Inhibitor Of Intracellular Calcium Mobilization Research Articles

Molecular mechanism of bitter taste receptor agonist-mediated relaxation of airway smooth muscle.

G-protein-coupled receptors (GPCRs) belonging to the type 2 taste receptors (TAS2Rs) family are predominantly present in taste cells to allow the perception of bitter-tasting compounds. TAS2Rs have also been shown to be expressed in human airway smooth muscle (ASM), and TAS2R agonists relax ASM cells and bronchodilate airways despite elevating intracellular calcium. This calcium "paradox" (calcium mediates contraction by pro-contractile Gq-coupled GPCRs) and the mechanisms by which TAS2R agonists relax ASM remain poorly understood. To gain insight into pro-relaxant mechanisms effected by TAS2Rs, we employed an unbiased phosphoproteomic approach involving dual-mass spectrometry to determine differences in the phosphorylation of contractile-related proteins in ASM following the stimulation of cells with TAS2R agonists, histamine (an agonist of the Gq-coupled H1 histamine receptor) or isoproterenol (an agonist of the Gs-coupled β2-adrenoceptor) alone or in combination. Our study identified differential phosphorylation of proteins regulating contraction, including A-kinase anchoring protein (AKAP)2, AKAP12, and RhoA guanine nucleotide exchange factor (ARHGEF)12. Subsequent signaling analyses revealed RhoA and the T853 residue on myosin light chain phosphatase (MYPT)1 as points of mechanistic divergence between TAS2R and Gs-coupled GPCR pathways. Unlike Gs-coupled receptor signaling, which inhibits histamine-induced myosin light chain (MLC)20 phosphorylation via protein kinase A (PKA)-dependent inhibition of intracellular calcium mobilization, HSP20 and ERK1/2 activity, TAS2Rs are shown to inhibit histamine-induced pMLC20 via inhibition of RhoA activity and MYPT1 phosphorylation at the T853 residue. These findings provide insight into the TAS2R signaling in ASM by defining a distinct signaling mechanism modulating inhibition of pMLC20 to relax contracted ASM.

Utilization of an Intracellular Calcium Mobilization Assay for the Screening of Transduced FK506-Binding Proteins.

FK506-binding proteins (FKBPs) belong to the immunophilin family and are linked to various disease states, including the inflammatory response. The inhibition of cytokine and chemokine expression in addition to positive effects of FKBPs on corneal inflammation in animal models suggests that they may be used for ophthalmic delivery in the treatment of dry eye disease. To pass the effective barriers protecting eye tissues, testing the transduction domains of FKBPs is essential. However, monitoring their transduction efficiencies is not a simple task. The quantitative measurement of FKBP interactions was performed using a cell model with a specific G protein-coupled receptor, as FKBPs had been known to act at the inositol 1,4,5-trisphosphate receptor (IP3R) leading to the inhibition of intracellular calcium mobilization. Because of its luminescence amplitude and stability, human urotensin II receptor was expressed in aequorin parental cells to measure the action of selected FKBPs. This luminescence-based functional assay platform exhibited a high signal-to-background ratio of more than 100 and a Z' factor at 0.6204. As expected, changes in the sequence of the transduction domain affected the function of the FKBPs. The intracellular calcium mobilization assay with selected FKBPs represented a robust and reliable platform to screen initial candidates. Although the precise nature of the control that FKBPs exert on the IP3R is uncertain, this approach can be used to develop innovative anti-inflammatory treatments for dry eye disease by optimizing protein transduction domain sequences.

Mechanisms of the antiplatelet and analgesic effects of dextromethorphan and its metabolites.

Objective:In the present study, we investigated the effects of dextromethorphan (DM) and its metabolites, including dextrorphan (LK2), 3-methoxymorphinan (LK3), and 3-hydroxymorphinan (LK4), on platelet aggregation in vitro and the inflammatory pain caused by carrageenan in rats, and their underlying mechanisms.Materials and Methods:Rabbit platelets were pretreated with DM or its metabolites to assess their effects on platelet aggregation and related target mediators. In addition, the analgesic activity and the underlying mechanisms of DM and LK3 were investigated in a carrageenan-evoked thermal hyperalgesia rat model.Results:The inhibitory potency of DM and its metabolites on platelet aggregation induced by arachidonic acid or collagen was LK3> DM > LK4>> LK2 as demonstrated by the half-maximal inhibitory concentration values. Moreover, the mechanisms of the antiplatelet effect of DM and LK3 may involve the inhibition of intracellular calcium mobilization, expression of platelet surface glycoprotein IIb/IIIa, the formation of thromboxane B2, and elevation of platelet membrane fluidity. DM and LK3 also exhibited analgesic effects on carrageenan-evoked thermal hyperalgesia by suppressing the production of pro-inflammatory cytokines, nitric oxide, prostaglandin E2, and neutrophil infiltration in inflammatory sites.Conclusion:DM and its metabolites, especially LK3, exhibit both antiplatelet and analgesic effects, and may, therefore, potentially ameliorate platelet hyperactivity and inflammatory-related diseases.

Synthesis and pharmacological evaluation of 2-aryloxy/arylamino-5-cyanobenzenesulfonylureas as novel thromboxane A2 receptor antagonists

New series of original 2-aryloxy/arylamino-5-cyanobenzenesulfonylureas were synthesized and evaluated as thromboxane A2 receptor (TP receptor) antagonists. A functional pharmacological test was used, which consisted of measuring the inhibition of intracellular calcium mobilization in a model of mammalian cell line that specifically over-expressed the individual TPα or TPβ isoforms. 2-Arylamino-5-cyanobenzenesulfonylureas exhibited virtually identical affinity and/or functional activity than 2-aryloxy-5-cyanobenzenesulfonylureas for both TPα and TPβ, but some 2-aryloxy-substituted compounds showed increased selectivity for TPβ relative to TPα. Several compounds were found to be as potent as the 2-arylamino-5-nitrobenzenesulfonylurea reference compound BM-573, supporting the view that the bioisosteric replacement of the nitro group by a cyano group was tolerated.TP receptor antagonist activity of the most promising molecules was confirmed in a platelet aggregation assay using the TP receptor agonist U-46619 as a proaggregant.Three compounds (7e, 7h and 8h) were identified as leads for further non-clinical pharmacological and toxicological studies.

Effects of membrane cholesterol depletion and GPI‐anchored protein reduction on osteoblastic mechanotransduction

We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y(2) puringeric receptors. However, the molecular mechanisms by which fluid flow initiates cellular responses are still unclear. Accumulating evidence suggests that lipid rafts, one of the important membrane structural components, may play an important role in transducing extracellular fluid shear stress to intracellular responses. Due to the limitations of current techniques, there is no direct approach to study the role of lipid rafts in transmitting fluid shear stress. In this study, we targeted two important membrane components associated with lipid rafts, cholesterol, and glycosylphosphatidylinositol-anchored proteins (GPI-anchored proteins), to disrupt the integrity of cell membrane structures. We first demonstrated that membrane cholesterol depletion with the treatment of methyl-β-cyclodextrin inhibits oscillatory fluid flow induced intracellular calcium mobilization and ERK1/2 phosphorylation in MC3T3-E1 osteoblastic cells. Secondly, we used a novel approach to decrease the levels of GPI-anchored proteins on cell membranes by overexpressing glycosylphosphatidylinositol-specific phospholipase D in MC3T3-E1 osteoblastic cells. This resulted in significant inhibition of intracellular calcium mobilization and ERK1/2 phosphorylation in response to oscillatory fluid flow. Finally, we demonstrated that cholesterol depletion inhibited oscillatory fluid flow induced ATP releases, which were responsible for the activation of calcium signaling pathways in MC3T3-E1 osteoblastic cells. Our findings suggest that cholesterol and GPI-anchored proteins, two membrane structural components related to lipid rafts, may play an important role in osteoblastic cell mechanotransduction.

CGMP-Dependent Protein Kinase I and Smooth Muscle Relaxation

See related article, pages 1096–1103 The maintenance of vascular tone is central to the regulation of blood pressure and tissue perfusion and plays a role in the pathogenesis of hypertension and atherosclerosis. Vascular tone is determined by the balance of vasodilator and vasoconstrictor stimuli. After several decades of research, the NO/cGMP/cGMP-dependent protein kinase (cGK) pathway is now recognized as an important mediator of vasodilation. However, the mechanisms by which cGK causes smooth muscle relaxation continue to be an important question. Smooth muscle contraction and relaxation are tightly coupled to the phosphorylation and dephosphorylation, respectively, of the regulatory myosin light chain.1 Myosin light chain phosphorylation state is determined by the relative activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). MLCK phosphorylates MLC leading to contraction,2 and MLCP dephosphorylates MLC, leading to relaxation3 (Figure). Both MLCK and MLCP activities are highly regulated. MLCK activity is activated by the binding of calcium/calmodulin and thus is the primary mechanism linking intracellular calcium concentration to smooth muscle contractility.4 MLCP activity is regulated by both vasodilator and vasoconstrictor stimuli, and is therefore responsible for much of the calcium-independent regulation of contractility (reviewed in5). Figure. MLC phosphorylation determines smooth muscle contractility. Contractile agonists lead to inositol 1,4,5 triphosphate (IP3) production or activation of RhoA (RhoA-GTP). IP3 binding to its receptor in the sarcoplasmic reticulum leads to release of Ca2+. Ca2+/calmodulin binds to and activates MLCK, which in turn phosphorylates MLC (calcium-dependent contraction). Activated RhoA binds to and activates ROCK, leading to phosphorylation and inhibition of MLCP, inhibiting MLC dephosphorylation (calcium-independent contraction). cGKI mediates relaxation by inhibiting both calcium-dependent and -independent contraction. cGKIα activates MLCP by a direct interaction and by inhibition of RhoA, and activates RGS2 to inhibit Gαq …

Pharmacological Study of Oyster Mushroom (Pleurotus Ostreatus) Extract on Isolated Guinea Pig Trachea Smooth Muscle

Mushroom farm workers suffer from respiratory symptoms during the farming of mushrooms. The objective of this study was to analyze the effects of oyster mushroom (Pleurotus ostreatus) extract (OME) on isolated guinea pig tracheal smooth muscle in vitro. Isolated guinea pig tracheal tissue from 27 nonsensitized guinea pigs were studied. The OME was obtained from indoor mushroom growing fields and prepared as a 1:10 w/v aqueous solution. Dose-related contractions of nonsensitized guinea pig trachea were demonstrated using these extracts. The OME contained significant quantities of bacterial components (eg., endotoxin: 43,072.92 EU/mg). Parallel, pharmacological studies were performed by pre-treating the tissues with mediator-modifying agents including atropine, indomethacin, pyrilamine, BPB, acivicin, NDGA, captopril, TMB8 and capsaicin. Atropine consistently and strikingly reduced the contractile effects of this extract. These observations suggest an interaction of the OME with parasympathetic nerves or more directly with muscarinic receptors. Pretreatment with TMB8 (inhibitor of intracellular calcium mobilization) also significantly blocked the constrictor effect of OME, indicating a role of calcium mobilization in the constricting effect of OME. Inhibition of contraction by blocking of other mediators was less effective and varied depending on the drug. We conclude that OME causes a dose-related airway smooth muscle constriction by nonimmunological mechanisms involving a variety of airway mediators and possibly cholinergic receptors. This effect is not dependent on pre-sensitization of the guinea pigs.

Activation of Endothelial Nitric-oxide Synthase by the p38 MAPK in Response to Black Tea Polyphenols

Black tea improves endothelial function in patients with coronary artery disease. We sought to determine the responsible components of black tea and elucidate the underlying cell signaling mechanisms. We exposed porcine aortic endothelial cells to components of black tea and found that the polyphenol fraction acutely enhanced nitric oxide bioactivity. This effect involved endothelial nitric-oxide synthase (eNOS) phosphorylation at Ser-1177 and dephosphorylation at Thr-495, consistent with increased eNOS activity. These effects were calcium-dependent, as removal of extracellular calcium prevented eNOS phosphorylation at Ser-1177, whereas inhibition of intracellular calcium mobilization with TMB-8 blunted Thr-495 dephosphorylation. Black tea polyphenol-induced eNOS activation appeared dependent upon the phosphatidylinositol 3-kinase-Akt pathway, as it was significantly inhibited by LY294002 and a dominant negative Akt, respectively. Pharmacological inhibition of p38 mitogen-activated protein kinase (p38 MAPK) with either SB202190 or SB203580 as well as overexpression of a dominant negative p38 MAPKalpha attenuated both eNOS activation and phosphorylation changes in response to black tea polyphenols. Inhibition of p38 MAPKalpha also blunted Akt activation in response to black tea polyphenols, suggesting that p38alpha MAPK is upstream of Akt in this pathway. Finally, a constitutively active mutant of MKK6bE, an upstream kinase for p38 MAPK, enhanced both the basal and stimulated activity of Akt, leading to increased eNOS activity. Taken together, these data identify the p38 MAPK as an upstream component of Akt-mediated eNOS activation.

Cellular cholesterol enrichment impairs T cell activation and chemotaxis

Human aging is associated with an increase in immune cell cholesterol levels, independent of circulating cholesterol levels. The effects of such an increase in membrane cholesterol on lipid raft-associated immune cell function have not been investigated. We sought to examine the effects of in vitro cholesterol loading on two known lipid raft-associated pathways of T cells, namely T cell activation and chemokine stimulation. Using β-cyclodextrin (BCD) as a vehicle, we were able to rapidly load cholesterol onto human T cell lines and primary peripheral blood T cells without inducing significant cell toxicity. Loading of cholesterol to four-fold that of normal levels induced significant inhibition of intracellular calcium mobilization by both αCD3 and SDF-1α. Cholesterol-loaded peripheral T cells were completely unresponsive to αCD3/αCD28 stimulation, demonstrating no increase in IL-2, GM1 expression or cell size. T cell polarization of lipid rafts to αCD3/αCD28 beads was also impaired. In addition, cholesterol loading potently inhibited SDF-1α-induced chemotaxis. We propose that excess membrane cholesterol could potentially disrupt raft-related cell functions downstream of receptor triggering and that the loss of cholesterol regulation of aging immune cells could contribute to immune cell senescence.

Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity

VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1, but not on other known adhesion molecules. Here we have investigated the signalling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilization are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho-family GTPase Rac1, and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity that are required for the activation of NADPH oxidase.

LAAE-14, a new in vitro inhibitor of intracellular calcium mobilization, modulates acute and chronic inflammation

A new lipidic acid-amido ether derivative (LAAE-14) able to reduce dose-dependently the calcium increases mediated either by calcium ionophore ionomycin, by the endoplasmic reticular Ca 2+-ATPase inhibitor thapsigargin, or by the chemotactic tripeptide N-formyl- l-methionyl- l-leucyl- l-phenylalanine (fMLP), in human neutrophils as well as in murine peritoneal macrophages, but not ATP, has been evaluated as a potential anti-inflammatory drug. This compound attenuated leukocyte activation by means of its inhibitory effect on the respiratory burst elicited in both types of cells by 12- O-tetradecanoyl phorbol 13-acetate, by inhibition of the degranulation process induced by cytochalasin B+fMLP or cytochalasin B+platelet activating factor, as well as by reduction of leukotriene B 4 synthesis induced by the calcium ionophore A23187. In addition, in zymosan-stimulated mouse peritoneal macrophages LAAE-14 caused a potent inhibition of nitrite and prostaglandin E 2 production. This compound exerted acute and chronic anti-inflammatory effects by oral route, that may be related with several mechanisms such as attenuation of leukocyte activation, inhibition of inducible nitric oxide synthase, cyclo-oxygenase-2 and cytosolic phospholipase A 2 expression as well as reduction in tumour necrosis factor-α production. Its anti-inflammatory profile is clearly correlated with its behavior as inhibitor of intracellular calcium mobilization. The profile and potency of this compound may have relevance for the inhibition of the inflammatory response at different levels and may represent a new approach to the development of new anti-inflammatory drugs.

Characterization of a novel NCAM ligand with a stimulatory effect on neurite outgrowth identified by screening a combinatorial peptide library.

The neural cell adhesion molecule, NCAM, plays a key role in neural development and plasticity mediating cell adhesion and signal transduction. By screening a combinatorial library of synthetic peptides with NCAM purified from postnatal day 10 rat brains, we identified a nonapeptide, termed NCAM binding peptide 10 (NBP10) and showed by nuclear magnetic resonance analysis that it bound the NCAM IgI module of NCAM. NBP10 modulated cell aggregation as well as neurite outgrowth induced specifically by homophilic NCAM binding. Moreover, both monomeric and multimeric forms of NBP10 stimulated neurite outgrowth from primary hippocampal neurons. The neurite outgrowth response to NBP10 was inhibited by a number of compounds previously shown to inhibit neurite outgrowth induced by homophilic NCAM binding, including voltage-dependent calcium channel antagonists, suggesting that NBP10 induced neurite outgrowth by activating a signal transduction pathway similar to that activated by NCAM itself. Moreover, an inhibitor of intracellular calcium mobilization, TMB-8, prevented NBP10-induced neurite outgrowth suggesting that NCAM-dependent neurite outgrowth also requires mobilization of calcium from intracellular calcium stores in addition to calcium influx from extracellular sources. By single-cell calcium imaging we further demonstrated that NBP10 was capable of inducing an increase in intracellular calcium in PC12E2 cells. Thus, the NBP10 peptide is a new tool for the study of molecular mechanisms underlying NCAM-dependent signal transduction and neurite outgrowth, and could prove to be a useful modulator of regenerative processes in the peripheral and central nervous system.

Increased intracellular calcium is required for neurite outgrowth induced by a synthetic peptide ligand of NCAM

We have recently identified a synthetic peptide, termed C3, capable of binding the first immunoglobulin-like module of neural cell adhesion molecule (NCAM) by means of combinatorial chemistry and shown that this NCAM ligand promotes neurite outgrowth. By means of single cell calcium imaging using the calcium-sensitive probe fura-2-acetomethyl ester, we here show that the C3-peptide induced an increase in intracellular calcium in primary hippocampal neurons and PC12-E2 cells, presumably requiring mobilization of calcium from both extracellular and intracellular stores. We further observed that C3-induced neurite outgrowth was inhibited by antagonists of voltage-dependent calcium channels as well as by an inhibitor of intracellular calcium mobilization, TMB-8. These findings demonstrate at the single cell level that a synthetic NCAM ligand directly can induce an increase in intracellular calcium and suggest that NCAM-dependent neurite outgrowth requires calcium mobilization from both extracellular and intracellular calcium stores. Thus, the C3-peptide may be regarded as a useful tool for the study of NCAM-dependent signal transduction. Furthermore, the peptide may be of considerable therapeutical interest for the treatment of neurodegenerative disorders.

8-(Diethylamino)octyl-3,4,5-trimethoxybenzoate, HCl], the inhibitor of intracellular calcium mobilization, blocked mitogen-induced T cell proliferation by interfering with the sustained phase of protein kinase C activation.

The physiological role of IP(3)-dependent Ca(2+) release in T cell activation was in question due to the contradictory findings that [8-(Diethylamino)octyl-3,4,5-trimethoxybenzoate, HCl] (TMB-8), an inhibitor of intracellular Ca(2+) mobilization, blocked T cell proliferation, curtailing specifically the level of released Ca(2+) did not affect T cell activation and T cell line lacking IP(3) receptor was defective in IL-2 production in response to TCR/CD3 ligand. In the present study we found that TMB-8 inhibited Concanavalin A (Con A)- but not PMA/Ionomycin-induced T cell proliferation in a reversible and dose-dependent manner. The kinetic study revealed that TMB-8 exerted the inhibitory effect at a very early step of T cell activation. The Ca(2+) ionophore ionomycin augmented instead of overcoming the inhibitory effect of TMB-8, although the same doses of ionomycin alone had no effect on Con A-induced T cell proliferation. PMA the metabolically stable, but not diacylglycerol (DAG) the metabolically labile, activator of protein Kinase C (PKC) completely overcome the antiproliferative effect of TMB-8. A specific DAG lipase inhibitor RHC80267 also overcome the effect of TMB-8. Taken together, these results showed that the process of Ca(2+) release through IP(3) receptor, not the released Ca(2+), is essential for the sustained phase of PKC activation during T cell proliferation.

Shear stress enhances prostacyclin release from endocardial endothelial cells

The effect of shear stress on the release of prostacyclin (PGI 2) from cultured endocardial endothelial cells (EECs) was investigated. EECs were harvested from the right ventricle (RV) and the left ventricle (LV) of porcine heart. Confluent EECs were incubated under various degrees of shear stress (0.2, 1, 4 and 6 dyne/cm 2) and PGI 2 release from each cell was measured. PGI 2 release from LV-EECs and RV-EECs was enhanced by the elevation of shear stress in a shear-dependent manner with a rapid increase at the onset of flow; however, there was no significant difference in PGI 2 production between RV-EECs and LV-EECs. production of PGI 2 was significantly inhibited from cells exposed to 8-(dimetilamino) octyl 3,4,5-trymrthoxybenzoate hydrochloride (10 and 100 μM: an inhibitor of intracellular calcium mobilization) or cyclopiazonic acid (10 μM: an endoplasmic reticulum Ca 2+-ATPase inhibitor). These results indicate that shear stress enhances PGI 2 release from cultured EECs and that mechanotransduction of shear stress depends on calcium mobilization in EECs.

Intracellular calcium in prothoracic glands of Manduca sexta

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP 3-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.

β-Amyloid-induced IL-1 β release from an activated human monocyte cell line is calcium- and G-protein-dependent

The proinflammatory cytokine, interleukin-1 (IL-1) is elevated in the Alzheimer's disease (AD) brain [1–3]. Studies from our laboratory have demonstrated that β-amyloid (A β) 1–42, fibrillar A β 1–40 and A β 25–35 induce the release of IL-1 β from activated THP-1 cells, a human monocyte cell line [4, 9]. A β also is chemotactic for primary rodent microglia and peritoneal macrophages [5]. We hypothesize that A β is a chemokine and induces these responses by interaction with chemotactic receptors. If this is true, then these A β-induced responses should be calcium-dependent and require activation of pertussis toxin-sensitive G-proteins. To test this hypothesis, THP-1 cells were grown in culture with lipopolysaccharide (LPS) and incubated with A β 1–42 (5 μM) in the presence and absence of a calcium chelator, an inhibitor of intracellular calcium mobilization, a calcium channel blocker, or pertussis toxin, a bacterial endotoxin which uncouples G proteins from receptors by catalyzing the ADP ribosylation of cysteine near the carboxy-terminus of the α subunit [6]. The media was collected and IL-1 β present in the media was measured using an ELISA. Treatment of LPS-activated THP-1 cells with A β 1–42 significantly elevated IL-1 β released into the media as previously shown. Addition of ethylene glycol-bis ( β-aminothyl ether) N, N, N′, N′-tetraacetic acid (EGTA) (0.5 mM), a calcium chelator, to the media blocked A β-induced IL-1 β release, but had no effect on LPS-activated THP-1 cell release of IL-1 β. The presence of 3,4,5-trimethoxybenzoic acid 8-(diethyl amino)-octyl ester (TMB-8), an inhibitor of intracellular calcium mobilization, as well as nickel chloride, a non-specific calcium channel blocker, in the media also inhibited A β-induced IL-1 release from LPS-activated THP-1 cells. IL-1 β release from activated THP-1 monocytes incubated with TMB-8 and nickel chloride without A β remained at baseline values. Pretreatment of THP-1 monocytes with pertussis toxin for 4 h, followed by LPS activation and incubation with A β, antagonized the release of IL-1 β from these cells, but did not alter IL-1 β release from activated THP-1 monocytes. These data suggest that A β-induced IL-1 β release from these cells is calcium-dependent and requires the activation of specific G-proteins. These findings are consistent with known second messengers that are activated following stimulation of chemotactic receptors.

Interaction between Calcium Ions and Bacillus thuringiensis Toxin Activity against Sf9 Cells (Spodoptera frugiperda, Lepidoptera)

The effects of calcium ions and modulators of calcium movement on Bacillus thuringiensis insecticidal protein toxicity were investigated with Sf9 cells (Spodoptera frugiperda, fall armyworm) by a new B. thuringiensis toxicity assay based on measurement of fluorescence of ethidium homodimer, a high-affinity DNA stain. CryIC toxicity was substantially stimulated by extracellular calcium in a dose-dependent way (in the millimolar range), while toxicity enhancement could not be replicated when calcium was replaced by barium. This incremental toxicity was reduced by cobalt and lanthanum ions, two inorganic-calcium transport inhibitors. Methoxyverapamil, a voltage-dependent calcium channel blocker, and nifedipine, an inhibitor of dihydropyridine-sensitive L-type calcium channels, had no effect on CryIC toxin activity, but BAY K 8644, an L-type calcium channel activator, increased CryIC activity at high concentrations of extracellular calcium. While A23187, a calcium ionophore, and TMB-8, an inhibitor of intracellular-calcium mobilization, did not change CryIC-induced mortality, thapsigargin, an inhibitor of calcium uptake in intracellular stores, and more particularly trifluoperazine, which inhibits calcium-calmodulin-dependent processes, increased CryIC-mediated toxicity. The incremental effect of extracellular calcium on CryIC-induced toxicity was consistent with an increased concentration of intracellular calcium.

培養ラット血管平滑筋細胞の遊走と増殖におよぼす抗アレルギー薬pemirolastの抑制効果

An antiallergic drug, pemirolast potassium (TBX), has been shown to inhibit the release of pro-inflammatory mediators from mast cells and eosinophils through the inhibition of intracellular calcium mobilization and modulation of phosphatidylinositol (PI) turnover.We have studied the effect of TBX on cell proliferation and migration of vascular smooth muscle cells (SMCs) in vitro. SMCs were cultured from rat aorta and were stimulated with fetal calf serum (FCS), platelet-derived growth factor (PDGF), angiotensin II (ATII) or endothelfin I (ET-I).In the cell proliferation assay. DNA synthesis was measured as BrdU incorporation into DNA at 36hr after FCS stimulation. TBX at concentrations from 10-7 to 10-4M inhibited BrdU incorporation into DNA. This finding was consistent with the observation that TBX dose-dependently decreased the number of cells at 24, 36 and 48hr after FCS stimulation. Furthermore, TBX (10-4M) inhibited cell proliferation stimulated with PDGF, ATII or ET-I.On the migration, the effect of TBX at concentrations from 10-7 to 10-4M was examined by Boyden's chamber method. TBX dose-dependently inhibited cell migration as determined by number of cells.Morever, we examined the inhibitory effect of TBX on 1, 4, 5-IP3 formation induced by ATII in SMCs. TBX also suppressed PI turnover.These findings indicated that TBX may intervene initiation and progression of atherosclerosis through the inhibition of proliferation and migration of arterial SMCs. Therefore, TBX may be clinically applicable for the prevention of restenosis after percutaneous transluminal coronary angioplasty.

Involvement of calcium in prothoracicotropic stimulation of ecdysone synthesis inGalleria mellonella

The cytosolic free calcium was measured with Fura-2 in single prothoracic gland cells of Galleria larvae. During the last two larval instars calcium concentration correlated with ecdysone secretion by the glands. Addition of prothoracicotropic hormone (PTTH) from brains of Galleria larvae to prothoracic glands in vitro induced a significant increase in calcium in the gland cells. This effect of PTTH was abolished by removal of extracellular calcium, or by the addition of lanthanum or of the calcium channel antagonists nicardipine and verapamil. The calcium channel agonist Bay K 8644 evoked an increase in intracellular calcium. TMB-8, an inhibitor of intracellular calcium mobilization, did not block the PTTH-stimulated rise in calcium concentration or ecdysone production, indicating that intracellular calcium stores are not involved in the calcium-mediated ecdysone synthesis. Moreover, PTTH seems to exert its action by influencing dihydropyridine-sensitive calcium channels in the plasma membrane. © 1996 Wiley-Liss, Inc.