Mobilization Of Intracellular Ca2 Research Articles

The Involvement of Voltage-Operated Calcium Channels in Somato-Dendritic Oxytocin Release

Magnocellular neurons of the supraoptic nucleus (SON) secrete oxytocin and vasopressin from axon terminals in the neurohypophysis, but they also release large amounts of peptide from their somata and dendrites, and this can be regulated both by activity-dependent Ca2+ influx and by mobilization of intracellular Ca2+. This somato-dendritic release can also be primed by agents that mobilise intracellular Ca2+, meaning that the extent to which it is activity-dependent, is physiologically labile. We investigated the role of different Ca2+ channels in somato-dendritic release; blocking N-type channels reduced depolarisation-induced oxytocin release from SONs in vitro from adult and post-natal day 8 (PND-8) rats, blocking L-type only had effect in PND-8 rats, while blocking other channel types had no significant effect. When oxytocin release was primed by prior exposure to thapsigargin, both N- and L-type channel blockers reduced release, while P/Q and R-type blockers were ineffective. Using confocal microscopy, we found immunoreactivity for Cav1.2 and 1.3 channel subunits (which both form L-type channels), 2.1 (P/Q type), 2.2 (N-type) and 2.3 (R-type) in the somata and dendrites of both oxytocin and vasopressin neurons, and the intensity of the immunofluorescence signal for different subunits differed between PND-8, adult and lactating rats. Using patch-clamp electrophysiology, the N-type Ca2+ current density increased after thapsigargin treatment, but did not alter the voltage sensitivity of the channel. These results suggest that the expression, location or availability of N-type Ca2+ channels is altered when required for high rates of somato-dendritic peptide release.

Expression of Novel Opsins and Intrinsic Light Responses in the Mammalian Retinal Ganglion Cell Line RGC-5. Presence of OPN5 in the Rat Retina

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca2+mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca2+ levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca2+ mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.

Mechanisms involved in endothelin‐1‐induced contraction of the pig urinary bladder neck

There is no information about the signaling pathways involved in the endothelin-1 (ET-1)-induced contraction of bladder neck. The current study investigates the mechanisms involved in the ET-1-elicited contraction in the pig bladder neck. Bladder neck strips were mounted in organ baths containing physiological saline solution at 37°C and gassed with 95% O(2) and 5% CO(2) , for isometric force recording to endothelin receptor agonists, noradrenaline (NA), and electrical field stimulation. Endothelin ET(A) receptor expression was also determined, by both immunohistochemistry and Western blot. ET(A) receptor expression (Western blot) was observed in the muscular layer and urothelium. A strong ET(A) -immunoreactivity (ET(A) -IR) was identified within nerve fibers among smooth muscle bundles. ET-1 and ET-2 evoked similar concentration-dependent contractions of urothelium-denuded preparations. ET-3 produced a slight response, whereas the ET(B) receptor agonist BQ3020 failed to promote contraction. BMS182874, an ET(A) receptor antagonist, reduced ET-1-induced contraction whereas BQ788, an ET(B) antagonist, did not change such responses. ET-1 contractions were reduced by extracellular Ca(2+) removal and by inhibition of voltage-gated Ca(2+) (VOC) (L-type) and non-VOC channels, Rho/Rho-kinase pathway, and neuronal VOC channels. NA produced contractions which were enhanced by ET-1 threshold concentrations. ET(A) receptor blockade enhanced nitric oxide-dependent nerve-mediated relaxations. These results suggest that ET-1 produces contraction via muscular ET(A) receptors coupled to extracellular Ca(2+) entry via VOC (L-type) and non-VOC channels. Intracellular Ca(2+) mobilization and a Rho/Rho-kinase pathway could also be involved in these responses. ET-1-evoked potentiation on noradrenergic contraction, and neuronal ET(A) receptors modulating nitrergic inhibitory neurotransmission, are also demonstrated.

Emodin, a naturally occurring anthraquinone derivative, suppresses IgE-mediated anaphylactic reaction and mast cell activation

The high-affinity receptor for IgE (FcɛRI)-mediated activation of mast cells plays an important role in allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. Emodin, a naturally occurring anthraquinone derivative in oriental herbal medicines, has several beneficial pharmacologic effects, such as anti-cancer and anti-diabetic activities. However, the anti-allergic effect of emodin has not yet been investigated. To assess the anti-allergic activity of emodin, in vivo passive anaphylaxis animal model and in vitro mouse bone marrow-derived mast cells were used to investigate the mechanism of its action on mast cells. Our results showed that emodin inhibited degranulation, generation of eicosanoids (prostaglandin D 2 and leukotriene C 4), and secretion of cytokines (TNF-α and IL-6) in a dose-dependent manner in IgE/Ag-stimulated mast cells. Biochemical analysis of the FcɛRI-mediated signaling pathways demonstrated that emodin inhibited the phosphorylation of Syk and multiple downstream signaling processes including mobilization of intracellular Ca 2+ and activation of the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and NF-κB pathways. When administered orally, emodin attenuated the mast cell-dependent passive anaphylactic reaction in IgE-sensitized mice. Thus, emodin inhibits mast cell activation and thereby the anaphylactic reaction through suppression of the receptor-proximal Syk-dependent signaling pathways. Therefore, emodin might provide a basis for development of a novel anti-allergic drug.

Human catestatin enhances migration and proliferation of normal human epidermal keratinocytes

Skin-derived antimicrobial peptides, such as human β-defensins and cathelicidins, actively contribute to host defense by inactivating microorganisms. Catestatin, a neuroendocrine peptide that affects human autonomic functions, has recently been detected in keratinocytes upon injury/infection where it inhibits the growth of pathogens. Human catestatin exhibits three single nucleotide polymorphisms: Gly364Ser, Pro370Leu, and Arg374Gln. To investigate the effects of human catestatin and its variants on keratinocyte migration and proliferation, and to elucidate the possible signaling mechanisms involved. The migration of normal human keratinocytes was analyzed using Boyden microchamber assay and in vitro wound closure assay. Cell proliferation was evaluated by BrdU incorporation, cell count assay and cell cycle analysis. Intracellular Ca(2+) mobilization was measured using a fluorescent calcium assay kit. The phosphorylation of epidermal growth factor receptor (EGFR), Akt, and MAPKs was determined by Western blotting. Catestatin and its variants dose-dependently enhanced keratinocyte migration and proliferation. Moreover, catestatin peptides increased intracellular Ca(2+) mobilization and induced the phosphorylation of EGFR, Akt, extracellular signal-regulated kinase (ERK), and p38 in keratinocytes. The induction of keratinocyte migration and proliferation by catestatin peptides involved G-proteins, phospholipase C, EGFR, PI3-kinase, ERK, and p38, as evidenced by the specific inhibitory effects of pertussis toxin (G-protein inhibitor), U-73122 (phospholipase C inhibitor), AG1478 (EGFR inhibitor), anti-EGFR antibody, wortmannin (PI3-kinase inhibitor), U0126 (ERK inhibitor), and SB203580 (p38 inhibitor), respectively. Besides inhibiting the growth of skin pathogens, catestatin peptides may also contribute to cutaneous wound closure by enhancing keratinocyte migration and proliferation at the wound site.

Dendritic cell maturation and chemotaxis is regulated by TRPM2‐mediated lysosomal Ca2+release

Chemokines induce calcium (Ca(2+)) signaling and chemotaxis in dendritic cells (DCs), but the molecular players involved in shaping intracellular Ca(2+) changes remain to be characterized. Using siRNA and knockout mice, we show that in addition to inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOCE), the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca(2+) release but not Ca(2+) influx in mouse DCs. Consistent with these findings, TRPM2 expression in DCs is restricted to endolysosomal vesicles, whereas in neutrophils, the channel localizes to the plasma membrane. TRPM2-deficient DCs show impaired maturation and severely compromised chemokine-activated directional migration as well as bacterial-induced DC trafficking to the draining lymph nodes. Defective DC chemotaxis is due to perturbed chemokine-receptor-initiated Ca(2+) signaling mechanisms, which include suppression of TRPM2-mediated Ca(2+) release and secondary modification of SOCE. DCs deficient in both TRPM2 and IP(3) receptor signaling lose their ability to perform chemotaxis entirely. These results highlight TRPM2 as a key player regulating DC chemotaxis through its function as Ca(2+) release channel and confirm ADP-ribose as a novel second messenger for intracellular Ca(2+) mobilization.

Somatodendritic Dopamine Release Requires Synaptotagmin 4 and 7 and the Participation of Voltage-gated Calcium Channels

Somatodendritic (STD) dopamine (DA) release is a key mechanism for the autoregulatory control of DA release in the brain. However, its molecular mechanism remains undetermined. We tested the hypothesis that differential expression of synaptotagmin (Syt) isoforms explains some of the differential properties of terminal and STD DA release. Down-regulation of the dendritically expressed Syt4 and Syt7 severely reduced STD DA release, whereas terminal release required Syt1. Moreover, we found that although mobilization of intracellular Ca(2+) stores is inefficient, Ca(2+) influx through N- and P/Q-type voltage-gated channels is critical to trigger STD DA release. Our findings provide an explanation for the differential Ca(2+) requirement of terminal and STD DA release. In addition, we propose that not all sources of intracellular Ca(2+) are equally efficient to trigger this release mechanism. Our findings have implications for a better understanding of a fundamental cell biological process mediating transcellular signaling in a system critical for diseases such as Parkinson disease.

Mitochondrial Pathway Mediates the Antileukemic Effects of Hemidesmus Indicus, a Promising Botanical Drug

BackgroundAlthough cancers are characterized by the deregulation of multiple signalling pathways, most current anticancer therapies involve the modulation of a single target. Because of the enormous biological diversity of cancer, strategic combination of agents targeted against the most critical of those alterations is needed. Due to their complex nature, plant products interact with numerous targets and influence several biochemical and molecular cascades. The interest in further development of botanical drugs has been increasing steadily and the FDA recently approved the first new botanical prescription drug. The present study is designed to explore the potential antileukemic properties of Hemidesmus indicus with a view to contributing to further development of botanical drugs. Hemidesmus was submitted to an extensive in vitro preclinical evaluation.Methodology/Principal FindingsA variety of cellular assays and flow cytometry, as well as a phytochemical screening, were performed on different leukemic cell lines. We have demonstrated that Hemidesmus modulated many components of intracellular signaling pathways involved in cell viability and proliferation and altered the protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential and increased Bax/Bcl-2 ratio. ADP, adenine nucleotide translocator and mitochondrial permeability transition pore inhibitors did not reverse Hemidesmus-induced mitochondrial depolarization. Hemidesmus induced a significant [Ca2+]i raise through the mobilization of intracellular Ca2+ stores. Moreover, Hemidesmus significantly enhanced the antitumor activity of three commonly used chemotherapeutic drugs (methotrexate, 6-thioguanine, cytarabine). A clinically relevant observation is that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemic patients.Conclusions/SignificanceThese results indicate the molecular basis of the antileukemic effects of Hemidesmus and identify the mitochondrial pathways and [Ca2+]i as crucial actors in its anticancer activity. On these bases, we conclude that Hemidesmus can represent a valuable tool in the anticancer pharmacology, and should be considered for further investigations.

P73 regulates cisplatin-induced apoptosis in ovarian cancer cells via a calcium/calpain-dependent mechanism

P73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in ovarian cancer (OVCA) is poorly understood. Furthermore, if and how the deregulation of p73-mediated apoptosis confers resistance to cisplatin (CDDP) treatment is unclear. Here we demonstrate that TAp73α over-expression enhanced CDDP-induced PARP cleavage and apoptosis in both chemosensitive (OV2008 and A2780s) and their resistant counterparts (C13* and A2780cp) and another chemoresistant OVCA cells (Hey); in contrast, the effect of ΔNp73α over-expression was variable. P73α downregulation attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008 cells. CDDP decreased p73α steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced p73α downregulation was mediated by a calpain-dependent pathway. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73α in OV2008, but not C13* cells. CDDP increased the intracellular calcium concentration ([Ca2+]i) in OV2008 but not C13* whereas cyclopiazonic acid (CPA), a Ca2+-ATPase inhibitor, caused this response and calpain activation, p73α processing and apoptosis in both cell types. CDDP-induced [Ca2+]i increase in OV2008 cells was not effected by the elimination of extracellular Ca2+, but this was attenuated by the depletion of internal Ca2+ store, indicating that mobilization of intracellular Ca2+] stores was potentially involved. These findings demonstrate that p73α and its regulation by the Ca2+-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that dysregulation of Ca2+/calpain/p73 signaling may in part be the pathophysiology of CDDP resistance. Understanding the cellular and molecular mechanisms of chemoresistance will direct the development of effective strategies for the treatment of chemoresistant OVCA.

Differential effects of vasodilators on the mobilization of calcium pools during contractions of rabbit ear artery induced by noradrenaline and high potassium.

The effects of some vasodilators on the mobilization of different Ca2+ pools during contractions produced by noradrenaline and high K+ medium in rabbit ear artery has been investigated. Sodium nitrite was much more effective in antagonizing high K+-stimulated Ca2+ fluxes through the potential-dependent Ca2+ channels than noradrenaline-induced mobilization of intracellular Ca2+ stores or Ca2+ fluxes through receptor-operated channels. Papaverine was only slightly more active on contractions sustained by influx of extracellular Ca2+ (through potential-dependent channels and receptor-operated channels) than on those sustained by intracellular Ca2+. Verapamil and nifedipine were much more effective in antagonizing contractions sustained by Ca2+ entry through potential-dependent channels than through receptor-operated channels. Nifedipine was completely ineffective in antagonizing noradrenaline-induced mobilization of intracellular Ca2+ stores while verapamil had a limited inhibitory action in high concentrations. Phentolamine was equieffective in antagonizing both types of noradrenaline-induced contractions while having no effect on high K+-induced tonic contraction.

The role of histamine H1, H2 and H3 receptors in the stimulation of NT‐3 synthesis in astrocytes

Neurotrophin‐3 (NT‐3) is produced by astrocytes, in addition to neurons, and monoamine neurotransmitters play a role in controlling NT‐3 synthesis. We evaluated the active involvement of histamine receptors and intracellular mechanisms in the regulation of NT‐3 production by HA.HA (1 μM) significantly and transiently elevated NT‐3 mRNA levels by 2.2‐fold after 30 min of incubation following by 2.1‐fold increase in NT‐3 intracellular levels after 6 h. Its stimulation was partly inhibited by selective H1, H2 and H3 antagonists. NT‐3 levels in astrocytes were increased by selective H1, H2 and H3 agonists as well as by adenylyl cyclase activation, PKA activation, PKC activation and mobilization of intracellular Ca2+. HA‐induced increase in NT‐3 cellular levels were significantly reduced by H‐89, staurosporin, KN‐62 and desensitizing pretreatment with TPA. MAP kinase cascade inhibitor PD98059 completely blocked the stimulatory action of HA and all selective agonists. Using quantitative RT‐PCR we confirmed that cultured rat cortical astrocytes express not only H1 and H2 receptors already identified by radioligand binding studies but also H3 receptors which were sensitive to pertussis toxin. In conclusion, the synthesis of astrocytic NT‐3 stimulated by HA is an adaptable process using several parallel histaminergic pathways using H1‐, H2‐, and H3‐receptor pathways. The observed H1, H2 and H3 receptor crosstalk in histaminergic regulation of NT‐3 leads to the convergence of these pathways at the level of MAP kinase activity which in the next step triggers the increased expression and subsequently the synthesis of NT‐3 in astrocytes.

Airway Epithelial Epidermal Growth Factor Receptor Mediates Hogbarn Dust–Induced Cytokine Release but Not Ca2+ Response

A subset of workers in swine confinement facilities develops chronic respiratory disease. An aqueous extract of dust from these facilities (hogbarn dust extract [HDE]) induces IL-6 and IL-8 release and several other responses in isolated airway epithelial cells. The cell membrane receptors by which HDE initiates these responses have not been identified. Because several other inhaled agents induce airway epithelial cell responses through epidermal growth factor receptor (EGFR) activation, we hypothesized that HDE would activate EGFRs and that EGFRs would be required for some of the responses to HDE. Exposure of Beas-2B cells to HDE caused EGFR phosphorylation and downstream ERK activation, and both responses were blocked by the EGFR-selective kinase inhibitor AG1478. AG1478 and EGFR-neutralizing antibody reduced HDE-stimulated IL-6 and IL-8 release by about half. Similar EGFR phosphorylation and requirement of EGFRs for maximal IL-6 and IL-8 release were found with primary isolates of human bronchial epithelial cells. Because HDE-stimulated IL-6 and IL-8 release involve the Ca(2+)-dependent protein kinase Cα, we hypothesized that HDE would induce intracellular Ca(2+) mobilization. HDE exposure induced intracellular Ca(2+) mobilization in Beas-2B cells and in primary cell isolates, but this response was neither mimicked by EGF nor inhibited by AG1478. Thus, HDE activates EGFRs and their downstream signaling, and EGFR activation is required for some but not all airway epithelial cell responses to HDE.

Costunolide Induces Apoptosis Through Nuclear Calcium2+ Overload and DNA Damage Response in Human Prostate Cancer

Costunolide is a natural sesquiterpene lactone. We elucidated what to our knowledge is a novel mechanism to highlight its potential in chemotherapy for prostate cancer, particularly androgen refractory prostate cancer. Several pharmacological and biochemical assays were used to characterize the apoptotic signaling pathways of costunolide (ChromaDex™) in prostate cancer cells. Costunolide showed effective antiproliferative activity against hormone dependent (LNCaP) and independent (PC-3 and DU-145) prostate cancer cells (ATCC®) by sulforhodamine B assay, clonogenic test and flow cytometric analysis of carboxyfluorescein succinimidyl ester labeling. In PC-3 cells data showed that costunolide induced a rapid overload of nuclear Ca(2+), DNA damage response and ATR phosphorylation. Costunolide induced G1-phase cell cycle arrest, which was supported by p21 up-regulation and its association with the cyclin dependent kinase 2/cyclin E complex. The association resulted in inhibition of the complex activity and inhibition of Rb phosphorylation. Costunolide mediated effects were substantially inhibited by glutathione, the reactive oxygen species scavenger and glutathione precursor N-acetylcysteine, and the Ca(2+) chelator BAPTA-AM other than the reactive oxygen species scavenger Trolox®. This indicated the crucial role of intracellular Ca(2+) mobilization and thiol depletion but not of reactive oxygen species production in apoptotic signaling. Data suggest that costunolide induces the depletion of intracellular thiols and overload of nuclear Ca(2+) that cause DNA damage and p21 up-regulation. The association of p21 with the cyclin dependent kinase 2/cyclin E complex blocks cyclin dependent kinase 2 activity and inhibits Rb phosphorylation, leading to G1 arrest of the cell cycle and subsequent apoptotic cell death in human prostate cancer cells.

Allosteric Modulation of the Calcium-sensing Receptor by γ-Glutamyl Peptides: INHIBITION OF PTH SECRETION, SUPPRESSION OF INTRACELLULAR cAMP LEVELS, AND A COMMON MECHANISM OF ACTION WITH l-AMINO ACIDS

γ-Glutamyl peptides were identified previously as novel positive allosteric modulators of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization in HEK-293 cells that bind in the calcium-sensing receptor VFT domain. In the current study, we investigated whether γ-glutamyl-tripeptides including γ-Glu-Cys-Gly (glutathione) and its analogs S-methylglutathione and S-propylglutathione, or dipeptides including γ-Glu-Ala and γ-Glu-Cys are positive allosteric modulators of Ca(2+)(o)-dependent Ca(2+)(i) mobilization and PTH secretion from normal human parathyroid cells as well as Ca(2+)(o)-dependent suppression of intracellular cAMP levels in calcium-sensing receptor (CaR)-expressing HEK-293 cells. In addition, we compared the effects of the potent γ-glutamyl peptide S-methylglutathione, and the amino acid L-Phe on HEK-293 cells that stably expressed either the wild-type CaR or the double mutant T145A/S170T, which exhibits selectively impaired responses to L-amino acids. We find that γ-glutamyl peptides are potent positive allosteric modulators of the CaR that promote Ca(2+)(o)-dependent Ca(2+)(i) mobilization, suppress intracellular cAMP levels and inhibit PTH secretion from normal human parathyroid cells. Furthermore, we find that the double mutant T145A/S170T exhibits markedly impaired Ca(2+)(i) mobilization and cAMP suppression responses to S-methylglutathione as well as L-Phe indicating that γ-glutamyl peptides and L-amino acids activate the CaR via a common mechanism.

Positive regulation of inositol 1,4,5‐trisphosphate‐induced ca2+ release by mammalian target of rapamycin (mTOR) in RINm5F cells

The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, is the main regulator of intracellular Ca(2+) mobilization in non-excitable cells. An emerging body of evidence suggests that specific regulatory control of the Ca(2+) signaling pathway is modulated by the activation of additional signaling pathways. In the present study, we investigated the influence of the PI3-kinase/mammalian target of rapamycin (mTOR) pathway on the activity of the IP(3)R/Ca(2+) signaling pathway in RINm5F cells. We used a co-immunoprecipitation approach to show that mTOR physically interacts with IP(3)R-3 in an mTOR activity-dependent manner. We also showed that IP(3)R is phosphorylated by mTOR in cellulo. All the conditions known to modulate mTOR activity (IGF-1, wortmannin, rapamycin, PP242, and nutrient starvation) were shown to modify carbachol-induced Ca(2+) signaling in RINm5F cells. Lastly, we used an assay that directly measures the activity of IP(3)R, to show that mTOR increases the apparent affinity of IP(3)R. Given that mTOR controls cell proliferation and cell homeostasis, and that Ca(2+) plays a key role in these two phenomena, it follows that mTOR facilitates IP(3)R-mediated Ca(2+) release when the nutritional status of cells requires it.

Native high-density lipoproteins inhibit platelet activation via scavenger receptor BI: Role of negatively charged phospholipids

ObjectivesHIGH-density lipoproteins (HDL) are a negative predictor of platelet-dependent thrombus formation and reduced platelet activation has been observed in vitro in the presence of HDL3, a major HDL fraction. However, mechanisms underlying the anti-thrombotic effects of HDL3 are poorly understood. Scavenger receptors class B represent possible HDL3 binding partners on platelets. We here investigated the role of scavenger receptor class B type I (SR-BI) and CD36 in mediating inhibitory effects of native HDL3 on thrombin-induced platelet activation. Methods and resultsRhodamine isothiocyanate-labeled HDL3 bound specifically to platelets and HDL3 binding was inhibited by scavenger receptor class B ligands such as phosphatidylserine (PS)- or phosphatidylinositol (PI)-containing liposomes or maleylated albumin (mBSA). By contrast, scavenger receptor class A ligands failed to displace HDL3 from platelets. HDL3, PS- and PI-liposomes, and mBSA inhibited thrombin-induced platelet aggregation, fibrinogen binding, P-selectin expression and mobilization of intracellular Ca2+. In addition, PS- and PI-liposomes emulated HDL3-induced intracellular signaling cascades including diacylglycerol production and protein kinase C activation. The reduction of platelet activation by liposomes was related to their PS or PI content. Moreover, inhibitory effects of native HDL3 were enhanced after enriching lipoproteins with PS, while PS- and PI-poor HDL2 failed to inhibit platelet aggregation and Ca2+ mobilization. Both, HDL3 and PS-containing liposomes failed to inhibit thrombin-induced activation of platelets obtained from SR-BI-deficient mice but not CD36-deficient mice. ConclusionWe suggest that SR-BI is a functional receptor for native HDL3 on platelets that generates an inhibitory signal for platelet activation. The content of negatively charged phospholipids (PS, PI) in HDL may be an important determinant of their anti-thrombotic potential.

Catestatin, a neuroendocrine antimicrobial peptide, induces human mast cell migration, degranulation and production of cytokines and chemokines

Catestatin, a neuroendocrine peptide with effects on human autonomic function, has recently been found to be a cutaneous antimicrobial peptide. Human catestatin exhibits three single nucleotide polymorphisms: Gly364Ser, Pro370Leu and Arg374Gln. Given reports indicating that antimicrobial peptides and neuropeptides induce mast cell activation, we postulated that catestatin might stimulate numerous functions of human mast cells, thereby participating in the regulation of skin inflammatory responses. Catestatin and its naturally occurring variants caused the human mast cell line LAD2 and peripheral blood-derived mast cells to migrate, degranulate and release leukotriene C(4) and prostaglandins D(2) and E(2). Moreover, catestatins increased intracellular Ca(2+) mobilization in mast cells, and induced the production of pro-inflammatory cytokines/chemokines such as granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1/CCL2, macrophage inflammatory protein-1α/CCL3 and macrophage inflammatory protein-1β/CCL4. Our evaluation of possible cellular mechanisms suggested that G-proteins, phospholipase C and the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) are involved in catestatin-induced mast cell activation as evidenced by the inhibitory effects of pertussis toxin (G-protein inhibitor), U-73122 (phospholipase C inhibitor) and U0126 (ERK inhibitor), respectively. We also found that human mast cells express the α7 subunit of the nicotinic acetylcholine receptor at both the mRNA and protein levels. Given that silencing the α7 receptor mRNA and an α7-specific inhibitor did not affect catestatin-mediated activation of mast cells, however, we concluded that this receptor is not likely to be functional in human mast cell stimulation by catestatins. Our finding that the neuroendocrine antimicrobial peptide catestatin activates human mast cells suggests that this peptide might have immunomodulatory functions, and provides a new link between neuroendocrine and cutaneous immune systems.

Intracellular Ca2+Mobilization and Beta-hexosaminidase Release Are Not Influenced by 60 Hz-electromagnetic Fields (EMF) in RBL 2H3 Cells

The effects of extremely low frequency electromagnetic fields (EMF) on intracellular Ca(2+) mobilization and cellular function in RBL 2H3 cells were investigated. Exposure to EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h did not produce any cytotoxic effects in RBL 2H3 cells. Melittin, ionomycin and thapsigargin each dose-dependently increased the intracellular Ca(2+) concentration. The increase of intracellular Ca(2+) induced by these three agents was not affected by exposure to EMF (60 Hz, 1 mT) for 4 or 16 h in RBL 2H3 cells. To investigate the effect of EMF on exocytosis, we measured beta-hexosaminidase release in RBL 2H3 cells. Basal release of beta-hexosaminidase was 12.3±2.3% in RBL 2H3 cells. Exposure to EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h did not affect the basal or 1 µM melittin-induced beta-hexosaminidase release in RBL 2H3 cells. This study suggests that exposure to EMF (60 Hz, 0.1 or 1 mT), which is the limit of occupational exposure, has no influence on intracellular Ca(2+) mobilization and cellular function in RBL 2H3 cells.

Epac2-dependent mobilization of intracellular Ca2+by glucagon-like peptide-1 receptor agonist exendin-4 is disrupted in β-cells of phospholipase C-ɛ knockout mice

Calcium can be mobilized in pancreatic β-cells via a mechanism of Ca(2+)-induced Ca(2+) release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in β-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C- (PLC-) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC- gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the β-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse β-cells were loaded with the photolabile Ca(2+) chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca(2+) generated CICR in only 9% of the β-cells tested, whereas CICR was generated in 82% of the β-cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6-Bnz-cAMP-AM) or Epac2 (8-pCPT-2'-O-Me-cAMP-AM) selectively. However, in β-cells of PLC- KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC- KO β-cells with recombinant PLC- rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC- with a mutated Ras association (RA) domain, or a H1640L PLC- that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT β-cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC- exists in β-cells, and that the activities of Epac2 and PLC- are key determinants of CICR in this cell type.

Normalizing effect of plant-originated glycoprotein (116 kDa) on G0/G1 arrest in cadmium chloride-induced primary cultured mouse myelocytes

Cadmium chloride is a well-known carcinogenic and immunotoxic metal chemical, which is commonly found in cigarette smoke and industrial effluent and which is able to cause cell cycle arrest in various cell lines. This study demonstrated that glycoprotein (116kDa) isolated from Ulmus Davidiana Nakai (UDN) is able to normalize cell cycle arrest caused by cadmium chloride (10μM, for indicated treatment time in the each experiment) in primary cultured mouse myelocytes. To assess cell cycle arrest, the parameters that are related to the cell cycle evaluated included cytotoxicity, production of intracellular reactive oxygen species (ROS), intracellular Ca(2+) mobilization, the activities of cell cycle-related proteins (p53, p21, and p27), and cyclin D1/cell cyclin-dependent kinase 4 (CDK4) using immunoblot analysis and fluorescence-activated cell sorter analysis. The results in this study showed that UDN glycoprotein (50μg/ml) inhibits the cytotoxicity, production of intracellular reactive oxygen species (ROS), and intracellular Ca(2+) mobilization brought about by cadmium chloride. With regard to cell cycle-related proteins, UDN glycoprotein (50μg/ml) significantly suppressed the expression of p53, p21, and p27, whereas it enhanced activity of cyclin D1/CDK4. Taken together, these findings suggest that UDN glycoprotein (50μg/ml) significantly normalizes arrest of G(0)/G(1) in the cell cycle. Thus, UDN glycoprotein appears to be one compound derived from natural products that is able normalize the calcium chloride-mediated arrest of cell cycle (G(0)/G(1)) in immune cells.