Free fatty acids, neutral and polar lipids in Hordeum vulgare exposed to long-term fumigation with SO2

Three 9-day-old cultivars of Hordeum vulgare L. (Barberousse, Gerbel and Panda) were exposed to low levels of SO₂ fumigation (40 ± 5 and 117 ± 20 ppb). After 48 days of treatment the seedlings were harvested for lipid analysis. In comparison to the control (plants exposed to charcoal-filtered air), the total lipid content of fumigated seedlings declined at 40 ppb SO₂ and even more so at 117 ppb in all three cultivars. A large reduction in diacylglycerols. polar lipids and free sterols was also observed after fumigations at both SO₂ concentrations, whereas the treatments resulted in an increase in triacylglycerols and free fatty acids. The percentage composition of total fatty acids and that of each lipid class were changed by the fumigations. resulting in an increase in the degree of unsaturation. No changes in the percentage composition of sterols occurred in the fumigated leaves. These results suggest that even if SO₂ may not directly oxidize unsaturated fatty acids at the low concentrations used here (which do not cause visible injury). it may alter lipid metabolism. This alteration. which was particularly evident in the polar lipids and sterols, could affect the functions associated with membrane stabilization, in which lipids plus sterols play a key part.     

Alkalinization Prolongs Recovery from Glutamate-Induced Increases in Intracellular Ca2+ Concentration by Enhancing Ca2+ Efflux Through the Mitochondrial Na+/Ca2+ Exchanger in Cultured Rat Forebrain Neurons

Abstract: Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 µ M , for 15 s)-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES-buffered saline solution (HBSS) was 126 ± 30 s, whereas recovery time was 216 ± 19 s when the pH was increased to 8.5. Removal of extracellular Ca²⁺ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca²⁺ recovery by affecting intraneuronal Ca²⁺ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca²⁺ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p -(trifluoromethoxyphenyl)hydrazone (FCCP; 750 n M ). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential-sensitive fluorescent indicator, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). We found that the effect of pH 8.5 HBSS on Ca²⁺ recovery was completely inhibited by the mitochondrial Na⁺/Ca²⁺ exchange inhibitor CGP-37157 (25 µ M ). This suggests that increased mitochondrial Ca²⁺ efflux via the mitochondrial Na²⁺/Ca²⁺ exchanger is responsible for the prolongation of [Ca²⁺]_i recovery caused by alkaline pH following glutamate exposure.     

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.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

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.     

Comparison of ion transportation before and after egg hatching in Amphinemura sp. (Plecoptera)

Abstract.  An increase in egg size with embryonic development in stoneflies is believed to result from the uptake of water by osmosis. The present study aims to investigate whether a selective ion transport through egg membranes exists before hatching, and whether ions are released after hatching. Viable and nonviable egg masses are incubated in Petri dishes filled with water, and the concentrations of the ions F⁻, Cl⁻, SO₄²⁻, NO₃⁻, Na⁺, K⁻, Mg²⁺ and Ca²⁺ in the water are determined. The ion transport of an egg mass before and after hatching and a nonviable egg mass is then calculated. Before hatching, Cl⁻, SO₄²⁻, NO₃⁻, Na⁺, Mg²⁺ and Ca²⁺ are taken up from the surrounding water into the inner egg. These ions are selectively taken into the egg. After hatching, Cl⁻, SO₄²⁻, Na⁺, Mg²⁺ and Ca²⁺ are released into the surrounding water. The amount of these ions released after hatching is lower than the amount taken up before hatching. Ions that are not released after hatching are considered to be used in embryonic development.     

5'-(N-Ethylcarboxamido)adenosine Inhibits Ca2+ Influx and Activates a Protein Phosphatase in Bovine Adrenal Chromaffin Cells

Abstract: We investigated the effect of the adenosine receptor agonist 5'-( N -ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A_2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30–40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 ± 3.3%), slowly developing inhibition of [Ca²⁺]_i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca²⁺ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca²⁺]_i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca²⁺/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca²⁺ influx pathway (e.g., L-type Ca²⁺ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.     

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.     

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.     

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.     

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.     

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.     

Diagnostic parameters for selecting against novel spruce (Picea abies) decline: IV. Response of photosynthesis and transpiration to SO2+NO2 exposures

The dose- and time-response effects of 3 days of 6 h day-time sequential exposures to NO₂, SO₂ and SO₂+NO₂ of 0.45–1.81 μl l⁻¹ (ppm) SO₂ and 1.50–7.65 μl l⁻¹ NO₂ on photosynthesis, transpiration and dark respiration were examined for nine Carpatho-Ukrainian half-sib families and a population from the GFR ('Westerhof') of Norway spruce [ Piecea abies (L.) Karst.], all in their 5th growing season.
SO₂+NO₂ inhibited photosynthesis and transpiration and stimulated dark respiration more than SO₂ alone. SO₂ and SO₂+NO₂ at the lowest concentrations inhibited night transpiration, but increased it at the highest concentration, the strongest effects being obtained with combined exposures. Photosynthesis of the different half-sib families was affected significantly differently by SO₂+NO₂ exposures. NO₂ alone had no effects.
Sensitivity to transpiration decline correlated negatively with branch density. Height of trees correlated postitively with decline sensitivity in the seed orchard. The distribution of photosynthesis and transpiration sensitivities over all tested half-sib families correlated negatively with the distribution of decline sensitivity of their parents in a rural Danish seed orchard. The relative photosynthesis and transpiration sensitivities may thus serve as diagnostic parameters for selecting against novel spruce decline.     

A rapid assay for aluminium phytotoxicity at submicromolar concentrations

Investigations of Al phytotoxicity, including the identification of the Al species responsible for toxicity, require a rapid assay procedure employing very low concentrations of Al and a chemically simple rooting medium. Root elongation in newly germinated red clover ( Trifolium pratense L. cv. Kenland) was inhibited by submicromolar concentrations of Al. Ca²⁺ at concentrations of at least 0.2 m M was essential for optimal elongation in control seedlings. Ca²⁺ also relieved Al toxicity with the net effect that maximum reduction of elongation by 1 μ M Al was achieved at 0.2 m M Ca²⁺. Elongation in control seedlings was at least 90% of maximum from pH 4.5 to 5.7. Increases in pH relieved Al toxicity so that maximum sensitivity to 1 μ M Al occurred at pH 4.7. As a consequence of these experiments and other considerations we chose for our basic assay a medium composed of 0.2 m M CaSO₄ adjusted to pH 4.5 with H₂SO₄, variously supplemented with Al₂(SO₄)₃.
Day-old seedlings were incubated in this aerated medium in the dark at 23°C for one day. No additions of other solutes increased the sensitivity of the assay, but amelioration of Al toxicity was effected by Mg²⁺, F^-, phosphate and citrate. Increases in ionic strength per se had comparatively little effect on the toxic effects of Al. Two barley cultivars ( Hordeum vulgare L. cv. Dayton and Kearney) and two wheat cultivars ( Triticum aestivum L. cv. Hart and Thorne) known to differ in sensitivity to Al were reliably separated at submicromolar Al concentrations by the assay procedure, which was slightly modified. Suggestions for the improvement of the assay and for applications to future research are offered.     

Germination and seedling growth of cotton: salinity-calcium interactions

Abstract. The effects of NaCl salinity on germination and early seedling growth of cotton were studied. Germination was both delayed and reduced by 200 mol m⁻³ NaCl in the presence of a complete nutrient medium. Seedlings, 7–9 d old, were greatly reduced in fresh weight by salinity. The addition of supplemental Ca²⁺ (10 mol m⁻³ as SO₄²⁻ or Cl⁻) to the medium did not improve germination but, to a large degree, offset the reduction in root growth caused by NaCl. Roots growing in the high salt medium without supplemental Ca²⁺ appeared infected by microbes. The cation specificity of the beneficial Ca²⁺ effect on growth was ascertained by testing additions of MgSO₄ or KCl to the NaCl treatments. The contents of K⁴ and Ca²⁺ were reduced in both roots and shoots by the NaCl treatments. Supplemental Ca²⁺ partially offset this effect for K⁴ in the roots and for Ca²⁺ in both roots and shoots. Sodium contents were not affected by the supplemental Ca²⁺. It is concluded that the beneficial effect of high Ca²⁺ concentrations on root growth of cotton seedlings in a saline environment may be due to maintenance of K/Na-selectivity and adequate Ca status in the root.     

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.     

Endothelin-1 Increases Arachidonic Acid Release in C6 Glioma Cells Through a Potassium-Modulated Influx of Calcium

Abstract: Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca²⁺, and the demonstration of an increase in phospholipase A₂ (PLA₂) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA₂ in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca²⁺]_i triggered by Et-1, whereas valinomycin, which causes K⁺ efflux, not only caused a rapid rise in [Ca²⁺]_i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA₂ in this cell type requires calcium influx dependent on K⁺ efflux.     

A Charybdotoxin-Sensitive, Ca2+-Activated K+ Channel with Inward Rectifying Properties in Brain Microvascular Endothelial Cells: Properties and Activation by Endothelins

Abstract: A charybdotoxin-sensitive, Ca²⁺-activated K⁺ channel was identified in cultured rat brain capillary endothelial cells by using conventional single-channel recording techniques and ⁸⁶Rb⁺-influx and efflux experiments. Channel activity was dependent on the presence of Ca²⁺ on the cytosolic face of the membrane with a threshold concentration of 100 n M . It was inhibited by charybdotoxin (IC₅₀ 30 n M ) and quinine (IC₅₀ 0.1 m M ) but not by apamin. K(Ca) channels showed unusual inward rectifying properties under asymmetrical ionic conditions. They were activated by endothelin-1 (EC₅₀ 0.7 n M ) and endothelin-3 (EC₅₀ 7–10 n M ). The actions of endothelins were prevented by BQ-123 ( K _i = 8 n M ) in a competitive fashion, hence suggesting the involvement of an ET_A-receptor subtype. The channel activity was unaffected by cyclic AMP- or cyclic GMP-elevating agents. The possible role of the intermediate conductance, Ca²⁺-activated K⁺ channels for mediating K⁺ movements across the blood-brain barrier is discussed.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

Early Events in Free Radical-Mediated Damage of Isolated Nerve Terminals: Effects of Peroxides on Membrane Potential and Intracellular Na+ and Ca2+ Concentrations

Abstract: The effects of peroxides were investigated on the membrane potential, intracellular Na⁺ ([Na⁺]_i) and intracellular Ca²⁺ ([Ca²⁺]_i) concentrations, and basal glutamate release of synaptosomes. Both H₂O₂ and the organic cumene hydroperoxide produced a slow and continuous depolarization, parallel to an increase of [Na⁺]_i over an incubation period of 15 min. A steady rise of the [Ca²⁺]_i due to peroxides was also observed that was external Ca²⁺ dependent and detected only at an inwardly directed Ca²⁺ gradient of the plasma membrane. These changes did not correlate with lipid peroxidation, which was elicited by cumene hydroperoxide but not by H₂O₂. Resting release of glutamate remained unchanged during the first 15 min of incubation in the presence of peroxides. These alterations may indicate early dysfunctions in the sequence of events occurring in the nerve terminals in response to oxidative stress.