Glibenclamide induces apoptosis through inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and intracellular Ca(2+) release in HepG2 human hepatoblastoma cells.

Glibenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels, induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoblastoma cells. Glibenclamide increased intracellular Ca(2+) concentration, which was significantly inhibited by Ca(2+) release blockers dantrolene and TMB-8. BAPTA/AM, an intracellular Ca(2+) chelator, and the Ca(2+) release blockers significantly inhibited glibenclamide-induced apoptosis. Glibanclamide also increased intracellular Cl(-) concentration, which was significantly blocked by CFTR Cl(-) channel activators levamisole and bromotetramisole. These activators also significantly inhibited both intracellular Ca(2+) release and apoptosis induced by glibenclamide. The expression of CFTR protein in the cells was confirmed by Western blot analysis. These results suggest that glibenclamide induced apoptosis through inhibition of CFTR Cl(-) channels and intracellular Ca(2+) release and that this protein may be a good target for treatment of human hepatomas.     

Growth inhibition of capsaicin on HeLa cells is not mediated by intracellular calcium mobilization

The effects of capsaicin on cellular growth and intracellular calcium mobilization were examined in human cervical carcinoma derivation, HeLa cells. Capsaicin inhibited cellular growth and increased intracellular calcium level in HeLa cells. This capsaicin-induced intracellular calcium concentration rise was blocked by capsazepin, vanilloid (capsaicin) receptor antagonist. But, an intracellular calcium chelator BAPTA/AM did not block the inhibitory effect of capsaicin on cellular growth. These observations suggest that intracellular calcium mobilization is not required for the capsaicin-induced inhibition of cellular growth.     

Inhibition of mechanical strain-induced fetal rat lung cell proliferation by gadolinium,a stretch-activated channel blocker

Normal growth of the fetal lung is dependent upon fetal breathing movements. We have previously demonsrated that mechanical strain, simulating fetal breathing movements, stimulated DNA synthesis and cell division by reaggregated alveolar-like structures of fetal rat lung cells. Herein, we report that both intracellular and extracellular calcium modulate strain-induced proliferative activity. Strain-induced cell proliferation was inhibited by BAPTA/AM, an intracellular calcium chelator. The intracellular calcium modulators, cyclopiazonic acid and 2,5-di-(tert-butyl)-1, 4-benzohydroquinone, increased DNA synthesis of unstrained cultures and partially reduced strain-induced cell growth activity. A similar effect was noted with the calcium ionophore A23187. Extracellular Ca²⁺ increased DNA synthesis in unstrained cultures in a concentration-dependent fashion. The stimulatory effect of strain on DNA synthesis was also dependent on the calcium concentration in the medium. Furthermore, strain-enhanced DNA synthesis was inhibited by the presence of a divalent ion chelator, EGTA, in the medium. Mechanical strain increased ⁴⁵Ca²⁺ influx within 1 min after the onset strain. This rapid entry of calcium was not affected by calcium channel blockers, such as verapamil or Ni²⁺. Calcium channel blockers verapamil, nifedipine, Ni²⁺, Co²⁺, or La³⁺ also did not inhibit strain-induced cell growth activity. In contrast, gadolinium, a stretch-activated channel blocker, inhibited strain-induced ⁴⁵Ca²⁺ influx and suppressed strain-enhanced DNA synthesis. We conclude that the entry of calcium into cells through stretch-activated ion channels plays a critical role in strain-induced fetal lung cell proliferation. © 1994 Wiley-Liss, Inc.     

小鼠卵母细胞体外成熟不同阶段对钙的依赖性

To determine the role of calcium and calmodulin in mouse oocyte maturation, we examined the distribution of intracellular calcium during mouse oocyte maturation by using Mira Cal Imaging System. The calcium was present homogeneously in oocytes with intact germinal vesicle (GV) and accumulated around the nuclear region after GV breakdown(GVBD). The high level of calcium disappeared 6 hours later after GVBD. In the presence of 50 mumol/L BAPTA/AM, we failed to observe this phenomena. All eggs treated with 20 mumol/L W7, an antagonist of calmodulin, 50 mumol/L BAPTA/AM, a calcium chelator, could not develop to metaphase II (MII), although GVBD was not affected. We also detected the activity of a cytoplasmic maturation-promoting factor (MPF). W7 and BAPTA/AM had no effects on the rise of MPF activity in the course of maturation. We suggest that compartment distribution of calcium around nuclear region plays an important role in mouse oocyte maturation.     

Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: evidence of its dependence on an initial calcium mobilization from intracellular stores

It has been repeatedly shown that stimulation of a human leukemic T-cell line, JURKAT, by lectins such as phytohaemagglutinin and anti-T3 antibody (OKT3) leads to an elevation in the concentration of cytosolic free Ca2. This Ca2+ transient results from both an intracellular mobilization and an influx of Ca2+ through specific membrane channels. The objective of this study was to investigate the mechanism by which receptor-mediated influx of Ca2+ is regulated in JURKAT cells, which demonstrably lack 'voltage-dependent calcium channels'. It was found that upon increased loading with quin2 or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) there was a pronounced decline of both phytohaemagglutinin-stimulated and OKT3-stimulated influx of 45Ca2+. Using 15 microM quin2/AM or 30 microM BAPTA/AM, agonist-stimulated 45Ca2+ influx was almost totally abolished. At these concentrations of both quin2/AM or BAPTA/AM, phytohaemagglutinin and OKT3 could still induce a rise of cytosolic free Ca2+ above 200 nM. In the presence of La3+ (200 microM), which completely inhibited the agonist-induced 45Ca2+ influx, both phytohaemagglutinin and OKT3 were able to raise the concentrations of cytosolic free Ca2+ to well above 200 nM by merely mobilizing Ca2+ from intracellular stores alone. The data suggest that an agonist-induced increase in the concentration of cytosolic free Ca2+, due to mobilization from intracellular stores, could either directly or indirectly, initiate receptor-mediated Ca2+ influx across the plasma membrane in JURKAT cells.     

Expression of a rapid,low-voltage threshold K current in insulin-secreting cells is dependent on intracellular calcium buffering

Summary Depolarization-activated outward currents ranging in amplitude from 100–1000 pA were studied in cultured, insulinsecreting HIT cells and mouse B-cells using the whole-cell patch clamp. Outward current was identified as a K current since it was blocked by K channel blockers and its tail current reversed nearE _K. The K currents of HIT cells dialyzed with internal solutions containing 0.1–10mm EGTA with no added calcium (Ca), or 10mm EGTA with 2mm added Ca, activated rapidly with depolarization. However, the stronger Ca buffer BAPTA (5mm; no added Ca) blocked the rapidly activating current to reveal an underlying more slowly activating K current. With intracellular EGTA, application of the Ca channel blocker cadmium mimicked the effect of intracellular BAPTA. These data suggest that the rapid K current was mediated by low-voltage threshold, Ca-activated K channels while the slower K current was mediated by high threshold delayed rectifier K channels. Mouse B-cells also had both K current components. Dialyzing these cells with either BAPTA (5mm, no added Ca) or high EGTA (10mm with 2mm Ca) blocked the rapid Ca-activated K current observed when cells were filled with 0.1 to 1mm EGTA. It is concluded that the extent of Ca-activated K current activation in either HIT or adult mouse B-cells depends on the degree of intracellular Ca buffering.     

Protein kinase C and intracellular calcium are involved in follicle-stimulating hormone-mediated meiotic resumption of cumulus cell-enclosed porcine oocytes in hypoxanthine-supplemented medium

The present experiments were conducted to examine the hypothesis that follicle-stimulating hormone (FSH) can stimulate the hydrolysis of phosphoinositide, generating the intracellular second messengers to activate protein kinase C and mobilizing intracellular calcium, thus inducing oocyte meiotic resumption. Pig cumulus cell-enclosed oocytes (CEO) were cultured for 24 hr in 4 mM hypoxanthine (HX)-supplemented medium and treated with different agents in the following designs: (1) CEO were treated with neomycin (an inhibitor of phosphoinositide hydrolysis) in the presence of FSH or only treated with 7,12-dimethylbenzin(a) anthracene (DMBA, a tumor promoter which can cause phosphorylation of phospholipase C (PLC), formation of inositol triphophate, and mobilization of intracellular calcium) to mimic the direct activation of PLC; (2) CEO were challenged by FSH, together with sphingosine or staurosporine (two kinds of PKC inhibitors); or treated with phorbol myristate acetate (PMA, an activator of PKC) separately; (3) CEO were primed with BAPTA/AM (an intracellular calcium chelator) or BAPTA/AM +FSH for 60 min, and then transferred into a new culture medium supplemented with FSH but without BAPTA/AM; total culture time was 24 hr. At the end of the culture, the incidence of germinal vesicle breakdown (GVBD) was calculated. The results showed that: (1) FSH (100 U/liter) could stimulate pig CEO to override the arrest of HX and resume meiosis; DMBA (10(-8)-10(-5) M) itself also had such a kind of effect; whereas neomycin, at the level of 10-20 mM, could dramatically inhibit the stimulatory effect of FSH. (2) Staurosporine (10(-9)-10(-6) M) or sphingosine (10(-8)-10(-5) M) could also inhibit the effect of FSH in a dose-dependent manner on stimulating CEO to resume meiosis. However, PMA (10(-8)-10(-5) M) alone had a dual effect on the meiotic resumption of pig CEO. PMA, at the level of 10(-8)-10(-6) M, could stimulate CEO to resume meiosis, and at high concentration of 10(-5) M , it could even enhance the inhibitory effect of HX. (3) Priming CEO with BAPTA/AM only or BAPTA/AM +FSH for 60 min could significantly inhibit the effect of FSH in a dose-dependent manner. These results indicate that in the process of ligand-mediated meiotic resumption of pig CEO, FSH can stimulate the hydrolysis of phosphoinositide leading to the activation of PKC and mobilization of intracellular calcium; and suggest that multiple signaling pathways and signal interaction are involved in this process.     

Influence of calcium ion on host cell invasion and intracellular replication by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of Ca2+ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and Ca2+ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the Ca2+ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular Ca2+ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.     

Regulation of juvenile hormone biosynthesis in Heliothis virescens by Manduca sexta allatotropin

In Heliothis virescens, reproduction is strictly dependent on juvenile hormone (JH). In females, mating induces a sharp increase in JH titers, which stimulates increased vitellogenin biosynthesis and higher rates of egg production. JH biosynthesis is presumably stimulated by production and/or release of stimulatory neuropeptides such as allatotropins. There is evidence that allatotropin of H. virescens may be structurally related to Manduca sexta allatotropin (Manse-AT). In a radiochemical in vitro assay, synthetic Manse-AT stimulated JH biosynthesis by corpora allata (CA) of virgin H. virescens females in a dose-dependent manner, but had no effect on CA activity in H. virescens males. In females, the CA showed a transient increase in sensitivity to Manse-AT shortly after mating. Several structurally related peptides stimulated CA activity to a similar extent as Manse-AT. Corpora allata activity was stimulated by a Ca2+ ionophore, A23187. A membrane-permeable Ca2+ chelator, BAPTA/AM, antagonized the stimulatory effects of Manse-AT, suggesting that Manse-AT may enhance CA activity by increasing intracellular Ca2+ concentration.     

Deficiency of presenilin-1 increases calcium-dependent vulnerability of neurons to oxidative stress in vitro

We examined the function of presenilin-1 (PS1) on neuronal resistance to oxidative stress. CNS neurons cultured from PS1-deficient mice exhibited increased vulnerability to H2O2 treatment compared with those from wild-type mice. Antioxidants protected the cultured neurons against the oxidative stress. An intracellular calcium chelator, BAPTA AM, as well as an L-type voltage-dependent calcium channel blocker, nifedipine, rescued the neurons from H2O2-induced death, while an N-type voltage-dependent calcium channel blocker, omega-conotoxin, or calcium release blockers from ER stores, dantrolene and xestospongin C, failed to rescue them. Wild-type and PS1-deficient neurons showed comparable increases of cytoplasmic free calcium levels after exposure to H2O2. Taken together with the data that PS1-deficient neurons exhibited increased vulnerability to glutamate, these findings imply that PS1 confers resistance to oxidative stress on neurons in calcium-dependent manners.     

Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-alpha-mediated apoptosis

Tumour necrosis factor-alpha (TNF-alpha) was found to be a cell cycle-independent apoptogenic cytokine in cultured fibroblast L929 cells. This assertion is based on the observations (1) TNF-alpha increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytometry, and (2) TNF-alpha induced DNA fragmentation as resolved by agarose gel electrophoresis. When cells were exposed to TNF-alpha (50 ng/ml), a slow rise in intracellular free Ca2+ level and a delayed increase in the production of reactive oxygen species (ROS) (both observed 3 h after the addition of TNF-alpha) were observed in fluo-3 and fura-red or dichlorofluorescein loaded cells, respectively. Interestingly, challenge of cells with TNF-alpha in the presence of BAPTA/AM, an intracellular Ca2+ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidinooxy (4OH-TEMPO) blocked the TNF-alpha-mediated Ca2+ rise. Moreover, when cells were exposed to TNF-alpha with both 4OH-TEMPO and BAPTA/AM, more viable cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca2+ are two cross-talk messengers important in TNF-alpha-mediated apoptosis.     

Depletion of intracellular Ca2+ store itself may be a major factor in thapsigargin-induced ER stress and apoptosis in PC12 cells

The mechanisms of intracellular calcium store depletion and store-related Ca²⁺ dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca²⁺ by BAPTA/AM (50 μM), or La³⁺ (100 μM) enhanced while dantrolene (100 μM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca²⁺ stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca²⁺ concentrations ([Ca²⁺]_i).     

Changes in cytosolic calcium monitored by inward currents during action potentials in guinea-pig ventricular cells

Action potentials were recorded from single cells isolated from guinea-pig ventricular muscle. Contraction was measured with an optical technique. Tail currents thought to be activated by cytosolic calcium were recorded when action potentials were interrupted by application of a voltage-clamp. A family of tail currents was recorded by interrupting the action potential at various times after the upstroke. The envelope of tail current amplitudes was taken as an index of changes in cytosolic calcium. Consistent with this interpretation, tail currents were negligible following intracellular loading with the calcium chelator BAPTA to suppress calcium transients. The cytosolic calcium transient estimated from the envelope of tails reached a peak approximately 50 ms after the upstroke of the action potential, and fell close to diastolic levels before repolarization was complete; 10 mM caffeine delayed the time to peak contraction, and caused a prolongation of the cytosolic calcium transient estimated from the envelope of tail currents. Caffeine also induced the appearance of a distinct late plateau phase of the action potential. Intracellular BAPTA suppressed the late plateau, contraction and tail currents in cells exposed to caffeine. Exposure to caffeine increased the time constant for decay of tail currents (from approximately 25 to 70 ms). When action potentials were greatly abbreviated by interruption with a voltage-clamp, a progressive decline occurred in the subsequent three contractions and tail currents. There was a progressive reversal of these effects over four responses when the full action potential duration was restored. None of these effects was observed in cells exposed to caffeine. Calcium-activated tail currents appear to be a useful qualitative index of changes in cytosolic calcium. The observations are consistent with the suggestion that cytosolic calcium is reduced during the plateau by a combination of calcium extrusion through Na-Ca exchange and calcium uptake into caffeine-sensitive stores. It also appears that reduction of stores loading during abbreviated action potentials reduces subsequent contraction in cells not exposed to caffeine.     

Intracellular calcium levels in the Plasmodium berghei ookinete

Ookinetes are the motile and invasive stages of Plasmodium parasites in the mosquito host. Here we explore the role of intracellular Ca2+ in ookinete survival and motility as well as in the formation of oocysts in vitro in the rodent malaria parasite Plasmodium berghei. Treatment with the Ca2+ ionophore A23187 induced death of the parasite, an effect that could be prevented if the ookinetes were co-incubated with insect cells before incubation with the ionophore. Treatment with the intracellular calcium chelator BAPTA/AM resulted in increased formation of oocysts in vitro. Calcium imaging in the ookinete using fluorescent calcium indicators revealed that the purified ookinetes have an intracellular calcium concentration in the range of 100 nm. Intracellular calcium levels decreased substantially when the ookinetes were incubated with insect cells and their motility was concomitantly increased. Our results suggest a pleiotropic role for intracellular calcium in the ookinete.     

Cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in hela cells

Results of recent studies using BAPTA/AM have raised a serious question on whether Ca(2+) signal is truly involved in regulating the progression of apoptosis. To resolve this question, we examined the differential effects of three different Ca(2+) signaling blockers (BAPTA/AM, membrane-impermeant BAPTA, and heparin) on UV-induced apoptosis in HeLa cells. We found that although the membrane-permeable form of BAPTA (i.e., BAPTA/AM) could not inhibit cell death, the membrane-impermeant form of BAPTA, loaded into the cytosol by electroporation, clearly protected cells from entering apoptosis. Furthermore, when we injected heparin to block Ca(2+) release from the endoplasmic reticulum (ER) to cytosol, apoptosis was greatly suppressed. These findings strongly suggest that elevation of cytosolic Ca(2+) is part of the signal that drives the progression of apoptosis. The negative result of BAPTA/AM is probably due to its dual effect on subcellular Ca(2+) distribution; besides suppressing the Ca(2+) elevation in cytosol, BAPTA/AM can also enter into the ER to reduce the free Ca(2+) level there. The depletion of Ca(2+) in ER is believed to stimulate apoptosis and thus would counterbalance the protection effect of BAPTA/AM in suppressing the cytosolic Ca(2+) elevation.     

Dexamethasone-mediated inhibition of calcium transients and ACTH release in a pituitary cell line (AtT-20).

In the corticotroph-like murine pituitary tumor cell line, AtT-20, adrenocorticotropic hormone release is triggered by corticotropin-releasing hormone and is attenuated by the synthetic adrenal steroid dexamethasone. The precise mechanisms by which dexamethasone inhibits secretion are under investigation. We examined whether dexamethasone can modulate release via regulation of calcium homeostasis. More specifically, we have evaluated the effects of dexamethasone on calcium current, intracellular calcium concentration, and adrenocorticotropic hormone release. Using perforated patch-clamp and calcium imaging with fura PE3/AM, we found that dexamethasone decreases calcium current and intracellular calcium levels. The inhibition of current by dexamethasone is not, however, altered by the calcium channel antagonists nifedipine (L-type) or omega-agatoxin IVA (P/Q-type), despite the presence of these calcium channel subtypes in AtT-20 cells and the exclusive coupling of adrenocorticotropic hormone release to the L-type channel in these cells. We also evaluated the temporal relationship between dexamethasone-mediated inhibition of secretion and calcium influx. Whereas a prolonged (2 h) incubation with dexamethasone inhibits corticotropin-induced release by approximately 40%, a rapid (10 min) incubation (a time interval sufficient for dexamethasone-mediated inhibition of calcium transients) does not inhibit release. These data suggest, therefore, that dexamethasone does, indeed, modulate calcium homeostasis in AtT-20 cells, but that this effect is not responsible for its inhibition of secretion.     

Barium ions enter chromaffin cells via voltage-dependent calcium channels and induce secretion by a mechanism independent of calcium

Barium ions enter chromaffin cells via voltage-sensitive calcium channels, although the intracellular site of barium action is distinct from that of calcium. The entry of barium primarily through voltage-sensitive channels was indicated by experiments showing inhibition of 133Ba2+ uptake by blockers of voltage-dependent calcium channels. In addition, 133Ba2+ uptake was stimulated by 50 mM KCl but not by nicotine. Furthermore, 133Ba2+ uptake was inhibited by hyperosmolarity, which specifically blocks the voltage-sensitive calcium channel but not the receptor-associated calcium channel. These conclusions from studies on barium uptake were also borne out by experiments measuring catecholamine secretion. Thus, blockers of voltage-dependent calcium channels which inhibited barium uptake also inhibited barium-induced catecholamine secretion. In other experiments, simultaneous stimulation with nicotine and barium in the presence of calcium evoked coincident and additive catecholamine secretion. By contrast, when 50 mM KCl was substituted for nicotine in the same experimental design, barium ions inhibited potassium-induced catecholamine secretion at low calcium concentrations. Only at high calcium concentrations were barium-induced and potassium-induced secretion additive. These data also indicate that barium and calcium compete at the voltage-sensitive pathway. Furthermore, these additivity data suggest that once inside the cell, barium and calcium have two distinct mechanisms of action. As predicted by this hypothesis, in digitonin-permeabilized chromaffin cells either calcium or barium stimulated catecholamine release, and in the presence of both cations catecholamine secretion was equivalent to the sum of secretion with either cation alone. Additional support of this concept comes from experiments showing that while calcium-mediated catecholamine secretion is sensitive to trifluoperazine and imipramine, barium-mediated secretion is not. Taken together, all these data indicate that there are two distinct intracellular sites of action for barium and calcium. In contrast to catecholamine secretion, non-exocytotic ascorbic acid secretion was induced by nicotine and potassium in the presence of calcium, but not by barium alone. These data provide additional evidence that barium acts by a different mechanism than calcium, in still another secretory system in chromaffin cells.     

肺腺癌A549/DDP细胞周期变化及其多药耐药性

用Fura 2 /AM标记药物敏感的肺腺癌细胞A54 9和抗顺铂药物的肺腺癌细胞A54 9/DDP两种细胞胞内游离Ca2 ,用碘化丙锭 (PI)标记细胞DNA ,检测其胞内Ca2 的变化及两种细胞增殖能力和细胞周期 .实验结果表明 ,抗药性细胞株A54 9/DDP胞浆内游离Ca2 的浓度仅为药物敏感细胞株A54 9的 1/ 3左右 ,同时前者的细胞增殖能力较后者明显增强 ,而且细胞周期也明显缩短 .当用BAPTA AM和EGTA或A2 3187和Thapsigargin处理细胞以降低或升高其胞内自由Ca2 浓度时可改变细胞的生长周期 ,二者也呈现明显差别 .这些结果表明 ,对顺铂产生耐药性的人肺腺癌A54 9/DDP细胞胞内Ca2 浓度的降低 ,可能影响细胞的增殖 ,缩短细胞的生长周期 ,特别是影响起决定作用的G1期 ,从而有利于肿瘤细胞多药耐药特性的维持     

Salmonella typhimurium induces an inositol phosphate flux in infected epithelial cells

Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion. During invasion it causes significant rearrangement of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane. We found that intracellular inositol phosphate concentrations in HeLa cells increased during S. typhimurium entry and returned to normal levels after bacterial internalization. A chelator of intracellular calcium (BAPTA/AM) blocked S. typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion. These results indicate that S. typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake.     

NADPH diaphorase and nitric oxide synthase in the corpus cardiacum-corpus allatum of the cockroach Diploptera punctata

Juvenile hormone synthesis by corpora allata is regulated partly by allatostatin containing nerves from the brain that innervate the corpora cardiaca and the corpora allata. To investigate whether NO also participates in the regulation of juvenile hormone synthesis, antibody against NO synthase and the histochemical test for NADPH diaphorase activity, a marker for NO synthase, were applied to the corpora cardiaca-corpora allata of Diploptera punctata. Strong NADPH diaphorase activity occurred in corpus allatum cells but not in nerve fibers in the corpora allata or corpora cardiaca. In contrast, NO immunoreactivity occurred in nerves in the corpora cardiaca but not within the corpora allata. NO and allatostatin were not colocalized. NO synthase and NADPH diaphorase activity were localized in similar areas of the subesophageal ganglion and cells in the pars intercerebralis of the brain. Positive correlation of the quantity of NADPH diaphorase activity with juvenile hormone synthesis during the gonadotrophic cycle and lack of such correlation in subesophageal ganglia suggest that NADPH diaphorase activity reflects the necessity of NADPH in the pathway of juvenile hormone synthesis. These data suggest that NO is unlikely to play a significant role in the regulation of the corpora allata.