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Keywords:

  • Cav3.2;
  • differentiation;
  • hydrogen sulfide;
  • neurite outgrowth;
  • neuroblastoma NG108-15 cell line;
  • T-type Ca2+ channel

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

We investigated if stimulation of T-type Ca2+ channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H2S), could cause neuronal differentiation of NG108-15 cells. Like dibutyryl cyclic AMP (db-cAMP), treatment with NaHS at 1.5–13.5 mM for 16 h enhanced neurite outgrowth in a concentration-dependent manner. Synergistic neuritogenic effect was obtained in the cells stimulated with NaHS in combination with db-cAMP at subeffective concentrations. Exposure to NaHS or db-cAMP for 2 days resulted in enhancement of expression of high-voltage-activated currents consisting of N-, P/Q-, L- and also other types, but not of T-type currents. Mibefradil, a pan-T-type channel blocker, abolished the neuritogenesis induced by NaHS, but not by db-cAMP. The NaHS-evoked neuritogenesis was also completely blocked by pretreatment with BAPTA/AM, a chelator of intracellular Ca2+, and by zinc chloride at a concentration known to selectively inhibit Cav3.2 isoform of T-type Ca2+ channels, but not Cav3.1 or Cav3.3. Further, l-ascorbate, recently proven to selectively inhibit Cav3.2, abolished the neuritogenic effect of NaHS, but not db-cAMP. Our data thus demonstrate that NaHS/H2S is a novel inducer of neuronal differentiation in NG108-15 cells, as characterized by neuritogenesis and expression of high-voltage-activated currents, and suggest the involvement of T-type Ca2+ channels, especially Cav3.2.

Abbreviations used
CBS

cystathionine-β-synthase

CSE

cystathionine-γ-lyase

db-cAMP

dibutyryl cyclic AMP

DTNB

5,5′-dithiobis(2-nitrobenzic acid)

DTT

dithiothreitol

FCS

fetal calf serum

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

H2S

hydrogen sulfide

HVA

high-voltage-activated

KATP channels

ATP-sensitive K+ channels

LTP

long-term potentiation

NaHS

sodium hydrosulfide

RT-PCR

reverse-transcribed-polymerase chain reaction

ZnCl2

zinc chloride

Hydrogen sulfide (H2S) is well known as a natural chemical hazard with a strong odor of rotten eggs. Given evidence for significant levels of endogenous H2S in rat and human brain tissues (Goodwin et al. 1989; Warenycia et al. 1989), H2S, like nitric oxide (NO) and carbon monoxide (CO), is now considered to function as a gasotransmitter in the mammalian body (Wang 2002; Moore et al. 2003; Lowicka and Beltowski 2007). H2S is formed endogenously from l-cysteine by cystathionine-γ-lyase and cystathionine-β-synthase (Moore et al. 2003), and H2S levels in mammalian tissues and blood are relatively high (10–160 μM). In the brain, H2S enhances NMDA receptor-mediated currents and facilitates the induction of hippocampal long-term potentiation (LTP) via increase in cyclic AMP (cAMP) levels (Abe and Kimura 1996; Kimura 2000). H2S also activates ATP-sensitive K+ (KATP) channels in rat aorta (Zhao et al. 2001; Kubo et al. 2007) and a rat insulin-secreting cell line, INS-1E (Yang et al. 2005). Most recently, we have shown that intraplantar or intracolonic administration of sodium hydrosulfide (NaHS), a donor of H2S causes prompt hyperalgesia, an effect being abolished by mibefradil, an inhibitor of T-type Ca2+ channels (Kawabata et al. 2007; Kawabata 2008). Our electrophysiological evidence has also demonstrated that NaHS actually enhances membrane currents through T-type channels in NG108-15 cells, neuroblastoma × glioma hybrid cells, as well as mouse dorsal root ganglion neurons (Kawabata et al. 2007; Kawabata 2008).

Neuroblast differentiation into neurons is judged by neurite outgrowth and synapse formation, and can be characterized by changes in electrophysiological properties, i.e. later appearance of high-voltage-activated (HVA) Ca2+ currents after the first appearance of T-type Ca2+ currents (Gottmann et al. 1988; Goodwin et al. 1989; McCobb et al. 1989; Chemin et al. 2002). NG108-15 cells are widely used in studies on neuronal development and differentiation, and known to be abundant in T-type channels but not HVA channels, unless differentiated (Nirenberg et al. 1983; Chemin et al. 2002). NG108-15 cells, when stimulated with dibutyryl cyclic AMP (db-cAMP), develop neuron-like properties, revealing neurite outgrowth, synapse formation and functional expression of HVA Ca2+ currents (Kleinman et al. 1988; Han et al. 1991; Kasai and Neher 1992; Taussig et al. 1992; Chemin et al. 2002). Chemin et al. (Chemin et al. 2002, 2004) have shown that blockade of T-type channels, particularly of Cav3.2 (α1H) isoform, partially inhibits db-cAMP-evoked neuritogenesis and abolishes concomitant HVA Ca2+ current expression in NG108-15 cells. Since H2S is capable of facilitating T-type currents, as mentioned above, it is likely that H2S might cause and/or promote neuronal differentiation in NG108-15 cells. Thus, in the present study, we asked if NG108-15 cells treated with NaHS, a donor for H2S, reveal neuron-like properties, by examining neurite outgrowth and expression of HVA Ca2+ currents.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Cell culture and assessment of neurite outgrowth

NG108-15 cells were cultured in high glucose-containing Dulbecco’s Modified Eagle’s Medium (Sigma, St. Louis, MO, USA) supplemented with 0.1 mM hypoxanthine, 1 μM aminopterin, 16 μM thymidine, 50 U/mL penicillin, 50 μg/mL streptomycin and 10% fetal calf serum (FCS) (Thermo Electron, Melbourne, Australia) (Imanishi et al. 2006). The cells were harvested and reseeded at a density of 1 × 104/mL in culture dishes (35 mm in diameter) coated with poly-l-ornithine, filled with 1 mL of the above medium containing 1% FCS. Three hours after plating, the cells were stimulated with NaHS at 0.3–13.5 mM and/or db-cAMP at 0.1–1.0 mM for 16 h (and for 48 h in the time-course experiment). Considering the short half-life time of H2S in the medium, cells were also repeatedly stimulated by repetitive application of NaHS at 1.5 mM at 15-min or 30-min intervals, nine times in total. Cells were also stimulated with dithiothreitol (DTT) at 0.3–4.5 mM for 2 h and cultured for additional 14 h after removal of DTT, since longer incubation with DTT than 2 h caused detachment of the cells from the dish bottom. Morphological observation was performed 16 h after the onset of stimulation. Eight to 20 fields of view from 4 to 10 dishes (1–2 fields from each dish) were examined under the microscope for each experiment. The average number of cells in each field was about 36 cells. Neurite outgrowth was evaluated by counting cells with neurites that were longer than the cell body diameter. Inhibitors were applied 30 min before addition of NaHS, db-cAMP or DTT in neurite outgrowth determination.

Determination of cell damage

NG108-15 cells were plated at a density of 2 × 104 cells/dish in culture dishes (35 mm in diameter) coated with poly-l-ornithine, filled with 1 mL of medium containing 1% FCS. Three hours after plating, the cells were stimulated with NaHS at 13.5 mM or the vehicle (H2O) for 16 or 48 h. Trypan blue solution (0.5% in phosphate-buffered saline, pH 7.4) in a volume of 2 mL was applied to the cells. One min after the addition, the supernatant in the culture dishes was sucked off, and the cells were covered with a cover glass. Cell damage was evaluated by counting cells stained with trypan blue under a microscope (10×). Effect of ionomycin at 3 μM for 2 h was observed as a positive control of cell damage.

Whole-cell patch-clamp recordings

For determining the expression levels of functional HVA channels and T-type Ca2+ channels after differentiation stimuli in NG108-15 cells, whole-cell patch-clamp recordings were performed as described previously (Kawabata et al. 2007). The cells were seeded and stimulated with NaHS or db-cAMP, as described above, for 2 days. The cells were washed with an extracellular solution for patch-clamp experiments containing (in mM): 97 N-methyl-d-glucamine (NMDG), 10 BaCl2, 10 HEPES, 40 tetraethylammonium (TEA)-Cl and 5.6 glucose, adjusted to pH 7.4. Ba2+ currents were recorded from randomly chosen cells at 20–25°C using a whole-cell patch-clamp amplifier. A patch pipette was filled with an intracellular solution containing (mM): 150 CsCl, 4 MgCl2, 5 EGTA and 10 HEPES, adjusted to pH 7.2. The resistance of patch electrodes ranged from 3 to 7 MΩ. Series-resistance was compensated by 80%, and current recordings were low-pass filtered (<5 kHz). The cell membrane voltage was held at −90 mV, and whole cell Ba2+ currents were elicited by step pulses from −120 to 40 mV with increments of 10 mV or a voltage ramp of 800-ms duration from −120 to 30 mV. T-type currents were measured as the difference between currents of the peak and 150 ms after the beginning of a step pulse at −20 mV. HVA currents were measured as persistent currents 75 ms after the beginning of a step pulse at +10 mV. In the experiments to determine the composition of the HVA-Ca2+ current component by using specific blockers, currents were measured at +10 mV from a holding potential of −60 mV to inactivate T-type currents. In the experiments to confirm the involvement of Cav3.2 in the low-voltage-activated currents in response to a voltage ramp in NG108-15 cells treated with NaHS at 13.5 mM for 2 days, the currents were measured before and 2 min after the application of zinc chloride (ZnCl2) at 10 μM, known to suppress the currents via Cav3.2, but not Cav3.1, Cav3.3 or HVA Ca2+ channels (Jeong et al. 2003). Then, the currents at −30 mV and at +10 mV of the voltage ramp were measured in the absence or presence of ZnCl2. Data were acquired and digitized with a Digidata interface (1322A, Axon Instruments, Foster City, CA, USA) and analyzed by a personal computer using pClamp8 software (Axon Instruments).

Reverse-transcribed-polymerase chain reaction (RT-PCR)

NG108-15 cells treated with NaHS at 13.5 mM or vehicle for 48 h were washed with phosphate-buffered saline and lysed in the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA extracted from the cell lysate was reverse-transcribed and then amplified by PCR using RNA LA PCR kit (AMV), version 1.1 (Takara, Otsu, Japan). PCR primers employed were: 5′-CCTGAGAATTTCAGCCTCCC-3′ (forward) and 5′-GATCGCATGCCGTTCTCC-3′ (reverse), amplifying 113-bp fragments for Cav3.1; 5′-ATGTACTCACTGGCTGTGACC-3′ (forward) and 5′-GAGTCCAAAAGAGTGTGGGC-3′ (reverse), amplifying 146-bp fragments for Cav3.2; 5′-CTGCACTTACCACGACTCCA-3′ (forward) and 5′-ATGTGAGTGACAGGCTGCTG-3′ (reverse), amplifying 92-bp fragments for Cav3.3; 5′-TGCATCCTGCACCACCAACT-3′ (forward) and 5′-AACACGGAAGGCCATGCCAG-3′ (reverse), amplifying 259-bp fragments for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Amplification was allowed to proceed for 30 cycles for GAPDH and 35 cycles for Cav3.1, 3.2 and 3.3, beginning with a 30-s denaturation period at 94°C followed by a 30-s reannealing time at 60°C and a primer extension period of 1 min at 72°C. The PCR products were verified by electrophoresis on 2% agarose gels and visualized under UV with ethidium bromide.

Chemicals

NaHS and zinc chloride (ZnCl2) were purchased from Kishida Chemical Co. Ltd. (Osaka, Japan). Mibefradil, nitrendipine, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), ionomycin and l-ascorbic acid (ascorbate) were obtained from Sigma-Aldrich. Dibutyryl cyclic AMP (db-cAMP) was from Fluka (Buchs, Switzerland), ω-conotoxin GVIA and ω-conotoxin MVIIC were from Peptide Inst. Inc. (Osaka, Japan), and (±)-DTT was from Wako Pure Chemicals (Osaka, Japan). DTNB and nitrendipine were dissolved in dimethylsulfoxide (Sigma-Aldrich), and other chemicals were in distilled water.

Statistics

Data are shown as the mean ± SEM. Statistical analysis was performed by Student’s t-test for two-group data and Tukey’s test for multiple comparisons. Significance was set at a p < 0.05 level.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

NaHS, a donor of H2S, promotes neurite outgrowth in NG108-15 cells

As with db-cAMP at 1 mM, a differentiation inducer (Tojima et al. 2003a,b), treatment with NaHS at 13.5 mM, a relatively high concentration, for 16 h enhanced neurite outgrowth in NG108-15 cells (Fig. 1a,b and d). Longer incubation of the cells with NaHS at the same concentration did not cause further enhancement of neurite outgrowth; the proportion (%) of cells with neurites was 35.6 ± 2.3 and 13.5 ± 1.4 in cells treated with NaHS and vehicle for 48 h, respectively (n = 16). The effects of NaHS and db-cAMP were concentration-dependent in ranges of 1.5–13.5 mM and of 0.5–1 mM, respectively (Fig. 1b and d). Dithiothreitol, a reducing reagent, at 1.5 mM, known to mimic the facilitating effect of NaHS on T-type Ca2+ channels (Kawabata et al. 2007; Nelson et al. 2007a), also caused neurite outgrowth (Fig. 1c). NaHS at 1.5 mM, a concentration capable of facilitating T-type Ca2+ channels (Kawabata et al. 2007), synergistically potentiated the neurite outgrowth induced by db-cAMP at 0.05 mM, a subeffective concentration, leading to a significant effect, albeit NaHS at the same concentration itself was without significant effect (Fig. 1e). It is to be noted that repetitive application of NaHS at 1.5 mM at 15-min or 30-min intervals, nine times in total, also significantly enhanced neurite outgrowth (Fig. 1f). When cytotoxicity of NaHS was evaluated by the trypan blue staining, stimulation with NaHS at 13.5 mM for 16 or 48 h did not cause cell death, whilst incubation with ionomycin at 3 μM for 2 h killed most cells (Fig. 1g and h). Also, the effect of NaHS on pH of the culture medium was checked. The pH value of the culture medium elevated by 0.5 immediately after application of NaHS at 13.5 mM, thereafter decreasing by 0.2 within 30 min and then returning to the original value within 60 min. On the other hand, the immediate elevation of pH by application of NaHS at 1.5 mM was only 0.2, disappearing within 30 min.

image

Figure 1.  Effects of NaHS, dithiothreitol (DTT) and dibutyryl cyclic AMP (db-cAMP) on neurite outgrowth in NG108-15 cells. (a) Representative photographs of NG108-15 cells treated with NaHS or db-cAMP for 16 h. Arrows indicate neurites. (b–f) Neurite outgrowth, determined as increase in proportion of cells with neurites longer than the cell diameter, caused by 16-h stimulation with NaHS and/or db-cAMP (b, d, and e), by 2-h stimulation with DTT followed by 14-h incubation without DTT (c), and by 16-h stimulation with repetitive application (nine times) of NaHS at 1.5 mM (total 13.5 mM) with 15 or 30 min intervals. (g and h) Determination of cell damage with trypan blue staining. (g) Arrows indicate stained cells. Data show the mean ± SEM from 8–12 (b–d and f), 20 (e) or 6 (h) experiments. NS, not significant.

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NaHS increases expression of HVA currents in NG108-15 cells

Appearance of HVA currents could be a criterion for neuronal differentiation in addition to neurite outgrowth in various cells including NG108-15 cells (Gottmann et al. 1988; McCobb et al. 1989; Chemin et al. 2002). When Ba2+ currents were analyzed by applying a voltage ramp (−120 to +30 mV, 800-ms duration) from a holding potential of −90 mV, only one clear current peak with a low threshold, most probably because of activation of T-type Ca2+ channels, was observed around −30 mV in vehicle-treated cells (undifferentiated cells), while two current peaks, possibly because of activation of HVA Ca2+ channels in addition to T-type Ca2+ channels, were found at −30 mV and at +10 mV in cells treated with NaHS at 13.5 mM (Fig. 2a) or db-cAMP at 1 mM (Fig. 2b) for 2 days. ZnCl2 at 10 μM, known to selectively suppress Cav3.2, but not Cav3.1, Cav3.3 or HVA Ca2+ channels (Jeong et al. 2003), significantly attenuated the currents at −30 mV, but not +10 mV, of ramp stimulation in the cells treated with NaHS at 13.5 mM for 2 days (Fig. 2c). Further, current–voltage relationship of Ba2+ currents was determined by applying step pulses (−120 to +40 mV) from a holding potential of −90 mV. In vehicle-treated cells, the current–voltage curve of Ba2+ currents measured at the peak (sum of T-type currents and HVA currents) (Fig. 3a) was clearly obtained (open symbols in Fig. 3b and c, left), whereas the persistent currents measured 75 ms after the beginning of the test pulse (mainly HVA currents) (Fig. 3a) were small, showing no remarkable peak in the current–voltage curves (open symbols in Fig. 3b and c, right). In contrast, the persistent currents in the cells treated with NaHS or db-cAMP for 2 days were relatively greater, particularly when stimulated with step pulses at 0 mV or higher voltage, peaking at +10 mV (closed symbols in Fig. 3b and c, right). HVA currents, determined as persistent currents 75 ms after the beginning of a test pulse at +10 mV from a holding potential of −90 mV (Fig. 4a, right), essentially as described previously (Chemin et al. 2002), were significantly augmented by differentiation stimuli with NaHS or db-cAMP for 2 days (Fig. 4b and c, right). For quantitative analysis, T-type currents were estimated as the difference between currents of the peak and 150 ms after the beginning of a test pulse at −20 mV from a holding potential of −90 mV (Fig. 4a, left), to minimize contamination of HVA currents, essentially as described previously (Chemin et al. 2002). T-type currents thus remained constant before and after treatment with NaHS or db-cAMP (Fig. 4b and c left).

image

Figure 2.  Effect of treatment with NaHS (a) or db-cAMP (b) for 2 days on Ba2+ currents through Ca2+ channels in response to ramp stimulation in NG108-15 cells. The cells were stimulated with NaHS or db-cAMP for 2 days, and thereafter, Ba2+ currents through Ca2+ channels were measured in randomly chosen cells in the absence of NaHS or db-cAMP. An 800-ms ramp from −120 to +30 mV was applied from a holding potential (HP) of −90 mV. Data show the mean ± SEM from 7–10 different cells. (c) Effect of zinc chloride (ZnCl2) on the low (−30 mV) and high (+10 mV) voltage-activated Ca2+ channel-dependent currents in response to ramp stimulation in NG108-15 cells stimulated with NaHS at 13.5 mM for 2 days. Pre and ZnCl2 10 μM indicate the currents before and 2 min after application of ZnCl2. Data show the mean ± SEM from 12 different cells.

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image

Figure 3.  Effect of treatment with NaHS or db-cAMP for 2 days on current–voltage relationship of Ca2+ channel-dependent currents in response to step-pulse stimulation in NG108-15 cells. Step pulses from −120 to +40 mV were applied from a holding potential (HP) of −90 mV. (a) A typical example for currents in response to a 200-ms test pulse at 0 mV. Peak currents and persistent currents 75 ms after the beginning of each test pulse were determined. (b and c) Current–voltage relationships for peak and for persistent currents in NG108-15 cells after incubation with NaHS (b), db-cAMP (c) or vehicle for 2 days. Patch-clamp recordings were performed in the absence of either NaHS or db-cAMP. Data show the mean ± SEM from 7–10 different cells. *, p < 0.05; **, p < 0.01 versus the values of vehicle.

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image

Figure 4.  Functional expression of T-type and HVA Ca2+ channels in NG108-15 cells treated with NaHS or db-cAMP. (a) T-type Ca2+ currents (T-currents) were measured as the difference between currents of the peak and 150 ms after the beginning of a test pulse (TP) at −20 mV (left) from a holding potential (HP) of −90 mV, while HVA currents were determined as currents 75 ms after the beginning of TP at +10 mV (right). (b and c) Cells exposed to NaHS (b) or db-cAMP (c) for 2 days were randomly chosen, and used for path-clamp recordings after their removal. Data show the mean ± SEM from 7–10 different cells. *, p < 0.05 and **, p < 0.01 versus undifferentiated.

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To examine the composition of HVA currents in the NaHS-differentiated cells, we tested effects of specific blockers for N-, P/Q- and L-type Ca2+ channels, ω-conotoxin GVIA at 1 μM, ω-conotoxin MVIIC at 1 μM and nitrendipine at 1 μM, respectively, on the HVA currents (persistent currents) induced by a test pulse at +10 mV from a holding potential of −60 mV. The estimated average composition was approximately: N-type, 23%; P/Q-type, 25%; L-type, 41%; other types, 11% (n = 10).

Involvement of T-type Ca2+ channels in NaHS-evoked neurite extension in NG108-15 cells

Given two independent studies in NG108-15 cells showing facilitation of T-type currents by NaHS (Kawabata et al. 2007) and involvement of T-type Ca2+ channels in neuronal differentiation (Chemin et al. 2002), we next asked whether T-type Ca2+ channels mediate neuronal differentiation induced by NaHS in NG108-15 cells, as evaluated by the extent of neurite outgrowth. Mibefradil, a pan-T-type channel blocker, even at 0.3 μM significantly suppressed the neurite outgrowth induced by NaHS and by DTT, but not by db-cAMP (Fig. 5a). Similarly, the effect of NaHS was abolished by BAPTA/AM, a chelator of intracellular Ca2+ (Fig. 5b). DTNB at 10 μM, an oxidant, capable of inhibiting activation of T-type channels by NaHS (Kawabata et al. 2007), also suppressed the NaHS-induced neurite outgrowth (Fig. 5c). Interestingly, ZnCl2 at 10 μM and ascorbate at 0.1–1 μM, known to selectively inhibit Cav3.2 among three forms of T-type channels (Nelson et al. 2007a,b; Traboulsie et al. 2007), mimicked the inhibitory effects of the pan-T-type channel blocker and the Ca2+ chelator, as described above (Fig. 5a,b,d and e). Of note is that ascorbate in the same concentration range failed to alter the neuritogenic effect of db-cAMP (Fig. 5f). Expression of Cav3.2 mRNA was most clearly detected among three isoforms of T-type Ca2+ channels in NG108-15 cells (Fig. 5g). Expression levels of any of T-type channel isoforms did not change after treatment with NaHS at 13.5 mM, compared with the cells treated with vehicle.

image

Figure 5.  Effects of various inhibitors or chemicals on the NaHS-enhanced neurite outgrowth in NG108-15 cells. Cells were treated with mibefradil, a pan-T-type Ca2+ channel blocker (a) or BAPTA/AM, an intracellular Ca2+ chelator (b), and with DTNB, an oxidant (c), zinc chloride (ZnCl2) (d) and ascorbate (e and f) at concentrations, known to block selectively inhibit Cav3.2, but not Cav3.1 and Cav3.3, 30 min before 16-h stimulation with NaHS (a–e), db-cAMP (a and f) or DTT (a). Neurite outgrowth was evaluated by counting cells with neurites that were longer than the cell body diameter. Data show the mean ± SEM from 8–19 different experiments. NS, not significant. (g) Detection of mRNA expression of Cav3.1, 3.2, 3.3 and GAPDH, a housekeeping gene, in NG108-15 cells stimulated with NaHS at 13.5 mM or vehicle for 48 h. Parentheses show the size of PCR products.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

The present study provides morphological and electrophysiological evidence that NaHS, a donor of H2S, induces neuronal differentiation of NG108-15 cells, as characterized by neurite outgrowth and expression of HVA Ca2+ currents. The differentiation-inducer-like activity of NaHS/H2S is considered predominantly dependent on Ca2+ influx through T-type Ca2+ channels, most probably of Cav3.2 isoform, differing from db-cAMP-induced neuronal differentiation in which activation of T-type Ca2+ channels plays limited roles.

Undifferentiated NG108-15 cells express T-type, but not HVA, Ca2+ channels, whereas, after differentiation, HVA currents consisting of any of L-, N-, P/Q- and R-types appear and dominate the total Ca2+ influx in the cells (Chemin et al. 2002). Similar phenomena have been reported in various neuron-related cells including hippocampal, motor and sensory neuronal systems (Gottmann et al. 1988; Goodwin et al. 1989; McCobb et al. 1989; Chemin et al. 2002). Therefore, our data that, like db-cAMP, NaHS not only caused rapidly developing neurite outgrowth but also resulted in delayed dramatic augmentation of HVA currents of L-, N-, P/Q- and other types, strongly suggest the induction of neuronal differentiation by NaHS. It would be interesting to ask, in future, if NaHS/H2S could cause neuronal differentiation in other neuron-related cells.

Most recently, we have reported that NaHS increases Ba2+ currents through T-type Ca2+ channels in undifferentiated NG108-15 cells (Kawabata et al. 2007), supporting the present evidence for the involvement of T-type Ca2+ channels in NaHS-evoked neurite outgrowth on the basis of the inhibitory effect of mibefradil, a pan-T-type Ca2+ channel blocker. Further, the inhibitory effects of ZnCl2 at 10 μM and ascorbate at 0.1–1 μM on the neuritogenesis caused by NaHS imply a central role of Cav3.2, since they, when applied at appropriate concentrations, selectively inhibit Cav3.2, but not Cav3.1 and Cav3.3 (Nelson et al. 2007a,b; Traboulsie et al. 2007). In the present study, neither mibefradil nor ascorbate attenuated db-cAMP-evoked neurite outgrowth, although they completely blocked the neuritogenic effect of NaHS. These findings are not consistent with the study of Chemin et al. (2002), since they have shown a possible role of Cav3.2 T-type Ca2+ channels in db-cAMP-induced differentiation of NG108-15 cells. In their study, however, the inhibition of db-cAMP-induced neurite outgrowth by mibefradil at 1 μM or by nickel at 30 μM, a concentration known to block Cav3.2, but not Cav3.1 or Cav3.3 (Perez-Reyes 2003), is limited, the magnitude of inhibitory effect being only 20–30% (Chemin et al. 2002). This is also similar to the extent (30%) of inhibitory effect of antisense oligodeoxynucleotides against Cav3.2 mRNA (Chemin et al. 2002). Together, db-cAMP-induced neuronal differentiation of NG108-15 cells should involve other major mechanisms beside T-type channels. In this context, it is of particular interest that NaHS/H2S-induced neuronal differentiation is fully dependent on the activation of T-type Ca2+ channels, differing from the db-cAMP-evoked differentiation.

The effective concentration range, 4.5–13.5 mM, of NaHS in facilitating neurite outgrowth is considered relatively high (see Fig. 1). Nonetheless, the facilitating effect of DTT on neurite outgrowth can be obtained even at 1.5 mM, being inconsistent to our previous evidence that either NaHS or DTT at the same concentration, 1.5 mM, facilitates T-type currents in NG108-15 cells (Kawabata et al. 2007). This discrepancy might be associated with distinct stability or degradation of DTT and NaHS in the culture medium, since H2S concentration rapidly decays in cell culture dishes and the half-life time in the culture medium is 6.2 min (Garcia-Bereguiain et al. 2008). Persistent activation of T-type channels might thus be required for neuritogenesis. This notion is supported by the significant effect of repetitive application of NaHS at 1.5 mM. It is to be noted that DTT at 1.5 mM had to be removed immediately after 2-h exposure to the cells, because of its cell toxicity, while 2-day exposure to NaHS even at 13.5 mM hardly caused cell death (Fig. 1g). The synergistic effect of NaHS at 1.5 mM, a relatively low concentration, in combination with db-cAMP at a subeffective concentration, as observed in the present study, could strengthen significance and/or usefulness of NaHS/H2S as a differentiation-inducer. Physiological significance of NaHS/H2S-induced neuronal differentiation in vivo is still open to question.

T-currents tended to increase in the cells treated with NaHS at 13.5 mM for 2 days (Figs 2a and 4b). Like the delayed induction of HVA channels, the tendency toward delayed induction of T-type Ca2+ channels would be a consequence of NaHS-evoked differentiation rather than causative. As mentioned above, prompt facilitation of T-currents possibly by direct interaction of H2S/NaHS with Cav3.2 protein (Kawabata et al. 2007), even seen in undifferentiated NG108-15 cells, should play more critical roles in the NaHS-induced neurite outgrowth. It is also noteworthy that expression of Cav3.2 at mRNA levels was not different between cells treated with NaHS (differentiated cells) and with vehicle (undifferentiated cells) (Fig. 5g).

It has been reported that the concentration of ascorbate in cerebrospinal fluid is 200–400 μM (Rice 2000), being much higher than its effective concentration used in our study (1 μM). Nelson et al. (Nelson et al. 2007b) have shown that ascorbate inhibition of T-currents is obtained at 1 μM in the Cav3.2-transfected HEK293 cells, whereas 300 μM of ascorbate is necessary for suppression of low-threshold Ca2+ spikes in rat thalamic slice. In this context, the effective concentration of ascorbate in inhibiting T-currents might vary with experimental conditions such as ‘in vitro versus in vivo’ and redox states. Nonetheless, it is open to question whether H2S-induced neurogenesis can be blocked by endogenous ascorbate at physiological concentrations.

In humans, H2S is known to be lethal at 750–1000 ppm in the atmosphere, and the presence of sulfide ion in blood or major organs at 1–5 mg/L or mg/kg (32–160 μmol/L or μmol/kg or 0.0001–0.0005%) might be corroboration of a diagnosis of sulfide-induced death (Milby and Baselt 1999). However, actual toxic concentrations of H2S in the blood or tissues during H2S inhalation in vivo are considered much higher than 5 mg/L or mg/kg, because H2S levels would rapidly decrease immediately after the death. Nonetheless, the effective concentrations (1.5–13.5 mM = 0.005–0.045%) of H2S as a differentiation inducer in the present study are considered quite high, although they were not toxic in NG108-15 cells, as assessed by the trypan blue staining. Further in-depth studies including examination of species differences would be necessary to determine the physiological significance of our findings.

The control values for neurite outgrowth greatly vary with distinct experiments (see Fig. 1). At present, we do not know why such fluctuation always happens, although differences in the passage number, etc. might affect the cell conditions. Nonetheless, the stimulating effect of NaHS on neurite outgrowth was reproducible regardless the extent of neurite outgrowth in the control cells.

In conclusion, our data reveal that NaHS/H2S is a novel inducer of neuronal differentiation in NG108-15 cells, and suggest involvement of T-type Ca2+ channels, especially Cav3.2. Since H2S is an endogenous gas, application of NaHS/H2S might be beneficial or useful to study roles of Cav3.2 T-type channels in neuronal differentiation of various cells including NG108-15 cells.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References