H+ Fluxes at Plasmalemma Level: In Vivo Evidence for a Significant Contribution of the Ca2+-ATPase and for the Involvement of its Activity in the Abscisic Acid-Induced Changes in Egeria Densa Leaves

Abstract: The features of Ca²⁺ fluxes, the importance of the Ca²⁺ pump‐mediated H⁺/Ca²⁺ exchanges at plasmalemma level, and the possible involvement of Ca²⁺‐ATPase activity in ABA‐induced changes of H⁺ fluxes were studied in Egeria densa leaves. The results presented show that, while in basal conditions no net Ca²⁺ flux was evident, a conspicuous Ca²⁺ influx (about 1.1 ìmol g^?1 FW h^?1) occurred. The concomitant efflux of Ca²⁺ was markedly reduced by treatment with 5 íM eosin Y (EY), a specific inhibitor of the Ca²⁺‐ATPase, that completely blocked the transport of Ca²⁺ after the first 20 ‐ 30 min. The decrease in Ca²⁺ efflux induced by EY was associated with a significant increase in net H⁺ extrusion (?ÄH⁺) and a small but significant cytoplasmic alkalinization. The shift of external [Ca²⁺] from 0.3 to 0.2 mM (reducing Ca²⁺ uptake by about 30 %) and the hindrance of Ca²⁺ influx by La³⁺ were accompanied by progressively higher ?ÄH⁺ increases, in agreement with a gradual decrease in the activity of a mechanism counteracting the Ca²⁺ influx by an nH⁺/Ca²⁺ exchange. The ABA‐induced decreases in ?ÄH⁺ and pH_cyt were accompanied by a significant increase in Ca²⁺ efflux, all these effects being almost completely suppressed by EY, in line with the view that the ABA effects on H⁺ fluxes are due to activation of the plasmalemma Ca²⁺‐ATPase. These results substantially stress the high sensitivity and efficacy of the plasmalemma Ca²⁺ pump in removing from the cytoplasm the Ca²⁺ taken up, and the importance of the contribution of Ca²⁺ pump‐mediated H⁺/Ca²⁺ fluxes in bringing about global changes of H⁺ fluxes at plasmalemma level.     

The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

Mechanisms of calcium sequestration by isolated Malpighian tubules of the house cricket Acheta domesticus

Hemolymph calcium homeostasis in insects is achieved by the Malpighian tubules, primarily by sequestering excess Ca²⁺ within internal calcium stores (Ca‐rich granules) most often located within type I (principal) tubule cells. Using both the scanning ion‐selective electrode technique and the Ramsay secretion assay this study provides the first measurements of basolateral and transepithelial Ca²⁺ fluxes across the Malpighian tubules of an Orthopteran insect, the house cricket Acheta domesticus. Ca²⁺ transport was specific to midtubule segments, where 97% of the Ca²⁺ entering the tubule is sequestered within intracellular calcium stores and the remaining 3% is secreted into the lumen. Antagonists of voltage‐gated (L‐type) calcium channels decreased Ca²⁺ influx ≥fivefold in adenosine 3′,5′‐cyclic monophosphate (cAMP)‐stimulated tubules, suggesting basolateral Ca²⁺ influx is facilitated by voltage‐gated Ca²⁺ channels. Increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca²⁺ had opposite effects on tubule Ca²⁺ transport. The adenylyl cyclase‐cAMP‐PKA pathway promotes Ca²⁺ sequestration whereas both 5‐hydroxytryptamine and thapsigargin inhibited sequestration. Our results suggest that the midtubules of Acheta domesticus are dynamic calcium stores, which maintain hemolymph calcium concentration by manipulating rates of Ca²⁺ sequestration through stimulatory (cAMP) and inhibitory (Ca²⁺) regulatory pathways.     

Cadmium impairs ion homeostasis by altering K+ and Ca2+ channel activities in rice root hair cells

Cadmium (Cd²⁺) interferes with the uptake, transport and utilization of several macro‐ and micronutrients, which accounts, at least in part, for Cd²⁺ toxicity in plants. However, the mechanisms underlying Cd²⁺ interference of ionic homeostasis is not understood. Using biophysical techniques including membrane potential measurements, scanning ion‐selective electrode technique for non‐invasive ion flux assays and patch clamp, we monitored the effect of Cd²⁺ on calcium (Ca²⁺) and potassium (K⁺) transport in root hair cells of rice. Our results showed that K⁺ and Ca²⁺ contents in both roots and shoots were significantly reduced when treated with exogenous Cd²⁺. Further studies revealed that three cellular processes may be affected by Cd²⁺, leading to changes in ionic homeostasis. First, Cd²⁺‐induced depolarization of the membrane potential was observed in root hair cells, attenuating the driving force for cation uptake. Second, the inward conductance of Ca²⁺ and K⁺ was partially blocked by Cd²⁺, decreasing uptake of K⁺ and Ca²⁺. Third, the outward K⁺ conductance was Cd²⁺‐inducible, decreasing the net content of K⁺ in roots. These results provide direct evidence that Cd²⁺ impairs uptake of Ca²⁺ and K⁺, thereby disturbing ion homeostasis in plants.     

Plasma membrane Ca²+ transporters mediate virus-induced acquired resistance to oxidative stress

This paper reports the phenomenon of acquired cross‐tolerance to oxidative stress in plants and investigates the activity of specific Ca²⁺ transport systems mediating this phenomenon. Nicotiana benthamiana plants were infected with Potato virus X (PVX) and exposed to oxidative [either ultraviolet (UV‐C) or H₂O₂] stress. Plant adaptive responses were assessed by the combined application of a range of electrophysiological (non‐invasive microelectrode ion flux measurements), biochemical (Ca²⁺‐ and H⁺‐ATPase activity), imaging (fluorescence lifetime imaging measurements of changes in intracellular Ca²⁺ concentrations), pharmacological and cytological transmission electrone microscopy techniques. Virus‐infected plants had a better ability to control UV‐induced elevations in cytosolic‐free Ca²⁺ and prevent structural and functional damage of chloroplasts. Taken together, our results suggest a high degree of crosstalk between UV and pathogen‐induced oxidative stresses, and highlight the crucial role of Ca²⁺ efflux systems in acquired resistance to oxidative stress in plants.     

Ce4+-stimulated ion fluxes are responsible for apoptosis and taxol biosynthesis in suspension cultures of Taxus cells

Ion fluxes across the plasma membrane activated by 1 mM Ce⁴⁺, cell apoptosis and taxol biosynthesis in suspension cultures ofTaxus cuspidata were studied. The extracellular pH sharply decreased upon the addition of 1 mM Ce⁴⁺, then increased gradually and exceeded the initial pH value over a time period of 12 h. The extracellular Ca²⁺ concentration decreased within the first 3 h after the addition of Ce⁴⁺, then gradually decreased to one third of initial value in control at about 72 h and remained unchanged afterwards. Experiments with an ion channel blocker and a Ca²⁺-channel blocker indicated that the dynamic changes in extracellular pH and the Ca²⁺ concentration resulted from the Ce⁴⁺-induced activation of H⁺ uptake and Ca²⁺ influx across the plasma membranevia ion channels. A pretreatment of the ion channel blocker initiated Ce⁴⁺-treated cells to undergo necrosis, and the prior addition of the Ca²⁺-channel blocker inhibited Ce⁴⁺-induced taxol biosynthesis and apoptosis. It is thus inferred that H⁺ uptake is necessary for cells to survive a Ce⁴⁺-caused acidic environment and is one of the mechanisms of Ce⁴-induced apoptosis. Furthermore, the Ca²⁺ influx across the plasma membrane mediated both the Ce⁴⁺-induced apoptosis and taxol biosynthesis.     

Calcium-dependent K current in plasma membranes of dermal cells of developing bean cotyledons

In developing seeds of bean (Phaseolus vulgaris L.), phloem‐imported assimilates (largely sucrose and potassium) are released from coats to seed apoplasm and subsequently retrieved by the dermal cell complexes of cotyledons. To investigate the mechanisms of K⁺ uptake by the cotyledons, protoplasts of dermal cell complexes were isolated and whole‐cell currents across their plasma membranes were measured with the patch‐clamp technique. A weakly rectified cation current displaying a voltage‐dependent blockade by external Ca²⁺ and acidic pH, dominated the conductance of the protoplasts. The P haseolus v ulgaris Cotyledon Dermal‐cell pH and Calcium‐dependent Cation Conductance (Pv‐CD‐pHCaCC) was highly selective for K⁺ over Ca²⁺ and Cl^–. For K⁺ current through Pv‐CD‐pHCaCC a sigmoid shaped current–voltage (I–V) curve was observed with negative conductance at voltages between ?200 and ?140 mV. This negative K⁺ conductance was Ca²⁺ dependent. With other univalent cations (Na⁺, Rb⁺, NH₄⁺) the currents were smaller and were not Ca²⁺ dependent. Reversal potentials remained constant when external K⁺ was substituted with these cations, suggesting that Pv‐CD‐pHCaCC channels were non‐selective. The Pv‐CD‐pHCaCC would provide a pathway for K⁺ and other univalent cation influx into developing cotyledons. These cation influxes could be co‐ordinated with sucrose influx via pH and Ca²⁺dependence.     

Effects of Ca2+ and Temperature on Potassium Uptake along Roots of Wheat, Rice and Cucumber

The differences in K⁺ uptake of different segments of excised roots of two thermophilic plants (rice, Oryza sativa L. cv. Dunghan Shali and cucumber, Cucumis sativus L. cv. Csemege Fürtös) and a non-thermophilic plant (wheat, Triticum aestivum L. cv. Aurora) were investigated in the presence and absence of Ca²⁺, at 0 and 25°C, using radiotracer K⁺(⁸⁶Rb⁺) technique. The K⁺ uptake exhibited different temperature- and Ca²⁺-dependent distributions along the root axis for the different species studied. In the case of rice and cucumber an extraordinarily large K⁺ uptake occurred in the apical root portion at 0°C if Ca²⁺ was omitted. The presence of Ca²⁺ diminished this anomaly. For wheat normal K⁺ uptake patterns were observed under similar conditions. At 25°C Ca²⁺-stimulated K⁺ uptake may appear in each root segment, depending upon species and composition of the uptake solution. The results indicate that there may be considerable differences in the compositions of the cell walls and membranes of root cells of thermophilic and non-thermophilic plants, and in their ion-exchange properties, especially in the apical region.     

The effect of short-term low-temperature treatments on gene expression in Arabidopsis correlates with changes in intracellular Ca2+ levels

The role of changes in intracellular calcium ion concentration ([Ca²⁺]_i) in low‐temperature signal transduction in plants has lately been supported by several studies. An analysis to determine whether the low‐temperature‐induced increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) could be correlated with a downstream response such as gene expression was carried out. The induction of the low‐temperature‐regulated gene LTI78 was used as an end point marker of the signal transduction pathway. It was found that this gene is induced by very brief low‐temperature exposures and that the induction does not depend on a continuous exposure to low temperature. By altering the cooling rate, different patterns of the Ca²⁺ response were obtained which could be correlated with different patterns of LTI78 induction. Furthermore, reducing the Ca²⁺ transients by pre‐treatment with the Ca²⁺ channel blocker La³⁺ also led to a reduced level of gene induction. The results show that brief exposures to low temperature results in the onset of a signalling pathway that leads to the induction of gene expression. This indicates the involvement of changes in [Ca²⁺]_cyt in low‐temperature signalling leading to LTI78 expression but the presence of multiple signalling pathways is suggested.     

Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles

Abstract: The inhibitory effects of Na⁺/Ca²⁺ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na⁺/Ca²⁺ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na⁺-dependent Ca²⁺ uptake in both rat brain [IC₅₀ = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC₅₀ = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ～80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na⁺-dependent Ca²⁺ release, and the inhibitory effect was enhanced by increasing the extravesicular Na⁺ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na⁺. When XIP was added at steady state (5 min after the initiation of intravesicular Na⁺-dependent Ca²⁺ uptake), it was found that the intravesicular Ca²⁺ content declined with time. Analysis of unidirectional fluxes for Ca²⁺ at steady state showed that 50 µM XIP inhibited Ca²⁺ influx and efflux ～85 and 70%, respectively. This result suggested that XIP inhibited both Na⁺/Ca²⁺ exchange and Ca²⁺/Ca²⁺ exchange but had no effect on the passive release pathway for Ca²⁺. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na⁺ or other monovalent cations, e.g., K⁺, is required for XIP to have its inhibitory effect on Ca²⁺ transport.     

A heat-activated calcium-permeable channel--Arabidopsis cyclic nucleotide-gated ion channel 6--is involved in heat shock responses

An increased concentration of cytosolic calcium ions (Ca²⁺) is an early response by plant cells to heat shock. However, the molecular mechanism underlying the heat‐induced initial Ca²⁺ response in plants is unclear. In this study, we identified and characterized a heat‐activated Ca²⁺‐permeable channel in the plasma membrane of Arabidopsis thaliana root protoplasts using reverse genetic analysis and the whole‐cell patch‐clamp technique. The results indicated that A. thaliana cyclic nucleotide‐gated ion channel 6 (CNGC6) mediates heat‐induced Ca²⁺ influx and facilitates expression of heat shock protein (HSP) genes and the acquisition of thermotolerance. GUS and GFP reporter assays showed that CNGC6 expression is ubiquitous in A. thaliana, and the protein is localized to the plasma membrane of cells. Furthermore, it was found that the level of cytosolic cAMP was increased by a mild heat shock, that CNGC6 was activated by cytosolic cAMP, and that exogenous cAMP promoted the expression of HSP genes. The results reveal the role of cAMP in transduction of heat shock signals in plants. The correlation of an increased level of cytosolic cAMP in a heat‐shocked plant with activation of the Ca²⁺ channels and downstream expression of HSP genes sheds some light on how plants transduce a heat stimulus into a signal cascade that leads to a heat shock response.     

Phaeomoniella chlamydospora‐induced Oxidative Burst in Vitis vinifera Cell Suspensions: Role of NADPH Oxidase and Ca2+

The biphasic oxidative burst induced by Phaeomoniella chlamydospora extract (Pce) in Vitis vinifera (Vv) cell suspensions was investigated. Treatment of cell suspensions with diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, prevented the Pce‐induced biphasic reactive oxygen species (ROS) accumulation, suggesting that NADPH oxidase is the primary ROS source in the oxidative burst induced by Pce elicitation of Vv cells. The role of Ca²⁺ in the oxidative burst was also investigated using a Ca²⁺ chelator and several Ca²⁺ channel blockers. The treatment of Vv cell suspensions with the Ca²⁺ chelator ethylene glycol‐bis(2‐aminoethylether)‐N, N, N’; N’‐tetraacetic acid (EGTA) completely inhibited Pce‐induced ROS accumulation, suggesting that Ca²⁺ availability is necessary for occurrence of the induced oxidative burst. However, only the Ca²⁺ channel blocker ruthenium red strongly inhibited the Pce‐induced ROS accumulation, suggesting that the specific Ca²⁺ channel types from which Ca²⁺ influx is originated also play an important role in the Pce‐induced oxidative burst. Furthermore, Ca²⁺ availability seems to be necessary for the Pce‐induced activity of NADPH oxidase.     

Modulation of dihydropyridine‐sensitive calcium channels in drosophila by a cAMP‐mediated pathway

Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca²⁺ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L‐type Ca²⁺ channels raise questions on the extent of conservation of Ca²⁺ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)‐sensitive Ca²⁺ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L‐type (DHP‐sensitive) Ca²⁺ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP‐specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8‐Br‐cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H‐89, an inhibitor of protein kinase‐A (PKA), reduced the current and inhibited the effect of 8‐Br‐cAMP. The data suggest modulation of L‐type Ca²⁺ channels of Drosophila via a cAMP‐PKA mediated pathway. While there are differences in L‐type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP‐sensitive Ca²⁺ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 491–500, 1999     

Relationship Between Ca2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla

Abstract: Carbachol or elevated K⁺ stimulated ⁴⁵Ca²⁺ uptake into chromaffin cells two- to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca²⁺ uptake and catecholamine secretion induced by the mixed nicotinic-muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca²⁺ uptake occurred within 15 s of stimulation by carbachol or elevated K⁺ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K⁺ was similar to that of Ca²⁺ uptake. There was a close correlation between Ca²⁺ uptake and catecholamine secretion at various concentrations of Ca²⁺. The concentration dependencies for inhibition of both processes by Mg²⁺ or Cd²⁺ were similar. Ca²⁺ uptake saturated with increasing Ca²⁺ concentrations, with an apparent K_m for both carbachol-induced and elevated K⁺-induced Ca²⁺ uptake of approximately 2 mM. The Ca²⁺ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca²⁺ entry and a presumed increase in cytosolic Ca²⁺ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca²⁺- dependent processes associated with catecholamine secretion. Ca²⁺ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage-sensitive Ca²⁺ channels.     

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca²⁺ and Na⁺ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac‐specific Sirt1 knockout (Sirt1^?/?). Sirt1^flox/flox mice were served as control. Sirt1^?/? mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca²⁺ transient amplitudes and sarcoplasmic reticulum (SR) Ca²⁺ stores, and increased SR Ca²⁺ leak were shown in the Sirt1^?/? mice. Electrophysiological measurements were performed using patch‐clamp method. While L‐type Ca²⁺ current (I_{Ca, L}) was smaller in Sirt1^?/? myocytes, reverse‐mode Na⁺/Ca²⁺ exchanger (NCX) current was larger compared with those in control myocytes. Late Na⁺ current (I_{Na, L}) was enhanced in the Sirt1^?/? mice, alongside with elevated cytosolic Na⁺ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1^?/? mice. Sirt1^?/? cardiomyocytes showed down‐regulation of L‐type Ca²⁺ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), but up‐regulation of Ca²⁺/calmodulin‐dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca²⁺ and Na⁺ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.     

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K+ channels in Arabidopsis guard cells

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca²⁺] ([Ca²⁺]_i), and also on mechanisms that are independent of [Ca²⁺]_i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca²⁺]_i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca²⁺]_i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca²⁺ sensitivity of anion and inward‐rectifying K⁺ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca²⁺]_i elevations and clamping [Ca²⁺]_i to resting levels. The absence of [Ca²⁺]_i elevations was confirmed by ratiometric [Ca²⁺]_i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca²⁺]_i elevations above the physiological resting [Ca²⁺]_i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca²⁺]_i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca²⁺]_i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca²⁺ to activate S‐type anion channels and down‐regulate inward‐rectifying K⁺ channels, providing strong evidence for a Ca²⁺ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca²⁺]_i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca²⁺]_i sensitivity of stomatal closure mechanisms.     

压力超负荷性心肌肥厚大鼠心肌细胞核钙转运的改变

通过腹主动脉缩窄(abdominalaorticcoarctation ,AAC)心肌肥厚大鼠模型制备、差速离心提纯心肌细胞核、酶学方法测定Ca2 +-ATPase活性、45Ca2 +同位素法测定核钙摄取和荧光分光光度计测定细胞核内自由钙浓度 ,初步揭示压力超负荷心肌肥厚大鼠心肌细胞核钙转导异常的环节。结果发现 :心肌细胞核上存在具有[Ca2 +]和ATP依赖性的高亲和力Ca2 +-ATPase ,以[Ca2 +]依赖的方式摄取45Ca2 +,并呈先升高后降低趋势。AAC术后4周大鼠心肌显著肥厚 ,伴有明显的血流动力学异常 ,与对照组比较 ,AAC大鼠心肌细胞核Ca2 +-ATPase活性减少51.93 %(p<0.001) ,但核45Ca2 +摄入量(核外[Ca2 +]浓度为800 -1600nmol/L时)和核内[Ca2 +](核外[Ca2 +]浓度为0 -1000nmol/L时)均明显增加(p<0.05) ;正常组离体心肌细胞核Ca2 +摄取受PKA刺激(p<0.05) ,而被PKC抑制剂和CaM抑制剂显著抑制(p<0.05) ,AAC大鼠心肌细胞核Ca2 +摄取仅受CaM抑制剂抑制(p<0.01) ,而PKA和PKC抑制剂对其无明显影响(p>0.05)。结论为心肌肥厚时 ,心肌细胞核Ca2 +转运系统及其磷酸化调节可能发生改变。     

Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx

Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux ‘signatures’ during these stress conditions are poorly characterized. We investigated the kinetics of K⁺, Ca²⁺ and H⁺ ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non‐invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K⁺ efflux was observed following acute ER stress with peak K⁺ efflux being ?30·6 and ?138·7 nmolm^?2 s^?1 for 10 and 50 µg ml^?1, respectively (p < 0·01). TM‐dependent Ca²⁺ uptake was more prolonged with peak values of 0·85 and 2·68 nmol m^?2 s^?1 for 10 and 50 µg ml^?1 TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K⁺ efflux and Ca²⁺ uptake. While K⁺ changes were significantly higher at each time point tested, Ca²⁺ uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K⁺ efflux and Ca²⁺ uptake. Functional assays to investigate the effect of inhibiting K⁺ efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K⁺, Ca²⁺ and H⁺ ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases. Copyright © 2012 John Wiley & Sons, Ltd.     

Long‐chain base phosphates modulate pollen tube growth via channel‐mediated influx of calcium

Long‐chain base phosphates (LCBPs) have been correlated with amounts of crucial biological processes ranging from cell proliferation to apoptosis in animals. However, their functions in plants remain largely unknown. Here, we report that LCBPs, sphingosine‐1‐phosphate (S1P) and phytosphingosine‐1‐phosphate (Phyto‐S1P), modulate pollen tube growth in a concentration‐dependent bi‐phasic manner. The pollen tube growth in the stylar transmitting tissue was promoted by SPHK1 overexpression (SPHK1‐OE) but dampened by SPHK1 knockdown (SPHK1‐KD) compared with wild‐type of Arabidopsis; however, there was no detectable effect on in vitro pollen tube growth caused by misexpression of SPHK1. Interestingly, exogenous S1P or Phyto‐S1P applications could increase the pollen tube growth rate in SPHK1‐OE, SPHK1‐KD and wild‐type of Arabidopsis. Calcium ion (Ca²⁺)‐imaging analysis showed that S1P triggered a remarkable increase in cytosolic Ca²⁺ concentration in pollen. Extracellular S1P induced hyperpolarization‐activated Ca²⁺ currents in the pollen plasma membrane, and the Ca²⁺ current activation was mediated by heterotrimeric G proteins. Moreover, the S1P‐induced increase of cytosolic free Ca²⁺ inhibited the influx of potassium ions in pollen tubes. Our findings suggest that LCBPs functions in a signaling cascade that facilitates Ca²⁺ influx and modulates pollen tube growth.