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

Oxidative stress appears to be implicated in the pathogenesis of various diseases including alcoholic liver injury. In this study we investigated the mechanism of apoptosis induced by tert-butyl hydroperoxide (TBHP) in HepG2 human hepatoblastoma cells. Treatment with TBHP significantly reduced glutathione content and glutathione reductase activity, and increased glutathione peroxidase activity, indicating that TBHP induced oxidative stress in the HepG2 cells. TBHP also induced reduction of cell viability and DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, TBHP induced a sustained increase in intracellular Ca2+ concentration, which was completely prevented by the extracellular Ca2+ chelation with EGTA. TBHP also induced Mn2+ influx. These results indicate that the intracellular Ca2+ increase by TBHP is exclusively due to Ca2+ influx from the extracellular site. Treatment with either an extracellular (EGTA) or an intracellular Ca2+ chelator (BAPTA/AM) significantly suppressed the TBHP-induced apoptosis. Taken together, these results suggest that TBHP induced the apoptotic cell death in the HepG2 cells and that Ca2+ influx may play an important role in the apoptosis induced by TBHP.
Apoptosis/drug effects* ; Calcium Signaling/drug effects* ; Chelating Agents/pharmacology ; Egtazic Acid/pharmacology ; Egtazic Acid/analogs & derivatives ; Hepatoblastoma/pathology* ; Hepatoblastoma/metabolism ; Hepatoblastoma/drug therapy ; Human ; Manganese/metabolism ; Oxidative Stress/drug effects ; Tumor Cells, Cultured ; tert-Butylhydroperoxide/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

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

We have shown that hyaluronic acid stimulates the proliferation of quiescent NIH 3T3 cells. We have shown that treatment of 1 mg/ml hyaluronic acid results in increase of tyrosine phosphorylation of two proteins, MW 124 kDa and 60 kDa as detected by anti-tyrosine antibodies by Western blot analysis. Maximum phosphorylation occurred within 2 h after addition of 1 mg/ml hyaluronic acid. Stimulation of proliferation was also accompanied by increase in c-Myc protein, which was inhibited by amlloride, an inhibitor of Na+/H+ antiporter and EGTA and increase in the steady state level of pRb, the RB gene product. These results suggest that the intracellular signal transduction pathways that mediate the stimulatory effects of hyaluronic acid on cellular proliferation are similar to those of growth factors.
3T3 Cells ; Amiloride/pharmacology ; Animal ; Cell Division ; Dose-Response Relationship, Drug ; Egtazic Acid/pharmacology ; Hyaluronic Acid/pharmacology* ; Mice ; Mitogens/pharmacology* ; Phosphoproteins/metabolism* ; Phosphorylation ; Proto-Oncogene Proteins c-myc/metabolism* ; Retinoblastoma Protein/metabolism* ; Signal Transduction ; Sodium-Hydrogen Antiporter/antagonists & inhibitors ; Tyrosine

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

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

Eukaryotic elongation factor eEF-2 mediates regulatory steps important for the overall regulation of mRNA translation in mammalian cells and is activated by variety of cellular conditions and factors. In this study, eEF-2 specific, Ca2+/CaM-dependent protein kinase III (CaM PK III), also called eEF-2 kinase, was examined under oxidative stress and cell proliferation state using CHO cells. The eEF-2 kinase activity was determined in the kinase buffer containing Ca2+ and CaM in the presence of eEF-2 and [gamma-32P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca2+ and CaM. Phosphorylation of eEF-2 was clearly identified in proliferating cells, but not detectable in CHO cells arrested in their growth by serum deprivation. The content of the eEF-2 protein, however, was equivalent in both cells. Using a phosphorylation state-specific antibody, we show that oxidant such as H2O2, which triggers a large influx of Ca2+, dramatically enhances the phosphorylation of eEF-2. In addition, H2O2-induced eEF-2 phosphorylation is dependent on Ca2+ and CaM, but independent of protein kinase C. In addition, okadaic acid inhibits phosphoprotein phosphatase 2A(PP2A)-mediated eEF-2 dephosphorylation. These results may provide a possible link between the elevation of intracellular Ca2+ and cell division and suggest that phosphorylation of eEF-2 is sensitive cellular reflex on stimuli that induces intracellular Ca2+ flux.
Animal ; CHO Cells ; Ca(2+)-Calmodulin Dependent Protein Kinase/*metabolism ; Cell Division ; Cells, Cultured ; Comparative Study ; Cytosol/enzymology ; Egtazic Acid/pharmacology ; Hamsters ; Human ; Hydrogen Peroxide/*pharmacology ; Mice ; Okadaic Acid/pharmacology ; Oxidants/*pharmacology ; Peptide Elongation Factors/metabolism ; Phosphoprotein Phosphatase/metabolism ; Phosphorylation ; Polyethylene Glycols/pharmacology ; Trifluoperazine/pharmacology

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*