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

Depending on the intracellular buffering of calcium by chelation, zinc has the following two apparent effects on neuronal excitability: enhancement or reduction. Zinc increased tonic activity in the depolarized state when neurons were intracellularly dialyzed with EGTA but attenuated the neuronal activity when BAPTA was used as an intracellular calcium buffer. This suggests that neuronal excitability can be modulated by zinc, depending on the internal calcium buffering capacity. In this study, we elucidated the mechanisms of zinc-mediated alterations in neuronal excitability and determined the effect of calcium-related channels on zinc-mediated alterations in excitability. The zinc-induced augmentation of firing activity was mediated via the inhibition of small-conductance calcium-activated potassium (SK) channels with not only the contribution of voltage-gated L-type calcium channels (VGCCs) and ryanodine receptors (RyRs), but also through the activation of VGCCs via melastatin-like transient receptor potential channels. We suggest that zinc modulates the dopaminergic neuronal activity by regulating not only SK channels as calcium sensors, but also VGCCs or RyRs as calcium sources. Our results suggest that the cytosolic calcium-buffering capacity can tightly regulate zinc-induced neuronal firing patterns and that local calcium-signaling domains can determine the physiological and pathological state of synaptic activity in the dopaminergic system.
Animals ; Calcium ; Calcium Channels, L-Type ; Cytosol ; Dopaminergic Neurons ; Egtazic Acid ; Electrophysiology ; Fires ; Neurons ; Potassium ; Rats ; Ryanodine Receptor Calcium Release Channel ; Transient Receptor Potential Channels ; Zinc

A comprehensive collection of proteins senses local changes in intracellular Ca²⁺ concentrations ([Ca²⁺](i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca²⁺ concentrations in cat esophageal smooth muscle cells. To measure [Ca²⁺](i) levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca²⁺](i) in the cells. Pretreatment with EGTA, an extracellular Ca²⁺ chelator, decreased the S1P-induced increase in [Ca²⁺](i), and an L-type Ca²⁺-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca²⁺ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP₃ receptor blocker, the S1P-evoked increase in [Ca²⁺](i) was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of G(i)-protein, suppressed the increase in [Ca²⁺](i) evoked by S1P. These results suggest that the S1P-induced increase in [Ca²⁺](i) in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca²⁺ from the InsP₃-sensitive Ca²⁺ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca²⁺ via the L type Ca²⁺ channel, which was dependent on activation of the S1P₄ receptor coupled to PTX-sensitive G(i) protein, via phospholipase C-mediated Ca²⁺ release from the InsP₃-sensitive Ca²⁺ pool in cat esophageal smooth muscle cells.
Animals ; Calcium ; Cats* ; Egtazic Acid ; Fura-2 ; Methods ; Microscopy, Fluorescence ; Muscle Contraction ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Nimodipine ; Pertussis Toxin ; Phospholipases ; Sarcoplasmic Reticulum ; Thapsigargin ; Type C Phospholipases

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*

Atopic dermatitis (AD) is characterized by disturbances in epidermal barrier functions and the hyperactive immune response. Staphylococcus aureus (S. aureus) can be cultured from 90% of AD skin lesions and can exacerbate or contribute to the persistent skin inflammation in AD by secreting toxins with superantigenic properties. Superantigens can induce mast cell (MC) degranulation after penetrating the epidermal barrier. The role of MCs in AD is suggested by the increase in the MC number and MC activation. MCs are activated for degranulation and mediator release by allergens that cross-link IgE molecules or by microbial products. Therefore, MCs may be critically involved in the pathogenesis of AD. However, the understanding mechanisms of MC degranulation by S. aureus in relation to AD have still not been fully elucidated. In this study, we found that live S. aureus or methicillin-resistant S. aureus (MRSA) but not heat-killed bacteria induced MC degranulation. The heat-treatment partially inhibited MC degranulation by conditioned media (CM) of S. aureus or MRSA. The calcium chelator ethylene glycol tetraacetic acid (EGTA) did not block MC degranulation induced by live S. aureus or MRSA, but EGTA-treatment partially inhibited MC degranulation by CM from S. aureus or MRSA. These results suggest that live S. aureus and MRSA can degranulate MCs via direct interaction which may be important role in AD.
Allergens ; Bacteria ; Calcium ; Culture Media, Conditioned ; Dermatitis, Atopic ; Egtazic Acid ; Humans* ; Immunoglobulin E ; Inflammation ; Mast Cells ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Skin ; Staphylococcus aureus ; Superantigens

Atopic dermatitis (AD) is characterized by disturbances in epidermal barrier functions and the hyperactive immune response. Staphylococcus aureus (S. aureus) can be cultured from 90% of AD skin lesions and can exacerbate or contribute to the persistent skin inflammation in AD by secreting toxins with superantigenic properties. Superantigens can induce mast cell (MC) degranulation after penetrating the epidermal barrier. The role of MCs in AD is suggested by the increase in the MC number and MC activation. MCs are activated for degranulation and mediator release by allergens that cross-link IgE molecules or by microbial products. Therefore, MCs may be critically involved in the pathogenesis of AD. However, the understanding mechanisms of MC degranulation by S. aureus in relation to AD have still not been fully elucidated. In this study, we found that live S. aureus or methicillin-resistant S. aureus (MRSA) but not heat-killed bacteria induced MC degranulation. The heat-treatment partially inhibited MC degranulation by conditioned media (CM) of S. aureus or MRSA. The calcium chelator ethylene glycol tetraacetic acid (EGTA) did not block MC degranulation induced by live S. aureus or MRSA, but EGTA-treatment partially inhibited MC degranulation by CM from S. aureus or MRSA. These results suggest that live S. aureus and MRSA can degranulate MCs via direct interaction which may be important role in AD.
Allergens ; Bacteria ; Calcium ; Culture Media, Conditioned ; Dermatitis, Atopic ; Egtazic Acid ; Humans* ; Immunoglobulin E ; Inflammation ; Mast Cells ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Skin ; Staphylococcus aureus ; Superantigens

The present study was designed to investigate the characteristics of gintonin, one of components isolated from Korean Ginseng on secretion of catecholamines (CA) from the isolated perfused model of rat adrenal gland and to clarify its mechanism of action. Gintonin (1 to 30 µg/ml), perfused into an adrenal vein, markedly increased the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. The gintonin-evoked CA secretion was greatly inhibited in the presence of chlorisondamine (1 µM, an autonomic ganglionic bloker), pirenzepine (2 µM, a muscarinic M₁ receptor antagonist), Ki14625 (10 µM, an LPA₁/₃ receptor antagonist), amiloride (1 mM, an inhibitor of Na⁺/Ca²⁺ exchanger), a nicardipine (1 µM, a voltage-dependent Ca²⁺ channel blocker), TMB-8 (1 µM, an intracellular Ca²⁺ antagonist), and perfusion of Ca²⁺-free Krebs solution with 5mM EGTA (a Ca²⁺chelater), while was not affected by sodium nitroprusside (100 µM, a nitrosovasodialtor). Interestingly, LPA (0.3~3 µM, an LPA receptor agonist) also dose-dependently enhanced the CA secretion from the adrenal medulla, but this facilitatory effect of LPA was greatly inhibited in the presence of Ki 14625 (10 µM). Moreover, acetylcholine (AC)-evoked CA secretion was greatly potentiated during the perfusion of gintonin (3 µg/ml). Taken together, these results demonstrate the first evidence that gintonin increases the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. This facilitatory effect of gintonin seems to be associated with activation of LPA- and cholinergic-receptors, which are relevant to the cytoplasmic Ca²⁺ increase by stimulation of the Ca²⁺ influx as well as by the inhibition of Ca²⁺ uptake into the cytoplasmic Ca²⁺ stores, without the increased nitric oxide (NO). Based on these results, it is thought that gintonin, one of ginseng components, can elevate the CA secretion from adrenal medulla by regulating the Ca²⁺ mobilization for exocytosis, suggesting facilitation of cardiovascular system. Also, these findings show that gintonin might be at least one of ginseng-induced hypertensive components.
Acetylcholine ; Adrenal Glands ; Adrenal Medulla* ; Amiloride ; Animals ; Cardiovascular System ; Catecholamines ; Chlorisondamine ; Cytoplasm ; Egtazic Acid ; Exocytosis ; Ganglia, Autonomic ; Nicardipine ; Nitric Oxide ; Nitroprusside ; Panax ; Perfusion ; Pirenzepine ; Rats ; Veins

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

Spinal dorsal horn nociceptive neurons have been shown to undergo long-term synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Here, we focused on the spinothalamic tract (STT) neurons that are the main nociceptive neurons projecting from the spinal cord to the thalamus. Optical technique using fluorescent dye has made it possible to identify the STT neurons in the spinal cord. Evoked fast mono-synaptic, excitatory postsynaptic currents (eEPSCs) were measured in the STT neurons. Time-based tetanic stimulation (TBS) was employed to induce long-term potentiation (LTP) in the STT neurons. Coincident stimulation of both pre- and postsynaptic neurons using TBS showed immediate and persistent increase in AMPA receptor-mediated EPSCs. LTP can also be induced by postsynaptic spiking together with pharmacological stimulation using chemical NMDA. TBS-induced LTP observed in STT neurons was blocked by internal BAPTA, or Ni2+, a T-type VOCC blocker. However, LTP was intact in the presence of L-type VOCC blocker. These results suggest that long-term plastic change of STT neurons requires NMDA receptor activation and postsynaptic calcium but is differentially sensitive to T-type VOCCs.
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ; Animals ; Calcium ; Depression ; Egtazic Acid ; Excitatory Postsynaptic Potentials ; Horns ; Long-Term Potentiation* ; N-Methylaspartate ; Neurons* ; Nociceptors ; Plastics ; Rats* ; Spinal Cord* ; Spinothalamic Tracts* ; Thalamus

The calcium-activated K+ (BKCa) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. Ca2+ is the main regulator of BKCa channel activation. The BKCa channel contains two high affinity Ca2+ binding sites, namely, regulators of K+ conductance, RCK1 and the Ca2+ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular Ca2+ levels through diverse G proteins such as Galphaq/11, Galphai, Galpha12/13, and Galphas and the related signal transduction pathway. In the present study, we examined LPA effects on BKCa channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated BKCa channel activation was also attenuated by the PLC inhibitor U-73122, IP3 inhibitor 2-APB, Ca2+ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated BKCa channel activation. The present study indicates that LPA-mediated activation of the BKCa channel is achieved through the PLC, IP3, Ca2+, and PKC pathway and that LPA-mediated activation of the BKCa channel could be one of the biological effects of LPA in the nervous and vascular systems.
Binding Sites ; Egtazic Acid ; Estrenes ; GTP-Binding Proteins ; Ion Channels ; Isoxazoles ; Lysophospholipids ; Naphthalenes ; Oocytes ; Potassium ; Potassium Channels ; Propionates ; Pyrrolidinones ; Receptors, Lysophosphatidic Acid ; Signal Transduction ; Xenopus