Calcium-free Medium Research Articles

Lactacidosis-induced glial cell swelling depends on extracellular Ca 2+

Cerebral tissue acidosis following ischemia or traumatic brain injury contributes to cytotoxic brain edema formation. In vitro lactacidosis induces swelling of glial cells by intracellular Na +- and Cl −-accumulation by the Na +/H +-antiporter, Cl −/HCO 3 −-antiporters and the Na +–K +–2Cl −-cotransport. The present study aimed to elucidate whether mechanisms of lactacidosis-induced glial swelling are dependent on intra- or extracellular Ca 2+-ions. Therefore, C6 glioma cells were exposed to a lactacidosis of pH 6.2 in standard or calcium-free medium and following intracellular calcium chelation. Cell volume and intracellular pH were assessed by flow cytometry. Lactacidosis of pH 6.2 induced a prompt and sustained swelling of suspended C6 glioma cells reaching a maximum of 128% within 60 min. Omission of Ca 2+ from the suspension medium strongly attenuated cell swelling while chelation of intracellular Ca 2+ had no effects. Intracellular acidosis was not affected by either treatment. The present data show a strong dependency of lactacidosis-induced glial swelling upon extracellular Ca 2+ while intracellular acidosis is not affected by omission of [Ca 2+] e. Therefore, our data suggest that the Na +–K +–2Cl −-cotransporter, the only so far known transporter involved in cell volume regulation but not in pH i regulation during lactacidosis, is activated in a [Ca 2+] e-dependent manner.

Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.

Mouse β-TC6 Insulinoma Cells: High Expression of Functional α3β4 Nicotinic Receptors Mediating Membrane Potential, Intracellular Calcium, and Insulin Release

Nicotine elicited membrane depolarization, elevation of intracellular calcium, rubidium efflux, and release of insulin from mouse beta-TC6 insulinoma cells. Such responses were blocked by the nicotinic antagonist mecamylamine but not by the muscarinic antagonist atropine. Neither the selective alpha4beta2 antagonist dihydro-beta-erythroidine nor the selective alpha7 antagonist methyllycaconitine significantly blocked the nicotine-elicited depolarization or the calcium response. The elevation of intracellular calcium did not occur in calcium-free media, indicating that the increase in intracellular calcium was due to the influx of calcium. The rank order of potency for nicotinic agonists was as follows: epibatidine > nicotine = 3-(azetidinylmethoxy)pyridine (A-85380), cytisine, dimethylphenylpiperazinium (DMPP). Cytisine and DMPP seemed to be partial agonists. The density of nicotinic receptors measured by [3H]epibatidine binding was 7-fold higher in membranes from beta-TC6 cells than in rat brain membranes. No binding of 125I-A-85380 was detected, indicating the absence of beta2-containing receptors. Reverse transcription-polymerase chain reaction analyses indicated the presence of mRNA for alpha3 and alpha4 subunits and beta2 and beta4 subunits in beta-TC6 cells. The binding and functional data suggest that the major nicotinic receptor is composed of alpha3 and beta4 subunits. The beta-TC6 cells thus provide a model system for pharmacological study of such nicotinic receptors.

An indirect enzyme-linked immunosorbent assay for rapid and quantitative assessment of Type III virulence phenotypes of Pseudomonas aeruginosa isolates

BackgroundThe presence of a Type III secretion system in clinical isolates of Pseudomonas aeruginosa is associated with severe disease and poor outcomes in infections caused by this pathogen. We describe an indirect enzyme-linked immunosorbent assay that rapidly and quantitatively detects two exotoxins, ExoU and ExoT, and two structural components, PopD and PcrV, of the P. aeruginosa Type III secretion system after in-vitro growth in a calcium-free minimal medium.MethodsWe used this assay to characterize the Type III secretion phenotype of 74 clinical isolates of P. aeruginosa. Findings were compared with results of standard immunoblotting and correlated with Type III secretion-dependent virulence of isolates toward cultured epithelial cells.ResultsResults of the ELISA assay were concordant with immunoblot detection of the secreted antigens for 73 of 74 isolates. The Type III secretion phenotype assessed by this immunoassay predicted bacterial virulence toward epithelial cells in vitro for all but five of the clinical isolates.ConclusionThe availability of an ELISA assay for rapid detection of Type III secreted virulence factors will facilitate large clinical studies to examine whether the Type III secretion phenotype of a P. aeruginosa isolate predicts the course of clinical disease in a patient and should be taken into account in determining optimal treatment strategies for infected patients.

EphA2 Phosphorylates the Cytoplasmic Tail of Claudin-4 and Mediates Paracellular Permeability

Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell-cell interaction. EphA2 is expressed in various cancer tissues and cell lines. Although the mechanism of action of EphA2 is unknown, its expression correlates with progression of the malignant phenotype of cancerous tissues. Here, we have shown that EphA2 modulates the localization and function of claudin-4, a constituent of tight junctions. EphA2 associates with claudin-4 via their extracellular domains. This association, in turn, leads to phosphorylation of the cytoplasmic carboxyl terminus of claudin-4 at Tyr-208. The tyrosine phosphorylation of claudin-4 attenuates association of claudin-4 with ZO-1, decreasing integration of claudin-4 into sites of cell-cell contact and enhancing paracellular permeability. These results indicate that EphA2 moderates the function of tight junctions via phosphorylation of claudin-4.

CPU 86017, p-chlorobenzyltetrahydroberberine chloride, attenuates monocrotaline-induced pulmonary hypertension by suppressing endothelin pathway1

To elucidate the involvement of the endothelin (ET) pathway in the pathogenesis of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and the therapeutic effect of CPU 86017 (p-chlorobenzyltetrahydroberberine chloride) in rats. Rats were injected with a single dose (60 mg/kg, sc) of MCT and given CPU 86017 (20, 40, and 80 mg/kg-1/d-1, po) or saline for 28 d. The hemodynamics, mRNA expression, and vascular activity were evaluated. Right ventricular systolic pressure and central venous pressures were elevated markedly in the PAH model and decreased by CPU 86017. In the PAH group, the endothelin-1 (ET-1) in serum and lungs was dramatically increased by 54% (79.9 pg/mL, P<0.01) and 93% (166.2 pg/mL, P<0.01), and mRNA levels of preproET-1, eNOS, and iNOS also increased dramatically compared with control. Compared with PAH group, CPU 86017 decreased the content of ET-1 to the normal level in lung tissue, but was less effective in serum. The level of NO was significantly increased in CPU 86017 at 80 and 40 mg/kg-1/d-1 groups in tissue, whereas the difference in serum was not significant. A significant reduction in MDA production and an increase in the SOD activity in the serum and lungs was observed in all three CPU 86017 groups. CPU 86017 80 mg/kg-1/d-1 po increased the activity of cNOS by 33% (P<0.01). The up-regulation of eNOS and iNOS mRNA levels induced by MCT was significantly reversed in 3 CPU 86017 groups, and preproET-1 mRNA abundance was also reduced notably in CPU 86017 80 mg/kg-1/d-1 group vs the PAH group. The KCl-induced vasoconstrictions in the calcium-free medium decreased markedly in PAH group but recovered partially after CPU 86017 intervention. The constrictions in the presence of Ca(2+) was not improved by CPU 86017. The phenylephrine-induced vasoconstrictions in the calcium-free medium decreased markedly in PAH group but not recovered after CPU 86017 intervention. The constrictions in the presence of Ca(2+) completely returned to the normal after CPU 86017 intervention. CPU 86017 suppressed MCT-induced PAH mainly through an indirect suppression of the ET-1 system, which was involved in the pathogenesis of the disease.

Cardiovascular Effects of the Essential Oil of Aniba canelilla Bark in Normotensive Rats

Cardiovascular effects of intravenous (i.v.) treatment with the essential oil of the bark of Aniba canelilla (EOAC) were investigated in normotensive rats. In both pentobarbital-anesthetized and conscious rats, i.v. bolus injections of EOAC (1 to 20 mg/kg) elicited similar and dose-dependent hypotension and bradycardia. Pretreatment of anesthetized rats with bilateral vagotomy significantly reduced the bradycardia without affecting the hypotension. In conscious rats, pretreatment with hexamethonium (30 mg/kg, i.v.) significantly reduced the EOAC-induced bradycardia without affecting the hypotension. The opposite effect was observed after i.v. pretreatment with the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl esther (L-NAME, 20 mg/kg). However, both EOAC-induced hypotension and bradycardia were significantly reduced by pretreatment with methylatropine (1 mg/kg, i.v.). In rat endothelium-containing aorta preparations, EOAC (1-600 microg/mL) induced a concentration-dependent reduction of potassium (60 mM)-induced contraction [IC50 (geometric mean+/-95% confidence interval)=64.5 (45.6-91.2) microg/mL)], an effect that was significantly reduced by the addition of atropine (10 microM) in the perfusion medium [IC50=109.5 (72.5-165.4) microg/mL)]. Furthermore, the vasorelaxant effects of the EOAC were also but significantly reduced [IC50=139.1 (105.2-183.9) microg/mL)] by removal of the vascular endothelium. Furthermore, the CaCl2-induced contractions in calcium-free medium were reduced and even fully abolished by EOAC (100 and 600 microg/mL), respectively. However, EOAC (600 microg/mL) was without significant effect on caffeine-induced contractions in calcium-free medium. These data show that i.v. treatment of rats with EOAC induces dose-dependent hypotension and bradycardia, which occurred independently. The bradycardia appears mainly dependent upon the presence of an operational and functional parasympathetic drive to the heart. However, the hypotension is due to an active vascular relaxation rather than withdrawal of sympathetic tone. This relaxation seems partly mediated by an endothelial L-arginine/nitric oxide pathway through peripheral muscarinic receptor activation (endothelium-dependent relaxation) and predominantly through an inhibition of calcium inward current (endothelium-independent relaxation).

TDZ-induced triple-response and shoot formation on intact seedlings of Linum, putative role of ethylene in regeneration

Intact seedlings of Linum regenerate to form shoots on hypocotyl on TDZ medium. Prior to regeneration, these seedlings were characterized by ‘triple-response’; inhibition of root elongation, shortening and swelling of hypocotyl and tightening of cotyledons towards apex. Triple-response due to TDZ is a new record, it is similar to ethylene-action and points toward the mode of TDZ action which results in production of ‘stress-ethylene’ and is followed by shoot regeneration. Putative role of ethylene in regeneration, became apparent when inhibitors of ethylene-action (AgNO 3, 10 μM) or ethylene-biosynthesis (CoCl 2, 10 μM) resulted in reduction of regeneration response. Also inclusion of KMnO 4, an effective absorber of ethylene in culture atmosphere, resulted in inhibition of regeneration. A correlation exists between root repression and shoot promotion by TDZ. BAP also resulted in root repression and shoot formation but at higher concentration (10 μM). TDZ-promoted regeneration is a calcium-mediated response. On calcium-free medium, TDZ induced a low frequency shoot-buds, which failed to develop into leafy shoots. However, this frequency increased on a corresponding increase in concentration of calcium. When seedlings were subjected to calcium-stress, by germinating seeds on calcium-free medium, and on transfer to calcium- and TDZ-fortified medium (1 μM) there was an increased frequency of shoot formation not only on hypocotyl but also on cotyledonary-node, which normally remains quiescent. Inhibitors of calmodulin (CPZ or TFP) inhibited shoot formation on TDZ medium but also promoted elongation of roots. Elongation of root by CPZ or TFP is new finding of this investigation. It also supports the suggestion that root repression and shoot promotion are inversely related.

Dynamics of calcium fluxes in human platelets assessed in calcium-free medium

Dynamics of changes in cytosolic calcium concentration resulting from facilitation of calcium leakage from the stores and (or) blocking the pathways of its reuptake back into the stores or extrusion out of the cell (or both) have been investigated experimentally. It has been found that: (a) no mechanisms other than the membrane leakage, PMCA or SERCA, are involved in the discharge of calcium stores and calcium extrusion or reuptake; (b) the discharge of calcium stores in the absence of both its extrusion and reuptake back into the stores depends only on membrane leakage, the asymptotic calcium concentration in cytosol depending only on the initial content of the stores and being independent of the leakage; (c) the dynamics of the activity of both PMCA and SERCA depend on the initial rate of calcium influx, the dynamics differing from each other at high initial rates of calcium influx; (d) whereas there is no observable background activity of PMCA, background activity of SERCA is observed.

Voltage‐gated calcium channels in developing GnRH‐1 neuronal system in the mouse

Migration of gonadotropin-releasing hormone-1 (GnRH-1) neurons from the nasal placode into the central nervous system occurs in all vertebrates. This study characterizes the expression of L- and N-type voltage-gated calcium channels (VGCCs) in migrating GnRH-1 neurons in mice. Class C (L-type) and class B (N-type) VGCGs were detected in GnRH-1 cells and cells in the olfactory and vomeronasal epithelium during prenatal development. This expression pattern was mimicked in a nasal explant model known to retain many characteristics of GnRH-1 development in vivo. Using this in vitro system, perturbation studies were performed to elucidate the role of VGCCs in GnRH-1 neuronal development. This report shows that olfactory axon outgrowth and GnRH-1 neuronal migration are attenuated when nasal explants are grown in calcium-free media, and that this effect is temporally restricted to an early developmental period. Blockade of either the L- or the N-type channel did not alter GnRH-1 cell number or overall olfactory axon outgrowth. However, blockade of N-type channels altered the distribution of GnRH-1 neurons in the periphery of the nasal explants. In these explants, more GnRH-1 neurons were located proximal to, and fewer GnRH-1 neurons distal to, the main tissue mass, suggesting a general decrease in the rate of GnRH-1 neuronal migration. These results indicate that extracellular calcium is required for initiating GnRH-1 neuronal migration and that these events are partially dependent on N-type VGCC signals.

Role of Calcium in Exocytosis Induced by Hypotonic Swelling

We studied the influence of Ca(2+) on exocytosis induced by hypotonic shock using the fluorescent dyes acridine orange FM1-43 and FM2-10. It was shown using acridine orange that lowering osmolarity to 230 mOsm/L induces exocytosis both in calcium-containing and calcium-free media. By contrast, we were able to demonstrate calcium dependence of exocytosis using styryl dyes. Lowering osmolarity leads to an increase of neurotransmitter release in a calcium-independent manner. Thus, our data suggest that hypotonic swelling induces calcium-independent exocytosis. Calcium influx mediated by stretch channels is able to switch mode of exocytosis from "kiss and run" to full fusion.

Membrane Depolarization Induces the Undulating Phosphorylation/Dephosphorylation of Glycogen Synthase Kinase 3β, and This Dephosphorylation Involves Protein Phosphatases 2A and 2B in SH-SY5Y Human Neuroblastoma Cells

Changes in plasma membrane electrical potential evoke signals that regulate the expressions of various genes in the nervous system. However, the role of glycogen synthase kinase 3beta (GSK-3beta) in this process has not been elucidated. Thus, this study was performed to examine whether membrane depolarization can regulate the phosphorylation of GSK-3beta and to identify the molecular mechanisms involved in this regulation. The depolarization by treating with 100 mm KCl for 5 min resulted in the undulating phosphorylation of GSK-3beta at Ser-9 in SH-SY5Y human neuroblastoma cells, in H19 -7/IGF-IR rat embryonic hippocampal cells, and in PC12 rat pheochromocytoma cells, but not in A172 human glioblastoma cells. Cellular beta-catenin contents showed a temporal pattern similar to that of the Ser-9 phosphorylation of GSK-3beta. Treatment with wortmannin or calphostin C or the expression of dominant negative Akt inhibited phosphorylation of GSK-3beta at Ser-9 following the KCl-induced depolarization of SH-SY5Y cells. Moreover, pretreatment with okadaic acid or cyclosporin A blocked the dephosphorylation of GSK-3beta at Ser-9 at 0, 15, and 30 min after KCl-induced depolarization, and the activity of protein phosphatases (PP) 2A and 2B increased at these times. Treatment with nifedipine or calcium-free medium inhibited GSK-3beta dephosphorylation following membrane depolarization, and the amounts of co-immunoprecipitated GSK-3beta and PP2A changed in parallel with GSK-3beta dephosphorylation. Our study demonstrated that KCl-induced depolarization caused undulating GSK-3beta phosphorylation/dephosphorylation, which was regulated for the most part by phosphatidylinositol 3-kinase and Akt (phosphorylation) and PP2A and PP2B (dephosphorylation), respectively.

Duration of In Vitro Maturation of Recipient Oocytes Affects Blastocyst Development of Cloned Porcine Embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.

Synaptogenesis on mature hippocampal dendrites occurs via filopodia and immature spines during blocked synaptic transmission

During development, dendritic spines emerge as stubby protrusions from single synapses on dendritic shafts or from retracting filopodia, many of which have more than one synapse. These structures are rarely encountered in the mature brain. Recently, confocal and two-photon microscopy have revealed a proliferation of new filopodia-like protrusions in mature hippocampal slices, especially when synaptic transmission was blocked. It was not known whether these protrusions have synapses nor whether they are accompanied by the other immature spine forms. Here, reconstruction from serial section electron microscopy (ssEM) was used to answer these questions. Acute hippocampal slices from mature male rats, ages 56 and 63 days, were maintained in vitro in control medium or in a nominally calcium-free medium with high magnesium, glutamate receptor antagonists, and sodium and calcium channel blockers. At the end of each 8-hour experiment, all slices were fixed, coded, and processed for ssEM. In agreement with light microscopy, there were more filopodia along dendrites in slices with blocked synaptic transmission. These filopodia were identified by their pointy tips and either the absence of synapses or presence of multiple synapses along them. There was also a proliferation of stubby spines. Filopodia along mature dendrites were typically shorter than developmental filopodia, with outgrowth likely being constrained by reduced extracellular space and compact neuropil, providing numerous candidate presynaptic partners in the vicinity of the mature dendrites. These findings suggest that synaptogenesis and spine formation are readily initiated under conditions of reduced activity in the mature brain.

Adrenomedullin Stimulates Nitric Oxide Release from SK-N-SH Human Neuroblastoma Cells by Modulating Intracellular Calcium Mobilization

We used SK-N-SH human neuroblastoma cells to test the hypothesis that adrenomedullin (ADM), a multifunctional neuropeptide, stimulates nitric oxide (NO) release by modulating intracellular free calcium concentration ([Ca2+]i) in neuron-like cells. We used a nitrite assay to demonstrate that ADM (10 pM to 100 nM) stimulated NO release from the cells, with a maximal response observed with 1 nM at 30 min. This response was blocked by 1 nM ADM(22-52), an ADM receptor antagonist or 2 microM vinyl-L-NIO, a neuronal NO synthase inhibitor. In addition, 5 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular calcium chelator, eliminated the ADM-induced NO release. Similar results were observed when the cells were incubated in calcium-free medium or when L-type calcium channels were inhibited with 5 microM nifedipine or 10 microM nitrendipine. Depletion of calcium stores in the endoplasmic reticulum (ER) with 1 microM cyclopiazonic acid or 150 nM thapsigargin, or inhibition of ryanodine-sensitive receptors in the ER with 10 microM ryanodine attenuated the ADM-induced NO release. NO responses to ADM were mimicked by 1 mM dibutyryl cAMP, a cAMP analog, and were abrogated by 5 microM H-89, a protein kinase A inhibitor. Furthermore, Fluo-4 fluorescence-activated cell sorter analysis showed that ADM (1 nM) significantly increased [Ca2+]i at 30 min. This response was blocked by nifedipine (5 microM) or H-89 (5 microM) and was reduced by ryanodine (10 microM). These results suggest that ADM stimulates calcium influx through L-type calcium channels and ryanodine-sensitive calcium release from the ER, probably via cAMP-protein kinase A-dependent mechanisms. These elevations in [Ca2+)]i cause activation of neuronal NO synthase and NO release.

Extracellular Ca2+ requirement for serotonin-induced release and meiosis reinitiation from prophase in oocytes of the scallop Argopecten purpuratus

Summary The requirement for Ca2+in the external medium for the release and reinitiation of meiosis in the scallop Argopecten purpuratus was examined. In vitro experiments demonstrated that the serotonin-induced release of oocytes is inhibited in a calcium-free medium. Caffeine (10 and 20 mM), a known activator of ryanodine channels, increased oocyte release by gonadal pieces and 50 μM ryanodine inhibited this release and blocked the serotonin-induced effect. The number of oocytes released under control conditions was not affected when ryanodine was used in concentrations in the nanomolar range. Germinal vesicle breakdown, indicative of meiosis reinitiation, was inhibited in gonadal pieces incubated in calcium-free normal or artificial seawater and serotonin does not overcome this inhibition in the same conditions. An increase in fluorescence on adding serotonin (10−5 M, final concentration) was detected under the confocal microscope when oocytes charged with the calcium fluorescent dye Fluo-3 AM were studied. We postulate that there is a rise in cellular calcium in the serotonin-induced spawning process and that the source for the increase is influx from the external medium and release from internal stores. We suggest that this calcium rise plays a role in germinal vesicle breakdown and in the contraction of gonadal smooth muscle.

ADAM10 Mediates Ectodomain Shedding of the Betacellulin Precursor Activated by p-Aminophenylmercuric Acetate and Extracellular Calcium Influx

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.

78 EFFECTS OF DIFFERENT ACTIVATION PROTOCOLS ON ACTIN FILAMENT DISTRIBUTION AND IN VITRO DEVELOPMENT OF MINIATURE PIG NT EMBRYOS

In the process of producing reconstructed oocytes nuclear transfer (NT) embryos by somatic cell nuclear transfer, in vitro-matured oocytes can be used as recipient ones. It, however, has been well documented that after IVF porcine embryos derived from in vitro-matured oocytes have a small number of cells and low viability compared from those in vivo. As one possible reason, abnormal actin filament distribution has been detected in abnormal embryo cleavage and small cell numbers (Wang et al. 1999 Biol. Reprod. 60, 1020-1028). Artificial activation, which is necessary for development of NT embryos, can affect actin filament distribution of porcine oocytes matured in vitro, resulting in fragmentation (Kawahara et al. 2002 Theriogenology 58, 1081-1095). In the present study, we investigated effects of different activation protocols on actin filament distribution and in vitro development of miniature pig NT embryos. Porcine oocytes collected from ovaries were matured in vitro for 40 to 44 h in NCSU-23. First, we compared different activation protocols in development rates to blastocysts of oocytes activated. We used three activation methods (15 �M ionomycin treatment for 20 min (I), double DC pulses of 1.2 kV/cm for 60 ms in intervals of 5 s (E), and 5 mg/mL cycloheximide treatment for 5 h (C)) to prepare seven activation protocols (I, E, C, I + C, I + E, E + C, and I + E + C). Second, we examined effects of different activation protocols on actin filament distribution and subsequent development of NT embryos activated by the different activation protocols. Matured oocytes were enucleated, and fused with miniature pig fetal fibroblasts in calcium-free medium; approximately 3 h later, the resultant NT embryos were activated with three activation protocols (E, I + C, or I + E + C). All data were analyzed by chi-square test. The developmental rates to blastocysts in the I, E, C, I + C, I + E, E + C, and I + E + C groups were 5.6, 11.1, 0.0, 36.1, 20.7, 14.6, and 24.7%, respectively, showing that the rate in oocytes activated with I + C was significantly higher (P &lt; 0.05) than the rates in oocytes activated by other treatments. In NT embryos, the developmental rates to blastocysts in the E, I + C, or I + E + C groups were 4.1, 14.3, and 4.6%, respectively, showing that the rate in NT embryos activated with I + C was significantly higher (P &lt; 0.05) than the rate in NT embryos activated with other treatments. The abnormal rate of actin filament distribution in NT embryos activated with E or I + E + C was significantly higher (P &lt; 0.05) than that in NT embryos activated with I + C (26.7% or 33.3% vs. 6.7%). The present results suggest that in miniature pig NT embryos an activation protocol by ionomycin combined with cycloheximide treatments may avoid physical damage to actin filaments with the resultant improvement of subsequent development.

BDNF Acutely Modulates Synaptic Transmission and Calcium Signalling in Developing Cortical Neurons

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, BDNF has been reported to exert an acute potentiation of synaptic activity and are critically involved in long-term potentiation(LTP). We found that BDNF rapidly induced potentiation of synaptic activity and an increase in the intracellular Ca²⁺ concentration in cultured cortical neurons. Within minutes of BDNF application to cultured cortical neurons, spontaneous firing rate was dramatically increased as was the frequency and amplitude of excitatory spontaneous postsynaptic currents (EPSCs). Fura-2 recordings showed that BDNF acutely elicited an increase in intracellular calcium concentration ([Ca²⁺]_i). This effect was partially dependent on extracellular Ca²⁺. In calcium-free perfusion medium a substantial calcium signal remained which disappeared after loading of cortical neurons with 5 µM U-73122. BDNF-induce Ca²⁺ transients were completely blocked by K252a and partially blocked by Cd²⁺. The results demonstrate that BDNF can enhance synaptic transmission and induce directly a rise in [Ca²⁺]_i that require two routes: the release of Ca²⁺ from intracellular calcium stores and influx of extracellular Ca²⁺ mainly through voltage-dependent Ca²⁺ channels in cultured cortical neurons.

Effects of haloperidol and clozapine on glutamate release from nerve terminals isolated from rat prefrontal cortex

The present study was conducted to understand the effect of haloperidol, a typical antipsychotic, and clozapine, an atypical one, on the release of endogenous glutamate in nerve terminals isolated from rat prefrontal cortex using an on-line enzyme-linked fluorometric assay. We found that both haloperidol and clozapine significantly inhibited 4-aminopyridine (4AP)-evoked and veratridine-evoked but not KCl-evoked glutamate release from prefrontocortical synaptosomes. This inhibition produced by these two drugs was concentration-dependent with different potency, and associated with a reduction both in the depolarization-evoked increase in the intrasynaptosomal free Na(+) concentration ([Na(+)](i)) and in 4AP or KCl-evoked depolarization of the synaptosomal plasma membrane potential. Additionally, in the presence of calcium-free medium containing 0.2 mM EGTA, the Ca(2+)-independent component of 4AP-evoked glutamate release was also inhibited by haloperidol or clozapine. Based on these results, we suggest that haloperidol and clozapine inhibit glutamate release from rat prefrontocortical nerve terminals by affecting ion-channel activities determining nerve terminal excitability, probably as a result of Na(+) channel blockage or K(+) channel activation.