- Top of page
- Materials and methods
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.
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.
- Top of page
- Materials and methods
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.