Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability

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钙释放激活钙通道调节分子1促进结肠癌细胞系SW480的增殖

目的 探讨钙释放激活钙通道调节分子1(ORAI1)对结肠癌细胞系SW480增殖的影响及其机制.方法 以ORAI1干扰慢病毒感染SW480细胞,分别用实时荧光定量PCR和Western blot检测细胞中ORAI1mRNA和蛋白的表达,通过MTT法、集落形成实验和倍增时间检测细胞的增殖,流式细胞仪检测其周期,激光共聚焦显微镜检测细胞钙内流(SOCE),Western blot法检测细胞中ERK1/2、p-ERK1/2和CyclinD1蛋白表达.结果 SW480转染ORAI1干扰慢病毒72 h后,可见明显的荧光表达;干扰组较空病毒组和对照组,ORAI1的表达降低(P＜0.01)、细胞生长减慢(P＜0.05)、集落形成能力减弱(P＜0.01)、倍增时间延长(P＜0.01)、G1期细胞比例增高(P＜0.05)、SOCE内流峰值降低(P＜0.05)、p-ERK1/2和CyclinD1的蛋白表达量降低(P＜0.01).结论 ORAI1可能通过SOCE调节胞内Ca2+浓度,并进一步通过MAPK信号通路促进SW480细胞的增殖.     

Aging does not affect calcium response to CCL2 and LPS in human monocytes

Monocytes play important roles in anti-microbial and anti-viral responses and chronic inflammatory diseases. Monocytes' functions are altered by aging. We investigated age-changes in calcium (Ca²⁺) response to CCL2 and LPS in human monocytes. CCL2 and LPS induced a slow increase of the cytosolic Ca²⁺ level, with a maximum response at ～360 s and ～300 s, respectively, in monocytes of young and older adults. No difference was observed in the magnitude and in the Ca²⁺ kinetic with both stimuli. Furthermore, store-operated Ca²⁺ entry and plasma membrane expression of ORAI1 showed no difference between both groups. In summary, monocytes from older adults maintained the capacity to mobilize calcium as their counterparts in young adults suggesting that the mechanisms underlying the dysfunctions in monocytes in aging might not involve alterations in Ca²⁺ flow through the plasma membrane.     

STIM1 but not STIM2 is an essential regulator of Ca influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells

Extracellular Ca²⁺ entry, primarily mediated through store-operated Ca²⁺ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca²⁺ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca²⁺ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca²⁺ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca²⁺ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca²⁺ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca²⁺ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development.     

Store-operated calcium entry modulates neuronal network activity in a model of chronic epilepsy

Store-operated Ca²⁺ entry (SOCE) over the plasma membrane is activated by depletion of intracellular Ca²⁺ stores and has only recently been shown to play a role in CNS processes like synaptic plasticity. However, the direct effect of SOCE on the excitability of neuronal networks in vitro and in vivo has never been determined. We confirmed the presence of SOCE and the expression of the calcium sensors STIM1 and STIM2, which convey information about the calcium load of the stores to channel proteins at the plasma membrane, in neurons and astrocytes. Inhibition of SOCE by pharmacological agents 2-APB and ML-9 reduced the steady-state neuronal Ca²⁺ concentration, reduced network activity, and increased synchrony of primary neuronal cultures grown on multi-electrode arrays, which prompted us to elucidate the relative expression of STIM proteins in conditions of pathologic excitability. Both proteins were increased in brains of chronic epileptic rodents and strongly expressed in hippocampal specimens from medial temporal lobe epilepsy patients. Pharmacologic inhibition of SOCE in chronic epileptic hippocampal slices suppressed interictal spikes and rhythmized epileptic burst activity. Our results indicate that SOCE modulates the activity of neuronal networks in vitro and in vivo and delineates SOCE as a potential drug target.     

Arachidonic acid and ion channels: an update

Arachidonic acid (AA), a polyunsaturated fatty acid with four double bonds, has multiple actions on living cells. Many of these effects are mediated by an action of AA or its metabolites on ion channels. During the last 10 years, new types of ion channels, transient receptor potential (TRP) channels, store-operated calcium entry (SOCE) channels and non-SOCE channels have been studied. This review summarizes our current knowledge about the effects of AA on TRP and non-SOCE channels as well as classical ion channels. It aims to distinguish between effects of AA itself and effects of AA metabolites. Lipid mediators are of clinical interest because some of them (for example, leukotrienes) play a role in various diseases, others (such as prostaglandins) are targets for pharmacological therapeutic intervention.     

Vasodilatory effects of ethanol extract of Radix Paeoniae Rubra and its mechanism of action in the rat aorta

Ethnopharmacological relevance

Radix Paeoniae Rubra (RPR) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China. RPR is the radix of either Paeonia lactiflora Pall. or Paeonia veitchii Lynch. RPR has a wide variety of pharmacological actions such as anti-thrombus, anti-coagulation, and anti-atherosclerotic properties, protecting heart and liver. However, the mechanisms involved are to be defined.

Aim of the study

The aim of the present study was to define the effect of Paeonia lactiflora Pall. extracts on vascular tension and responsible mechanisms in rat thoracic aortic rings.

Materials and methods

Ethanol extract of Paeonia lactiflora Pall. (EPL) was examined for their vascular relaxant effects in isolated phenylephrine-precontracted rat thoracic aorta.

Results

EPL induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Vascular relaxation induced by EPL was significantly inhibited by removal of the endothelium or pretreatment of the rings with N^G-nitro-l-arginine methylester (l-NAME) or 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ). Extracellular Ca²⁺ depletion or diltiazem significantly attenuated EPL-induced vasorelaxation. Modulators of the store-operated Ca²⁺ entry (SOCE), thapsigargin, 2-aminoethyl diphenylborinate and Gd³⁺, and an inhibitor of Akt, wortmannin, markedly attenuated the EPL-induced vasorelaxation. Further, the EPL-induced vasorelaxation was significantly attenuated by pretreatment with tetraethylammonium, a non-selective K_Ca channels blocker, or glibenclamide, an ATP-sensitive K⁺ channels inhibitor, respectively. Inhibition of cyclooxygenases with indomethacin, and adrenergic and muscarinic receptors blockade had no effects on the EPL-induced vasorelaxation.

Conclusions

The present study suggests that EPL relaxes vascular smooth muscle via endothelium-dependent and Akt- and SOCE-eNOS-cGMP-mediated pathways through activation of both K_Ca and K_ATP channels and inhibition of L-type Ca²⁺ channels.     

Biphasic effects of nitric oxide on calcium influx in human platelets

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca²⁺]_i) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca²⁺]_i which suggests that a small portion of the thrombin mediated increase in [Ca²⁺]_i was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca²⁺]_i by stimulating store-operated Ca²⁺ entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca²⁺]_i however it inhibited the ability of thrombin to increase [Ca²⁺]_i and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca²⁺]_i whereas higher concentrations (> 200 nM) of SNP inhibited thrombin induced increases in [Ca²⁺]_i. Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba²⁺ influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (> 200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba²⁺ influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.