leu-Phe (fMLP) stimulation of human PMNs at concentrations that primed or activated the NADPH oxidase (10⁻⁹ to 10⁻⁶ M), becoming maximal after 30 s. Pretreatment with pertussis toxin (Ptx) or tyrosine kinase inhibitors abrogated this phosphorylation and inhibited fMLP activation of the oxidase. The fMLP concentrations employed also caused a rapid increase in cytosolic Ca²⁺ but chelation negated the effects, including the cytosolic Ca²⁺ flux, oxidase activation, and the tyrosine phosphorylation of Rel-1. Conversely, chelation of extracellular Ca²⁺ decreased the fMLP-mediated Ca²⁺ flux, had no affect on the oxidase, and augmented tyrosine phosphorylation of Rel-1. Phosphorylation of Rel-1 was inhibited when PMNs were preincubated with a p38 MAP kinase (MAPK) inhibitor (SB203580). In addition, fMLP elicited rapid activation of p38 MAPK which was abrogated by chelation of cytosolic Ca²⁺. Thus, fMLP concentrations that prime or activate the oxidase cause a rapid Ca²⁺-dependent tyrosine phosphorylation of Rel-1 involving p38 MAPK activation.     

Mechanisms for L-channel-mediated increase in [Ca]i and its reduction by anti-bipolar drugs in cultured astrocytes combined with its mRNA expression in freshly isolated cells support the importance of astrocytic L-channels

The importance of Ca²⁺ signaling in astrocytes is undisputed but a potential role of Ca²⁺ influx via L-channels in the brain in vivo is disputed, although expression of these channels in cultured astrocytes is recognized. This study shows that an increase in free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in astrocytes in primary cultures in response to an increased extracellular K⁺ concentration (45 mM) is inhibited not only by nifedipine (confirming previous observations) but also to a very large extent by ryanodine, inhibiting ryanodine receptor-mediated release of Ca²⁺, known to occur in response to an elevation in [Ca²⁺]_i. This means that the actual influx of Ca²⁺ is modest, which may contribute to the difficulty in demonstrating L-channel-mediated Ca²⁺ currents in astrocytes in intact brain tissue. Chronic treatment with any of the 3 conventional anti-bipolar drugs lithium, carbamazepine or valproic acid similarly causes a pronounced inhibition of K⁺-mediated increase in [Ca²⁺]_i. This is shown to be due to an inhibition of capacitative Ca²⁺ influx, reflected by decreased mRNA and protein expression of the ‘transient receptor potential channel’ (TRPC1), a constituent of store-operated channels (SOCEs). Literature data are cited (i) showing that depolarization-mediated Ca²⁺ influx in response to an elevated extracellular K⁺ concentration is important for generation of Ca²⁺ oscillations and for the stimulatory effect of elevated K⁺ concentrations in intact, non-cultured brain tissue, and (ii) that Ca²⁺ channel activity is dependent upon availability of metabolic substrates, including glycogen. Finally, expression of mRNA for Ca_v1.3 is demonstrated in freshly separated astrocytes from normal brain.     

Modeling of the Modulation by Buffers of Ca Release through Clusters of IP3 Receptors

Intracellular Ca²⁺ release is a versatile second messenger system. It is modeled here by reaction-diffusion equations for the free Ca²⁺ and Ca²⁺ buffers, with spatially discrete clusters of stochastic IP₃ receptor channels (IP₃Rs) controlling the release of Ca²⁺ from the endoplasmic reticulum. IP₃Rs are activated by a small rise of the cytosolic Ca²⁺ concentration and inhibited by large concentrations. Buffering of cytosolic Ca²⁺ shapes global Ca²⁺ transients. Here we use a model to investigate the effect of buffers with slow and fast reaction rates on single release spikes. We find that, depending on their diffusion coefficient, fast buffers can either decouple clusters or delay inhibition. Slow buffers have little effect on Ca²⁺ release, but affect the time course of the signals from the fluorescent Ca²⁺ indicator mainly by competing for Ca²⁺. At low [IP₃], fast buffers suppress fluorescence signals, slow buffers increase the contrast between bulk signals and signals at open clusters, and large concentrations of buffers, either fast or slow, decouple clusters.     

Polyamine uptake in carrot cell cultures

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca²⁺. The effects of Ca²⁺ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca²⁺ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca²⁺ concentration range between 10 micromolar and 1 millimolar. La³⁺ nullified the stimulatory effect of 10 micromolar Ca²⁺ on putrescine uptake and that of 1 millimolar Ca²⁺ on spermidine uptake. La³⁺ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule.     

Regulation by ca of a cytosolic fructose-1,6-bisphosphatase from spinach leaves

Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6-bisphosphate in the presence of either Mg²⁺ or Mn²⁺, and was free of interfering enzyme activities. Ca²⁺ was an effector of fructose-1,6-bisphosphatase activity, and showed different kinetics, depending on whether Mg²⁺ or Mn²⁺ was used as cofactor. In the presence of 5 millimolar Mg²⁺, Ca²⁺ appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6-bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca²⁺. However, with Mn²⁺ (60 micromolar) as cofactor, Ca²⁺ exhibited the Michaelis-Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca²⁺ behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg²⁺ or Mn²⁺ as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg²⁺ as cofactor, but this effect was low with Mn²⁺. From these results we suggest that Ca²⁺ plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase.     

Calcium signaling mediates the response to cadmium toxicity in Saccharomyces cerevisiae cells

The involvement of Ca²⁺ in the response to high Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Hg²⁺ was investigated in Saccharomyces cerevisiae. The yeast cells responded through a sharp increase in cytosolic Ca²⁺ when exposed to Cd²⁺, and to a lesser extent to Cu²⁺, but not to Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, or Hg²⁺. The response to high Cd²⁺ depended mainly on external Ca²⁺ (transported through the Cch1p/Mid1p channel) but also on vacuolar Ca²⁺ (released into the cytosol through the Yvc1p channel). The adaptation to high Cd²⁺ was influenced by perturbations in Ca²⁺ homeostasis. Thus, the tolerance to Cd²⁺ often correlated with sharp Cd²⁺-induced cytosolic Ca²⁺ pulses, while the Cd²⁺ sensitivity was accompanied by the incapacity to rapidly restore the low cytosolic Ca²⁺.