Capsaicin selectively stimulates nociceptive neurons and has been widely used to study pain-related events. In this topic, we will highlight some aspects of how capsaicin induces pain and its importance to the current understanding of neuronal mechanisms of pain.

Before the discovery of the capsaicin-activated receptor, intradermal injection of capsaicin was used to produce primary and secondary hypersensitivity to noxious and innocuous stimuli in both monkeys and rats [ 22 23 ]. Seminal works demonstrated that capsaicin excites nociceptors by increasing the influx of ions, such as calcium, in dorsal root ganglion (DRG) neurons [ 24 25 ]. Years later, cloning transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor shed light on the mechanism by which capsaicin induces pain [ 26 ]. This work is a landmark in the mechanisms of pain since demonstrated that capsaicin induces pain-like behavior by activation of TRPV1 receptors expressed by nociceptors. At that time, TRPV1 receptors were denominated vanilloid receptor 1 (VR1) [ 26 ]. More importantly, this discovery has changed our understanding of pain mechanisms since it demonstrates that a receptor-coupled channel expressed by nociceptors detects environment stimuli resulting in nociceptor depolarization and consequently producing pain. Also, this discovery opened avenues to the development of new drugs since Mendelian disorders in these proteins can produce pain [ 27 ]. After that, in vivo evidence demonstrated that mice lacking TRPV1 receptors exhibit reduced thermal noxious response and capsaicin-induced paw licking [ 28 ]. Whole patch-clamp technique demonstrated that mice lacking TRPV1 receptors present impaired calcium influx in DRG neurons [ 28 ]. Therefore, administration of capsaicin in animals was important to elucidate the function of TRPV1 as well as to aid our knowledge about pain processing and modulation. Therefore, the discovery of TRPV1 was essential to validate capsaicin-induced pain models, which can now be used to study neuronal mechanisms of pain, in addition to testing new TRPV1 antagonists and drugs that target the consequences of TRPV1 activation before clinical trials.

2.2. Mechanisms of Capsaicin-Induced Pain

One of the first evidence of a selective action of capsaicin on C-polymodal nociceptors was obtained by the capsaicin-evoked response of C-fibers in the cat saphenous nerve. In addition, injection of capsaicin reduces the thermal threshold in both rats and humans [ 29 ]. This seminal work demonstrates that capsaicin selectively acts on C-polymodal nociceptors and the thermodependency of sensory effects on animals and humans [ 29 ]. Spinal cord mechanisms of capsaicin-evoked mechanical allodynia depend on G-protein and protein kinases (PKA and PKC) and could be reversed by both G-protein and protein kinase inhibitors. For instance, kinase activity may result in an increase of receptor activity as well as an increase of trafficking and cell-surface expression of molecules [ 23 ]. In fact, capsaicin activates PKA and PKC that phosphorylate NMDA receptor subunit NR1 at serine residue 890 and 897, and serine residue 896, respectively, which enhances receptor activity [ 30 31 ]. Alongside with this, mitogen-activated protein kinase (MAPK) family has been involved in pain-related states and, indeed, capsaicin administration increases the phosphorylation of p38 MAPK in the periphery and spinal cord dorsal horn [ 32 ]. Therefore, inhibition of these kinases has helped to define some of the intracellular mechanisms involved in capsaicin-induced central sensitization. In addition to these kinases, the neuropeptide CGRP is another important component in central sensitization. Capsaicin-induced TRPV1 activation stimulates the release of CGRP in the spinal cord, and intrathecal treatment with CGRP antagonist reduces the development and maintenance of mechanical hyperalgesia and secondary allodynia [ 33 ].

N-tert -butylnitrone) reduces the activation of neurons in the dorsal horn as observed by the reduction of electrophysiological activity detected by the number of neuronal spikes [ Capsaicin-induced pain model was also useful to demonstrate the role of reactive oxygen species (ROS) in central sensitization. Despite their pro-hyperalgesic effect per se [ 34 35 ], ROS can also be a source of post-translational modification due to their action on redox-sensitive protein residues such as cysteine and serine [ 36 ]. In fact, treatment with the ROS scavenger Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) and PBN (-butylnitrone) reduces the activation of neurons in the dorsal horn as observed by the reduction of electrophysiological activity detected by the number of neuronal spikes [ 37 ]. As a consequence of that, there is reduction of primary and secondary hyperalgesia, and reduction of neuron responsiveness induced by capsaicin, suggesting a role of ROS in the maintenance of persistent pain [ 37 ]. Keratinocytes are in proximity to nociceptors, which may imply a role for these cells in pain. Using Cre-lox technique to promote expression of TRPV1 in keratinocytes demonstrated that capsaicin stimulates TRPV1-expressing keratinocytes inducing c-fos expression in laminae I and II of the ipsilateral spinal cord dorsal horn, which contributes to evoke acute paw-licking nociceptive behavior [ 38 ]. This addresses the interaction between keratinocytes and nociceptors in pain-state.

Capsaicin has helped us to understand the mechanisms related to abdominal pain, a condition inherent of patients with irritable bowel syndrome (IBS). Intracolonic injection of capsaicin induces abdominal mechanical hyperalgesia, and pain-related behaviors such as abdominal licking in a morphine-sensitive manner suggesting its nociceptive nature instead of a normal grooming behavior [ 39 ]. IBS patients present abdominal mechanical hyperalgesia and allodynia [ 40 ]. Nociceptive fibers present in the colon respond to TRPV1 agonist, and, therefore, highlight these receptors as potential targets for abdominal pain [ 35 ]. In fact, TRPV1 co-localizes with substance P and calcitonin gene-related peptide (CGRP) in a model of DSS (dextran sulfate sodium)-induced colitis. Substance P and CGRP are two important neuropeptides in pain signaling that together with TRPV1 mediate visceral pain [ 41 ]. This is important considering that TRPV1/CGRP pathway is considered an attractive pharmacological approach to treat visceral pain [ 42 ]. In vivo functional magnetic resonance imaging (fMRI) further corroborates the importance of TRPV1 receptors and demonstrates the activity of supraspinal mechanisms in capsaicin-induced pain. Injection of capsaicin in wild-type (WT) rats activates putative pain neural circuit, such as Papez circuit, and the habenular system; and TRPV1 receptor deficiency reduces the activation in these same brain regions in response to capsaicin [ 43 ]. This is important since it points out to the supraspinal modulation of TRPV1 in pain. And additionally to these mechanisms, TRPV1 also modulates the emotional component of visceral pain [ 44 ]. Modulation of TRPV1/CGRP pathway is important in arthritis as well [ 45 ]. In fact, intra-articular injection of CGRP in normal or mono-iodoacetate (MIA)-induced arthritis rats reduces the mechanical threshold and increases percentage of sensitized fibers [ 46 ], and treatment with CGRP antagonist reduces CGRP- and MIA-induced sensory neuron firing [ 46 ], suggesting that peripheral release of CGRP contributes to inflammation and sensitization of joint nociceptors [ 45 ].

In the past few years, efforts have been made to identify ligand-receptor and receptor-receptor interactions and their role with pain. Among the first interactions that were shown, we can highlight the capsaicin-TRPV1. In fact, co-administration of capsaicin with QX-314 (a membrane-impermeable sodium channel blocker) facilitates the access of the QX-314 that blocks sodium inward currents in capsaicin-responsive DRG neurons producing analgesia [ 47 ]. Nevertheless, in this work, neither the potentially dynamic of TRPV1 permeability to different ions size or charges (unknown at the moment), nor the effect of pore size of the TRPV1 was addressed. TRPV1 receptor was considered a nonselective cation channel with higher affinity for calcium than sodium. TRPV1 agonists such as capsaicin, changes TRPV1 pore size leading to time-dependent discrimination between monovalent and divalent cations over a time frame of seconds that can persist for several minutes [ 48 ]. Another striking feature was that phosphorylation of TRPV1 serine 800 residue by PKC allows neurons to discriminate the size of cations by increasing permeability to large cation, and proportionating sensitization of the TRPV1, and enhancement of inward currents [ 48 ]. In fact, PKC phosphorylates TRPV1 at serine 800 residues, but not at serine 502, in DRG neurons of rats and contributes to pain in MIA-induced osteoarthritis model [ 49 ] ( Figure 2 ). Inhibition of PKC, but not PKA, reduces capsaicin-induced pain-related behavior in MIA-induced osteoarthritis rats [ 49 ]. TRPV1 agonists such as N-arachidonoyldopamine (NADA), piperine and resiniferatoxin (RTX) provide distinct pattern of ion selectivity and discrimination [ 48 ]. Thus, suggesting that different TRPV1 agonist change the selectivity to inward ions, and the activity of different kinases (such as PKA and PKC) [ 48 49 ] could provide different inward ion. Recent data further advanced in this topic by demonstrating that capsaicin binds to TRPV1 pocket as a unique molecule [ 50 ].

Capsaicin has a very high affinity, sensitivity, and selectivity for TRPV1 and does not activate the homologous TRPV2–TRPV6 receptors [ 50 ]. In addition, an elegant work demonstrated how capsaicin binds to TRPV1 and which amino acid residues are involved in this binding. Capsaicin binds to TRPV1 in a “tail-up, head-down configuration” (as coined by the authors). The aliphatic “tail” interacts with the channel through nonspecific van der Waals forces and contributes to binding affinity. Hydrogen bonds between its vanillyl “head” and amide “neck” with residues of glutamic acid E571 and T551 of the channel, respectively, grant specificity for ligand binding [ 50 ] ( Figure 3 ). Other interactions with TRPV1, such as Tyr511, Glu570, and Ile569; with the vanillyl “head” allows capsaicin accommodation in this specific pocket (called as vanilloid pocket). On the other hand, RTX (a TRPV1 agonist) molecule is bigger than capsaicin, and possesses a different electron cloud, which does not allow its accommodation in the same vanilloid pocket because this pocket is too shallow for RTX [ 53 ]. Therefore, this spatial allocation of both molecules accounts to the distinct agonist pattern and potency explaining the increased potency of RTX compared to capsaicin [ 53 ]. In addition to the spatial allocation, structure-activity relationship study demonstrates the functional groups that are essential to these difference. For instance, the amide group is essential for capsaicin activity, while for RTX the five-membered diterpene ring fulfills this role [ 54 ]. These studies had an enormous impact because they demonstrated the fundamental pockets to capsaicin or other agonist binding and activation of TRPV1. Therefore, these studies enable future pharmacological approaches based on this knowledge since these agonists can act both as pro-hyperalgesic and anti-hyperalgesic as we will discuss in the next topic.

Regarding receptor-receptor interaction, TRPV1-TRPA1 is a well-documented one [ 58 ]. This interaction is attributed to the formation of a heterodimer between TRPV1-TRPA1 receptors [ 59 ], which is possible due to lipid raft movement and formation of a cluster of receptors in neurons [ 60 ]. Recent evidence demonstrated that a trans-membrane receptor called Tmem100 is co-expressed with both TRPV1-TRPA1 complex in DRG neurons and is essential to modulate their activity by acting as an adaptor molecule [ 51 ]. Nevertheless, forming TRPV1-TRPA1 complex without Tmem100 is also possible [ 51 52 ]. In the TRPV1-TRPA1 complex without Tmem100, TRPV1 inhibits TRPA1 activity since TRPV1-TRPA1 positive DRG neurons present reduction of inward current after mustard oil (TRPA1 agonist) as stimulus, but not to capsaicin. On the other hand, in the presence of Tmem100 TRPV1 increases TRPA1 activity and potentiates pain perception [ 51 ] ( Figure 2 ). Additionally, TRPA1-initiated calcium influx promotes PKA activation, thereby sensitizing TRPV1 channels [ 61 ].

Therefore, there is a complex interaction of capsaicin and other agonists with TRPV1 that shed light in the complex pathway to understand TRPV1 modulation. TRPV1 crosstalks with other receptors build up an entirely different pharmacology adding up complexity.