**5. SP-Mediated Anti-Nociception in Muscle**

Accumulating evidence has shown a role for SP in anti-nociception in the PNS, especially in muscle. Despite much evidence indicating that SP can cause cutaneous pain, applying SP to muscle induced neither neurogenic inflammation nor painful perception in humans and rats [54–56]. In contrast, Lin et al. showed that SP had an anti-nociceptive role in muscle rather than causing pain [57]. With whole-cell patch clamp recordings on dissociated muscle-afferent dorsal root ganglion (DRG) neurons, the authors revealed that SP attenuated the acid sensing ion channel 3 (ASIC3)-induced inward current by enhancing M-channel-like potassium current. ASIC3 is a voltage-independent sodium channel activated by the extracellular protons. It has been found as a molecular determinant involved in pain-associated tissue acidosis [58,59]. As well, a recent study showed that ASIC3 can detect extracellular acidification and also respond to mechanical stimuli [60,61].

The in vivo antinociceptive role of SP was demonstrated in a rodent model of chronic widespread muscle pain induced by dual intramuscular acid injections, one of the fibromyalgia pain models developed by Sluka et al., in 2001 [62]. Two injections, separated by one to five days, of pH-4 acidic saline in the unilateral gastrocnemius muscle in rodents produced chronic and bilateral mechanical hyperalgesia of hind paws and muscle that required activation of ASIC3 [63]. Blocking ASIC3 activation, at the first or the second or both acid injections, abolished the induction and development of chronic muscle hyperalgesia. Furthermore, in the dual acid-injection model, the first acid injection could depolarize ASIC3-expressing muscle nociceptors and also simultaneously trigger SP release, which further enhanced the M-channel-like potassium current to attenuate ASIC3-induced depolarization in gastrocnemius muscle-afferent DRG neurons. In mice lacking *TAC1* (no SP and neurokinin A production), chronic pain could be induced by a single acid injection, which suggests that the anti-nociceptive effect was produced by the first acid injection but was diminished with the second injection [57]. The reason for ineffective SP in a second acid injection is still unclear and requires further investigation.

Regarding the acid-induced anti-nociception via SP release, the other important question is what types of acid sensors contribute to the release of SP as an anti-nociceptive acid sensor. The anti-nociceptive acid sensors are still unknown. Although previous study indicated that an acid sensor other than ASIC3 and transient receptor potential cation channel subfamily V member 1 (TRPV1) could trigger SP release [64], the possibility of the co-contribution of ASIC3, TRPV1, and other acid sensors such as other ASIC subtypes and/or proton-sensing G protein-coupled receptors is still not excluded. A recent study demonstrated that low-level laser therapy (LLLT) was effective in reducing mechanical hyperalgesia in the dual acid-injection model. The analgesic mechanism is associated with activation of TRPV1 to release SP in muscle [65]. This study provides new insights regarding the involvement of TRPV1 in acid-mediated anti-nociception. Furthermore, it reveals the involvement of SP in LLLT analgesia, which is widely used in pain control for musculoskeletal pain in the field of physical medicine and rehabilitation. In light of the antinociceptive role of SP in muscle, NK1R agonists might be promising candidates for pain relief in intractable musculoskeletal pain, such as fibromyalgia.

#### **6. Ion Channels Involved in SP Signaling**

SP can modulate a variety of ion channels (Table 1) resulting in an increase or decrease of neuronal excitability [66]. In most studies, SP excites neurons by increasing the function of excitatory ion channels and decreasing that of inhibitory ion channels. For example, SP has been shown to excite neurons by elevating the conductance of sodium channels and decreasing that of potassium channels in locus coeruleus neurons [67]. SP also inhibits inwardly rectifying K+ channels in nucleus basalis neurons via Gq/11 [68,69], and inhibits Ca2+-activated potassium channels [IK(Ca)] in stellate ganglion neurons via pertussis toxin-insensitive G proteins [70]. Other studies showed that SP can inhibit the N-type calcium channel in sympathetic neurons via pertussis toxin-insensitive G proteins [71,72]. The above studies suggest that SP mainly modulates ion channel activity via the G-protein-dependent pathway. However, non-G-protein effector systems are also reported in SP-mediated signaling. Lu

and colleagues revealed that SP-induced increase of sodium conductance was mediated by activating the sodium ion-permeable cation channel complex of NALCN (sodium leak channel, non-selective) and UNC-80 in mouse hippocampal and ventral tegmental area neurons independent of G-protein but mediated by Src family tyrosine kinase [73]. Accordingly, SP can modulate diverse channels and activate the neurons by G-protein-dependent or -independent signaling.

A few studies showed that SP hyperpolarizes neurons in the PNS. SP hyperpolarized vagal sensory neurons of ferrets by inducing a Ca2+-dependent outward potassium current [74]. SP decreased non-selective cation channel conductance in outer hair cells of guinea pig cochlea [75]. SP enhanced the M-type potassium current independent of G-protein but dependent on tyrosine kinase in half of muscle-afferent DRG neurons [57]. Similarly, an SP-mediated Gi/o-dependent pathway could augment the M-type potassium current in DRG neurons and trigeminal ganglion (TG) neurons [76]. Finally, SP could inhibit T-type calcium channels in DRG and TG neurons [77]. Together, SP can modulate a variety of ion channels via different signaling pathways, which are cell type-specific.



Abbreviations: NMDAR, *N*-methyl-*D*-aspartate receptor; Kir, inward rectifier potassium channel; KCa, calciumactivated potassium channel; Ih, hyperpolarization-activated channel; PLC, phospholipase C; PTX, pertussis toxin; PKC, Ca2+/phospholipid-dependent protein kinase; DRG, dorsal root ganglion; TG, trigeminal ganglion; Symbols: ↑, increase; ↓, decrease.
