Commentary: Serotonin Receptor 2B Mediates Mechanical Hyperalgesia by Regulating Transient Receptor Potential Vanilloid 1

Serotonin [5-hydroxytryptamine (5-HT)] is an inflammatory mediator which contributes to inflammatory pain. We previously demonstrated that 5-HTinduced mechanical hyperalgesia is mediated by 5-HT2B, but not by other 5-HT receptors. Our recent article provided further evidence how 5-HT2B regulates 5-HT-induced mechanical hyperalgesia, and suggested, that 5-HT2B mediates mechanical hyperalgesia through Gq/11-phospholipase Cβ (PLCβ)-protein kinase Cε (PKCε) pathway. Interestingly, transient receptor potential vanilloid 1 (TRPV1) also involves in 5-HT2B-mediated hyperalgesia. It was the first evidence that 5-HT receptor regulates TRP channel to affect mechanical hyperalgesia. It is a commentary on the recent article that suggests distinct roles of peptidergic (IB4-negative) and non-peptidergic (IB4-positive) nociceptors in regulating 5-HTinduced mechanical hyperalgesia. In IB4-negative neurons, 5-HT2B in response to 5-HT mediates PLCβ-PKCε to regulate TRPV1 function. In IB4-positive neurons, 5-HT2B may control 5-HT3 or other channels to regulate mechanical hyperalgesia. Commentary: Serotonin Receptor 2B Mediates Mechanical Hyperalgesia by Regulating Transient Receptor Potential Vanilloid 1 Yeu-Shiuan Su1*, and Wei-Hsin Sun1,2* 1Department of Life Sciences, National Central University, Jhongli, Taiwan 2Center for Biotechnology and Biomedical Engineering, National Central University, Jhongli, Taiwan

Serotonin [5-hydroxytryptamine (5-HT)] is known as a neurotransmitter, which is involved in regulating transmission of nociceptive signals in central nerve system 1 . 5-HT is also an inflammatory mediator, released from immune cells, platelets and epithelial cells in the peripheral tissues. Mounting evidence has suggested that 5-HT is a pro-inflammatory and pro-nociceptive agent which can cause pain and hyperalgesia through activating various subtypes of 5-HT receptors which are present in primary afferents 2,3 . We previously demonstrated that 5-HT-induced mechanical hyperalgesia is attributed to 5-HT 2B activation 4 . Our recent article provided more detail mechanism that 5-HT 2B mediates G q/11 -phospholipase Cβ (PLCβ)-protein kinase Cε (PKCε) pathway to control mechanical hyperalgesia in both IB 4 -negative and -positive neurons. Interestingly, transient receptor potential vanilloid 1 (TRPV1) and 5-HT 3 are also regulated by 5-HT 2B to participate in 5-HT-induced mechanical hyperalgesia.

The distinct roles of IB 4 -negative and IB 4 -positive neurons in 5-HT-induced mechanical hyperalgesia
cell-derived neurotrophic factor receptors (GDNF) and P2X3 receptors 6 . IB 4 -negative neurons have low action potential (AP) threshold and shorter AP duration than IB 4positive neurons 7 . IB 4 -negative neurons are more important in transducing information about stimuli. Consistent with that 5-HT injection dramatically enhanced 5-HT-evoked intracellular calcium signals in IB4-negative neurons, but not in IB 4 -positive neurons. Therefore, IB 4 -negative neurons are the major neurons responsible for transducing 5-HT stimuli to induce mechanical hyperalgesia. Blocking 5-HT 2B , PLCβ or PKCε before 5-HT injection inhibited the enhanced calcium signals in IB 4 -negative neurons. It is correlated with behavioral results that blocking of 5-HT 2B -PLCβ-PKCε pathway inhibited mechanical hyperalgesia.
Although IB 4 -positive neurons did no increases intracellular calcium signals after 5-HT injection, the number of the neurons responding to 5-HT was increased. IB 4 -positive neurons with higher density of tetrodotoxin ((TTX)-resistant Na+ channel and longer AP could lead more efficient calcium influx into the presynaptic terminal, resulting in more transmitter release 8 . IB 4 -positive neurons mediating more reliable synaptic connection could participate maintenance of hyperalgesia. As expected, blocking 5-HT 2B , PLCβ or PKCε before 5-HT injection also inhibited the calcium signals in IB 4 -positive neurons.
In IB 4 -negative neurons, 5-HT-induced calcium signals were inhibited by removal of extracellular calcium; while only some of IB 4 -positive neurons were sensitive to calcium-free condition. It indicated that channels allow calcium influx may participate in 5-HT-induced calcium signals. TRPV1 and 5-HT 3 are identified to participate in the downstream of 5-HT 2B -mediated signaling pathway in IB 4 -positive and IB 4 -negative neurons, respectively.

Participation of TRPV1 in 5-HT signaling transduction in IB 4 -negative neurons: contribution to induction of mechanical hyperalgesia
TRPV1 is known as a heat and capsaicin receptor, which is widely expressed in sensory neurons, especially in c-fiber nociceptors. TRPV1 is expressed both in peptidergic and non-peptidergic c-fiber in rat but predominantly in peptidergic c-fiber in mouse by immunohistochemical analysis 9,10 . Although capsaicin-induced calcium signals were greater in IB 4 -positive neurons than in IB 4 -negative neurons before 5-HT injection, capsaicin-evoked calcium signals were significantly enhanced in IB 4 -negative neurons after 5-HT injection. Therefore, capsaicin-sensitive IB 4 -negative neurons may play a role in 5-HT-induced mechanical hyperalgesia under regulation of 5-HT 2B -PKCε.
As confirmed in animal behavioral studies, administration of a TRPV1 antagonist before 5-HT injection in mice inhibited 5-HT-induced mechanical hyperalgesia. Mice lacking TRPV1 genes also showed the absence of mechanical hyperalgesia after 5-HT injection. Even though TRPV1 participates in thermal hyperalgesia, several lines of evidence have also suggested that the involvement of TRPV1 in capsaicin, acid or CFA-induced mechanical hyperalgesia which indicated the involvement of TRPV1 in mechanical hyperalgesia 11,12 . In those studies, TRPV1 is activated either by acid or by capsaicin. Despite that TRPV1 function is enhanced by 5-HT through PKA and PKC phosphorylation 13,14 . However, no evidence demonstrated that TRPV1 can be activated by 5-HT. How 5-HT 2B regulates TRPV1 function remains unclear.
In addition to being activated by allyl-isothiocyanate, capsaicin, acid, noxious heat and the pungent compound in mustard and wasabi, TRPV1 can be activated by some endogenous ligands. Anandamide (AEA), an endogenous fatty acid neurotransmitter derived from arachidonic acid (AA), can bind to and activate cannabinoid CB1 and CB2 receptors 15 . The AEA is generated from N-acylphosphotidylethanolamides (NAPE) through phospholipase C-mediated hydrolysis 16 and is reported to activate TRPV1 17 . The data give one possible way that activation of 5-HT 2 receptor may mediate PLC leading AEA formation to activate TRPV1. Arachidonic acid (AA) is the precursor that can be metabolized by various enzymes. The products of lipoxygenase include 12-and 15-HEPETE, and 5-HETE that are also TRPV1 agonists 18 . 5-HT 2B receptor activation activate phospholipase A2, leading the neuronal secretion of AA 19 . Therefore, peripheral 5-HT 2B activation by 5-HT may relieve phosphatidylinositol 4,5-bisphosphatedependent channel inhibition and generates endogenous ligands AEA or AA to activate and regulate peripheral TRPV1 function, resulting in mechanical hyperalgesia.

Participation of 5-HT 3 in 5-HT-induced calcium signal in IB 4 -positive neurons: contribution to maintenance of mechanical hyperalgesia
The calcium signals in IB 4 -negative neurons were completely dependent on 5-HT 2B -PLCβ-PKCε signaling and TRPV1 activation as described above. 5-HT-induced calcium signals in IB 4 -positive neurons were partially sensitive to removal of extracellular calcium, suggesting that the calcium signals may be from channels in both the plasma membrane and the endothelium reticulum (ER). 5-HT 3 receptor antagonist (Granisetron) specifically inhibited 5-HT-induced calcium signals in a small set of IB 4positive population, explaining sensitivity of these neurons to removal of extracellular calcium. Thus, there are at least two distinct pathways in IB 4 -positive neurons in response to 5-HT stimulation. One is 5-HT 2B -PLCβ-PKCε pathway and the other is 5-HT 2B -PLCβ-PKCε/5-HT 3 pathway.

5-HT 3 was found in pain-related regions and is involved
in pain processing 20,21 . In our previous study, mice with preinjection of 5-HT 3 antagonist did not inhibit mechanical hyperalgesia but shortened the duration of pain after 5-HT injection 4 . Data from the recent article are consistent with the previous study by Lin et al., that 5-HT 3 is not involved in 5-HT-induced mechanical hyperalgesia 1 . However, the shortening of mechanical hyperalgesia suggests that 5-HT 3 may have an influence on modulating the maintenance of hyperalgesia. Stucky and Lewin 7 suggested that IB 4 -positive neurons have a higher action potential (AP) threshold and longer AP duration than IB 4 -negative neurons. Also, there are some reports showing that IB 4 -positive neurons can exhibit sustained responses, but not transient or mixed responses to low pH 22 . Therefore, the responses in IB 4positve neurons that are sensitive to granisetron are thought to be responsible for extending the duration of 5-HT-induced mechanical hyperalgesia.

The GPCR-TRP channel axis in 5-HT-induced pain and itch-like sensation
In previous studies, 5-HT can induce pain and itch sensations in mice and humans 23 . A subset of 5-HTsensitive neurons is also sensitive to histamine and chloroquine, suggesting that these neurons are involved in itch perception 24 . A distinct subgroup of c-fibers that were preferentially excited by pruritic compounds has been reported 25 . 5-HT 2 receptors were reported to respond for 5-HT-induced itch through activating Gq/11-PLC pathway, which leads to mitogen-activate protein kinase (MAPK) and PKC activation 26 . GPCR-TRP channel pathways are the major pathways to itch responses. Similarly, 5-HT-induced mechanical hyperalgesia is also mediated by 5-HT 2B -TRPV1 pathway. Our recent article also provides more detailed mechanisms about transduction and maintenance of pain signals. Transduction of noxious stimulation could be located at IB 4 -positive nociceptors and mediated by 5-HT 2B -5-HT 3 channel axis and 5-HT 2B -second messenger pathways. The similarity of GPCR-TRP channel axis between pain and itch sensation suggests that mechanisms used in pain sensation are possibly involved in itch sensation.

Conclusions
The recent article demonstrated that peptidergic and non-peptidergic nociceptors mediate 5-HT signaling through distinct mechanisms to induce mechanical hyperalgesia. The axis of 5-HT 2B -Gq-PLCβ-PKCε-TRPV1 used in peptidergic neurons contributes to induction of hyperalgesia, while the axis of 5-HT 2B -Gq-PLCβ-PKCε-5-HT 3 in non-peptidergic neurons participates in maintenance of hyperalgesia. The GPCR-TRP channel axis used in pain sensation could be also involved in itch sensation.