Evidence for Ca-dependent protein phosphorylation in vitro in guard cells from Vicia faba L.

Protein phosphorylation in vitro was investigated in guard cells from Vicia faba. A number of proteins with apparent molecular masses of 72, 67, 57, 52, 49, 44, 37, and 26 kDa were phosphorylated when guard-cell extract was incubated with [γ-³²P]ATP under Ca²⁺-free conditions. In the presence of Ca²⁺ at 1 μM, several proteins with apparent molecular masses of 125, 83, 41, 31, and 25 kDa were newly phosphorylated. These Ca²⁺-dependent protein phosphorylations were suppressed by (8R^*,9S^*,11S^*)-(−)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a- triazadibenzo[a,g]cycloocta[cde]trinden-1-one (K-252a), a wide-range inhibitor of protein kinases, suggesting that the protein phosphorylations were mediated by protein kinases. Several proteins were phosphorylated in vitro in mesophyll extract from Vicia. In contrast to guard cells, there was no detectable Ca²⁺-dependent protein phosphorylation in mesophyll cells. 1-(5-Indonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), an inhibitor of myosin light chain kinase (MLCK), and an antagonist of calmodulin (CaM), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited Ca²⁺-dependent phosphorylation of 41- and 25-kDa proteins in guard cells. Fractionation experiments revealed that the Ca²⁺-dependent phosphorylated proteins with molecular masses of 41 and 25 kDa were present in the mitochondria, and the 125- and 31-kDa proteins in the cytosol. These results suggest that Ca²⁺-dependent protein phosphorylation occurs markedly in guard cells, and that Ca²⁺-dependent phosphorylation of 41- and 25-kDa proteins may be catalyzed by MLCK or MLCK-like protein kinase in guard cells.     

Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Mechanical stimulation induces Ca2+i transients and membrane depolarization in cultured endothelial cells. Effects on Ca2+i in co-perfused smooth muscle cells

Cytosolic Ca²⁺ concentration and membrane potential were monitored in individual cultured enothelial cells mechanically stimulated with a micropipette attached to the stage of a microscope. Both dimpling and poking of endothelial cells resulted in Ca²⁺_i transients (from 63 ± 12 to 397 ± 52 nM, characterized by a refractory period of approx. 2 min) and cell depolarization. Ca²⁺_i transients of the reduced amplitude (201 ± 41 nM) were evoked by mechanical stimulation of endothelial cells incubated in a Ca²⁺-free medium. Dimpling-induced Ca²⁺_i transients were refractory to the pretreatments with pertussis toxin, colchicine, or cytochalasin B, and were not mimicked by an increase in the hydrodynamic pressure. In a co-perfusion system (endothelium: smooth muscle), both the KCl-induced depolarization and ionomycin-induced increase in Ca²⁺_i in the endothelial cells resulted in the reduction of Ca²⁺_i in the smooth muscle cells. The data reported are consistent with the phenomenon of vascular relaxation in response to the increased blood flow. We hypothesize that the mechanical interaction of the formed elements with the microvascular endothelium can serve as a pacemaker for the sustained relaxation of vascular smooth muscle.     

Mechanisms of ATP-induced Ca signaling in osteoclasts

We invetigate the mechanisms underlying the intracellular calcium pulse that occurs in response to extracellular adenosine triphosphate (ATP) in osteoclasts. We find that pre-loading of GDP-β-S abolishes the response in Ca²⁺-free medium, demonstrating an internal release of Ca²⁺ via a pathway that involves a G protein. GDP-β-S does not block in normal Ca²⁺-containing medium, suggesting that ATP also induces a Ca²⁺ influx across the cell membrane. We confirmed this using the Mn²⁺ quenching technique, which shows significant opening of Ca²⁺ channels. We find a smaller response to adenosine diphosphate (ADP) and 2-methylthio-ATP (2-MeSATP), but no response to β, γ-methylene-ATP (AMP-PCP), adenosine monophosphate (AMP) or uridine triphosphate (UTP). Prior application of AMP and UTP, but not AMP-PCP, blocks the response to ATP. Our resutls indicate that the receptor is a P₂ subtype that is not characteristic of any previously reported P₂ receptor or combination of P₂ receptors.     

Low frequency and low intensity pulsed electromagnetic field exerts its antiinflammatory effect through restoration of plasma membrane calcium ATPase activity

Rheumatoid arthritis (RA) is a chronic inflammatory disorder affecting 1% of the population worldwide. Pulsed electromagnetic field (PEMF) has a number of well-documented physiological effects on cells and tissues including antiinflammatory effect. This study aims to explore the antiinflammatory effect of PEMF and its possible mechanism of action in amelioration of adjuvant induced arthritis (AIA). Arthritis was induced by a single intradermal injection of heat killed Mycobacterium tuberculosis at a concentration of 500 μg in 0.1 ml of paraffin oil into the right hind paw of rats. The arthritic animals showed a biphasic response regarding changes in the paw edema volume. During the chronic phase of the disease, arthritic animals showed an elevated level of lipid peroxides and depletion of antioxidant enzymes with significant radiological and histological changes. Besides, plasma membrane Ca²⁺ ATPase (PMCA) activity was inhibited while intracellular Ca²⁺ level as well as prostaglandin E₂ levels was noticed to be elevated in blood lymphocytes of arthritic rats. Exposure of arthritic rats to PEMF at 5 Hz × 4 μT × 90 min, produced significant antiexudative effect resulting in the restoration of the altered parameters. The antiinflammatory effect could be partially mediated through the stabilizing action of PEMF on membranes as reflected by the restoration of PMCA and intracellular Ca²⁺ levels in blood lymphocytes subsequently inhibiting PGE₂ biosynthesis. The results of this study indicated that PEMF could be developed as a potential therapy for RA in human beings.     

Ca entry through the store-mediated pathway directly activates only the K current but the subsequent Ca release from the store activates both K and Cl currents in submandibular gland acinar cells of the rat

The store-mediated Ca²⁺ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca²⁺ activated ionic membrane currents. In the cells where intracellular Ca²⁺ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca²⁺-free extracellular condition, an employment of external Ca²⁺ in the absence of ACh caused a sustained increase of the K⁺ current without affecting the Cl⁻ current. A renewed ACh challenge without external Ca²⁺ caused repetitive spikes of both K⁺ and Cl⁻ currents due to the Ca²⁺ release. SK & F 96365 inhibited the generation of the sustained K⁺ current and refilling of the Ca²⁺ store following the Ca²⁺ readmission. It is suggested that the Ca²⁺ enters the cell through the store-mediated pathway near the K⁺ channels and is taken up by the store. Thus, only Ca²⁺ released from the store can activate both the K⁺ and Cl⁻ currents.     

Effects of calcium on the thermal stability, stability in organic solvents and resistance to hydrogen peroxide of artichoke (Cynara scolymus L.) peroxidase: A potential method of enzyme control

The effects of calcium ions (Ca²⁺) on the stability of artichoke (Cynara scolymus L.) peroxidase (AKPC) have been studied. The thermal stability of AKPC was improved by the addition of Ca²⁺; the melting temperature increased by 20 °C and the deactivation energy by 26 kJ mol⁻¹. AKPC was stable in a selection of organic solvents but was less active with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) than under aqueous conditions. Ca²⁺-free AKPC retained more activity in the presence of organic solvents due to its better maintenance of the rate of compound I formation with hydrogen peroxide (H₂O₂) compared to AKPC-Ca²⁺. AKPC retained at least 75% activity over 24 h in the pH range 3.0–10.5 and about 50% over 1 month at pH 7.0 or 5.5, irrespective of the Ca²⁺ content. AKPC-Ca²⁺ was considerably more resistant to inactivation by H₂O₂ than Ca²⁺-free AKPC suggesting that the presence of Ca²⁺ boosts turnover under oxidizing conditions. AKPC has been applied as an alternative to horseradish peroxidase (HRP) in glucose concentration assays; the presence of Ca²⁺ or of the Ca²⁺ chelating agent ethylenediaminetetraacetic acid made no difference to the final result. The possibility is discussed that addition and removal of a labile Ca²⁺ from AKPC could be used to control enzyme activity both in vivo and in vitro.     

Interaction of ovokinin(2-7) with vascular bradykinin 2 receptors

Intravenous administration of ovokinin(2–7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1–5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2–7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B₂ receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B₁ receptor antagonist [des-Arg¹⁰]-HOE140 (26.3±15.5%). Intracellular Ca²⁺ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2–7). The increases were abolished by HOE140 and unaffected by [des-Arg¹⁰]-HOE140. The elevation of intracellular Ca²⁺ by ovokinin(2–7) was dependent on Ca²⁺ entry from extracellular space as it was reduced in a Ca²⁺-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca²⁺ increase by the peptide. PA phosphohydrolase and phospholipase A₂ inhibitors significantly reduced the responses as well. Our results show that ovokinin(2–7) modulates cardiovascular activity by interacting with B₂ bradykinin receptors.     

Intracellular Calcium Response of ACL and MCL Ligament Fibroblasts to Fluid-Induced Shear Stress

This study examines the real-time intracellular calcium concentration, [Ca²⁺]_i, response of canine medial collateral ligament (MCL) and anterior cruciate ligament (ACL) fibroblasts subjected to a fluid-induced shear stress of 25 dynes/cm². In experiments using a modified Hanks' Balanced Salt Solution (HBSS) perfusate, both cell types demonstrated a significant increase in peak [Ca²⁺]_i compared to respective no-flow controls, the response of MCL fibroblasts being nearly 2-fold greater than that of ACL fibroblasts. In studies where the cells were bathed in a medium of HBSS supplemented with 2% newborn bovine serum (NBS) and then introduced to flow with the same medium, ACL fibroblasts responded nearly 3-fold greater than MCL fibroblasts. Neomycin (10 mM), thapsigarigin (1 μM) and Ca²⁺-free media supplemented with EGTA (1 mM) were able to inhibit significantly the [Ca²⁺]_i response to flow with HBSS in both fibroblasts. Thapsigargin also blocked the NBS flow response in both cell types, while neomycin and Ca²⁺-free media significantly inhibited the ACL response. Our findings demonstrate that ACL and MCL cells are not the same. These differences may be related to the disparate healing capacity of the ACL and MCL observed clinically.     

Reactive oxidants regulate membrane repolarization and store-operated uptake of calcium by formyl peptide-activated human neutrophils

Although the rapid and considerable membrane depolarization response which accompanies activation of the phagocyte NADPH oxidase is due to transmembrane electron fluxes, little is known about the involvement of reactive oxidant species (ROS) in the subsequent repolarization response. In the current study, we have investigated the effects of superoxide dismutase (SOD), catalase, methionine, and the myeloperoxidase (MPO) inhibitors, sodium azide and 4-aminobenzoyl hydrazide (ABAH), as well as those of H₂O₂ and HOCl (both at 100 μM) on the alterations in membrane potential which accompany activation of human neutrophils with the chemoattractant, FMLP (1 μM), and on store-operated uptake of Ca²⁺. The generation of ROS by FMLP-activated neutrophils was monitored according to the magnitude of oxygen consumption and autoiodination, while spectrofluorimetric procedures were used to measure alterations in membrane potential and influx of Ca²⁺. Treatment of the cells with H₂O₂, and HOCl, significantly impeded membrane repolarization, while sodium azide, ABAH, methionine, and catalase exerted the opposite effects, potentiating both the rates and the magnitudes of membrane repolarization and store-operated uptake of Ca²⁺. These observations demonstrate that NADPH oxidase regulates neutrophil membrane potential and Ca²⁺ influx not only via its electrogenic activity, but also as a consequence of the generation of ROS.     

Hsp90 Interaction with INrf2(Keap1) Mediates Stress-induced Nrf2 Activation

INrf2(Keap1) functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to stress, Nrf2 is released from INrf2 and translocates inside the nucleus leading to activation of cytoprotective proteins critical in protection against adverse effects including cancer. We demonstrate here a novel role of heat shock protein 90 (Hsp90) in control of the INrf2 and Nrf2 activation. Hsp90 interacted with INrf2 that leds to stabilization of INrf2 during heat shock stress. Domain mapping showed the requirement of INrf2-NTR and the Hsp90-CLD region for interaction of Hsp90 with INrf2. Heat shock and antioxidants induced Hsp90, and casein kinase 2 (CK2) phosphorylated INrf2Thr55. This led to increased Hsp90-INrf2 interaction, dissociation of the Rbx1/Cul3·INrf2·Nrf2 complex, and activation of Nrf2. Inhibitors of CK2 and Hsp90, and mutation of INrf2Thr55 abolished the Hsp90-INrf2 interaction and downstream signaling. INrf2 is released from Hsp90 once the heat shock or antioxidant stress subsidized, thereby allowing INrf2 to interact with Nrf2 and facilitate Nrf2 ubiquitination and degradation. The results together demonstrate a novel role for the stress-induced Hsp90-INrf2 interaction in regulation of Nrf2 activation and induction of cytoprotective proteins.     

Ca2+参与H2O2促进盐碱混合胁迫下燕麦种子的萌发和成苗

盐碱胁迫是制约作物高产优质的重要因素,Ca²⁺和H₂O₂作为信号分子参与作物逆境响应调节。为了解Ca²⁺是否参与H₂O₂对盐碱胁迫下植物种子萌发和成苗的调控,以燕麦（Avena nude）为试验材料,采用隶属函数分析方法,研究了胞外游离Ca²⁺螯合剂EGTA、质膜Ca²⁺通道阻断剂LaCl₃和液泡膜Ca²⁺释放抑制剂钌红（RR）与H₂O₂共处理对盐碱混合（NaCl：Na₂SO₄：NaHCO₃：Na₂CO₃=12：8：9：1）胁迫下种子萌发和成苗的影响。结果表明,25~200 mmol·L^-1盐碱混合胁迫显著抑制燕麦的种子萌发和成苗,抑制程度随浓度提高而增强;0.001~2 mmol·L^-1 H₂O₂能够促进燕麦种子的萌发和成苗,且0.5 mmol·L^-1 H₂O₂可以显著缓解75 mmol·L^-1盐碱混合胁迫对燕麦种子萌发和成苗的抑制作用;而EGTA、LaCl₃和RR均能消减H₂O₂对盐碱混合胁迫下燕麦种子萌发和成苗的促进作用。表明Ca²⁺参与H₂O₂促进盐碱混合胁迫下燕麦种子萌发和成苗的信号转导过程。     

Oxidative stress and Mrp2 internalization

Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Gö6850, a Ca²⁺ chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca²⁺-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 μM). Moreover, an increase in the intracellular Ca²⁺ level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca²⁺ elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.     

苜蓿体内H2S信号与Ca2+调节气孔运动的作用机制

为探究H₂S信号在苜蓿（Medicago sativa）体内调节气孔运动的作用,及在此过程中H₂S与Ca²⁺的关系,以蒺藜苜蓿（Medicago truncatula）的野生型和钙离子转运体突变体为试验材料,分别从转录水平、细胞水平和生理水平开展研究。采用qRT-PCR比较相关基因的表达量变化、荧光探针显示体内Ca²⁺含量、电极法测定H₂S含量、光学显微镜观察和测量气孔孔径等。结果表明：蒺藜苜蓿突变体NF3011和NF2734体内H₂S的含量与野生型相比极显著降低（P<0.01）;H₂S信号在一定程度上抑制钙离子转运体编码基因MTR_6g027580的表达;外源生理浓度H₂S熏蒸可诱导蒺藜苜蓿气孔关闭,与Ca²⁺通道阻断剂LaCl₃联合处理对野生型气孔运动未产生影响,而在突变体中的结果截然相反;利用荧光探针测定保卫细胞内的Ca²⁺含量,所得结果与气孔孔径的变化规律完全一致。综上所述,H₂S信号促进叶片保卫细胞内Ca²⁺的含量增加,最终表现为植物气孔孔径变小,在此过程中胞内Ca²⁺含量变化主要通过Ca²⁺转运体进行,少部分依赖Ca²⁺离子通道。该研究结果不仅在理论上丰富了H₂S信号的作用机制,更具应用于苜蓿生产实践并推广于其他作物的潜力。     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Release of endogenous GABA can occur through Ca-dependent and Ca-independent processes

Release of γ-aminobutyric acid (GABA) can be elicited by electrical field stimulation even in the absence of external Ca²⁺. Indeed, the release of GABA under such conditions is even higher than in the presence of Ca²⁺. To investigate the underlying mechanism of this phenomenon, the release of endogenous GABA from rat striatal slices was measured by high performance liquid chromatography with electro- chemical detection. Electrical stimulation at 2 Hz for 3 min elevated GABA efflux by 4.5-fold. Withdrawing external Ca²⁺ and adding 1 mM EGTA produced a small, transient increase in the basal efflux of GABA and increased electrically-evoked overflow 3-fold. Tetrodotoxin (5 μM) did not affect basal efflux in either normal or Ca²⁺-free conditions, but abolished electrically-evoked release. In the presence of normal Ca²⁺, nipecotic acid (1 mM) enhanced both spontaneous efflux and evoked overflow. Nipecotic acid also increased spontaneous release when external Ca²⁺ was removed. However, in the absence of Ca²⁺, nipecotic acid failed to increase electrically evoked GABA overflow. These results suggest that there exists a Ca²⁺-independent process for GABA release via the same carrier system that is utilized for high affinity GABA uptake.     

Dissociation of Intracellular Ca Release and Ca Entry Response to 5-Hydroxytryptamine in Cultured Canine Tracheal Smooth Muscle Cells

The relationship between the agonist-sensitive Ca²⁺ pool and those discharged by the Ca²⁺-ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca²⁺ ([Ca²⁺]_i), followed by a sustained plateau phase that was dependent on extracellular Ca²⁺. In such cells, TG produced a concentration-dependent increase in [Ca²⁺]_i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca²⁺. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca²⁺]_i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca²⁺-ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca²⁺-influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca²⁺ stores is sufficient for activation of Ca²⁺ influx. Some characteristics of the Ca²⁺-influx activated by depletion of internal Ca²⁺ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca²⁺ influx was inhibited by La³⁺, membrane depolarisation, and the novel Ca²⁺-influx blocker 1-{β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca²⁺ influx by TG also was blocked by La³⁺, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca²⁺ stores in TSMCs is sufficient for activation of Ca²⁺ influx, and (2) the agonist-activated Ca²⁺ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ pool are indistinguishable.     

胞质Ca2+参与外源H2S促进盐碱胁迫下裸燕麦种子萌发

硫化氢（Hydrogen sulfide,H₂S）是植物新型气体信号分子,钙离子（Calcium,Ca²⁺）为重要的第二信使,两者在植物逆境响应中分别发挥着重要作用。为明确胞质Ca²⁺在外源H₂S促进盐碱胁迫下作物种子萌发中的作用,以裸燕麦（Avena nude）为材料,采用培养皿培养,以混合盐碱（NaCl、Na₂SO₄、Na₂CO₃、NaHCO₃的摩尔比为12：8：1：9）模拟甘肃裸燕麦种植地盐碱环境,蒸馏水为对照,测定了胞外Ca²⁺螯合剂乙二醇-双-（2-氨基乙醚）四乙酸（EGTA）、质膜Ca²⁺通道阻断剂氯化镧（LaCl₃）、液泡Ca²⁺释放抑制剂钌红（RR）和内质网钙泵阻断剂毒胡萝卜素（Thaps）分别与H₂S供体硫氢化钠（NaHS）共处理下种子的发芽势、发芽率、发芽指数、活力指数、平均发芽速率、胚根长和胚芽长7个发芽指标,利用隶属函数分析方法综合评价胞质Ca²⁺对H₂S缓解盐碱胁迫抑制种子萌发的影响。结果表明,随着盐碱胁迫浓度增大,裸燕麦种子的发芽势、发芽率、发芽指数、活力指数、平均发芽速率、胚根长和胚芽长显著下降。与对照相比,15~75 mmol·L^-1盐碱胁迫导致裸燕麦种子萌发的隶属函数综合评价值（D）显著降低,30 mmol·L^-1盐碱胁迫下D值下降了73.1%;100~1 000 μmol·L^-1 NaHS不同程度提高了裸燕麦种子萌发的D值,且100 μmol·L^-1 NaHS缓解30 mmol·L^-1盐碱胁迫下D值下降的作用最大;EGTA、LaCl₃和RR均显著逆转了100 μmol·L^-1 NaHS对30 mmol·L^-1盐碱胁迫下D值下降的缓解作用,而Thaps对NaHS的作用无显著影响。表明胞质Ca²⁺参与外源H₂S促进盐碱胁迫下裸燕麦种子萌发的信号传导过程,且胞质Ca²⁺主要来源于胞外Ca²⁺的内流和液泡中Ca²⁺的释放。     

神经元钙信号系统异常与阿尔茨海默病的“钙假说”

钙信号是细胞调节各项生命活动的重要机制。神经元通过胞外钙离子(calcium ion, Ca²⁺)内流、内质网Ca²⁺释放以及Ca²⁺释放介导的Ca²⁺内流等方式产生具有时空特异性的钙信号,用于调控多种生物学过程,例如动作电位的调节、神经递质的释放、轴突的生长以及突触可塑性等。神经元胞内Ca²⁺浓度因受到细胞精确调控而处于动态平衡之中。若钙信号失调导致平衡被打破,则会造成神经元功能异常甚至死亡。近年来多项研究表明,钙稳态失衡与神经退行性疾病,例如阿尔茨海默病等的产生和发展密切相关,由此发展出关于阿尔茨海默病的钙假说。该假说认为,神经元钙稳态调节机制的持续性改变是神经元功能失常、大脑产生慢性疾病的重要因素。阿尔茨海默病发生发展过程中,神经元胞浆钙水平异常增高,致使多种钙依赖性酶的活性异常,进而影响基因转录。虽然内质网钙稳态的变化目前仍存在一定的争议,但较为确定的是线粒体中存在着钙超载的现象,导致氧化磷酸化反应下调,活性氧的产量增加,进而引发细胞凋亡。本文主要介绍了神经元钙信号系统及其功能,简要梳理了阿尔茨海默病钙假说的相关研究,并对后续研究进行了展望。