胞内钙动员在硝普钠增加豚鼠胃窦环行肌细胞钙激活钾电流中的作用

AIM: To investigate the role of calcium mobilization in the calcium-activated potassium currents [IK(ca)] increasedby sodium nitroprusside (SNP), a nitric oxide (NO) donor, in gastric antral circular myocytes of the guinea pig.METHODS- A perforated patch-clamp technique was used, and the myocytes were isolated by collagenase.RESULTS: SNP 100 lamol/L significantly increased IK(Ca), and enhanced the spontaneous transient outward currents (STOC). SNP-induced increase of IK(Ca) was not blocked by extracellular calcium-free solution (containingegtazic acid 10μmol/L and nicardipine 5μmol/L, an L-type calcium channel blocker. And SNP 100μmol/Lsuppressed the L-type calcium currents (Ica). SNP-induced increase of STOC was inhibited by heparin 3g/L, apotent inhibitor of inositol triphosphate receptor (InsP3R). However, ryanodine 10μrnol/L, an inhibitor of calciuminduced calcium release (CICR), did not inhibit the effect of SNP-induced increase of STOC. Methylene blue (1μmol/L), an inhibitor of soluble guanylate cyclase, also inhibited such an effect. CONCLUSION: The increase ofIK(Ca) caused by SNP may be mediated by cGMP via IP3-sensitive calcium pools, however, extracellular Ca^2^ maynot be involved in the process.     

MEK inhibitor PD98059 acutely inhibits synchronized spontaneous Ca^2＋ oscillations in cultured hippocampal networks

AIM: To investigate the changes in synchronized spontaneous Ca2+ oscillations induced by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 at different concentrations in cultured hippocampal network. METHODS: Hippocampal neurons in culture for 1-2 weeks were used for this study. Spontaneous synaptic activities of these hippocampal neurons were examined by Ca2+ imaging using calcium-sensitive dye. MEK inhibitor PD98059 (10, 30, and 60 micromol/L) and SB202474 (10 and 60 micromol/L), a negative control for mitogen-activated protein kinase (MAPK) cascade study, were applied to the cells under the microscope while imaging was taking place. RESULTS: PD98059 at a lower concentration of 10 micromol/L had little effect on the Ca2+ oscillation. At the higher concentration of 30 micromol/L, 5 min after application of PD98059, the spike frequency was decreased to 25.38% +/-7.40% (mean+/-SEM, n=16, P<0.01 vs medium control) of that of the control period. At an even higher concentration of 60 micromol/L, 5 min after application of PD98059, the spike frequency was decreased to 14.53%+/-5.34% (mean+/-SEM, n=16, P< 0.01 vs medium control) of that of the control period. The spike amplitude underwent a corresponding decrease. However, the negative control SB202474 at concentrations of 10 and 60 micromol/L had little inhibition effect on the Ca2+ oscillation. CONCLUSION: These results indicate that PD98059 inhibits synchronized spontaneous Ca2+ oscillation through inhibition of MEK, which hints that the MAPK cascade is required to maintain synchronized spontaneous Ca2+ oscillation.     

Slow depletion of endoplasmic reticulum Ca(2+) stores and block of store-operated Ca(2+) channels by 2-aminoethoxydiphenyl borate in mouse pancreatic acinar cells

The compound 2-aminoethoxydiphenyl borate (2-APB) has been used as either a specific membrane-permeable inhibitor for InsP(3) receptors or a store-operated Ca(2+) channel blocker in some cells. In this study, we have investigated actions of 2-APB on Ca(2+) signalling in mouse pancreatic acinar cells by measuring Ca(2+) concentration in the cytosol ([Ca(2+)]c) and in the endoplasmic reticulum (ER). Although 2-APB (50 microM) inhibited or modulated [Ca(2+)]c oscillations generated by a low dose of ACh, it did not block InsP(3)-mediated Ca(2+) release from the ER elicited by high doses of acetylcholine (ACh). 2-APB alone more than 70 microM tended to increase [Ca(2+)]c. When we directly measured Ca(2+) concentration in the lumen of the ER with a low affinity Ca(2+) dye, Mag-fluo-4, 2-APB itself slowly lowered ER Ca(2+) concentration and ACh could further release Ca(2+) from the ER in the presence of 2-APB, suggesting its lack of potency to block InsP(3) receptors. When store-operated Ca(2+) entry was evoked by addition of external Ca(2+) (5 mM) after depletion of Ca(2+) stores, 2-APB (50 microM) substantially blocked the Ca(2+) influx in a reversible manner. We conclude that (a) 2-APB is a good blocker for store-operated Ca(2+) channels, (b) 2-APB could not effectively block InsP(3) receptors, and (c) low doses of 2-APB lower Ca(2+) concentration in the lumen of the ER without a significant elevation of [Ca(2+)]c.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

1. In muscle, intracellular calcium concentration, hence skeletal muscle force and cardiac output, is regulated by uptake and release of calcium from the sarcoplasmic reticulum. The ryanodine receptor (RyR) forms the calcium release channel in the sarcoplasmic reticulum. 2. The free [Ca2+] in the sarcoplasmic reticulum regulates the excitability of this store by stimulating the Ca2+ release channels in its membrane. This process involves Ca2+-sensing mechanisms on both the luminal and cytoplasmic sides of the RyR. In the cardiac RyR, these have been shown to be a luminal Ca2+ activation site (L-site; 60 micromol/L affinity), a cytoplasmic activation site (A-site; 0.9 micromol/L affinity) and a cytoplasmic Ca2+ inactivation site (I2-site; 1.2 micromol/L affinity). 3. Cardiac RyR activation by luminal Ca2+ occurs by a multistep process dubbed 'luminal-triggered Ca2+ feed-through'. Binding of Ca2+ to the L-site initiates brief (1 msec) openings at a rate of up to 10/s. Once the pore is open, luminal Ca2+ has access to the A-site (producing up to 30-fold prolongation of openings) and to the I2-site (causing inactivation at high levels of Ca2+ feed-through). 4. The present paper reviews the evidence for the principal aspects of the 'luminal-triggered Ca2+ feed-through' model, the properties of the various Ca2+-dependent gating mechanisms and their likely role in controlling sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle. 5. The model makes the following important predictions: (i) there will be a close link between luminal and cytoplasmic regulation of RyRs and any cofactor that prolongs channel openings triggered by cytoplasmic Ca2+ will also promote RyR activation by luminal Ca2+; (ii) luminal Mg2+ (1 mmol/L) is essential for the control of SR excitability in cardiac muscle by luminal Ca2+; and (iii) the different RyR isoforms in skeletal and cardiac muscle will be controlled quite differently by the luminal milieu. For example, Mg2+ in the SR lumen (approximately 1 mmol/L) can strongly inhibit RyR2 by competing with Ca2+ for the L-site, whereas RyR1 is not affected by luminal Mg2+.     

Tonic potentiation and attenuation produced by membrane depolarization in guinea-pig trachealis

1. We studied how membrane depolarization directly affected intracellular Ca2+ signalling when voltage-operated Ca2+ channels (VOCC) were not available in guinea-pig tracheal smooth muscle. To block VOCC, we used 3 micromol/L verapamil, which completely abolished high K+ (20-60 mmol/L)-induced contraction, and elevation of fura-2 signal. 2. Muscle tone was generated by adding Ca2+ to the extracellular Ca2+-free solution containing prostaglandin (PG)E2 (100 nmol/L) after abolishing basal tone with indomethacin (1 micromol/L). 3. In the absence of verapamil, high K+ (20-60 mmol/L) solution potentiated 2.4 mmol/l Ca2+-induced sustained contractions. Even in the presence of 3 micromol/L verapamil, replacement with 20 and 40 mmol/L K+ solution induced tonic potentiation, which was changed to attenuation with a higher K+ solution (60 mmol/L), lower extracellular Ca2+ concentration ([Ca2+]o) and pretreatment with cyclopiazonic acid (10 micromol/L), a Ca2+ sequestration inhibitor. 4. These results indicate that the balance between depolarization-dependent Ca2+ release and receptor-operated cation channel inhibition may determine whether tonic potentiation or attenuation is manifested, depending on the availability of VOCC, the magnitude of the depolarization, [Ca2+]o and Ca2+ content in the sarcoplasmic reticulum.     

粉防己碱对培养乳牛基底动脉平滑肌细胞内游离钙浓度的影响

目的：研究粉防己碱对培养乳牛基底动脉平滑肌细胞游离钙浓度（[Ca^2 ]i)的影响。方法：利用AR－CM－MIC阳离子测定系统,采用Fura 2-AM为指示剂,测量单个细胞内[Ca^2 ]i。结果：粉防己碱10－100μmol/L对培养乳牛基底动脉平滑肌细胞静息[Ca^2 ]i无明显影响。在细胞外钙为1．3mmol/L,粉防己碱可浓度依赖性地抑制KC1引起[Ca^2 ]i的升高。咖啡因10mmol/L可诱导一次[Ca^2 ]i瞬间快速升高,随后自发回复到静息水平,粉防己碱10和30μmol/L对咖啡因诱导的[Ca^2 ]i瞬间升高没有作用,但高浓度（100μmol/L）粉防己碱抑制了[Ca^2 ]i瞬间升高。在细胞外钙为1.3mmol/L,苯肾上腺素10μmol/L可引起双相[Ca^2 ]i变化,包括快速升高相和持续升高相。在细胞外钙为零,苯肾上腺素仅引起[Ca^2 ]i的快速升高相。粉防己碱可浓度依赖性地抑制苯肾上腺素引起[Ca^2 ]i快速升高相。结论：在培养乳牛基底动脉平滑肌细胞,粉防己碱可能通过影响电压依赖性和苯肾上腺素受体介导的钙通道而抑制钙内流。高浓度粉防己碱也可能影响肌浆网钙释放或钙摄取。     

Effect of calcium on the vascular contraction induced by cobra venom cardiotoxin

1. The cytotoxic effects of cardiotoxin (CTX) purified from Cobra venom were tested in endothelium-denuded rat aortic ring preparations in tissue organ baths and the effect of extracellular Ca2+ on the cytotoxic effect of CTX was investigated using a digital dynamic calcium imaging technique. 2. At 10 micromol/L, CTX induced a slowly developing and sustained contraction that amounted to approximately 50% of the maximal contraction induced by 80 mmol/L KCl. At high concentrations (> 15 micromol/L), CTX caused irreversible damage to the smooth muscle contractile function. However, washout of CTX at its peak contraction did not affect the subsequent contraction to either KCl or phenylephrine. 3. Contraction induced by CTX was dependent on the Ca2+ concentration in the external solution. A maximal contractile response to CTX was obtained in medium containing 1-2.5 mmol/L Ca2+. This contractile response induced by CTX decreased with higher Ca2+ concentrations and was completely diminished when 7 mmol/L Ca2+, 3 mmol/L Ni2+ or 30 micromol/L tetrandrine (a non-selective calcium channel blocker) was present in the external solution before addition of CTX to the bath. 4. The above observations were supported by the calcium imaging work performed with cultured aortic smooth muscle cells from Wistar-Kyoto rats, in which CTX was shown to induce the elevation of cytosolic Ca2+ in the presence, but not in the absence, of 2.5 mmol/L extracellular Ca2+. Increasing the extracellular Ca2+ concentration to 7 mmol/L, the addition of 3 mmol/L Ni2+ or inclusion of 30 micro mol/L tetrandrine inhibited the elevation of cytosolic Ca2+ induced by CTX. 5. These results suggest that: (i) a CTX-sensitive internal calcium store does not exist in rat aortic smooth muscle; (ii) the contractile effect CTX is associated with a Ca2+ influx process; and (iii) CTX interacts extracellularly with the plasma membrane at the level of the calcium channels, as well as anionic sites to which Ca2+ and other inorganic cations bind.     

Effect of Zn^2＋ ions on ryanodine binding to sarcoplasmic reticulum of striated muscles in the presence of pyrithione

AIM: To explore whether the differential effects of Zn^2 on ryanodine binding to the sarcoplasmic reticulum (SR)of skeletal and cardiac muscles resulted from different permeability of the SR to Zn^2 . METHODS: [^3H]ryanodinebinding assays were performed to examine the effect of Zn^2 on ryanodine binding to the SR in the presence ofpyrithione sodium (PyNa), a specific Zn^2 ionophore. RESULTS: As a control, PyNa up to 50 μmol/L did notinduce any effect on ryanodine binding to the SR of cardiac muscle. But PyNa 1-100 μnol/L increased ryanodinebinding in skeletal muscle with maximum binding (222.2 % 20.9 % of the control) and inhibited ryanodine bindingto 50 % of the control at about 500 μmol/L. In the presence of PyNa 10 and 50 μnol/L the dose-dependence of theeffect of Zn^2 in cardiac muscle was still monophasic and not changed by PyNa, while the biphasic effect of Zn^2 in skeletal muscle became monophasic. CONCLUSION: Different permeability of the SR to Zn^2 may account forthe differential effects of Zn^2 on ryanodine binding in skeletal and cardiac muscles. PyNa is not a strictly specific Zn^2 ionophore.     

Fendiline mobilizes intracellular Ca2+ in Chang liver cells

1. The effects of the antianginal drug fendiline (N-[3,3-diphenylpropyl]-alpha-methyl-benzylamine) on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. 2. Fendiline (1-100 micromol/L) increased [Ca2+](i) in a concentration-dependent manner, with an EC50 of 25 micromol/L. 3. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase. Removal of extracellular Ca2+ partly reduced the [Ca2+](i) signals. 4. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was reduced by 65% following pretreatment with 1 micromol/L thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete Ca2+ stored in the endoplasmic reticulum. 5. After pretreatment with 10 micromol/L fendiline in Ca2+-free medium for several minutes, addition of 3 mmol/L Ca2+ induced an increase in [Ca2+](i) of a magnitude four-fold greater than control. This increase in [Ca2+](i) was not reduced by 10 micromol/L SKF96365, econazole, nifedipine or verapamil. 6. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was not altered by inhibition of phospholipase C with 2 micromol/L 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U73122). 7. The results of the present study show that fendiline induces an increase in [Ca2+](i) in Chang liver cells by releasing stored Ca2+ in an inositol 1,4,5-trisphosphate-independent manner and by causing extracellular Ca2+ influx.     

Role of InsP3 and ryanodine receptors in the activation of capacitative Ca2+ entry by store depletion or hypoxia in canine pulmonary arterial smooth muscle cells

BACKGROUND AND PURPOSE: Experiments were performed to determine if capacitative Ca(2+) entry (CCE) in canine pulmonary arterial smooth muscle cells (PASMCs) is dependent on InsP(3) receptors or ryanodine receptors as induction of CCE is dependent on simultaneous depletion of the functionally separate InsP(3)- and ryanodine-sensitive sarcoplasmic reticulum (SR) Ca(2+) stores in these cells. EXPERIMENTAL APPROACH: Myocytes were isolated from canine pulmonary arteries using enzymatic procedures and were used within 8 h of preparation. Measurements of cytosolic Ca(2+) were made by imaging fura-2 loaded individual myocytes that were perfused with physiological buffered saline solution with or without Ca(2+). KEY RESULTS: Treating myocytes with 10 microM cyclopiazonic acid (CPA), removing extracellular Ca(2+), and briefly applying 10 mM caffeine and 10 microM 5-hydroxytryptamine (5-HT) depleted SR Ca(2+) stores. Extracellular Ca(2+) reintroduction caused cytosolic [Ca(2+)] to elevate above baseline signifying CCE. The InsP(3) receptor inhibitors 2-aminobiphenylborate (50-75 microM; 2-APB) and xestospongin-C (20 microM; XeC) abolished CCE. Yet, CCE was unaffected by 10 microM or 300 microM ryanodine or 10 microM dantrolene, which modify ryanodine receptor activity. Higher dantrolene concentrations (50 microM), however, can inhibit both ryanodine receptors and InsP(3) receptors, did reduce CCE. In contrast, CCE activated by hypoxia was unaffected by XeC (20 microM). CONCLUSIONS AND IMPLICATIONS: The results provide evidence that CCE activated by depletion of both InsP(3) and ryanodine SR Ca(2+) stores in canine PASMCs is dependent on functional InsP(3) receptors, whereas the activation of CCE by hypoxia appears to be independent of functional InsP(3) receptors.     

新鲜分离的新生大鼠皮层神经元的钙振荡

目的:研究新鲜分离的新生大鼠皮层神经元胞内钙离子浓度([Ca~(2 )]_i)发生振荡的机制。方法:采用酶解结合机械分离法从6—7日龄大鼠分离皮层神经元,用M40钙离子测量系统(PTI)测量细胞内钙离子浓度的变化。用Fura-2作为钙离子指示剂。结果:在观察到的82个神经元细胞中,47个产生了自发钙振荡。自发钙振荡依赖于胞外钙离子浓度。去除外钙后自发钙振荡立即停止。四乙铵1mmol/L引起钙振荡振幅增大,频率变快。CsCl 1mmol/L主要引起频率增加。BaCl_2 1mmol/L可使振幅、频率增高,并有明显的高台样基线增加。结论:皮层神经元在无突触联系的情况下具有产生自发[Ca~(2 )]_i振荡的特性,K~ 通道在决定钙振荡的幅值和频率方面起重要作用。     

Inositol 1,4,5-trisphosphate- and guanosine 5''-O-(3-thiotriphosphate)-induced Ca2+ release in cultured airway smooth muscle.

1. The interaction between inositol 1,4,5-trisphosphate (InsP3) and guanosine 5'-O-(3-thio triphosphate) (GTP gamma S) releasable calcium (Ca2+) pools was examined using 45Ca effluxes in permeabilized cultured airway smooth muscle cells from rabbit trachea. 2. Addition of InsP3 or GTP gamma S caused a concentration-dependent release of intracellular Ca2+. The release of Ca2+ by InsP3 was much greater than with GTP gamma S. Pretreatment with maximally effective InsP3 (10 microM) abolished the GTP gamma S-induced Ca2+ release, whereas pretreatment with 100 microM GTP gamma S reduced the InsP3-induced Ca2+ release by 25%. 3. Ryanodine (100 microM), also gave a large release of intracellular Ca2+. After pretreatment with 100 microM ryanodine, GTP gamma S did not induce Ca2+ release, and InsP3-induced Ca2+ release was reduced by 76%. 4. Caffeine (50 mM), produced a slow release of intracellular Ca2+. Pre-exposure to 50 mM caffeine had no effect on the GTP gamma S-induced Ca2+ release but reduced the InsP3 releasable Ca2+ by 58%. 5. Pretreatment with ryanodine abolished the caffeine-induced Ca2+ release, and addition of caffeine before ryanodine reduced the ryanodine-induced Ca2+ release by 64.4%. 6. These results suggest that there are at least three pools of Ca2+ present within airway smooth muscle cells. The largest pool is released by InsP3 or ryanodine, another is released either by a high concentration of InsP3 or on application of GTP gamma S, and the third by InsP3 alone. Ca2+ may be able to move from the GTP gamma S-sensitive pool into the InsP3- and ryanodine-sensitive pool when this becomes depleted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence against inhibition of sarcoplasmic reticulum Ca2+-pump as mechanism of H2O2-induced contraction of rat aorta.

AIM: To test whether inhibition of sarcoplasmic reticulum (SR) Ca2+-pump is involved in H2O2-induced contraction of endothelium-denuded rat aorta. METHODS: Isometric tension recording of H2O2 and cyclopiazonic acid (CPA)-induced contractions of rat aortic rings were compared in the absence or presence of various pharmacological tools to discriminate their signaling pathways involved. RESULTS: Both H2O2 and CPA contracted rat aortic rings, but with different contractile patterns. H2O2 triggered a fast and phasic contraction, whereas CPA elicited a slow and sustained contraction. In Ca2+-free medium, pretreatment of aortic rings with CPA 30 micromol/L but not with H2O2 30 micromol/L nearly abolished phenylephrine (10 micromol/L)-induced contraction. In addition, upon the maximal contraction induced by thapsigargin 30 micromol/L, H2O2 but not CPA further contracted aortic rings. On the other hand, H2O2 (30 micromol/L)- but not CPA (10 micromol/L)-induced contraction could be inhibited by suramin and RB-2 (each 100 micromol/L), two P2-purinoceptor antagonists. Furthermore, although pretreatment with 2-APB, a membrane permeable IP3 receptor blocker, inhibited both H2O2- and CPA-induced contractions, only H2O2 (30 micromol/L)-induced contraction could be depressed, to different degree, by various inhibitors of receptor-coupled or downstream signaling enzymes, including PLC, PKC, PLA2, COX, and protein tyrosine kinases. CONCLUSION: Inhibition of smooth muscle SR Ca2+-pump is unlikely the mechanism responsible for H2O2-induced contraction of endothelium-denuded rat aorta.     

Effect of osmotic stress on spontaneous calcium sparks in rat ventricular myocytes

AIM: To study whether the volume of cardiomyocytes and their functions would change under severe pathological conditions or osmotic stress. To clarify the role of ryanodine receptors/calcium release channels (RyRs) in the functional change, the effect of osmotic stress on spontaneous Ca2+ sparks in rat ventricular myocytes was investigated. METHODS: A laser scanning confocal microscope was used to detect spontaneous Ca2+ sparks of intact or saponin permeabilized myocytes loaded with Fluo-4. High and low tonicity was obtained by adding sucrose and reducing NaCl concentration in the external medium, respectively. RESULTS: In intact myocytes the frequency of Ca2+ sparks was increased and decreased by hyperosmotic (1.5 T) and hyposmotic (0.6 T) exposure, respectively. In addition, hyperosmotic exposure increased the temporal parameters and decreased the spatial parameter of Ca2+ sparks, while opposite changes occurred with hyposmotic exposure. The spatio-temporal properties of Ca2+ sparks were slightly affected by altering [K+]i (50-200 mmol/L) in saponin permeabilized myocytes in the presence of 8% dextran. It was observed that the spatio-temporal parameters of the Ca2+ sparks in permeabilized myocytes were dose-dependently altered by dextran. The propagating velocity of Ca2+ waves in intact and permeabilized myocyte was also affected by osmotic pressure or dextran. CONCLUSION: The effect of osmotic stress on the frequency of spontaneous Ca2+ sparks might be ascribed to the change of myoplasmic Ca2+ and Ca2+ content in the sarcoplasmic reticulum, while the effect on the spatio-temporal properties is caused by the alteration of Ca2+ diffusion mainly resulting from the morphological change of the myocytes.     

灯盏花素对谷氨酸诱导大鼠海马神经细胞毒性的保护作用

选用培养的海马神经细胞研究灯盏花素(breviscapine，Bre)对谷氨酸(glutamate，Glu)诱导神经细胞毒性的保护作用及其机制。新生大鼠海马神经细胞体外培养8 d后， 用L-谷氨酸(0.1， 0.5及1.0 mmol·L^-1)处理30 min； 灯盏花素处理组在加入L-谷氨酸的同时给予不同剂量的灯盏花素(10， 20及40 μmol·L^-1)； 继续正常培养24 h后， 用Annexin V联合流式细胞仪检测细胞的凋亡和坏死率； RT-PCR分析凋亡蛋白抑制剂XIAP mRNA的表达。结果表明， L-谷氨酸浓度依赖性地诱导神经细胞发生凋亡和坏死， 并使细胞XIAP mRNA表达发生浓度依赖性的双向变化： 0.1 mmol·L^-1 L-谷氨酸使神经细胞XIAP mRNA表达增强， 而较高浓度使XIAP mRNA表达下调。灯盏花素(20和40 μmol·L^-1)可有效抑制谷氨酸诱导的神经细胞死亡， 分别使细胞凋亡率下降30.4%和40.1%， 坏死率下降32.5%和38.8%； 并上调XIAP mRNA表达45.1%和54.9%。激光共聚焦显微技术联合Fluo-3荧光标记检测表明，L-谷氨酸处理过程中海马神经细胞内Ca²⁺水平显著升高， 而灯盏花素可抑制谷氨酸引起的细胞内Ca²⁺超载(P<0.01)。以上结果提示， 灯盏花素可能通过抑制细胞Ca²⁺超载， 调节凋亡抑制因子XIAP的表达而有效保护谷氨酸对神经细胞的兴奋性毒性作用。     

Synergistic actions of diacylglycerol and inositol 1,4,5 trisphosphate for Ca^2＋-dependent inactivation of TRPC7 channel

AIM: The aim of the present study was to explore the mechanism for the Ca2+- dependent inactivation of the canonical transient receptor potential (TRPC) 7 channel expressed in human embryonic kidney 293 cells. METHOD: The whole-cell patch-clamp technique was used in the study. RESULTS: With Ca2+-free external solution, the perfusion of 100 micromol/L carbachol to, or dialysis of the cell with 100 micromol/L guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), induced large inward currents, respectively. These currents were rapidly inhibited by the addition of 1 mmol/L Ca2+ into the bath, and recovery from this inhibition was only partial after the Ca2+ removal, unless vigorous intracellular Ca2+ buffering with 10 mmol/L 1,2 bis(2- aminophenoxy)ethane-N,N,No,No-tetraacetic acid (BAPTA) (plus 4 mmol/L Ca2+) was employed. In contrast, the current induced by a membrane-permeable analog of diacylglycerol (DAG), 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 micromol/L) did not undergo the inhibition persisting after Ca2+ removal. Interestingly, the inclusion of inositol 1,4,5 trisphosphate (IP3; 100 micromol/L) in the patch pipette rendered the OAG-induced current susceptible to the persistent Ca2+-mediated inhibition independent of the IP3 receptor in the majority of the tested cells, as evidenced by the inability of heparin and thapsigargin in reversing the effect of IP3. CONCLUSION: The present results suggest that Ca2+ entry via the activated TRPC7 channel plays a critical role in inactivating the channel where the cooperative actions of DAG and IP3 are essentially involved.     

Astragaloside IV inhibits spontaneous synaptic transmission and synchronized Ca^2＋ oscillations on hippocampal neurons

AIM: To investigate the changes in the spontaneous neuronal excitability induced by astragaloside IV (AGS-IV) in the cultured hippocampal network. METHODS: Hippocampal neurons in culture for 9-11 d were used for this study. The spontaneous synaptic activities of these hippocampal neurons were examined by Ca2+ imaging and whole-cell patch-clamp techniques. In total, 40 mg/L AGS-IV dissolved in DMSO and 2 mL/L DMSO were applied to the neurons under a microscope while the experiments were taking place. RESULTS: AGS-IV inhibited the frequencies of synchronized spontaneous Ca2+ oscillations to 59.39%+/- 3.25%(mean+/-SEM), the spontaneous postsynaptic currents to 43.78%+/- 7.72%(mean+/-SEM), and the spontaneous excitatory postsynaptic currents to 49.25%+/- 7.06%(mean+/-SEM) of those of the control periods, respectively, at 16 min after the AGSIV applications. AGS-IV also decreased the peak values of the voltage-gated K+ and Na+ channel currents at that time point. CONCLUSION: These results indicate that AGS-IV suppresses the spontaneous neuronal excitabilities effectively. Such a modulation of neuronal activity could represent new evidence for AGS-IV as a neuroprotector.     

Aortas isolated from sinoaortic-denervated rats exhibit rhythmic contractions that are regulated by pharmacologically distinct calcium sources

Sinoaortic denervation is characterized by arterial pressure lability, without sustained hypertension. Aortas isolated from rats with sinoaortic denervation present rhythmic contractions. We studied the participation of distinct Ca(2+) sources in the maintenance of the oscillations. Three days after the surgeries, aortic rings were placed in an organ chamber, and the incidence of aortas presenting rhythmic contractions was measured. Specific drugs were employed to analyse the participation of the Ca(2+) released from the sarcoplasmic reticulum [2-APB (diphenylborinic acid 2-aminoethyl ester), thapsigargin and ryanodine] and external Ca(2+) entry [Bay K 8644, verapamil and DMB (dimethylbenzyl amiloride)] on the rhythmic contractions. Additionally, we verified the effects of chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] on the maintenance of the rhythmic contractions. Under phenylephrine stimulus, sinoaortic-denervated rat aortas exhibited rhythmic contractions in the frequency of 4.5 +/- 0.50 cycles/min. and an amplitude of 0.465 +/- 0.05 g. 2-APB, thapsigargin and ryanodine inhibited the rhythmic contractions. Bay K 8644 increased the oscillations, reaching maximum values with a concentration of 50 nM (18.5 +/- 2.5 cycles/min.). The rhythmic contractions were inhibiting by verapamil and Ca(2+)-free solution. DMB and NPPB did not alter the oscillations. In conclusion, we observed that aorta isolated from sinoaortic-denervated rats present rhythmic contractions. Moreover, drugs that impaired intracellular Ca(2+) release from sarcoplasmic reticulum interrupted the oscillations. The oscillations also depend on the extracellular Ca(2+) entry through L-type Ca(2+).