Fusicoccin Counteracts the n-Ethylmaleimide and Silver-Induced Stimulation of Oxygen Uptake in Egeria densa Leaves

It was previously shown that a number of sulfhydryl [SH] group reagents (N-ethylmaleimide [NEM], iodoacetate, Ag⁺, HgCl₂, etc.) can induce a marked, transitory stimulation of O₂ uptake (QO₂) in Egeria densa leaves, insensitive to CN⁻ and salicylhydroxamic acid and inhibited by diphenylene iodonium and quinacrine. The phytotoxin fusicoccin (FC) also induces a marked increase in O₂ consumption in E. densa leaves, apparently independent of the recognized stimulating action on the H⁺-ATPase. In this investigation we compared the FC-induced increase in O₂ consumption with those induced by NEM and Ag⁺, and we tested for a possible interaction between FC and the two SH blockers in the activation of QO₂. The results show (a) the different nature of the FC- and NEM- or Ag⁺-induced increases of QO₂; (b) that FC counteracts the NEM- (and Ag⁺)-induced respiratory burst; and (c) that FC strongly reduces the damaging effects on plasma membrane permeability observed in E. densa leaves treated with the two SH reagents. Two alternative models of interpretation of the action of FC, in activating a CN⁻-sensitive respiratory pathway and in suppressing the SH blocker-induced respiratory burst, are proposed.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells

Anoxia induces a rapid elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca²⁺ release is important for gene activation and survival in O₂-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca²⁺ to the anoxic [Ca²⁺]_cyt elevation in maize cells. Imaging of intramitochondrial Ca²⁺ levels showed that a majority of mitochondria released their Ca²⁺ in response to anoxia and took up Ca²⁺ upon reoxygenation. We also investigated whether the mitochondrial Ca²⁺ release contributed to the increase in [Ca²⁺]_cyt under anoxia. Analysis of the spatial association between anoxic [Ca²⁺]_cyt changes and the distribution of mitochondrial and other intracellular Ca²⁺ stores revealed that the largest [Ca²⁺]_cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca²⁺]_cyt under aerobic conditions but prevented a [Ca²⁺]_cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca²⁺]_cyt and participate in the signaling of O₂ deprivation.     

Inhibition of the Gravitropic Response of Snapdragon Spikes by the Calcium-Channel Blocker Lanthanum Chloride

The putative Ca²⁺-channel blocker LaCl₃ prevented the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes (S. Philosoph-Hadas, S. Meir, I. Rosenberger, A.H. Halevy [1996] Plant Physiol 110: 301–310) and inhibited stem curvature to a greater extent than vertical and horizontal stem elongation at the bending zone. This might indicate that LaCl₃, which modulates cytosolic Ca²⁺, does not influence general stem-growth processes but may specifically affect other gravity-associated processes occurring at the stem-bending zone. Two such specific gravity-dependent events were found to occur in the bending zone of snapdragon spikes: sedimentation of starch-containing chloroplasts at the bottom of stem cortex cells, as seen in cross-sections, and establishment of an ethylene gradient across the stem. Our results show that the lateral sedimentation of chloroplasts associated with gravity sensing was prevented in cross-sections taken from the bending zone of LaCl₃-treated and subsequently gravistimulated spikes and that LaCl₃ completely prevented the gravity-induced, asymmetric ethylene production established across the stem-bending zone. These data indicate that LaCl₃ inhibits stem curvature of snapdragon spikes by preventing several gravity-dependent processes. Therefore, we propose that the gravitropic response of shoots could be mediated through a Ca²⁺-dependent pathway involving modulation of cytosolic Ca²⁺ at various stages.     

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca²⁺-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca²⁺]_cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca²⁺]_cyt and inhibition of growth that was similar to the effect of the Ca²⁺ channel blocker La³⁺ and the Ca²⁺ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca²⁺ channel blockers verapamil and nifedipine did not induce a decrease in [Ca²⁺]_cyt in these cells and also failed to inhibit growth. Al and La³⁺, but not verapamil or nifedipine, reduced the rate of Mn²⁺ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca²⁺- and Mn²⁺-permeable channels. These results suggest that Al may act to block Ca²⁺ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.     

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles

The initial rate of Ca²⁺ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca²⁺-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca²⁺-selective minielectrodes to determine pCa values. The initial rate of Ca²⁺ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca²⁺ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K⁺. In addition, Ca²⁺ was shown to move across the membrane vesicles in the presence of a K⁺ diffusion potential gradient. The potential-stimulated rate of Ca²⁺ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl₃, verapamil, and nifedipine had little or no effect. These results indicate that Ca²⁺ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.     

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca²⁺]_cyt response. Cyclic-nucleotide-mediated elevations in [Ca²⁺]_cyt involved both internal and external Ca²⁺ stores. Both cAMP- and cGMP-mediated [Ca²⁺]_cyt elevations could be inhibited by the Ca²⁺-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca²⁺]_cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca²⁺]_cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca²⁺ signaling.     

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells

Inactivation of inward-rectifying K⁺ channels (I_K,in) by a rise in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca²⁺]_i action on I_K,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca²⁺]_i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording I_K,in under a voltage clamp and [Ca²⁺]_i by Fura-2 fluorescence ratiophotometry. I_K,in inactivation correlated positively with [Ca²⁺]_i and indicated a K_i of 329 ± 31 nm with cooperative binding of four Ca²⁺ ions per channel. I_K,in was promoted by the Ca²⁺ channel antagonists Gd³⁺ and calcicludine, both of which suppressed the [Ca²⁺]_i rise, but the [Ca²⁺]_i rise was unaffected by the K⁺ channel blocker Cs⁺. We also found that ryanodine, an antagonist of intracellular Ca²⁺ channels that mediate Ca²⁺-induced Ca²⁺ release, blocked the [Ca²⁺]_i rise, and Mn²⁺ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca²⁺]_i control of I_K,in and to roles for discrete Ca²⁺ flux pathways in feedback control of the K⁺ channels by membrane voltage.Ca²⁺ underlies many fundamental regulatory processes in plants, including adaptive responses to abiotic environmental stress (Knight et al., 1996; Russell et al., 1996; McAinsh et al., 1997) and programmed cell death evoked by pathogen attack (Low and Merida, 1996; Hammondkosack and Jones, 1997). Coordination of changes in [Ca²⁺]_i and its integration with downstream response elements are central in coupling stimulus input to cellular response in these processes.In stomatal guard cells, the best characterized higher-plant cell model, major downstream targets of [Ca²⁺]_i and their roles in stomatal function have been identified. Increasing [Ca²⁺]_i is known to inactivate I_K,in and to activate Cl⁻ channels, events that bias plasma membrane transport for net efflux of osmotically active solute and a loss of turgor, which drives stomatal closure (Blatt and Grabov, 1997). Furthermore, changes in [Ca²⁺]_i are associated with ABA, CO₂, and the growth hormone auxin (Blatt and Grabov, 1997; McAinsh et al., 1997). These [Ca²⁺]_i signals have been observed to oscillate (McAinsh et al., 1995; Webb et al., 1996), characteristics that may constitute “Ca²⁺ signatures” to encode specific downstream responses (Berridge, 1996). Yet, despite the evidence for [Ca²⁺]_i signaling in guard cells, surprisingly little detail is known about the link between [Ca²⁺]_i changes and ion channel activity at the plasma membrane or about the mechanisms mediating such [Ca²⁺]_i changes. To our knowledge, in no instance have the characteristics of ion channel regulation by Ca²⁺ been quantified directly in any higher-plant cell.We recently described the coupling of membrane voltage to [Ca²⁺]_i, demonstrating that hyperpolarization, whether under a voltage clamp or in the presence of low [K⁺]_o, evoked [Ca²⁺]_i increases in guard cells, and that the voltage threshold for [Ca²⁺]_i rise was profoundly altered by ABA (Grabov and Blatt, 1998). Our observations indicated a link to Ca²⁺ influx across the plasma membrane and raised questions about the efficacy of [Ca²⁺]_i in inactivating I_K,in and about the contributions of intracellular Ca²⁺ release to the [Ca²⁺]_i signal. We have used membrane voltage to experimentally manipulate [Ca²⁺]_i and report that I_K,in is strongly dependent on [Ca²⁺]_i, consistent with a cooperative binding of four Ca²⁺ ions to effect inactivation. Additional experiments indicate that voltage-evoked [Ca²⁺]_i increases depend both on Ca²⁺ influx and on release of Ca²⁺ from intracellular stores. These results underscore the role of [Ca²⁺]_i as a high-gain “switch” in the control of I_K,in, and implicate [Ca²⁺]_i in feedback control linking membrane voltage to the activity of the K⁺ channels.     

Divalent Cation Block of Inward Currents and Low-Affinity K+ Uptake in Saccharomyces cerevisiae

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Δ trk2Δ strain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K⁺ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K⁺ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K⁺ with Rb⁺, Cs⁺, or Na⁺ only slightly modulated the magnitude of the inward current, whereas replacement with Li⁺ reduced the inward current by approximately 50%, and tetraethylammonium (TEA⁺) and choline were relatively impermeant. The inward current was blocked by extracellular Ca²⁺ and Mg²⁺ with apparent K_is (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K⁺ increased the magnitude of the inward current independently of the electrochemical driving force for K⁺ influx, consistent with regulation of the inward current by cytosolic K⁺. Uptake of ⁸⁶Rb⁺ by intact trk1Δ trk2Δ cells was inhibited by extracellular Ca²⁺ with a K_i within the range observed for the inward current. Furthermore, increasing extracellular Ca²⁺ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.     

Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography

The Ca²⁺-ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl β-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca²⁺ concentrations, the Ca²⁺-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca²⁺-ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca²⁺-ATPase by ¹²⁵I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca²⁺-ATPase cross-reacted with an antiserum against a putative Ca²⁺-ATPase of the Arabidopsis thaliana chloroplast envelope.     

Ca2+ Homeostasis in the Endoplasmic Reticulum: Coexistence of High and Low [Ca2+] Subcompartments in Intact HeLa Cells

Two recombinant aequorin isoforms with different Ca²⁺ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca²⁺ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca²⁺]_er or [Sr²⁺]_er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca²⁺] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca²⁺ concentration, the [Ca²⁺]_er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca²⁺]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr²⁺ was used as a Ca²⁺ surrogate), the bulk of the organelle was shown to accumulate free [cation²⁺]_er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca²⁺], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca²⁺ or Sr²⁺, is presented. Moreover, a few other key problems concerning the ER Ca²⁺ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP₃ vary depending on their initial [Ca²⁺]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca²⁺] the cation is simultaneously accumulated; (b) stimulation of Ca²⁺ release by receptor-generated InsP₃ is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation²⁺]_er of the InsP₃ receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca²⁺], observed when the cells are incubated in Ca²⁺-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).     

Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca²⁺-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca²⁺ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca²⁺ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca²⁺ is quenched to or below resting intracellular Ca²⁺ concentration ([Ca²⁺]_e [Ca²⁺]_i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca²⁺]_e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca²⁺]_i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca²⁺ mobilization from stores (providing close to threshold Ca²⁺ levels) and Ca²⁺ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca²⁺ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.     

Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat

Enhanced Cl⁻ efflux during acidosis in plants is thought to play a role in cytosolic pH (pH_c) homeostasis by short-circuiting the current produced by the electrogenic H⁺ pump, thereby facilitating enhanced H⁺ efflux from the cytosol. Using an intracellular perfusion technique, which enables experimental control of medium composition at the cytosolic surface of the plasma membrane of charophyte algae (Chara corallina), we show that lowered pH_c activates Cl⁻ efflux via two mechanisms. The first is a direct effect of pH_c on Cl⁻ efflux; the second mechanism comprises a pH_c-induced increase in affinity for cytosolic free Ca²⁺ ([Ca²⁺]_c), which also activates Cl⁻ efflux. Cl⁻ efflux was controlled by phosphorylation/dephosphorylation events, which override the responses to both pH_c and [Ca²⁺]_c. Whereas phosphorylation (perfusion with the catalytic subunit of protein kinase A in the presence of ATP) resulted in a complete inhibition of Cl⁻ efflux, dephosphorylation (perfusion with alkaline phosphatase) arrested Cl⁻ efflux at 60% of the maximal level in a manner that was both pH_c and [Ca²⁺]_c independent. These findings imply that plasma membrane anion channels play a central role in pH_c regulation in plants, in addition to their established roles in turgor/volume regulation and signal transduction.     

Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone

Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca²⁺- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca²⁺], suggesting that it inhibited GA action downstream of the Ca²⁺ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca²⁺ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 μm), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca²⁺-dependent regulator of the GA response in these cells.     

Oxidative burst and electrolyte leakage induced by sulfhydryl blockers and by membrane permeabilizing reagents in different organs ofEgeria densa

Summary Sulfhydryl blockers, such as N-ethylmaleimide, iodoacetate and heavy metals induce a transitory stimulation of O₂ consumption and H₂O₂ production (oxidative burst) and a rapid release of electrolytes in leaves of various aquatic plants. The correlation between these two responses to N-ethylmaleimide or to Ag⁺ in separate organs and stages of leaf development was investigated inEgeria densa. Only adult leaves were able to respond to the sulfhydryl blockers with an oxidative burst, whereas this response was absent in immature growing leaves and in stem and root segments. In N-ethyl-maleimide- as well as in Ag⁺-treated adult leaves the oxidative burst was constantly associated with a relevant electrolyte leakage. These data are consistent with a model in which the SH reagent would first interact with a plasmalemma protein, leading to an increase in passive permeability to ions and to the activation of an oxidative enzyme of the type of the superoxide synthase described for granulocytes. In its turn, active-oxygen species produced by the activated oxidase might further damage the plasma membrane, increasing its passive permeability. Digitonin and nystatin, two reagents known to cause a permeabilization of lipid membranes, induced in adultE. densa leaves a transient increase in the rate of O₂ consumption and H₂O₂ production and an electrolyte leakage very similar to those induced by sulfhydryl blockers. These effects, however, were not influenced by the flavin analogues diphenylene iodonium and quinacrine, and were partially inhibited by the presence of CN^– and salicylhydroxamic acid, thus suggesting the involvement of a different oxidase in the oxidative burst elicited by these reagents.Abbreviations BTP 1,3-bis-tris(hydroxymethyl)methylaminopropane - CCCP carbonylcyanide-chlorophenylhydrazone - DCMU 3-(3,4 dichloropheny 1)-1,1-dimethylurea - DPI diphenylene iodonium - NEM N-ethylmaleimide - QO₂ O₂ uptake     

Three Mechanisms for the Calcium Alleviation of Mineral Toxicities

Ca²⁺ in rooting medium is essential for root elongation, even in the absence of added toxicants. In the presence of rhizotoxic levels of Al³⁺, H⁺, or Na⁺ (or other cationic toxicants), supplementation of the medium with higher levels of Ca²⁺ alleviates growth inhibition. Experiments to determine the mechanisms of alleviation entailed measurements of root elongation in wheat (Triticum aestivum L. cv Scout 66) seedlings in controlled medium. A Gouy-Chapman-Stern model was used to compute the electrical potentials and the activities of ions at the root-cell plasma membrane surfaces. Analysis of root elongation relative to the computed surface activities of ions revealed three separate mechanisms of Ca²⁺ alleviation. Mechanism I is the displacement of cell-surface toxicant by the Ca²⁺-induced reduction in cell-surface negativity. Mechanism II is the restoration of Ca²⁺ at the cell surface if the surface Ca²⁺ has been reduced by the toxicant to growth-limiting levels. Mechanism III is the collective ameliorative effect of Ca²⁺ beyond mechanisms I and II, and may involve Ca²⁺-toxicant interactions at the cell surface other than the displacement interactions of mechanisms I and II. Mechanism I operated in the alleviation of all of the tested toxicities; mechanism II was generally a minor component of alleviation; and mechanism III was toxicant specific and operated strongly in the alleviation of Na⁺ toxicity, moderately in the alleviation of H⁺ toxicity, and not at all in the alleviation of Al³⁺ toxicity.     

Loss of Pde6 reduces cell body Ca2+ transients within photoreceptors

Modulation of Ca²⁺ within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca²⁺ indicators to define changes in perikaryal Ca²⁺ concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca²⁺]_i levels within the cell body. Arguing against Ca²⁺ overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca²⁺ fluxes revealed that degeneration of pde6c^w59 mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca²⁺]_i. Analysis of [Ca²⁺]_i in dissociated Pde6β^rd1mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca²⁺ elevations (‘flashes'') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca²⁺ fluxes diminished. This study thus provides insight into Ca²⁺ dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca²⁺]_i that occurs in normal cones.     

Capacitative Ca2+ Entry Is Closely Linked to the Filling State of Internal Ca2+ Stores: A Study Using Simultaneous Measurements of ICRAC and Intraluminal [Ca2+]

I_CRAC (the best characterized Ca²⁺ current activated by store depletion) was monitored concurrently for the first time with [Ca²⁺] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca²⁺] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca²⁺ chelator N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca²⁺] within the ER can be lowered and restored to its original level within 10–15 s without modifications of Ca²⁺ pumps or release channels. Using these new tools, we demonstrate here that Ca²⁺ release–activated Ca²⁺ current (I_CRAC) is activated (a) solely by reduction of free [Ca²⁺] within the ER and (b) by any measurable decrease in [Ca²⁺]_ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.     

Sulfhydryl modification induces calcium entry through IP₃-sensitive store-operated pathway in activation-dependent human neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT₁MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca²⁺]_c) without external calcium, followed by a second [Ca²⁺]_c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca²⁺]_c change of Ca²⁺-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca²⁺]_c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP₃-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.     

Hydrogen-Deuterium Exchange Effects on β-Endorphin Release from AtT20 Murine Pituitary Tumor Cells

Abundant evidences demonstrate that deuterium oxide (D₂O) modulates various secretory activities, but specific mechanisms remain unclear. Using AtT20 cells, we examined effects of D₂O on physiological processes underlying β-endorphin release. Immunofluorescent confocal microscopy demonstrated that 90% D₂O buffer increased the amount of actin filament in cell somas and decreased it in cell processes, whereas β-tubulin was not affected. Ca²⁺ imaging demonstrated that high-K⁺-induced Ca²⁺ influx was not affected during D₂O treatment, but was completely inhibited upon D₂O washout. The H₂O/D₂O replacement in internal solutions of patch electrodes reduced Ca²⁺ currents evoked by depolarizing voltage steps, whereas additional extracellular H₂O/D₂O replacement recovered the currents, suggesting that D₂O gradient across plasma membrane is critical for Ca²⁺ channel kinetics. Radioimmunoassay of high-K⁺-induced β-endorphin release demonstrated an increase during D₂O treatment and a decrease upon D₂O washout. These results demonstrate that the H₂O-to-D₂O-induced increase in β-endorphin release corresponded with the redistribution of actin, and the D₂O-to-H₂O-induced decrease in β-endorphin release corresponded with the inhibition of voltage-sensitive Ca²⁺ channels. The computer modeling suggests that the differences in the zero-point vibrational energy between protonated and deuterated amino acids produce an asymmetric distribution of these amino acids upon D₂O washout and this causes the dysfunction of Ca²⁺ channels.