This study was designed to determine the inhibitory effect of astragaloside Ⅳ(AS-Ⅳ), a principal bioactive component extracted from the Chinese medicinal Astragali Radix, on the inflammatory response of vascular endothelial cells induced by angiotensin Ⅱ(Ang Ⅱ), the most major pathogenic factor for cardiovascular diseases, and to clarify the role of calcium(Ca~(2+))/phosphatidylinosi-tol-3-kinase(PI3K)/protein kinase B(Akt)/endothelial nitric oxide synthase(eNOS)/nitric oxide(NO) pathway in the process. To be specific, human umbilical vein endothelial cells(HUVECs) were cultured in the presence of AS-Ⅳ with or without the specific inhibitor of NO synthase(NG-monomethyl-L-arginine, L-NMMA), inhibitor of PI3K/Akt signaling pathway(LY294002), or Ca~(2+)-chelating agent(ethylene glycol tetraacetic acid, EGTA) prior to Ang Ⅱ stimulation. The inhibitory effect of AS-Ⅳ on Ang Ⅱ-induced inflammatory response and the involved mechanism was determined with enzyme-linked immunosorbent assay(ELISA), cell-based ELISA assay, Western blot, and monocyte adhesion assay which determined the fluorescently labeled human monocytic cell line(THP-1) adhered to Ang Ⅱ-stimulated endothelial cells. AS-Ⅳ increased the production of NO by HUVECs in a dose-and time-dependent manner(P<0.05) and raised the level of phosphorylated eNOS(P<0.05). The above AS-Ⅳ-induced changes were abolished by pretreatment with L-NMMA, LY294002, or EGTA. Compared with the control group, Ang Ⅱ obviously enhanced the production and release of cytokines(tumor necrosis factor-α, interleukin-6), chemokines(monocyte chemoattractant protein-1) and adhesion molecules(intercellular adhesion molecule-1, vascular cellular adhesion molecule-1), and the number of monocytes adhered to HUVECs(P<0.05), which were accompanied by the enhanced levels of phosphorylated inhibitor of nuclear factor-κBα protein and activities of nuclear factor-κB(NF-κB)(P<0.05). This study also demonstrated that Ang Ⅱ-induced inflammatory response was inhibited by pretreatment with AS-Ⅳ(P<0.05). In addition, the inhibitory effect of AS-Ⅳ was abrogated by pretreatment with L-NMMA, LY294002, or EGTA(P<0.05). This study provides a direct link between AS-Ⅳ and Ca~(2+)/PI3K/Akt/eNOS/NO pathway in AS-Ⅳ-mediated anti-inflammatory actions in endothelial cells exposed to Ang Ⅱ. The results indicate that AS-Ⅳ attenuates endothelial cell-mediated inflammatory response induced by Ang Ⅱ via the activation of Ca~(2+)/PI3K/Akt/eNOS/NO signaling pathway.
Humans ; Angiotensin II/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; omega-N-Methylarginine/pharmacology* ; Egtazic Acid/pharmacology* ; Human Umbilical Vein Endothelial Cells ; NF-kappa B/metabolism* ; Nitric Oxide/metabolism* ; Cells, Cultured

This study aims to validate our hypothesis that acid-sensing ion channels (ASICs) may contribute to the symptom of pain in patients with chronic prostatitis (CP). We first established a CP rat model, then isolated the L5-S2 spinal dorsal horn neurons for further studies. ASIC1a was knocked down and its effects on the expression of neurogenic inflammation-related factors in the dorsal horn neurons of rat spinal cord were evaluated. The effect of ASIC1a on the Ca²⁺ ion concentration in the dorsal horn neurons of rat spinal cord was measured by the intracellular calcium ([Ca²⁺]i) intensity. The effect of ASIC1a on the p38/mitogen-activated protein kinase (MAPK) signaling pathway was also determined. ASIC1a was significantly upregulated in the CP rat model as compared with control rats. Acid-induced ASIC1a expression increased [Ca²⁺]i intensity in the dorsal horn neurons of rat spinal cord. ASIC1a also increased the levels of neurogenic inflammation-related factors and p-p38 expression in the acid-treated dorsal horn neurons. Notably, ASIC1a knockdown significantly decreased the expression of pro-inflammatory cytokines. Furthermore, the levels of p-p38 and pro-inflammatory cytokines in acid-treated dorsal horn neurons were significantly decreased in the presence of PcTx-1, BAPTA-AM, or SB203580. Our results showed that ASIC1a may contribute to the symptom of pain in patients with CP, at least partially, by regulating the p38/MAPK signaling pathway.
Acid Sensing Ion Channel Blockers/pharmacology* ; Acid Sensing Ion Channels/genetics* ; Animals ; Calcium/metabolism* ; Chelating Agents/pharmacology* ; Chronic Disease ; Cytokines/metabolism* ; Disease Models, Animal ; Egtazic Acid/pharmacology* ; Gene Knockdown Techniques ; Imidazoles/pharmacology* ; Inflammation/metabolism* ; MAP Kinase Signaling System/genetics* ; Male ; Pain/genetics* ; Peptides/pharmacology* ; Phosphorylation/drug effects* ; Posterior Horn Cells/metabolism* ; Prostatitis/complications* ; Protein Kinase Inhibitors/pharmacology* ; Pyridines/pharmacology* ; Rats ; Spider Venoms/pharmacology* ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/metabolism*

The dynamic polar polymers actin filaments and microtubules are usually employed to provide the structural basis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the ability to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly understood. Here we show that Ca(2+) oscillations induced by the Ca(2+) release from intracellular Ca(2+) store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation. The chelation of cytosolic Ca(2+) suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing exogenous Ca(2+) into sperm cells. Ca(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca(2+) promotes MSP disassembly by activating Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase calcineurin. In addition, Ca(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane, a regulated exocytosis required for sperm motility. Thus, Ca(2+) plays multifunctional roles during sperm activation in Ascaris suum.
Animals ; Ascaris suum ; metabolism ; Calcineurin ; metabolism ; Calcium ; metabolism ; Calmodulin ; metabolism ; Cytoskeleton ; metabolism ; Cytosol ; metabolism ; Egtazic Acid ; analogs & derivatives ; pharmacology ; Helminth Proteins ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; metabolism ; Male ; Membrane Potential, Mitochondrial ; physiology ; Mitochondria ; metabolism ; Pseudopodia ; metabolism ; Signal Transduction ; Sperm Motility ; Spermatids ; drug effects ; physiology ; Spermatogenesis ; Type C Phospholipases ; metabolism

In the present study, the modulatory effect of AMPA receptors on gamma-aminobutyric acid (GABA) transporter current was investigated on enzymatically isolated horizontal cells of carp retina. The GABA transporter current elicited by 1 mmol/L GABA was decreased immediately after pre-application of AMPA (30 mumol/L or 3 mmol/L) for 50 s. Application of 10 mmol/L BAPTA in intracellular solution inhibited the suppression effect of AMPA on GABA transporter current. The suppression effect induced by co-application of 3 mmol/L AMPA and 3 mmol/L NMDA was similar to that of 3 mmol/L AMPA or 3 mmol/L NMDA alone. These results suggest that the activation of AMPA receptors inhibits GABA transporter-mediated current by affecting intracellular Ca(2+) processes in the retinal horizontal cells, which is identical with the modulatory effect of NMDA receptors on GABA transporters.
Animals ; Carps ; Egtazic Acid ; analogs & derivatives ; pharmacology ; GABA Plasma Membrane Transport Proteins ; metabolism ; Receptors, Ionotropic Glutamate ; metabolism ; Retinal Horizontal Cells ; metabolism ; gamma-Aminobutyric Acid ; pharmacology

6-Hydroxydopamine (6-OHDA) is a neurotoxin and is commonly used to generate experimental models of Parkinson's disease (PD). In this study, we investigated the signaling molecules involved in the 6-OHDA-induced cell death using a neuronal catecholaminergic cell line (SK-N-SH cells), and the protective effect of fustin, a flavonoid from Rhus verniciflua Stokes, on 6-OHDA-induced neuronal death. 6-OHDA significantly increased levels of reactive oxygen species (ROS), intracellular Ca2+ ([Ca2+](i)), and p38 phosphorylation. In addition, this ROS increase by 6-OHDA was reduced by pretreatment with N-acetylcysteine (NAC), a free radical scavenger, but not by bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA), a Ca2+ chelator. However, the [Ca2+](i) increase induced by 6-OHDA was suppressed by NAC. Moreover, pretreatment with NAC or BAPTA significantly prevented the 6-OHDA-induced increases in p38 phosphorylation, Bax/Bcl-2 ratio, and caspase-3 activity. Although 6-OHDA-increased phosphorylation of p38 was prevented by NAC or BAPTA, inhibition of p38 by SB203580 did not suppress ROS, Bax/Bcl-2 ratio, or caspase-3 activity increases, and only partially prevented 6-OHDA-induced cell death, thus demonstrating that p38 activation is a component of a signaling pathway leading to the initiation of 6-OHDA-induced cell death, which acts in parallel with an ROS-Ca2+ -Bcl-2-caspase-3 pathway. Moreover, fustin not only suppressed 6-OHDA-induced cell death in a concentration-dependent manner but also blocked 6-OHDA-induced increases in ROS, [Ca2+](i), Bax/Bcl-2 ratio, caspase-3 activity, and p38 phosphorylation. These results suggest that fustin exerts neuroprotection against 6-OHDA-induced cell death.
Acetylcysteine/pharmacology ; Apoptosis ; Calcium/metabolism ; Caspase 3/metabolism ; Cell Death/drug effects ; Cell Line, Tumor ; Cytoprotection ; Egtazic Acid/analogs & derivatives/pharmacology ; Enzyme Activation ; Flavonoids/*pharmacology ; Humans ; Imidazoles/pharmacology ; Neurons/cytology/*drug effects ; Oxidopamine/*toxicity ; Phosphorylation ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Pyridines/pharmacology ; Reactive Oxygen Species/metabolism ; Rhus/*chemistry ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism

A previous report by this laboratory demonstrated that bacterial iron chelator (siderophore) triggers inflammatory signals, including the production of CXC chemokine IL-8, in human intestinal epithelial cells (IECs). Microarray-based gene expression profiling revealed that iron chelator also induces macrophage inflammatory protein 3 alpha (MIP-3alpha)/ CC chemokine-ligand 20 (CCL20). As CCL20 is chemotactic for the cells involved in host adaptive immunity, this suggests that iron chelator may stimulate IECs to have the capacity to link mucosal innate and adaptive immunity. The basal medium from iron chelator deferoxamine (DFO)-treated HT-29 monolayers was as chemotactic as recombinant human CCL20 at equivalent concentrations to attract CCR6+ cells. The increase of CCL20 protein secretion appeared to correspond to that of CCL20 mRNA levels, as determined by real-time quantitative RT-PCR. The efficacy of DFO at inducing CCL20 mRNA was also observed in human PBMCs and in THP-1 cells, but not in human umbilical vein endothelial cells. Interestingly, unlike other proinflammatory cytokines, such as TNF-alpha and IL-1beta, a time-dependent experiment revealed that DFO slowly induces CCL20, suggesting a novel mechanism of action. A pharmacologic study also revealed that multiple signaling pathways are differentially involved in CCL20 production by DFO, while some of those pathways are not involved in TNF-alpha-induced CCL20 production. Collectively, these results demonstrate that, in addition to some bacterial products known to induce host adaptive immune responses, direct chelation of host iron by infected bacteria may also contribute to the initiation of host adaptive immunity in the intestinal mucosa.
Calcium/metabolism ; Cell Movement/drug effects ; Chemokines, CC/genetics/*metabolism ; Deferoxamine/*pharmacology ; Egtazic Acid/analogs & derivatives/pharmacology ; HT29 Cells ; Humans ; Immunity, Mucosal/*drug effects ; Intestinal Mucosa/*drug effects/immunology/metabolism ; Iron Chelating Agents/*pharmacology ; Macrophage Inflammatory Proteins/genetics/*metabolism ; NF-kappa B/metabolism ; Phosphoprotein Phosphatase/physiology ; Protein Transport/drug effects ; Protein-Serine-Threonine Kinases/physiology ; RNA, Messenger/genetics/metabolism ; Receptors, Chemokine/metabolism ; Research Support, Non-U.S. Gov't

OBJECTIVETo study the effect of extremely low frequency magnetic fields on intracellular calcium concentration ([Ca(2+)]i).

METHODSFura-2 loaded HepG2 cells were exposed to 1.55 mT (average value), 16 Hz pulsed magnetic fields for 60 min and to 300 mT, 2 Hz rotating magnetic fields for 5 min, and then [Ca(2+)]i was measured by fluorescence spectrophotometer. [Ca(2+)]i of HepG2 cells was also measured when they were exposed to 0.9 mT [root mean square (rms)], 16 Hz sinusoidal magnetic fields in real time.

RESULTSThe R values (F(340) nm/F(380) nm) of the control and the exposed group were 2.4519 +/- 0.2378 and 2.5266 +/- 0.2915 respectively after HepG2 cells were exposed to 1.55 mT, 16 Hz magnetic fields, 1.365 0 +/- 0.0626 and 1.3602 +/- 0.0771 respectively to 300 mT, 2 Hz rotating magnetic fields. The ratios of the trendline slope [r((501 - 1,000)) / r((0 - 500))] from the data of R values were 1.1213 +/- 0.4559 and 1.0727 +/- 0.1971 respectively (P > 0.05), and the ratios of the intercept [b((501 - 1,000)) / b((0 - 500))] from the trendline were 0.9912 +/- 0.0098 and 0.9979 +/- 0.0060 (P > 0.05) when HepG2 cells were exposed to the 0.9 mT, 16 Hz sinusoidal magnetic fields.

CONCLUSIONThe effect of extremely low frequency magnetic fields on [Ca(2+)]i of HepG2 cells under the experimental condition has not been found.

Calcium ; metabolism ; Cell Line, Tumor ; drug effects ; metabolism ; radiation effects ; Chelating Agents ; pharmacology ; Egtazic Acid ; pharmacology ; Electromagnetic Fields ; Humans ; Ion Transport ; drug effects ; radiation effects ; Octoxynol ; pharmacology ; Spectrometry, Fluorescence ; Time Factors

Firing patterns of injured nerve fibers were recorded using the single-fiber firing recording technique. Under the same background firing pattern, three types of bursting were induced separately by EGTA, veratridine or high [Ca(2+)](o) in the same type of nerve fibers. The results suggest that different firing patterns are related to different stimuli, which means that each firing pattern carries corresponding neural information.
Action Potentials ; Animals ; Calcium ; pharmacology ; Egtazic Acid ; pharmacology ; Nerve Fibers ; drug effects ; pathology ; Rats ; Veratridine ; pharmacology

Characterization of ceramide-effector(s), which includes protein phosphatase 2A (PP2A) is an important prelude to understanding the molecular basis of sphingolipid-mediated biological effects such as cell growth, differentiation and apoptosis. Recently, the existence of a metal-dependent form of PP2A has been reported (Cai et al., 1995). In this study, we investigated the effects of metal ions and chelators on ceramide-activated PP2A (CAPP). Our study demonstrates that at 0.5 mM concentration, Mg2+ appears to have no significant effect on either basal or ceramide-stimulated phosphatase activities, whereas Ca2+ stimulated the basal phosphatase activity, but was inhibitory towards CAPP. Moreover, the divalent cations Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ and Zn2+ were tested and all were found to be inhibitory towards both CAPP and basal phosphatase activities. By contrast, Cs+ and Li+ had almost no effect on CAPP, although both stimulated basal phosphatase activity. The effects of EDTA and EGTA were tested and it was observed that EDTA decreased CAPP activity in a dose-dependent fashion, but had no effect upon basal phosphatase activity. These results suggest that CAPP is a metal-dependent protein, but, because Ca2+ inhibitied CAPP and EGTA was much less potent than EDTA in inhibiting CAPP, Ca2+ is unlikely to be its metal cofactor.
Cations, Divalent/*pharmacology ; Cell Line ; Edetic Acid/pharmacology ; Egtazic Acid/pharmacology ; Enzyme Activation ; Human ; Lymphocytes/cytology ; Phosphoprotein Phosphatase/drug effects/isolation & purification/*metabolism

Characterization of ceramide-effector(s), which includes protein phosphatase 2A (PP2A) is an important prelude to understanding the molecular basis of sphingolipid-mediated biological effects such as cell growth, differentiation and apoptosis. Recently, the existence of a metal-dependent form of PP2A has been reported (Cai et al., 1995). In this study, we investigated the effects of metal ions and chelators on ceramide-activated PP2A (CAPP). Our study demonstrates that at 0.5 mM concentration, Mg2+ appears to have no significant effect on either basal or ceramide-stimulated phosphatase activities, whereas Ca2+ stimulated the basal phosphatase activity, but was inhibitory towards CAPP. Moreover, the divalent cations Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ and Zn2+ were tested and all were found to be inhibitory towards both CAPP and basal phosphatase activities. By contrast, Cs+ and Li+ had almost no effect on CAPP, although both stimulated basal phosphatase activity. The effects of EDTA and EGTA were tested and it was observed that EDTA decreased CAPP activity in a dose-dependent fashion, but had no effect upon basal phosphatase activity. These results suggest that CAPP is a metal-dependent protein, but, because Ca2+ inhibitied CAPP and EGTA was much less potent than EDTA in inhibiting CAPP, Ca2+ is unlikely to be its metal cofactor.
Cations, Divalent/*pharmacology ; Cell Line ; Edetic Acid/pharmacology ; Egtazic Acid/pharmacology ; Enzyme Activation ; Human ; Lymphocytes/cytology ; Phosphoprotein Phosphatase/drug effects/isolation & purification/*metabolism