Exocytotic steps in cell-free system after cholesterol deprivation in synaptosomal plasma membranes and synaptic vesicles

Using a cell-free system we investigated a specific role of cholesterol in exocytotic processes. To modulate the cholesterol content in membrane methyl-beta-cyclodextrin was used as a cholesterol binding agent. The experimental conditions for cholesterol depletion from synaptosomal membrane structures were determined and depended on methyl-beta-cyclodextrin concentration, time and mediums temperature. The role of cholesterol was studied on the stages of synaptic vesicles docking and Ca(2+)-stimulated fusion which are the components of multivesicular compound exocytosis. Using dynamic light scattering technique we have found that after cholesterol depletion from synaptic vesicles the process of their aggregation (docking) remains unchanged. It was found that the rate of calcium-triggered fusion of synaptic vesicles depends on the membrane level of cholesterol. The decreasing level of synaptosomal plasma membrane cholesterol by 8% leads to suppression of the Ca(2+)-dependent membrane fusion with synaptic vesicles. But, under 25% reduction of plasma membrane cholesterol the level of membrane merging with synaptic vesicles did not differ from control; probably this is due to changes in physical properties of lipid bilayer and/ or disturbances in function of membrane proteins driving this process. In cholesterol depleted synaptosomes the exocytotic release of glutamate stimulated by calcium was decreased by 32%. Obtained data suggest that the cholesterol concenration in synaptosomal plasma membranes or synaptic vesicles is the crucial determinant for synaptic transmission efficiency in nerve terminals.     

Negative control of store-operated Ca2+ influx by B cell receptor cross-linking

An increase in the intracellular Ca(2+) concentration by B cell receptor (BCR) cross-linking plays important roles in the regulation of B cell functions. [Ca(2+)](i) is regulated by Ca(2+) release from the Ca(2+) store as well as store-operated Ca(2+) influx (SOC). Protein tyrosine kinases downstream of BCR cross-linking were shown to regulate the mechanism for Ca(2+) release. However, it remains elusive whether BCR cross-linking regulates SOC or not. In this study, we examined the effect of BCR cross-linking on thapsigargin-induced SOC in the DT40 B cells. We found that the SOC-mediated increase in intracellular Ca(2+) concentration was inhibited by BCR cross-linking. Using a membrane-potential-sensitive dye, we found that BCR cross-linking induced depolarization, which is expected to decrease the driving force of Ca(2+) influx and SOC channel conductance. When membrane potential was held constant by the transmembrane K(+) concentration gradient in the presence of valinomycin, the BCR-mediated inhibition of SOC was still observed. Thus, the BCR-mediated inhibition of SOC involves both depolarization-dependent and depolarization-independent mechanisms of SOC inhibition. The depolarization-independent inhibition of the SOC was abolished in Lyn-deficient, but not in Bruton's tyrosine kinase-, Syk- or SHIP (Src homology 2 domain containing phosphatidylinositol 5'-phosphatase)-deficient cells, indicating that Lyn is involved in the inhibition. These results show novel pathways of BCR-mediated SOC regulations.     

The effect of rottlerin in calcium regulation in HMC-1(560) cells is mediated by a PKC-delta independent effect

The human mast cell line (HMC-1(560)) is a good model for Ca(2+) signaling studies, because intracellular alkalinization is the mainly histamine release stimulus without changes in the intracellular Ca(2+) levels. This fact allows us to study Ca(2+) changes without degranulation, since this process can affected cellular viability. Ionomycin and thapsigargin have been fully used for induced Ca(2+) influx across SOC channels. When HMC-1(560) cells are incubated with rottlerin, 5 microM, for 5 min a strong inhibition of ionomycin-induced Ca(2+) influx is observed. However, when thapsigargin stimulates Ca(2+) influx, rottlerin did not show any effect on Ca(2+) levels. This fact point two possibilities, ionomycin and thapsigargin might activate different SOC channels or that these drugs might activate the same channel but in a different way in HMC-1(560) cells. The rottlerin inhibition of ionomycin-induced Ca(2+) influx is PKC-delta independent and this effect is not related with the store depletion, since rottlerin has the same effect when it is added before or after the stores are empty. FCCP, a know uncoupler of oxidative phosphorylation in mitochondria, induces the same inhibition in ionomycin Ca(2+) influx than rottlerin which point to the mitochondria as a cellular target to rottlerin.     

Spatial organization of Ca(2+) entry and exocytosis in mouse pancreatic beta-cells

Secretion from single pancreatic beta-cells was imaged using a novel technique in which Zn(2+), costored in secretory granules with insulin, was detected by confocal fluorescence microscopy as it was released from the cells. Using this technique, it was observed that secretion from beta-cells was limited to an active region that comprised approximately 50% of the cell perimeter. Using ratiometric imaging with indo-1, localized increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by membrane depolarization were also observed. Using sequential measurements of secretion and [Ca(2+)](i) at single cells, colocalization of exocytotic release sites and Ca(2+) entry was observed when cells were stimulated by glucose or K(+). Treatment of cells with the Ca(2+) ionophore 4-Br-A23187 induced large Ca(2+) influx around the entire cell circumference. Despite the nonlocalized increase in [Ca(2+)](i), secretion evoked by 4-Br-A23187 was still localized to the same region as that evoked by secretagogues such as glucose. It is concluded that Ca(2+) channels activated by depolarization are localized to specific membrane domains where exocytotic release also occurs; however, localized secretion is not exclusively regulated by localized increases in [Ca(2+)](i), but instead involves spatial localization of other components of the exocytotic machinery.     

Store-operated Ca2+ entry in porcine airway smooth muscle

Ca(2+) influx triggered by depletion of sarcoplasmic reticulum (SR) Ca(2+) stores [mediated via store-operated Ca(2+) channels (SOCC)] was characterized in enzymatically dissociated porcine airway smooth muscle (ASM) cells. When SR Ca(2+) was depleted by either 5 microM cyclopiazonic acid or 5 mM caffeine in the absence of extracellular Ca(2+), subsequent introduction of extracellular Ca(2+) further elevated [Ca(2+)](i). SOCC was insensitive to 1 microM nifedipine- or KCl-induced changes in membrane potential. However, preexposure of cells to 100 nM-1 mM La(3+) or Ni(2+) inhibited SOCC. Exposure to ACh increased Ca(2+) influx both in the presence and absence of a depleted SR. Inhibition of inositol 1,4,5-trisphosphate (IP)-induced SR Ca(2+) release by 20 microM xestospongin D inhibited SOCC, whereas ACh-induced IP(3) production by 5 microM U-73122 had no effect. Inhibition of Ca(2+) release through ryanodine receptors (RyR) by 100 microM ryanodine also prevented Ca(2+) influx via SOCC. Qualitatively similar characteristics of SOCC-mediated Ca(2+) influx were observed with cyclopiazonic acid- vs. caffeine-induced SR Ca(2+) depletion. These data demonstrate that a Ni(2+)/La(3+)-sensitive Ca(2+) influx via SOCC in porcine ASM cells involves SR Ca(2+) release through both IP(3) and RyR channels. Additional regulation of Ca(2+) influx by agonist may be related to a receptor-operated, noncapacitative mechanism.     

Mechanisms of integrin-mediated calcium signaling in MDCK cells: regulation of adhesion by IP3- and store-independent calcium influx.

Peptides containing Arg-Gly-Asp (RGD) immobilized on beads bind to integrins and trigger biphasic, transient increases in intracellular free Ca2+ ([Ca2+]i) in Madin-Darby canine kidney epithelial cells. The [Ca2+]i increase participates in feedback regulation of integrin-mediated adhesion in these cells. We examined influx pathways and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ store release as possible sources of the [Ca2+]i rise. The RGD-induced [Ca2+]i response requires external Ca2+ (threshold approximately 150 microM), and its magnitude is proportional to extracellular calcium. RGD-induced transients were attenuated by Ca2+ channel inhibitors (Ni2+ and carboxy-amidotriazole) or by plasma membrane depolarization, indicating that Ca2+ influx contributes to the response. Loading cells with heparin reduced the size of RGD-induced [Ca2+]i transients, indicating that IP3-mediated release of Ca2+ from stores may also contribute to the RGD response. Depletion of Ca2+ stores with thapsigargin activated Ni(2+)-sensitive Ca2+ influx that might also be expected to occur after IP3-mediated depletion of stored Ca2-. However, RGD elicited a Ni(2+)-sensitive Ca2+ influx even after pretreatment with thapsigargin, indicating that Ca2+ influx is controlled by a mechanism independent of IP3-mediated store depletion. We conclude that RGD-induced [Ca2+]i transients in Madin-Darby canine kidney cells result primarily from the combination of two distinct mechanisms: 1) IP3-mediated release of intracellular stores, and 2) activation of a Ca2+ influx pathway regulated independently of IP3 and Ca2+ store release. Because Ni2+ and carboxy-amidotriazole inhibited adhesion, whereas store depletion with thapsigargin had little effect, we suggest that the Ca2+ influx mechanism is most important for feedback regulation of integrin-mediated adhesion by increased [Ca2+]i.     

A pyrazole derivative,YM-58483, potently inhibits store-operated sustained Ca2+ influx and IL-2 production in T lymphocytes

In nonexcitable cells, Ca(2+) entry is mediated predominantly through the store depletion-dependent Ca(2+) channels called store-operated Ca(2+) (SOC) or Ca(2+) release-activated Ca(2+) channels. YM-58483, a pyrazole derivative, inhibited an anti-CD3 mAb-induced sustained Ca(2+) influx in acute T cell leukemia, Jurkat cells. But it did not affect an anti-CD3 mAb-induced transient intracellular Ca(2+) increase in Ca(2+)-free medium, nor anti-CD3 mAb-induced phosphorylation of phospholipase Cgamma1. It was suggested that YM-58483 inhibited Ca(2+) influx through SOC channels without affecting the TCR signal transduction cascade. Furthermore, YM-58483 inhibited thapsigargin-induced sustained Ca(2+) influx with an IC(50) value of 100 nM without affecting membrane potential. YM-58483 inhibited by 30-fold the Ca(2+) influx through SOC channels compared with voltage-operated Ca(2+) channels, while econazole inhibited both SOC channels and voltage-operated Ca(2+) channels with an equivalent range of IC(50) values. YM-58483 potently inhibited IL-2 production and NF-AT-driven promoter activity, but not AP-1-driven promoter activity in Jurkat cells. Moreover, this compound inhibited delayed-type hypersensitivity in mice with an ED(50) of 1.1 mg/kg. Therefore, we concluded that YM-58483 was a novel store-operated Ca(2+) entry blocker and a potent immunomodulator, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 would be a candidate for the study of capacitative Ca(2+) entry mechanisms through SOC/CRAC channels and for identification of putative Ca(2+) channel genes.     

A spirochete surface protein uncouples store-operated calcium channels in fibroblasts: a novel cytotoxic mechanism

The cytotoxicity of infectious agents can be mediated by disruption of calcium signaling in target cells. Outer membrane proteins of the spirochete Treponema denticola, a periodontal pathogen, inhibit agonist-induced Ca(2+) release from internal stores in gingival fibroblasts, but the mechanism is not defined. We determined here that the major surface protein (Msp) of T. denticola perturbs calcium signaling in human fibroblasts by uncoupling store-operated channels. Msp localized in complexes on the cell surface. Ratio fluorimetry showed that in cells loaded with fura-2 or fura-C18, Msp induced cytoplasmic and near-plasma membrane Ca(2+) transients, respectively. Increased conductance was confirmed by fluorescence quenching of fura-2-loaded cells with Mn(2+) after Msp treatment. Calcium entry was blocked with anti-Msp antibodies and inhibited by chelating external Ca(2+) with EGTA. Msp pretreatment reduced the amplitude of [Ca(2+)](i) transients upon challenge with ATP or thapsigargin. In experiments using cells loaded with mag-fura-2 to report endoplasmic reticulum Ca(2+), Msp reduced Ca(2+) efflux from endoplasmic reticulum stores when ATP was used as an agonist. Msp alone did not induce Ca(2+) release from these stores. Msp inhibited store-operated influx of extracellular calcium following intracellular Ca(2+) depletion by thapsigargin and also promoted the assembly of subcortical actin filaments. This actin assembly was blocked by chelating intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester. The reduced amplitude of agonist-induced transients and inhibition of store-operated Ca(2+) entry due to Msp were reversed by latrunculin B, an inhibitor of actin filament assembly. Thus, Msp retards Ca(2+) release from endoplasmic reticulum stores, and it inhibits subsequent Ca(2+) influx by uncoupling store-operated channels. Actin filament rearrangement coincident with conformational uncoupling of store-operated calcium fluxes is a novel mechanism by which surface proteins and toxins of pathogenic microorganisms may damage host cells.     

Diverse effects of sphingosine on calcium mobilization and influx in differentiated HL-60 cells

Sphingosine induces a biphasic increase in cytosolic-free Ca(2+)([Ca(2+)](i)) with an initial peak followed by a sustained increase in HL-60 cells differentiated into neutrophil-like cells. The initial peak is not affected by the presence of ethylene glycol bis (beta-aminoethyl ether) N, N, N', N-tetraacetic acid (EGTA) in the buffer and appears to be dependent on conversion of sphingosine to sphingosine -1-phosphate (S1P) by sphingosine kinase, since it is blocked by the presence of N, N-dimethylsphingosine (DMS), which, like sphingosine, causes a sustained increase itself. The sustained increase that is elicited by sphingosine or DMS is abolished by the presence of EGTA in the buffer. The sustained sphingosine-induced Ca(2+)influx does not appear due to Ca(2+)influx through store-operated Ca(2+)(SOC) channels, since the influx is not inhibited by SKF 96365, nor is it augmented by loperamide. In addition, sphingosine and DMS attenuate the Ca(2+)influx through SOC channels that occurs after depletion of intracellular stores by ATP or thapsigargin. Both the initial peak and the sustained increase in [Ca(2+)](i)elicited by sphingosine can be blocked by phorbol 12-myristate 13-acetate (PMA)-elicited activation of protein kinase C. Thus, in HL-60 cells sphingosine causes a mobilization of Ca(2+)from intracellular Ca(2+)stores, which requires conversion to S1P, while both sphingosine and DMS elicit a Ca(2+)influx through an undefined Ca(2+)channel and cause a blockade of SOC channels.     

Two types of store-operated channels in A431 cells

Activation of phospholipase C-coupled receptors leads to the release of Ca2+ from Ca2+ stores, and subsequent activation of store-operated cation (SOC) channels, promoting sustained Ca2+ influx. The most studied SOC channels are CRAC ("calcium-release activated calcium") channels exhibiting a very high selectivity for Ca2+. However, there are many SOC channels permeable for Ca2+ but having a lower selectivity. And while Ca2+ influx is important for many biological processes, little is known about the types of SOC channels and mechanisms of SOC channel activation. Previously, we described store-operated Imin channels in A431 cells. Here, by whole-cell recordings, we demonstrated that the store depletion activates two types of current in A431 cells--highly selective for divalent cations (presumably, ICRAC), and moderately selective (ISOC supported by Imin channels). These currents can be registered separately and have different developing time and amplitude. Coexisting of two different types of SOC channels in A431 cells seems to facilitate the control of intracellular Ca(2+)-dependent processes.     

STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.     

Phosphatidylserine externalization in caveolae inhibits Ca2+ efflux through plasma membrane Ca2+-ATPase in ECV304

It has been evidenced that plasma membrane Ca(2+)-ATPase (PMCA) is localized at caveolae. However, the caveolar function of PMCA in living cells has never been demonstrated. In the present study, PMCA is exclusively localized at caveolae from ECV 304 cells demonstrated by sucrose gradient fractionation and the co-localization of PMCA with caveolin-1 was visualized by confocal microscopy. We found that PMCA is the main mechanism involved in Ca(2+) efflux in ECV 304 cells. Treatment of cells with MbetaCD to disrupt caveolae significantly reduced the Ca(2+) efflux, and the rate of decay is 4.45+/-0.14 min(-1) in the absence of MbetaCD and 1.99+/-0.038 min(-1) in the presence of MbetaCD. Moreover, the replenishment of cholesterol restored the reduction of the PMCA-mediated Ca(2+) efflux in the presence of MbetaCD. Consistent with Ca(2+) efflux in living cells, the activity of the reconstituted PMCA in membranes extracted from cells in vitro was decreased in the presence of MbetaCD. It was found that phosphatidylserine, which is normally in the inner leaflet of plasma membranes and is able to stimulate PMCA was relatively enriched in caveolae. Importantly, the treatment of cells with MbetaCD concomitantly increased the phosphatidylserine externalization. Taken together, our results suggest that activation of PMCA in caveolae is modulated by phosphatidylserine, and phosphatidylserine externalization induced by MbetaCD reduced the interaction of phosphatidylserine with PMCA, subsequently PMCA-mediated Ca(2+) efflux in ECV 304 cells.     

Nongenomic mechanism of glucocorticoid inhibition of bradykinin-induced calcium influx in PC12 cells: possible involvement of protein kinase C

Many stimulants, including bradykinin (BK), can induce increase in [Ca(2+)](i) in PC12 cells. Bradykinin induces an increase in [Ca(2+)](i) via intracellular Ca(2+) release and extracellular Ca(2+) influx through the transduction of G protein, but not through voltage-sensitive calcium channels. In this experiment, We analyzed how corticosterone (Cort) influences BK-induced intracellular Ca(2+) release and extracellular Ca(2+) influx, and further studied the mechanism of glucocorticoid's action. To dissociate the intracellular Ca(2+) release and extracellular Ca(2+) influx induced by BK, the Ca(2+)-free/Ca(2+)- reintroduction protocol was used. The results were as follows: (1) The Ca(2+) influx induced by BK could be rapidly inhibited by Cort, but intracellular Ca(2+) release could not be affected significantly. (2) The inhibitory effect of Cort-BSA (BSA -conjugated Cort) on Ca(2+) influx induced by BK was the same as the effect of free Cort. (3) Protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) could mimic and PKC inhibitor G?6976 could reverse the inhibitory effect of Cort. (4) There was no inhibitory effect of Cort on Ca(2+) influx induced by BK when pretreated with pertussis toxin. The results suggested, for the first time, that Cort might act via a putative membrane receptor and inhibit the Ca(2+) influx induced by BK through the pertussis toxin -sensitive G protein-PKC pathway.     

Lysophosphatidic acid is a mediator of Trp-Lys-Tyr-Met-Val-d-Met-induced calcium influx

Intracellular calcium (Ca(2+)) homeostasis is very strictly regulated, and the activation of G-protein-coupled receptor (GPCR) can cause two different calcium changes, intracellular calcium release, and calcium influx. In this study, we investigated the possible role of lysophosphatidic acid (LPA) on GPCR-induced Ca(2+) signaling. The addition of exogenous LPA induced dramatic Ca(2+) influx but not intracellular Ca(2+) release in U937 cells. LPA-induced Ca(2+) influx was not affected by pertussis toxin and phospholipase C inhibitor (U73122), ruling out the involvement of pertussis toxin-sensitive G-proteins, and phospholipase C. Stimulation of U937 cells with Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), which binds to formyl peptide receptor like 1, enhanced phospholipase A(2) and phospholipase D activation, indicating LPA formation. The inhibition of LPA synthesis by phospholipase A(2)-specific inhibitor (MAFP) or n-butanol significantly inhibited WKYMVm-induced Ca(2+) influx, suggesting a crucial role for LPA in the process. Taken together, we suggest that LPA mediates WKYMVm-induced Ca(2+) influx.     

Cytosolic organelles shape calcium signals and exo-endocytotic responses of chromaffin cells

The concept of stimulus-secretion coupling was born from experiments performed in chromaffin cells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca(2+)) entry and this generates a transient elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that triggers the exocytotic release of catecholamines. The control of the [Ca(2+)](c) signal is complex and depends on various classes of plasmalemmal calcium channels, cytosolic calcium buffers, the uptake and release of Ca(2+) from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca(2+) extrusion mechanisms, such as the plasma membrane Ca(2+)-stimulated ATPase, and the Na(+)/Ca(2+) exchanger. Computation of the rates of Ca(2+) fluxes between the different cell compartments support the proposal that the chromaffin cell has developed functional calcium tetrads formed by calcium channels, cytosolic calcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca(2+) transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytotic responses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca(2+)](c) transients occurring at different cell compartments, and generated by redistribution and release of Ca(2+) by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo-endocytotic activities with energy production and protein synthesis.     

Altered membrane lipid domains limit pulmonary endothelial calcium entry following chronic hypoxia

Agonist-induced Ca(2+) entry into the pulmonary endothelium depends on activation of both store-operated Ca(2+) (SOC) entry and receptor-operated Ca(2+) (ROC) entry. We previously reported that pulmonary endothelial cell SOC entry and ROC entry are reduced in chronic hypoxia (CH)-induced pulmonary hypertension. We hypothesized that diminished endothelial Ca(2+) entry following CH is due to derangement of caveolin-1 (cav-1) containing cholesterol-enriched membrane domains important in agonist-induced Ca(2+) entry. To test this hypothesis, we measured Ca(2+) influx by fura-2 fluorescence following application of ATP (20 μM) in freshly isolated endothelial cells pretreated with the caveolar-disrupting agent methyl-β-cyclodextrin (mβCD; 10 mM). Cholesterol depletion with mβCD attenuated agonist-induced Ca(2+) entry in control endothelial cells to the level of that from CH rats. Interestingly, endothelial membrane cholesterol was lower in cells isolated from CH rats compared with controls although the density of caveolae did not differ between groups. Cholesterol repletion with a cholesterol:mβCD mixture or the introduction of the cav-1 scaffolding peptide (AP-cav; 10 μM) rescued ATP-induced Ca(2+) entry in endothelia from CH arteries. Agonist-induced Ca(2+) entry assessed by Mn(2+) quenching of fura-2 fluorescence was also significantly elevated by luminal AP-cav in pressurized intrapulmonary arteries from CH rats to levels of controls. Similarly, patch-clamp experiments revealed diminished inward current in response to ATP in cells from CH rats compared with controls that was restored by AP-cav. These data suggest that CH-induced pulmonary hypertension leads to reduced membrane cholesterol that limits the activity of ion channels necessary for agonist-activated Ca(2+) entry.     

Inhibitory effects of polymethoxy flavones isolated from Citrus reticulate on degranulation in rat basophilic leukemia RBL-2H3: enhanced inhibition by their combination

Polymethoxy flavones (PMFs) are present in fruit tissues of Citrus species. It has been reported that flavonoids isolated from several Citrus have been shown to suppress the degranulation as inferred by histamine release in rat basophilic leukemia RBL-2H3 cells. In this study, we examined the effect of PMFs (PMF-1: 6,7,4',5'-tetramethoxy-5-monohydroxyflavone, PMF-2: 5,6,8,3',6'-pentamethoxy flavone, PMF-3: 5,6,7,3',4',5'-hexamethoxy flavone) on the degranulation in RBL-2H3 cells. All the PMFs suppressed the degranulation from Ag-stimulated RBL-2H3 cells. Interestingly, PMF-combination (PMF-1+PMF-2; PMF-1+PMF-3) treatment enhanced the inhibition of degranulation compared with PMF-single treatment. In order to clarify the inhibitory mechanism of degranulation by PMFs, we examined the activation of intracellular signaling molecules such as Lyn, Syk, and PLCgammas. All the PMFs significantly suppressed the activation of Syk and PLCgammas. In Ag-mediated activation of Fc epsilonRI on mast cells, three major subfamilies of mitogen-activated protein kinases, especially ERK44/42, were activated. These PMFs reduced the level of phospho-ERKs. The intracellular free Ca(2+) concentration ([Ca(2+)]i) was elevated by Fc epsilonRI activation, and PMF treatment reduced the elevation of [Ca(2+)]i by suppressing Ca(2+) influx. Thus, it was suggested that the suppression of Ag-stimulated degranulation by these PMFs mainly is due to the Syk/PLCgammas/PKC pathway and Ca(2+) influx. Furthermore, to be noted in the PMF-combination treatment, inactivation of Syk was enhanced compared with PMF-single treatment. But the inhibitory effect of degranulation by PMF-combination treatment was not associated with the suppression of Ca(2+) influx.     

Downregulation of TRPV6 channel activity by cholesterol depletion in Jurkat T cell line

Transient receptor potential vanilloid 6 (TRPV6) channels are key players in calcium metabolism of healthy and cancerous cells. Nevertheless, the mechanisms controlling abundance of these channels in plasma membrane of the cells to regulate Ca²⁺ transport is still poorly understood. In this study, we provide the first evidence that TRPV6 calcium channels and Ca ²⁺ influx in Jurkat T cell line are modulated by cholesterol, a main lipid component of the plasma membrane. Using patch‐clamp technique, we found that activity of TRPV6 channels decreased by cholesterol sequestration with methyl‐β‐cyclodextrin (MβCD). Continuous measurement of intracellular Ca²⁺ revealed a reduction of Ca²⁺ influx into Jurkat cells following cholesterol depletion. Immunofluorescence and immunoelectron microscopy analyses of MβCD‐treated cells detected the lower surface expression of the TRPV6 proteins in comparison with control cells. In general, our data showed that cholesterol regulates TRPV6 channel activity and TRPV6‐mediated Ca²⁺ influx in cells, apparently affecting the localization and density of the calcium channels in the plasma membrane of Jurkat T cells.