Effects of exogenous calcium ions on tip growth,intracellular Ca2+ concentration,and actin arrays in hyphae of the fungus Saprolegnia ferax

The maintenance of growth of hyphae of Saprolegnia ferax was dependent on the presence of external Ca²⁺ and the growth rate increased with increased external Ca²⁺ up to 5 × 10⁻² m Ca²⁺. When Ca²⁺ was greater than 5 × 10⁻² m, growth rates decreased. Internal membrane-associated Ca²⁺ was localized with chlortetracycline. Internal Ca²⁺ became depleted in hyphae grown in the absence of Ca²⁺ and was increased in hyphae grown in high concentrations of Ca²⁺, showing that internal Ca²⁺ can be modulated by external Ca²⁺. However, the range of the internal change was not as great as the range of external concentration used, indicating that the hyphae are capable of regulating Ca²⁺ in the presence of a large concentration gradient. In the absence of external Ca²⁺, growth can occur for a limited time through use of internal Ca²⁺. The actin cytoskeleton was altered in hyphae grown in both high and low Ca²⁺. Hyphae grown in 10⁻³ m Ca²⁺ had more actin in their apical network and peripheral plaques of actin were further from the apex than in more slowly growing hyphae in 10⁻¹ m and 0 Ca²⁺. The tips of hyphae growing in low Ca²⁺ also had a tendency to swell, giving these hyphae irregular shapes. Ca²⁺ is known to affect cell wall rigidity and the consistency of actin gels, two factors that can be expected to affect hyphal growth. External Ca²⁺ does play a role in hyphal growth possibly directly by acting on the cell wall and indirectly by altering internal Ca²⁺, thus affecting the actin cytoskeleton and possibly other growth processes.     

Ca2+‐dependent activation of guard cell anion channels,triggered by hyperpolarization,is promoted by prolonged depolarization

Rapid stomatal closure is driven by the activation of S‐type anion channels in the plasma membrane of guard cells. This response has been linked to Ca²⁺ signalling, but the impact of transient Ca²⁺ signals on S‐type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca²⁺ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S‐type anion channels were monitored using intracellular triple‐barrelled micro‐electrodes. In cells kept at a holding potential of ?100 mV, voltage steps to ?180 mV triggered elevation of the cytosolic free Ca²⁺ concentration. The increase in the cytosolic Ca²⁺ level was accompanied by activation of S‐type anion channels. Guard cell anion channels were activated by Ca²⁺ with a half maximum concentration of 515 nm (SE = 235) and a mean saturation value of ?349 pA (SE = 107) at ?100 mV. Ca²⁺ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to ?100 mV, a transient increase in the cytosolic Ca²⁺ level was observed, activating S‐type channels without measurable delay. These data show that cytosolic Ca²⁺ elevation can activate S‐type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca²⁺ transport proteins, resulting in an overshoot of the cytosolic Ca²⁺ level after returning the membrane potential to ?100 mV.     

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.).     

Cytosolic Ca2+ and H+ buffers in green algae: A comment

Summary Recently Plieth et al. [Protoplasma (1997) 198: 107–124; 199: 223] gave a quantitative picture of the Ca²⁺ and H⁺ buffers in green algae which we would like to comment. In that paper a mechanistic model was derived which describes the relationship between cytosolic Ca²⁺ and H⁺ assuming that Ca²⁺ and H⁺ interact with the same binding site of a Ca²⁺-H⁺-exchange buffer. But the increase of the cytosolic free Ca²⁺ concentration observed upon acidification can alternatively be described by a co-operative (n=2) protonation of a Ca²⁺/H⁺-binding buffer pointing to an allosteric mechanism of Ca²⁺ liberation. Furthermore we present evidences that the cytosolic buffer capacities for H⁺ (90 mM/pH) and Ca²⁺ (20 mM/pCa) given for Eremosphaera viridis were overestimated by a factor of three and three orders of magnitude, respectively.Abbreviations [Ca²⁺]_c free cytosolic - Ca²⁺ concentration     

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

cAMP activates hyperpolarization-activated Ca2+ channels in the pollen of Pyrus pyrifolia

Many signal-transduction processes in plant cells have been suggested to be triggered by signal-induced opening of calcium ion (Ca²⁺) channels in the plasma membrane. Cyclic nucleotides have been proposed to lead to an increase in cytosolic free Ca²⁺ in pollen. However, direct recordings of cyclic-nucleotide-induced Ca²⁺ currents in pollen have not yet been obtained. Here, we report that cyclic AMP (cAMP) activated a hyperpolarization-activated Ca²⁺ channel in the Pyrus pyrifolia pollen tube using the patch-clamp technique, which resulted in a significant increase in pollen tube protoplast cytosolic-Ca²⁺ concentration. Outside-out single channel configuration identified that cAMP directly increased the Ca²⁺ channel open-probability without affecting channel conductance. cAMP-induced currents were composed of both Ca²⁺ and K⁺. However, cGMP failed to mimic the cAMP effect. Higher cytosolic free-Ca²⁺ concentration significantly decreased the cAMP-induced currents. These results provide direct evidence for cAMP activation of hyperpolarization-activated Ca²⁺ channels in the plasma membrane of pollen tubes, which, in turn, modulate cellular responses in regulation of pollen tube growth.     

Intestinal secretagogues increase cytosolic free Ca2+ concentration and K+ conductance in a human intestinal epithelial cell line

Summary A human intestinal epithelial cell line (Intestine 407) is known to retain receptors for intestinal secretagogues such as acetylcholine (ACh), histamine, serotonin (5-HT) and vasoactive intestinal peptide (VIP). The cells were also found to possess separate receptors for secretin and ATP, the stimulation of which elicited transient hyperpolarizations coupled to decreased membrane resistances. These responses were reversed in polarity at the K⁺ equilibrium potential. The hyperpolarizing responses to six agonists were reversibly inhibited by quinine or quinidine. By means of Ca²⁺-selective microelectrodes, increases in the cytosolic free Ca²⁺ concentration were observed in response to individual secretagogues. The time course of Ca²⁺ responses coincided with that of hyperpolarizing responses. The responses to ACh and 5-HT were abolished by a reduction in the extracellular Ca²⁺ concentration down to pCa 7 or by application of Co²⁺. Thus, in Intestine 407 cells, not only the intestinal secretagogues, which are believed to act via increased cytosolic Ca²⁺ (ACh, 5-HT and histamine), but also those which elevate cyclic AMP (VIP, secretin and ATP) induce increases in cytosolic Ca²⁺, thereby activating the K⁺ conductance. It is likely that the origin of increased cytosolic Ca²⁺ is mainly extracellular for ACh- and 5-HT-induced responses, whereas histamine, VIP, secretin and ATP mobilize Ca²⁺ from the internal compartment.     

Na+/Ca2+ Exchange and Cellular Ca2+ Homeostasis

The Na⁺/Ca²⁺ exchange system is the primary Ca²⁺ efflux mechanism in cardiac myocytes, and plays an important role in controlling the force of cardiac contraction. The exchanger protein contains 11 transmembrane segments plus a large hydrophilic domain between the 5th and 6th transmembrane segments; the transmembrane regions are reponsible for mediating ion translocation while the hydrophilic domain is responsible for regulation of activity. Exchange activity is regulated in vitro by interconversions between an active state and either of two inactive states. High concentrations of cytosolic Na⁺ or the absence of cytosolic Ca²⁺ promote the formation of the inactive states; phosphatidylinositol-(4,5)bisphosphate (or other negatively charged phospholipids) and cytosolic Ca²⁺ counteract the inactivation process. The importance of these mechanisms in regulating exchange activity under normal physiological conditions is uncertain. Exchanger function is also dependent upon cytoskeletal interactions, and the exchanger's location with respect to intracellular Ca²⁺-sequestering organelles. An understanding of the exchanger's function in normal cell physiology will require more detailed information on the proximity of the exchanger and other Ca²⁺-transporting proteins, their interactions with the cytoskeleton, and local concentrations of anionic phospholipids and transported ions.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Cold-sensitive Ca2+ Influx in Paramecium

The concentration of intracellular calcium, [Ca²⁺] _i , in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca²⁺] _i at the anterior region of the cell. Increase in [Ca²⁺] _i was not observed at any region in Ca²⁺-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K⁺ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca²⁺. The cold-induced inward current was lost upon replacing extracellular Ca²⁺ with equimolar concentration of Co²⁺, Mg²⁺ or Mn²⁺, but it was not affected significantly by replacing with equimolar concentration of Ba²⁺ or Sr²⁺. These results indicate that Paramecium cells have Ca²⁺ channels that are permeable to Ca²⁺, Ba²⁺ and Sr²⁺ in the anterior soma membrane and the channels are opened by cooling. Received: 1 April 1996/Revised: 23 July 1996     

Human platelets use a cytosolic Ca2+ nanodomain to activate Ca2+-dependent shape change independently of platelet aggregation

Human platelets use a rise in cytosolic Ca²⁺ concentration to activate all stages of thrombus formation, however, how they are able to decode cytosolic Ca²⁺ signals to trigger each of these independently is unknown. Other cells create local Ca²⁺ signals to activate Ca²⁺-sensitive effectors specifically localised to these subcellular regions. However, no previous study has demonstrated that agonist-stimulated human platelets can generate a local cytosolic Ca²⁺ signal. Platelets possess a structure called the membrane complex (MC) where the main intracellular calcium store, the dense tubular system (DTS), is coupled tightly to an invaginated portion of the plasma membrane called the open canalicular system (OCS). Here we hypothesised that human platelets use a Ca²⁺ nanodomain created within the MC to control the earliest phases of platelet activation. Dimethyl-BAPTA-loaded human platelets were stimulated with thrombin in the absence of extracellular Ca²⁺ to isolate a cytosolic Ca²⁺ nanodomain created by Ca²⁺ release from the DTS. In the absence of any detectable rise in global cytosolic Ca²⁺ concentration, thrombin stimulation triggered Na⁺/Ca²⁺ exchanger (NCX)-dependent Ca²⁺ removal into the extracellular space, as well as Ca²⁺-dependent shape change in the absence of platelet aggregation. The NCX-mediated Ca²⁺ removal was dependent on the normal localisation of the DTS, and immunofluorescent staining of NCX3 demonstrated its localisation to the OCS, consistent with this Ca²⁺ nanodomain being formed within the MC. These results demonstrated that human platelets possess a functional Ca²⁺ nanodomain contained within the MC that can control shape change independently of platelet aggregation.     

Influx of extracellular Ca2+ involved in jasmonic-acid-induced elevation of [Ca2+]cyt and JR1 expression in Arabidopsis thaliana

The changes in cytosolic Ca²⁺ levels play important roles in the signal transduction pathways of many environmental and developmental stimuli in plants and animals. We demonstrated that the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) of Arabidopsis thaliana leaf cells was induced by exogenous application of jasmonic acid (JA). The elevation of [Ca²⁺]_cyt was detected within 1 min after JA treatment by the fluorescence intensity using laser scanning confocal microscopy, and the elevated level of fluorescence was maintained during measuring time. With pretreatment of nifedipine (Nif), a nonpermeable L-type channel blocker, the fluorescence of [Ca²⁺]_cyt induced by JA was inhibited in a dose-dependent manner. In contrast, verapamil, another L-type channel blocker, had no significant effect. Furthermore, Nif repressed JA-induced gene expression of JR1 but verapamil did not. JA-induced gene expression could be mimicked by higher concentration of extracellular Ca²⁺. W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an antagonist of calmodulin (CaM), blocked the JA induction of JR1 expression while W-5 [N-(6-aminohexyl)-1-naphthalenesulfonamide], its inactive antagonist, had no apparent effect. These data provide the evidence that the influx of extracellular Ca²⁺ through Nif sensitive plasma membrane Ca²⁺ channel may be responsible for JA-induced elevation of [Ca²⁺]_cyt and downstream gene expression, CaM may be also involved in JA signaling pathway.     

Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs

On mammalian fertilization, long-lasting Ca²⁺ oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca²⁺ increase in Ca²⁺ concentration ([Ca²⁺]) in the cytosol is due to Ca²⁺ release from the endoplasmic reticulum (ER), Ca²⁺ influx from outside is required for Ca²⁺ oscillations to persist. In this study, we investigated how Ca²⁺ influx is interrelated to the cycle of Ca²⁺ release and uptake by the intracellular Ca²⁺ stores during Ca²⁺ oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca²⁺] with fura-2, the influx rate was evaluated using Mn²⁺ quenching technique, and the change in [Ca²⁺] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca²⁺ release and then diminished gradually to the basal level, and demonstrated that the ER Ca²⁺ stores once depleted by Ca²⁺ release were gradually refilled until the next Ca²⁺ transient to be initiated. Experiments altering extracellular [Ca²⁺] in the middle of Ca²⁺ oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca²⁺ influx, indicating the crucial role of Ca²⁺ influx in determining the intervals of Ca²⁺ transients. As for the influx pathway supporting Ca²⁺ oscillations to persist, STIM1/Orai1-mediated store-operated Ca²⁺ entry (SOCE) may not significantly contribute, since neither known SOCE blockers nor the expression of protein fragments that interfere the interaction between STIM1 and Orai1 inhibited the oscillation frequency or the influx rate.     

Mitochondrial free Ca2+ concentration in living cells

Evidence has accrued during the past two decades that mitochondrial Ca²⁺ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca²⁺ transport systems might be able to relay the changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca²⁺ concentration ([Ca²⁺]_m) become essential. In this article, we review the results from recent studies attempting to measure [Ca²⁺]_m in living cells. In addition, the significance of each study is discussed.     

Participation of Intracellular Ca2+ Stores in Ca2+ Signalling in Neurons of the Thalamic Laterodorsal Nucleus of Rats

Experiments were carried out on isolated neurons of the thalamic nucleus lateralis dorsalis (LD) from 12-day-old rats. According to the morphological characteristics, LD neurons were classified as relay thalamo-cortical units and interneurons. The concentration of free Ca²⁺ ions in the cytoplasm ([Ca²⁺] _i ) was measured by a fluorescent calcium indicator, fura-2AM. Application of 30 mM caffeine caused a transient change in the [Ca²⁺] _i in 8 of 15 and in 6 of 11 of the thalamo-cortical units and interneurons under study, respectively. After stimulation of a cell with application of 50 mM KCl, a caffeine-induced increase in the [Ca²⁺] _i was observed in all tested neurons. To study the contribution of Ca²⁺-induced Ca²⁺ release (CICR) to the calcium transient evoked by depolarization of the neuronal membrane, caffeine in a subthreshold concentration was pre-applied. After 50 mM KCl had been added to the medium following pre-application of 0.5 mM caffeine, the calcium transient amplitude in thalamo-cortical neurons increased by 51 ± 7% (n = 16). In interneurons this effect was not observed (n = 11). The data obtained allow us to hypothesize that CICR contributes to the depolarization-evoked calcium transient only in the relay (thalamo-cortical) neurons. Differences in the pattern of calcium signalling, which were detected in two types of neurons of the thalamic LD, can be a factor determining distinctions in the physiological characteristics of these neurons.     

A critical appraisal of the role of intracellular Ca2+-signaling pathways in Kawasaki disease

Kawasaki disease is a multi-systemic vasculitis that generally occurs in children and that can lead to coronary artery lesions. Recent studies showed that Kawasaki disease has an important genetic component. In this review, we discuss the single-nucleotide polymorphisms in the genes encoding proteins with a role in intracellular Ca²⁺ signaling: inositol 1,4,5-trisphosphate 3-kinase C, caspase-3, the store-operated Ca²⁺-entry channel ORAI1, the type-3 inositol 1,4,5-trisphosphate receptor, the Na⁺/Ca²⁺ exchanger 1, and phospholipase Cß4 and Cß1. An increase of the free cytosolic Ca²⁺ concentration is proposed to be a major factor in susceptibility to Kawasaki disease and disease outcome, but only for polymorphisms in the genes encoding the inositol 1,4,5-trisphosphate 3-kinase C and the Na⁺/Ca²⁺ exchanger 1, the free cytosolic Ca²⁺ concentration was actually measured and shown to be increased. Excessive cytosolic Ca²⁺ signaling can result in hyperactive calcineurin in T cells with an overstimulated nuclear factor of activated T cells pathway, in hypersecretion of interleukin-1ß and tumor necrosis factor-α by monocytes/macrophages, in increased urotensin-2 signaling, and in an overactivation of vascular endothelial cells.     

Oscillations of cytoplasmic concentrations of Ca2+ and K+ in fused L cells

Summary Using Ca²⁺- and K⁺-selective microelectrodes, the cytosolic free Ca²⁺ and K⁺ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca²⁺ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca²⁺ concentration were observed in the submicromolar ranges. During the Ca²⁺ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca²⁺ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca²⁺ concentration down to 10^–7 m or by applying a Ca²⁺ channel blocker, nifedipine (50 m). In the presence of 0.5mm quinine, an inhibitor of Ca²⁺-activated K⁺ channel, sizable Ca²⁺ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K⁺ concentration between about 145 and 140mm were often associated with the potential oscillations. The minimum phase of the K⁺ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca²⁺ level, which, in turn, periodically stimulate Ca²⁺-activated K⁺ channels.     

Review article. Light-dependent signal transduction and transient changes in cytosolic Ca2+ in a unicellular green alga

The physiological function and the molecular mechanisms of Ca²⁺-mediated signal transduction processes were studied in the unicellular green alga Eremosphaera viridis by different electrophysiological and microfluorimetric techniques. A sudden blockage of photosynthetic electron transport by darkening or inhibitors causes a transient hyperpolarization of the plasma membrane. For the alga this transient hyperpolarization seems to be an important mechanism to release monovalent ions and to drive the uptake of divalent cations. The transient hyperpolarization is due to the opening of K⁺ channels and is caused by a rapid transient elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]cy spike). Different agonists like caffeine or InsP3 which are known to release Ca²⁺ from internal stores in animal cells, also cause a transient hyperpolarization and a [Ca²⁺]cy spike, similar to darkening. In Eremosphaera the transient hyperpolarization can be used as an indicator for [Ca²⁺]cy spikes. The InsP3 gated and the ryanodine/cADPR gated Ca²⁺ channels which obviously both mediate Ca²⁺ release from internal stores in Eremosphaera do not seem to be involved in the dark-induced [Ca²⁺]cy spikes. Besides single [Ca²⁺]cy spikes, the addition of Sr²⁺ (or caffeine in the absence of divalent cations) causes repetitive [Ca²⁺]cy spikes which may last hours and resemble [Ca²⁺]cy oscillations observed in excitable animal cells. These observations suggest that some principal molecular mechanisms causing single or repetitive [Ca²⁺]cy spikes are conserved from animal to plant cells.     

Intracellular Ca2+ Chelators Prevent Dna Damage And Protect Hepatoma 1Clc7 Cells From Quinone-Induced Cell Killing

Exposure of hepatoma lclc7 cells to 2,3-drniethoxy-1.4-naphthoquinone (DMNQ) resulted in a sustained elevation of cytosolic Ca²⁺. DNA single strand breaks and cell killing. DNA single strand break formation was prevented when cells were preloaded with either of the intracellular Ca²⁺ chelators. Quin 2 or BAPTA, to buffer the increase in cytosolic Ca²⁺ concentration induced by the quinone. DMNQ caused marked NAD⁺ depletion which was prevented when cells were preincubated with 3-aminobenzamide. an inhibitor of nuclear poly-(ADP-ribose)-synthetase activity. or with either of the two Ca²⁺ chelators. However. 3-aminobenzamide did not protect the hepatoma cells from loss of viability. Our results indicate that quinone-induced DNA damage. NAD⁺ depletion and cell killing are mediated by a sustained elevation of cytosolic Ca²⁺