Activity Of Ca2 Research Articles

Erythrocyte Shrinkage and Cell Membrane Scrambling after Exposure to the Ionophore Nonactin

The ionophore antibiotic nonactin permeabilizes cell membranes to NH4+ and K(+) . Treatment of erythrocytes with nonactin is expected to trigger cellular K(+) loss with subsequent cell shrinkage, which in turn is known to trigger suicidal death of a wide variety of cells including erythrocytes. This study explored whether nonactin exposure induces eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signalling of eryptosis includes increase in cytosolic Ca(2+) activity [(Ca(2+) )i ] and stimulation of protein kinase C (PKC) as well as p38 mitogen-activated protein kinase. Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter (FSC) and (Ca(2+) )i from Fluo3-fluorescence. A 48-hr treatment of human erythrocytes with nonactin significantly decreased FSC (≥10ng/ml) and significantly increased the percentage of annexin-V-binding cells (≥10ng/ml), effects paralleled by increase in (Ca(2+) )i (≥50ng/ml) and virtually abrogated by increase in extracellular K(+) concentration to 120mM at the expense of Na(+) . The up-regulation of annexin-V-binding after nonactin treatment was significantly blunted but not abolished by the removal of extracellular Ca(2+) and by addition of either PKC inhibitor staurosporine (0.4μM) or p38 kinase inhibitor SB203580 (2μM). In conclusion, exposure of erythrocytes to the K(+) ionophore nonactin induces erythrocyte shrinkage and subsequent erythrocyte membrane scrambling, effects involving cellular K(+) loss, Ca(2+) entry and activation of staurosporine as well as SB203580-sensitive kinases.

Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases.

Caffeic acid (CA), one of the active constituents of Radix Salvia miltiorrhizae, exhibits antioxidant and anti-inflammatory activities. However, few studies have assessed the ability of CA to inhibit platelet mediated thrombus generation in vivo. In this study, we investigated the antithrombotic effect of CA in mouse cerebral arterioles and venules using intravital microscopy. The antiplatelet activity of CA in ADP stimulated mouse platelets in vitro was also examined in attempt to explore the underlying mechanism. Our results demonstrated that CA (1.25–5 mg/kg) significantly inhibited thrombus formation in vivo. In vitro, CA (25–100 μM) inhibited ADP-induced platelet aggregation, P-selectin expression, ATP release, Ca2+ mobilization, and integrin αIIbβ3 activation. Additionally, CA attenuated p38, ERK, and JNK activation, and enhanced cAMP levels. Taken together, these data provide evidence for the inhibition of CA on platelet-mediated thrombosis in vivo, which is, at least partly, mediated by interference in phosphorylation of ERK, p38, and JNK leading to elevation of cAMP and down-regulation of P-selectin expression and αIIbβ3 activation. These results suggest that CA may have potential for the treatment of aberrant platelet activation-related diseases.

Thermodynamic Properties and Phase Equilibria of the Secondary Copper Minerals Libethenite, Olivenite, Pseudomalachite, Kröhnkite, Cyanochroite, and Devilline

The thermodynamic properties of libethenite [Cu 2 (PO 4 )(OH)], olivenite [Cu 2 (AsO 4 )(OH)], pseudomalachite [Cu 5 (PO 4 ) 2 (OH) 4 ], cyanochroite [K 2 Cu(SO 4 ) 2 · 6H 2 O], krohnkite [Na 2 Cu(SO 4 ) 2 · 2H 2 O], and devilline [CaCu 4 (SO 4 ) 2 (OH) 6 · 3H 2 O] were determined by a combination of acid-solution calorimetry (enthalpy of formation) and relaxation calorimetry (heat capacity and entropy). The calculated Gibbs free energies (in kJ · mol −1 ) for these phases are −1229.3 ± 4.5, −848.7 ± 4.8, −2837.9 ± 10.8, −3441.4 ± 3.9, −2442.3 ± 3.6, −3843.2 ± 8.4, respectively. The phases studied were characterized by powder X-ray diffraction, infrared and Raman spectroscopy, and electron microprobe, as needed. We have also determined the crystal structure of devilline by single-crystal X-ray diffraction. Both of the copper phosphates investigated, libethenite and pseudomalachite, have a stability fieldinthe pH–p∈ space, but olivenite appears to be the only copper arsenate among those considered here (cornubite, clinoclase, euchroite) that has a stability field at T = 298.15 K and P = 1 bar. Chemical data from natural solid-solutions between libethenite–olivenite and pseudomalachite–cornwallite suggest that both solid solutions deviate slightly from ideality. The arsenic-rich members of these solutions tend to accept zinc in larger amounts. We have performed thermodynamic simulations showing that devilline crystallizes only when gypsum is nearby, which elevates the activity of Ca 2+ in the aqueous solutions.

GABA-ergic interneurons involved in transcallosal inhibition of the visual cortices in vivo in mice

In the current study we investigated the role of the corpus callosum, particularly the gamma-aminobutyric acid-ergic (GABAergic) projection neurons involved in interhemispheric inhibition (IHI). In order to explore IHI in primary visual cortices, we adopted a protocol whereby we performed a direct current lesion of the unilateral primary visual cortex with or without posterior callosotomy, and used two-photon Ca(2+)in vivo imaging on the opposite unaffected region to detect neural activities in mice. Following this procedure, the numbers of vesicular GABAergic transporters (VGATs) and GABAergic interneurons in the unaffected primary cortex were determined using immunofluorescence staining. Results indicated that following unilateral visual cortical lesioning without callosotomy, the neuronal Ca(2+) activities in the opposite side were significantly increased. However, the neuronal activities of the unaffected visual cortex in animals with unilateral cortical lesion with callosotomy were not significantly different. Additionally, there was no significant difference in the numbers of GABAergic interneurons in the unaffected region between each group, while the number of VGATs in the unaffected region was significantly decreased following unilateral visual cortical lesion without callosotomy, which was unchanged once with callosotomy. Finally, callosotomy alone without cortical lesioning produced no change in neuronal activities, the number of GABAergic interneurons or VGATs. Our results demonstrate that IHI between the homologous primary visual cortices occurs via the corpus callosum, and further indicate the important involvement of long-range GABAergic interneurons in transcallosal inhibition.

Triggering of Erythrocyte Death by Triparanol.

The cholesterol synthesis inhibitor Triparanol has been shown to trigger apoptosis in several malignancies. Similar to the apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress which may activate erythrocytic Ca2+ permeable unselective cation channels with subsequent Ca2+ entry and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether and how Triparanol induces eryptosis. To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and ROS formation from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. As a result, a 48 h exposure of human erythrocytes to Triparanol (20 µM) significantly increased DCFDA fluorescence and significantly increased Fluo3-fluorescence. Triparanol (15 µM) significantly increased the percentage of annexin-V-binding cells, and significantly decreased the forward scatter. The effect of Triparanol on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. In conclusion, Triparanol leads to eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane. Triparanol is at least in part effective by stimulating ROS formation and Ca2+ entry.

UCP2 modulates single-channel properties of a MCU-dependent Ca(2+) inward current in mitochondria.

The mitochondrial Ca2+ uniporter is a highly Ca2+-selective protein complex that consists of the pore-forming mitochondrial Ca2+ uniporter protein (MCU), the scaffolding essential MCU regulator (EMRE), and mitochondrial calcium uptake 1 and 2 (MICU1/2), which negatively regulate mitochondrial Ca2+ uptake. We have previously reported that uncoupling proteins 2 and 3 (UCP2/3) are also engaged in the activity of mitochondrial Ca2+ uptake under certain conditions, while the mechanism by which UCP2/3 facilitates mitochondrial Ca2+ uniport remains elusive. This work was designed to investigate the impact of UCP2 on the three distinct mitochondrial Ca2+ currents found in mitoplasts isolated from HeLa cells, the intermediate- (i-), burst- (b-) and extra-large (xl-) mitochondrial/mitoplast Ca2+ currents (MCC). Using the patch clamp technique on mitoplasts from cells with reduced MCU and EMRE unveiled a very high affinity of MCU for xl-MCC that succeeds that for i-MCC, indicating the coexistence of at least two MCU/EMRE-dependent Ca2+ currents. The manipulation of the expression level of UCP2 by either siRNA-mediated knockdown or overexpression changed exclusively the open probability (NPo) of xl-MCC by approx. 38 % decrease or nearly a 3-fold increase, respectively. These findings confirm a regulatory role of UCP2 in mitochondrial Ca2+ uptake and identify UCP2 as a selective modulator of just one distinct MCU/EMRE-dependent mitochondrial Ca2+ inward current.

Cadmium inhibits motility, activities of plasma membrane Ca(2+)-ATPase and axonemal dynein-ATPase of human spermatozoa.

Cd(2+) has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the result of inhibition exerted by Cd(2+) on the activity of the sperm ATPases associated with sperm motility. In this study, we evaluated the plasma membrane Ca(2+)-ATPase and the axonemal dynein-ATPase activities as well as sperm motility, in the presence of different free Cd(2+) concentrations in the assay media. It was found that spermatozoa incubated for 5 h in a medium containing 25 nm free Cd(2+) showed a significant inhibition of progressive motility, reaching values even lower at higher Cd(2+) concentrations. In addition, it was found that the activity of the plasma membrane Ca(2+)-ATPase reached maximal inhibition at 50 nm free Cd(2+), with a K50% inhibition of 18.3 nm free Cd(2+). The dynein-ATPase activity was maximally inhibited by 25 nm free Cd(2+) in the assay medium, with a K50% inhibition of 11.3 nm Cd(2+). Our results indicate that the decreased activity of the sperm ATPases might have a critical importance in the biochemical mechanisms underlying the decreased sperm motility of individuals exposed to Cd(2+).

Hydrothermal alteration of a chevkinite-group mineral to a bastnäsite-(Ce)-ilmenite- columbite-(Fe) assemblage: interaction with a F-, CO2-rich fluid

The results are presented of a textural and mineral chemical study of a previously undescribed type of hydrothermal alteration of chevkinite-(Ce) which occurs in a syenitic pegmatite from the Vishnevye Mountains, Urals Region, Russia. The progressive alteration of the chevkinite to a bastnäsite-(Ce)-ilmenite-columbite-(Fe) assemblage through a series of texturally complex intermediate stages is described and electron microprobe analyses are given of all the major phases. Unusual Nb ± Th-rich phases formed late in the alteration sequence provide evidence of local Nb mobility. The main compositional fluxes are traced, especially of the REE, HFSE, Th and U. It appears that almost all elements, with the exception of La, released from the chevkinite-(Ce) were reincorporated into later phases, such that they did not leave the alteration crust in significant amounts. The hydrothermal fluids are inferred to have been F- and CO2-rich, with variable levels of Ca activity, and with fO2 mainly between the nickel-nickel oxide and magnetite-hematite buffers. This occurrence represents a new paragenesis for a columbite-group mineral.

Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle: Implications for Neuronal Energy Metabolism.

The malate-aspartate NADH shuttle (MAS) operates in neurons and other cells to translocate reducing equivalents from the cytosol to the mitochondrial matrix, thus allowing a continued flux through the glycolytic pathway and metabolism of extracellular lactate. Recent discoveries have taught us that MAS is regulated by fluctuations in cytosolic Ca(2+) levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca(2+) fluctuations below the threshold of the mitochondrial calcium uniporter, there is a positive correlation between Ca(2+) and MAS activity; however, if cytosolic Ca(2+) increases above the threshold, MAS activity is thought to be reduced by an intricate mechanism. The latter forces the neurons to partly rely on anaerobic glycolysis producing lactate that may be metabolized subsequently, by neurons or other cells. In this review, we will discuss the evidence for Ca(2+)-mediated regulation of MAS that have been uncovered over the last decade or so, together with the need for further verification, and examine the metabolic ramifications for neurons.

The effect of modulators of large-conductance Ca2+-modulated K+ channels on rat AS-30D ascites hepatoma cells and isolated liver mitochondria treated with Cd2+

ATP-producing cell organelles, mitochondria, are a primary target of heavy metals, major environmental pollutants causing a variety of diseases and pathologies. The mechanism of heavy metal toxic effect was established to include changes both in the intracellular production of reactive oxygen species (ROS) and mitochondrial dysfunction due to respiratory chain disorders and activation of the Ca2+-dependent non-selective pore in the mitochondrial inner membrane. Te role of other ion channels including such selective potassium channels as large-conductance Ca2+-activated K+ channels, BK(Ca), supposed to be cytoprotective, remains poorly studied. In the present work conducted on rat AS-30D ascites hepatoma cells and liver mitochondria, we studied the effect of different BK(Ca) effectors in the presence or absence of Cd2+ ions in the incubation medium, specifically of two activators, NS1619 and NS004, and one blocker, paxilline. After 24-h incubation of AS-30D cells with 10 μM of both NS1619 and NS004, the number of apoptized cells was found to increase significantly versus control; moreover, the presence of these BK(Ca) activators in the incubation medium exerted an additive effect on Cd2+-induced apoptosis of AS-30D cells. The same concentration of NS1619 and NS004 did not affect significantly AS-30D cellular respiration after 3, 24 and 48 h of incubation, although increasing after 3 h the intracellular production of ROS. In experiments on isolated rat liver mitochondria, NS1619 and NS004 added at the same concentration to the KCl-containing medium did not affect the rates of respiratory State 3 (after Chance) and maximally uncoupled respiration (both in the presence and absence of Cd2+); concurrently, they induced a weak uncoupling effect by increasing both basal and resting-state (State 3 after Chance) respirations, and also enhanced a high-amplitude mitochondrial swelling induced by Cd2+ in this medium. Paxilline (at 1μM) was shown to reduce the mortality of AS-30D cells after 3-, 24- and 48 h-incubation in the presence of Cd2+ and to increase the intracellular ROS production in control after 3 and 24 h of exposure. Paxilline added at a concentration exerting a long-term protective effect did not affect cellular respiration of rat AS-30D cells and isolated liver mitochondria (both in the presence and absence of Cd2+) and did not reduce mitochondrial swelling observed in the presence of Cd2+ and BK(Ca) activators. Possible molecular action mechanisms of BK(Ca) modulators are discussed.

Estimation of Functional Activity of Ionized Calcium for Complement System Reactivity Assessment.

We proposed a new indicator for evaluation of functional activity of Ca(2+) in human blood serum based on lysis of sheep erythrocytes with 10% human blood serum in the presence of 0.55 mM ethylene glycol tetraacetic acid at 37°C for 10 min. After incubation, the degree of sheep erythrocytes lysis inhibition is estimated: inhibition of complement hemolytic activity <30% is considered as high functional Са(2+) activity, inhibition by 31-70% corresponds to normal activity, and >71% indicates low activity. The comparative studies of complement activating function of heterophilic antibodies, complement system reactivity in the presence of 0.29 M NaCl, and functional activity of ionized Ca(2+) make possible estimation of the individual's immune status.

Sulfonamide bearing pyrazolylpyrazolines as potent inhibitors of carbonic anhydrase isoforms I, II, IX and XII.

A series of pyrazolylpyrazolines was designed, synthesized and evaluated for carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity against cytosolic human (h) isozymes hCA I and hCA II as well as transmembrane tumor associated isozymes, hCA IX and hCA XII. All the tested compounds exhibited an excellent CA activity profile against hCA I, hCA II and hCA XII when compared to the reference drug acetazolamide (AZA). Compounds 6d, 6f and 7a-7f have exhibited better inhibition profile against hCA XII (Ki = 0.47-5.1 nM) as compared with AZA (Ki = 5.7 nM) especially, compounds 6a, 7a, 7c and 7d which were nearly 10-fold better than reference drug. Against hCA II, all of the tested compounds were better than the standard drug especially compounds 6c, 6d, 7c and 7d (Ki = 1.1-1.7 nM) were many fold better inhibitors than AZA (Ki = 12.1 nM). In addition, they acted as selective CA inhibitors of isoform hCA XII over the physiological isoform hCA I.

The Effects of Aerobic Exercise and Melatonin on Ca Activity in Rats

The Effects of Aerobic Exercise and Melatonin on Ca Activity in Rats

Earthworm assemblages in different intensity of agricultural uses and their relation to edaphic variables.

The objective of this study was to relate earthworm assemblage structure with three different soil use intensities, and to indentify the physical, chemical, and microbiological soil variables that are associated to the observed differences. Three soil uses were evaluated: 1-Fifty year old naturalized grasslands, low use intensity; 2-Recent agricultural fields, intermediate use intensity, and 3-Fifty year old intensive agricultural fields, high use intensity. Three different sites for each soil use were evaluated from winter 2008 through summer 2011. Nine earthworm species were identified across all sampling sites. The sites shared five species: the native Microscolex dubius, and the introduced Aporrectodea caliginosa, A. rosea, Octalasion cyaneum, and O. lacteum, but they differed in relative abundance by soil use. The results show that the earthworm community structure is linked to and modulated by soil properties. Both species abundance and diversity showed significant differences depending on soil use intensity. A principal component analysis showed that species composition is closely related to the environmental variability. The ratio of native to exotic species was significantly lower in the intensive agricultural system when compared to the other two, lower disturbance systems. Microscolex dubius abundance was related to naturalized grasslands along with soil Ca, pH, mechanical resistance, and microbial respiration. Aporrectodea caliginosa abundance was related to high K levels, low enzymatic activity, slightly low pH, low Ca, and appeared related to the highly disturbed environment. Eukerria stagnalis and Aporrectodea rosea, commonly found in the recent agricultural system, were related to high soil moisture condition, low pH, low Ca and low enzymatic activity. These results show that earthworm assemblages can be good indicators of soil use intensities. In particular, Microscolex dubius, Aporrectodea caliginosa, and Aporrectodea rosea, showed different temporal patterns and species associations, due to the changes in soil properties attributable to soil use intensity, defined as the amount and type of agricultural operations.

Antimicrobial 2-hydroxyisocaproic acid and chlorhexidine resist inactivation by dentine.

To compare the antibacterial activity of 2-hydroxyisocaproic acid (HICA) with currently used root canal medicaments and to examine their interactions with potential inhibitors in nutrient-deficient and nutrient-rich conditions. First, the antibacterial activity of single concentrations of HICA, calcium hydroxide solution or slurry, chlorhexidine digluconate or acetate was tested against Enterococcus faecalis with and without potential inhibitors: dentine powder (DP), hydroxyapatite or bovine serum albumin, in a low concentration of peptone water. Relative viable counts were determined by culture at 1, 24 and 48 h. In the second set of experiments, the activity of three concentrations of HICA was evaluated against two isolates of E. faecalis with and without potential inhibitors in nutrient-rich thioglycollate broth using a modification of a standard microdilution method. The minimum bactericidal concentration was determined by culture at 1, 24 and 48 h. Concentrations of ≥33 mg mL(-1) of HICA were found to be bactericidal against E. faecalis in both nutrient-deficient and nutrient-rich environments at 24- to 48-h incubation, whereas the initial activity of Ca(OH)2 slurry was lost at 48-h incubation. HICA tolerated well all tested potential inhibitors up to 19 mg mL(-1) . DP concentrations higher than this inhibited its activity in a dose-dependent manner in both environments. DP demonstrated moderate antibacterial activity, and it enhanced the otherwise limited activity of Ca(OH)2 slurry and solution. DP did not impact on the activity of chlorhexidine. These results support the long-term antibacterial activity of HICA and indicate its tolerance to clinically relevant concentrations of dentine and other inhibitors commonly present in the root canal system. Therefore, HICA may have potential as an interappointment medication in the treatment of root canal infections.

Potential protection of taurine on antioxidant system and ATPase in brain and blood of rats exposed to aluminum.

Taurine (Tau) is used in clinical treatments but its protection of brains against aluminum-induced oxidative damage has been explored for the first time. The positive effect of Tau was studied on the activities of antioxidant enzymes and ATPase. Aluminum (Al) intake caused significant increasement of malondialdehyde (MDA) but reduction of the activities of superoxide dismutase, glutathione peroxidase (GSH-Px), Na(+)K(+)-ATPase and Mg(2+)-ATPase in brain compared to control group (P < 0.05). Tau administration, however, significantly reduced the MDA content, and increased the activities of aforementioned enzymes (P < 0.05). The MDA content was higher and the activities of GSH-Px, Ca(2+)-ATPase and Mg(2+)-ATPase lower in the blood of the Al-treated group' (P < 0.05), while Tau markedly decreased MDA content and improved the activities of GSH-Px and Ca(2+)-ATPase (P < 0.05). Tau may play a crucial role in the protection of antioxidant system and ATPase against Al-induced toxicity in brain and blood of rats.

Effects of stromal interacting molecule 1 gene silencing by short hairpin RNA on the biological behavior of human gastric cancer cells.

Gastric cancer is one of the most common types of cancer worldwide. It has been reported that stromal interacting molecule 1 (STIM1) is associated with tumor progression and metastatic spread, including in cervical cancer, breast carcinoma and prostatic cancer. The present study investigated whether STIM1, an endoplasmic reticulum Ca(2+) sensor and activator of store-operated channel entry, contributed to SGC7901 cell progression. The pGPU6-shSTIM1 recombinant plasmid was constructed, and the effects of downregulation of STIM1 on the proliferation, apoptosis, migration and invasion of SGC7901 cells were examined. Western blot analysis revealed that transfection with the pGPU6-shSTIM1 plasmid successfully inhibited the expression of STIM1. STIM1 silencing in the gastric cancer cells significantly inhibited cell proliferation by arresting the cell cycle at the G0/G1 phase, and increasing the apoptotic rate following treatment of the SGC7901 cells with pGPU6-shSTIM1, indicated using an MTT cell viability assay and flow cytometery, respectively. As expected, STIM1 knock down also reduced the migration and invasion of the SGC7901 cells, demonstrated using a Transwell assay. The possible molecular mechanism involved the regulation of several signaling pathways involved in the biological behavior of cell survival, apoptosis, migration and metastasis. Together, these finding suggested that the expression of STIM1 is crucial for the proliferation and invasion of SGC7901 cells, providing a foundation for the development of novel type‑specific diagnostic strategies and treatments for gastric cancer.

Intracellular calcium dynamics in cortical microglia responding to focal laser injury in the PC::G5-tdT reporter mouse.

Microglia, the resident immune cells of the brain parenchyma, are highly responsive to tissue injury. Following cell damage, microglial processes redirect their motility from randomly scouting the extracellular space to specifically reaching toward the compromised tissue. While the cell morphology aspects of this defense mechanism have been characterized, the intracellular events underlying these responses remain largely unknown. Specifically, the role of intracellular Ca2+ dynamics has not been systematically investigated in acutely activated microglia due to technical difficulty. Here we used live two-photon imaging of the mouse cortex ubiquitously expressing the genetically encoded Ca2+ indicator GCaMP5G and fluorescent marker tdTomato in central nervous system microglia. We found that spontaneous Ca2+ transients in microglial somas and processes were generally low (only 4% of all microglia showing transients within 20 min), but baseline activity increased about 8-fold when the animals were treated with LPS 12 h before imaging. When challenged with focal laser injury, an additional surge in Ca2+ activity was observed in the somas and protruding processes. Notably, coherent and simultaneous Ca2+ rises in multiple microglial cells were occasionally detected in LPS-treated animals. We show that Ca2+ transients were pre-dominantly mediated via purinergic receptors. This work demonstrates the usefulness of genetically encoded Ca2+ indicators for investigation of microglial physiology.

Assessing Ca2+-Removal Pathways in Cardiac Myocytes

The decline of an intracellular calcium ([Ca(2+)]i) transient during a single excitation-contraction coupling (ECC) cycle reflects the combined activity of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) pump and the sarcolemmal Na(+)-Ca(2+) exchanger (NCX), along with minor contributions of the plasma membrane Ca(2+)-ATPase and mitochondrial Ca(2+) uniporter, in removing Ca(2+) from the cytosol. A traditional approach for assessing the individual components is to fit the decline of the [Ca(2+)]i transient evoked during electrical stimulation with an exponential. This reflects mostly the SERCA-dependent rate of uptake, which can be properly deduced after correcting for a component of NCX removal. As NCX function is an important determinant of the membrane potential as well as the Ca(2+) balance, we present here several detailed protocols for assessing NCX function. As the reversal potential and the amplitudes of the current are highly dependent on the prevailing concentrations of Na(+) and Ca(2+), we show how NCX function can be assessed under highly controlled conditions, with Ca(2+) and Na(+) clamped, as well as under more physiological conditions, with freely changing Ca(2+) and Na(+).

Vacuolar CBL-CIPK12 Ca2+-Sensor-Kinase Complexes Are Required for Polarized Pollen Tube Growth

Polarized tip growth is a fundamental process of specialized eukaryotic cells like neuronal axons, fungal hyphae, and plant root hairs and pollen tubes. In pollen tubes, a tip-focused oscillating Ca(2+) gradient governs ions fluxes, vesicle transport, and cytoskeleton dynamics to ensure proper polarized cell growth [1, 2]. While a crucial role of vacuolar Ca(2+) signaling is established for cellular movements like guard cell dynamics [3-5], its contribution to polarized growth remains to be defined. Here we identified the two closely related tonoplast-localized Ca(2+)-sensor proteins CBL2 and CBL3 as crucial regulators of vacuolar dynamics and polarized pollen tube growth. Overexpression of CBL2 or CBL3 in Arabidopsis and tobacco pollen tubes affected vacuolar morphology, pollen germination, and tube growth, but did not alter actin organization, PI(4,5)P2 distribution, ortip-focused Ca(2+) oscillations. Similarly, loss of function of each single Ca(2+) sensor and cbl2/cbl3 double mutants exhibited impaired pollen tube growth invitro and invivo. Both Ca(2+) sensors interacted with the kinase CIPK12, which translocated from the cytoplasm to the vacuolar membrane upon this interaction. Also, overexpression of CIPK12 induced severe vacuolar phenotypes, and loss of function of CIPK12 lead to impairment of polar growth. Remarkably, co-expression of CBL2 or CBL3 with CIPK12 resulted in a phosphorylation-dependent, massively enhanced vacuolar inflation and further disruption of polar growth. Together, these findings identify an essential role of the vacuole and vacuolar Ca(2+) signaling for polarized tip growth. We propose that a faithfully balanced activity of Ca(2+)-activated CBL2/3-CIPK12 complexes fulfills fundamental functions to enable the fast growth of pollen tubes in higher plants.