Ca2+ -induced cell injury of cardiomyocyte

Pathogenesis of ischemia/reperfusion injury involves Ca(2+) -induced cell injury. Elevated intracellular Ca(2+) concentration at the reperfusion activates the Ca(2+) dependent protease, calpain and increases the generation of reactive oxygen species (ROS) in mitochondria, which cause cell injury in ischemia/reperfusion.     

Adverse bioenergetic consequences of Na+-Ca2+ exchanger-mediated Ca2+ influx in cardiac myocytes

BACKGROUND: In heart failure, the Na+-Ca2+ exchanger (NCX) is upregulated and mediates Ca2+ influx (instead of efflux) during the cardiac action potential. Although this partly compensates for impaired sarcoplasmic reticulum Ca2+ release and supports inotropy, the energetic consequences have never been considered. Because NCX-mediated Ca2+ influx is rather slow and mitochondrial Ca2+ uptake (which stimulates NADH production by the Krebs cycle) is thought to be facilitated by high Ca2+ gradients in a "mitochondrial Ca2+ microdomain," we speculated that NCX-mediated Ca2+ influx negatively affects the bioenergetic feedback response. Methods and Results- With the use of a patch-clamp-based approach in guinea-pig myocytes, cytosolic and mitochondrial Ca2+ ([Ca2+](c) and [Ca2+](m), respectively) was determined within the same cell after varying Ca2+ influx via L-type Ca2+ channels (I(Ca,L)) or the NCX. The efficiency of mitochondrial Ca2+ uptake, indexed by the slope of plotting [Ca2+](m) against [Ca2+](c) during each Ca2+ transient, was maximal during I(Ca,L)-triggered sarcoplasmic reticulum Ca2+ release. Depletion of sarcoplasmic reticulum Ca2+ load and increased contribution of the NCX to cytosolic Ca2+ influx independently reduced the efficiency of mitochondrial Ca2+ uptake. The upstroke velocity of cytosolic Ca2+ transients closely correlated with the efficiency of mitochondrial Ca2+ uptake. Despite comparable [Ca2+](c), sarcoplasmic reticulum Ca2+ release, but not NCX-mediated Ca2+ influx, led to stimulation of Ca2+-sensitive dehydrogenases of the Krebs cycle. Conclusions- Increased contribution of the NCX to cytosolic Ca2+ transients, which occurs in cardiac myocytes from failing hearts, impairs mitochondrial Ca2+ uptake and the bioenergetic feedback response. This mechanism could contribute to energy starvation of failing hearts.     

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.     

Molecular coupling of a Ca2+-activated K+ channel to L-type Ca2+ channels via alpha-actinin2

Cytoskeletal proteins are known to sculpt the structural architecture of cells. However, their role as bridges linking the functional crosstalk of different ion channels is unknown. Here, we demonstrate that a small conductance Ca(2+)-activated K(+) channels (SK2 channel), present in a variety of cells, where they integrate changes in intracellular Ca(2+) concentration [Ca(2+)(i)] with changes in K(+) conductance and membrane potential, associate with L-type Ca(2+) channels; Ca(v)1.3 and Ca(v)1.2 through a physical bridge, alpha-actinin2 in cardiac myocytes. SK2 channels do not physically interact with L-type Ca(2+) channels, instead, the 2 channels colocalize via their interaction with alpha-actinin2 cytoskeletal protein. The association of SK2 channel with alpha-actinin2 localizes the channel to the entry of external Ca(2+) source, which regulate the channel function. Furthermore, we demonstrated that the functions of SK2 channels in atrial myocytes are critically dependent on the normal expression of Ca(v)1.3 Ca(2+) channels. Null deletion of Ca(v)1.3 channel results in abnormal function of SK2 channel and prolongation of repolarization and atrial arrhythmias. Our study provides insight into the molecular mechanisms of the coupling of SK2 channel with voltage-gated Ca(2+) channel, and represents the first report linking the coupling of 2 different types of ion channels via cytoskeletal proteins.     

Ca2+ blinks: rapid nanoscopic store calcium signaling

Luminal Ca(2+) in the endoplasmic and sarcoplasmic reticulum (ER/SR) plays an important role in regulating vital biological processes, including store-operated capacitative Ca(2+) entry, Ca(2+)-induced Ca(2+) release, and ER/SR stress-mediated cell death. We report rapid and substantial decreases in luminal [Ca(2+)], called "Ca(2+) blinks," within nanometer-sized stores (the junctional cisternae of the SR) during elementary Ca(2+) release events in heart cells. Blinks mirror small local increases in cytoplasmic Ca(2+),orCa(2+) sparks, but changes of [Ca(2+)] in the connected free SR network were below detection. Store microanatomy suggests that diffusional strictures may account for this paradox. Surprisingly, the nadir of the store depletion trails the peak of the spark by about 10 ms, and the refilling of local store occurs with a rate constant of 35 s(-1), which is approximately 6-fold faster than the recovery of local Ca(2+) release after a spark. These data suggest that both local store depletion and some time-dependent inhibitory mechanism contribute to spark termination and refractoriness. Visualization of local store Ca(2+) signaling thus broadens our understanding of cardiac store Ca(2+) regulation and function and opens the possibility for local regulation of diverse store-dependent functions.     

Reoxygenation-induced Ca2+ rise is mediated via Ca2+ influx and Ca2+ release from the endoplasmic reticulum in cardiac endothelial cells

OBJECTIVE: Conditions of ischemia-reperfusion disturb the homoeostasis of cytosolic Ca2+ in cardiac microvascular endothelial cells (CMEC), leading to numerous malfunctions of the endothelium. Reperfusion specifically aggravates the Ca2+ overload developed during sustained ischemia. The aim of this study was to identify the origin of the reperfusion-induced part of the Ca2+ overload. Our hypotheses were that this is either due to a Na+-dependent process, e.g. involving the Na+/H+ exchanger (NHE) and/or the Na+/Ca2+ exchanger (NCX), or a process involving the endoplasmic reticulum (ER) and store-operated channels (SOC). METHODS AND RESULTS: Cultured CMEC from rats were exposed to conditions of simulated ischemia (hypoxia, pH 6.4) and reperfusion (reoxygenation, pH 7.4). Cytosolic Ca2+ ([Ca2+]i) and cytosolic Na+ ([Na+]i) concentrations and cytosolic pH (pHi) were measured with the use of fluorescent indicators. Removal of Ca2+ from the extracellular media during reoxygenation prevented the [Ca2+]i rise. Neither the activation of the NHE nor of the NCX in reoxygenated CMEC caused a change in this [Ca2+]i rise. Complete or partial removal of Na+ from the external media also had no effect on the [Ca2+]i rise. In contrast, specific inhibition of the inositol trisphosphate (InsP3) receptor by xestospongin C (3 micromol/l), of phospholipase (PLC) by U73122 (1 micromol/l), or of SOC by the inhibitors gadolinium (10 micromol/l) or 2-APB (50 micromol/l) lowered or abolished the reoxygenation-induced [Ca2+]i rise. CONCLUSION: In CMEC exposed to reperfusion conditions, the enhanced Ca2+ overload is due to Ca2+ influx. The influx is not mediated by a Na+-dependent mechanism, but rather is due to activation of the InsP3 receptor of the ER and activation of SOC.     

Alternative splicing of ryanodine receptors modulates cardiomyocyte Ca2+ signaling and susceptibility to apoptosis

Ca(2+) release via type 2 ryanodine receptors (RyR2) regulates cardiac function. Molecular cloning of human RyR2 identified 2 alternatively spliced variants, comprising 30- and 24-bp sequence insertions; yet their role in shaping cardiomyocyte Ca(2+) signaling and cell phenotype is unknown. We profiled the developmental regulation and the tissue and species specificity of these variants and showed that their recombinant expression in HL-1 cardiomyocytes profoundly modulated nuclear and cytoplasmic Ca(2+) release. All splice variants localized to the sarcoplasmic reticulum, perinuclear Golgi apparatus, and to finger-like invaginations of the nuclear envelope (nucleoplasmic reticulum). Strikingly, the 24-bp splice insertion that was present at low levels in embryonic and adult hearts was essential for targeting RyR2 to an intranuclear Golgi apparatus and promoted the intracellular segregation of this variant. The amplitude variability of nuclear and cytoplasmic Ca(2+) fluxes were reduced in nonstimulated cardiomyocytes expressing both 30- and 24-bp splice variants and were associated with lower basal levels of apoptosis. Expression of RyR2 containing the 24-bp insertion also suppressed intracellular Ca(2+) fluxes following prolonged caffeine exposure (1 mmol/L, 16 hours) that protected cells from apoptosis. The antiapoptotic effects of this variant were linked to increased levels of Bcl-2 phosphorylation. In contrast, RyR2 containing the 30-bp insertion, which was abundant in human embryonic heart but was decreased during cardiac development, did not protect cardiomyocytes from caffeine-evoked apoptosis. Thus, we provide the first evidence that RyR2 splice variants exquisitely modulate intracellular Ca(2+) signaling and are key determinants of cardiomyocyte apoptotic susceptibility.     

Increased cardiomyocyte function and Ca2+ transients in mice during early congestive heart failure

End-stage heart failure is believed to involve depressed cardiomyocyte contractility and Ca2+ transients. However, the time course of these alterations is poorly understood. We examined alterations in myocyte excitation-contraction coupling in a mouse model of early congestive heart failure (CHF) following myocardial infarction. One week after myocardial infarction was induced by ligation of the left coronary artery, CHF mice were selected based on established criteria (increased left atrial diameter, increased lung weight). Sham-operated animals (SHAM) served as controls. Echocardiographic measurements showed decreased global function in early CHF relative to SHAM, but increased local function in viable regions of the myocardium which deteriorated with time. Cardiomyocytes isolated from the non-infarcted septum also exhibited larger contractions in early CHF than SHAM (CHF=219.6+/-15.3% of SHAM values, P<0.05; 1 Hz field stimulation), and relaxation was more rapid (time to 50% relaxation=82.9+/-5.5% of SHAM values, P<0.05). Ca2+ transients (fluo-4 AM) were larger and decayed more rapidly in CHF than SHAM during both field stimulation (1 Hz) and voltage-clamp steps. Sarcoplasmic reticulum (SR) Ca2+ content was increased. Western blots showed that while SR Ca2+ ATPase (SERCA) expression was unaltered in CHF, phospholamban (PLB) was downregulated (60+/-11% of SHAM values, P<0.05). Thus, an increased SERCA/PLB ratio in CHF may promote SR Ca2+ re-uptake. Additionally, peak L-type Ca2+ current and Na+/Ca2+ exchanger expression were increased in CHF, suggesting increased sarcolemmal Ca2+ flux. Thus, in early CHF, alterations in Ca2+ homeostasis improve cardiomyocyte contractility which may compensate for loss of function in the infarction area.     

Oleic acid glucose-independently stimulates glucagon secretion by increasing cytoplasmic Ca2+ via endoplasmic reticulum Ca2+ release and Ca2+ influx in the rat islet alpha-cells

The effect of long-chain free fatty acids on glucagon secretion from islet alpha-cells has been a controversial issue. This study examined direct effects of oleic acid (OA) on glucagon release from rat pancreatic islets and on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in single alpha-cells by fura-2 fluorescence imaging. OA at 30 microM increased glucagon release from isolated islets in the presence of low (2.8 mM) and elevated (8.3 mM) glucose concentrations. OA at 6-10 microm concentration-dependently increased [Ca(2+)](i) in alpha-cells, irrespective of glucose concentrations (1.4, 2.8, and 8.3 mM). OA at 10 mum increased [Ca(2+)](i) in 90% of alpha-cells. OA-induced [Ca(2+)](i) increases were strongly inhibited by the endoplasmic reticulum Ca(2+)-pump inhibitors cyclopiazonic acid and thapsigargin and by 2-aminoethoxydiphenyl borate, the blocker of both inositol 1,4,5-trisphosphate receptors and store-operated Ca(2+) channels. Furthermore, the amplitude, but not incidence, of OA-induced [Ca(2+)](i) increases was reduced substantially by Ca(2+)-free conditions and mildly by an L-type Ca(2+) channel blocker, nitrendipine, and an ATP-sensitive K(+) channel activator, diazoxide. OA-induced glucagon release was also inhibited mildly by nitrendipine and strongly by 2-aminoethoxydiphenyl borate. These results indicate that OA glucose-independently stimulates glucagon release by increasing [Ca(2+)](i) in rat pancreatic alpha-cells and that the [Ca(2+)](i) increase is triggered by Ca(2+) release from endoplasmic reticulum and amplified by Ca(2+) influx possibly via store-operated channels and via voltage-dependent L-type Ca(2+) channels. The glucose-independent action of OA to stimulate glucagon release from alpha-cells may operate under hypoglycemic conditions when plasma free fatty acids levels are elevated, possibly playing a role in maintaining glucose metabolism.     

钙调神经磷酸酶信号通路与心肌细胞肥大

目的　探讨不同来源的细胞内Ca2 ([Ca2 ]i)在钙调神经磷酸酶 (CaN) 活化T细胞核因子 3 (NFAT3 )介导的心肌肥大中的作用。方法　分别用血管紧张素Ⅱ (AngⅡ )或雷尼丁刺激培养的大鼠心肌细胞外Ca2 跨膜内流或细胞内Ca2 释放 ,检测CaN、NFAT3、锌指转录因子 (GATA4)蛋白量、NFAT3定位以及氚 亮氨酸 (3H Leu)掺入量 ,环孢素A作为CaN特异抑制剂。结果　AngⅡ、雷尼丁刺激 1、3d ,心肌细胞CaN、NFAT3、GATA4蛋白表达及3H Leu掺入量较对照组明显增高(P值 <0 0 5或 <0 0 1)。AngⅡ和雷尼丁刺激第 1天 ,心肌细胞NFAT3表达由胞质转入胞核表达为主。环孢素A可抑制上述作用 ,与刺激组相比差异有显著性 (P <0 0 5或 <0 0 1)。结论　刺激心肌细胞Ca2 内流及Ca2 释放 ,均可激活CaN NFAT3信号通路。CaN NFAT3信号通路的激活与[Ca2 ]i增加有关 ,而与 [Ca2 ]i的来源无关。环孢素A能够抑制AngⅡ和雷尼丁介导的CaN NFAT 3 GATA 4表达的增加和蛋白质合成     

Role of the reverse mode of the Na+/Ca2+ exchanger in reoxygenation-induced cardiomyocyte injury

OBJECTIVE: We have recently shown that spontaneous Ca2+ oscillations elicit irreversible hypercontracture of cardiomyocytes during reoxygenation. The aim of this study was to investigate whether influx of exterior Ca2+ through the reverse mode of the Na+/Ca2+ exchanger (NCE) contributes to the development of these oscillations and, therefore, to reoxygenation-induced hypercontracture. METHODS: Isolated cardiomyocytes and hearts from rats were used as models. Cardiomyocytes were exposed to 60 min simulated ischemia (pH(o) 6.4) and 10 min reoxygenation (pH(o) 7.4). During reoxygenation cardiomyocytes were superfused with medium containing 1 mmol/l Ca2+ (control), with nominally Ca2+-free medium or with medium containing 10 micromol/l KB-R 7943 (KB), a selective inhibitor of the reverse mode of the NCE. RESULTS: In reoxygenated cardiomyocytes rapid Ca2+ oscillations occurred which were reduced under Ca2+-free conditions or in presence of KB. Hypercontracture was also significantly reduced under Ca2+-free conditions or in presence of KB. After 30 min of normoxic perfusion isolated rat hearts were subjected to 60 min global ischemia and reperfusion. KB (10 micromol/l) was present during the first 10 min of reperfusion. LVEDP, LVdevP and lactate dehydrogenase (LDH) release were measured. Presence of KB reduced post-ischemic LVEDP and improved left ventricular function (LVdevP). In KB treated hearts the reperfusion induced release of LDH was markedly reduced from 81.1 +/- 9.9 (control) to 49.3 +/- 8.8 U/60 min/g dry weight. CONCLUSION: Our study shows that inhibition of the reverse mode of the NCE, during reperfusion only, protects cardiomyocytes and whole hearts against reperfusion injury.     

Heterogeneous responses of cell Ca2+ in human airway epithelium.

The Ca(2+)-mobilizing actions of adenosine 5'-triphosphate (ATP), bradykinin, and histamine were compared in phenotypically distinct human nasal epithelial (HNE) cell types and as a function of time in cell culture. Single-cell measurements of intracellular free Ca2+ (Ca2+i, Fura-2 fluorescence) were recorded in ciliated cells 1-2 days in primary culture, and in nonciliated cells 1-2 days (keratin 14-positive) or 4-5 days (keratin 18-positive) after seeding. No difference in basal Ca2+i was noted between ciliated and nonciliated cell preparations. For ciliated and nonciliated cells studied 1-2 days in culture, ATP, bradykinin, and histamine elicited a cytosolic Ca2+ response in 100% of the cells examined. For nonciliated HNE cells maintained 4-5 days in culture, ATP (10(-4) M) increased cytosolic Ca2+ in all cells tested, but only 85% of the cells responded to bradykinin (10(-5) M) addition, and 65% to histamine (10(-4) M) stimulation. In terms of the absolute change of Ca2+i (delta Ca2+i, peak-basal value), the efficacy was ATP > bradykinin > histamine for the 3 HNE cell preparations. However, the delta Ca2+i in response to agonists was smaller in nonciliated HNE cells studied 1-2 days or 4-5 days in culture as compared to the ciliated cell preparation. Thapsigargin (300 nM), an agent that mobilizes Ca2+i, was equally effective in raising cytosolic Ca2+ in nonciliated (1-2 days and 4-5 days in culture) and ciliated HNE cells. These data show that ciliated cells consistently respond to all agonists, whereas the cytosolic Ca2+ response to ATP, bradykinin, and histamine in nonciliated cells was quantitatively reduced at a comparable time period (1-2 days) and became smaller and less frequent in nonciliated cell preparations maintained 4-5 days in culture. These results demonstrate time-dependent differences in the magnitude and frequency of cytosolic Ca2+ responses to certain agonists, strongly indicating that measurements of Ca2+i in HNE cells must account for the heterogeneity of the cell types and the time cells are maintained in primary culture.     

Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.     

Mitochondrial Ca2+ flux and respiratory enzyme activity decline are early events in cardiomyocyte response to H2O2

Oxidative stress is involved in mitochondrial apoptosis, and plays a critical role in ischemic heart disease and cardiac failure. Exposure of cardiomyocytes to H₂O₂ leads to oxidative stress and mitochondrial dysfunction. In this study, we investigated the temporal order of mitochondrial-related events in the neonatal rat cardiomyocyte response to H₂O₂ treatment. At times ranging from 10 to 90 min after H₂O₂ treatment, levels were determined for respiratory complexes I, II, IV and V, and citrate synthase activities, mitochondrial Ca²⁺ flux, intracellular oxidation, mitochondrial membrane potential and apoptotic progression. Complexes II and IV activity levels were significantly reduced within 20 min of H₂O₂ exposure while complexes I and V, and citrate synthase were unaffected. Mitochondrial membrane potential declined after 20 and 60 min of H₂O₂ exposure while intracellular oxidation, declining complex I activity and apoptotic progression were detectable only after 60 min. Measurement of mitochondrial Ca²⁺ ([Ca²⁺]_m) using rhodamine 2 detected an early accumulation of [Ca²⁺]_m occurring between 5 and 10 min. Pretreatment of cardiomyocytes with either ruthenium red or cyclosporin A abrogated the H₂O₂-induced decline in complexes II and IV activities, indicating that [Ca²⁺]_m flux and onset of mitochondrial permeability transition pore opening likely precede the observed early enzymatic decline. Our findings suggest that [Ca²⁺]_m flux represents an early pivotal event in H₂O₂-induced cardiomyocyte damage, preceding and presumably leading to reduced mitochondrial respiratory activity levels followed by accumulation of intracellular oxidation, mitochondrial membrane depolarization and apoptotic progression concomitant with declining complex I activity.     

Epinephrine enhances Ca2+ current-regulated Ca2+ release and Ca2+ reuptake in rat ventricular myocytes.

The voltage dependence of the intracellular Ca2+ transients was measured in single rat ventricular myocytes with the fluorescent Ca2+ indicator dye fura-2. The whole-cell voltage clamp technique was used to measure the membrane current, and 0.9 mM fura-2 was loaded into the cell by including it in the dialyzing solution of the patch electrode. A mechanical light chopper operating at 1200 Hz was used to obtain simultaneous measurements of the intracellular Ca2+ activity with fluorescence excitation on either side of the isosbestic point (330 nm and 410 nm). The symmetry of the two optical Ca2+ signals was used as a criterion to guard against artifacts resulting, for instance, from motion. The voltage dependence of peak Ca2+ current and the Ca2+ transient measured 25 ms after depolarizing clamps from a holding potential of -40 mV were bell-shaped and virtually identical. The Ca2+ entry estimated from the integral of the Ca2+ current (0 mV, 25 ms) corresponds to a 5-10 microM increase in the total intracellular Ca2+ concentration, whereas the optical signal indicated a 100 microM increase in total intracellular Ca2+. Repolarization of clamp pulses from highly positive potentials were accompanied by a second Ca2+ transient, the magnitude of which, when summed with that measured during depolarization, was nearly constant. Ryanodine (10 microM) had little or no effect on the peak Ca2+ current but reduced the magnitude of the early Ca2+ transients by 70-90%. Epinephrine (1 microM) increased the Ca2+ current and the Ca2+ transients, accelerated the rate of decline of the Ca2+ transients at potentials between -30 and +70 mV, and reduced the intracellular [Ca2+] below baseline at potentials positive to +80 or negative to -40 mV, where clamp pulses did not elicit any Ca2+ release. Elevation of intracellular cAMP mimicked the relaxant effect of epinephrine at depolarizing potentials, whereas elevation of extracellular [Ca2+] did not. These results suggest that most of the activator Ca2+ in rat ventricular cells is released from the sarcoplasmic reticulum as a graded response to sarcolemmal Ca2+ influx. Consistent with a graded Ca2+-induced Ca2+ release we find that epinephrine increases the internal Ca2+ release by increasing the Ca2+ current. Epinephrine may also increase the Ca2+ content of the sarcoplasmic reticulum that may, in turn, increase the Ca2+-induced Ca2+ release. The relaxant effect of epinephrine appears to be caused by enhanced rate of Ca2+ resequestration and is mediated by adenylate cyclase system.     

Bergmann glia modulate cerebellar Purkinje cell bistability via Ca2+-dependent K+ uptake

Recent studies have shown that cerebellar Bergmann glia display coordinated Ca(2+) transients in live mice. However, the functional significance of Bergmann glial Ca(2+) signaling remains poorly understood. Using transgenic mice that allow selective stimulation of glial cells, we report here that cytosolic Ca(2+) regulates uptake of K(+) by Bergmann glia, thus providing a powerful mechanism for control of Purkinje cell-membrane potential. The decline in extracellular K(+) evoked by agonist-induced Ca(2+) in Bergmann glia transiently increased spike activity of Purkinje cells in cerebellar slices as well as in live anesthetized mice. Thus, Bergmann glia play a previously unappreciated role in controlling the membrane potential and thereby the activity of adjacent Purkinje cells.     

醛固酮对介导大鼠肝星状细胞收缩的非钙离子依赖信号通路的影响

目的 探讨醛固酮(Aldo)对介导大鼠肝星状细胞(HSC)收缩的非Ca2+依赖性信号传导通路的影响.方法 对HSC-T6细胞给予Aldo 10 μmol/L处理,用聚硅酮膜法检测HSC-T6细胞的收缩性;在激光共聚焦显微镜下动态观察HSC-T6细胞胞内游离钙离子浓度的变化.RT-PCR检测Aldo受体阻断剂安体舒通、蛋白激酶C特异性抑制剂Stauro、Rho激酶特异性抑制剂Y27632、肌球蛋白轻链激酶特异性抑制剂ML-7对Rho-Rock通路中Rock2、RhoAGTP、RhoGEFmRNA表达水平的影响.对各组间数据行单因素方差分析,两两比较采用LSD法.结果 Aldo可诱导HSC-T6细胞收缩;Aldo对HSC-T6细胞胞内游离钙离子浓度变化无明显影响.Aldo可诱导Rock2、RhoAGTP、RhoGEF mRNA的表达增强(0.770±0.049、0.960±0.096、0.180±0.006,P值均<0.01),而其阻断剂安体舒通可明显抑制这三种元件mRNA的表达(0.440±0.166、0.370±0.180、0.050±0.001,P值均<0.01).Aldo+Y27632组上述三种元件mRNA的表达较Aldo组减弱.Aldo+ML-7+Stauro组三种元件的mRNA表达水平(0.940±0.066、1.330±0.192、0.160±0.007)较对照组(0.140±0.023、0.540±0.111、0.110±0.012)增强(P<0.05),Aldo+Y27632+ML-7+Stauro组RhoGEF(0.290±0.004,P<0.01)的表达较ML-7+Stauro两者联合抑制组(0.160±0.007)增强.结论 Aldo可诱导HSC的收缩,这与Rho激酶介导的非Ca2+依赖性信号通路相关.

Abstract：

Objective To investigate the mechanisms of Aldosterone stimulating hepatic stellate cells(HSCs) contraction via Ca2+-independent pathways. Methods HSC-T6 cell line was pre-disposed with Aldo 10 μmol/L. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The concentration variation of intracellular free calcium in rat HSC was observed by laser confocal microscopy.Besides, HSC-T6 cell line was under pre-disposal treatment with the blocking agents of Aldo receptor -antisterone, protein kinase C (PKC) special blocking agent-Stauro, Rho kinase blocking agent-Y27632 and MLCK special blocking agent-ML-7 respectively prior to stimulation with aldosterone. RT-PCR was used to detect the expression of Rock2, RhoAGTP and RhoGEF in Ca2+- independent pathways mediated by Rho-kinase. Results Aldo could induce HSCs contraction. The concentration of intracellular free calcium in rat HSCs had no change after pre-disposal treatment with Aldo. The mRNA expressions of Rock2, RhoAGTP and RhoGEF increased significantly after treatment with Aldo (0.770 ± 0.049, 0.960 ± 0.096, 0.180 ±0.006, P < 0.01).When inhibited with antisterone, the mRNA expressions of the three elements were (0.440 ± 0.166, 0.370 ± 0.180 and 0.050 ± 0.001, P < 0.01), lower than that of Aldo group, but higher in ML-7+Stauro + Aldo groups (0.940 ± 0.066, 1.330 ±0.192 and 0.160 ± 0.007, P < 0.05) as compared to the control group (0.140 ± 0.023,0.540 ± 0.111 and 0.110 ± 0.012). In the Y27632 + ML-7 + Stauro+Aldo group, the mRNA expression of RhoGEF (0.290 ± 0.004, P < 0.01)was higher than that of the ML-7 + Stauro + Aldo group (0.160 ± 0.007). Conclusion Aldo could induce HSCs contraction via Ca2+-independent pathways and Rho-Rock pathway involved in the process.