Inhibition Of Calcium Signaling Research Articles

Salt-Induced Early Changes in Photosynthesis Activity Caused by Root-to-Shoot Signaling in Potato.

Salinity is one of the most dangerous types of stress in agriculture. Acting on the root, salinity causes changes in physiological processes in the shoot, especially photosynthesis, which is crucial for plant productivity. In our study, we used potato plants, the most important crop, to investigate the role of salt-induced signals in changes in photosynthesis activity. We found a salt-induced polyphasic decrease in photosynthesis activity, and the earliest phase started several minutes after salt addition. We found that salt addition triggered rapid hydraulic and calcium waves from root to shoot, which occurred earlier than the first phase of the photosynthesis response. The inhibition of calcium signals by lanthanum decreased with the formation of rapid changes in photosynthesis. In addition to this, a comparison of the characteristic times of signal propagation and the formation of a response revealed the role of calcium waves in the modulation of rapid changes in photosynthesis. Calcium waves are activated by the ionic component of salinity. The salt-induced decrease in transpiration corresponds in time to the second phase of the photosynthetic response, and it can be the cause of this change. The accumulation of sodium in the leaves occurs a few hours after salt addition, and it can be the cause of the long-term suppression of photosynthesis. Thus, salinity modulates photosynthetic activity in plants in different ways: both through the activation of rapid distant signals and by reducing the water input and sodium accumulation.

Survival Outcomes of Calcium Channel Blocker Therapy in Patients With Non-Small Cell Lung Cancer Treated With Epidermal Growth Factor Receptor Inhibitors: A Retrospective Study.

Non-cancer drugs are currently being repurposed for cancer treatment. Mounting evidence highlights the influence of calcium channels on tumorigenesis and progression. Hence, inhibition of calcium signaling may be a promising cancer treatment strategy. In this study, we aimed to examine whether calcium channel blockers (CCBs) affect the efficacy of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in patients with non-small cell lung cancer (NSCLC). We conducted a retrospective analysis. In this study, conducted between January 2009 and June 2021, patients with NSCLC treated with erlotinib, or gefitinib for at least 1 week were enrolled and divided into 2 groups: CCBs-/EGFR-TKIs+ and CCBs+/EGFR-TKIs+, depending on whether they received CCB therapy. Progression-free survival (PFS) and overall survival (OS) were determined as the primary and secondary endpoints, respectively. : The estimated median PFS and OS for the CCBs-/EGFR-TKIs+group were 7.70 and 12.17 months, respectively, and they were significantly different from those of the CCBs+/EGFR-TKIs+ group (10.43 and 18.07 months, respectively). CCB use was associated with improved PFS (adjusted hazard ratios [HR] 0.77, 95% confidence interval [CI]: 0.61-0.98; P = .035) and OS (adjusted HR 0.66, 95% CI: 0.51-0.84; P < .001). Calcium channels have been implicated in cancer pathogenesis. Our findings revealed the potential additive anticancer effects of CCBs when used concomitantly with EGFR-TKIs. However, study limitations, including the retrospective nature and small number of patients, necessitate large-scale prospective studies on the therapeutic potential of CCB as an adjunctive therapy with EGFR-TKIs in patients with NSCLC.

Novel Algorithm of Network Calcium Dynamics Analysis for Studying the Role of Astrocytes in Neuronal Activity in Alzheimer's Disease Models.

Accumulated experimental data strongly suggest that astrocytes play an important role in the pathogenesis of neurodegeneration, including Alzheimer's disease (AD). The effect of astrocytes on the calcium activity of neuron-astroglia networks in AD modelling was the object of the present study. We have expanded and improved our approach's capabilities to analyze calcium activity. We have developed a novel algorithm to construct dynamic directed graphs of both astrocytic and neuronal networks. The proposed algorithm allows us not only to identify functional relationships between cells and determine the presence of network activity, but also to characterize the spread of the calcium signal from cell to cell. Our study showed that Alzheimer's astrocytes can change the functional pattern of the calcium activity of healthy nerve cells. When healthy nerve cells were cocultivated with astrocytes treated with Aβ42, activation of calcium signaling was found. When healthy nerve cells were cocultivated with 5xFAD astrocytes, inhibition of calcium signaling was observed. In this regard, it seems relevant to further study astrocytic-neuronal interactions as an important factor in the regulation of the functional activity of brain cells during neurodegenerative processes. The approach to the analysis of streaming imaging data developed by the authors is a promising tool for studying the collective calcium dynamics of nerve cells.

Phosphoproteomic profiling of skeletal muscle twitch torque potentiation in ovarian hormone deficient female mice

Skeletal muscle makes up ~40% of total body mass in a healthy adult. Muscle strength begins to decline with age. In females, preclinical and clinical studies have shown that reduction in estrogen reduces muscle force (i.e., strength). Previously, we showed the skeletal muscle phosphoproteome in a basal, non‐contracting state was remodeled in estrogen‐deficient females. Therefore, we questioned how estrogen deficiency would impact the skeletal muscle phosphoproteome after force generation. We performed two label‐free quantification phosphoproteomic analyses of the tibialis anterior muscle in ovariectomized (Ovx/Sham) and ovarian senescent (Old/Young) female mice after in vivo post‐tetanic potentiation protocol. We identified 25 and 60 differentially regulated phosphoproteins (p‐value&lt;0.05 and ≥1.4‐fold change) in Ovx/Sham and Old/Young mice, respectively. Comparative analysis using Ingenuity Pathway Analysis’s activation Z‐score found similar patterns of predicted inhibition and activation of canonical pathways, such as inhibition of calcium signaling and activation of 14‐3‐3‐mediated signaling in both datasets. Likewise, parallel patterns in functional analysis were found relating to muscle contraction, fibrogenesis, etc. Overall, our findings suggest that the similarities identified in both datasets could elucidate the molecular characteristics of muscle proteins that might contribute to decrements in muscle function observed in Ovx and Old females due to the loss of estrogen.

Calcium Signaling Regulates Valvular Interstitial Cell Alignment and Myofibroblast Activation in Fast-Relaxing Boronate Hydrogels.

The role viscoelasticity in fibrotic disease progression is an emerging area of interest. Here, a fast-relaxing hydrogel system is exploited to investigate potential crosstalk between calcium signaling and mechanotransduction. Poly(ethylene glycol) (PEG) hydrogels containing boronate and triazole crosslinkers are synthesized, with varying ratios of boronate to triazole crosslinks to systematically vary the extent of stress relaxation. Valvular interstitial cells (VICs) encapsulated in hydrogels with the highest levels of stress relaxation (90%) exhibit a spread morphology by day 1 and are highly aligned (80 ± 2%) by day 5. Key myofibroblast markers, including α-smooth muscle actin (αSMA) and collagen 1a1 (COL1A1), are significantly elevated. VIC myofibroblast activation decreases by 42 ± 18% through inhibition of mechanotransduction, independently of VIC morphology and alignment. Calcium signaling through a transient receptor potential vanilloid 4 (TRPV4) is found to regulate VIC spreading, alignment, and activation in a time dependent manner. Inhibition of calcium signaling at early time points results in disturbed cell alignment, decreased mechanotransduction, and diminished activation, while inhibition at later time points only causes partially reduced myofibroblast activation. These results suggest a potential crosstalk mechanism, where calcium signaling acts upstream of mechanosensing and can regulate VIC myofibroblast activation independently of mechanotransduction.

Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

BackgroundMicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level and are key modulators in neurodegenerative diseases. Overexpressed miRNAs play an important role in ALS; however, the pathogenic mechanisms of deregulated miRNAs are still unclear.MethodsWe aimed to assess the dysfunction of RNAs or miRNAs in fALS (SOD1 mutations). We compared the RNA-seq of subcellular fractions in NSC-34 WT (hSOD1) and MT (hSOD1 (G93A)) cells to find altered RNAs or miRNAs. We identified that Hif1α and Mef2c were upregulated, and Mctp1 and Rarb were downregulated in the cytoplasm of NSC-34 MT cells.ResultsSOD1 mutations decreased the level of miR-18b-5p. Induced Hif1α which is the target for miR-18b increased Mef2c expression as a transcription factor. Mef2c upregulated miR-206 as a transcription factor. Inhibition of Mctp1 and Rarb which are targets of miR-206 induces intracellular Ca2+ levels and reduces cell differentiation, respectively. We confirmed that miR-18b-5p pathway was also observed in G93A Tg, fALS (G86S) patient, and iPSC-derived motor neurons from fALS (G17S) patient.ConclusionsOur data indicate that SOD1 mutation decreases miR-18b-5p, which sequentially regulates Hif1α, Mef2c, miR-206, Mctp1 and Rarb in fALS-linked SOD1 mutation. These results provide new insights into the downregulation of miR-18b-5p dependent pathogenic mechanisms of ALS.

Projection length stimulated by oxytocin is modulated by the inhibition of calcium signaling in U-87MG cells.

Neuropeptide oxytocin contributes to the regulation of glial cell morphology. The precise mechanisms, however, are not yet fully understood. In the present study, we have investigated whether an oxytocin-induced increase of intracellular calcium is required for cell extension in astrocyte-like U-87MG cells. Oxytocin (1µM) significantly increased the length of the cell projections measured by the green-fluorescent protein labeled microtubule-associated protein after 48h. The knockdown of oxytocin receptors (OXTR) in U-87MG cells prevented the elongation of the projections. Incubation of U-87MG cells in the presence of oxytocin, resulted in a significant increase of intracellular calcium, specifically blocked by the OXTR antagonist L-371,257. Both quercetin, which is a phosphoinositide 3-kinase inhibitor, and the phospholipase C inhibitor U-73122 reduced oxytocin-induced elevation of intracellular calcium concentration. Conversely, neither diltiazem, an L-type voltage-gated calcium channel blocker nor tetracaine, which is a blocker of the ryanodine receptors, showed an effect on intracellular calcium levels. Treatment of cells with quercetin, U-73122 and the voltage-gated calcium channel blockers cilnidipine, ω-agatoxin and mibefradil prevented the elongation of projections stimulated by oxytocin. Oxytocin treatment resulted in a significant increase in gene and protein expression of the scaffolding protein SHANK3. Our results clearly show that activation of OXTRs contributes to the elongation of cell projections in astrocyte-like U-87MG cells and that this effect is mediated by an extracellular calcium influx accompanied by an increase in scaffolding proteins expression.

Hypo-osmotic Stress Drives IL-33 Production in Human Keratinocytes—An Epidermal Homeostatic Response

Although inflammation has traditionally been considered a response to either exogenous pathogen-associated signals or endogenous signals of cell damage, other perturbations of homeostasis, generally referred to as stress, may also induce inflammation. The relationship between stress and inflammation is, however, not well defined. Here, we describe a mechanism of IL-33 induction driven by hypo-osmotic stress in human keratinocytes and also report interesting differences when comparing the responsiveness of other inflammatory mediators. The induction of IL-33 was completely dependent on EGFR and calcium signaling, and inhibition of calcium signaling not only abolished IL-33 induction but also dramatically changed the transcriptional pattern of other cytokines upon hypo-osmotic stress. IL-33 was not secreted but instead showed nuclear sequestration, conceivably acting as a failsafe mechanism whereby it is induced by potential danger but released only upon more extreme homeostatic perturbations that result in cell death. Finally, stress-induced IL-33 was also confirmed in an exvivo human skin model, translating this mechanism to a potential tissue-relevant signal in the human epidermis. In conclusion, we describe hypo-osmotic stress as an inducer of IL-33 expression, linking cellular stress to nuclear accumulation of a strong proinflammatory cytokine.

Dietary potassium regulates vascular calcification and arterial stiffness.

Vascular calcification is a risk factor that predicts adverse cardiovascular complications of several diseases including atherosclerosis. Reduced dietary potassium intake has been linked to cardiovascular diseases such as hypertension and incidental stroke, although the underlying molecular mechanisms remain largely unknown. Using the ApoE-deficient mouse model, we demonstrated for the first time to our knowledge that reduced dietary potassium (0.3%) promoted atherosclerotic vascular calcification and increased aortic stiffness, compared with normal (0.7%) potassium-fed mice. In contrast, increased dietary potassium (2.1%) attenuated vascular calcification and aortic stiffness. Mechanistically, reduction in the potassium concentration to the lower limit of the physiological range increased intracellular calcium, which activated a cAMP response element-binding protein (CREB) signal that subsequently enhanced autophagy and promoted vascular smooth muscle cell (VSMC) calcification. Inhibition of calcium signals and knockdown of either CREB or ATG7, an autophagy regulator, attenuated VSMC calcification induced by low potassium. Consistently, elevated autophagy and CREB signaling were demonstrated in the calcified arteries from low potassium diet-fed mice as well as aortic arteries exposed to low potassium ex vivo. These studies established a potentially novel causative role of dietary potassium intake in regulating atherosclerotic vascular calcification and stiffness, and uncovered mechanisms that offer opportunities to develop therapeutic strategies to control vascular disease.

Vesicle-Mediated Steroid Hormone Secretion in Drosophila melanogaster

Steroid hormones are a large family of cholesterol derivatives regulating development and physiology in both the animal and plant kingdoms, but little is known concerning mechanisms of their secretion from steroidogenic tissues. Here, we present evidence that in Drosophila, endocrine release of the steroid hormone ecdysone is mediated through a regulated vesicular trafficking mechanism. Inhibition of calcium signaling in the steroidogenic prothoracic gland results in the accumulation of unreleased ecdysone, and the knockdown of calcium-mediated vesicle exocytosis components in the gland caused developmental defects due to deficiency of ecdysone. Accumulation of synaptotagmin-labeled vesicles in the gland is observed when calcium signaling is disrupted, and these vesicles contain an ABC transporter that functions as an ecdysone pump to fill vesicles. We propose that trafficking of steroid hormones out of endocrine cells is not always through a simple diffusion mechanism as presently thought, but instead can involve a regulated vesicle-mediated release process.

Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells

BackgroundAdvanced prostate cancer commonly metastasizes to bone leading to osteoblastic and osteolytic lesions. Although an osteolytic component governed by activation of bone resorbing osteoclasts is prominent in prostate cancer metastasis, the molecular mechanisms of prostate cancer-induced osteoclastogenesis are not well-understood.MethodsWe studied the effect of soluble mediators released from human prostate carcinoma cells on osteoclast formation from mouse bone marrow and RAW 264.7 monocytes.ResultsSoluble factors released from human prostate carcinoma cells significantly increased viability of naïve bone marrow monocytes, as well as osteoclastogenesis from precursors primed with receptor activator of nuclear factor κ-B ligand (RANKL). The prostate cancer-induced osteoclastogenesis was not mediated by RANKL as it was not inhibited by osteoprotegerin (OPG). However inhibition of TGFβ receptor I (TβRI), or macrophage-colony stimulating factor (MCSF) resulted in attenuation of prostate cancer-induced osteoclastogenesis. We characterized the signaling pathways induced in osteoclast precursors by soluble mediators released from human prostate carcinoma cells. Prostate cancer factors increased basal calcium levels and calcium fluctuations, induced nuclear localization of nuclear factor of activated t-cells (NFAT)c1, and activated prolonged phosphorylation of ERK1/2 in RANKL-primed osteoclast precursors. Inhibition of calcium signaling, NFATc1 activation, and ERK1/2 phosphorylation significantly reduced the ability of prostate cancer mediators to stimulate osteoclastogenesis.ConclusionsThis study reveals the molecular mechanisms underlying the direct osteoclastogenic effect of prostate cancer derived factors, which may be beneficial in developing novel osteoclast-targeting therapeutic approaches.

801 Calcium-Dependent Signaling, via Calcineurin and NFAT, Is Key to Steatosis in Alcoholic Liver Disease (ALD) in Mice

Background: Alcohol abuse leads to alcoholic liver disease (ALD). In ALD hepatic steatosis is a prerequisite of disease progresses to steatohepatitis (SH) at which stage the liver injury becomes evident. The mechanisms of steatosis in ALD are noit fully understood. Calciumdependent signaling is activated in ALD in mice. The Aim of our study was to evaluate the role of calcium-dependent signaling in development of steatosis in ALD. Methods: We fed alcohol (Lieber-deCarli) or control diet to control C57Bl6 and NFAT-KO mice or cyclosporine-treated C57Bl6 mice. Results: Alcohol, but not control, diet led to significant ALD, revealed by elevated liver Tg content and significant OilRedO liver tissue staining suggestive of steatosis, increased serum ALT suggestive of liver injury, and serum cytokines TNFa, IL1, IL6, suggestive of inflammation, in C57Bl6 mice. There was significant elevation of calcium signaling in livers of alcohol-fed animals compared to control diet, as revealed by higher expression of Calcineurin, PLC, PKC, and MAPKp38 and elevated NFAT activity. Alcohol, but not control, diet lead to significant induction ACS, SCD1, ELOV16, GPAT and DGAT, LDLR HMG-CoA reductase mRNA in the livers of ethanol-fed animals. Further, the amount of mature SREBP-1protein, suggestive of SREBP activation, was increased in liver of alcoholfed animals. Inhibition of calcium signaling by either Cyclosporine treatment (at the level of Calcineurin) or by genetic NFAT deficiency partially prevented alcohol diet-induced upregulation of ACS, SCD1, ELOV16, GPAT and DGAT; more important, inhibition of calcium signaling led to partial protective against alcohol diet-induced liver injury and steatosis. NFAT protein was detected in both KCs and Hpt. In vitro, palmitic acid-exposed Hepa1.6 cells, used as surrogate of Hpt, developed steatosis; this process was significantly impaired when the cells were treated with cyclosporine and in cells made NFAT deficient by specific siRNA treatment. Co-culture of Hepa 1.6 cells+palmitic acid with inflammatory (LPS-pretreated) KCs lead to further upregulation of lipid uptake; sole exposure of KCs to cyclosporine did not prevent steatosis in co-culture. These data suggested that calciumdependent signaling mechanisms are involved in lipid synthesis in hepatocytes at different levels, including lipogenesis and lipolysis, in a KC-dependent manner. In conclusion, we report novel finding that calcium signaling is, in part, responsible for development of steatosis component of ALD in mice. It remains to be determined if inhibition of calcium signaling may be beneficial for delaying of steatosis and/or blunting of progression from HS to SH phases of ALD.

802 Tgfb Signaling Participates in Steatohepatitis Through the Induction of Cell Death and Suppression of Beta-Oxidation in Mice

Background: Alcohol abuse leads to alcoholic liver disease (ALD). In ALD hepatic steatosis is a prerequisite of disease progresses to steatohepatitis (SH) at which stage the liver injury becomes evident. The mechanisms of steatosis in ALD are noit fully understood. Calciumdependent signaling is activated in ALD in mice. The Aim of our study was to evaluate the role of calcium-dependent signaling in development of steatosis in ALD. Methods: We fed alcohol (Lieber-deCarli) or control diet to control C57Bl6 and NFAT-KO mice or cyclosporine-treated C57Bl6 mice. Results: Alcohol, but not control, diet led to significant ALD, revealed by elevated liver Tg content and significant OilRedO liver tissue staining suggestive of steatosis, increased serum ALT suggestive of liver injury, and serum cytokines TNFa, IL1, IL6, suggestive of inflammation, in C57Bl6 mice. There was significant elevation of calcium signaling in livers of alcohol-fed animals compared to control diet, as revealed by higher expression of Calcineurin, PLC, PKC, and MAPKp38 and elevated NFAT activity. Alcohol, but not control, diet lead to significant induction ACS, SCD1, ELOV16, GPAT and DGAT, LDLR HMG-CoA reductase mRNA in the livers of ethanol-fed animals. Further, the amount of mature SREBP-1protein, suggestive of SREBP activation, was increased in liver of alcoholfed animals. Inhibition of calcium signaling by either Cyclosporine treatment (at the level of Calcineurin) or by genetic NFAT deficiency partially prevented alcohol diet-induced upregulation of ACS, SCD1, ELOV16, GPAT and DGAT; more important, inhibition of calcium signaling led to partial protective against alcohol diet-induced liver injury and steatosis. NFAT protein was detected in both KCs and Hpt. In vitro, palmitic acid-exposed Hepa1.6 cells, used as surrogate of Hpt, developed steatosis; this process was significantly impaired when the cells were treated with cyclosporine and in cells made NFAT deficient by specific siRNA treatment. Co-culture of Hepa 1.6 cells+palmitic acid with inflammatory (LPS-pretreated) KCs lead to further upregulation of lipid uptake; sole exposure of KCs to cyclosporine did not prevent steatosis in co-culture. These data suggested that calciumdependent signaling mechanisms are involved in lipid synthesis in hepatocytes at different levels, including lipogenesis and lipolysis, in a KC-dependent manner. In conclusion, we report novel finding that calcium signaling is, in part, responsible for development of steatosis component of ALD in mice. It remains to be determined if inhibition of calcium signaling may be beneficial for delaying of steatosis and/or blunting of progression from HS to SH phases of ALD.

Calcium Influx Blockade as Cancer Therapy

Copyright: © 2012 Cox JL. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Metastasis is the major reason for treatment failure when it comes to therapy for different cancers. Here I would like to give a brief overview of some research that has attempted to block metastasis through inhibition of calcium signaling and metabolism. This strategy to inhibit metastasis through blocking calcium channels/calcium function is not yet being applied clinically, but could be within several years following clinical trials. It remains an open question whether this calcium strategy will lead to durable improvements for the management of cancer. A number of recent advances in understanding calcium regulation in cancer cells has also brought us closer to possible clinical application of this strategy, as new calcium interfering drugs are being developed based on new molecular and cellular physiological information.

Grape seed proanthocyanidin extract inhibits glutamate-induced cell death through inhibition of calcium signals and nitric oxide formation in cultured rat hippocampal neurons

BackgroundProanthocyanidin is a polyphenolic bioflavonoid with known antioxidant activity. Some flavonoids have a modulatory effect on [Ca2+]i. Although proanthocyanidin extract from blueberries reportedly affects Ca2+ buffering capacity, there are no reports on the effects of proanthocyanidin on glutamate-induced [Ca2+]i or cell death. In the present study, the effects of grape seed proanthocyanidin extract (GSPE) on glutamate-induced excitotoxicity was investigated through calcium signals and nitric oxide (NO) in cultured rat hippocampal neurons.ResultsPretreatment with GSPE (0.3-10 μg/ml) for 5 min inhibited the [Ca2+]i increase normally induced by treatment with glutamate (100 μM) for 1 min, in a concentration-dependent manner. Pretreatment with GSPE (6 μg/ml) for 5 min significantly decreased the [Ca2+]i increase normally induced by two ionotropic glutamate receptor agonists, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). GSPE further decreased AMPA-induced response in the presence of 1 μM nimodipine. However, GSPE did not affect the 50 mM K+-induced increase in [Ca2+]i. GSPE significantly decreased the metabotropic glutamate receptor agonist (RS)-3,5-Dihydroxyphenylglycine-induced increase in [Ca2+]i, but it did not affect caffeine-induced response. GSPE (0.3-6 μg/ml) significantly inhibited synaptically induced [Ca2+]i spikes by 0.1 mM [Mg2+]o. In addition, pretreatment with GSPE (6 μg/ml) for 5 min inhibited 0.1 mM [Mg2+]o- and glutamate-induced formation of NO. Treatment with GSPE (6 μg/ml) significantly inhibited 0.1 mM [Mg2+]o- and oxygen glucose deprivation-induced neuronal cell death.ConclusionsAll these data suggest that GSPE inhibits 0.1 mM [Mg2+]o- and oxygen glucose deprivation-induced neurotoxicity through inhibition of calcium signals and NO formation in cultured rat hippocampal neurons.

Negative Feedback Regulation of Raf/MEK/ERK Cascade After Sublethal Cerebral Ischemia in the Rat Hippocampus

Sublethal preconditioning ischemia protects neurons from lethal ischemia, and activation of ERK is associated with this protection. However, sublethal ischemia and reperfusion also results in rapid inactivation of ERK, which contributes to the dual-phase activation profile of ERK. In the present study, we observed sublethal ischemia-induced rapid inactivation of ERK was accompanied by phosphorylation of Raf-1 at Ser289/296/301 sites. Inhibition of calcium signaling by ketamine resulted in down-regulation of the Raf-1/ERK cascade and decreased phosphorylation of Raf-1 at Ser289/296/301. The MEK inhibitor U0126 suppressed ERK activity and phosphorylation of Raf-1 at Ser289/296/301 but not Raf-1 activation elicited by its dephosphorylation at S259 following ischemia. The PP2A inhibitor cantharidin but not Pin1 inhibitor juglone blocked Raf-1 dephosphorylation at Ser289/296/301 and ERK dephosphorylation and led to ERK sustained activation, which is associated with transcriptional up-regulation of genes related to differentiation. Furthermore, dual-phase activation of ERK did not alter the mRNA levels of genes related to proliferation or differentiation. These results indicate the initial robust activation of ERK phosphorylates Raf-1 at Ser289/296/301, resulting in Raf-1inhibition and then prompt inactivation of ERK following sublethal preconditioning ischemia. Dual-phase activation of ERK may exert its neuroprotection against lethal ischemia through blocking cell proliferation and differentiation.

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca2+]i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5 μM Fura-2/AM and [Ca2+]i transients for stimulation with 30 mM KCl (Hi K+) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380 nm, emission was recorded at 510 nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca2+]i transients in a reversible manner. [Ca2+]i increase evoked by Hi K+ was significantly reduced to 99.9 ± 5.1% (n = 18) and 62.5 ± 4.2% (n = 15, P < 0.05) after the application of 5 and 50 µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K+-induced [Ca2+]i responses, reduced to 98.7 ± 4.8% (n = 10) and 69.5 ± 4.5% (n = 9, P < 0.05) inhibition of fluorescence ratio values of Hi K+-induced responses at 5 and 50 μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.

The effects of PM10particles and oxidative stress on macrophages and lung epithelial cells: modulating effects of calcium-signaling antagonists

We have previously examined the ability of air pollution particles (PM(10)) to promote release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) from human peripheral blood mononuclear cells and demonstrated a role for calcium as a signaling molecule in this process. We have now studied the ability of oxidative stress induced by a synthetic oxidant tert-butyl hydroperoxide (tBHP) to induce TNF-alpha production via calcium signaling in the mouse macrophage cell line (J774). The oxidant tBHP significantly increased intracellular calcium and the release of TNF-alpha in J774 cells, an effect that was reduced to control levels by inhibition of calcium signaling with verapamil, BAPTA-AM, and W-7. This study also investigated interactions between PM(10)-treated macrophages and epithelial cells by using conditioned medium (CM) from PM(10)-treated mononuclear cells to stimulate the release of the neutrophil chemoattractant chemokine IL-8 from A549 lung epithelial cells. TNF-alpha protein release was demonstrated in human mononuclear cells after PM(10) treatment, an effect that was inhibited by calcium antagonists. Treatment of A549 cells with monocyte/PM(10) CM produced increased IL-8 release that was reduced with CM from monocyte/PM(10)/calcium antagonist treatments. The expression of ICAM-1 was increased after incubation with CM from monocyte/PM(10) treatment, and this increase was prevented by treatment with CM from monocyte/PM(10)/calcium antagonist. These data demonstrate a link between oxidative stress, calcium, and inflammatory mediator production in macrophages and lung epithelial cells.

The anti-apoptotic effects of nordihydroguaiaretic acid: Inhibition of cPLA 2 activation during TNF-induced apoptosis arises from inhibition of calcium signaling

Nordihydroguaiaretic acid (NDGA) is a plant lignan produced by Larrea tridentata, the creosote bush of the American southwest. In this report we examine the mechanism underlying the ability of NDGA to inhibit TNF-induced apoptosis. Our results show that NDGA blocks many key indicators of apoptosis. Caspase cleavage, mitochondrial inactivation, externalization of phosphatidyl serine, and 51Cr-release were all blocked by low micromolar concentrations of NDGA. NDGA also inhibited the cPLA 2-dependent release of 3H-arachidonic acid. We investigated this activity and found that NDGA prevented the rise in intracellular calcium necessary for the apoptotic activation of cPLA 2. On the other hand, NDGA did not interfere with the TNF-induced phosphorylation of cPLA 2, indicating that NDGA does not block all TNF-dependent signaling. Finally, we asked whether the anti-apoptotic effect of NDGA could be attributed to its anti-oxidant activity. Comparison with the effects of butylated hydroxyanisole (BHA) did not completely support this hypothesis. While BHA strongly inhibited caspase activation and partially blocked the release of 51Cr, it was unable to significantly block the calcium response or the release of 3H-arachidonic acid associated with TNF-induced apoptosis. The anti-oxidant activity of NDGA may, therefore, explain some but not all of its anti-apoptotic activity.

Calcium-ROS Feedback Loop

Stimulation of immune cell receptors, such as the B cell receptor (BCR), can lead to multiple distinct cellular responses, making these cells excellent models for studying signal plasticity. Singh et al . found that inhibition of either release of calcium from the endoplasmic reticulum (with TMB-8) or uptake of calcium from the extracellular medium (with EGTA, a calcium chelator) produced a global dampening of BCR-dependent phosphorylation, including phosphorylation events that occurred within the first minute of BCR activation. Indeed, one of the events that was inhibited was the phosphorylation of Lyn, a Src family tyrosine kinase, which is believed to be the first enzyme activated in response to BCR ligand binding. This was surprising because calcium signals are downstream events; thus, this result suggested that calcium can feed back on the early signaling events. Formation of reactive oxygen species (ROS) in response to BCR activation occurred within seconds and was inhibited by pharmacological inhibition of protein tyrosine kinase activity or phosphoinositide 3-kinase activity. Importantly, ROS production was also inhibited by TMB-8 or EGTA, and inhibition of ROS production attenuated BCR-stimulated increases in intracellular calcium concentration. These effects were dose-dependent, which is consistent with a graded regulatory mechanism rather than a bistable switch mechanism. ROS can inhibit protein tyrosine phosphatase activity, and pharmacological inhibition of calcium signaling or ROS production suppressed BCR-mediated inhibition of two phosphatases, BCR-associated SHP-1 and CD45, which both exhibit a rapid and transient decrease in activity within seconds of BCR stimulation. Finally, the activity of a murine dual oxidase, DUOX1, may provide a mechanism for the regulation of ROS production by calcium, because enzymes in this family have two EF hand motifs, which are calcium-binding motifs. Using siRNA to suppress DUOX1 expression, the authors showed that ROS and calcium signaling were inhibited and that phosphorylation of Lyn was decreased. The authors suggest that low-intensity activation of the BCR produces an initial calcium signal that feeds back and regulates the amplitude of a rapidly produced ROS signal, which then transiently inhibits protein tyrosine phosphatase activity, thereby controlling the amplitude and kinetics of the BCR signal. D. K. Singh, D. Kumar, Z. Siddiqui, S. K. Basu, V. Kumar, K. V. S. Rao, The strength of receptor signaling is centrally controlled through a cooperative loop between Ca 2+ and an oxidant signal. Cell 121 , 281-293 (2005). [Online Journal]