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Q-type calcium channels and N-methyl-d -aspartate receptors in rat cortical neurons
Brain Research 1063 (2005) 9 – 14 www.elsevier.com/locate/brainres
Research Report
Nitric oxide modulates calcium entry through P/Q-type calcium channels and N-methyl-d-aspartate receptors in rat cortical neurons Gabor C. Petzold a,b,*,1, Franziska Scheibe a, Johann S. Braun a,b, Dorette Freyer a, Josef Priller a,c, Ulrich Dirnagl a,b, Jens P. Dreier a,b a
Department of Experimental Neurology, Charite´-University Medicine Berlin, Schumannstr. 20-21, 10117 Berlin, Germany b Department of Neurology, Charite´-University Medicine Berlin, Schumannstr. 20-21, 10117 Berlin, Germany c Department of Psychiatry, Charite´-University Medicine Berlin, Schumannstr. 20-21, 10117 Berlin, Germany Accepted 24 September 2005 Available online 7 November 2005
Abstract Voltage-gated calcium channels (VGCC) and N-methyl-d-aspartate receptors (NMDAR) account for most of the depolarization-induced neuronal calcium entry. The susceptibility of individual routes of calcium entry for nitric oxide (NO) is largely unknown. We loaded cultured rat cortical neurons with fluo-4 acetoxymethylester to study the effect of the NO synthase inhibitor NN-nitro-l-arginine and the NO donor Snitroso-N-acetylpenicillamine on the intracellular calcium concentration ([Ca2+]i). The potassium-induced [Ca2+]i increase was amplified by NN-nitro-l-arginine and attenuated by S-nitroso-N-acetylpenicillamine. This modulation was abolished by either the P/Q-type VGCC antagonist N-agatoxin IVA or by the NMDAR antagonist MK-801, but not by N-type (N-conotoxin GVIA) or L-type (nimodipine) VGCC blockers. These results suggest that NO can modulate neuronal calcium entry during depolarization by interacting with P/Q-type VGCC and NMDAR. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Other neurotransmitters Keywords: Nitric oxide; Voltage-gated calcium channel; Neocortex; N-methyl-d-aspartate receptor; Excitotoxicity; Cell culture
1. Introduction Intracellular calcium is critically involved in almost all aspects of the neuronal life cycle, including development, differentiation, migration, communication, plasticity, and death. Moreover, changes of intracellular calcium pathways have been implicated in a plethora of neurological diseases, such as migraine, epilepsy, ischemia, cerebral hemorrhage, and Alzheimer’s disease [21]. Nitric oxide (NO) shows a
* Corresponding author. Department of Neurology, Charite´-University Medicine Berlin, Schumannstr. 20-21, 10117 Berlin, Germany. Fax: +49 30 450 560932. E-mail address: [email protected] (G.C. Petzold). 1 Current address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.09.048
similarly ubiquitous role in health and disease of the central nervous system, and, perhaps not surprisingly, it has been shown that NO is fundamentally involved in many aspects of calcium signaling in the neuroglial network [12,20,30]. However, the effect of the tissue NO level on the intracellular calcium level following neuronal activation and the individual calcium channels that govern this effect are largely unknown. In this study, we focused on the susceptibility for NO of different routes of calcium entry into cultured rat cortical neurons following depolarization. Using the intracellular calcium indicator fluo-4 acetoxymethylester (fluo-4 AM), we applied inhibitors of voltagegated calcium channels (VGCC) and N-methyl-d-aspartate (NMDA) receptors since most of the intracellular calcium surge following neuronal depolarization is mediated by these channels [25,28]. Our data indicate that depolariza-
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tion-induced neuronal calcium entry is modulated by NO interacting with P/Q-type VGCC and NMDA receptors, but not with N-type or L-type VGCC.
2. Materials and methods All experiments were performed according to institutional guidelines and were approved by the local ethics committee. 2.1. Cell culture and calcium measurements All cell culture media and supplements were purchased from Biochrom (Berlin, Germany). Primary rat cortical neurons were isolated from fetal rats at embryonic day 16– 18 as described previously [1]. Neuronal cultures were kept for 11 days in vitro at 36.5 -C and 5% CO2. To evaluate changes in the relative intracellular calcium concentration ([Ca2+]i), cells were loaded with fluo-4 AM (10 AM; Molecular Probes, Leiden, Netherlands) for 60 min and then rinsed three times with PBS. Prior to recording, the culture medium was exchanged for a modified HBSS consisting of (in mM): NaCl 137, KCl 5, NaHCO3 3, Na2HPO4 0.6, KH2PO4 0.4, CaCl2 1.4, MgSO4 0.8, HEPES 20, glucose 5.6, glycine 0.005 (pH 7.4). Fluorimetric recordings were performed using the CytoFluor multi-well plate reader (Applied Biosystems, Darmstadt, Germany). Baseline fluorescence was recorded at the beginning of the experiments, and subsequent [Ca2+]i changes were determined in relation to baseline ( F/F 0). Each group consisted of n = 36 wells from n = 6 plates. Following determination of basal [Ca2+]i levels, the cells were depolarized for 3 min by switching to HBSS containing increased extracellular potassium concentration ([K+]o) (35 mM; [Na+]o was lowered reciprocally to maintain isoosmolarity). [Ca2+]i changes were determined for at least 10 min at intervals of 30 s following depolarization. Statistical comparisons were performed by comparing the peak and basal fluo-4 fluorescence values ( F/F 0) using ANOVA followed by Bonferroni t test. P < 0.05 was accepted as statistically significant. NN-nitro-l-arginine (l-NNA; Sigma, Deisenhofen, Germany), S-nitroso-N-acetylpenicillamine (SNAP; Sigma), dizocilpine (MK-801; Sigma), N-conotoxin GVIA (Bachem, Heidelberg, Germany), NMDA (Sigma), and N-agatoxin IVA (Peptide Institute, Osaka, Japan) were solubilized in HBSS. Nimodipine (Sigma) was solubilized in DMSO (final concentration <0.1%) and then added to HBSS. The concentrations of all reagents were adopted from those reported in the literature [3 –6,9,13 –19,23,24,27,28,32]. The cell cultures were preincubated with these reagents for 30 min before depolarization. 2.2. Immunocytochemistry Cell cultures were fixed (4% PFA, 15 min at RT) and incubated overnight at 4 -C with primary antibodies against
MAP-2 (1:500; Sigma), TUJ1 (1:500; Covance, Berkeley, CA), glial fibrillary acidic protein (GFAP, 1:500; Dako, Hamburg, Germany), CNPase (1:100; Sternberger, Lutherville, MD), and ED1 (1:1000; Serotec, Du¨ sseldorf, Germany). Secondary antibodies conjugated with Alexa Fluor 488 or Texas Red (Molecular Probes) were added at a dilution of 1:250 for 1 h at RT; omission of primary antibodies served as negative control. The sections were coverslipped with Immuno-fluore (MP Biomedicals, Eschwege, Germany) and examined under a conventional fluorescence microscope (DMRA; Leica, Bensheim, Germany) equipped with standard fluorescein and Texas Red filter sets. Cells from four independent preparations were counted at 20 magnification (10 visual fields/preparation). Data are given as mean T SD.
3. Results 3.1. Characterization of cortical cell cultures We used antibodies that specifically recognize neuronal (MAP-2, TUJ1), oligodendroglial (CNPase), astroglial (GFAP), and microglial (ED1) antigens in order to determine the purity of our cultures. Immunocytochemical analysis revealed that neurons by far exceeded any other cell population in our preparations (neurons: 77.2 T 5.8%; astrocytes: 18.5 T 2.1%; oligodendrocytes 2.3 T 8.1%; microglia: 0.2 T 1.0%). Fig. 1 shows a representative example. Thus, only astrocytes could have added measurably to changes in fluo-4 AM fluorescence besides cortical neurons. However, the astrocytic expression of VGCC and NMDA receptors is, if at all, very low. Thus, it is assumed that the [Ca2+]i changes were predominantly related to neurons.
Fig. 1. Neurons are the predominant cell type in rat primary cortical cultures. Immunocytochemical analysis was performed using antibodies that specifically recognize neuronal (MAP-2, arrows) and astroglial (GFAP, arrowheads) antigens. Secondary antibodies conjugated with Alexa Fluor 488 (green) and Texas Red (red) were used for visualization. Note that the number of neurons exceeded the number of astrocytes.
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3.2. The NO level modulates the [Ca2+]i increase following depolarization In order to identify the potassium concentration that elicited a robust fluorescence increase, we performed dose –response experiments using different [K+]o (Fig. 2). We chose to perform all subsequent experiments at 35 mM as a strong and reproducible F/F 0 increase was observed when cells were depolarized at this concentration. No significant fluorescence changes were observed when the experiments were performed in calcium-free solution (Fig. 2), indicating that calcium entry in these cultured cortical neurons appeared to be mediated by the influx of extracelllular calcium rather than by the release from intracellular stores. We used this potassium concentration to investigate the effect of the NO level on the calcium changes. K+-induced depolarization resulted in a significant [Ca2+]i-induced F/F 0 increase to 1.67 T 0.11 (Fig. 3A). To estimate the effect of the NO level, [Ca2+]i was measured in cultures preincubated with either the NO synthase inhibitor l-NNA (1 mM) or with the NO donor SNAP (100 AM). No significant changes in [Ca2+]i-induced fluorescence were observed during the incubation period preceding depolarization. Following depolarization, l-NNA significantly amplified the depolarization-induced [Ca2+]i increase (2.18 T 0.17; Fig. 3A) compared to increased [K+]o alone, indicating enhanced depolarization-induced calcium influx at low basal NO level. In contrast, calcium influx was significantly attenuated compared to high [K+]o alone in cultures preincubated with SNAP (1.27 T 0.15; Fig. 3A), suggesting that NO decreased neuronal calcium influx during depolarization.
Fig. 2. [Ca2+]i changes following K+-induced depolarization are mediated by influx of extracellular calcium. Ascending concentrations of potassium ([K+]o) were applied to neuronal cell cultures (closed circles), inducing a concentration-dependent fluorescence increase compared to [K+]o at 3 mM ( F/F 0). In contrast, no significant fluorescence increase was detected when cells were depolarized in calcium-free medium, indicating that release from intracellular calcium stores did not significantly contribute to the fluorescence changes. *P < 0.05.
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These results suggest that the intracellular calcium surge during neuronal depolarization is sensitive to changes of the extracellular NO level. 3.3. The depolarization-induced [Ca2+]i increase is mediated by VGCC and NMDAR In order to identify the membrane channels involved in the depolarization-induced calcium influx, we incubated the cultures with different VGCC inhibitors and an NMDA receptor antagonist. Compared to raised [K+]o alone, the Ntype VGCC (Cav2.2(a´1B)) blocker N-conotoxin GVIA (1 AM) significantly attenuated the [Ca2+]i-related fluorescence increase induced by raised [K+]o (1.41 T 0.15). Similarly, the depolarization-induced [Ca2+]i increases were significantly reduced by the l-type VGCC (Cav1.3(a´1D)) blocker nimodipine (2 AM; 1.57 T 0.18), the P/Q-type VGCC (Cav2.1(a´1A)) blocker N-agatoxin IVA (300 nM; 1.35 T 0.12), and by the NMDA receptor antagonist MK-801 (10 AM, 1.39 T 0.1). No significant changes in [Ca2+]i-induced fluorescence were observed in cultures incubated with the VGCC inhibitors or MK-801 at baseline [K+]o. These results suggest that calcium entry appeared to be mediated by all four VGCC types and the NMDA receptor in these cultured cortical neurons. 3.4. The modulatory potential of NO is mainly attributable to NMDA receptors and P/Q-type VGCC Next, we studied whether the modulation of the calcium signal by NO was attributable to these individual channel types. Co-application of increased [K+]o with N-conotoxin GVIA (1 AM) and either SNAP or l-NNA did not affect the ability of NO to modulate the calcium signals (Fig. 3B), indicating that the effect of NO on [Ca2+]i did not depend on N-type VGCC activity modulation. Similarly, application of the L-type VGCC inhibitor nimodipine (2 AM) resulted in a reduction of [Ca2+]i, but the modulation of the calcium signal by NO was still present (Fig. 3C). The combination of nimodipine and N-conotoxin GVIA decreased the calcium signal, but the modulation by different NO levels was still detectable. In contrast, the ability of l-NNA to amplify the depolarization-induced [Ca2+]i changes was abolished when the neuronal cultures were preincubated with MK-801 (10 AM), whereas the ability of SNAP to significantly reduce the [Ca2+]i signal was still apparent (Fig. 3D). This suggested that the modulation of [Ca2+]i changes by NO was partially attributable to the NMDA receptor. Similar results were obtained when neurons were preincubated with N-agatoxin IVA (300 nM; Fig. 3E). Furthermore, preincubation with both MK-801 and N-agatoxin IVA abolished the ability of SNAP and l-NNA, respectively, to modulate the [Ca2+]i changes induced by depolarization (Fig. 3F). To further confirm the involvement of NMDA receptors, we investigated the [Ca2+]i response to increasing concentra-
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Fig. 3. NO modulates calcium influx through VGCC and the NMDA receptor in rat primary cortical neurons. Following preincubation with the indicated inhibitors, cells were depolarized by either high [K+]o alone (closed circles), high [K+]o and the NO synthase inhibitor l-NNA (open circles) or high [K+]o and the NO donor SNAP (triangles). [Ca2+]i changes are presented as relative fluorescence values compared to baseline ( F/F 0) over time. Asterisks denote statistically significant differences at the corresponding time points ( P < 0.05). (A) Depolarization resulted in a [Ca2+]i increase that was further amplified by lNNA and attenuated by SNAP. (B – C) The N-type VGCC blocker N-conotoxin GVIA (Cono) and the L-type VGCC blocker nimodipine (Nimo) attenuated the [Ca2+]i increase but had no effect on the modulation of [Ca2+]i by l-NNA and SNAP. (D – F) The NMDA receptor inhibitor MK-801 and the P/Q-type VGCC inhibitor N-agatoxin IVA (Aga) significantly attenuated the potential of l-NNA to modulate the [Ca2+]i changes. (G) Co-application of MK-801 and N-agatoxin IVA abolished the [Ca2+]i changes induced by l-NNA or SNAP.
tions of NMDA (Fig. 4). Compared to NMDA alone, the fluorescence increase was significantly attenuated by SNAP, whereas no significant changes were observed in cultures preincubated with l-NNA. These results indicate that both the NMDA receptor and the P/Q-type VGCC are both involved in the modulation of intracellular calcium changes following depolarization and that this modulation can be abrogated by the combined blockade of both channels.
4. Discussion The second messengers NO and calcium are involved in almost all aspects of neuronal function. NO plays a fundamental role in the intricate network that fine-tunes calcium homeostasis, and vice versa. Thus, it has been shown that NO strongly modulates calcium influx through membrane channels [7,12]. In turn, calcium influx following neuronal activation—either presynaptically through VGCC
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Fig. 4. The NMDA-evoked [Ca2+]i increase is modulated by the NO level. Ascending concentrations of NMDA were applied to cortical neuronal cultures. Compared to NMDA alone (closed circles), the fluorescence increase was significantly attenuated by the NO donor SNAP (triangles) at 200 AM and 400 AM NMDA, whereas no significant changes were observed in cultures preincubated with the NO synthase inhibitor l-NNA (open circles). *P < 0.05.
or postsynaptically through NMDA receptors—directly activates NO synthesis [12]. In this study, we have investigated whether these two major calcium gateways in neurons are also sensitive to the extracellular NO level. We found that the relative amount of neuronal calcium influx depends on the NO level of the environment and that NMDA receptors and P/Q-type VGCC, but not N-type and L-type VGCC, are critically involved in this effect. Our data implicate that NMDA receptors and P/Q-type channels do not only activate neuronal NO production but that NO reciprocally also affects the activity of these channels. Thus, a mutual modulation of these channels may help to regulate and fine-tune both neuronal calcium and NO homeostasis by this feedback loop. The exact mechanisms by which NO modulates the activities of different Ca2+ channels remain elusive. However, the ability of NO to alter ion channel activity is well known. It has been shown that NO down-regulates NMDA receptor activity [6,16,18,19], whereas contradictory results have been obtained regarding the influence of NO on VGCC activity. Although most studies have demonstrated that NO inhibits VGCC [3,9,13,17,23,27,32], some reports have suggested that NO may also activate these channels [4,5,14]. These discrepancies could partially be explained by the different types and concentrations of NO donors used, the cell types studied, and different experimental procedures (e.g., cell culture vs. patch clamp recordings). Confirmatory studies using confocal microscopy or single cell studies will be needed to further address these issues. Moreover, direct effects (e.g., by S-nitrosylation [6]) and indirect effects of NO (e.g., by guanylyl cyclase activation) can differentially affect channel activity. Thus, it has been shown that NO directly activates P/Q-type VGCC [5],
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whereas the NO-induced production of cyclic guanosine monophosphate inhibits these channels [13]. Furthermore, it has been shown that NO induces a sustained increase of neuronal [Ca2+]i when NO donor concentrations several fold higher than those applied in the present study are used [2], indicating that the [Ca2+]i changes can be reverted under pathophysiological conditions. The modulatory effect of NO on NMDA receptors and P/ Q-type VGCC might also have important implications for the actions of glutamate in health and disease. P/Q-type VGCC mainly reside on neuronal presynaptic terminals [29] and are a major gateway for the synaptic release of glutamate [31]. Postsynaptically, NMDA receptors are a main target of glutamatergic action, e.g., in synaptic transmission, plasticity, or excitotoxicity [8]. Hence, our results suggest that the basal NO level under physiological conditions may indirectly modulate the actions of glutamate by reducing its presynaptic release and postsynaptic action. Under pathological conditions, rise of NO, as during epileptic seizure-like events, could contribute to terminate hypersynchronous neuronal activity [26], but a decline in the basal NO level could augment glutamate excitotoxicity. This may be particularly relevant in cerebral hemorrhage, in which the NO concentration may be reduced by decreased NO production due to shortage of molecular oxygen [10], by endogenous NO synthase inhibitors [22], by dysfunction of NO producing sources such as neurons or endothelial cells [22], or by extracellular hemoglobin, a potent NO scavenger [11].
Acknowledgments This study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 507 to J.P., U.D., and J.P.D.; PE 1193/1-1 to G.C.P.) and by the Hermann and Lilly Schilling Foundation (U.D.).
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Research Report
Nitric oxide modulates calcium entry through P/Q-type calcium channels and N-methyl-d-aspartate receptors in rat cortical neurons Gabor C. Petzold a,b,*,1, Franziska Scheibe a, Johann S. Braun a,b, Dorette Freyer a, Josef Priller a,c, Ulrich Dirnagl a,b, Jens P. Dreier a,b a
Department of Experimental Neurology, Charite´-University Medicine Berlin, Schumannstr. 20-21, 10117 Berlin, Germany b Department of Neurology, Charite´-University Medicine Berlin, Schumannstr. 20-21, 10117 Berlin, Germany c Department of Psychiatry, Charite´-University Medicine Berlin, Schumannstr. 20-21, 10117 Berlin, Germany Accepted 24 September 2005 Available online 7 November 2005
Abstract Voltage-gated calcium channels (VGCC) and N-methyl-d-aspartate receptors (NMDAR) account for most of the depolarization-induced neuronal calcium entry. The susceptibility of individual routes of calcium entry for nitric oxide (NO) is largely unknown. We loaded cultured rat cortical neurons with fluo-4 acetoxymethylester to study the effect of the NO synthase inhibitor NN-nitro-l-arginine and the NO donor Snitroso-N-acetylpenicillamine on the intracellular calcium concentration ([Ca2+]i). The potassium-induced [Ca2+]i increase was amplified by NN-nitro-l-arginine and attenuated by S-nitroso-N-acetylpenicillamine. This modulation was abolished by either the P/Q-type VGCC antagonist N-agatoxin IVA or by the NMDAR antagonist MK-801, but not by N-type (N-conotoxin GVIA) or L-type (nimodipine) VGCC blockers. These results suggest that NO can modulate neuronal calcium entry during depolarization by interacting with P/Q-type VGCC and NMDAR. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Other neurotransmitters Keywords: Nitric oxide; Voltage-gated calcium channel; Neocortex; N-methyl-d-aspartate receptor; Excitotoxicity; Cell culture
1. Introduction Intracellular calcium is critically involved in almost all aspects of the neuronal life cycle, including development, differentiation, migration, communication, plasticity, and death. Moreover, changes of intracellular calcium pathways have been implicated in a plethora of neurological diseases, such as migraine, epilepsy, ischemia, cerebral hemorrhage, and Alzheimer’s disease [21]. Nitric oxide (NO) shows a
* Corresponding author. Department of Neurology, Charite´-University Medicine Berlin, Schumannstr. 20-21, 10117 Berlin, Germany. Fax: +49 30 450 560932. E-mail address: [email protected] (G.C. Petzold). 1 Current address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.09.048
similarly ubiquitous role in health and disease of the central nervous system, and, perhaps not surprisingly, it has been shown that NO is fundamentally involved in many aspects of calcium signaling in the neuroglial network [12,20,30]. However, the effect of the tissue NO level on the intracellular calcium level following neuronal activation and the individual calcium channels that govern this effect are largely unknown. In this study, we focused on the susceptibility for NO of different routes of calcium entry into cultured rat cortical neurons following depolarization. Using the intracellular calcium indicator fluo-4 acetoxymethylester (fluo-4 AM), we applied inhibitors of voltagegated calcium channels (VGCC) and N-methyl-d-aspartate (NMDA) receptors since most of the intracellular calcium surge following neuronal depolarization is mediated by these channels [25,28]. Our data indicate that depolariza-
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tion-induced neuronal calcium entry is modulated by NO interacting with P/Q-type VGCC and NMDA receptors, but not with N-type or L-type VGCC.
2. Materials and methods All experiments were performed according to institutional guidelines and were approved by the local ethics committee. 2.1. Cell culture and calcium measurements All cell culture media and supplements were purchased from Biochrom (Berlin, Germany). Primary rat cortical neurons were isolated from fetal rats at embryonic day 16– 18 as described previously [1]. Neuronal cultures were kept for 11 days in vitro at 36.5 -C and 5% CO2. To evaluate changes in the relative intracellular calcium concentration ([Ca2+]i), cells were loaded with fluo-4 AM (10 AM; Molecular Probes, Leiden, Netherlands) for 60 min and then rinsed three times with PBS. Prior to recording, the culture medium was exchanged for a modified HBSS consisting of (in mM): NaCl 137, KCl 5, NaHCO3 3, Na2HPO4 0.6, KH2PO4 0.4, CaCl2 1.4, MgSO4 0.8, HEPES 20, glucose 5.6, glycine 0.005 (pH 7.4). Fluorimetric recordings were performed using the CytoFluor multi-well plate reader (Applied Biosystems, Darmstadt, Germany). Baseline fluorescence was recorded at the beginning of the experiments, and subsequent [Ca2+]i changes were determined in relation to baseline ( F/F 0). Each group consisted of n = 36 wells from n = 6 plates. Following determination of basal [Ca2+]i levels, the cells were depolarized for 3 min by switching to HBSS containing increased extracellular potassium concentration ([K+]o) (35 mM; [Na+]o was lowered reciprocally to maintain isoosmolarity). [Ca2+]i changes were determined for at least 10 min at intervals of 30 s following depolarization. Statistical comparisons were performed by comparing the peak and basal fluo-4 fluorescence values ( F/F 0) using ANOVA followed by Bonferroni t test. P < 0.05 was accepted as statistically significant. NN-nitro-l-arginine (l-NNA; Sigma, Deisenhofen, Germany), S-nitroso-N-acetylpenicillamine (SNAP; Sigma), dizocilpine (MK-801; Sigma), N-conotoxin GVIA (Bachem, Heidelberg, Germany), NMDA (Sigma), and N-agatoxin IVA (Peptide Institute, Osaka, Japan) were solubilized in HBSS. Nimodipine (Sigma) was solubilized in DMSO (final concentration <0.1%) and then added to HBSS. The concentrations of all reagents were adopted from those reported in the literature [3 –6,9,13 –19,23,24,27,28,32]. The cell cultures were preincubated with these reagents for 30 min before depolarization. 2.2. Immunocytochemistry Cell cultures were fixed (4% PFA, 15 min at RT) and incubated overnight at 4 -C with primary antibodies against
MAP-2 (1:500; Sigma), TUJ1 (1:500; Covance, Berkeley, CA), glial fibrillary acidic protein (GFAP, 1:500; Dako, Hamburg, Germany), CNPase (1:100; Sternberger, Lutherville, MD), and ED1 (1:1000; Serotec, Du¨ sseldorf, Germany). Secondary antibodies conjugated with Alexa Fluor 488 or Texas Red (Molecular Probes) were added at a dilution of 1:250 for 1 h at RT; omission of primary antibodies served as negative control. The sections were coverslipped with Immuno-fluore (MP Biomedicals, Eschwege, Germany) and examined under a conventional fluorescence microscope (DMRA; Leica, Bensheim, Germany) equipped with standard fluorescein and Texas Red filter sets. Cells from four independent preparations were counted at 20 magnification (10 visual fields/preparation). Data are given as mean T SD.
3. Results 3.1. Characterization of cortical cell cultures We used antibodies that specifically recognize neuronal (MAP-2, TUJ1), oligodendroglial (CNPase), astroglial (GFAP), and microglial (ED1) antigens in order to determine the purity of our cultures. Immunocytochemical analysis revealed that neurons by far exceeded any other cell population in our preparations (neurons: 77.2 T 5.8%; astrocytes: 18.5 T 2.1%; oligodendrocytes 2.3 T 8.1%; microglia: 0.2 T 1.0%). Fig. 1 shows a representative example. Thus, only astrocytes could have added measurably to changes in fluo-4 AM fluorescence besides cortical neurons. However, the astrocytic expression of VGCC and NMDA receptors is, if at all, very low. Thus, it is assumed that the [Ca2+]i changes were predominantly related to neurons.
Fig. 1. Neurons are the predominant cell type in rat primary cortical cultures. Immunocytochemical analysis was performed using antibodies that specifically recognize neuronal (MAP-2, arrows) and astroglial (GFAP, arrowheads) antigens. Secondary antibodies conjugated with Alexa Fluor 488 (green) and Texas Red (red) were used for visualization. Note that the number of neurons exceeded the number of astrocytes.
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3.2. The NO level modulates the [Ca2+]i increase following depolarization In order to identify the potassium concentration that elicited a robust fluorescence increase, we performed dose –response experiments using different [K+]o (Fig. 2). We chose to perform all subsequent experiments at 35 mM as a strong and reproducible F/F 0 increase was observed when cells were depolarized at this concentration. No significant fluorescence changes were observed when the experiments were performed in calcium-free solution (Fig. 2), indicating that calcium entry in these cultured cortical neurons appeared to be mediated by the influx of extracelllular calcium rather than by the release from intracellular stores. We used this potassium concentration to investigate the effect of the NO level on the calcium changes. K+-induced depolarization resulted in a significant [Ca2+]i-induced F/F 0 increase to 1.67 T 0.11 (Fig. 3A). To estimate the effect of the NO level, [Ca2+]i was measured in cultures preincubated with either the NO synthase inhibitor l-NNA (1 mM) or with the NO donor SNAP (100 AM). No significant changes in [Ca2+]i-induced fluorescence were observed during the incubation period preceding depolarization. Following depolarization, l-NNA significantly amplified the depolarization-induced [Ca2+]i increase (2.18 T 0.17; Fig. 3A) compared to increased [K+]o alone, indicating enhanced depolarization-induced calcium influx at low basal NO level. In contrast, calcium influx was significantly attenuated compared to high [K+]o alone in cultures preincubated with SNAP (1.27 T 0.15; Fig. 3A), suggesting that NO decreased neuronal calcium influx during depolarization.
Fig. 2. [Ca2+]i changes following K+-induced depolarization are mediated by influx of extracellular calcium. Ascending concentrations of potassium ([K+]o) were applied to neuronal cell cultures (closed circles), inducing a concentration-dependent fluorescence increase compared to [K+]o at 3 mM ( F/F 0). In contrast, no significant fluorescence increase was detected when cells were depolarized in calcium-free medium, indicating that release from intracellular calcium stores did not significantly contribute to the fluorescence changes. *P < 0.05.
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These results suggest that the intracellular calcium surge during neuronal depolarization is sensitive to changes of the extracellular NO level. 3.3. The depolarization-induced [Ca2+]i increase is mediated by VGCC and NMDAR In order to identify the membrane channels involved in the depolarization-induced calcium influx, we incubated the cultures with different VGCC inhibitors and an NMDA receptor antagonist. Compared to raised [K+]o alone, the Ntype VGCC (Cav2.2(a´1B)) blocker N-conotoxin GVIA (1 AM) significantly attenuated the [Ca2+]i-related fluorescence increase induced by raised [K+]o (1.41 T 0.15). Similarly, the depolarization-induced [Ca2+]i increases were significantly reduced by the l-type VGCC (Cav1.3(a´1D)) blocker nimodipine (2 AM; 1.57 T 0.18), the P/Q-type VGCC (Cav2.1(a´1A)) blocker N-agatoxin IVA (300 nM; 1.35 T 0.12), and by the NMDA receptor antagonist MK-801 (10 AM, 1.39 T 0.1). No significant changes in [Ca2+]i-induced fluorescence were observed in cultures incubated with the VGCC inhibitors or MK-801 at baseline [K+]o. These results suggest that calcium entry appeared to be mediated by all four VGCC types and the NMDA receptor in these cultured cortical neurons. 3.4. The modulatory potential of NO is mainly attributable to NMDA receptors and P/Q-type VGCC Next, we studied whether the modulation of the calcium signal by NO was attributable to these individual channel types. Co-application of increased [K+]o with N-conotoxin GVIA (1 AM) and either SNAP or l-NNA did not affect the ability of NO to modulate the calcium signals (Fig. 3B), indicating that the effect of NO on [Ca2+]i did not depend on N-type VGCC activity modulation. Similarly, application of the L-type VGCC inhibitor nimodipine (2 AM) resulted in a reduction of [Ca2+]i, but the modulation of the calcium signal by NO was still present (Fig. 3C). The combination of nimodipine and N-conotoxin GVIA decreased the calcium signal, but the modulation by different NO levels was still detectable. In contrast, the ability of l-NNA to amplify the depolarization-induced [Ca2+]i changes was abolished when the neuronal cultures were preincubated with MK-801 (10 AM), whereas the ability of SNAP to significantly reduce the [Ca2+]i signal was still apparent (Fig. 3D). This suggested that the modulation of [Ca2+]i changes by NO was partially attributable to the NMDA receptor. Similar results were obtained when neurons were preincubated with N-agatoxin IVA (300 nM; Fig. 3E). Furthermore, preincubation with both MK-801 and N-agatoxin IVA abolished the ability of SNAP and l-NNA, respectively, to modulate the [Ca2+]i changes induced by depolarization (Fig. 3F). To further confirm the involvement of NMDA receptors, we investigated the [Ca2+]i response to increasing concentra-
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Fig. 3. NO modulates calcium influx through VGCC and the NMDA receptor in rat primary cortical neurons. Following preincubation with the indicated inhibitors, cells were depolarized by either high [K+]o alone (closed circles), high [K+]o and the NO synthase inhibitor l-NNA (open circles) or high [K+]o and the NO donor SNAP (triangles). [Ca2+]i changes are presented as relative fluorescence values compared to baseline ( F/F 0) over time. Asterisks denote statistically significant differences at the corresponding time points ( P < 0.05). (A) Depolarization resulted in a [Ca2+]i increase that was further amplified by lNNA and attenuated by SNAP. (B – C) The N-type VGCC blocker N-conotoxin GVIA (Cono) and the L-type VGCC blocker nimodipine (Nimo) attenuated the [Ca2+]i increase but had no effect on the modulation of [Ca2+]i by l-NNA and SNAP. (D – F) The NMDA receptor inhibitor MK-801 and the P/Q-type VGCC inhibitor N-agatoxin IVA (Aga) significantly attenuated the potential of l-NNA to modulate the [Ca2+]i changes. (G) Co-application of MK-801 and N-agatoxin IVA abolished the [Ca2+]i changes induced by l-NNA or SNAP.
tions of NMDA (Fig. 4). Compared to NMDA alone, the fluorescence increase was significantly attenuated by SNAP, whereas no significant changes were observed in cultures preincubated with l-NNA. These results indicate that both the NMDA receptor and the P/Q-type VGCC are both involved in the modulation of intracellular calcium changes following depolarization and that this modulation can be abrogated by the combined blockade of both channels.
4. Discussion The second messengers NO and calcium are involved in almost all aspects of neuronal function. NO plays a fundamental role in the intricate network that fine-tunes calcium homeostasis, and vice versa. Thus, it has been shown that NO strongly modulates calcium influx through membrane channels [7,12]. In turn, calcium influx following neuronal activation—either presynaptically through VGCC
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Fig. 4. The NMDA-evoked [Ca2+]i increase is modulated by the NO level. Ascending concentrations of NMDA were applied to cortical neuronal cultures. Compared to NMDA alone (closed circles), the fluorescence increase was significantly attenuated by the NO donor SNAP (triangles) at 200 AM and 400 AM NMDA, whereas no significant changes were observed in cultures preincubated with the NO synthase inhibitor l-NNA (open circles). *P < 0.05.
or postsynaptically through NMDA receptors—directly activates NO synthesis [12]. In this study, we have investigated whether these two major calcium gateways in neurons are also sensitive to the extracellular NO level. We found that the relative amount of neuronal calcium influx depends on the NO level of the environment and that NMDA receptors and P/Q-type VGCC, but not N-type and L-type VGCC, are critically involved in this effect. Our data implicate that NMDA receptors and P/Q-type channels do not only activate neuronal NO production but that NO reciprocally also affects the activity of these channels. Thus, a mutual modulation of these channels may help to regulate and fine-tune both neuronal calcium and NO homeostasis by this feedback loop. The exact mechanisms by which NO modulates the activities of different Ca2+ channels remain elusive. However, the ability of NO to alter ion channel activity is well known. It has been shown that NO down-regulates NMDA receptor activity [6,16,18,19], whereas contradictory results have been obtained regarding the influence of NO on VGCC activity. Although most studies have demonstrated that NO inhibits VGCC [3,9,13,17,23,27,32], some reports have suggested that NO may also activate these channels [4,5,14]. These discrepancies could partially be explained by the different types and concentrations of NO donors used, the cell types studied, and different experimental procedures (e.g., cell culture vs. patch clamp recordings). Confirmatory studies using confocal microscopy or single cell studies will be needed to further address these issues. Moreover, direct effects (e.g., by S-nitrosylation [6]) and indirect effects of NO (e.g., by guanylyl cyclase activation) can differentially affect channel activity. Thus, it has been shown that NO directly activates P/Q-type VGCC [5],
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whereas the NO-induced production of cyclic guanosine monophosphate inhibits these channels [13]. Furthermore, it has been shown that NO induces a sustained increase of neuronal [Ca2+]i when NO donor concentrations several fold higher than those applied in the present study are used [2], indicating that the [Ca2+]i changes can be reverted under pathophysiological conditions. The modulatory effect of NO on NMDA receptors and P/ Q-type VGCC might also have important implications for the actions of glutamate in health and disease. P/Q-type VGCC mainly reside on neuronal presynaptic terminals [29] and are a major gateway for the synaptic release of glutamate [31]. Postsynaptically, NMDA receptors are a main target of glutamatergic action, e.g., in synaptic transmission, plasticity, or excitotoxicity [8]. Hence, our results suggest that the basal NO level under physiological conditions may indirectly modulate the actions of glutamate by reducing its presynaptic release and postsynaptic action. Under pathological conditions, rise of NO, as during epileptic seizure-like events, could contribute to terminate hypersynchronous neuronal activity [26], but a decline in the basal NO level could augment glutamate excitotoxicity. This may be particularly relevant in cerebral hemorrhage, in which the NO concentration may be reduced by decreased NO production due to shortage of molecular oxygen [10], by endogenous NO synthase inhibitors [22], by dysfunction of NO producing sources such as neurons or endothelial cells [22], or by extracellular hemoglobin, a potent NO scavenger [11].
Acknowledgments This study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 507 to J.P., U.D., and J.P.D.; PE 1193/1-1 to G.C.P.) and by the Hermann and Lilly Schilling Foundation (U.D.).
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