Alain Bloc

Zinc inhibits glutamate release via activation of pre‐synaptic KATP channels and reduces ischaemic damage in rat hippocampus

Zinc is concentrated in certain CNS excitatory tracts, especially in hippocampal mossy fibres where it has been suggested to modulate synaptic transmission and plasticity. Using rat mossy fibre synaptosomes depolarized by 4‐aminopyridine, we show here that low zinc concentrations restore the membrane potential and reduce glutamate release. Both effects arose from activation of ATP‐sensitive potassium channels (KATP), since they were mimicked by the KATP opener diazoxide and antagonized by the KATP blocker tolbutamide. Using recombinant channels expressed in COS‐7 cells, we confirmed that micromolar zinc did activate KATP of the type found in hippocampus. We tested the hypothesis that this action of zinc could be beneficial during an ischaemic challenge by using organotypic hippocampal slice cultures. When zinc was applied at micromolar concentrations during a brief anoxic‐hypoglycaemic episode, it significantly attenuated the ensuing neuronal death, whereas chelation of endogenous zinc markedly aggravated cell damage. Protective effect of zinc was mediated through KATP, as was shown by using the opener diazoxide and the blocker tolbutamide. Thus, by activating pre‐synaptic KATP channels, zinc protects neurones from hyper‐excitation, excessive transmitter release and exitotoxicity, and may thus act as an endogenous neuroprotector in conditions such as epilepsy or stroke.

Zinc is both an intracellular and extracellular regulator of KATP channel function

Extracellular Zn2+ has been identified as an activator of pancreatic KATP channels. We further examined the action of Zn2+ on recombinant KATP channels formed with the inward rectifier K+ channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn2+, applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of KATP channels. External Zn2+, at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn2+ dose‐dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half‐maximal effects at 1.8 and 60 μm, respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn2+ on the SUR subunit. Effects of internal Zn2+ were not due to Zn2+ leaking out, since they were unaffected by the presence of a Zn2+ chelator on the external side. Similarly, internal chelators did not affect activation by external Zn2+. Therefore, Zn2+ is an endogenous KATP channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting KATP channels, and suggest a new role for Zn2+ as an intracellular signalling molecule.

Zinc‐induced changes in ionic currents of clonal rat pancreatic β‐cells: activation of ATP‐sensitive K+ channels

1 The effects of zinc (Zn2+) on excitability and ionic conductances were analysed on RINm5F insulinoma cells under whole‐cell and outside‐out patch‐clamp recording conditions. 2 We found that extracellular application of 10‐20 μM Zn2+ induced a reversible abolition of Ca2+ action potential firing, which was accompanied by an hyperpolarisation of the resting membrane potential. 3 Higher concentrations of Zn2+, in the tens to hundreds micromolar range, induced a reversible reduction of voltage‐gated Ca2+ and, to a lesser extent, K+ currents. Low‐voltage‐activated Ca2+ currents were more sensitive to Zn2+ block than high voltage‐activated Ca2+ currents. 4 The Zn2+‐induced hyperpolarisation arose from a dose‐dependent increase in a voltage‐independent K+ conductance that was pharmacologically identified as an ATP‐sensitive K+ (KATP) conductance. The effect was rapid in onset, readily reversible, voltage independent, and related to intracellular ATP concentration. In the presence of 1 mM intracellular ATP, half‐maximal activation of KATP channels was obtained with extracellular application of 1.7 μM Zn2+. 5 Single channel analysis revealed that extracellular Zn2+ increased the KATP channel open‐state probability with no change in the single channel conductance. 6 Our data support the hypothesis that Zn2+ binding to KATP protein subunits results in an activation of the channels, therefore regulating the resting membrane potential and decreasing the excitability of RINm5F cells. Taken together, our results suggest that Zn2+ can influence insulin secretion in pancreatic β‐cells through a negative feedback loop, involving both KATP and voltage‐gated conductances.

Defective Tumor Necrosis Factor-α-dependent Control of Astrocyte Glutamate Release in a Transgenic Mouse Model of Alzheimer Disease

The cytokine tumor necrosis factor-α (TNFα) induces Ca2+-dependent glutamate release from astrocytes via the downstream action of prostaglandin (PG) E2. By this process, astrocytes may participate in intercellular communication and neuromodulation. Acute inflammation in vitro, induced by adding reactive microglia to astrocyte cultures, enhances TNFα production and amplifies glutamate release, switching the pathway into a neurodamaging cascade (Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. (2001) Nat. Neurosci. 4, 702–710). Because glial inflammation is a component of Alzheimer disease (AD) and TNFα is overexpressed in AD brains, we investigated possible alterations of the cytokine-dependent pathway in PDAPP mice, a transgenic model of AD. Glutamate release was measured in acute hippocampal and cerebellar slices from mice at early (4-month-old) and late (12-month-old) disease stages in comparison with age-matched controls. Surprisingly, TNFα-evoked glutamate release, normal in 4-month-old PDAPP mice, was dramatically reduced in the hippocampus of 12-month-old animals. This defect correlated with the presence of numerous β-amyloid deposits and hypertrophic astrocytes. In contrast, release was normal in cerebellum, a region devoid of β-amyloid deposition and astrocytosis. The Ca2+-dependent process by which TNFα evokes glutamate release in acute slices is distinct from synaptic release and displays properties identical to those observed in cultured astrocytes, notably PG dependence. However, prostaglandin E2 induced normal glutamate release responses in 12-month-old PDAPP mice, suggesting that the pathology-associated defect involves the TNFα-dependent control of secretion rather than the secretory process itself. Reduced expression of DENN/MADD, a mediator of TNFα-PG coupling, might account for the defect. Alteration of this neuromodulatory astrocytic pathway is described here for the first time in relation to Alzheimer disease.

The lectin-like domain of tumor necrosis factor-α increases membrane conductance in microvascular endothelial cells and peritoneal macrophages

Herein, we show that TNF exerts a pH‐dependent increase in membrane conductance in primary lung microvascular endothelial cells and peritoneal macrophages. This effect was TNF receptor‐independent, since it also occurred in cells isolated from mice deficient in both types of TNF receptors. A TNF mutant in which the three amino acids critical for the lectin‐like activity were replaced by an alanine did not show any significant effect on membrane conductance. Moreover, a synthetic 17‐amino acid peptide of TNF, which was previously shown to exert lectin‐like activity, also increased the ion permeability in these cells. The amiloride sensitivity of the observed activity suggests a binding of TNF to an endogenousion channel rather than channel formation by TNF itself. This may have important implications in mechanisms of TNF‐mediated vascular pathology.

Nicotine-induced and depolarisation-induced glutamate release from hippocampus mossy fibre synaptosomes: two distinct mechanisms

Hippocampus mossy fibre terminals activate CA3 pyramidal neurons via two distinct mechanisms, both quantal and glutamatergic: (i) rapid excitatory transmission in response to afferent action potentials and (ii) delayed and prolonged release following nicotinic receptor activation. These processes were analysed here using rat hippocampus mossy fibres synaptosomes. The relationships between synaptosome depolarisation and glutamate release were established in response to high‐KCl and gramicidin challenges. Half‐maximal release corresponded to a 52 mV depolarisation step. KCl‐induced release was accompanied by transient dissipation of the proton gradient across synaptic vesicle membrane. Nicotine elicited a substantial glutamate release from mossy fibre synaptosomes (EC50 3.14 μM; Vmax 12.01 ± 2.1 nmol glutamate/mg protein; Hill’s coefficient 0.99). However, nicotine‐induced glutamate release was not accompanied by any change in the membrane potential or in the vesicular proton gradient. The effects of acetylcholine (200 μM) were similar to those of nicotine (25 μM). Nicotinic α7 receptors were evidenced by immuno‐cytochemistry on the mossy fibre synaptosome plasma membrane. Therefore, the same terminals can release glutamate in response to two distinct stimuli: (i) rapid neurotransmission involving depolarisation‐induced activation of voltage‐gated Ca2+ channels and (ii) a slower nicotinic activation which does not involve depolarisation or dissipation of the vesicular proton gradient.

Membrane interaction of TNF is not sufficient to trigger increase in membrane conductance in mammalian cells

Tumor necrosis factor (TNF) can trigger increases in membrane conductance of mammalian cells in a receptor‐independent manner via its lectin‐like domain. A lectin‐deficient TNF mutant, lacking this activity, was able to bind to artificial liposomes in a pH‐dependent manner, but not to insert into the bilayer, just like wild type TNF. A peptide mimicking the lectin‐like domain, which can still trigger increases in membrane currents in cells, failed to interact with liposomes. Thus, the capacity of TNF to trigger increases in membrane conductance in mammalian cells does not correlate with its ability to interact with membranes, suggesting that the cytokine does not form channels itself, but rather interacts with endogenous ion channels or with plasma membrane proteins that are coupled to ion channels.

Voltage‐gated K+ current: a marker for apoptosis in differentiating neuronal progenitor cells?

We investigated apoptosis during early stages of in vitro differentiation of neuronal precursors generated by embryonic day 14 (E14) mouse striata stem cells. Differentiation was in conditions of suboptimal growth factor supply. Apoptosis reached 10–15% of cells and affected proliferating as well as postmitotic cells, including TUJ1‐positive cells. Inhibition of apoptosis led to an increased proportion of TUJ1‐positive cells generated by stem cells. K+ current was reported to be related to apoptosis. Outward K+ currents were present in differentiating neuronal precursors that were consistent with delayed rectifier and transient A‐type currents. The amplitude of the delayed rectifier current varied during the first 4 days of stem cell differentiation. Current amplitude was greatly increased in the presence of staurosporine but reduced at elevated extracellular K+ concentration. In addition, the amplitude of the current was significantly diminished by inhibiting several caspases, but not caspase 8. In Bax knock‐out transgenic neuronal precurors, K+ current was not decreased after the first day but at later stages of cell differentiation. At this early stage, apoptosis of proliferating cells and of TUJ1‐positive cells was not reduced by the absence of Bax, but was by caspase 9 inhibition. Thus, activation of a delayed rectifier K+ current in differentiating stem cells is related to apoptosis. Recordings of this current revealed that apoptosis at early stages of neuronal differentiation occurred in two phases that did not exhibit similar dependence on the proapoptotic protein Bax and that probably used different pathways.

An invertebrate defense molecule activates membrane conductance in mammalian cells by means of its lectin-like domain

The invertebrate defense molecule Coelomic Cytolytic Factor-1 (CCF-1) and the mammalian cytokine Tumor Necrosis Factor (TNF) share a similar lectin-like domain that, upon interaction with specific sugars, causes lysis of African trypanosomes. In contrast to TNF, CCF-1 does not require an acidification of a lysosomal compartment for this activity. Moreover, we could demonstrate using the whole cell patch clamp technique that both TNF and CCF-1 activate amiloride-sensitive channels in mammalian cells, in a TNF receptor-independent way, but, unlike TNF, CCF-1 does not require acidic conditions for this activity. These data confirm the functional analogies of an invertebrate defense molecule and a mammalian cytokine, based on a similar lectin-like interaction.

An improved approach to freeze-fracture morphology of monolayer cell cultures

Much work is currently done on cell cultures to elucidate membrane processes associated with different cell functions. We describe here a modified freeze-fracture method to obtain systematically large fractured areas of the plasma membrane from monolayer cell culture in situ. Cells are grown until confluence on a Thermanox coverslip overlaid with poly-L-ornithine. After chemical fixation, the culture is flattened overnight by sandwiching it between the Thermanox coverslip, a Falcon membrane and a glass coverslip, under a 5 g weight. After freeze-fracture, vast pictures of the protoplasmic leaflets are obtained in a reproducible manner. Our approach was applied to cultures which were stimulated to release acetylcholine; it has been found very appropriate for studying modifications affecting intramembrane particles and vesicles openings in the plasmalemma. Accurate quantifications were performed and correlations were established between the membrane changes and the data revealed by thin sections. The present sandwich method can be applied to a variety of cell preparations, allowing for quantitative study of structure-function relationships.