Involvement of Src kinase in T-type calcium channel-dependent neuronal differentiation of NG108-15 cells by hydrogen sulfide

Authors


Address correspondence and reprint requests to Atsufumi Kawabata, Division of Pharmacology and Pathophysiology, Kinki University School of Pharmacy, 3-4-1 Kowakae, Higashi-Osaka 577-8502, Japan. E-mail: kawabata@phar.kindai.ac.jp

Abstract

J. Neurochem. (2010) 114, 512–519.

Abstract

Hydrogen sulfide (H2S), a gasotransmitter, induces neuronal differentiation characterized by neuritogenesis and functional up-regulation of high voltage-activated Ca2+ channels, via activation of T-type Ca2+ channels in NG108-15 cells. We thus analyzed signaling mechanisms for the H2S-evoked neuronal differentiation. NaHS, a donor for H2S, facilitated T-type Ca2+ channel-dependent membrane currents, an effect blocked by ascorbic acid that selectively inhibits Cav3.2 among three T-type channel isoforms. NaHS, applied once at a high concentration (13.5 mM) or repetitively at a relatively low concentration (1.5 mM), as well as ionomycin, a Ca2+ ionophore, evoked neuritogenesis. The neuritogenesis induced by NaHS, but not by ionomycin, was abolished by mibefradil, a T-type Ca2+ channel blocker. PP2, a Src kinase inhibitor, completely suppressed the neuritogenesis caused by NaHS or ionomycin, while it only partially blocked neuritogenesis caused by dibutyryl cAMP, a differentiation inducer. NaHS, but not dibutyryl cAMP, actually caused phosphorylation of Src, an effect blocked by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl, an intracellular Ca2+ chelator, mibefradil or ascorbic acid. The up-regulation of high voltage-activated currents in the cells treated with NaHS was also inhibited by PP2. Together, our data reveal that Src kinase participates in the T-type Ca2+ channel-dependent neuronal differentiation caused by NaHS/H2S in NG108-15 cells.

Abbreviations used:
BAPTA/AM

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl

CaMKII

calmodulin-dependent protein kinase II

db-cAMP

dibutyryl cAMP

FCS

fetal calf serum

HVA

high voltage-activated

TBS

Tris-buffered saline

T-currents

T-type calcium channel-dependent current

TRPA1

transient receptor potential ankyrin-1

Hydrogen sulfide (H2S), a gasotransmitter, is generated endogenously from l-cysteine by cystathionine-γ-lyase and/or cystathionine-β-synthase in mammals (Stipanuk and Beck 1982; Erickson et al. 1990; Bukovska et al. 1994). The concentration of H2S in the mammalian tissue or humor is relatively high (e.g. 10–160 μM in human, rat and bovine brain, and 200–300 μM in rat blood plasma) (Li and Moore 2008), and may increase in certain pathological conditions such as endotoxemia and permanent occlusion of the middle cerebral artery (Hui et al. 2003; Qu et al. 2006). H2S plays extensive and complex roles throughout the mammalian body including the cardiovascular system (Zhao et al. 2001; Kubo et al. 2007; Yang et al. 2008), gastrointestinal tract (Wallace et al. 2007, 2009; Yonezawa et al. 2007; Taniguchi et al. 2009) and neuronal system (Abe and Kimura 1996; Kimura 2000; Kawabata et al. 2007; Nagasawa et al. 2009). H2S targets multiple molecules, such as ATP-sensitive K+ (KATP) channels (Tang et al. 2005), T-type Ca2+ channels (Kawabata et al. 2007), L-type Ca2+ channels (Garcia-Bereguiain et al. 2008), transient receptor potential ankyrin-1 (TRPA1) (Streng et al. 2008), glyceraldehydes-3-phosphate dehydrogenase, actin (Mustafa et al. 2009), etc.

Among three isoforms of T-type Ca2+ channels, Cav3.1, Cav3.2 and Cav3.3, H2S appears to target Cav3.2 (Maeda et al. 2009; Nagasawa et al. 2009) that is abundantly expressed in the primary afferents and participates in transmission of pain information (Jevtovic-Todorovic and Todorovic 2006). Exogenous and endogenous H2S thus facilitates somatic and visceral pain most probably by targeting Cav3.2 (Kawabata et al. 2007; Maeda et al. 2009; Matsunami et al. 2009; Nishimura et al. 2009). T-type Ca2+ channels, particularly of the Cav3.2 isoform, are also involved in neuronal differentiation (Chemin et al. 2002, 2004). Most recently, we have reported that NaHS, a donor for H2S, increases T-type channel-dependent membrane currents (T-currents) in NG108-15 cells, neuroblastoma × glioma hybrid cells (Kawabata et al. 2007), and consequently causes neuronal differentiation of the cells, characterized by neurite outgrowth and functional up-regulation of high voltage-activated (HVA) Ca2+ channels (Nagasawa et al. 2009). In the present study, we investigated signal transduction mechanisms for neuronal differentiation of NG108-15 cells induced by NaHS. Here we show that Src kinase is a downstream signal of the H2S/T-type Ca2+ channel pathway in neuronal differentiation.

Materials and methods

Cell culture and assessment of neurite outgrowth

As described previously (Nagasawa et al. 2009), NG108-15 cells were cultured in high glucose-containing Dulbecco’s Modified Eagle’s Medium (Wako Pure Chemicals, Osaka, Japan) 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). 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 reseeding, the cells were stimulated with NaHS at 13.5 mM, ionomycin at 0.1–0.3 μM or dibutyryl cAMP (db-cAMP) at 1.0 mM. Considering the short half-life time (6.2 min) of H2S and the rapid decay of H2S concentration in the culture dishes (Garcia-Bereguiain et al. 2008), NaHS at a relatively low concentration, 1.5 mM, was also repetitively added at 30-min intervals, three or five times in total, essentially as described previously (Nagasawa et al. 2009). Morphological observation was performed 16 h after the onset of stimulation. Neurite outgrowth was estimated by counting cells with neurites that were longer than the cell body diameter. Inhibitors were applied 30 min before addition of NaHS, ionomycin or db-cAMP in neurite outgrowth determination.

Western blot analysis for detection of Src and phosphorylated Src

NG108-15 cells (1.2 × 105 cells/well) were seeded in 6-well plates, filled with 1 mL of the above medium containing 1% FCS. Three hours after seeding, the cells were stimulated with NaHS at 13.5 mM, db-cAMP at 1 mM for 5 or 30 min. NaHS at 1.5 mM was also repeatedly added at 30-min intervals and the cells were collected 30 min after three-time or five-time applications. Inhibitors were added 30 min before stimulation. After aspirating the medium at scheduled time points for collection of cells, the cells were rinsed with Ca2+-/Mg2+-free phosphate-buffered saline, and lysed in 30 μL of a lysis buffer containing 2% sodium dodecyl sulfate, 62.5 mM Tris–HCl, and 10% glycerol (pH 6.8). The cell lysate was harvested with a cell scraper and exposed to freeze-thawing. The samples were then sonicated for 10 s and denatured by heating at 95°C for 5 min. Protein samples (20–30 μg/lane) were separated by electrophoresis on a 12.5% sodium dodecyl sulfate-polyacrylamide gel (Wako Pure Chemicals) and transferred onto polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation, Billerica, MA, USA). The membrane was blocked with a blocking solution containing 5% skim milk, 137 mM NaCl, 0.1% Tween 20, and 20 mM Tris–HCl (pH 7.6) for 1 h at 20–24°C. After being washed three times with Tris-buffered saline (TBS) containing 0.1% Tween 20, the membrane was incubated with the primary polyclonal antibodies at appropriate dilution with gentle agitation overnight at 4°C. The primary antibodies employed were rabbit anti-Src kinase antibody (1 : 2000) and rabbit anti-phospho-Src family kinase (Tyr416) antibody (1 : 1000) (Cell Signaling Technology, Beverly, MA, USA). After incubation with the primary antibody, the membrane was washed three times with the above-mentioned TBS solution and then incubated with a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Chemicon International, Billerica, MA, USA) as the secondary antibody for 1 h at 20–24°C. The membrane was washed three times with the TBS solution again, and positive bands were detected by the enhanced chemiluminescence staining (GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Whole-cell patch-clamp recording

Whole-cell patch-clamp recordings were performed as described previously (Kawabata et al. 2007; Nagasawa et al. 2009). In the experiments using undifferentiated NG108-15 cells, the cells (1 × 104 cells) were seeded in plastic dishes (35 mm in diameter) coated with poly-l-ornithine, and cultured for a day in the above-mentioned culture medium containing 10% FCS. In the differentiation experiments, the cells were seeded in the medium containing 1% FCS and stimulated with NaHS for 2 days in the absence or presence of PP2 that was added 30 min before application of NaHS. These cells were washed with an extracellular solution for patch-clamp experiments containing (in mM): 97 N-methyl-d-glucamine, 10 BaCl2, 10 HEPES, 40 tetraethylammonium-chloride and 5.6 glucose, adjusted to pH 7.4. Ba2+ currents were recorded from randomly chosen cells at 20–24°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 of 200 ms duration from −120 mV to +40 mV with increments of 10 mV or a voltage ramp of 850-ms duration from −120 mV to +40 mV. T-currents were measured as the peak currents elicited by a square pulse at −20 mV. HVA currents were measured as persistent currents 75 ms after the beginning of a square pulse at +10 mV. 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). In inhibition experiments using ascorbic acid in undifferentiated cells, after the control T-current measurements, ascorbic acid at 1 μM was added 2–3 min before the test T-current measurements, and the test T-current relative to the control T-current was calculated.

Chemicals

NaHS was purchased from Kishida Chemicla Co. Ltd. (Osaka, Japan). l-Ascorbic acid (ascorbate), mibefradil, nitrendipine, glibenclamide, KT5720, U0126 and genistein were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ionomycin, PP2, KN-93 and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl (BAPTA/AM) were provided from Calbiochem (Darmstadt, Germany), db-cAMP and LY294002 were from Fluka (Buchs, Switzerland), and Tocris Cookson Ltd. (Ballwin, MO, USA), respectively. Both ω-conotoxin GVIA and ω-conotoxin MVIIC were obtained from Peptide Inst. Inc. (Osaka, Japan), allyl isothiocyanate was from Tokyo Chem. Ind. Co. Ltd. (Tokyo, Japan), and AP-18 was from Enzo Life Sci. Int., Inc. (Farmingdale, NY, USA). NaHS, ascorbic acid, mibefradil, ω-conotoxin GVIA and ω-conotoxin MVIIC were dissolved in distilled water, and other chemicals were in dimethyl sulfoxide (DMSO) (Sigma-Aldrich).

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

Ascorbic acid inhibition of NaHS-induced sensitization of T-type Ca2+ channels in undifferentiated NG108-15 cells

Given the recent evidence that ascorbic acid inhibits Cav3.2, but not Cav3.1 or Cav3.3 (Nelson et al. 2007), we asked whether ascorbic acid blocks the NaHS facilitation of T-currents, using a whole-cell patch-clamp technique. T-currents were clearly detected and facilitated by NaHS at 1.5 mM in NG108-15 cells (Fig. 1). The facilitating effect of NaHS was abolished by ascorbic acid at 1 μM (Fig. 1), indicating the involvement of Cav3.2, in agreement with our previous evidence for ascorbic acid inhibition of the neurite outgrowth in NG108-15 cells (Nagasawa et al. 2009).

Figure 1.

 NaHS-evoked facilitation of T-type Ca2+ channel-dependent currents (T-currents) and its reversal by ascorbic acid in undifferentiated NG108-15 cells. After the control T-current was measured, NaHS was applied at 2 min. Ascorbic acid, known to selectively inhibit Cav3.2 among three forms of T-type Ca2+ channels, was applied 10 s before addition of NaHS at 1.5 mM or vehicle. Data show the mean with SEM from 4 to 10 different cells.

Mibefradil, a T-type Ca2+ channel blocker, inhibits the neurite outgrowth induced by NaHS, but not by ionomycin, in NG108-15 cells

NaHS at 1.5 mM, applied repetitively at 30-min intervals, five, but not three, times in total, mimicked the facilitating effect of a single application of NaHS at a very high concentration, 13.5 mM, on neurite outgrowth (Fig. 2a), reflecting the rapid decay of NaHS/H2S concentrations in the culture medium, as reported previously (Nagasawa et al. 2009). Interestingly, ionomycin, a Ca2+ ionophore, at 0.1–0.3 μM itself also significantly increased neuritogenesis (Fig. 2b). Mibefradil, a pan-T-type Ca2+ channel inhibitor, at 0.3 μM inhibited the neurite outgrowth induced by NaHS at 13.5 mM (Fig. 2c), but not by ionomycin at 0.1 μM (data not shown). On the other hand, neither the mixture of inhibitors of HVA Ca2+ channels (ω-conotoxin GVIA at 1 μM for N-type; ω-conotoxin MVIIC at 1 μM for P/Q-type; nitrendipine at 1 μM for L-type Ca2+ channels) nor glibenclamide, an inhibitor of ATP-sensitive K+ (KATP) channels, at 30 μM affected the NaHS-induced neurite outgrowth (Fig. 2d and e). AP-18, an inhibitor of TRPA1, at 15 μM exhibited a slight tendency toward suppression of the neuritogenesis (Fig. 2f), whereas allyl isothiocyanate, an agonist of TRPA1, did not induce neurite outgrowth by itself (Fig. 2g), indicating no involvement of TRPA1 in the NaHS-induced neuritogenesis.

Figure 2.

 Mibefradil-sensitive neurite outgrowth caused by NaHS in NG108-15 cells. Proportion of cells with neurites longer than the cell diameter was determined 16 h after the onset of stimulation with NaHS or ionomycin. (a) NaHS was applied at 13.5 mM once or applied at 1.5 mM repetitively at 30-min intervals three or five times in total. (b) Ionomycin, a Ca2+ ionophore, was applied once to the cells. (c–f) Mibefradil, a pan-T-type Ca2+ channel inhibitor, (c) a mixture of inhibitors of HVA Ca2+ channels (HVA inhibitors, ω-conotoxin GVIA at 1 μM for N-type; ω-conotoxin MVIIC at 1 μM for P/Q-type; nitrendipine at 1 μM for L-type Ca2+ channels) (d), glibenclamide (Gliben), a KATP channel inhibitor, (e), or AP-18, a TRPA1 inhibitor, (f) were added 30 min before stimulation with NaHS. (g) Allyl isothiocyanate (AITC), an agonist of TRPA1, had no increase in neurite outgrowth. Data show the mean with SEM from 6 to 12 different experiments. n.s., not significant.

Involvement of Src in the neurite outgrowth induced by NaHS or ionomycin in NG108-15 cells

To clarify signaling mechanisms for the NaHS-evoked neuritogenesis, we performed inhibition experiments. PP2, a Src kinase inhibitor, at 1 μM abolished neurite outgrowth caused by NaHS or ionomycin (Fig. 3a and b), and partially inhibited the db-cAMP-evoked neurite outgrowth (Fig. 3c). In contrast, neither KN-93 at 10 μM, a calmodulin-dependent protein kinase II (CaMKII) inhibitor, nor KT5720 at 1 μM, a protein kinase A inhibitor, notably affected the NaHS-induced neurite outgrowth (Table 1). It is to be noted that KT5720 at 10 μM, a higher concentration, increased the neurite outgrowth by itself (data not shown). U0126, an MAP kinase/ERK kinase (MEK) inhibitor, LY294002, a phosphatidylinositol 3-kinase inhibitor, and genistein, a non-selective tyrosine kinase inhibitor, each evoked neurite outgrowth in the presence and absence of stimulation with NaHS (Table 1).

Figure 3.

 Effects of PP2, a Src inhibitor, on the neurite outgrowth induced by NaHS, ionomycin or db-cAMP in NG108-15 cells. PP2 was added 30 min before stimulation with NaHS, ionomycin or dibutyryl cAMP (db-cAMP) for 16 h. Data show the mean with SEM from 8 (a), 8–12 (b) or 14 (c) different experiments.

Table 1.   Effects of inhibitors on the NaHS-induced neurite outgrowth in NG108-15 cells
StimulatorsInhibitorsProportion of cells with neurites (%)
  1. Cells were stimulated with NaHS, dibutyryl cAMP (db-cAMP) or vehicle (water) for 16 h in the absence or presence of inhibitors. Inhibitors were applied 30 min before stimulation with NaHS or db-cAMP. Data show the mean with SEM from 6 to 18 different experiments. **p < 0.01, *p < 0.05 vs. vehicle + vehicle, p < 0.05 vs. vehicle + NaHS. Statistical analysis was performed by using the Tukey’s test.

Vehicle (water)Vehicle (DMSO)20.3 ± 1.5
Vehicle (water)KN-93 (10 μM)25.9 ± 4.0
Vehicle (water)KT5720 (1 μM)16.9 ± 1.8
Vehicle (water)U0126 (10 μM)44.9 ± 1.7**
Vehicle (water)LY294002 (10 μM)34.7 ± 1.7**
Vehicle (water)Genistein (30 μM)45.7 ± 1.9**
NaHS (13.5 mM)Vehicle (DMSO)34.3 ± 2.1**
NaHS (13.5 mM)KN-93 (10 μM)34.4 ± 7.2*
NaHS (13.5 mM)KT5720 (1 μM)27.1 ± 4.1
NaHS (13.5 mM)U0126 (10 μM)46.8 ± 2.4**,
NaHS (13.5 mM)LY294002 (10 μM)32.2 ± 4.1
NaHS (13.5 mM)Genistein (30 μM)43.7 ± 0.8**
db-cAMP (1 mM)Vehicle (DMSO)42.0 ± 2.6**
db-cAMP (1 mM)KN-93 (10 μM)39.4 ± 2.3**

NaHS causes phosphorylation of Src via activation of T-type Ca2+ channels in NG108-15 cells

As PP2, the Src inhibitor, inhibited the NaHS-evoked neurite outgrowth (see Fig. 3a), we asked if NaHS causes/facilitates activation of Src. A single application of NaHS at 13.5 mM caused clear phosphorylation of Src in 5–30 min, an effect at 30 min being significant (Fig. 4a). NaHS at 1.5 mM, a lower concentration, applied repetitively at 30-min intervals, three or five times in total, also facilitated phosphorylation of Src (Fig. 4b). In contrast, db-cAMP at 1 mM did not lead to notable Src phosphorylation (Fig. 4c).

Figure 4.

 Phosphorylation of Src in response to NaHS, but not dibutyryl cyclic AMP (db-cAMP), in NG108-15 cells. Phosphorylation of Src was determined by western blot analysis of Src and phosphorylated Src. The top panels show the representative data and the bottom panels show the quantified data by densitometry. (a) The cells were stimulated with NaHS at 13.5 mM or vehicle for 5 or 30 min. (b) NaHS at 1.5 mM were repetitively applied at 30 min intervals, one, three or five times in total, and the cells were collected for the analysis 30 min after the final application. (c) The cells were stimulated with db-cAMP at 1 mM for 5 or 30 min. Data show the mean with SEM from 4 (a), 3 (b) or 8 (c) different experiments. **p < 0.01 vs. vehicle. n.s., not significant.

The NaHS-evoked phosphorylation of Src was blocked by BAPTA/AM, a chelator of intracellular Ca2+, mibefradil, an inhibitor of T-type Ca2+ channels, and ascorbic acid that selectively inhibits Cav3.2 among three isoforms of T-type channels (Nelson et al. 2007) (Fig. 5).

Figure 5.

 Effects of BAPTA/AM, mibefradil or ascorbic acid on the NaHS-caused phosphorylation of Src in NG108-15 cells. The cells were stimulated with NaHS for 30 min in the absence or presence of BAPTA/AM, an intracellular calcium chelator, mibefradil, a T-type Ca2+ channel inhibitor, or ascorbic acid, a selective inhibitor of Cav3.2. All three reagents were added 30 min before stimulation with NaHS. Data show the mean with SEM from three to six different experiments.

Participation of Src in the NaHS-induced expression of HVA currents in NG108-15 cells

In addition to neurite outgrowth, expression of HVA currents can be another criterion for neuronal differentiation (Gottmann et al. 1988; McCobb et al. 1989; Chemin et al. 2002), and is induced by NaHS in NG108-15 cells (Nagasawa et al. 2009). We thus asked whether Src participates in NaHS-evoked expression of HVA currents. When the cells were stimulated with a voltage ramp (−120 mV to +40 mV, 850-ms duration) from a holding potential of −90 mV, the currents at +10 mV, mainly attributable to activation of HVA channels, clearly increased in the cells treated with NaHS at 13.5 mM for 2 days (Fig. 6a). Such a current increase was not found in the cells treated with NaHS in combination with PP2, the Src inhibitor (Fig. 6a). The HVA currents, when defined as the currents detected 75 ms after the beginning of a test pulse of +10 mV from a holding potential of −90 mV (Fig. 6b), significantly increased in the cells exposed to NaHS for 2 days (Fig. 6c). This increase in HVA currents was completely suppressed by PP2 (Fig. 6c).

Figure 6.

 Effects of PP2 on the up-regulation of HVA Ca2+ channel-dependent currents in NG108-15 cells exposed to prolonged treatment with NaHS. The cells were treated with NaHS at 13.5 mM for 2 days in the absence or presence of PP2, a Src kinase inhibitor, at 1 μM. PP2 was applied 30 min before treatment with NaHS. The randomly chosen cells were stimulated with a voltage ramp (from −120 mV to +40 mV, 850-ms duration) (a) or a square pulse of +10 mV (200-ms duration) (b, c) from a holding potential of −90 mV. HVA currents (c) were determined as the currents at 75 ms after the beginning of the test pulse (b). Data show the mean with SEM from 32 to 36 (a) and 31 to 39 (c) different cells.

Discussion

The main findings in the present study were that the Src inhibitor, PP2, prevented NaHS-evoked neuritogenesis followed by functional up-regulation of HVA currents and that NaHS actually caused phosphorylation of Src, indicating a critical role of Src signaling in the neuronal differentiation of NG108-15 cells. The Src signal is considered downstream of the NaHS-triggered extracellular Ca2+ influx through T-type Ca2+ channels, especially of the Cav3.2 isoform, as the NaHS-induced phosphorylation of Src was blocked by the intracellular Ca2+ chelator, BAPTA/AM, the pan-T-type Ca2+ channel inhibitor, mibefradil, and ascorbic acid known to selectively inhibit the Cav3.2 isoform of the T-type Ca2+ channels (Nelson et al. 2007). Together, our data suggest that NaHS sensitizes/activates T-type Ca2+ channels, particularly of the Cav3.2 isoform, and subsequently causes the downstream activation of Src, resulting in neuronal differentiation characterized by neurite outgrowth and functional up-regulation of HVA channels in NG108-15 cells (see Fig. 1).

Members of the Src family of tyrosine kinases are involved in cellular processes, including cell growth, neuronal signaling and cell differentiation (Superti-Furga 1995). There is evidence that depolarization-induced neuronal differentiation is mediated by Ca2+-dependent activation of Src in PC12 cells (Banno et al. 2008), being consistent with the neuronal differentiation of NG108-15 cells mediated by Ca2+-dependent activation of Src following activation of T-type Ca2+ channels by H2S in the present study. However, it is to be noted that CaMKII participates in activation of Src in PC12 cells (Banno et al. 2008), whereas the NaHS-evoked neurite outgrowth was resistant to the CaMKII inhibitor, KN-93, in NG108-15 cells (see Table 1). The precise mechanisms by which intracellular Ca2+ activates Src in NG108-15 cells have yet to be clarified. The finding that PP2 partially inhibited the db-cAMP-caused neurite outgrowth (see Fig. 3c), might imply some contribution of Src to the db-cAMP-induced neuronal differentiation of NG108-15 cells. However, our results that db-cAMP was incapable of increasing phosphorylation of Src (see Fig. 4c) suggest that Src is not downstream of the db-cAMP-triggered signals. As some phosphorylated Src was detected in the vehicle-treated cells (see Fig. 4), the db-cAMP-induced neurite outgrowth might require the background activation of Src. As the IC50 value of KN-93 is 1 μM according to the manufacturer’s information, the concentration, 10 μM, used in the present study should be enough to inhibit CaMKII, suggesting no involvement of CaMKII in the NaHS/H2S-triggered neuronal differentiation. The IC50 value of KT5720 is 56 nM in the presence of low concentrations of ATP, but 3 μM in the presence of high concentrations of ATP. In the present experiments, KT5720 had no effect at 1 μM and enhanced neurite outgrowth by itself at 10 μM, suggesting no contribution of protein kinase A to the NaHS/H2S-triggered neuronal differentiation in NG108-15 cells. It is clear from our inhibition experiments (see Fig. 2) that the NaHS-evoked neuritogenesis in NG108-15 cells is not mediated by known H2S-targeted molecules other than T-type Ca2+ channels, such as KATP channels (Tang et al. 2005), HVA Ca2+ channels (Garcia-Bereguiain et al. 2008) and TRPA1 (Streng et al. 2008). It is to be noted that TRPC5 channel activity is also implicated in neurite outgrowth in NG108-15 cells (Wu et al. 2007), although its involvement in the NaHS-evoked effect is still open to question.

In our study, the neurite-outgrowth effects of NaHS varied among experiments (see Figs 2 and 3. Such fluctuations in the extent of neurite outgrowth, however, often occurs in distinct experiments, even after stimulation with not only NaHS but also db-cAMP, a well-known differentiation-inducer, as reported previously (Nagasawa et al. 2009). Although we still do not know the exact reason, the differences in cell conditions including the passage number of the cells might affect the levels of expression of the key molecules, such as Cav3.2 channels and Src, that are responsible for the neurite outgrowth.

Our previous study has demonstrated that a single application of NaHS even at 13.5 mM is not toxic in NG108-15 cells, and that NaHS, when repetitively applied at 1.5 mM at 15-min or 30-min intervals, nine times in total, causes neuritogenesis equivalent to that caused by a single application of NaHS at 4.5–13.5 mM (Nagasawa et al. 2009). In the present study, we found that even five-time repetitive applications of NaHS at 1.5 mM at 30-min intervals were capable of causing neurite outgrowth (see Fig. 2a) and that repetitive, but not single, applications of NaHS at 1.5 mM caused phosphorylation of Src, as the single application of NaHS at 13.5 mM did (see Fig. 4a and b). H2S concentration rapidly decays in cell culture dishes possibly mainly because of vaporization, and the half-life is about 6.2 min in such conditions (Garcia-Bereguiain et al. 2008). Hence, the interval, 30 min, for repeated addition of NaHS may be still too long to maintain the effective concentration of NaHS, and we assume that the neurite outgrowth might be induced by NaHS at concentrations even lower than 1.5 mM in vivo in which the decay time should be prolonged. In neutral solution at 37°C, 18.5% of NaHS exists as H2S (Li and Moore 2008), indicating that the concentration of H2S in 1.5 mM NaHS solution is approximately 300 μM. This is comparable to the reported physiological concentration of H2S in the mammalian body (10–300 μM) (Li and Moore 2008). Nonetheless, considering that the concentration of H2S in human, rat and bovine brain is 10–160 μM (Garcia-Bereguiain et al. 2008), H2S might not contribute to normal neuronal development under physiological conditions. There is abundant evidence that H2S formation is dramatically elevated following up-regulation of H2S-forming enzymes in response to inflammatory stimuli (Li and Moore 2008; Nishimura et al. 2009; Wallace et al. 2009). Therefore, we hypothesize that the H2S-evoked neurite outgrowth/neuronal differentiation might be involved in the healing and/or development of inflammatory or traumatic diseases in the central and peripheral nervous systems. In addition to the facilitating effect of H2S on neuritogenesis, as described in the present study, H2S plays various roles in modulation of neuronal functions; e.g. H2S is involved in hippocampal long-term potentiation, neurodegeneration/neuroprotection and processing of somatic and visceral pain (Kawabata et al. 2007; Li and Moore 2008; Maeda et al. 2009; Matsunami et al. 2009; Nishimura et al. 2009; Kimura 2010). H2S-targeted multiple signaling molecules including T-type Ca2+ channels would mediate those distinct effects of H2S (Li and Moore 2008; Kimura 2010). Thus, the present study demonstrating that NaHS/H2S causes neuronal differentiation via the Cav3.2/Ca2+/Src cascade in NG108-15 cells, would add a novel aspect for understanding of the neuronal functions of H2S.

Acknowledgement

This work was supported in part by ‘Antiaging Center Project’ for Private Universities from Ministry of Education, Culture, Sports, Science and Technology, 2008-2012.

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