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Gap junction communication is crucial for myelination and axonal survival in both the peripheral nervous system (PNS) and central nervous system (CNS). This review examines the different types of gap junctions in myelinating glia of the PNS and CNS (Schwann cells and oligodendrocytes respectively), including their functions and involvement in neurological disorders. Gap junctions mediate intercellular communication among Schwann cells in the PNS, and among oligodendrocytes and between oligodendrocytes and astrocytes in the CNS. Reflexive gap junctions mediating transfer between different regions of the same cell promote communication between cellular compartments of myelinating glia that are separated by layers of compact myelin. Gap junctions in myelinating glia regulate physiological processes such as cell growth, proliferation, calcium signaling, and participate in extracellular signaling via release of neurotransmitters from hemijunctions. In the CNS, gap junctions form a glial network between oligodendrocytes and astrocytes. This transcellular communication is hypothesized to maintain homeostasis by facilitating restoration of membrane potential after axonal activity via electrical coupling and the re-distribution of potassium ions released from axons. The generation of transgenic mice for different subsets of connexins has revealed the contribution of different connexins in gap junction formation and illuminated new subcellular mechanisms underlying demyelination and cognitive defects. Alterations in metabolic coupling have been reported in animal models of X-linked Charcot–Marie–Tooth disease (CMTX) and Pelizaeus–Merzbarcher-like disease (PMLD), which are caused by mutations in the genes encoding for connexin 32 and connexin 47 respectively. Future research identifying the expression and regulation of gap junctions in myelinating glia is likely to provide a better understanding of myelinating glia in nervous system function, plasticity, and disease. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

Evidence is provided to show that synaptic vesicles have an internal matrix. Suspensions of cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata fish were permeabilized in solutions containing low concentrations of Na(+) or Ca(2+). The release of ATP from the vesicular matrix was 10 times more effective with Ca(2+) than with Na(+). We ascertained whether these two cations induced a different velocity of release of ATP from the matrix. The release of ATP was monitored with the chemiluminescent reaction of luciferin-luciferase. The light signal generated was the result of the kinetics of ATP release of the enzymatic reaction. To overcome the kinetics of the enzymatic reaction, the light records were deconvoluted. The actual kinetics of ATP release of vesicles containing Na(+) or Ca(2+) were coincident. To validate this result, comparison was made with ATP release from intact nerve terminals which were already deconvoluted. The results show that the real time course of release is longer than that obtained from synaptic vesicles. This was as expected given that the release of neurotransmitters is due to successive molecular steps of synaptic vesicle exocytosis.

Voltage-dependent K + (Kv) channels are involved in the immune response. Kv1.3 is highly expressed in activated macrophages and T-effector memory cells of autoimmune disease patients. Macrophages are actively involved in T-cell activation by cytokine production and antigen presentation. However, unlike T-cells, macrophages express Kv1.5, which is resistant to Kv1.3-drugs. We demonstrate that mononuclear phagocytes express different Kv1.3/Kv1.5 ratios, leading to biophysically and pharmacologically distinct channels. Therefore, Kv1.3-based treatments to alter physiological responses, such as proliferation and activation, are impaired by Kv1.5 expression. The presence of Kv1.5 in the macrophagic lineage should be taken into account when designing Kv1.3-based therapies. 2006 Elsevier Inc. All rights reserved.

A field-stimulated electric organ generated an electric field and release of a large quantity of ATP. Oocytes injected with mRNA from a Torpedo electric organ released ATP when stimulated with carbamylcholine. We set up a configuration recording that let us simultaneously monitor the oocyte ionic currents and ATP release from a single oocyte. Hyperpolarization pulses applied to Xenopus oocytes induced an inward current and release of ATP independently of the expression of the CIC-0 chloride channel. The current was insensitive to substitution of ions in the extracellular saline solution, either anions or cations. The reversal potential of the current was established between +40 and +100 mV, which is in the same range as the equilibrium potential of ATP -4 . There was a correlation between the magnitude of the inward current and the amount of ATP released. The inward current closed slowly. During this closing period there was still release of ATP, which is sensitive to the holding potential. Positive potentials cut the amount of ATP released. The evoked current and ATP release were both sensitive to inhibitors of E-NTPDase activity, such as suramin, 4-acetamido-4'isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'diisothiocyanatostilbene-2'-disulfonic acid (DIDS), and a high concentration of calcium and gadolinium ions. The expression of CD39 increased the size of the hyperpolarizing induced current. By reverse transcription polymerase chain reaction, we found a protein homologous to CD39. The relationship between the results obtained in the electric organ and oocytes is that, in both cases, stimulation or hyperpolarization causes a change in the intracellular ion concentration, producing an osmotic stress, which, in turn, triggers ATP release. Stimulation or hyperpolarization may alter the intercellular ion concentration, leading to osmotic stress and the release of ATP in both electric organ and oocytes. Drug Dev.

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.

The diadenosine polyphosphate hydrolase present in presynaptic plasma membranes from the Torpedo electric organ has been characterized using fluorogenic substrates of the form di-(1, N6-ethenoadenosine) 5',5'''-P1,Pn-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (ApnA, where n=3-5), producing AMP and the corresponding adenosine (n-1) 5'-phosphate, Ap(n-1). The Km values of the enzyme were 0.543+/-0.015, 0.478+/-0.043 and 0. 520+/-0.026 microM, and the Vmax values were 633+/-4, 592+/-18 and 576+/-45 pmol/min per mg of protein, for the etheno derivatives of Ap3A (adenosine 5',5'''-P1,P3-triphosphate), Ap4A (adenosine 5',5"'-P1,P4-tetraphosphate) and Ap5A (adenosine 5',5'''-P1,P5-pentaphosphate) respectively. Ca2+, Mg2+ and Mn2+ are enzyme activators, with EC50 values of 0.86+/-0.11, 1.35+/-0.24 and 0.58+/-0.10 mM respectively. The fluoride ion is an inhibitor with an IC50 value of 1.38+/-0.19 mM. The ATP analogues adenosine 5'-tetraphosphate and adenosine 5'-[gamma-thio]triphosphate are potent competitive inhibitors and adenosine 5'-[alpha,beta-methylene]diphosphate is a less potent competitive inhibitor, the Ki values being 0.29+/-0.03, 0.43+/-0.05 and 7.18+/-0.8 microM respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulphonic acid behaves as a non-competitive inhibitor with a Ki value of 29.7+/-3.1 microM, and also exhibits a significant inhibitory effect on Torpedo apyrase activity. The effect of pH on the Km and Vmax values, together with inhibition by diethyl pyrocarbonate, strongly suggests the presence of functional histidine residues in Torpedo diadenosine polyphosphate hydrolase. The enzyme from Torpedo shows similarities with that of neural origin from neurochromaffin cells, and significant differences compared with that from endothelial vascular cells.

Ecto-apyrase is a widespread enzymatic activity that hydrolyses tri- and diphosphonucleotides and consequently controls the amount of available extracellular ATP and ADP. In the nervous system, purines have important neuromodulatory actions, acting at pre- and postsynaptic sites, and consequently, ecto-apyrase may play an indirect role in the modulation of nucleotide- and nucleoside-mediated processes. The azido-nucleotides have been largely employed to characterize the nucleotide binding sites of several proteins. In the present work the azido-nucleotides are described as putative substrates for apyrase activity in a presynaptic plasma membrane preparation (PSPM) from the Torpedo electric organ. Both 8-N 3 -ATP and 8-N 3 -ADP were hydrolyzed in a calcium-dependent manner showing V max of 23.8 ± 4.8 and 14.5 ± 3 U/mg of protein, and K m values (in μM) of 116 ± 39 and 119 ± 4, respectively. V max for calcium-dependent hydrolysis of ATP and ADP were significantly higher: 59.2 ± 3.9 and 32.9 ± 3.5 U/mg of protein respectively, while K m values did not show any significant differences regarding azido-nucleotides: 83.8 ± 12 μM for Ca 2+ -ATP and 121 ± 34 μM for Ca 2+ -ADP. The photoactivation of the PSPM in the presence of the azido-derivatives results in an irreversible inactivation of apyrase activity, showing an IC 50 of 10 μM and a maximal inhibitory effect of 38 and 60% on Ca 2+ -ATPase and Ca 2+ -ADPase activities. Apyrase was protected from inactivation by nucleotides that are natural substrates for this enzymatic activity and also by AMP while adenosine did not protect from apyrase inhibition.

1. The role of ATP, which is co-released with acetylcholine in synaptic contacts of Torpedo electric organ, was investigated by use of suramin. Suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulphoni c acid], a P2 purinoceptor antagonist, potently inhibited in a non-competitive manner the ecto-apyrase activity associated with plasma membrane isolated from cholinergic nerve terminals of Torpedo electric organ. The Ki was 30 microM and 43 microM for Ca(2+)-ADPase and Ca(2+)-ATPase respectively. 2. In Torpedo electric organ, repetitive stimulation decreased the evoked synaptic current by 51%. However, when fragments of electric organ were incubated with suramin the evoked synaptic current declined by only 14%. Fragments incubated with the selective A1 purinoceptor antagonist, DPCPX, showed 5% synaptic depression. 3. The effects of suramin and DPCPX on synaptic depression were not addictive. Synaptic depression may thus be linked to endogenous adenosine formed by dephosphorylation of released ATP by an ecto-apyrase. The final effector in synaptic depression, adenosine, acts via the A1 purinoceptor. 4. ATP hydrolysis is prevented in the presence of suramin. It slightly increased (20%) the mean amplitude of spontaneous miniature endplate currents. The frequency distribution of the amplitude of spontaneous events was shifted to the right, indicating that ATP, when not degraded, may modulate the activation of nicotinic acetylcholine receptors activated by the quantal secretion of acetycholine.

It has been shown previously that ATP is released into extracellular space from pre- and postsynaptic sources in peripheral synapses. The extracellular metabolism of ATP is likely to affect nucleotide- and nucleoside-mediated regulation of neurotransmission. The enzymes responsible for ATP breakdown are nucleotidases whose active site faces the extracellular space. ADPase and ADPase Ca 2+ -dependent activities were characterized in presynaptic plasma membrane preparation from the electric organ of Torpedo . Features described were in accordance with the presence of an ATP-diphosphohydrolase (apyrase EC 3.6.1.5) in this fraction. Active site studies using the affinity label 5′-fluorosulfonylbenzoyladenosine were performed on Torpedo apyrase. ADPase and ADPase Ca 2+ -dependent activities were inhibited with 5′-fluorosulfonylbenzoyladenosine. From this study it is concluded: (1) 5′-fluorosulfonylbenzoyladenosine binds specifically to the active site of apyrase. (2) Divalent cations accelerate the apyrase inactivation rate. (3) Divalent cations are not required for the binding of either the substrate or the inhibitor to the active site. (4) The apyrase active site is more specific for highly phosphorylated nucleotides. The results presented may be extrapolated to apyrases from other sources. The importance of this enzyme and its regulation are discussed.

We report here on the synthesis and characterization of a fully active biotinylated derivative of the botulinum neurotoxin type A. Different ratios of biotin:botulinum toxin were tested to optimize derivatizing conditions and a ratio of 35:1 was selected for further experiments. The average number of biotin groups per toxin molecule was estimated to be 7.8, occurring at both heavy and light chains, and almost all externally located and easily accessible to recognition by streptavidin. The modified toxin retained its toxicity and its ability to interact with biological membranes. Apart from its suitability for detection in Western blots and in microtiter well plates, biotinylated botulinum toxin proved to be adequate for morphological labeling studies at both light and electron microscopy. Peroxidase histochemistry in cryostat sections of intoxicated rat hemidiaphragm muscles showed a distinct labeling of end-plates. Electron microscopy studies were performed on the electric organ of Torpedo marmorata using colloidal gold-conjugated streptavidin for detection. After intoxication of electric organ fragments with the modified toxin, gold labels were found associated with the presynaptic plasma membrane of nerve terminals and with the membrane of synaptic vesicles. Moreover, the distribution of biotinylated botulinum toxin binding sites over the membrane of synaptosomes isolated from the electric organ of Torpedo and their relationship with intramembrane particles were analyzed using the replica-staining label-fracture technique. It was found that the toxin is never associated with intramembrane particles. © 1993 Wiley-Liss, Inc.

Amyloid beta (Aβ) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which Aβ oligomers cause neurotoxicity remain unknown. We recently reported that Aβ oligomers cause intracellular Ca(2+) overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether Aβ oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, Aβ oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to Aβ oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca(2+) induced by Aβ oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that Aβ oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent Aβ damage to neurons in Alzheimeŕs disease.

Torpedo electric organ has been used to study the binding of botulinum neurotoxin type A to pure cholinergic synaptosomes and presynaptic plasma membrane. 125I-labeled botulinum neurotoxin type A exhibits specific binding to cholinergic fractions. Two binding sites have been determined according to data analysis: a high affinity binding site (synaptosomes: Kd = 0.11 +/- 0.03 nM, Bmax = 50 +/- 10 fmol.mg prot-1; presynaptic plasma membrane: Kd = 0.2 +/- 0.05 nM, Bmax = 150 +/- 15 fmol.mg prot-1) and a low affinity binding site (synaptosomes: Kd approximately 26 nM, Bmax approximately 7.5 pmol.mg prot-1; presynaptic plasma membrane: Kd approximately 30 nM, Bmax approximately 52 pmol.mg prot-1). The binding of 125I-botulinum neurotoxin type A is decreased by previous treatment of synaptosomes by neuraminidase and trypsin, and by a preincubation with bovine brain gangliosides or antiserum raised against Torpedo presynaptic plasma membrane. When presynaptic plasma membranes are blotted to nitrocellulose sheet, either 125I-botulinum neurotoxin or botulinum toxin-gold complexes bind to a M(r) approximately 140,000 protein. Botulinum toxin-gold complexes have also been used to study the toxin internalization process into Torpedo synaptosomes. The images fit the three step sequence model in the pathway of botulinum neurotoxin poisoning.

We have examined the effect of several blockers of voltage-sensitive calcium channels on the release of acetylcholine and ATP from synaptosomes isolated from Torpedo marmorata electric organ. Depolarization of these nerve terminals with high K + -containing solutions resulted in a calcium-dependent release of both molecules. Cadmium ions (10 −6 to 10 −3 M) inhibited similarly both releases whereas nickel ions (10 −4 M) in the external medium did not affect either neurotransmitter or nucleotide release. Both releases were completely resistant to the effect of 1,4-dihydropyridines (antagonists nimodipine, nifedipine and agonist Bay K 8644) and of a related compound (diltiazem) at concentrations up to 10 −5 M. These drugs failed to cause any effect even when synaptosomes were submaximally depolarized during incubation. Omega-conotoxin (10 −8 to5 × 10 −5 M) showed a differential effect on acetylcholine and ATP releases. Nucleotide release was inhibited 90% ai the highest concentration tested (50 μm) while acetylcholine release was only moderately decreased (30%). ec 50 values for acetylcholine and ATP were of 167 and 2 μM respectively. The results suggest the implication of different types of calcium channels in the release of these molecules.

The X-linked Charcot Marie Tooth (CMTX) is an inherited disease due to mutations in connexin 32 (Cx32) gene expressed in Schwann cells (SC) of peripheral nerves. In SC, Cx32 localizes in the paranodes, in the Schmidt Lanterman incisures and in the cell surface. Cx32 can form “reflexive” gap junction channels as well as functional hemichannels open upon membrane depolarization. We have explored the permeability of Cx32 hemichannels to ATP, in SC and in a heterologous expression system. Murine sciatic nerve trunks release ATP under electrical or mechanical stimulation, as determined by the luciferase reaction. Luminescence imaging revealed that ATP release is especially intense at the SC paranodes, which contain the highest immunofluorescent label for Cx32. Cultured adult SC have a high expression of Cx32 and under mechanical stimulus release ATP being insensitive to exocytosis blockers like brefeldin A. In Xenopus oocytes expressing human Cx32, we measured simultaneously the hemichannel currents and the release of ATP elicited by a square depolarizing pulse up to +100 mV. Depolarizing pulses induced characteristic slowly activating outward currents and when the membrane potential returned to the holding voltage tail currents coinciding with the peak of ATP release. The deconvolution of the light signal revealed that the time courses of the tail current and the ATP release were coincident. We established a direct relationship between the amount of ATP released and the amplitude of tail current. Applying positive voltages closer to the ATP reversal potential during the tail current reduced the amount of ATP released. Five different single amino acid mutants of Cx32, described in CMTX, affecting intracellular, extracellular or transmembrane domains, were tested. Those mutations deeply inhibited or abolished the hemichannel currents and the ATP release.

Kv1.3 activity is determined by raft association. In addition to Kv1.3, leukocytes also express Kv1.5, and both channels control physiological responses. Because the oligomeric composition may modify the channel targeting to the membrane, we investigated heterotetrameric Kv1.3/Kv1.5 channel traffic and targeting in HEK cells. Kv1.3 and Kv1.5 generate multiple heterotetramers with differential surface expression according to the subunit composition. FRET analysis and pharmacology confirm the presence of functional hybrid channels. Raft association was evaluated by cholesterol depletion, caveolae colocalization, and lateral diffusion at the cell surface. Immunoprecipitation showed that both Kv1.3 and heteromeric channels associate with caveolar raft domains. However, homomeric Kv1.3 channels showed higher association with caveolin traffic. Moreover, FRAP analysis revealed higher mobility for hybrid Kv1.3/Kv1.5 than Kv1.3 homotetramers, suggesting that heteromers target to distinct surface microdomains. Studies with lipopolysaccharide-activated macrophages further supported that different physiological mechanisms govern Kv1.3 and Kv1.5 targeting to rafts. Our results implicate the traffic and localization of Kv1.3/Kv1.5 heteromers in the complex regulation of immune system cells.

The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.

During synaptic transmission large amounts of ATP are released from pre- and post-synaptic sources of Torpedo electric organ. A chain reaction sequentially hydrolyses ATP to adenosine, which inhibits acetylcholine secretion. The first enzyme implicated in this extracellular ATP hydrolysis is an ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) that dephosphorylates both ATP and ADP to AMP. This enzyme has been biochemically characterized in the synaptosomal fraction of Torpedo electric organ, having almost equal affinity for ATP as for ADP, a fact that pointed to the type-1 NTPDase enzyme. In the present work we describe the cloning and molecular characterization of the cDNA for an NTPDase from Torpedo marmorata electric organ. The clone, obtained using the RACE-PCR technique, contains and open-reading frame of 1506 bp and encodes a 502 amino acids protein that exhibits high homology with other NTPDases1 from vertebrates previously identified, including those of zebrafish and Xenopus, as well as human, rat and mouse. Topology analyses revealed the existence of two transmembrane regions, two short cytoplasmic tails and a long extracellular domain containing five apyrase-conserved regions. Gene expression studies revealed that this gene is expressed in all the Torpedo tissues analyzed. Finally, activity and cellular localization of the protein encoded by this newly cloned cDNA was assessed by heterologous expression experiments involving COS-7 and HeLa cells.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are widely expressed enzymes implicated in the modulation of nucleotide cell signaling. They dephosphorylate either ATP or ADP in the presence of divalent cations, and efforts have been made to identify efficient inhibitors. E-NTPDase activity has been described in Torpedo electric organ electrocytes. We show here that gadolinium, an established blocker of stretch-activated channels, efficiently inhibits E-NTPDase activity of Torpedo electric organ (Ki = 3 microM for ATPase) as well as apyrase from potato tuber, frequently used in inhibition experiments. To our knowledge, gadolinium is the most potent inhibitor described to date for both membrane-bound and soluble E-NTPDases.

Voltage-dependent K+ channels (VDPC) are expressed in most mammalian cells and involved in the proliferation and activation of lymphocytes. However, the role of VDPC in macrophage responses is not well established. This study was undertaken to characterize VDPC in macrophages and determine their physiological role during proliferation and activation. Macrophages proliferate until an endotoxic shock halts cell growth and they become activated. By inducing a schedule that is similar to the physiological pattern, we have identified the VDPC in non-transformed bone marrow-derived macrophages and studied their regulation. Patch clamp studies demonstrated that cells expressed outward delayed and inwardly rectifying K+ currents. Pharmacological data, mRNA, and protein analysis suggest that these currents were mainly mediated by Kv1.3 and Kir2.1 channels. Macrophage colony-stimulating factor-dependent proliferation induced both channels. Lipopolysaccharide (LPS)-induced activation differentially regulated VDPC expression. While Kv1.3 was further induced, Kir2.1 was down-regulated. TNF-alpha mimicked LPS effects, and studies with TNF-alpha receptor I/II double knockout mice demonstrated that LPS regulation mediates such expression by TNF-alpha-dependent and -independent mechanisms. This modulation was dependent on mRNA and protein synthesis. In addition, bone marrow-derived macrophages expressed Kv1.5 mRNA with no apparent regulation. VDPC activities seem to play a critical role during proliferation and activation because not only cell growth, but also inducible nitric-oxide synthase expression were inhibited by blocking their activities. Taken together, our results demonstrate that the differential regulation of VDPC is crucial in intracellular signals determining the specific macrophage response.

The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 +/- 0.3 pmol/oocyte) generated a slow inward current (2.3 +/- 0.1 microA). During hyperpolarizing pulses, the permeability for ATP(4-) was more than 4000 times higher than that for Cl(-). The sensitivity to GdCl(3) (0. 2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP.

Injection of chick cerebellar membranes, rich in kainate binding sites, into Xenopus oocytes resulted in the structural integration of chick membrane patches into the oocyte plasma membrane that could be easily identified by specific immunofluorescent staining. Application of kainate to the oocyte perfusion medium, under voltage-clamp conditions, induced dose-dependent (EC50 = 87+/-14 microM) inward currents, confirming the functional incorporation to the oocyte of kainate-driven channels. Responses to kainate were consistently nondesensitizing and strongly potentiated by cyclothiazide, suggesting the selective involvement of alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA)-preferring receptors. Binding experiments with (S)-[3H]AMPA confirmed the presence in the chick membrane preparation of low-affinity AMPA receptors (K(D) = 278 nM) amounting to

Tetanus toxin decreased the frequency of spontaneous events at the electric organ of Torpedo marmorata. This reduction was up to 70% in poisoned electric organ. According to distribution analysis of miniature end plate currents, only a subpopulation of events which have small amplitudes were recorded after poisoning. Furthermore, isolated cholinergic nerve terminals showed a decrease in VAMP/synaptobrevin when poisoned with tetanus toxin under similar conditions. The relationship between the two effects of the toxin, i.e. inhibition of vesicle exocytosis and peptidase activity on synaptobrevin, is discussed.

Botulinum neurotoxin type A blocks acetylcholine release from the peripheral nervous system. We have previously described a putative botulinum neurotoxin type A receptor of presynaptic plasma membranes from Torpedo. The electric organ of Torpedo, which is largely enriched in cholinergic nerve endings, is homologous to the neuromuscular junction, allowing us to isolate large scale of presynaptic components. In order to characterize this protein we have raised a polyclonal antibody (a-P140) against this receptor. The antiserum a-P140 recognizes a 140,000 mol. wt band in non-reducing conditions and an 80,000 band in reducing conditions. The immunohistochemistry assay reveals the P140 protein on the ventral face of the electrocytes where the nerve terminals are localized. Moreover, a-P140 antiserum recognizes the P140-BoNT/A complex after binding and cross-linking experiments. In addition, we have immunoprecipitated an in vitro translated product which is closely coincident in mol. wt to the 80,000 band of the receptor.

Giant liposomes were made from a mixture of asolectin phospholipid vesicles and presynaptic plasma membranes isolated from Torpedo cholinergic nerve; endings. Acetylcholine filled giant liposomes were able to release neurotransmitter upon stimulation by the Ca2+ ionophore A23187 and Ca2+. Botulinum neurotoxin type A inhibited this Ca2+-dependent acetylcholine transport. Additionally, Botulinum toxin type A decreased membrane fluidity of liposomes. These results suggest that Botulinum toxin can interact directly with components of the presynaptic plasma membrane and inhibit acetylcholine translocation. Furthermore, since the reconstituted liposomes do not have synaptic vesicle components, the observed effects may account for the action of Botulinum toxin on the non-quantal release of acetylcholine from motor nerve terminals., This work is supported by grants from DGICYT (Ministerio de Educacion, Spain) to C.S., J.M. and J.M.G.R. Elena Lopez-Alonso is a fellow of Generalitat de Valencia.

It is well known that acetylcholine (ACh) and ATP are co-stored and co-released in nerve terminals of the electric organ of Torpedo. Cholinergic synaptosomes were subjected to a cycle of freezing and thawing showing that ATP is distributed in two operational pools like those described for ACh. The bound pool is resistant to freezing and thawing, and it is presumably protected by membranes. When metabolically active ATP was prelabelled with [3H]adenosine, 76% of the radioactivity was associated with the free pool of ATP. When the preparation was depolarized in a calcium containing medium, there was a decrease in the specific radioactivity of ATP in the free pool and an increase in the bound pool. These results reflect that the patterns of distribution of ACh and ATP, in this synaptosomal preparation, are similar in resting conditions and during K+ depolarization.

Acetylcholine and ATP are costored and coreleased during synaptic activity at the electric organ of Torpedo. It has been suggested that released ATP is converted to adenosine at the synaptic cleft, and in turn this nucleoside would depress the evoked release of acetylcholine. In the present communication we have used a chemiluminescent reaction that let us to monitor continuously the presence of adenosine in this preparation. The chemiluminescent reaction is based on the conversion of adenosine into uric acid and H2O2 by adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzymes. The hydrogen peroxide has been detected by peroxidase-luminol mixture. The reaction has a sensitivity on the picomol range and discerned between Adenosine, AMP, ADP, and ATP. We have developed this technique in the hope of understanding whether adenosine is released during synaptic activity or it comes from the released ATP. We have studied the release or formation of adenosine in fragments of the electric organ and in isolated cholinergic nerve terminals obtained from it. In both conditions we have followed the effect of potassium stimulation upon the detection of adenosine. Potassium stimulation increased the extracellular adenosine either in slices or the synaptosomal fraction of Torpedo electric organ. The presence of alpha, beta-methylene ADP, an inhibitor of 5'-nucleotidase, inhibits the detection of adenosine, suggesting that extracellular adenosine is a consequence of ectocellular dephosphorylation of released ATP.

We have studied the action of tetanus toxin on the release of acetylcholine from a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. We have also studied the morphological changes induced by chemical stimulation on the presynaptic plasma membrane of poisoned synaptosomes. These changes were studied by means of freeze-fracture techniques. We found that tetanus toxin blocks the release of acetylcholine from isolated nerve terminals in a dose-dependent manner. The maximal inhibition is achieved at a concentration of 12.5 nM in 10 min. This effect is prevented by tetanus toxin antiserum. Tetanus toxin also blocks the rearrangement of intramembrane particles at plasma membrane of poisoned synaptosomes, specifically the decrease of small (less than or equal to 9.5 nm diameter) intramembranous particles at the protoplasmic hemimembrane leaflet and the increase of large (greater than 9.5 nm diameter) intramembrane particles at the external hemimembrane leaflet induced by potassium stimulation. These results suggest that intramembrane particle rearrangement could be related to acetylcholine secretion.

The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release, was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Acetylcholine and ATP release were continuously monitored by chemiluminescent methods. To catch the membrane morphological changes, the quick-freezing method was applied. Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected. The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min. This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity. Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization. In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation. The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata.

We have studied the correlation between [3H]ouabain binding sites, (Na+ + K+)ATPase (EC 3.6.1.3) activity and acetylcholine (ACh) release in different subcellular fractions of Torpedo marmorata electric organ (homogenate, synaptosomes, presynaptic plasma membranes). Presynaptic plasma membranes contained the greater number of [3H]ouabain binding sites, in good agreement with the high (Na+ + K+)ATPase activity found in this fraction. Blockade of this enzymatic activity by ouabain dose-dependently induced ACh release from pure cholinergic synaptosomes, either in the presence or absence of extracellular calcium ions. We suggest that one of the mechanisms involved in the ouabain-induced ACh release in the absence of Ca2+o may be an increase in Na+i that could (a) evoke Ca2+ release from internal stores and (b) inhibit ATP-dependent Ca2+ uptake by synaptic vesicles.

Pure cholinergic synaptosomes from the electric organ of Torpedo marmorata have a saturable adenosine uptake mechanism (calculated K m , 3 μM; V max , 24 pmol/min/mg prot.). In this preparation, high intracellular calcium elicited by increasing external calcium concentration, potassium depolarization, sodium-calcium exchange inhibition or divalent cation ionophore A23187 action, inhibits adenosine uptake into synaptosomes. The data presented in this paper are consistent with the interpretation that high intracellular calcium participates in the regulation of the high-affinity adenosine uptake system.

Pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata were stimulated by calcium ionophore A-23187. The effect of time course of stimulation on the changes in intramembrane particles (IMPs) on presynaptic membranes was studied by quick-freezing and aldehyde-fixation freeze-fracture. We showed that the decrease of small-particle density at the P-face and the increase of large-particle density at the E-face was maximum after 30 sec of A-23187 stimulation. Later, the density of synaptic vesicles decreased. We suggest that the redistribution of IMPs on the presynaptic membrane and acetylcholine (ACh) release from pure cholinergic synaptosomes have a similar time course when triggered by A-23187.

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

Isolated pure cholinergic synaptosomes from Torpedo electric organ were incubated in vitro with β-bungarotoxin for 15, 30 and 60 min and processed for electron microscopy. It was found that no morphological damage was seen after 15 min but by contrast, severe disruption of synaptosomes was present at 30 or 60 min after incubation with toxin. Synaptosomes were incubated also for 15 min in the presence of 125 I-labelled β-bungarotoxin and the binding was evaluated by electron microscopic autoradiography. The toxin was found to bind to the presynaptic membrane. The surface density of toxin binding sites was calculated to be around 3000/μm 2 . In a minor population of synaptosomes, the toxin was translocated into large vesicles suggesting that the toxin-receptor complexes underwent endocytosis in such vesicles. These results give further support to the view that inhibition of transmitter release by the toxin is produced by its action on plasma membrane.

Botulinum neurotoxin, a strong blocker of acetylcholine release at peripheral cholinergic synapses, inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata. Moreover, tetrodotoxin has the same effect on protein phosphorylation when cholinergic synaptosomes are depolarized by veratridine. Correlation between presynaptic protein phosphorylation and acetylcholine release is suggested by the fact that botulinum neurotoxin blocks specifically neurotransmitter release without affecting membrane depolarization and calcium fluxes in our synaptosomal preparation.

We report here the effects of Botulinum Toxin type A on the release of ATP and Acetylcholine from Torpedo electric organ synaptosomes. Our results show that Botulinum Toxin type A inhibits specifically the K + -induced release of Acetylcholine from synaptosomes without affecting the release of ATP. Membrane potential and calcium uptake into cholinergic nerve terminals are not modified after Botulinum Toxin poisoning. It is suggested that either most of the ATP released during the depolarization of the cholinergic synaptosomes does not originate from cholinergic synaptic vesicles or that there are two populations of synaptic vesicles, Acetylcholine-enriched synaptic vesicles and ATP-enriched synaptic vesicles. However, the possibility that the ACh and ATP released could come from different intrasynaptosomal compartments cannot be excluded.

Tetanus toxin (TeTx) is a neurotransmission impairing toxin that acts on several neurotransmitter systems. TeTx also inhibits the K+-induced release of acetylcholine (ACh) from synaptosomes isolated from the electric organ of Torpedo. Neither the membrane potential and depolarization, nor the depolarization-induced calcium uptake into cholinergic nerve terminals is modified after TeTx poisoning. On the other hand, it is known that, when cholinergic nerve terminals are stimulated, there is a release of ATP associated with the release of ACh. We have explored the action of TeTx on this co-release, and have found that there is no action of TeTx on the nucleotide release. Thus, TeTx blocks ACh release without modifying ATP release.

We have investigated the redistribution of filipin-cholesterol complexes at freeze-fractured presynaptic membrane of pure cholinergic synaptosomes isolated from Torpedo electric organ during acetylcholine release. After chemical depolarization, filipin-induced lesions increase at the presynaptic membrane. These changes do not take place when synaptosomes are stimulated in a calcium-free medium. Botulinum neurotoxin type A blocks both acetylcholine release and the rearrangement of filipin-induced lesions induced by depolarization. Since botulinum neurotoxin type A does not block either membrane depolarization or calcium entry into the nerve terminal, our results suggest that the redistribution of filipin-cholesterol complexes is linked to the acetylcholine release process.

In previous reports, we have shown that botulinum neurotoxin inhibits acetylcholine release from Torpedo marmorata electric organ and from its synaptosomal fraction. Here, we have focussed our attention on the study of the effect of botulinum neurotoxin on the metabolism of acetylcholine, namely, the precursors supply, the synthesis activity and the storage of the neurotransmitter into nerve endings isolated from Torpedo electric organ. Radiolabelled acetylcholine precursors (acetate and choline) uptake, choline O-acetyltransferase activity, and the compartmentalization of the transmitter into the synaptosomes were not modified by botullinum neurotoxin. When labelled nerve ending were depolarized by K+, the specific radioactivity of acetylcholine in the free pool fell markedly, but the specific radioactivity in the bound pool remained constant. Botulinum neurotoxin prevented this K+-induced decrease of specific radioactivity in the free pool.

Cholinergic synaptosomes obtained from the electric organ of Torpedo marmorata have been used to study chemical stimulation-stimulated protein phosphorylation. Cholinergic synaptosomes were exposed to elevated K+0 concentrations or other chemical depolarizing agents such as gramicidin or secretagogues as the calcium ionophore A23187. During depolarization several synaptosomal proteins increase their state of phosphorylation. This phenomenon depends on the presence of Ca2+ in the external medium. These results suggest that stimulation of protein phosphorylation may be implicated in the acetylcholine release process and could represent a modulation mechanism in the neurotransmitter release machinery at this cholinergic synapse.