Mechanism Underlying the ATP-Induced Increase in the Cytosolic Ca2+ Concentration in Chick Ciliary Ganglion Neurons

Abstract: We examined the mechanism underlying the ATP-induced increase in the cytosolic Ca²⁺ concentration ([Ca]_in) in acutely isolated chick ciliary ganglion neurons, using fura-2 microfluorometry. The ATP-induced increase in [Ca]_in was dependent on external Ca²⁺, was blocked in a dose-dependent manner by reactive blue 2, and was substantially inhibited by both L- and N-type Ca²⁺ channel blockers. ATP was effective in increasing [Ca]_in in the presence of a desensitizing concentration of nicotine (100 µ M ), and simultaneous addition of maximal doses of ATP and nicotine caused an additive increase in [Ca]_in, suggesting that ATP acts on a site distinct from nicotinic acetylcholine receptors. ATP also increased the cytosolic Na⁺ concentration as determined by sodium-binding benzofuran isophthalate microfluorometry. These results suggest that ATP increases Na⁺ influx through P₂ purinoceptor-associated channels resulting in membrane depolarization, which in turn increases Ca²⁺ influx through voltage-dependent Ca²⁺ channels. However, ATP still caused a small increase in [Ca]_in under Na⁺-free conditions, and this [Ca]_in increase was little affected by Ca²⁺ channel blockers. ATP also increased Mn²⁺ influx under Na⁺-free conditions, as indicated by quenching of fura-2 fluorescence. These results suggest that nonselective cationic channels activated by ATP are permeable not only to Ca²⁺ but also to Mn²⁺, in addition to monovalent cations.     

Signaling Pathway Downstream of GABAA Receptor in the Growth Cone

Abstract: The growth cone is responsible for axonal elongation and pathfinding by responding to various modulators for neurite growth, including neurotransmitters, although the sensor mechanisms are not fully understood. Among neurotransmitters, GABA is most likely to demonstrate activity in vivo because GABA and the GABA_A receptor appear even in early stages of CNS development. We investigated the GABA_A receptor-mediated signaling pathway in the growth cone using isolated growth cones (IGCs). Both the GABA_A binding site and the benzodiazepine modulatory site were enriched in the growth cone membrane. In the intact IGC, GABA induced picrotoxin-sensitive Cl⁻ flux (not influx but efflux) and increased the intracellular Ca²⁺ concentration in a picrotoxin- and verapamil-sensitive manner. Protein kinase C (PKC)-dependent phosphorylation of two proteins identified as GAP-43 and MARCKS protein was enhanced in the intact IGC stimulated by GABA, resulting in the release of MARCKS protein and GAP-43 from the membrane. Collectively, our results suggest the following scheme: activation of the functional GABA_A receptor localized in the growth cone membrane → Cl⁻ efflux induction through the GABA_A-associated Cl⁻ channel → Ca²⁺ influx through an L-type voltage-sensitive Ca²⁺ channel → Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein by PKC.     

Modulatory Action of Taurine on the Release of GABA in Cerebellar Slices of the Guinea Pig

Abstract: For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [³H]γ-aminobutyric acid (pH]GABA) and l -[³H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [³H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of l -[³H]glutamate was little affected by taurine. The release of [³H]GABA was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [³H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [³H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca²⁺ into cerebellar tissues was tentatively suggested.     

Protein Phosphorylation and Calcium Uptake into Rat Forebrain Synaptosomes: Modulation by the σ Ligand, 1,3-Ditolylguanidine

Abstract: The σ ligand 1,3-di- O -tolylguanidine (DTG) increased basal dynamin and decreased depolarization-stimulated phosphorylation of the synaptosomal protein synapsin Ib without having direct effects on protein kinases or protein phosphatases. DTG dose-dependently decreased the basal cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and blocked the depolarization-dependent increases in [Ca²⁺]_i. These effects were inhibited by the σ antagonists rimcazole and BMY14802. The nitric oxide donors sodium nitroprusside (SNP) and 8-( p -chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate decreased basal [Ca²⁺]_i and the KCl-evoked rise in [Ca²⁺]_i to an extent similar to DTG. SNP, but not DTG, produced a rise in cyclic GMP levels, suggesting that the effect of DTG on [Ca²⁺]_i was not mediated via downstream regulation of cyclic GMP levels. DTG increased ⁴⁵Ca²⁺ uptake and efflux under basal conditions and inhibited the ⁴⁵Ca²⁺ uptake induced by depolarization with KCl. The KCl-evoked rise in [Ca²⁺]_i was inhibited by ω-conotoxin (ω-CgTx)-GVIA and -MVIIC but not nifedipine and ω-agatoxin-IVA. The effect of DTG on decreasing the KCl-evoked rise in [Ca²⁺]_i was additive with ω-CgTx-MVIIC but not with ω-CgTx-GVIA. These data suggest that DTG was producing some of its effects on synapsin I and dynamin phosphorylation and intrasynaptosomal Ca²⁺ levels via inhibition of N-type Ca²⁺ channels.     

Neuropeptide Y inhibits [Ca2+]i changes in rat retinal neurons through NPY Y1, Y4, and Y5 receptors

Neuropeptide Y (NPY) and NPY receptors are widely distributed in the CNS, including the retina, but the role of NPY in the retina is largely unknown. The aim of this study was to investigate whether NPY modulates intracellular calcium concentration ([Ca²⁺]_i) changes in retinal neurons and identify the NPY receptors involved. As NPY decreased the [Ca²⁺]_i amplitudes evoked by 30 mM KCl in only 50% of neurons analyzed, we divided them in two populations: NPY-non-responsive neurons (Δ2/Δ1 ≥ 0.80) and NPY-responsive neurons (Δ2/Δ1 < 0.80), being the Δ2/Δ1 the ratio between the amplitude of [Ca²⁺]_i increase evoked by the second (Δ2) and the first (Δ1) stimuli of KCl. The NPY Y₁/Y₅, Y₄, and Y₅ receptor agonists (100 nM), but not the Y₂ receptor agonist (300 nM), inhibited the [Ca²⁺]_i increase induced by KCl. In addition, the inhibitory effect of NPY on evoked-[Ca²⁺]_i changes was reduced in the presence of the Y₁ or the Y₅ receptor antagonists. In conclusion, NPY inhibits KCl-evoked [Ca²⁺]_i increase in retinal neurons through the activation of NPY Y₁, Y₄, and Y₅ receptors. This effect may be viewed as a potential neuroprotective mechanism of NPY against retinal neurodegeneration.     

Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells

Abstract: Using a range of Ca²⁺ channel blockers we have investigated the Ca²⁺ channel subtypes that mediate the depolarisation-induced elevation of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release from cultured rat cerebellar granule cells. ω-Conotoxin-GVIA had little effect on either the transient or plateau phase of the depolarisation-induced [Ca²⁺]_i rise or on glutamate release, ruling out a significant role for N-type Ca²⁺ channels. Nifedipine substantially inhibited the initial transient rise in [Ca²⁺]_i and the plateau phase of the [Ca²⁺]_i rise and glutamate release, suggesting the involvement of L-type Ca²⁺ channels. Both ω-agatoxin and ω-conotoxin-MVIIC also inhibited the transient rise in [Ca²⁺]_i and glutamate release but not the plateau phase of the [Ca²⁺]_i rise. The inhibitions by nifedipine were not increased by coaddition of ω-conotoxin-MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca²⁺ currents that are sensitive to nifedipine, ω-agatoxin-IVA, and also ω-conotoxin-MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca²⁺]_i rise or glutamate release to distinct P-, Q-, or O-type Ca²⁺ currents.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Zinc Inhibition of t-[3H]Butylbicycloorthobenzoate Binding to the GABAA Receptor Complex

Abstract: The effect of Zn²⁺ on t -[³H]butylbicycloorthobenzoate ([³H]TBOB) binding to the GABA_A receptor complex was studied autoradiographically in rat brain. Zn²⁺ inhibited [³H]TBOB binding in a dose-dependent manner at physiological concentrations. Saturation analysis revealed noncompetitive inhibition in various brain regions. The inhibitory effect of Zn²⁺ had regional heterogeneity; regions showing the greatest inhibition of [³H]TBOB binding were cortical laminae I–III, most areas of hippocampus, striatum, septum, and cerebellar cortex. Regions with relatively less inhibition of [³H]TBOB binding included cortical laminae V–VI, thalamus, superior colliculus, inferior colliculus, and central gray matter. The effect of Zn²⁺ and those of other GABA_A ligands, such as benzodiazepines, bicuculline, isoguvacine, and picrotoxin, on [³H]TBOB binding seemed to be additive. Ni²⁺, Cd²⁺, and Cu²⁺ also inhibited [³H]TBOB binding with a regional heterogeneity similar to that produced by Zn²⁺. These results are consistent with Zn²⁺ acting at the previously detected recognition site on the GABA_A receptor complex, distinct from the picrotoxin, GABA, and benzodiazepine sites. The regional heterogeneity of the Zn²⁺ effect may reflect differential regional distribution of GABA_A receptor subtypes among brain regions. Other divalent cations probably act at the Zn²⁺ binding site.     

Nitric Oxide Disrupts Ca2+ Homeostasis in Hippocampal Neurons

Abstract: Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In [Ca²⁺]_i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide-releasing agent S -nitrosocysteine produced a delayed rise in [Ca²⁺]_i over a 20-min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization-induced [Ca²⁺]_i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca²⁺]_i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP-regenerating system was supplied via the patch pipette in combined whole-cell patch-clamp-[Ca²⁺]_i fluorimetry experiments, S -nitrosocysteine had no effect on the resting [Ca²⁺]_i or on the recovery kinetics of [Ca²⁺]_i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca²⁺]_i homeostasis in hippocampal neurons by impairing Ca²⁺ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca²⁺]_i may contribute to the delayed neurotoxicity of nitric oxide.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

Systemin triggers an increase of cytoplasmic calcium in tomato mesophyll cells: Ca2+ mobilization from intra- and extracellular compartments

We show here that, within 1–2 min of application, systemin triggers a transient increase of cytoplasmic free calcium concentration ([Ca²⁺]_c) in cells from Lycopersicon esculentum mesophyll. The systemin-induced Ca²⁺ increase was slightly but not significantly reduced by L-type Ca²⁺ channel blockers (nifedipine, verapamil and diltiazem) and the Ca²⁺ chelator [ethylene glycol tetraacetic acid (EGTA)], whereas inorganic Ca²⁺ channel blockers (LaCl₃, CdCl₂ and GdCl₃) and compounds affecting the release of intracellular Ca²⁺ from the vacuole (ruthenium red, LiCl, neomycin) strongly reduced the systemin-induced [Ca²⁺]_c increase. By contrast, no inhibitory effect was seen with the potassium and chloride channel blockers tested. Unlike systemin, other inducers of proteinase inhibitor (PI) and of wound-induced protein synthesis, such as jasmonic acid (JA) and bestatin, did not trigger an increase of cytoplasmic Ca²⁺. The systemin-induced elevation of cytoplasmic Ca²⁺ which might be an early step in the systemin signalling pathway, appears to involve an influx of extracellular Ca²⁺ simultaneously through several types of Ca²⁺ permeable channels, and a release of Ca²⁺ from intracellular stores sensitive to blockers of inositol 1,4,5-triphosphate (IP₃)- and cyclic adenasine 5'-diphosphoribose (cADPR)-mediated Ca²⁺ release.     

Modulation of [3H]Acetylcholine Release from Cultured Amacrine-Like Neurons by Adenosine A1 Receptors

Abstract: We have investigated the effect of endogenous adenosine on the release of [³H]acetylcholine ([³H]ACh) in cultured chick amacrine-like neurons. The release of [³H]ACh evoked by 50 m M KCl was mostly Ca²⁺ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist. The effect of adenosine on [³H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca²⁺ channels. Ligand binding studies using [³H]DPCPX confirmed the presence of adenosine A₁ receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A₁ receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca²⁺-dependent release of [³H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A₁ receptors. The effect of adenosine on the [³H]ACh release may be due to a direct inhibition of N-type Ca²⁺ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Calcium and plant action potentials

Abstract. Under normal conditions the action potential in Characeae is dependent on the presence of both Cl⁻ and Ca²⁺. Cl⁻ seems to play a straightforward part as a transient depolarizing flow. The role of Ca²⁺, however, is emerging as an increasingly complex one: there are Ca²⁺ concentration changes in the cytoplasm, as well as transient Ca²⁺ currents across the plasmalemma and possibly the tonoplast. In most Characeae Ca²⁺ is necessary for the Cl⁻ channel to function, and it is also involved in the cessation of the cytoplasmic streaming observed at the time of excitation.
The function of Ca²⁺ at the time of the action potential is being revealed by experimental techniques of increasing sophistication. The development of these methods and possible associated artefacts are considered.     

Tachykinins Potentiate N-Methyl-D-Aspartate Responses in Acutely Isolated Neurons from the Dorsal Horn

Abstract: Substance P and neurokinin A both potentiated N -methyl- d -aspartate (NMDA)-induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca²⁺]_i in these cells. Substance P, but not neurokinin A, increased [Ca²⁺]_i in a subpopulation of neurons. The increase in [Ca²⁺]_i was found to be due to Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Substance P and neurokinin A also potentiated the increase in [Ca²⁺]_i produced by NMDA, but not by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, or 50 m M K⁺. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca²⁺]_i can be dissociated from their effects on NMDA receptors.     

Disrupted [Ca2+]i Homeostasis Contributes to the Toxicity of Nitric Oxide in Cultured Hippocampal Neurons

Abstract: Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca²⁺ concentration ([Ca²⁺]_i) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca²⁺]_i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S -nitrosocysteine (SNOC; 300 µ M ) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca²⁺]_i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca²⁺]_i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca²⁺ during the 20-min SNOC treatment failed to prevent the late [Ca²⁺]_i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca²⁺ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca²⁺-activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca²⁺]_i homeostatic mechanisms, and that the resulting persistent [Ca²⁺]_i elevations contribute to the delayed neurotoxicity of NO.     

AMPA Receptor-Mediated Excitotoxicity in Human NT2-N Neurons Results from Loss of Intracellular Ca2+ Homeostasis Following Marked Elevation of Intracellular Na+

Abstract: Human NT2-N neurons express Ca²⁺-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptors (AMPA-GluRs) and become vulnerable to excitotoxicity when AMPA-GluR desensitization is blocked with cyclothiazide. Although the initial increase in intracellular Ca²⁺ levels ([Ca²⁺]_i) was 1.9-fold greater in the presence than in the absence of cyclothiazide, Ca²⁺ entry via AMPA-GluRs in an early phase of the exposure was not necessary to elicit excitotoxicity in these neurons. Rather, subsequent necrosis was caused by a >40-fold rise in [Na⁺]_i, which induced a delayed [Ca²⁺]_i rise. Transfer of the neurons to a 5 m M Na⁺ medium after AMPA-GluR activation accelerated the delayed [Ca²⁺]_i rise and intensified excitotoxicity. Low-Na⁺ medium-enhanced excitotoxicity was partially blocked by amiloride or dizocilpine (MK-801), and completely blocked by removal of extracellular Ca²⁺, suggesting that Ca²⁺ entry by reverse operation of Na⁺/Ca²⁺ exchangers and via NMDA glutamate receptors was responsible for the neuronal death after excessive Na⁺ loading. Our results serve to emphasize the central role of neuronal Na⁺ loading in AMPA-GluR-mediated excitotoxicity in human neurons.     

Lysophosphatidic Acid Induces a Sustained Elevation of Neuronal Intracellular Calcium

Abstract: Lysophosphatidic acid (LPA) is a lipid biomediator enriched in the brain. A novel LPA-induced response in rat hippocampal neurons is described herein, namely, a rapid and sustained elevation in the concentration of free intracellular calcium ([Ca²⁺]_i). This increase is specific, in that the related lipids phosphatidic acid and lysophosphatidylcholine did not induce an alteration in [Ca²⁺]_i. Moreover, consistent with a receptor-mediated process, there was no further increase in [Ca²⁺]_i after a second addition of LPA. The LPA-induced increase in [Ca²⁺]_i required extracellular calcium. However, studies with Cd²⁺, Ni²⁺, and nifedipine and nystatin-perforated patch clamp analyses did not indicate involvement of voltage-gated calcium channels in the LPA-induced response. In contrast, glutamate appears to have a significant role in the LPA-induced increase in [Ca²⁺]_i, because this increase was inhibited by NMDA receptor antagonists and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonists. Thus, LPA treatment may result in an increased extracellular glutamate concentration that could stimulate AMPA/kainate receptors and thereby alleviate the Mg²⁺ block of the NMDA receptors and lead to glutamate stimulation of an influx of calcium via NMDA receptors.     

Intracellular water volume and internal pH of Streptomyces antibioticus spores

Abstract In the present study we compared the intracellular level of free calcium ([Ca²⁺]_i) and monomeric (G)/total (G + F) actin ratio in HeLa cells infected with diffuse (DAEC) and localised adherent Escherichia coli (LAEC). The level of [Ca²⁺]_i was increased in both DAEC- and LAEC-infected HeLa cells. However, studies with EGTA- and dantrolene-treated cells and also suspension of cells in Ca²⁺-free buffer suggested that the rise of [Ca²⁺]_i in DAEC-infected cells was due to the influx of Ca²⁺ from extracellular medium, whereas Ca²⁺ mobilisation from the intracellular stores was responsible for the enhancement of [Ca²⁺]_i in LAEC-infected cells. It was also evident that the infection of HeLa cells with DAEC and LAEC caused alteration of G / G + F actin ratio as compared to that of control cells. The ratio was much lower in LAEC-infected cells than that of DAEC-infected ones. Moreover, cytochalasin B inhibited both DAEC and LAEC invasion to HeLa cells, suggesting further the role of microfilaments in the invasion process.     

Differential Effects of the Egg Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca2+ and pH in Sea Urchin Spermatozoa

We examined the effects of two egg jelly components, a fucose sulfate glycoconjugate (FSG) and sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), on the intracellular pH (pH_i) and Ca²⁺ ([Ca²⁺]_i) of spermatozoa of the sea urchin Hemicentrotus pulcherrimus . FSG and/or SAP-I induced elevations of [Ca²⁺]; and pH_i in the spermatozoa at pH 8.0. At pH 8.0, a second addition of FSG did not induced further elevation of the [Ca²⁺]_i or pH_i of spermatozoa treated with FSG, but addition or FSG after SAP-I or of SAP-I after FSG induced further increases of [Ca²⁺]_i and pH_i, At pH 6.6, FSG and/or SAP-I did not induce significant elevation of the [Ca²⁺]_i, although SAP-I elevated the pH_i, its half-maximal effective concentration being 10 to 100 pM. At pH 8.0, tetraethyl-ammonium, a voltage-sensitive K⁺-channel blocker, inhibited induction of the acrosome reaction and elevations of [Ca²⁺]_i and pH_i by FSG, but did not affect those by SAP-I. These results suggest that FSG and SAP-I activate different Ca²⁺ and H⁺ transport systems.     

Control of Granule Mobility and Exocytosis by Ca2+-Dependent Formation of F-Actin in Pancreatic Duct Epithelial Cells

Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers exocytosis of secretory granules in pancreatic duct epithelia. In this study, we find that the signal also controls granule movement. Motions of fluorescently labeled granules stopped abruptly after a [Ca²⁺]_i increase, kinetically coincident with formation of filamentous actin (F-actin) in the whole cytoplasm. At high resolution, the new F-actin meshwork was so dense that cellular structures of granule size appeared physically trapped in it. Depolymerization of F-actin with latrunculin B blocked both the F-actin formation and the arrest of granules. Interestingly, when monitored with total internal reflection fluorescence microscopy, the immobilized granules still moved slowly and concertedly toward the plasma membrane. This group translocation was abolished by blockers of myosin. Exocytosis measured by microamperometry suggested that formation of a dense F-actin meshwork inhibited exocytosis at small Ca²⁺ rises <1 μ m . Larger [Ca²⁺]_i rises increased exocytosis because of the co-ordinate translocation of granules and fusion to the membrane. We propose that the Ca²⁺-dependent freezing of granules filters out weak inputs but allows exocytosis under stronger inputs by controlling granule movements.