Our study demonstrates for the first time that endogenous H2S producing enzyme cystathionine β-synthase (CBS) is abundantly expressed by trigeminal ganglion neurons and co-localized in Kv1.1 and Kv1.4 positive neurons (Figure 1). CBS and systathionine γ-lyase (CSE) are two important endogenous enzymes for generation of H2S in mammals, and have been found in many types of mammalian cells in the central nervous system as well as in the peripheral tissues [28–30]. We have previously reported that CBS was predominately expressed by small and medium sized dorsal root ganglion neurons and co-localized well with purinergic P2X3 receptors and TRPV1 in colon specific neurons, suggesting a role for CBS in visceral hypersensitivity . In this study, we showed that administration of NaHS, which mimics CBS production of H2S, reduced mechanical escape threshold in a dose-dependent manner (Figure 5), indicating that endogenous CBS-H2S signaling pathway might be involved in the trigeminal nociceptive processing.
H2S, formed by these two enzymes, has been found to regulate key neuronal functions, including induction of long-term potentiation and modulation of NMDA receptor currents in the hippocampus under physiological conditions . In addition, H2S donor NaHS enhanced the excitability of dorsal root ganglion neurons in vitro. In this study, we provide direct evidence that H2S donor NaHS sensitized TG neurons. This conclusion is based on several findings shown in Figures 2 and 3. First, NaHS application led to a marked depolarization of RP of TG neurons (Figure 2B). Secondly, these neurons exhibited lower current thresholds for initiating an action potential (AP, Figure 2C) and lower AP thresholds (Figure 2D). Finally, these neurons had an enhanced firing frequency in response to a standardized current stimulation after NaHS application (Figure 3). Together with our previous report that H2S enhanced the neuronal excitability of colon-specific DRG neurons , the present study indicates that H2S modulates membrane properties of rat primary sensory neurons.
Another interesting finding is that NaHS application significantly suppressed IK density in TG neurons (Figure 4). To the best of our knowledge, it is the first report that H2S play an inhibitory effect on voltage-gated potassium channels of TG neurons. At least 6 types of K+ currents have been detected in sensory neurons; including both rapidly inactivating, A-type K+ currents (IA) and slow-inactivating sustained (K-current; IK) [20, 21], and several different isoforms of K+ currents with different kinetics, and their related protein subunits, have been identified in primary sensory neurons. These voltage-gated K+ (Kv) channels are important physiological regulators of membrane potentials, action potential shape and firing frequencies in nociceptive sensory neurons and often shown to be decreased during injury-induced hyperalgesia [33, 34]. Since the opening of K+ channels leads to hyperpolarization of cell membrane and a consequent decrease in neuronal excitability , the reduction in IK density induced by NaHS may well contribute to the enhanced excitability of TG neurons. Although we do not know the intracellular pathway(s) for these effects of H2S, the acute inhibition of channel activity would likely explain these effects. However, other possibilities such as regulation of protein trafficking or endocytosis cannot be excluded. In addition, amplification of TRPV1 currents by H2S occurs via releasing tachykinins , and if this is also the pathway for H2S’s modulation of IK, then H2S acts on different receptors to activate separate intracellular pathways that converge on common targets. Their combined actions in the setting of peripheral tissue injury or inflammation probably ensures a strong, acute and prolonged response in nociceptor firing. When added to the amplified response of TRPV1 or purinergic receptors caused by H2S, and possibly others, e.g., nerve growth factors, the net effect will integrate a heightened transduction of nociceptive stimuli with reduced threshold and elevated repetitive firing to produce a powerful sensitization of TG nociceptors. Of note is that we do not know whether extracellular or intracellular or both sides of H2S mediate the modulation of potassium channels since H2S are membrane permeable. Future studies are needed to investigate the detailed mechanisms.
In addition to the effect of IK, the contribution of other K+ channels to the enhanced excitability of TG neurons has also to be considered. Transient K+ currents (IA) have been reported to be one of the major players in control of neuronal excitability . In this study, however, NaHS incubation had no effect on IA, indicating a specific effect of H2S. TG neurons after NaHS application depolarized resting membrane potentials without changes in membrane input resistance. Changes in RP may be mediated by modulation of hyperpolarization-activated cation current (IH; an inwardly rectifying current)  or leakage current (IL; an outward resting current) . However, the depolarization of RP in our study is unlikely due to an increase in IH, as this would be expected to result in a decrease in input resistance. Thus, depolarization of RPs in TG neurons after NaHS application may likely have resulted from a decrease in IL. However, in our experiments, when the RPs of TG neurons were corrected to the normal level (i.e., -55 mV) after NaHS application, the rheobase and cell spike frequency were not significantly changed compared with those before the correction of RPs (data not shown). Thus, even if there were changes in IL, they had a minimal effect on spike frequency and current threshold in this study. At this time we cannot rule out the possibility that H2S modulates other voltage-gated ion channels, including NaV and the various Ca2+ channels. Previous studies have shown that NaHS/H2S activates or sensitizes Cav3.2 T-type Ca2+ channels expressed in the primary afferents  and TRPV1 and TRPA1 in nonvascular smooth muscles . H2S is now thought to join the set of agents that enhance excitability; it too enhances TRPV1 currents and suppresses IK. These combined changes in the activities of different ion channels will have a strong, possibly synergistic effect in enhancing excitability, particularly for inducing repetitive firings to produce a powerful sensitization of TG nociceptors under pathophysiological conditions such as TMJ inflammation.
In conclusion, our data demonstrate that H2S enhanced the neuronal excitability and suppressed the IA potassium current density, indicating that CBS-H2S signaling pathways may play a role in trigeminal physiology and pathophysiology. Previous studies in different trigeminal nerve inflammation/injury models suggested that the hyperexcitability of primary afferent neurons contributes to the hyperalgesia and allodynia. Further experiments are warranted to determine changes in CBS-H2S signaling under chronic pain conditions such as temporomandibular joint and related disorders.