GSK3beta mediates the induced expression of synaptic acetylcholinesterase during apoptosis

Besides its role in terminating acetylcholine-mediated neurotransmission, acetylcholinesterase (AChE) is found to be expressed and participate in the process of apoptosis in various cell types. However, the mechanisms underlying AChE up-regulation in neuronal cells remain elusive. Herein we demonstrated that glycogen synthase kinase-3β (GSK3β) mediates induced AChE-S expression during apoptosis. In this study, A23187 and thapsigargin (TG) were employed to induce apoptosis in neuroendocrine PC12 cells. The results showed that exposure of PC12 cells to A23187 and TG up-regulated AChE activity significantly. The same treatment also led to activation of GSK3β. Two different inhibitors of GSK3β (lithium and GSK3β-specific inhibitor VIII) could block A23187- or TG-induced up-regulation of AChE activity, AChE-S mRNA level and protein expression. However, lithium could not inhibit the induction of AChE-R mRNA and protein under similar conditions. Taken together, our results show that GSK3β is specifically involved in the induction of AChE-S expression in PC12 cells during apoptosis.     

大鼠结肠平滑肌细胞的钙库操纵性通道

目的:探讨大鼠结肠平滑肌细胞是否存在钙库操纵性通道(SOC)。方法:荧光探针Fura-2/AM标记细胞内游离Ca2+后,用荧光分光光度计检测毒胡萝卜素(thapsigargin)和咖啡因(caffeine)耗竭胞内钙库后激活的SOC通道对酶解分离的大鼠结肠平滑肌细胞[Ca2+]i的影响。结果:在无Ca2+缓冲液中,thapsigargin(1μmol/L)以及caf-feine(10 mmol/L)分别使[Ca2+]i由静息时(68.32±3.43)nmol/L升高至(240.85±12.65)nmol/L(、481.25±34.77)nmol/L,继之,向细胞外液中引入两种浓度的Ca2+(1.5 mmol/L和3.0 mmol/L),导致[Ca2+]i进一步升高,分别为(457.55±19.80)nmol/L、(1005.93±54.62)nmol/L;(643.88±34.65)nmol/L、(920.16±43.25)nmol/L。且上述升高效应对维拉帕米(verapamil,5μmol/L)以及KCl引起的细胞膜去极化不敏感,但可被La3+(1 mmol/L)抑制。结论:在酶解分离的大鼠结肠平滑肌细胞上,存在胞内钙库耗竭激活的SOC通道,为支持在电兴奋性细胞上存在库容性Ca2+内流提供了实验和理论依据。     

Intracellular Ca<Superscript>2+</Superscript> Pools and Fluxes in Cardiac Muscle-Derived H9c2 Cells

Relevant Ca²⁺ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca²⁺-mobilizing agents. Vasopressin produced a cytoplasmic Ca²⁺ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca²⁺ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca²⁺. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca²⁺ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca²⁺ pool was not. The vasopressin-induced cytoplasmic Ca²⁺ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca²⁺ pool, was sensed as a mitochondrial Ca²⁺ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca²⁺ peak was affected by cyclosporin A when the Ca²⁺ signal was induced by irreversible discharge of the intracellular Ca²⁺ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca²⁺ signals generated in the reticular store.     

An improved system for the <Emphasis Type="Italic">in vitro</Emphasis> regeneration of <Emphasis Type="Italic">Thapsia garganica</Emphasis> via direct organogenesis – influence of auxins and cytokinins

An improved protocol for mass multiplication directly from leaf material of Thapsia garganicawas developed. Using factorial experimentation, auxins (NAA, IAA, 2,4-D) and cytokinin (BA, kinetin) combinations at 0–2 mg l⁻¹ added to Murashige and Skoog (MS) medium with 30 g l⁻¹ sucrose and 8 g l⁻¹ agar (pH 5.8) were tested for their effect on direct regeneration on leaflet and petiole explants. Of the media tested, the 0.5:1.5 NAA:BA medium was comparable for direct shoot organogenesis to the 2 mg l⁻¹ kinetin supplemented medium. However, when shoots were multiplied on these media, the 2 mg l⁻¹ kinetin without auxins was most effective as it kept the percentage of callus-derived plantlets to 3% and the number of hyperhydric shoots were minimal at a frequency of 2% compared to 25% on the 0.5:1.5 NAA:BA medium. The 2 mg l⁻¹ kinetin medium induced adventitious bud formation in 36% of the explants after 30 days. When the cultures were transferred to the same medium for multiplication, an average of six shoots (4.3 cm) were derived from each shoot base. Other combinations resulted in callus formation that either preceded shoot production or occurred together with adventitious shoot induction; whereas the 2 mg l⁻¹ medium resulted solely in adventitious buds that readily converted and elongated to shoots. On the 0.5:1.5 NAA:BA medium which tended to induce hyperhydric shoots in culture, agar (0.8, [w/v]) (15% hyperhydric plantlets) was more useful in maintaining a high health status in regenerating plantlets than gellan gum (Gelrite^®; 0.25, [w/v]) (60% hyperhydric plantlets). Although rooting in vitro was difficult, 58% of the propagules were successfully acclimatized when plants were exposed to fungicidal solutions as pre- and post-acclimatization treatments. A comprehensive protocol that allows for a reduction in mortality due to damping-off diseases during ex vitro transplantation of the in vitro-derived T. garganica plantlets is reported. The acclimatization procedure presented here is potentially suited to other umbelliferous species where fungal rots hamper ex vitro establishment.     

Release of intracellular calcium stores leads to retraction of membrane sheets and cell death in mature mouse oligodendrocytes

The ability of mature oligodendrocytes (OLs) to recover from insult is important in repair of damage following demyelination. Since regulation of Ca²⁺ levels within cells plays a critical role in function and survival, this study investigates the effects of changes in cytoplasmic Ca²⁺ on the viability of cultured mouse OLs and their ability to maintain membrane sheets. Mature OLs in culture respond rapidly to the calcium ionophore A23187 and promptly return to resting Ca²⁺ levels when the ionophore is removed. Longer exposure to 0.1–1.0 μM A23187 leads to microtubule disruption, membrane sheet retraction and eventual cell death; nuclear lysis occurs in many of the OLs, as reported by Scolding, et al. (1) for rat OLs. In our cultures, mature OLs were more susceptible to nuclear lysis than were immature OLs or astroglia. Release of intracellular Ca²⁺ stores with thapsigargin at 5–10 μM also leads to retraction of membrane sheets. Following 6 hours of continuous exposure to thapsigargin, the effects on membrane sheets are reversed over the next 12 hours. After 18 hours of continuous exposure to thapsigargin, only occasional nuclear lysis is observed, but a number of the mature OLs show signs of DNA fragmentation, indicating that apoptotic death is occurring. Our results suggest that mature OLs cannot survive a prolonged influx of extracellular calcium as readily as immature OLs and astroglia, but have mechanisms to withstand similar increases in cytoplasmic Ca²⁺ following sustained release of intracellular stores. Special issue dedicated to Dr. Marion E. Smith.     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

Structure of histamine releasing guaianolides from Thapsia species

The structures of six hexaoxygenated guaianolides esterified with four non isomeric carboxylic acids have been established by ¹H NMR and ¹³C NMR spectroscopy. The acyl residues have been located by chemical ionisation mass spectrometry. Some of the acyl residues are novel for sesquiterpene lactones. The acyl residues in two additional guaianolides esterified with isomeric acids have been located by partial hydrolysis.     

The role of the sarcoplasmic reticulum in various types of cardiomyocytes

The relative importance of the sarcoplasmic reticulum (SR) as a source of Ca²⁺ in the excitation-contraction coupling of mammalian myocytes was tested. Shortening and intracellular Ca²⁺ transients of electrically paced, isolated,adult rat myocytes were found to be absolutely dependent on the presence of a functional SR and were completely abolished by the SR Ca²⁺-ATPase inhibitors cyclopiazonic acid and thapsigargin or by the Ca²⁺-release channel opener ryanodine.Neonatal rat cardiomyocytes, on the other hand, elicited consistent intracellular Ca²⁺-transients even after complete functional inhibition of the SR. The transients, however, were markedly prolonged. Also isolatedadult guinea pig myocytes maintained the ability to shorten after a complete inhibition of the SR Ca²⁺-ATPase by either thapsigargin or cyclopiazonic acid. The twitches and the intracellular Ca²⁺-transients, however, were considerably longer after inhibition of the SR Ca²⁺-ATPase. Different results were obtained after preincubation of the cells with 10 M ryanodine to induce emptying of the SR Ca²⁺ pool. In this case, Ca²⁺ spikes and twitches were also markedly reduced in size, in addition to being prolonged. When a SR Ca²⁺-pump inhibitor was added to ryanodine-treated cells, the size of the Ca²⁺-transients and the capacity of the cells to shorten increased. Ryanodine leaves the activity of the Ca²⁺-pump of the SR intact and thus leads to an underestimation of the amount of excitatory Ca²⁺-flowing into the cell.The results show that, while the significance of the SR in regulating the Ca²⁺-transients and shortening of cardiomyocytes varies depending on the species and the stage of development, SR function is of paramount importance for the occurrence of rapid twitches.Abbreviations EGTA ethylene glycol-bis-(beta amino ethyl ether)N,N,N,N tetraacetic acid - MOPS morpholinopropane sulfonic acid - SR sarcoplasmic reticulum - BSA bovine serum albumin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid     

EGF receptor—mediated intracellular calcium increase in human hepatoma BEL—7404 cells

Epidermal growth factor(EGF) induced intracellular free calcium ([Ca^2 ]i) response was studied in fura-2- or fluo-3-loaded human hepatoma cells of BEL-7404 cell line.Single cell[Ca^2 ]i analysis and [Ca^2 ]i measurement in cell populations revealed that EGF triggered a rapid[Ca^2 ]i increase in the dose-dependent and time-dependent manner.Pretreatment of cells with an endoplasmic reticulum(ER) Ca^2 -ATPase inhibitor,thapsigargin(TG) at 100nM concentration for 20 min,completely abolished EGF-induced [Ca^2 ]i increase,and chelating extracellular calcium by excess EGTA partially inhibited the increase.Furthermore,the expression of antisense EGF receptor sequence in BEL-7404 cells suppressed the [Ca^2 ]i response to EGF.The results suggest that EGF receptor-mediated [Ca^2 ]i increase in the human hepatoma cells is essentially dependent on the Ca^2 storage in ER.     

Rab7a modulates ER stress and ER morphology

The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called “ER matrices” together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22?N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells.