**2. SP-Mediated Signaling**

There are three tachykinin receptors: Neurokinin 1 (NK1), 2 (NK2), and 3 (NK3). SP preferentially binds to NK1 receptor (NK1R). NKRs are G-protein-coupled receptors located in both the central

nervous system (CNS) and peripheral nervous system (PNS). G proteins have Gα, Gβ, and Gγ subunits. The Gα subunit is classified into 5 families (Gs, Gi, Go, Gq/11, and G12/13). In most cases, NK1R is coupled to the pertussis toxin-insensitive Gq/11 cascade [12,13]; nevertheless, crosstalk with other G proteins such as G12/13 [14], Go, and Gs [15] has been reported. In Gq/11-mediated signaling, phospholipase C is activated by Gβγ binding, which in turn results in hydrolysis of membrane phospholipid PtdIns(4,5)P2 to form diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 further triggers calcium release in cytoplasm from sarcoplasmic reticulum, whereas DAG activates protein kinase C (PKC) and results in calcium influx from the L-type calcium channel in the plasma membrane [16]. The increase in calcium ion in the cytoplasm further leads to an array of cell responses.

There are two natural forms of NK1R: A full-length receptor with 407 amino acids and a truncated receptor with 311 amino acids, lacking 96 amino acids in the carboxyl terminal [17,18]. The human *NK1R* gene contains five exons. The truncated version is generated when the intron between exons 4 and 5 is not removed that encounters a premature stop codon before the start of exon 5. These two types of NK1Rs have differential features. The binding affinity of SP is 10 times less to the truncated NK1R than the full-length form. Furthermore, the underlying signaling of the two different NK1Rs differs. The carboxyl terminus of full-length NK1R is a crucial element for G-protein coupling. The truncated form of NK1R impairs the ability of G-protein binding and results in G-protein-independent mechanisms. Indeed, the truncated form of NK1R fails to interact with β-arrestin, an important protein mediating the desensitization and internalization of activated G-protein-coupled receptors [19]. Furthermore, activation of the truncated NK1R has different effects on calcium mobilization, phosphorylation of PKCδ, extracellular signal-regulated kinase 1/2, and regulation of interleukin 8 mRNA expression as compared with the full-length NK1R [20].

The two NK1Rs are differentially distributed in peripheral tissues and in the nervous system. In the brain, full-length form is abundantly expressed in striatum, caudate nucleus, putamen, globus pallidus, nucleus accumbent, and hypothalamus; whereas the truncated NK1R expression is relatively low in the brain, and most represented in the PNS and peripheral tissues including heart, lung, prostate, and bone [21]. To sum up, the different NK1R subtypes could trigger differential effector systems and further result in distinct cellular responses in different tissues and organs.

#### **3. SP and Pain**

Pain is an unpleasant sensation and can be divided into two major categories: Acute and chronic pain. Acute pain lasts only short time and can be ameliorated over time. Acute pain helps us avoid the physical damage to our body and serves as a warning signal. In contrast, chronic pain has no biological function and is a disease itself rather than symptom of a disease. Chronic pain usually lasts for more than six months. People with chronic pain represent about 10.1% to 55.2% of the population in various countries according to epidemiological study [22]. People with chronic pain often show other symptoms, such as fatigue, sleep disorder, memory problems, anxiety, and depression, which greatly affect their quality of life.

Chronic pain can be further divided into two subtypes: Inflammatory and neuropathic pain. Inflammatory pain is related to tissue injuries, which lead to inflammatory reactions. In contrast, neuropathic pain results from nerve injury and neuronal sensitization in the CNS and PNS. However, inflammatory pain and neuropathic pain are also closely correlated. Nerve injuries result in tissue inflammatory responses, which lead to inflammatory pain. Likewise, tissue inflammation triggers an inflammatory cascade releasing a variety of inflammatory mediators such as SP, calcitonin gene-related peptide, and neurokinin A to damage nerves [23,24]. Clinical studies have demonstrated that many forms of chronic pain have mixed components of inflammation and neuropathy [25].

Substantial evidence suggests that SP is the key element in neurogenic inflammation and has an important role in eliciting pain sensation in both the CNS and PNS. In the CNS, SP results in central sensitization by activating excitatory post-synaptic potential [26]. In the PNS, releasing SP from peripheral nociceptive nerve fibers can cause neurogenic inflammation in the skin [27,28]. In addition, people with fibromyalgia, a chronic pain disorder, showed elevated SP level in cerebrospinal

fluid [29,30]. Chemical ablation of neurons expressing SP receptors in lamina I [31] and genetic disruption of the encoding gene of SP [32] or its receptor [33] reduced pain responses. Furthermore, a recent study showed that selectively ablating *TAC1*-expressing neurons in the spinal cord abolished the sustained pain but not the reflexive defensive response [34]. Together, these studies support that SP is an important signal molecule in pain transmission.

Thus, many studies have focused on developing selective NK1R antagonists as a potential analgesic drug. Although several preclinical studies showed an anti-nociceptive effect of NK1R antagonists, most clinical trials failed to show analgesia effects. The reasons for failure of most NK1R antagonists in clinical trials are still elusive. Several reasons for discrepancy between preclinical and clinical results have been proposed such as species differences in NKRs distribution [35], species differences in affinities to antagonists for NK1R [36–38], and the ability of animal models in predicting clinical pain [39]. The discovery of an antinociceptive effect of SP may also explain in part the failure of those clinical trials [10]. One hypothesis is that neurons innervating distinct locations may respond differently to SP—some neurons excited by SP and some inhibited by SP. Thus, elucidation of SP-mediated responses in different tissues and organs becomes a crucial step in developing specific NK1 antagonists as analgesic drugs.
