Osmotic stabilizers differentially inhibit permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin

Using a sensitive Vero (African green monkey kidney) cell model system, studies were performed to further investigate whether Clostridium perfringens enterotoxin acts via disruption of the colloid-osmotic equilibrium of sensitive cells. Enterotoxin was shown to cause a rapid loss of intracellular 86Rb+ (Mr approx. 100) with time- and dose-dependent kinetics. The enterotoxin-induced release of intracellular 86Rb+ preceded the loss of two larger labels, 51Cr label (Mr approx. 3500) and 3H-labeled nucleotides (Mr less than 1000). The osmotic stabilizers, sucrose and poly(ethylene glycol), differentially inhibited enterotoxin-induced larger label loss versus 86Rb+ loss. Further, enterotoxin was shown to cause a rapid influx of 24Na+ that was not significantly inhibited by osmotic stabilizers. Additional studies demonstrated that lysosomotropic agents were not protective against characteristic enterotoxin-induced membrane permeability alterations or morphological damage. Taken collectively, these results are consistent with an action for enterotoxin which involves a disruption of the osmotic equilibrium.     

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase ofRhodospirillum rubrum: Effects of pH and divalent cations

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores ofRhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly theK _m of Mg²⁺ or Pi for the enzyme; Mn²⁺ and Co²⁺ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg²⁺; other divalent cations like Zn²⁺ or Ca²⁺ do not support the exchange reaction. In the hydrolytic reaction, Zn²⁺, at low concentration, substitutes for Mg²⁺ as substrate, and Co²⁺ also substitutes in limited amount (50%). Other cations (Ca²⁺, Cu²⁺, Fe²⁺, etc.) do not act as substrates in complex with PPi. The Zn²⁺ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn²⁺. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn²⁺>Mn²⁺>Ca²⁺Co²⁺>Fe²⁺>Cu²⁺>Mg²⁺. The data in this work suggest that H⁺ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.     

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity. I. General requirements of the reaction and temporal relationship between lethal hits and cytolysis.

The effect of various physical and chemical parameters on the cytotoxic reaction was studied in a ⁵¹Cr-release assay in order to analyze the mechanism by which human blood mononuclear cells (MC) damage antibody-sensitized target cells infected with herpes simplex virus. Centrifugation of the target cell-MC mixture consistently increased the velocity of the reaction. In addition, uncentrifuged target cell-MC cultures showed a sigmoidal kinetic curve of ⁵¹Cr release with an initial lag phase of at least 10 min, whereas ⁵¹Cr release in centrifuged cultures followed a linear pattern with time without an initial lag. These findings indicate that direct contact between target and effector cells is necessary for cytotoxicity to occur. The reaction as a whole was temperature dependent, proceeding well at 37 °C and not at all at 4 °C. Incubation of the MC at 46 °C for 10 min abolished their cytotoxic potential without affecting their viability; similar heating of the target cells did not affect their background isotope release or sensitivity to the lytic process. Heating target cell-MC mixtures at 46 °C for 10 min thus provided a tool by which the temporal relationship between the mounting of “lethal hits” and specific isotope release, or cell lysis, could be studied. Using this technique, we observed virtually simultaneous occurrence of lethal hits and cell lysis, measured at various intervals between 10 and 360 min postincubation. Likewise, we were unable to demonstrate a transient period of increased osmotic fragility in target cells after contact with MC but before actual cell lysis. Taken together, these findings imply either that cell lysis, as indicated by ⁵¹Cr release, results from a sudden nonosmotic injury to the target cell membrane or, alternatively, osmotic damage leading to ⁵¹Cr release occurs too rapidly to be detected by the methods employed in this study. These findings imply either a qualitative or a quantitative difference between antibody-dependent cellular cytotoxicity (ADCC) mediated by K cells and cytotoxicity mediated by sensitized T cells.The cytotoxic reaction was completely inhibited by 10 mM EDTA and did not occur in a Ca²⁺- and Mg²⁺-free medium. Neither Ca²⁺ nor Mg²⁺ alone produced as much cytotoxicity as the two cations in tandem; in addition, when added to the culture medium in suboptimal amounts, the two cations were either additive or synergistic. These observations suggest that both cations are necessary in ADCC and also that there may be separate Ca²⁺- and Mg²⁺-dependent events in the lytic pathway.     

Cation-dependent uptake of zinc in human fibroblasts

The influence of K⁺ and Ca²⁺ on Zn²⁺ transport into cultured human fibroblasts was investigated. Zn²⁺ uptake was markedly reduced in the presence of both valinomycin and nigericin (electrogenic and electroneutral K ⁺ ionophores, respectively), and by reduction in the transmembrane K⁺ gradient produced by replacement of extracellular K⁺ with Na⁺, suggesting that Zn²⁺ may be driven by a Zn²⁺/K⁺ counter-transport system. To test the counter-transport hypothesis, we used ⁸⁶Rb as an analog of K ⁺ for efflux studies. The rate of Rb⁺ efflux was 3760 times that of Zn²⁺ uptake, thus the component of K⁺ involved in the Zn²⁺ counter-transport system was only a small proportion of the total K⁺ efflux. In investigating the effect of Ca²⁺ on Zn²⁺ uptake, we identified two components: (1) a basal Zn²⁺ uptake pathway, independent of hormonal or growth factors which does not require extracellular Ca²⁺ and (2) a Ca²⁺-dependent mechanism. The absence of Ca²⁺ decreased Zn²⁺ uptake, while increasing extracellular C+a²⁺ stimulated Zn²⁺ uptake. The effect was mediated by Ca²⁺ influx as the ionophores A23187 and ionomycin also stimulated Zn²⁺ uptake. We could not ascribe the Ca²⁺ effect to known Ca²⁺ influx pathways. We conclude that Zn²⁺ uptake occurs by a K⁺-dependent process, possibly by Zn²⁺/K⁺ counter-transport and that a component of this is also Ca²⁺-dependent.     

Toxicity of chromium and tin toAnabaena doliolum Interaction with bivalent cations

Summary The toxicity of chromium and tin on growth, photosynthetic carbon-fixation, oxygen evolution, heterocyst differentiation and nitrogenase activity ofAnabaena doliolum and its interaction with bivalent cations has been studied. Some interacting cations, viz. Ca²⁺, Mg²⁺ and Mn²⁺, substantially antagonised the toxic effects of chromium and tin with reference to growth, heterocyst differentiation and nitrogenase activity in the following hierarchal sequence: Ca²⁺ > Mg²⁺ > Mn²⁺. However, the sequence of hierarchy was Mg²⁺ > Ca²⁺ > Mn²⁺ for carbon fixation and Mn²⁺ > Mg²⁺ > Ca²⁺ for photosynthetic oxygen evolution. Synergistically inhibitory patterns were noticed for all the parameters, viz. growth,¹⁴CO₂ uptake, oxygen evolution, heterocyst differentiation and nitrogenase activity ofA. doliolum when Ni²⁺, Co²⁺ and Zn²⁺ were combined with the test metals in the growth medium. These cations followed the following sequence of synergistic inhibition: Ni²⁺ > Co²⁺ > Zn²⁺. Among all the interacting cations, Ca²⁺, Mg²⁺ and Mn²⁺ exhibited antagonistic effects which relieved the test cyanobacterium from metal toxicity. In contrast to this, Ni²⁺, CO²⁺ and Zn²⁺ showed synergistic inhibition which potentiating the toxicity of test metals in the N₂-fixing cyanobacteriumA. doliolum. It is evident from the present study that bivalent cations, viz. Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Co²⁺ and Zn²⁺, may appreciably regulate the toxicity of heavy metals in N₂-fixing cyanobacteria if present in aquatic media.     

Magnesium-Dependent stimulation of protein synthesis by the insulin mimic,pervanadate

The insulin mimic, peroxide of vanadate (pervanadate), stimulated ³⁵S-methionine incorporation into Xenopus oocyte protein in a Mg²⁺-dependent manner. Reducing the extracellular Mg²⁺ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg²⁺ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg²⁺ to 0.01 mM inhibited about 50% of the insulinstimulated component of methionine incorporation. Mg²⁺ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn²⁺ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg²⁺. Reducing extracellular Ca²⁺ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 m?I droplet of buffer containing the Mg²⁺ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg²⁺ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg²⁺. In contrast, apparent extracellular Mg²⁺ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg²⁺, but not Ca²⁺, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg²⁺, and this change in membrane transport may be an early event in signalling the increase in translation. © 1995 Wiley-Liss, Inc.     

Characterization of membrane permeability alterations induced in vero cells by Clostridium perfringens enterotoxin

Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were α-amino[¹⁴C]isobutyric acid (M_r 103), ³H-labeled nucleotide (M_r approx. 300), ⁵¹Cr label (M_r approx. 3000) and [³H]RNA (M_r > 25 000). Over a 2 h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and ⁵¹Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of M_r 3000 but not 25 000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional ‘holes’ of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and non-confluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional ‘holes’ of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death.     

Characterization of cell surface material removed by water and NaCl washing ofParacoccus denitrificans grown under conditions of divalent cation deficiency and sufficiency

Paracoccus denitrificans grown on complex medium deficient in Mg²⁺ and Ca²⁺ are rendered lysozyme susceptible by washing with NaCl, whereas cells grown in a succinate-salts medium (Mg²⁺ and Ca²⁺ sufficient) or complex medium supplemented with Mg²⁺+Ca²⁺ are not. The material released by water washing of cells grown on complex medium and complex medium supplemented with Mg²⁺ and Ca²⁺ was characterized by a high protein content. There was a high lipid: protein ratio and an appreciable amount of 3-deoxyoctulosonic acid in the material released by NaCl washing of cells grown under all conditions, indicating release of outer membrane material. The lipid ornithine: lipid phosphorous ratios of NaCl wash from cells grown on complex medium and complex medium supplemented with Mg²⁺ and Ca²⁺ were 0.54 and 0.34, respectively. Although NaCl washing removed outer membrane material from cells grown under all conditions, only divalent cation deficient cells were rendered lysozyme susceptible. This might be explained by the increased outer membrane ornithine-containing lipid to phospholipid ratio in these cells yielding a more permeable outer membrane.     

A Cell Permeable NPE Caged ADP-Ribose for Studying TRPM2

Transient potential receptor melastatin-2 (TRPM2) is a non-selective Ca²⁺-permeable cation channel of the TRPM channel subfamily and is mainly activated by intracellular adenosine diphosphate ribose (ADPR). Here we synthesized a 1-(2-nitrophenyl)ethyl caged ADPR (NPE-ADPR) and found that uncaging of NPE-ADPR efficiently stimulated Ca²⁺, Mg²⁺, and Zn²⁺ influx in a concentration-dependent manner in intact human Jurkat T-lymphocytes. The cation influx was inhibited by inhibitors or knockdown of TRPM2. Likewise, uncaging of NPE-ADPR markedly induced cation entry in HEK 293 cells that overexpress TRPM2. As expected, high temperature increased the ability of the photolyzed NPE-ADPR to induce cation entry, whereas acidic pH inhibited. Moreover, the absence of extracellular Ca²⁺ significantly inhibited Mg²⁺ and Zn²⁺ influx after uncaging NPE-ADPR. On the other hand, the absence of extracellular Na⁺ or Mg²⁺ had no effect on photolyzed NPE-ADPR induced Ca²⁺ entry. Taken together, our results indicated that NPE-ADPR is a cell permeable ADPR analogue that is useful for studying TRPM2-mediated cation entry in intact cells.     

Activation,but not inhibition,by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell

The effect of glucose on the Ca²⁺-activated K⁺ permeability in pancreatic islet cells was investigated by measuring the rate of ⁸⁶Rb efflux, ⁴⁵Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in ⁸⁶Rb outflow, ⁴⁵Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased ⁸⁶Rb and ⁴⁵Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in ⁸⁶Rb and ⁴⁵Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca²⁺ of the Ca²⁺-sensitive modality of K⁺ extrusion. On the contrary, as a result of its effect upon Ca²⁺ handling, glucose stimulates the Ca²⁺-activated K⁺ permeability.     

Stimulation of ethylene production in the mung bean hypocotyls by cupric ion, calcium ion, and kinetin

The synergistic stimulation of ethylene production by kinetin and Ca²⁺ in hypocotyl segments of mung bean (Phaseolus aureus Roxb.) seedling was further studied. The requirement for Ca²⁺ in this system was specific. Except for Sr²⁺, which mimicked the effect of Ca²⁺, none of the following divalent cations, including Ba²⁺, Mg⁶⁺, Cu²⁺, Hg²⁺, Co²⁺, Ni²⁺, Sn²⁺, and Zn²⁺, showed synergism with kinetin on ethylene production. Fe²⁺, however, showed a slight synergism with kinetin. Some of them (Hg²⁺, Co²⁺, and Ni²⁺) had a strong inhibitory effect, while others (Zn²⁺, Mg²⁺, Sn²⁺, and Ba²⁺) had a slight or no inhibitory effect on ethylene production in the absence or presence of kinetin.     

Interaction of metal ions with polynucleotides and related compounds. XXII. Effect of divalent metal ions on the conformational changes of polyribonucleotides

Changes in the conformation of poly(G), poly(C), poly(U), and poly(I) in the presence of divalent metal ions Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺, and Zn²⁺ have been measured by means of ORD and u.v. spectra. Mg²⁺ and Ca²⁺ ions stabilize helical structures of all the polynucleotides very effectively at concentrations several orders of magnitude lower than the effective concentration of Na⁺ion. Cu²⁺ and Cd²⁺ destabilize the helical structure of polynucleotides to form random coils. Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ions do not behave in such a clear-cut manner: they selectively stabilize some ordered structures, while destabilizing others, depending on the ligand strength of the nucleotide base as well as the preferred conformation of that polynucleotide.     

Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A ^1% ₂₈₀) of acutolyisn D have been determined to be 47,850 ± 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca²⁺-binding sites and two Zn²⁺-binding sites determined by atomic absorption spectrophotometer. Zn²⁺ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn²⁺ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)₂ precipitate formation. Ca²⁺ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca²⁺ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu²⁺, Co²⁺, Mn²⁺ or Tb³⁺ and inhibited completely by Cd²⁺, is enhanced by Mg²⁺. The fluorescence intensity of the protein decreases in the presence of Cu²⁺, Co²⁺, Cd²⁺ or Mn²⁺, but neither for Ca²⁺, Mg²⁺ nor for Tb³⁺. Zn²⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Co²⁺ and Tb³⁺ have slight effects on its secondary structure contents. In addition, Cd²⁺ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.     

INFLUENCE OF DIVALENT CATIONS ON REGULATION OF CYCLIC GMP AND CYCLIC AMP LEVELS IN BRAIN TISSUE

—Depolarizing concentrations of K⁺ elevate levels of both adenosine 3′,5′monophosphate (cyclic AMP) and guanosine 3′,5′monophosphate (cyclic GMP) in incubated slices of mouse cerebellum. Calcium is an essential requirement for the K⁺ -induced accumulation of cyclic GMP. Barium and Sr²⁺, but not Mn²⁺ or Co²⁺, can substitute for Ca²⁺ in this process. Relatively high concentrations of Mg²⁺ inhibit the effect of Ca²⁺ on K⁺-induced accumulation of cyclic GMP. In contrast, depolarizing concentrations of K⁺ are capable of elevating cyclic AMP levels in brain slices suspended in media containing Mg²⁺ and no other divalent cations. High concentrations of Ca²⁺ (1 mm or greater) augment this Mg²⁺ -dependent, K⁺-induced accumulation of cyclic AMP, however. Strontium and Mn²⁺, but not Ba²⁺ or Co²⁺, can substitute for Ca²⁺ in this process, and high concentrations of Mg²⁺ are not inhibitory. The divalent cation ionophore, A-23187 (10 μm ), in the presence of extracellular Ca²⁺ elevates the level of cyclic GMP, but not cyclic AMP, in incubated mouse cerebellum slices. The results of this study indicate that intracellular Ca²⁺ concentration is a major factor regulating cyclic GMP levels in brain. In addition the present results suggest that, in brain tissue, depolarization-induced accumulation of cyclic GMP, but not cyclic AMP, is closely linked to some Ca²⁺-dependent mechanism(s) mediating release of intracellular substances.     

Transition metal ions induce cell growth in NRK cells synchronized in G1 by picolinic acid

The G₁ arrest induced in NRK cells by picolinic acid could be prevented by addition of Fe³⁺, Zn²⁺ or Co²⁺ to the tissue culture media. Ca²⁺, Mg²⁺, Mn²⁺, Sr²⁺ or Ba²⁺ were ineffective. Complete and synchronous reversal of the G₁ block, however, was achieved by Fe³⁺ at lower concentration from that of Zn²⁺. Co²⁺ reversed the block but cells divided asynchronously. Thymidine incorporation, mitotic index and relative DNA content per cell, verified that G₁ arrested cells proceeded through the cell cycle after addition of Fe³⁺ or Zn²⁺. These observations afford a valuable model system for elucidating the biochemical events that occur between addition of a defined proliferative signal and stimulation of DNA synthesis in G₁ arrested cells.     

Magnesium-dependent induction of phagocytosis-associated chemiluminescence of adherent human polymorphonuclear leukocytes by non-opsonized zymosan

A severe dysfunction in the cellular response of human polymorphonuclear leukocytes (PMNL) to non-opsonized zymosan was observed under a deficiency of extracellular Mg²⁺. The phagocytosis-association native (luminol-independent) luminescence (NL), as well as luminol-dependent luminescence (LDL) (detected simultaneously and discriminated by spectral methods), was strongely inhibited. Apart from a general decrease of total light production, a Mg²⁺-concentration-dependent delay of the maximum of NL and LDL was observed. A disorder in recruitment of activated membrane-bound NADPH-oxidase of PMNL is suggested. The presence of extracellular Ca²⁺ did not compensate for the Mg²⁺ deficit. In the presence of Mg²⁺ only a slight Ca²⁺-dependent reduction of NL was obtained, but Ca²⁺ seemed to selectively promote LDL. This may indicate a positive influence of Ca²⁺ on the myeloperoxidase release from the cells. Experiments with the metalions-chelating agents EDTA and EGTA, which complex Mg²⁺ to differing extents, confirmed the important role of Mg²⁺ in PMNL-activation by non-opsonized zymosan.     

Production of constitutive, thermostable, hyper active exo-pectinase from Bacillus GK-8

Summary A new alkalophilic Bacillus GK-8 overproduced three thermostable extracellular pectinases. The temperature optima was 60°C for all the three enzymes which retained full activity for 2 h. They had pH optima 5.4, 7.0 and 10.4 and were designated as PI, PII and PIII respectively. The half life at 80°C of PI, PII and PIII was 18 min., 12 min. and 12 min., respectively. The enzyme activity was stimulated by Mg²⁺, Ca²⁺, Zn²⁺, Co²⁺ and Mn²⁺ ions.     

Effect of Liposomes Containing Various Divalent Cations on the Release of Acetylcholine from Synaptosomes

Abstract: The effect of increasing the cytoplasmic levels of various divalent cations on the release of [³H]acetylcholine ([³H]ACh) from synaptosomes was investigated. Synaptosomes prepared from rat brain and prelabeled with [³H]choline were incubated with liposomes containing Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Sr²⁺, or Ba²⁺. This treatment allows the transfer of the aqueous contents of the liposomes to the cytoplasm of the synaptosomes. The efflux of radioactivity subsequent to this treatment was measured, and the relative proportions of [³H]ACh and [³H]choline were determined. The release of radioactivity from synaptosomes incubated with liposomes containing Mg²⁺, Mn²⁺, or Co²⁺ was not altered when compared with synaptosomes incubated either without liposomes or with liposomes containing isotonic K⁺/Na⁺. Synaptosomes incubated with liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺, however, released significantly more radioactivity than did controls. Moreover, the released radioactivity consisted almost entirely of [³H]ACh. Liposomes containing either Ca²⁺ or Sr²⁺ were equally effective in promoting the release of [³H]ACh from synaptosomes, whereas liposomes containing Ba²⁺ were 2.5 times more effective in promoting the release of [³H]ACh than were liposomes containing either Ca²⁺ or Sr²⁺. Since liposomes introduce their aqueous contents into cytoplasm via a mechanism not involving plasma membrane channels, the increased release of [³H]ACh caused by liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺ is attributable to an increase in the intrasynaptosomal concentration of these ions, and not to their passage through calcium channels.     

Cytopathic Action of Naegleria fowleri Amoebae on Rat Neuroblastoma Target Cells

The axenically cultured, weakly pathogenic Naegleria fowleri LEE and the highly pathogenic, mouse passaged N. fowleri LEEmp are cytopathic for B103 rat nerve cells in culture. Cytopathogenicity was measured by release of radiolabeled rubidium or radiolabeled chromium from B103 target cells. Cytopathogenicity was time-dependent for up to 18 h and dependent upon amoebae effector to nerve cell target ratios of less than 1:1. Release of⁵¹ Cr from B103 cells by either LEE or LEEmp amoebae was enhanced by addition of calcium or magnesium to medium free of these divalent cations but the ion-channel inhibitor, verapamil, or the ionophore A23187 and phorbol myristate acetate did not alter release of ⁵¹ Cr from B103 cells cocultured with the amoebae. Cycloheximide or actinomycin D impaired release of ⁵¹ Cr from B103 target cells injured by either LEE or LEEmp amoebae. Both strains of amoebae were fractionated by glass bead disruption and high speed centrifugation into membrane and soluble fractions. Each fraction was incubated with either ⁸⁶Rb or ⁵¹ Cr labeled nerve cells. The membrane fraction from LEEmp was more active than the soluble fraction in facilitating rubidium and chromium release. In contrast, the soluble fraction from LEE was more active than the membrane fraction in facilitating rubidium release from radiolabeled target cells. The sequential release of ⁸⁶Rb and ⁵¹ Cr from target cells rather than the simultaneous release of the two isotopes indicates that target cell death is due to the release of ions followed later by the release of large macromolecules. The results indicate that N. fowleri amoebae injure nerve cells by two alternate mechanisms, trogocytosis or contact-dependent lysis.     

K+-stimulated amino acid release from cultured cerebellar neurons: Comparison of static and dynamic stimulation paradigms

The release of several endogenous amino acids and adenosine from rat cerebellar neuronal cultures following elevated K⁺ exposure in the presence and absence of added Ca²⁺ was studied. The amino acids aspartate (ASP), glutamate (GLU) and GABA were released from the cultures in a dose- and Ca²⁺-dependent manner. Taurine (TAU) and the nucleoside adenosine (ADN) efflux rates were dose-dependent but Ca²⁺-independent, and basal levels increased in the absence of Ca²⁺. The K⁺ depolarization induced release of serine (SER), alanine (ALA) and proline (PRO), was not dose-dependent and in the absence of extracellular Ca²⁺ (with added Mg²⁺) higher basal release of SER and ALA, but not PRO, was noted. These findings demonstrate that in addition to known cerebellar neurotransmitters, other neuroactive and neutral amino acids are released from cultured cerebellar neurons in response to K⁺ depolarization. Their observed efflux suggests they may have as yet unidentified roles in neuronal function with different classes of efflux corresponding to: neurotransmitter-type release (ASP, GLU, GABA), and osmoregulatory, possibly neuromodulatory-type release (TAU), a Ca²⁺-insensitive, possibly neuromodulatory-type release (ADN), and a depolarization-sensitive release (SER, ALA, PRO) of which SER and ALA are partially Ca²⁺-sensitive.