Alteration of expression of Ca2+ signaling proteins and adaptation of Ca2+ signaling in SERCA2+/- mouse parotid acini

PURPOSE: The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca2+ signaling. However, whether the changes in Ca2+ signaling and Ca2+ signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known. MATERIALS AND METHODS: In SERCA2+/- mouse parotid gland acinar cells, Ca2+ signaling, expression levels of Ca2+ signaling proteins, and amylase secretion were investigated. RESULTS: SERCA2+/- mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca2+ ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP3Rs), but the localization and activities of IP3Rs were not altered. In SERCA2+/- mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice. CONCLUSION: These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca2+ signaling proteins in the parotid gland acini, however, overall Ca2+ signaling is unchanged.     

Calcium entry via ORAI1 regulates glioblastoma cell proliferation and apoptosis

Introduction

Calcium entry plays a critical role in the proliferation and survival of certain tumors. Ca²⁺ release activated Ca²⁺ (CRAC) channels constitute one of the most important pathways for calcium entry especially that of store-operated calcium entry (SOCE). ORAI1 and stromal interaction molecule1 (STIM1) are essential protein components of CRAC channels. In this study we tested the effect of inhibiting CRAC through ORAI1 and STIM1 on glioblastoma multiforme (GBM) tumor cell proliferation and survival.

Methods

Two glioblastoma cell lines, C6 (rat) and U251 (human), were used in the study. ORAI1 and STIM1 expressions were examined using Western blot and immunohistochemistry. CRAC channel activity and its components were inhibited with ion channel blockers and using siRNA knockdown. Changes in intracellular calcium concentration were recorded using Fura-2 fluorescent calcium imaging. Cell proliferation and apoptosis were examined using MTS and TUNEL assays, respectively.

Results

CRAC blockers, such as SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole), 2-aminoethoxydiphenyl borate (2-APB) and Diethylstilbestrol (DES), inhibited cell proliferations and SOCE in GBM cells. Knockdown of ORAI1 and STIM1 proteins using siRNA significantly inhibited C6 cell proliferation and SOCE compared with those in control cells, and a more significant effect was observed in cells with ORAI1 siRNA knockdown than that of STIM1-treated cells. Both CRAC blockers and siRNA treatments increased apoptosis in C-6 cells compared with control.

Conclusion

Calcium entry via ORAI1 and CRAC channels are important for GBM proliferation and survival.     

Cycloastragenol mediates activation and proliferation suppression in concanavalin A-induced mouse lymphocyte pan-activation model

Context: Cycloastragenol (CAG) is a molecule isolated from various species in the genus Astragalus. Although the regulatory activity of Astragalus on immune system has been investigated, the effect of CAG on activated lymphocytes is poorly understood.

Objective: We aimed to biologically address the possible anti-inflammation potential of CAG on concanavalin A (Con A)-mediated mouse lymphocyte pan-activation model.

Materials and methods: Mouse lymphocytes were obtained from spleens and subjected to Con A for 24?h. Herein, the cells were treated with different concentrations of CAG. Cell viability was assayed by MTT. Pretreated by CAG and stimulated by Con A, the expression of CD69 and CD25, Th1/Th2/Th17 cytokines, cell cycle, proliferation and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were analyzed by flow cytometry.

Results: The results declared that CAG significantly downregulated both CD69 and CD25 expressed on Con A activated CD3?+?T cells’ surface, as well as inhibiting proliferation of activated lymphocytes. In addition, CAG blocked the Con A-induced mitogenesis, exhibiting lymphocyte G₀/G₁-phase cell-cycle arrest with significant reduction of cells in S and G₂/M phases. Meanwhile, pretreated by CAG, a significant decline in [Ca²⁺]_i was observed. Furthermore, CAG significantly inhibited the production of Th1 cytokines IFN-γ, TNF, IL-2, Th2 cytokines IL-4, IL-6, IL-10 and Th17 cytokine IL-17?A on Con A-activated lymphocytes.

Conclusion: Our results reinforce that CAG has important anti-inflammatory activity by inhibiting lymphocytes activation, proliferation and cytokines expression, and shows, that this effect may be related to reduction of overall intracellular Ca²⁺ overload.     

rAAV1-SERCA2a转染改善心力衰竭犬心功能及其机制探讨

 目的:研究重组1型腺相关病毒(rAAV1)介导的肌浆/内质网Ca2+-ATP酶2a（SERCA2a）基因转移对心力衰竭（HF）犬心功能的作用并探讨其机制。方法:采用快速右心室起搏建立比格犬的HF模型，设对照组、HF组、HF+EGFP组和HF+SERCA2a组（均n＝4）。后2组经开胸心肌内注射rAAV1-EGFP或rAAV1-SERCA2a，剂量为1×1012病毒基因组。结果:基因转移30 d后，HF+SERCA2a组超声心动图左室射血分数接近对照组，较HF组明显改善（P＜0.05)，SERCA2a mRNA较HF组显著提高（P＜0.05），SERCA2a蛋白在心肌组织中的表达显著高于HF组（P＜0.05)，心肌细胞凋亡指数和基质金属蛋白酶9（MMP-9）表达较HF组显著降低（P＜0.05）。HF+EGFP组各项观察指标接近HF组。但是，受磷蛋白mRNA的水平没有改变。结论:rAAV1-SERCA2a转染上调HF犬心肌组织SERCA2a的表达，能改善心功能，抑制心室重塑；其机制可能与抑制心肌细胞凋亡、下调MMP-9表达有关。     

B-lymphocyte calcium inFlux

Summary:  Dynamic changes in cytoplasmic calcium concentration dictate the immunological fate and functions of lymphocytes. During the past few years, important details have been revealed about the mechanism of store-operated calcium entry in lymphocytes, including the molecular identity of calcium release-activated calcium (CRAC) channels and the endoplasmic reticulum (ER) calcium sensor (STIM1) responsible for CRAC channel activation following calcium depletion of stores. However, details of the potential fine regulation of CRAC channel activation that may be imposed on lymphocytes following physiologic stimulation within an inflammatory environment have not been fully addressed. In this review, we discuss several underexplored aspects of store-operated (CRAC-mediated) and store-independent calcium signaling in B lymphocytes. First, we discuss results suggesting that coupling between stores and CRAC channels may be regulated, allowing for fine tuning of CRAC channel activation following depletion of ER stores. Second, we discuss mechanisms that sustain the duration of calcium entry via CRAC channels. Finally, we discuss distinct calcium permeant non-selective cation channels (NSCCs) that are activated by innate stimuli in B cells, the potential means by which these innate calcium signaling pathways and CRAC channels crossregulate one another, and the mechanistic basis and physiologic consequences of innate calcium signaling.     

The role of SERCA2a/PLN complex, Ca2+ homeostasis, and anti-apoptotic proteins in determining cell fate

Intracellular calcium is a major coordinator of numerous aspects of cellular physiology, including muscle contractility and cell survival. In cardiac muscle, aberrant Ca²⁺ cycling has been implicated in a range of pathological conditions including cardiomyopathies and heart failure. The sarco(endo)plasmic reticulum Ca²⁺ transport adenosine triphosphatase (SERCA2a) and its regulator phospholamban (PLN) have a central role in modulating Ca²⁺ homeostasis and, therefore, cardiac function. Herein, we discuss the mechanisms through which SERCA2a and PLN control cardiomyocyte function in health and disease. Emphasis is placed on our newly identified PLN-binding partner HS-1-associated protein X-1 (HAX-1), which has an anti-apoptotic function and presents with numerous similarities to Bcl-2. Recent evidence indicates that proteins of the Bcl-2 family can influence ER Ca²⁺ content, a critical determinant of cellular sensitivity to apoptosis. The discovery of the PLN/HAX-1 interaction therefore unveils an important new link between Ca²⁺ homeostasis and cell survival, with significant therapeutic potential.     

Inhibition of SERCA2 Ca2+-ATPases by Cs+

Replacement of K⁺ with Cs⁺ on the cytoplasmic side of the sarcoplasmic reticulum (SR) membrane reduces the maximum velocity (V_max) of Ca²⁺ uptake into the SR of saponin-permeabilized rat ventricular myocytes. To compare the sensitivity of the cardiac and smooth muscle/non-muscle forms of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a and -2b respectively) to replacement of K⁺ with Cs⁺, SERCA2a and SERCA2b were expressed in HEK-293 cells. Ca²⁺ uptake into HEK cell microsomes was inhibited by replacement of extravesicular K⁺ with Cs⁺ (V_max of SERCA2a-mediated Ca²⁺ uptake in CsCl was 80% of that in KCl; V_max of SERCA2b-mediated uptake was 70% of that in KCl). The Ca²⁺ sensitivity of uptake was decreased for both SERCA2a- and SERCA2b-mediated uptake and the Hill coefficients were increased in the presence of CsCl. The effects of Cs⁺ on uptake were associated with direct inhibition of the ATPase activity of SERCA2a and SERCA2b. Our results indicate that cation binding sites are present in both SERCA2 isoforms, although the extent to which SERCA2b is inhibited by K⁺ replacement is greater than that of SERCA2a or SERCA1. Consideration of these results and the recent molecular modeling work of others suggests that monovalent cations could interact with the Ca²⁺ binding region of SERCA.     

Increased O<Subscript>2</Subscript> consumption in excitation–contraction coupling in hypertrophied rat heart slices related to increased Na<Superscript>+</Superscript>–Ca<Superscript>2+</Superscript> exchange activity

The goal of our study was to evaluate the origin of the increased O₂ consumption in electrically stimulated left ventricular slices of isoproterenol-induced hypertrophied rat hearts with normal left ventricular pressure. O₂ consumption per minute (mVO₂) of mechanically unloaded left ventricular slices was measured in the absence and presence of 1-Hz field stimulation. Basal metabolic mVO₂, i.e., mVO₂ without electrical stimulation, was significantly smaller, but mVO₂ for the total Ca²⁺ handling in excitation–contraction coupling (E–C coupling mVO₂), i.e., delta mVO₂ (=mVO₂ with stimulation − mVO₂ without stimulation), was significantly larger in the hypertrophied heart. Furthermore, the fraction of E–C coupling mVO₂ was markedly altered in the hypertrophied heart. Namely, mVO₂ consumed by sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) was depressed by 40%; mVO₂ consumed by the Na⁺/K⁺-ATPase (NKA)-Na⁺/Ca²⁺ exchange (NCX) coupling was increased by 100%. The depressed mVO₂ consumption by SERCA2 was supported by lower protein expressions of phosphorylated-Ser¹⁶ phospholamban and SERCA2. The increase in NKA–NCX coupling mVO₂ was supported by marked augmentation of NCX current. However, the increase in NCX current was not due to the increase in NCX1 protein expression, but was attributable to attenuation of the intrinsic inactivation mechanisms. The present results demonstrated that the altered origin of the increased E–C coupling mVO₂ in hypertrophy was derived from decreased SERCA2 activity (1ATP: 2Ca²⁺) and increased NCX activity coupled to NKA activity (1ATP: Ca²⁺). Taken together, we conclude that the energetically less efficient Ca²⁺ extrusion pathway evenly contributes to Ca²⁺ handling in E–C coupling in the present hypertrophy model.     

Initiation site of Ca(2+) entry evoked by endoplasmic reticulum Ca(2+) depletion in mouse parotid and pancreatic acinar cells

Purpose

In non-excitable cells, which include parotid and pancreatic acinar cells, Ca²⁺ entry is triggered via a mechanism known as capacitative Ca²⁺ entry, or store-operated Ca²⁺ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca²⁺ stores, which acts as an important factor triggering Ca²⁺ entry. However, both the mechanism of store-mediated Ca²⁺ entry and the molecular identity of store-operated Ca²⁺ channel (SOCC) remain uncertain.

Materials and Methods

In the present study we investigated the Ca²⁺ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.

Results

Treatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca²⁺-ATPase, in an extracellular Ca²⁺ free state, and subsequent exposure to a high external calcium state evoked Ca²⁺ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca²⁺ channel, completely blocked Tg-induced Ca²⁺ entry. Microfluorometric imaging showed that Tg-induced Ca²⁺ entry started at a basal membrane, not a apical membrane.

Conclusion

These results suggest that Ca²⁺ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.     

Arrhythmogenesis Toxicity of Aconitine Is Related to Intracellular Ca2+ Signals

Aconitine is a well-known arrhythmogenic toxin and induces triggered activities through cardiac voltage-gated Na⁺ channels. However, the effects of aconitine on intracellular Ca²⁺ signals were previously unknown. We investigated the effects of aconitine on intracellular Ca²⁺ signals in rat ventricular myocytes and explored the possible mechanism of arrhythmogenic toxicity induced by aconitine. Ca²⁺ signals were evaluated by measuring L-type Ca²⁺ currents, caffeine-induced Ca²⁺ release and the expression of NCX and SERCA2a. Action potential and triggered activities were recorded by whole-cell patch-clamp techniques. In rat ventricular myocytes, the action potential duration was significantly prolonged by 1 µM aconitine. At higher concentrations (5 µM and 10 µM), aconitine induced triggered activities and delayed after-depolarizations (6 of 8 cases), which were inhibited by verapamil. Aconitine (1 µM) significantly increased the I_Ca-L density from 12.77 ± 3.12 pA/pF to 18.98 ± 3.89 pA/pF (n=10, p<0.01). The activation curve was shifted towards more negative potential, while the inactivation curve was shifted towards more positive potential by 1 μM aconitine. The level of Ca²⁺ release induced by 10 mM caffeine was markedly increased. Aconitine (1 µM) increased the expression of NCX, while SERCA2a expression was reduced. In conclusion, aconitine increased the cytosolic [Ca²⁺]_i by accelerating I_Ca-L and changing the expression of NCX and SERCA2a. Then, the elevation of cytosolic [Ca²⁺]_i induced triggered activities and delayed after-depolarizations. Arrhythmogenesis toxicity of aconitine is related to intracellular Ca²⁺ signals.     

Abnormal intracellular distribution of NFAT1 in T lymphocytes from patients with systemic lupus erythematosus and characteristic clinical features

Systemic lupus erythematosus (SLE) presents various clinical features; however, underlying mechanisms remain unclear. In the immunity of SLE, impaired T cell receptor (TCR) signaling and altered cytokine production are in the center of pathogenesis, although, little is known about NFAT (nuclear factor of activated T cells) in lupus T lymphocytes. TCR stimulation activates NFAT1 through Ca2+/calcineurin (Cn) pathway, facilitating nuclear translocation of NFAT1 from cytosol. Therefore, we investigated relationship of disease activity/features and intracellular NFAT1 localization in T lymphocytes from active lupus patients by fractionation. Results showed no significant relationship between disease activity and NFAT1 distribution. However, interestingly, we observed skewed NFAT1 distribution in pellet in patients with active lupus nephritis or pleuritis. In vitro cyclosporin A treatment suggested autonomously activated Ca2+/Cn pathway in lupus T lymphocytes. Considering these results, NFAT1 might be presenting the clinical heterogeneity in SLE.     

Ca2+ and Sr2+ activation: Comparison of cardiac and skeletal muscle contraction models

The mechanism of contraction in rabbit fast-twitch, and bovine and rabbit cardiac muscle was examined using functionally skinned fibers, ATPase activity of myofibrils, and cardiac or skeletal troponintropomyosin regulated actin heavy meromyosin. The Ca²⁺ and Sr²⁺ activation properties for the different measures of contraction were evaluated. (1) Tension in rabbit and bovine cardiac skinned fibers and rabbit cardiac myofibrillar ATPase were activated equally well by either Ca²⁺ or Sr²⁺. By contrast, rabbit adductor magnus (fast-twitch) skinned fibers required substantially higher [Sr²⁺] than [Ca²⁺] for activation, as did rabbit myofibrils from back muscle (fast-twitch). (2) Substantially more Sr²⁺ than Ca²⁺ was also required for activation of skeletal muscle actin heavy meromyosin ATPase, controlled by either the skeletal or cardiac troponin-tropomyosin complex, similar to the activation of fast-twitch muscle. (3) The absence of correlation between the divalent cation selectivity properties of actin heavy meromyosin ATPase controlled by cardiac troponin-tropomyosin and cardiac muscle tension or myofibrillar ATPase activation by Ca²⁺ and Sr²⁺ suggests that troponin, if primarily responsible for the activation of cardiac muscle, has very different in vivo and in vitro binding properties. (4) The close correlation between percentage of maximal Ca²⁺- and Sr²⁺-activated myofibrillar ATPase and tension in skinned fibers strongly justifies the use of myofibrillar ATPase, in contrast to a reconstituted troponin-tropomyosin actin heavy meromyosin ATPase system, as a biochemical measure of contraction.     

ATP depletion induces translocation of STIM1 to puncta and formation of STIM1–ORAI1 clusters: translocation and re-translocation of STIM1 does not require ATP

Depletion of the endoplasmic reticulum (ER) calcium store triggers translocation of stromal interacting molecule one (STIM1) to the sub-plasmalemmal region and formation of puncta—structures in which STIM1 interacts and activates calcium channels. ATP depletion induced the formation of STIM1 puncta in PANC1, RAMA37, and HeLa cells. The sequence of events triggered by inhibition of ATP production included a rapid decline of ATP, depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and a slow calcium leak from the ER followed by formation of STIM1 puncta. STIM1 puncta induced by ATP depletion were co-localized with clusters of ORAI1 channels. STIM1–ORAI1 clusters that developed as a result of ATP depletion were very poor mediators of Ca²⁺ influx. Re-translocation of STIM1 from puncta back to the ER was observed during total ATP depletion. We can therefore conclude that STIM1 translocation and re-translocation as well as formation of STIM1–ORAI1 clusters occur in an ATP-independent fashion and under conditions of PI(4,5)P₂ depletion. Michael Chvanov and Ciara M. Walsh are considered as equal first authors.     

Myofibril and sarcoplasmic reticulum changes with exercise and growth

Summary The intent of this study was to observe the effects of different treadmill running programs upon selected biochemical properties of soleus muscle from young rats. Young 10 day litter-mates were assigned to endurance (E), sprint (S) and control (C) groups. Each was partitioned into either 21 or 51 day exercising groups and 10 day controls. For C the myofibril ATPase activity at 21 and 51 days were lower than 10 day activity (p0.05). In the 51 day E group ATPase activity (0.378±0.009 mol Pi·mg^–1·min^–1) was greater than at 10 and 21 days (0.307±0.006 and 0.323±0.008 mol Pi·mg^–1·min^–1) (p0.05). No change occurred in the S group from 10 to 21 and 51 days (p0.05). Both the 21 and 51 day S (0.318±0.011 and 0.399±0.010 mol Pi·mg^–1·min^–1) and E (0.323±0.008 and 0.378±0.009 mol Pi·mg^–1·min^–1) groups had higher activity compared to the C group (0.193±0.029 and 0.172±0.031 mol Pi·mg^–1·min^–1) (p0.05). Maturation (10–51 day) resulted in a lowered sarcoplasmic reticulum (SR) yield and Ca²⁺ binding (p0.05) while Ca²⁺ uptake ability did not change (p0.05). SR yield, Ca²⁺ binding and uptake were not altered with S training (p0.05). The E training resulted in greater Ca²⁺ uptake at 51 days compared to C and S (p0.05), with no change in Ca²⁺ binding (p0.05). The data suggest that E training alters the normal development pattern of young rat soleus muscle.Supported by grants A-6449 and A-0425 from the Natural Sciences and Engineering Research Council of Canada     

Calcium uptake and release through sarcoplasmic reticulum in the inferior oblique muscles of patients with inferior oblique overaction

We characterized and compared the characteristics of Ca2+ movements through the sarcoplasmic reticulum of inferior oblique muscles in the various conditions including primary inferior oblique overaction (IOOA), secondary IOOA, and controls, so as to further understand the pathogenesis of primary IOOA. Of 15 specimens obtained through inferior oblique myectomy, six were from primary IOOA, 6 from secondary IOOA, and the remaining 3 were controls from enucleated eyes. Ryanodine binding assays were performed, and Ca2+ uptake rates, calsequestrins and SERCA levels were determined. Ryanodine bindings and sarcoplasmic reticulum Ca2+ uptake rates were significantly decreased in primary IOOA (p < 0.05). Western blot analysis conducted to quantify calsequestrins and SERCA, found no significant difference between primary IOOA, secondary IOOA, and the controls. Increased intracellular Ca2+ concentration due to reduced sarcoplasmic reticulum Ca2+ uptake may play a role in primary IOOA.     

Trifluoperazine protects brain plasma membrane Ca<Superscript>2+</Superscript>-ATPase from oxidative damaging

In the central nervous system (CNS), a number of different pathological processes such as necrosis, Parkinson’s and Alzheimer’s diseases are related to disturbance in calcium homeostasis associated with oxidative stress. Here we compare the susceptibility of rat brain plasma membrane Ca²⁺-ATPase (PMCA) and sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) isoforms to in vitro oxidative stress, and investigate a putative role of trifluoperazine (TFP), an antipsychotic drug that is also a powerful inhibitor of Ca²⁺-transporter proteins, in protecting these enzymes. It is shown that, in rat brain, PMCA is very sensitive to the damage induced by preincubation with Fe²⁺-ascorbate, or Fe²⁺-ascorbate plus H₂O_2, while SERCA is resistant. Inhibition of PMCA activity promoted by Fe²⁺/ascorbate medium is fully prevented by the presence of μM concentrations of either butylated hydroxytoluene (BHT) or TFP, but only partially protected, or reversed, by dithiothreitol (DTT), pointing to some protein cysteine(s) as one of the main targets for a lipid peroxidation-dependent damaging mechanism. However, when 0.5–1 mM H₂O₂ is added together with Fe²⁺/ascorbate, both BHT and TFP only partially prevent ATPase activity inhibition, and DTT does not confer any protection, suggesting two possible additional mechanisms involving both lipid peroxidation and direct damage to PMCA at amino acid residues other than cysteines. A possible use of μmolar concentrations of TFP as a direct antioxidant protector for PMCA under oxidative stress conditions is discussed.     

Role of sarco/endoplasmic reticulum calcium content and calcium ATPase activity in the control of cell growth and proliferation

Ca²⁺, the main second messenger, is central to the regulation of cellular growth. There is increasing evidence that cellular growth and proliferation are supported by a continuous store-operated Ca²⁺ influx. By controlling store refilling, the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) also controls store-operated calcium entry and, thus, cell growth. In this review, we discuss data showing the involvement of SERCA in the regulation of proliferation and hypertrophy. First, we describe the Ca²⁺-related signaling pathways involved in cell growth. Then, we present evidence that SERCA controls proliferation of differentiated cells and hypertrophic growth of cardiomyocytes, and discuss the role of SERCA isoforms. Last, we consider the potential therapeutic applications of increasing SERCA activity for the treatment of cardiovascular diseases and of modulating SERCA and SR content for the treatment of cancer.     

Adenosine receptors control HIV-1 Tat-induced inflammatory responses through protein phosphatase

Recently, adenosine has been proposed to be a "metabolic" switch that may sense and direct immune and inflammatory responses. Inflammation and pro-inflammatory cytokine production are important in development of HIV-1 associated dementia, a devastating consequence of HIV-1 infection of the CNS. The HIV-1 protein Tat induces cell death in the CNS and activates local inflammatory responses partially by inducing calcium release from the endoplasmic reticulum. Because activation of adenosine receptors decreases production of the pro-inflammatory cytokine TNF-alpha in several experimental paradigms both in vitro and in vivo, we hypothesized that adenosine receptor activation would control both increased intracellular calcium and TNF-alpha production induced by Tat. Treatment of primary monocytes with Tat significantly increased the levels of intracellular calcium released from IP3 stores. Activation of adenosine receptors with CGS 21680 inhibited Tat-induced increases of intracellular calcium by 90 +/- 8% and was dependent on protein phosphatase activity because okadaic acid blocked the actions of CGS 21680. Tat-induced TNF-alpha production was inhibited 90 +/- 6% by CGS 21680 and concurrent treatment with okadaic acid blocked the inhibitory actions of CGS 21680. Using a model monocytic cell line, CGS 21680 treatment increased cytosolic serine/threonine phosphatase. Together, these data indicate that A2A receptor activation increases protein phosphatase activity, which blocks IP3 receptor-regulated calcium release and reduction of intracellular calcium inhibits TNF-alpha production in monocytes.     

Interleukin-4 inhibits cyclo-oxygenase-2 expression and prostaglandin E production by human mature dendritic cells

Interleukin-4 (IL-4) is considered the key cytokine for inducing T helper type 2 (Th2) cell differentiation, while interferon-gamma and IL-12 are pivotal cytokines for Th1 immune responses. Paradoxically, IL-4 has also been demonstrated to enhance IL-12 production by dendritic cells, suggesting an IL-4-dependent regulatory feedback of the Th1/Th2 system. In addition, prostaglandin E(2) (PGE(2)), a lipid mediator of inflammation, has been implicated in the enhancement of Th2-type responses acting directly on T and B lymphocytes. PGE(2) synthesis is dependent on the serial engagement of various enzymes, among which the inducible cyclo-oxygenase-2 (COX-2) exerts a critical role in monocytes and dendritic cells. In this study we demonstrate that IL-4 inhibits COX-2 gene expression and consequently prevents secretion of PGE(2) by mature human dendritic cells. We also show that PGE(2) does not regulate IL-12 and IL-10 production by dendritic cells in an autocrine fashion. Hence, we suggest that IL-4 may exploit an IL-12-independent regulatory feedback of the Th1/Th2 system through PGE(2) inhibition.