The Impact of the Antipsychotic Medication Chlorpromazine on Cytotoxicity through Ca2+ Signaling Pathway in Glial Cell Models.

Chlorpromazine, an antipsychotic medication, is conventionally applied to cope with the psychotic disorder such as schizophrenia. In cellular studies, chlorpromazine exerts many different actions through calcium ion (Ca2+) signaling, but the underlying pathways are elusive. This study explored the effect of chlorpromazine on viability, Ca2+ signaling pathway and their relationship in glial cell models (GBM 8401 human glioblastoma cell line and Gibco® Human Astrocyte (GHA)). First, chlorpromazine between 10 and 40 µM induced cytotoxicity in GBM 8401 cells but not in GHA cells. Second, in terms of Ca2+ homeostasis, chlorpromazine (10-30 µM) increased intracellular Ca2+ concentrations ([Ca2+]i) rises in GBM 8401 cells but not in GHA cells. Ca2+ removal reduced the signal by approximately 55%. Furthermore, chelation of cytosolic Ca2+ with BAPTA-AM reduced chlorpromazine (10-40 µM)-induced cytotoxicity in GBM 8401 cells. Third, in Ca2+-containing medium of GBM 8401 cells, chlorpromazine-induced Ca2+ entry was inhibited by the modulators of store-operated Ca2+ channel (2-APB and SKF96365). Lastly, in Ca2+-free medium of GBM 8401 cells, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin completely inhibited chlorpromazine-increased [Ca2+]i rises. Conversely, treatment with chlorpromazine abolished thapsigargin-increased [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished chlorpromazine-increased [Ca2+]i rises. Together, in GBM 8401 cells but not in GHA cells, chlorpromazine increased [Ca2+]i rises by Ca2+ influx via store-operated Ca2+ entry and PLC-dependent Ca2+ release from the endoplasmic reticulum. Moreover, the Ca2+ chelator BAPTA-AM inhibited cytotoxicity in chlorpromazine-treated GBM 8401 cells. Therefore, Ca2+ signaling was involved in chlorpromazine-induced cytotoxicity in GBM 8401 cells.

Clostridium perfringens α-toxin inhibits myogenic differentiation of C2C12 myoblasts.

Clostridium perfringens type A is the causative agent of clostridial myonecrosis, and α-toxin has been reported to be responsible for the pathogenesis. Recently, it was reported that regeneration of skeletal muscle after C. perfringens-induced muscle disorders is delayed, but the detailed mechanisms have not been elucidated. Here, we tested whether α-toxin impairs the differentiation of C2C12 myoblasts, a useful cell line to study muscle growth, maturation, and regeneration in vitro. α-Toxin dose-dependently inhibited myotube formation in C2C12 cultures after induction of their differentiation by horse serum. Also, immunoblot analysis revealed that α-toxin dose-dependently decreases the expressions of two skeletal muscle differentiation markers, myogenic differentiation 1 (MyoD) and myogenin. These results demonstrate that α-toxin impairs the myogenic differentiation of C2C12 myoblasts. To reveal the mechanism behind α-toxin-mediated impairment of myogenic differentiation, we focused on ceramide production since α-toxin is known to promote the formation of ceramide by its sphingomyelinase activity. Immunofluorescent analysis revealed that ceramide production is accelerated by treatment with α-toxin. Furthermore, a synthetic cell-permeable ceramide analog, C2-ceramide, inhibited myotube formation in C2C12 cells and decreased the expressions of MyoD and myogenin, suggesting that accelerated ceramide production is involved in the α-toxin-mediated blockage of myogenic differentiation. Together, our results illustrate that the impairment of myogenic differentiation by α-toxin might be crucial for the pathogenesis of C. perfringens to delay regeneration of severely damaged skeletal muscles.

The soluble fraction of Neospora caninum treated with PI-PLC is dominated by NcSRS29B and NcSRS29C.

Neospora caninum is an obligate intracellular parasite related to cases of abortion and fertility impairment in cattle. The control of the parasite still lacks an effective protective strategy and the understanding of key mechanisms for host infection might be crucial for identification of specific targets. There are many proteins related to important mechanisms in the host cell infection cycle such as adhesion, invasion, proliferation and immune evasion. The surface proteins, especially SRS (Surface Antigen Glycoprotein - Related Sequences), have been demonstrated to have a pivotal role in the adhesion and invasion processes, making them potential anti-parasite targets. However, several predicted surface proteins were not described concerning their function and importance in the parasite life cycle. As such, a novel SRS protein, NcSRS57, was described. NcSRS57 antiserum was used to detect SRS proteins by immunofluorescence in parasites treated or not with phosphatidylinositol-specific phospholipase C (PI-PLC). The treatment with PI-PLC also allowed the identification of NcSRS29B and NcSRS29C, which were the most abundant SRS proteins in the soluble fraction. Our data indicated that SRS proteins in N. caninum shared a high level of sequence similarity and were susceptible to PI-PLC. In addition, the description of the SRS members, regarding abundance, function and immunogenicity will be useful in guiding specific methods to control the mechanism of adhesion and invasion mediated by these surface proteins.

Expression of the alpha toxin of Clostridium perfringens in Lactobacillus casei genome and evaluation of its immune effects in mice.

We previously developed a stable and marker-free Lactobacillus casei strain (PPαT Δupp) that contained a chromosomally integrated expression cassette (PPαT) that enabled the surface expression of the Clostridium perfringens alpha toxin. To measure immune responses against the alpha toxin, specific-pathogen-free BALB/c mice were inoculated with L. casei PPαT Δupp by oral gavage. Then, specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM). The results showed that alpha toxin-specific IgA and IgG antibodies and cytokines were markedly increased following immunization. Natural alpha toxin challenge and neutralization tests were performed. The results showed that immunized mice can fully resist 1.5 minimum lethal doses of toxin. These results indicated that the immunized mice can produce not only humoral immunity, but also cellular immunity. These results provide a new pathway for the development of a safe, effective, and food-grade vaccine.

Non-toxic perfringolysin O and α-toxin derivatives as potential vaccine candidates against bovine necrohaemorrhagic enteritis.

Bovine necrohaemorrhagic enteritis is a fatal Clostridium perfringens type A-induced disease that is characterised by sudden death. Recently the involvement of perfringolysin O and α-toxin in the development of necrohaemorrhagic lesions in the gut of calves was suggested, and thus derivatives of these toxins are potentially suitable as vaccine antigens. In the current study, the perfringolysin O derivative PFOL491D, alone or in combination with α-toxin derivative GST-cpa247-370, was evaluated as possible vaccine candidate, using in vitro assays. PFOL491D showed no haemolytic effect on horse red blood cells and no cytotoxic effect on bovine endothelial cells. Furthermore, calves immunised with PFOL491D raised antibodies against perfringolysin O that could inhibit the perfringolysin O-associated haemolytic activity on horse red blood cells. Antisera from calves immunised with PFOL491D had a significantly higher neutralising capacity against the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells than serum from control calves (P <0.05). Immunisation of calves with PFOL491D in combination with GST-cpa247-370 elicited antibodies against perfringolysin O and α-toxin and consequently inhibited both the perfringolysin O-associated haemolytic activity and the α-toxin-associated lecithinase activity in vitro. Additionally, the neutralising ability of these antisera on the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells was significantly higher than that from calves immunised with PFOL491D (P <0.001). In conclusion, perfringolysin O derivative PFOL491D is an immunogenic antigen that can potentially be used to produce vaccine against bovine necrohaemorrhagic enteritis. Including α-toxin derivative GST-cpa247-370 has an additional protective effect and therefore vaccination of calves with a combination of both antigens seems even more promising.

Clostridium perfringens α-Toxin Impairs Lipid Raft Integrity in Neutrophils.

Clostridium perfringens type A, a Gram-positive, anaerobic bacterium, causes gas gangrene. Recently, we reported that C. perfringens α-toxin blocked neutrophil differentiation in an enzyme activity-dependent manner to impair host innate immunity, which should be crucial for the pathogenesis of C. perfringens. However, the detailed mechanism remains unclear. Lipid rafts have been reported to be platforms for signaling molecules involved in the regulation of cell differentiation in many different cell types. In this study, we found that cell surface expression of a lipid raft marker, GM1 ganglioside, decreased in association with neutrophil differentiation by flow cytometry analysis and morphological observation. In vitro treatment of isolated mouse bone marrow cells with α-toxin or an α-toxin variant lacking phospholipase C and sphingomyelinase activities revealed that α-toxin increased the cell surface expression of GM1 ganglioside in an enzyme activity-dependent manner. C. perfringens infection also increased GM1 ganglioside levels in bone marrow myeloid cells. Moreover, treatment of bone marrow cells with methyl-ß-cyclodextrin, a lipid raft-disrupting agent, impaired neutrophil differentiation. Together, our results suggest that the integrity of lipid rafts should be properly maintained during granulopoiesis, and α-toxin might perturb lipid raft integrity leading to the impairment of neutrophil differentiation.

Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane.

Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA.

Sphingosylphosphorylcholine potentiates vasoreactivity and voltage-gated Ca2+ entry via NOX1 and reactive oxygen species.

AIMS: Sphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (≤1 µmol/L), however, it does not constrict intrapulmonary arteries (IPAs), but strongly potentiates vasoreactivity. Our aim was to determine whether this also occurs in a systemic artery and to delineate the signalling pathway. METHODS AND RESULTS: Rat mesenteric arteries and IPAs mounted on a myograph were challenged with ∼25 mmol/L [K+] to induce a small vasoconstriction. SPC (1 µmol/L) dramatically potentiated this constriction in all arteries by ∼400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C (PLC; U73122), PKCε (inhibitory peptide), Src (PP2), and NADPH oxidase (VAS2870), and also by Tempol (superoxide scavenger), but not by inhibition of Rho kinase (Y27632). Potentiation was lost in mesenteric arteries from p47(phox-/-), but not NOX2(-/-), mice. The intracellular superoxide generator LY83583 mimicked the effect of SPC. SPC elevated reactive oxygen species (ROS) in vascular smooth muscle cells, and this was blocked by PP2, VAS2870, and siRNA knockdown of PKCε. SPC (1 µmol/L) significantly reduced the EC50 for U46619-induced vasoconstriction, an action ablated by Tempol. In patch-clamped mesenteric artery cells, SPC (200 nmol/L) enhanced Ba2+ current through L-type Ca2+ channels, an action abolished by Tempol but mimicked by LY83583. CONCLUSION: Our results suggest that low concentrations of SPC activate a PLC-coupled and NOX1-mediated increase in ROS, with consequent enhancement of voltage-gated Ca2+ entry and thus vasoreactivity. We speculate that this pathway is not specific for SPC, but may also contribute to vasoconstriction elicited by other G-protein coupled receptor and PLC-coupled agonists.

The glycosylphosphatidylinositol-anchored protein repertoire of Babesia bovis and its significance for erythrocyte invasion.

Glycosylphosphatidylinositol-anchored proteins are abundant on the surface of pathogenic protozoans and might play an important role for parasite survival. In the present work, the relevance of GPI-anchored proteins for erythrocyte invasion of the cattle hemoparasite Babesia bovis was studied. We show that cleavage of GPI-anchored antigens from the merozoite parasite stage by phosphatidylinositol-specific phospholipase C abolished invasion of erythrocytes demonstrating the importance of this class of molecules for parasite propagation. In addition, the repertoire of GPI-anchored proteins of B. bovis was predicted with high fidelity by searching its genome with available web-based bioinformatic tools. Altogether 17 GPI-anchored proteins were identified, 5 of which represent the already characterized variable merozoite surface antigens (VMSAs). Fifteen of the identified GPI-anchored proteins contain 2-26 amino acid repeats indicating that they are likely involved in functions of recognition, adhesion, or transport. Repeats were found to contain an increased frequency of proline, indicative of unstructured regions; and were estimated to be 3.21 times more hydrophilic than non-repeat regions. This suggests that they might represent eminent antibody epitopes. The majority of the putative GPI-anchored antigens reported in this work have so far remained unnoticed, though they may represent potential candidates for inclusion in a subunit vaccine.

Clostridium perfringens phospholipase C induced ROS production and cytotoxicity require PKC, MEK1 and NFκB activation.

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.

PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels.

Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C-insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.

Removal of GPI-anchored membrane proteins causes clustering of lipid microdomains in the apical head area of porcine sperm.

The release of extracellular proteins is a part of the sperm capacitation process; this allows the sperm surface reorganization that enables the sperm to fertilize an oocyte. Some of the components released are 'decapacitation factors', an uncoordinated or early release of which may cause inappropriate surface destabilization and premature capacitation. We studied the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in sperm capacitation, and reported that CD52 and CD55 exhibit bicarbonate-dependent release during in vitro sperm capacitation. Treating sperm with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the enzymatic cleavage of CD55, in both capacitating and noncapacitating conditions. Moreover, PIPLC treatment in noncapacitating conditions caused surface reorganization events that included exposure of the ganglioside GM1, aggregation of flotillin-1, and the swelling of the apical acrosome region; all of which have been reported to be associated with sperm capacitation. The acrosomal swelling was monitored using wet mount atomic force microscopy, a new imaging technique that allows nanometer-level sperm surface measurements in samples hydrated with physiological buffer rather than dried. Despite these surface changes, PIPLC treatment in identical incubation conditions did not stimulate hyperactive sperm motility or protein tyrosine phosphorylation (other hallmarks of sperm capacitation in vitro). In full capacitating conditions (i.e., the presence of bicarbonate and albumin), PIPLC treatment caused sperm deterioration. The possible role of GPI-APs removal from the sperm surface during sperm capacitation is discussed.

[New insight from basic research of Clostridium perfringens alpha-toxin].

Clostridium perfringens causes gas gangrene with inflammatory myopathies and infrequently septicemia associated with massive intravascular hemolysis. The microorganism is known to produce a variety of toxins and enzymes that are responsible for severe myonecrotic lesions. Notably, alpha-toxin, which possesses hemolytic, necrotic and lethal activities, and phospholipase C and sphingomyelinase activities, is an important agent for the diseases. The cytokine storm induced by alpha-toxin, mainly the release of TNF-alpha, plays an important role in the death and massive hemolysis. The toxin-induced release of TNF-alpha from neutrophils and macrophages is dependent on the activation of ERK1/2 signal transduction via TrkA receptor. In addition, 14- and 15-membered macrolides specifically block the toxin-induced events through the activation of neutrophils and macrophages.

Clostridium perfringens alpha-toxin induces the release of IL-8 through a dual pathway via TrkA in A549 cells.

A characteristic feature of gas gangrene with Clostridium perfringens (C. perfringens) is the absence of neutrophils within the infected area and the massive accumulation of neutrophils at the vascular endothelium around the margins of the necrotic region. Intravenous injection of C. perfringens alpha-toxin into mice resulted in the accumulation of neutrophils at the vascular endothelium in lung and liver, and release of GRO/KC, a member of the CXC chemokine family with homology to human interleukin-8 (IL-8). Alpha-toxin triggered activation of signal transduction pathways causing mRNA expression and production of IL-8, which activates migration and binding of neutrophils, in A549 cells. K252a, a tyrosine kinase A (TrkA) inhibitor, and siRNA for TrkA inhibited the toxin-induced phosphorylation of TrkA and production of IL-8. In addition, K252a inhibited the toxin-induced phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). PD98059, an ERK1/2 inhibitor, depressed phosphorylation of ERK1/2 and nuclear translocation of nuclear factor kappa B (NF-κB) p65, but SB203580, a p38 MAPK inhibitor, did not. On the other hand, PD98059 and SB203580 suppressed the toxin-induced production of IL-8. Treatment of the cells with PD98059 resulted in inhibition of IL-8 mRNA expression induced by the toxin and that with SB203580 led to a decrease in the stabilization of IL-8 mRNA. These results suggest that alpha-toxin induces production of IL-8 through the activation of two separate pathways, the ERK1/2/NF-κB and p38 MAPK pathways.

Induction of neurite-outgrowth in PC12 cells by alpha-toxin from Clostridium perfringens.

Alpha-toxin-induced phosphorylation of PDK1 via the tyrosine kinase A (TrkA) receptor signaling pathway plays an important role in the activation of rabbit neutrophils. The relation between the toxin and TrkA, however, remains poorly understood. Here, we show that the toxin-induced phosphorylation of TrkA is closely related to the induction of neurite-outgrowth in PC12 cells. The toxin induced neurite-outgrowth and phosphorylation of TrkA in the cells in a dose-dependent manner. K252a, a TrkA inhibitor, and shRNA for TrkA inhibited the toxin-induced neurite-outgrowth, and phosphorylation of TrkA and ERK1/2. PD98059, an inhibitor of the ERK1/2 cascade, inhibited phosphorylation of ERK1/2 and the neurite-outgrowth induced by alpha-toxin. The wild-type toxin induced the formation of diacylglycerol, and neurite-outgrowth, but H148G, a variant toxin which binds to cell membranes and has lost the enzymatic activity did not. We demonstrated that the phosphorylation of TrkA through the phospholipid metabolism induced by the toxin synergistically play a key role in neurite-outgrowth.

Impact of luteolin on the production of alpha-toxin by Staphylococcus aureus.

AIMS: To investigate the influence of subinhibitory concentrations of luteolin on the production of α-toxin in Staphylococcus aureus. METHODS AND RESULTS: The minimal inhibitory concentrations (MICs) were determined using a broth microdilution method, and the MICs of luteolin against the tested Staph. aureus strains ranged from 16 to 64 µg ml(-1). Haemolysis, Western blot and real-time reverse transcription (RT)-PCR assays were used to evaluate the effect of luteolin on Staph. aureusα-toxin secretion and on the level of gene expression, respectively. The data indicated that subinhibitory concentrations of luteolin dose dependently decreased the production of α-toxin in both meticillin-sensitive Staph. aureus (MSSA) and meticillin-resistant Staph. aureus (MRSA). Furthermore, the transcriptional levels of agr (accessory gene regulator) in Staph. aureus were also inhibited by luteolin. CONCLUSIONS: Luteolin decreases the production and/or secretion of α-toxin in Staph. aureus; the reduced production may be dependent, in part, upon the luteolin-induced inhibition of the agr locus. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings indicate that luteolin may be used as a basis for the development of antimicrobial agents aimed at bacterial virulence factors.

Activation of muscarinic acetylcholine receptors induces a nitric oxide-dependent long-term depression in rat medial prefrontal cortex.

Cholinergic neurotransmission in the medial prefrontal cortex (mPFC) is critical for normal processing of cue detection and cognitive performance. However, the mechanism by which cholinergic system modifies mPFC synaptic function remains unclear. Here we show that activation of muscarinic acetylcholine receptors (mAChRs) by carbamoylcholine (CCh) induces long-term depression (CCh-LTD) of excitatory synaptic transmission on mPFC layer V pyramidal neurons. The induction of CCh-LTD is dependent on M(1) mAChR activation but does not require N-methyl-D-aspartate receptor activation or coincident synaptic stimulation. Activation of phospholipase C (PLC), protein kinase C (PKC), and postsynaptic Ca(2+) release from inositol 1,4,5-triphosphate (IP(3)) receptor-sensitive internal stores are required for CCh-LTD induction. The expression of CCh-LTD is likely to be presynaptic because it is accompanied by a decrease in 1/(coefficient of variance)(2) and an increase in synaptic failure and paired-pulse ratio of synaptic responses. CCh-LTD is blocked by nitric oxide (NO) synthase inhibitors, soluble guanylyl cyclase (sGC) inhibitor, and protein kinase G (PKG) inhibitor. Synaptic stimulation of M(1) mAChRs with prolonged paired-pulse low-frequency stimulation also triggers LTD. These results suggest that activation of M(1) mAChRs can induce LTD on mPFC layer V pyramidal neurons through the activation of postsynaptic PLC/PKC/IP(3) receptor- and subsequently presynaptic NO/sGC/PKG-dependent signaling processes.

Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca signaling.

The synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals are characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons. The synaptic vesicle accumulation and axon terminal remodeling are regulated by protein kinase A and calcineurin signaling, respectively. To investigate upstream signals of presynaptic differentiation, we focused on Ca(2+) signaling as Ca(2+)/calmodulin is required for the activation of both calcineurin and some adenylyl cyclases. We here showed that application of Ca(2+)/calmodulin inhibitor or olfactory sensory neuron-specific expression of calmodulin inhibitory peptide suppressed both synaptic vesicle accumulation and axon terminal remodeling. Thus, the trigger of presynaptic differentiation could be Ca(2+) release from intracellular stores or Ca(2+) influx. Application of a phospholipase C inhibitor or olfactory sensory neuron-specific expression of inositol 1,4,5-trisphosphate (IP(3)) 5-phosphatase suppressed synaptic vesicle accumulation, but not morphological remodeling. In contrast, application of a voltage-gated Ca(2+) channel blocker or expression of Kir2.1 inward rectifying potassium channel prevented the morphological remodeling. We also provided evidence that IP(3) signaling acted upstream of protein kinase A signaling. Our results suggest that IP(3)-mediated Ca(2+)/calmodulin signaling stimulates synaptic vesicle accumulation and subsequent neuronal activity-dependent Ca(2+)/calmodulin signaling induces the morphological remodeling of axon terminals.

Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol.

Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and "matrix" cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells.