Chapter 5 Nociceptive Signals to TRPV1 and its Clinical Potential

Publisher Summary

Nociceptive neurons are a peripherally located subset of sensory neurons that transmit pain signals to the spinal cord. Pain signal conducting sensory neurons are classified as unmyelinated or as small‐myelinated nerve fibers as compared with the large, rapidly conducting fibers that transmit tactile information. Nociceptive signals originate in nociceptive sensory neuron terminals, where many ion channels and receptors are expressed to transduce various stimuli to neural signals. These receptors or channels are activated by specific stimuli and serve as molecular sensors. The chapter presents different types of endogenous activators or endovanilloids and provides structural comparisons between endovanilloids and capsaicin. Mutagenic studies on transient receptor potential vanilloid 1(TRPV1) have identified three ligand‐binding sites. TRPV1 is implicated in the mediation of inflammatory pain; the signaling pathways that link TRPV1 and inflammatory mediators, such as bradykinin (BK) and histamine, are discussed in the chapter. Because of its implication as a molecular target for a new class of analgesics, TRPV1 antagonists are used in pharmaceutical companies as development bases for potential analgesics.

Introduction

Nociceptive neurons are a peripherally located subset of sensory neurons that transmit pain signals to the spinal cord. Pain signal conducting sensory neurons are classified as unmyelinated or as small‐myelinated nerve fibers, as compared with the large, rapidly conducting fibers that transmit tactile information. Nociceptive (painful) signals originate in nociceptive sensory neuron terminals, where many ion channels and receptors are expressed to transduce various stimuli to neural signals. These receptors or channels are activated by specific stimuli and serve as molecular sensors. Advances in basic biomedical technology have found that many ion channels are specifically activated by different types of stimuli. Of the channels discovered to date, TRPV1 has been studied most extensively because of its functional significance in the pain sensory system. In the present chapter, different types of endogenous activators or endovanilloids are introduced, and structural comparisons between endovanilloids and capsaicin are discussed. Mutagenic studies on TRPV1 have identified three ligand‐binding sites. One of these sites is located in transmembrane domain 3 and the other two are represented by Arg 114 and Glu 761 in N‐ and C‐termini, respectively. The critical region in transmembrane domain 3 is conserved in the capsaicin‐sensitive orthologs of some mammalian TRPV1s, whereas the latter two sites are highly conserved throughout species whether or not the TRPV1 orthologs are sensitive to capsaicin.

Because TRPV1 is implicated in the mediation of inflammatory pain, we discuss the signaling pathways that link TRPV1 and inflammatory mediators such as bradykinin (BK) and histamine. Because of its implication as a molecular target for a new class of analgesics, TRPV1 antagonists have drawn the attentions of pharmaceutical companies as development bases for potential analgesics. In the present chapter, we introduce some newly developed TRPV1 antagonists.

Hot chili peppers are a popular food additive. They were first grown in South America, and were transported to the Western World during the 15th century (Szolcsanyi, 1993). The major ingredient of peppers when first extracted was called “capsicol,” which was then described to have a “neuro‐selective” action because of its specificity for sensory nerves (Szolcsanyi, 1993). The canonical action of capsaicin in terms of its excitation of sensory nerves was well described by a group of Hungarian scientists, Jancso and Szolcsanyi (see also Chapter 4). Capsaicin, a vanilloid analog, as it possesses the vanillin moiety (Fig. 1), causes severe pain and pain‐related reflexes in man and animals (Simone 1987, Simone 1989). The pain elicited by capsaicin is due to its excitatory action on sensory neurons. Capsaicin excites a subset of sensory nerve fibers, especially, unmyelinated (C‐) or small myelinated (Aδ‐) fibers that are considered to mediate the sensation of pain (Szolcsanyi, 1993). Thus, capsaicin is also called an “excitotoxin” or a “neurotoxin” (Winter 1990, Oh 1996, Holzer 1998).

The excitation of sensory neurons by capsaicin suggests the presence of cation channels that are activated by capsaicin. In early biochemical studies it was found that capsaicin caused influxes of Rb⁺ or Ca²⁺ in cultured dorsal root ganglia (Wood et al., 1988). The actual currents activated by capsaicin were first described by Bevan and Szolcsanyi (Bevan and Szolcsanyi, 1990), who demonstrated that capsaicin opens a nonselective cation channel because capsaicin induces inward currents and a concomitant increase in membrane conductance that reverses at a membrane potential of 0 mV (Bevan and Szolcsanyi, 1990). Subsequently, Bevan and Docherty reported that capsaicin induced inward currents are dose‐dependent (Bevan and Docherty, 1993). Furthermore, in this same study it was found that the acidification of extracellular fluid also caused inward currents in the sensory neurons. Later, Liu and Simon (1994) confirmed that inward currents are induced by capsaicin in trigeminal neurons.

A comprehensive description of characteristics of capsaicin‐activated currents was obtained from single‐channel current recordings produced using patch‐clamp techniques. Initially, Bevan and his colleagues produced traces of single‐channel currents in sensory neurons activated by capsaicin (Bevan and Docherty, 1993). Later, a complete description of single‐channel currents activated by capsaicin was presented by Oh and colleagues (Oh et al., 1996). According to this study, capsaicin‐activated channels are permeable to various monovalent cations, namely, Na⁺, K⁺, Cs⁺, and Ca²⁺. Moreover, single‐channel conductances were found to be 45 pS and 80 pS at −60 and +60 mV, respectively, when Na⁺ is the major charge carrier (Oh et al., 1996). Thus, single‐channel currents activated by capsaicin are known to be outwardly rectifying, which was later found to be a universal feature of TRPV channels. One important finding of the study was that the channel involved was a ligand‐gated ion channel as channel currents were observed in isolated membrane patches. Capsaicin‐activated channel is also weakly dependent on membrane potential. Channel open probability (P_o) is much greater at depolarized than at hyperpolarized potentials. Capsaicin channel is activated in patches when depolarized without capsaicin application (unpublished data) with a half‐maximal voltage of −8.8 mV. Furthermore, for every 28.8 mV increase in membrane potential, an e‐fold increase in P_o is observed (Oh et al., 1996). Voltage dependence was later confirmed in TRPV1, a cloned capsaicin receptor, and is now considered a universal property of the TRPV subfamily (Nilius et al., 2004).

After a full description of capsaicin‐activated channels in sensory neurons was made available (Oh et al., 1996), David Julius and his colleagues at UCSF cloned a gene in a rat model, using functional expression cloning technique, which encoded a capsaicin‐activated channel (Caterina et al., 1997). The cloned gene was initially named vanilloid receptor 1 (VR1), because it was activated by capsaicin or resiniferatoxin (RTX), the vanilloid analogs (Caterina et al., 1997). VR1 was later termed TRPV1 according to the new taxonomy of TRP channels (Montell 2002, Clapham 2003). Rat TRPV1 encodes an 838 amino acid protein of molecular weight ∼95 kDa. The predicted topology of TRPV1 shows that it has six transmembrane domains and two intracellular cytosolic tails in N‐ and C‐termini with three ankyrin repeats in the N‐terminus (Fig. 3) (see also Chapters 4 and 6). To our surprise, the cloned channel was found to have near identical channel properties to those of native capsaicin‐activated channel in sensory neurons, that is, in terms of channel conductance, ion selectivity, and current–voltage relationship (Caterina 1997, Shin 2001). The most striking and interesting aspect of TRPV1 is its activation by heat and acid in addition to capsaicin (Tominaga et al., 1998). Thus, TRPV1 is capable of detecting several noxious stimuli, such as heat and acid, and is referred to as a “polymodal” molecular sensor that transduces these adverse stimuli to nociceptive neural signals in sensory neurons.

Another property unique to TRPV1 is its activation via intracellular capsaicin. Unlike many other ligand‐gated ion channels, capsaicin acts on TRPV1 from the cytosolic side. However, normally capsaicin is applied to a bath of outside‐out patches or to whole cells (extracellular side), though it is assumed that binding occurs at the extracellular side (Oh 1996, Jung 1999). Thus, capsaicin appears to act on TRPV1 from both sides of the channel. However, this is only made possible by the highly lipophilic nature of capsaicin, which easily traverses the cell membrane, to access binding sites from the intracellular side. Moreover, this lipophilic nature made it difficult to determine initially whether capsaicin binds TRPV1 intracellularly or extracellularly. Luckily, Jung et al. (1999) found a water‐soluble analog of capsaicin, DA‐5018•HCl, which cannot cross the plasma membrane easily. When this hydrophilic analog of capsaicin was applied to the intracellular side in sensory neurons, it produced single‐channel currents in a capsazepine‐reversible manner. However, DA‐5018HCl fails to activate capsaicin channel when applied to the extracellular side (Jung et al., 1999), thus, demonstrating that capsaicin and its analogs act on the intracellular side of TRPV1. This finding becomes important for determining the location of ligand‐recognition sites or identifying intracellular signals upstream of TRPV1 (see in a later section).