Instituto de Bioquímica (Centro Mixto CSIC-UCM), Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain
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Abstract |
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Introduction |
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Materials and Methods |
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Fura 2AM and bis-[1,3-diethylthiobarbiturate]trimethine oxonol (bis-oxonol) were obtained from Molecular Probes (Eugene, OR). Eagle's minimum essential medium (EMEM) was supplied by Bio-Whittaker and foetal calf serum (FCS) and horse serum (HS) by Sera-Lab (Sussex, UK). Nicotine, hexamethonium and muscimol came from Sigma (St Louis, MA). The remaining chemicals were reactive grade products from Merck (Darmstadt, Germany).
Cell Isolation and Culture
Brain neurons were obtained from foetal rat brains at gestation day 19 following the procedure described by Segal (Segal, 1983) with minor modifications. Isolated neurons were suspended in EMEM containing 0.3 g/l glutamine, 0.6% glucose, 10% FCS, 10% HS, 100 U/ml penicillin, 100 µg/ml streptomycin, 40 µg/ml gentamycin and 5 µg/ml imipenem. Cells, at a density of 2 x 106 cells/well, were plated onto plastic Petri dishes treated with 10 µg/ml poly-L-lysine to aid attachment. The plates were incubated in a humidified incubator in an atmosphere of 5% CO2/95% air at 37°C. After 72 h the incubation medium was replaced with fresh medium to which 10 µM cytosine arabinoside was added to prevent overgrowth of contaminating glial cells. Cells were used after 1015 days culture. Cell viability was checked by the trypan blue exclusion method. Viability was routinely >95%. Cell purity was checked by both cell staining with cresyl violet to identify neurons and with a specific anti-GFAP antibody to identify glial cells.
Glial Contamination
After 1015 days culture the cortical neurons were detached from the culture plates with trypsin, as indicated below. Cells were the fixed (for 30 min) in 2% paraformaldehyde and washed in phosphate-buffered saline (PBS) followed by treatment (1 h) with anti-rabbit GFAP antibody (diluted 1/500). Cells were once again washed in PBS and treated with anti-rabbit FITC-conjugated IgG at a dilution of 1/100 for 30 min and identified by flow cytometry. Under these conditions the glial cells in the cultures were estimated at 9 ± 3% of the total cell population (neural + glial cells).
Cytosolic [Ca2+]
Changes in intracellular calcium concentration, [Ca2+]i, were monitored by Fura 2AM fluorescence. Six day cultured cells were detached from the plates using trypsin (0.25% trypsin and 0.02% EDTA in Dulbecco's phosphate-buffered saline without calcium or magnesium) and washed twice using 1 ml of a Krebs HEPES solution (Locke medium) containing 140 mM NaCl, 4.4 mM KCl, 2.5 mM CaCl2, 1.2 mM Mg(SO4)2, 1.2 mM KH2PO4, 4.0 mM NaHCO3, 5.5 mM glucose, 0.58 mM ascorbic acid and 10 mM HEPES, adjusted to pH 7.5 and incubated with 5 µM Fura 2AM for 45 min at 37°C. Excessive dye was removed by washing the cells twice with fresh Locke medium followed by suspension in this medium at 1 x 106 cells/ml. After Fura 2AM treatment, cell viability was checked as indicated previously. Fluorescence (excitation wavelength 340/380 nm, emission 510 nm) was monitored at 37°C in a well stirred cuvette containing 1 ml of this suspension using a Perkin Elmer LS-50 spectro-fluorimeter (slits 5 nm excitation, 10 nm emission). At the end of each experiment 1% Triton X-100 was added to make the cells permeable and permit the dye to gain access to the extracellular Ca2+ (2.5 mM). This Ca2+ concentration saturated the dye and provided a measure of the maximum fluorescence signal (Fmax). To determine the minimum fluorescence signal (Fmin), 20 mM Tris base was added to raise the pH above 8.2, followed by 5 mM EGTA which reduced the Ca2+ to <1 nM. [Ca2+]i was calculated with the Grynkiewicz equation (Grynkiewicz et al., 1985):
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where F is the ratio between fluorescence values at 340 and 380 nm, SF2 is the maximum fluorescence at 380 nm and SB2 is the minimum fluorescence at 380 nm. The equilibrium dissociation constant (Kd) for the complex [Ca2+]Fura 2AM was of 224 nM.
Single Cell [Ca2+]i
Cells cultured on glass coverslips were loaded for 45 min with Fura 2AM (5 µM) dissolved in Locke medium. After incubation at 37°C in the dark, cells were washed with Locke medium and the coverlips placed under a Nikon inverted stage microscope under continuous perfusion with Locke medium. Light from a xenon lamp was filtered through two different band-pass filters (340 or 380 nm) in the excitation path and the specimen illuminated on the microscope stage by a dichroic mirror. Excitation wavelengths of 340 and 380 nm were alternately applied to the cells. The fluorescence emitted by the cells was passed through a band-pass filter (510 nm) and video images were obtained using a cold intensified camera. Ratios corresponding to 340/380 nm are given as grey levels. Output from the camera was digitalized and stored in a computerized imaging system (MiraCal). The reagents dissolved in Locke medium were applied in the perfusion medium. When tetrodotoxin (TTX) was used the order of nicotine and TTX applications was as follow. First neurons were stimulated with 10 µM nicotine and images were taken over 80 s. Then, cells were perfused for 10 min to permit recovery of the nicotinic receptor. Subsequently, neurons were stimulated with 10 µM nicotine plus 50 nM TTX and images taken for 80 s as before.
Membrane potential
Changes in the membrane potential of neurons were monitored using the fluorescent dye bisoxonol. This is a lipophilic anion whose distribution across the membrane is dependent upon the membrane potential. Thus, an increase in bisoxonol fluorescence indicates that the membrane has been depolarized, allowing more of this negatively charged dye to enter the cells (Waggoner, 1979). Washed cells, as used for [Ca2+]i determination, were suspended in Locke medium at a density of 5 x 105 cells. Neurons in suspension were incubated with 0.2 µM bisoxonol for 1020 min and placed in a fluorimeter. Fluorescence was measured at an excitation wavelength of 540 nm and emission wavelength of 565 nm and monitored at 37°C in a well stirred cuvette using a Perkin Elmer spectrofluorimeter. Drugs were added at the indicated concentrations. Controls were performed using Locke medium in place of the drug. Fluorescence intensity is reported in arbitrary units.
Amino Acid Secretion
High performance liquid chromatography (HPLC) of amino acids was performed according to the methods described by Márquez et al. (Márquez et al., 1986). Cells were washed twice at 10 min intervals with 1 ml of Locke medium. After removal of the medium cells were stimulated for 15 min periods at 37°C with 0.5 ml of fresh Locke medium containing the different secretagogues. The stimulating medium was then withdrawn and cells ruptured by the addition of 0.5 ml of distilled water. The concentration of amino acids was determined by reversed phase HPLC using pre-column derivation with dansyl chloride and UV detection at 254 nm. Integration of peaks was achieved using a Sprectraphysis integrator. Peaks were quantified by comparison with those obtained using simultaneously prepared amino acid standards. Separation of dansyl derivatives was performed using a 5 µM Spherisorb-ODS-2 column (15 x 0.46 cm).
Proteins were identified according to Bradford (Bradford, 1976). Results were expressed as nmol neurotransmitter/mg protein/well or as the percentage of amino acids released into the incubation medium with respect to the total amino acid content (incubation medium + cells).
Data Presentation
Data are presented as the means of three or four separate experiments performed on different cell cultures. Each experiment was performed in duplicate using different batches of cells. Student's t-test was used to statistically compare data.
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Results |
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Discussion |
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The data presented not only demonstrate the presence of nicotinic receptors in cultured cortical neurons, but also indicate their specificity and functionality. These findings are in accordance with those obtained by Vidal and Changeus, who reported an increase in the negative wave of field potentials reflecting increased excitability of cortical neurons when neocortical slices were treated with acetylcholine or dimethylphenyl piperazinium (Vidal and Changeus, 1989).
In the present study nicotine was also able to induce the release of amino acid neurotransmitters in a calcium-free medium (Ca2+-independent release). This Ca2+-independent amino acid release may be attributable to the reverse action of amino acid transporters, given that the nicotinic receptor is a Na+-permeable channel and, when open, the resulting increased intracellular Na+ level is a condition required to activate amino acid transporters.
Our data seem to indicate that different Ca2+ channels might be involved in release of the different amino acid neurotrans-mitters evoked by nicotine, since blockers of L, N and P/Q type Ca2+ channels inhibited release of the inhibitory amino acid neurotransmitters (glycine and GABA). However, N type Ca2+ channel blockers did not affect aspartate release and P/Q type Ca2+ channel blockers did not affect release of glutamate. These data would appear to suggest that opening of one Ca2+ channel or another is important in secretion of the different amino acid neurotransmitters mediated by nicotine.
The fact that nicotine-stimulated cortical neurons release excitatory (aspartate and glutamate) and inhibitory (glycine and GABA) amino acids would seem to indicate that nicotinic receptors are able to modulate excitatory and inhibitory synapses in cortical neurons.
An observation that warrants particular attention was the high release of aspartate produced when cortical neurons were stimulated with high concentrations of nicotine. This might indicate that in this part of the brain the toxic effect attributed to overexcitability produced by nicotine may be due not only to glutamate release but also to aspartate release that, like glutamate, is able to bind to NMDA receptors. The high release of glycine mediated by nicotine is also of note. Glycine release was some four times higher than GABA release. As both these amino acids are inhibitory, it may be considered that in nicotine-stimulated cortical neurons glycine may mediate inhibition or modulate the response of the NMDA receptor. This matter requires further study.
It may be inferred from the study of Ca2+ entry mediated by nicotine in single neurons that most cortical neurons have nicotinic receptors, although the possibility that Ca2+ entry could be mediated by excitatory amino acids released by nicotine should not be discarded. The increment in intracellular Ca2+ mediated by nicotine could be due to Ca2+ entry through: (i) voltage-dependent Ca2+ channels, since nicotine induced membrane depolarization, and/or (ii) Ca2+ entry through the nicotinic receptors, since, according to some authors, neuronal AchRs show significant permeability to Ca2+ (Adams and Nulter, 1992; Vernino et al., 1992
, 1994
). This Ca2+ permeation is sufficient to activate Ca2+-dependent cellular processes. However, in the presence of TTX, which completely blocked the Ca2+ entry mediated by 10 µM nicotine, the intracellular Ca2+ increment evoked by nicotine appeared to be mediated by the opening of Ca2+ channels and not by the nicotinic receptor. In this case the nicotinic receptors in cortical neurons seems to be impermeable to Ca2+ ions. These results agree with those of Lena and Changeux, which showed that the depolarizing effect of nicotine in thalamic neurons appears to be mediated through entry of calcium through voltage-sensitive calcium channels (Lena and Changeux, 1997). This is not surprising given the two major functional properties of neuronal nAChRs established by Vernino et al. (Vernino et al., 1992
). These authors described nAChRs which show substantial permeability to Ca2+ and a further population of receptors which do not appear to permit Ca2+ movement. These differences may be attributed to the high degree of heterogeneity of neuronal nAChRs brought about by the combination of different subunits to give rise to many structural and functional variants of this neuronal receptor (Boulter et al., 1987
; Duvoisin et al., 1989
; Couturier et al., 1990
).
Herrero et al. recently demonstrated that the Ca2+ channel blockers used in the present study also block 7 and
3ß4 AchRs in chromaffin cells (Herrero et al., 1999
), giving rise to the possibility that the present effect could be due to blockade of the nAchR. However, our results indicate that the effect of the toxins is mediated by Ca2+ channel blockade given that (i) the toxins show different sensitivity in inhibiting release of the different amino acid neurotransmitters and (ii) TTX, a voltage dependent Na+ channel blocker, completely abolished the Ca2+ entry mediated by 10 µM nicotine.
It may be concluded that: (i) cortical neurons contain functional nicotinic receptors since when stimulated with nicotine these neurons release aspartate, glutamate, glycine and GABA; (ii) the mechanism by which nicotine induces amino acid release is exocytotic or dual, depending on the amino acid; (iii) the effect of nicotine is specific since it is blocked by hexamethonium; (iv) L, N and P/Q type Ca2+ channels are involved in the nicotine effect.
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Notes |
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Address correspondence to M.P. González, Instituto de Bioquímica (Centro Mixto CSIC-UCM), Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain. Email: pilarg{at}eucmax.sim.ucm.es
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