Mast cell activation involves plasma membrane potential- and thapsigargin-sensitive intracellular calcium pools

Summary— The regulation and role of the intracellular Ca²⁺ pools were studied in rat peritoneal mast cells. Cytosolic free calcium concentration ([Ca²⁺]i) was monitored in fura-2 loaded mast cells. In the presence of Ca²⁺ and K+, compound 48/80 induced a biphasic increase in [Ca²⁺]i composed of a fast transient phase and an apparent sustained phase. The sustained phase was partially inhibited by the addition of Mn²⁺. DTPA, a cell-impermeant chelator of Mn²⁺, reversed this inhibition, suggesting that a quenching of fura-2 fluorescence occurs in the extracellular medium. In the absence of extracellular Ca²⁺, the transient phase, but not the sustained one, could be preserved, provided that mast cells were depolarized. The transient phase was completely abolished by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor. Maximum histamine release induced by either compound 48/80 or antigen was obtained in the absence of added Ca²⁺ only when mast cells were depolarized. These histamine releases were inhibited by low doses (< 30 nM) of thapsigargin. Thapsigargin at higher doses induced histamine release which was unaffected by changing the plasma membrane potential, but was completely dependent on extracellular Ca²⁺, showing that a Ca²⁺ influx is required for thapsigargin-induced exocytosis. Together, these results suggest that the mobilization of Ca²⁺ from thapsigargin sensitive-intracellular pools induced by compound 48/80 or antigen is sufficient to trigger histamine release. The modulation of these pools by the plasma membrane potential suggest their localization is close to the plasma membrane.     

反义ClC-3寡核苷酸促进thapsigargin诱导的PC12细胞凋亡

目的　研究反义ClC 3寡核苷酸对thapsigargin诱导的细胞凋亡的影响。方法　蛋白免疫印迹法检测ClC 3蛋白的表达 ;MTT法检测反义ClC 3寡核苷酸对TG诱导的细胞生长的影响 ;形态学方法、流式细胞仪和DNA琼脂糖凝胶电泳观察和分析PC12细胞在转染ClC 3反义寡核苷酸后 ,TG诱导的细胞形态、DNA含量的变化和DNA断裂的情况。结果　与对照组相比 ,反义ClC 3寡核苷酸呈浓度和时间依赖性地抑制ClC 3蛋白的表达 ;使TG诱导的PC12细胞存活率降低 ,凋亡程度加强 ,差异有显著性 ,而正义及随义ClC 3寡核苷酸对此没有影响。结论　反义ClC 3寡核苷酸对TG诱导的PC12细胞凋亡有促进作用。     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

Melatonin enhances thapsigargin-induced apoptosis through reactive oxygen species-mediated upregulation of CCAAT-enhancer-binding protein homologous protein in human renal cancer cells

Melatonin (N-acetyl-5-methoxytryptamine) has differentiated the effects on apoptosis in normal and cancer cells. The mechanisms that account for the opposite effects on these cells are not adequately understood. In this study, we investigated the combined effect of melatonin and thapsigargin (TG) on apoptosis of renal cancer cells. Cotreatment with melatonin (1mm) and TG (50nm) induced approximately 10-fold expression levels of CCAAT-enhancer-binding proteins homologous protein (CHOP) compared with that of TG (50nm) alone. Downregulation of CHOP expression using small interfering RNAs markedly attenuated melatonin plus TG-mediated apoptosis. In addition, cotreatment with TG- and melatonin-induced CHOP upregulation likely relates to melatonin's antioxidant capacity because we proved that this CHOP upregulation is melatonin receptor independent. Our results collectively demonstrate that the upregulation of CHOP contributes to the enhancing effect of melatonin plus TG on apoptosis in cancer cells.     

Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus,Respiratory Syncytial Virus and Influenza A Virus

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca²⁺ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.     

毒胡萝卜素诱导大鼠皮层神经元内质网应激及丹红注射液的干预作用

目的：探讨毒胡萝卜紊诱导大鼠皮层神经元内质网应激凋亡的机制及丹红注射液的干预作用。方法：体外培养SD乳鼠皮层神经元，免疫组织化学、免疫荧光染色鉴定神经元纯度。流式细胞术Annexin V、PI双标检测凋亡率及活性caspase-3、caspase-8、caspase-7、caspase-9表达，Western Noting免疫印迹分析caspase-12、GRP78、Bcl-2、细胞色素C蛋白表达，Fura-2／AM法荧光分光光度计检测细胞内钙浓度（[Ca^2＋]i）。结果：SD乳鼠皮层神经元可纯化体外培养。2μmol／L毒胡萝卜素作用神经元24、48h细胞凋亡率分别是17.88％、21.38％，丹红治疗组分别是6．30％、6．11％，两组比较差异有统计学意义（P〈0．05）。毒胡萝卜素诱导神经元GRP78表达上调，剪切活化caspase-3、caspase-8、caspase-9、caspase-12，使细胞色素C表达增加，Bcl-2表达减少。丹红注射液促进细胞Bcl-2表达，抑制细胞色素C释放，减少活化的caspase-3、caspase-8、caspase-9含量，稳定游离钙浓度。结论：毒胡萝卜素诱导神经元内质网应激反应性凋亡。丹红注射液能抑制体外培养神经元内质网应激所致凋亡。     

Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets

Redox regulation is important for the modulation of cytosolic Ca(2+) concentration. Hence, we have investigated the effect of H(2)O(2) on store-mediated Ca(2+) entry (SMCE). In fura-2-loaded human platelets treatment with H(2)O(2) resulted in a concentration-dependent increase in Ca(2+) release from intracellular stores, while the effect on Ca(2+) entry was biphasic. In addition, 1mM H(2)O(2) reduced SMCE induced by agonists. The inhibitory effect of 1mM H(2)O(2) was prevented by inhibition of actin polymerization with cytochalasin D. Consistent with this, we found that 10microM H(2)O(2) and store depletion by treatment with thapsigargin plus ionomycin induced a similar temporal sequence of actin reorganization, while exposure to 1mM H(2)O(2) shifted the dynamics between polymerization and depolymerization in favor of the former. One millimolar H(2)O(2)-induced polymerization was reduced by treatment with methyl 2,5-dihydroxycinnamate and farnesylthioacetic acid, inhibitors of tyrosine kinases and Ras superfamily proteins, respectively. Finally, exposure to 1mM H(2)O(2) significantly increased store depletion-induced p60(src) activation. We conclude that H(2)O(2) exerted a biphasic effect on SMCE. The inhibitory role of high H(2)O(2) concentrations is mediated by an abnormal actin reorganization pattern involving both Ras- and tyrosine kinases-dependent pathways.     

Effects of Ca-ATPase inhibitors on the intracellular calcium activity and motility of human spermatozoa

Although evidence suggests that high intracellular calcium activity ([Ca2+]i) inhibits sperm motility, data concerning [Ca2+]i within, or slightly above, the physiological range are sparse, particularly in mammalian sperm. We investigated inhibitors of the sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) and the plasma membrane Ca-ATPase with the objective of increasing the intracellular calcium ion activity in human spermatozoa to study its effect on motility and other functions. Thapsigargin (20 micromol/L) increased [Ca2+]i from 140 +/- 7 nmol/L over an approximately 2-min period to reach a plateau of 530 +/- 84 nmol/L (mean +/- SEM, n = 3, p < 0.05). In sperm suspended in calcium-free medium thapsigargin increased [Ca2+]i from 13 +/- 3.3 to 35 +/- 7.5 nmol/L (p < 0.01), consistent with the release of calcium from intracellular stores. Cyclopiazonic acid (60 micromol/L) caused a transient decrease in [Ca2+]i. Quercetin, (200 micromol/L) caused a rapid increase in [Ca2+]i to 1280 +/- 90 nmol/L, after which [Ca2+]i fell quickly at first but then more slowly. Thapsigargin (20 micromol/L) caused approximately 70% of sperm to acrosome react in < or = 5 min, but once acrosome reacted, many sperm died over the next 30 min. Lower concentrations of thapsigargin caused fewer acrosome reactions but were less toxic. Both thapsigargin and quercetin caused rapid dose-dependent decreases in sperm motility. The results are consistent with high [Ca2+]i in the range observed in caput epididymal or cryopreserved spermatozoa inhibiting motility, but might be confounded by other events following the acrosome reaction.     

Thapsigargin对SLE患者T细胞TCR／CD3复合物介导的[Ca^2＋]i反应的影响

目的证实SLE患者T细胞功能异常是否与TCR/CD3复合物介导的[Ca2+]i反应异常有关,以及[Ca2+]i反应异常的原因.方法用CD3单抗与羊抗鼠二抗IgG相关联刺激T细胞,并用Thapsigargin干预后,分别用粘附细胞仪连续观察10 min T细胞[Ca2+]i的变化,并评价[Ca2+]i反应与CD3分子表达的相关性.结果正常人和SLE患者T细胞[Ca2+]i反应的基准值相似(P=0.105);SLE患者T细胞的[Ca2+]i反应高峰值、平台值明显高于正常对照(P＜0.001,P＜0.001);加入Thapsigargin后二者[Ca2+]i反应无显著差异,二者的T细胞CD3阳性率无差异(P=0.665).结论SLE患者T细胞TCR/CD3介导的[Ca2+]i反应存在异常,并且这种异常不是因为胞内Ca2+库排空后跨膜Ca2+内流不同所致.     

Nitric oxide selectively depletes macrophages in atherosclerotic plaques via induction of endoplasmic reticulum stress

BACKGROUND AND PURPOSE: Macrophages in atherosclerotic plaques have a tremendous impact on atherogenesis and plaque destabilization. We previously demonstrated that treatment of plaques in cholesterol-fed rabbits with the nitric oxide (NO) donor molsidomine preferentially eliminates macrophages, thereby favouring features of plaque stability. In this study, we investigated the underlying mechanism. EXPERIMENTAL APPROACH: Macrophages and smooth muscle cells (SMCs) were treated in vitro with the NO donors, spermine NONOate or S-nitroso-N-acetylpenicillamine (SNAP) as well as with the well-known endoplasmic reticulum (ER) stress inducers thapsigargin, tunicamycin, dithiothreitol or brefeldin A. Cell viability was analysed by Neutral Red viability assays. Cleavage of caspase-3, DNA fragmentation and ultrastructural changes were examined to characterize the type of macrophage death. Induction of ER stress was evaluated by measuring C/EBP homologous protein (CHOP) expression, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a), splicing of X-box binding protein 1 (XBP1) and inhibition of protein synthesis. KEY RESULTS: Macrophages and SMCs treated with spermine NONOate or SNAP showed several signs of ER stress, including upregulation of CHOP expression, hyperphosphorylation of eIF2 alpha, inhibition of de novo protein synthesis and splicing of XBP1 mRNA. These effects were similar in macrophages and SMCs, yet only macrophages underwent apoptosis. Plaques from molsidomine-treated atherosclerotic rabbits showed a 2.7-fold increase in CHOP expression as compared to placebo. Beside NO, selective induction of macrophage death could be initiated with thapsigargin and tunicamycin. CONCLUSIONS AND IMPLICATIONS: Induction of ER stress explains selective depletion of macrophages in atherosclerotic plaques by a NO donor, probably via inhibition of protein synthesis.