Inhibition of T-type calcium channels protects neurons from delayed ischemia-induced damage.

Intracellular calcium increase is an early key event triggering ischemic neuronal cell damage. The role of T-type voltage-gated calcium channels in the neuronal response to ischemia, however, has never been studied. Using an in vitro model of ischemia-induced delayed cell death in rat organotypic hippocampal slice cultures, we show that T-type calcium channels inhibitors drastically reduce ischemic cell damage. Immunostaining studies reveal the existence of Ca(V)3.1 and Ca(V)3.2 types of low-voltage-activated calcium channels in rat organotypic hippocampal cultures. Low extracellular calcium (100 nM) or increase of intracellular calcium buffering ability by BAPTA-acetoxymethyl ester significantly reduced ischemia-induced neuronal damage. Pharmacological inhibition of the T-type calcium current by mibefradil, kurtoxin, nickel, zinc, and pimozide during the oxygen-glucose deprivation episode provided a significant protection against delayed neuronal death. Mibefradil and nickel exerted neuroprotective effects, not only if administrated during the oxygen-glucose deprivation episode but also in conditions of postischemic treatment. These data point to a role of T-type calcium currents in ischemia-induced, calcium-mediated neuronal cell damage and suggest a possible new pharmacological approach to stroke treatment.

Quantal transmitter release by glioma cells: quantification of intramembrane particle changes.

Glial cells in situ are able to release neurotransmitters such as glutamate or acetylcholine (ACh). Glioma C6BU-1 cells were used to determine whether the mechanisms of ACh release by a glial cell line are similar or not to quantal release from neurones. Individual C6BU-1 cells, pre-filled with ACh, were moved into contact with a Xenopus myocyte that was used as a real-time ACh detector. Upon electrical stimulation, C6BU-1 cells generated evoked ACh impulses which were Ca(2+)-dependent and quantal (quantal steps of ca. 100 pA). Changes in plasma membrane ultrastructure were investigated by using a freeze-fracture technique designed for obtaining large and flat replicas from monolayer cell cultures. A transient increase in the density of medium and large size intramembrane particles--and a corresponding decrease of small particles--occurred in the plasma membrane of C6BU-1 cells stimulated for ACh release. Changes in interaction forces between adjacent medium and large particles were investigated by computing the radial distribution function and the interaction potential. In resting cells, the radial distribution function revealed a significant increase in the probability to find two particles separated by an interval of 24 nm; the interaction potential suggested repulsive forces for intervals shorter than 24 nm and attractive forces between 24 and 26 nm. In stimulated cells, this interaction was displaced to 21 nm and made weaker, despite of the fact that the overall particle density increased. The nature of this transient change in intramembrane particles is discussed, particularly with regard to the mediatophore proteolipid which is abundant in the membranes C6-BU-1 like in those of cholinergic neurones. In conclusion, evoked ACh release from pre-filled C6-BU-1 glioma cells is quantal and Ca(2+)-dependent. It is accompanied by a transient changes in the size distribution and the organisation of intramembrane particles in the plasma membrane. Thus, for the release characteristics, glioma cells do not differ fundamentally from neurones.

Calretinin and calbindin D-28k delay the onset of cell death after excitotoxic stimulation in transfected P19 cells.

In some neurological diseases, injury to neurones reflects an over-stimulation of their receptors for excitatory amino acids. This response may disturb the Ca(2+)-homeostasis and lead to a pronounced and sustained increase in the intracellular concentration of this ion. On the basis of data derived from correlative studies, calcium-binding proteins have been postulated to play a protective role in these pathologies. We tested, directly, the capacity of the three calcium-binding proteins calretinin (CR), calbindin D-28k (CB) and parvalbumin (PV) to buffer [Ca(2+)], and to protect cells against excitotoxic death. We used P19 murine embryonic carcinoma cells, which can be specifically induced (by retinoic acid) to transform into nerve-like ones. The differentiated cells express functional glutamate-receptors and are susceptible to excitotoxic shock. Undifferentiated P19-cells were stably transfected with the cDNA for CR, CB or PV, induced to differentiate, and then exposed to NMDA, a glutamate-receptor agonist. The survival rates of clones expressing CR, CB or PV were compared with those of untransfected P19-cells using the lactate-dehydrogenase assay. CR- and CB-expressing cells were protected from death during the first 2 h of exposure to NMDA. This protection was, however, transient, and did not suffice to rescue P19-cells after prolonged stimulation. Two of the three PV-transfected clones raised were vulnerable to NMDA-induced excitotoxicity; the third, which expressed the lowest level of PV, was protected to a similar degree as that found for the CR- and CB-transfected clones. Our results indicate that in the P19-cell model, CR and CB can help to delay the onset of cell death after excitotoxic stimulation.

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

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. We found that extracellular application of 10-20 microM Zn2+ induced a reversible abolition of Ca2+ action potential firing, which was accompanied by an hyperpolarisation of the resting membrane potential. 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. 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 microM Zn2+. Single channel analysis revealed that extracellular Zn2+ increased the KATP channel open-state probability with no change in the single channel conductance. 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 beta-cells through a negative feedback loop, involving both KATP and voltage-gated conductances.

Reconstitution of mediatophore-supported quantal acetylcholine release.

Synaptic transmission of a nerve impulse is an extremely rapid event relying on transfer of brief chemical impulses from one cell to another. This transmission is dependent upon Ca2+ and known to be quantal, which led to the widely accepted vesicular hypothesis of neurotransmitter release. However, at least in the case of rapid synaptic transmission the hypothesis has been found difficult to reconcile with a number of observations. In this article, we shall review data from experiments dealing with reconstitution of quantal and Ca2+-dependent acetylcholine release in: i) proteoliposomes, ii) Xenopus oocytes, and iii) release-deficient cell lines. In these three experimental models, release is dependent on the expression of the mediatophore, a protein isolated from the plasma membrane of cholinergic nerve terminals of the Torpedo electric organ. We shall discuss the role of mediatophore in quantal acetylcholine release, its possible involvement in morphological changes affecting presynaptic membrane during the release, and its interactions with others proteins of the cholinergic nerve terminal.

Morphological changes related to reconstituted acetylcholine release in a release-deficient cell line.

The membrane changes accompanying Ca(2+)-dependent acetylcholine release were investigated by comparing release-competent and release-incompetent clones of mouse neuroblastoma N18TG-2 cells. No release could be elicited in native N18 cells or in a N18-choline acetyltransferase clone in which acetylcholine synthesis was induced by transfection with the gene for rat choline acetyltransferase. However, acetylcholine release was operative in a To/9 clone which was co-transfected with complementary DNAs from rat choline acetyltransferase and Torpedo mediatophore 16,000 mol. wt subunit. In thin sections, the aspect of resting N18 and To/9 cells was identical: a very dense cytoplasm with practically no vesicle-like organelles. Cells were chemically fixed at different times during a stimulation using A-23187 and Ca2+, and examined following both freeze-fracture and thin section. Stimulation of To/9 cells induced a marked change affecting the intramembrane particles. The number of medium-sized particles (9.9-12.38 nm) increased, while that of the small particles decreased. This change was not observed in control, release-incompetent cell lines. In the To/9 clone (but not in control clones), this was followed by occurrence of a large new population of pits which initially had a large diameter, but subsequently became smaller as their number decreased. Coated depressions and invaginations became abundant after stimulation, suggesting an endocytosis process. By considering the succession of events and by comparison with data from experiments performed on synapses in situ, it is proposed that a particle alteration was the counterpart of acetylcholine release in co-transfected To/9 cells; this was followed by a massive endocytosis.

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.

The lectin-like domain of tumor necrosis factor-alpha 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 endogenous ion channel rather than channel formation by TNF itself. This may have important implications in mechanisms of TNF-mediated vascular pathology.

Evoked acetylcholine release by immortalized brain endothelial cells genetically modified to express choline acetyltransferase and/or the vesicular acetylcholine transporter.

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.

Acetylcholine synthesis and quantal release reconstituted by transfection of mediatophore and choline acetyltranferase cDNAs.

Neuroblastoma N18TG-2 cells cannot synthesize or release acetylcholine (ACh), and do not express proteins involved in transmitter storage and vesicle fusion. We restored some of these functions by transfecting N18TG-2 cells with cDNAs of either rat choline acetyltransferase (ChAT), or Torpedo mediatophore 16-kDa subunit, or both. Cells transfected only with ChAT synthesized but did not release ACh. Cells transfected only with mediatophore expressed Ca2+-dependent ACh release provided they were previously filled with the transmitter. Cell lines produced after cotransfection of ChAT and mediatophore cDNAs released the ACh that was endogenously synthesized. Synaptic-like vesicles were found neither in native N18TG-2 cells nor in ChAT-mediatophore cotransfected clones, where all the ACh content was apparently cytosolic. Furthermore, restoration of release did not result from enhanced ACh accumulation in intracellular organelles consecutive to enhanced acidification by V-ATPase, as Torpedo 16 kDa transfection did not increase, but decreased the V-ATPase-driven proton transport. Using ACh-sensitive Xenopus myocytes for real-time recording of evoked release, we found that cotransfected cells released ACh in a quantal manner. We compared the quanta produced by ChAT-mediatophore cotransfected clones to those produced by clones transfected with mediatophore alone (artificially filled with ACh). The time characteristics and quantal size of currents generated in the myocyte were the same in both conditions. However, cotransfected cells released a larger proportion of their initial ACh store. Hence, expression of mediatophore at the plasma membrane seems to be necessary for quantal ACh release; the process works more efficiently when ChAT is operating as well, suggesting a functional coupling between ACh synthesis and release.

A simple, low-cost and fast Peltier thermoregulation set-up for electrophysiology.

Most of the parameters recorded in electrophysiology are strongly temperature dependent. In order to control temperature fluctuations we have built a system that ensures an accurate thermoregulation of the recording chamber. Temperature of physiological preparations can be changed relatively quickly (about 8 degrees C/min) and with a good accuracy (+/- 0.5 degrees C) without inducing thermal oscillations. Contrary to other thermoregulating devices, the temperature regulation is not carried out through the perfused medium but directly at the bottom of the chamber where a 3-cm2 Peltier element has been placed. The element is driven by a dedicated electronic device which controls the amount and the direction of the current flowing across the Peltier thermocouple. All construction details and the appropriate electrical circuits are provided. Using this home-made device, the steady-state chamber temperature could be precisely monitored with a resolution of +/- 0.1 degrees C in a range of 0-40 degrees C. This set-up was tested in experiments designed to evaluate the temperature dependence of synaptic transmission in the Torpedo nerve electroplate synapses and of calcium currents recorded from isolated nerve cells. This low-cost method is suitable for a wide range of applications.

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.

Quantal acetylcholine release induced by mediatophore transfection.

Mediatophore is a protein of approximately 200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.

Effects of ionotropic excitatory amino acid receptor antagonists on glutamate transport and transport-mediated changes in extracellular excitatory amino acids in the rat striatum.

This study examined the effects of intrastriatal administration of ionotropic excitatory amino acid receptor antagonists on biochemical markers of excitatory amino acid transmission in the rat striatum. High-affinity glutamate uptake was measured ex vivo on striatal homogenates 15 min after the local administration of either 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, or DL-2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA antagonist, at various doses (10-500 pmol injected). DNQX induced a dose-dependent increase in glutamate uptake rate, related to an increase in the Vmax of the transport process, whereas no significant change in glutamate uptake was detected after AP5 administration. Similar results were obtained from animals subjected to excitotoxic lesion of striatal neurons by kainate administration 15 days before the injection of DNQX or AP5. In a parallel series of experiments using in vivo microdialysis we showed that DNQX (10(-5) M) in the dialysis probe diminished by approximately 30-40% the increases in the concentrations of glutamate and aspartate elicited by L-trans-pyrrolidine-2,4-dicarboxylic acid (1 mM). These data suggest that presynaptic glutamate transmission in the rat striatum may undergo facilitatory autoregulatory processes involving ionotropic non-NMDA receptors and highlight the view that transporters for glutamate may be potent regulatory sites for glutamatergic transmission.

Local injections of excitatory amino acid agonists alter the glutamatergic and dopaminergic transmissions in the rat striatum.

This study examined the effects of kainic, ibotenic, and quisqualic acid-induced lesions of the rat striatum on biochemical markers of the glutamatergic corticostriatal and dopaminergic nigrostriatal afferent transmissions. Fifteen to 21 days after striatal injections of these various compounds, significant reductions in the high-affinity glutamate uptake rate, due to decreases in the Vmax of the transport process, were measured. Interestingly, the relationship between these decreases in the Vmax and the decreases in the levels of biochemical markers for the intrinsic striatal cholinergic and GABAergic neurons differed depending on the excitotoxin used. These findings suggest that excitatory amino acid agonists-induced alterations of the glutamatergic terminal activity may not depend only on the loss of cholinergic and GABAergic striatal neurons. In contrast, the observed changes in the dopamine and metabolite contents seemed to be related to the extent of the striatal neuronal degeneration induced by each excitotoxin. All in all, these results indicate that excitatory amino acid agonists can impair the activity and/or the integrity of the two main striatal afferent pathways, through presumably different mechanisms.

Intracerebroventricular administration of neuropeptide Y affects parameters of dopamine, glutamate and GABA activities in the rat striatum.

The effects of intracerebroventricular (ICV) injection of neuropeptide Y (NPY) on parameters of dopamine (DA), glutamate (Glu) and gamma-aminobutyric acid (GABA) activities were investigated in the rat striatum. NPY (1.17-4.70 nmol) induced a dose-dependent increase in the striatal endogenous DA release monitored in freely moving animals by means of a voltammetric method. Maximal increase was observed about one hour after the peptide injection. This result is consistent with the hypothesis that NPY may influence striatal DA turnover in a facilitatory manner by activating DA release. DA, DOPAC, Glu and GABA endogenous contents as well as 3H-Glu and 3H-GABA synaptosomal high affinity uptakes were examined one hour after NPY ICV administration at the same dose range in chloral hydrate-anesthetized animals. Depending on the NPY dose injected, opposite changes in Glu uptake were observed, suggesting that NPY has a bimodal influence on glutamatergic transmission. The Glu uptake rate increased markedly at 1.17 nmol NPY and decreased at 4.70 nmol, which may reflect an activation and an inhibition of the striatal Glu transmission, respectively. In parallel, the GABA uptake was found to decrease slightly at the higher doses of NPY tested, whereas no significant alteration of the striatal concentrations of either DA, DOPAC, Glu or GABA was observed. These results indicate that NPY may be involved in regulating the activity of nigral dopaminergic and cortical glutamatergic afferent pathways and that of intrinsic GABA neurons in the rat striatum.