Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.     

Na+ entry via glutamate transporter activates the reverse Na+/Ca2+ exchange and triggers Ca(i)2+-induced Ca2+ release in rat cerebellar Type-1 astrocytes

We have previously demonstrated that rat cerebellar Type-1 astrocytes express a very active genistein sensitive Na(+)/Ca(2+) exchanger, which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Cai(2+) with Fluo-3 demonstrate that the Cai(2+) signals associated long (> 20 s) periods of reverse operation of the Na(+)/Ca(2+) exchange are amplified by a mechanism compatible with calcium-calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4-chloro-m-cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co-localization of immunofluorescent labeled Na(+)/Ca(2+) exchanger and RyRs. Low (< 50 micromol/L) or high (> 500 micromol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in which is completely blocked by the Na(+)/Ca(2+) exchange inhibitors KB-R7943 and SEA0400. The most important novel finding presented in this work is that L-Glu activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing Na(+) entry through the electrogenic Na(+)-Glu-co-transporter and not through the ionophoric L-Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric L-Glu receptors and the electrogenic Glu transporter.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

The Na+/Ca2+ exchanger is active and working in the reverse mode in human umbilical artery smooth muscle cells

The data presented in this work suggest that in human umbilical artery (HUA) smooth muscle cells, the Na(+)/Ca(2+) exchanger (NCX) is active and working in the reverse mode. This supposition is based on the following results: (i) microfluorimetry in HUA smooth muscle cells in situ showed that a Ca(2+)-free extracellular solution diminished intracellular Ca(2+) ([Ca(2+)](i)), and KB-R7943 (5microM), a specific inhibitor of the Ca(2+) entry mode of the exchanger, also decreased [Ca(2+)](i) (40.6+/-4.5% of Ca(2+)-free effect); (ii) KB-R7943 produced the relaxation of HUA rings (-24.7+/-7.3gF/gW, n=8, p<0.05); (iii) stimulation of the NCX by lowering extracellular Na(+) increases basal [Ca(2+)](i) proportionally to Na(+) reduction (Delta fluorescence ratio=0.593+/-0.141 for Na(+)-free solution, n=8) and HUA rings' contraction (peak force=181.5+/-39.7 for 130mM reduction, n=8), both inhibited by KB-R7943 and a Ca(2+)-free extracellular solution. In conclusion, the NCX represents an important Ca(2+) entry route in HUA smooth muscle cells.     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

AMPA-mediated excitotoxicity in oligodendrocytes: role for Na(+)-K(+)-Cl(-) co-transport and reversal of Na(+)/Ca(2+) exchanger

We investigated the role of Na(+)-K(+)-Cl(-) co-transporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchanger (NCX(rev)) in glutamate-mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6-8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na(+)/Ca(2+) exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular (), which was followed by a sustained overload, NKCC1 phosphorylation, and a NKCC1-mediated Na(+) influx. In the presence of a specific AMPA receptor inhibitor 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), the AMPA/CTZ failed to elicit any changes in . The AMPA/CTZ-induced sustained rise led to mitochondrial Ca(2+) accumulation, release of cytochrome c from mitochondria, and cell death. The AMPA/CTZ-elicited increase, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCX(rev). These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1-mediated Na(+) influx. The latter triggers NCX(rev) and NCX(rev)-mediated overload and compromises mitochondrial function and cellular viability.     

Intracellular lithium and cyclic AMP levels are mutually regulated in neuronal cells

In this work, we studied the effect of intracellular 3',5'-cyclic adenosine monophosphate (cAMP) on Li+ transport in SH-SY5Y cells. The cells were stimulated with forskolin, an adenylate cyclase activator, or with the cAMP analogue, dibutyryl-cAMP. It was observed that under forskolin stimulation both the Li+ influx rate constant and the Li+ accumulation in these cells were increased. Dibutyryl-cAMP also increased Li+ uptake and identical results were obtained with cortical and hippocampal neurons. The inhibitor of the Na+/Ca2+ exchanger, KB-R7943, reduced the influx of Li+ under resting conditions, and completely inhibited the effect of forskolin on the accumulation of the cation. Intracellular Ca2+ chelation, or inhibition of N-type voltage-sensitive Ca2+ channels, or inhibition of cAMP-dependent protein kinase (PKA) also abolished the effect of forskolin on Li+ uptake. The involvement of Ca2+ on forskolin-induced Li+ uptake was confirmed by intracellular free Ca2+ measurements using fluorescence spectroscopy. Exposure of SH-SY5Y cells to 1 mm Li+ for 24 h increased basal cAMP levels, but preincubation with Li+, at the same concentration, decreased cAMP production in response to forskolin. To summarize, these results demonstrate that intracellular cAMP levels regulate the uptake of Li+ in a Ca(2+)-dependent manner, and indicate that Li+ plays an important role in the homeostasis of this second messenger in neuronal cells.     

Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.     

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.     

KB-R7943 inhibits store-operated Ca(2+) entry in cultured neurons and astrocytes

We have studied cyclopiazonic acid (CPA)-sensitive store-operated Ca(2+) entry (SOCE) in cultured neurons and astrocytes and examined the effect of 2-[2-[4-(4-nitrobenzyloxy)phenyl]]isothiourea (KB-R7943), which is often used as a selective inhibitor of the Na(+)-Ca(2+) exchanger (NCX), on the SOCE. CPA increased transiently intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a sustained increase in [Ca(2+)](i) in neurons and astrocytes. The sustained increase in [Ca(2+)](i) depended on the presence of extracellular Ca(2+) and inhibited by SOCE inhibitors, but not by a Ca(2+) channel inhibitor. CPA also caused quenching of fura-2 fluorescence when the cells were incubated in Mn(2+)-containing medium. KB-R7943 at 10 microM inhibited significantly CPA-induced sustained increase in [Ca(2+)](i) in neurons and astrocytes. KB-R7943 also inhibited CPA-induced quenching of fura-2 fluorescence in the presence of extracellular Mn(2+). These results indicate that cultured neurons and astrocytes possess SOCE and that KB-R7943 inhibits not only NCX but also SOCE.     

Relative contribution of the Na(+)/Ca(2+) exchanger, mitochondria and endoplasmic reticulum in the regulation of cytosolic Ca(2+) and catecholamine secretion of bovine adrenal chromaffin cells

The relative importance of mitochondria, the Na(+)/Ca(2+) exchanger (NCX) and the endoplasmic reticulum (ER) in the regulation of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were examined in bovine chromaffin cells using fura-2 for average [Ca(2+)](i) and amperometry for secretory activity, which reflects the local Ca(2+) concentration near the exocytotic sites. Chromaffin cells were stimulated by a high concentration of K(+) when the three Ca(2+) removal mechanisms were individually or simultaneously inhibited. When the mitochondrial Ca(2+) uptake was inhibited, the [Ca(2+)](i) decayed at a significantly slower rate and the secretory activity was higher than the control cells. The NCX appears to function only in the initial phase of [Ca(2+)](i) decay and when the ER Ca(2+) pump is blocked. Similarly, the ER had a significant effect on the [Ca(2+)](i) decay and on the secretion only when the NCX was blocked. Inhibition of all three mechanisms leads to a substantial delay in [Ca(2+)](i) recovery and an increase in the secretion. The results suggest that the three mechanisms work together in the regulation of the Ca(2+) near the Ca(2+) channels and exocytotic sites and therefore modulate the secretory activity. When Ca(2+) diffuses away from the exocytotic sites, the mitochondrial Ca(2+) uptake becomes the dominant mechanism.     

Instrumental role of Na+ in NMDA excitotoxicity in glucose-deprived and depolarized cerebellar granule cells

In glucose-deprived cerebellar granule cells, substitution of extracellular Na+ with Li+ or Cs+ prevented N-methyl-D-aspartate (NMDA)-induced excitotoxicity. NMDA stimulated 45Ca2+ accumulation and ATP depletion in a Na-dependent manner, and caused neuronal death, even if applied while Na,K-ATPase was inhibited by 1 mM ouabain. The cells treated with NMDA in the presence of ouabain accumulated sizable 45Ca2+ load but most of them failed to elevate cytosolic [Ca2+] upon mitochondrial depolarization. Na/Ca exchange inhibitor, KB-R7943, inhibited Na-dependent and NMDA-induced 45Ca2+ accumulation but only if Na,K-ATPase activity was compromised by ouabain. In cells energized by glucose and exposed to NMDA without ouabain, KB-R7943 reduced NMDA-elicited ionic currents by 19% but failed to inhibit 45Ca2+ accumulation. It appears that a large part of NMDA-induced Ca2+ influx in depolarized and glucose-deprived cells is mediated by reverse Na/Ca exchange. A high level of reverse Na/Ca exchange operation is maintained by a sustained Na+ influx via NMDA channels and depolarization of the plasma membrane. In cells energized by glucose, however, most Ca2+ enters directly via NMDA channels because Na,K-ATPase regenerating Na+ and K+ concentration gradients prevents Na/Ca exchange reversal. Since under these conditions Na/Ca exchange extrudes Ca2+, its inhibition destabilizes Ca2+ homeostasis.     

胍丁胺对大鼠心室肌细胞内游离钙浓度的影响

本研究旨在观察胍丁胺 (agmatine ,Agm)对分离大鼠心室肌细胞内游离钙浓度 ( [Ca2 +]i)的影响。用酶解方法分离大鼠心室肌细胞 ,用Fluo 3 AM负载 ,然后用激光共聚焦法测定单个心室肌细胞 [Ca2 +]i 的荧光强度 (fluorescenceintensity ,FI) ,结果以FI或相对荧光强度 (F/F0 % )表示。实验结果表明 ,在正常台氏液 (含钙 1 0mmol/L)和无钙台氏液中 ,单个大鼠心室肌细胞的荧光密度分别为 12 8 8± 13 8和 119 6± 13 6,两者无差异。Agm 0 1、1和 10mmol/L浓度依赖性地显著降低细胞的钙浓度 ;在正常台氏液中加入EGTA 3mmol/L ,Agm同样降低细胞的钙浓度。KCl 60mmol/L ,PE 3 0 μmol/L ,和Bay K 864 410 μmol/L均升高心室肌细胞的[Ca2 +]i。Agm同样降低高浓度KCl、Bay K 864 4和PE诱发的心室肌细胞 [Ca2 +]i 升高。当细胞外液钙浓度由 1mmol/L增加到 10mmol/L时 ,诱发心室肌细胞钙超载 ,同时部分心室肌细胞产生可传播的钙波 (Ca2 +wave) ,Agm 1mmol/L降低钙波的传播速度和持续时间 ,最终阻断钙波。以上结果提示 ,Agm对心室肌细胞的胞浆[Ca2 +]i具有抑制作用 ,此作用通过阻断电压依赖性钙通道而实现 ;并可能与抑制大鼠心室肌细胞内钙释放有关     

Differential contribution of plasmalemmal Na/Ca exchange isoforms to sodium-dependent calcium influx and NMDA excitotoxicity in depolarized neurons

Inhibition of Na(+),K(+)-ATPase during NMDA applications greatly increased NMDA-induced excitotoxicity in primary cultures of forebrain neurons (FNs), but not in cerebellar granule cells (CGCs). Because Na(+),K(+)-ATPase inhibition promotes reversal of plasmalemmal Na(+)/Ca(2+) exchangers, we compared the activities of reversed K(+)-independent (NCX) and K(+)-dependent (NCKX) Na(+)/Ca(2+) exchangers in these cultures. To this end, we measured gramicidin-induced and Na(+)-dependent elevation in cytosolic [Ca(2+)] ([Ca(2+)](c)) that represents Ca(2+) influx via reversed NCX and NCKX; NCX activity was dissected out by removing external K(+). The [Ca(2+)](c) elevations mediated by NCX alone, and NCX plus NCKX combined, were 17 and 6 times more rapid in FNs than in CGCs, respectively. Northern blot analysis showed that FNs preferentially express NCX1 whereas CGCs expressed NCX3. Differences in expression of other isoforms (NCX2, NCKX2, NCKX3 and NCKX4) were less pronounced. We tested whether the NCX or NCKX family of exchangers contributes most to the toxic NMDA-induced Ca(2+) influx in depolarized neurons. We found that in FNs, inhibition of NCX alone was sufficient to significantly limit NMDA excitotoxicity, whereas in CGCs, inhibition of both NCX and NCKX was required. The data suggest that the high activity of NCX isoforms expressed in FNs, possibly NCX1, sensitizes these neurons to NMDA excitotoxicity.     

Calcium elevation elicited by reverse mode Na+/Ca2+ exchange activity is facilitated by intracellular calcium stores in bovine chromaffin cells

The Na(+)/Ca(2+) exchanger (NCX) in plasma membranes either moves Ca(2+) out of (forward mode) or into (reverse mode) cells depending on the electrochemical gradient of these ions across the membrane. In this report, we characterize the sources responsible for the elevation in [Ca(2+)](i) elicited by reverse mode NCX activity. The elevation in [Ca(2+)](i) elicited by reverse mode NCX activity was significantly diminished by thapsigargin. KB-R7943 could only partially suppress the [Ca(2+)](i) change. Measurement of the [Ca(2+)](i) concurrent with reverse mode NCX current by perforated whole-cell patch showed that elevation in [Ca(2+)](i), but not the current, was inhibited by thapsigargin. The change in [Ca(2+)](i) response elicited by nicotinic acetylcholine receptor agonist was inhibited by thapsigargin. These suggest the importance of intracellular Ca(2+) stores in facilitating the [Ca(2+)](i) elevation elicited by reverse mode NCX activity under physiological condition.     

Mobilizing store Ca(2+) in the presence of La(3+) evokes exocytosis in bovine chromaffin cells

The effect on exocytosis of La(3+), a known inhibitor of plasma membrane Ca(2+)-ATPases and Na(+)/Ca(2+) exchangers, was studied using cultured bovine adrenal chromaffin cells. At high concentrations (0.3-3 mM), La(3+) substantially increased histamine-induced catecholamine secretion. This action was mimicked by other lanthanide ions (Nd(3+), Eu(3+), Gd(3+), and Tb(3+)), but not several divalent cations. In the presence of La(3+), the secretory response to histamine became independent of extracellular Ca(2+). La(3+) enhanced secretion evoked by other agents that mobilize intracellular Ca(2+) stores (angiotensin II, bradykinin, caffeine, and thapsigargin), but not that due to passive depolarization with 20 mM K(+). La(3+) still enhanced histamine-induced secretion in the presence of the nonselective inhibitors of Ca(2+)-permeant channels SKF96365 and Cd(2+), but the enhancement was abolished by prior depletion of intracellular Ca(2+) stores with thapsigargin. La(3+) inhibited (45)Ca(2+) efflux from preloaded chromaffin cells in the presence or absence of Na(+). It also enhanced and prolonged the rise in cytosolic [Ca(2+)] measured with fura-2 during mobilization of intracellular Ca(2+) stores with histamine in Ca(2+)-free buffer. The results suggest that the efficacy of intracellular Ca(2+) stores in evoking exocytosis is enhanced dramatically by inhibiting Ca(2+) efflux from the cell.     

八肽胆囊收缩素激活钙离子通道和酪氨酸激酶诱导豚鼠心肌细胞内的游离钙增高

探讨八肽胆囊收缩素(CCK-8)对豚鼠单个心肌细胞内游离钙浓度([Ca2+]i的影响及其信号转导机制.Fluo 3-AM标记酶消化法分离的单个心室肌细胞,用激光共聚焦显微镜测定细胞内[Ca2+]i的浓度.[Ca2+]i的变化用荧光强度(Fi)和相对荧光强度(Fi/F0%)表示.实验结果如下(1)在含Ca2+1.0 mmol/L的Tyrode's液中,CCK-8(1～104pmoVL)均可引起[Ca2+]i快速显著上升(P＜0.01).(2)用钙离子鳌合剂EGTA(3 mmol/L)和钙离子通道阻断剂nisoldipine(0.5μmol/L)预孵育心肌细胞5 min,CCK-8(102pmol/L)仅可引起[Ca2+]i缓慢轻度上升(P＜0.01).(3)用非选择性CCK受体拮抗剂丙谷胺(proglumide 6μmo1/L)或酪氨酸激酶抑制剂genistein(1 μmol/L)预孵育心肌细胞5 min,则完全抑制CCK-8诱导的[Ca2+]i升高(P＜0.01).CCK-8可通过激活其受体控制的Ca2+通道,引起Ca2+内流,诱导细胞内Ca2+释放,引起豚鼠单个心肌细胞内[Ca2+]i上升,此作用可能由酪氨酸激酶介导.     

The Na+/Ca2+ exchange inhibitor KB-R7943 potently blocks TRPC channels

Na(+)/Ca(2+) exchangers (NCXs) and members of the canonical transient receptor potential (TRPC) channels play an important role in Ca(2+) homeostasis in heart and brain. With respect to their overlapping expression and their role as physiological Ca(2+) influx pathways a functional discrimination of both mechanisms seems to be necessary. Here, the effect of the reverse-mode NCX inhibitor KB-R7943 was investigated on different TRPC channels heterologously expressed in HEK293 cells. In patch-clamp recordings KB-R7943 potently blocked currents through TRPC3 (IC(50)=0.46 microM), TRPC6 (IC(50)=0.71 microM), and TRPC5 (IC(50)=1.38 microM). 1-Oleoyl-2-acetyl-sn-glycerol-induced Ca(2+) entry was nearly completely suppressed by 10 microM KB-R7943 in TRPC6-transfected cells. Thus, KB-R7943 is able to block receptor-operated TRP channels at concentrations which are equal or below those required to inhibit reverse-mode NCX activity. These data further suggest that the protective effects of KB-R7943 in ischemic tissue may, at least partly, be due to inhibition of TRPC channels.     

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate--the role of pH and sodium

Although Zn(2+) homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn(2+) concentration ([Zn(2+) ](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn(2+)](i) and intracellular Ca(2+) concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na(+)-free conditions (Na(+) substituted with N-methyl-D-glucamine(+)) caused Ca(2+) influx, pH(i) drop, and Zn(2+) release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na(+) -dependent pathways of H(+) efflux, forcing H(+) efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn(2+)](i) elevations. In the absence of Na(+), the rate of [Zn(2+)](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na(+), the rate of [Zn(2+) ](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn(2+) ](i) elevations and that Na(+) counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn(2+) clearance.