Pain TRP‐ed up by PARs

Studies of proteolytic enzymes (proteinases) like thrombin and trypsin have long been the province of cardiovascular and gastrointestinal physiologists: thrombin as the key regulator of the coagulation cascade and trypsin and other pancreatic enzymes as the means for breaking down proteins in the small intestine. Conforming to a long-standing pattern of ‘where GI physiologists tread, neuroscientists inevitably follow’, the identification of proteinase-activated receptors (PARs) on peripheral and central neurones has generated increasing interest in how they may function in the nervous system (Noorbakhsh et al. 2003; Ossovskaya & Bunnett, 2004). PARs are a four-membered family of G-protein coupled receptors (PAR1–4) whose activation by serine proteinases occurs by enzymatic cleavage at N-terminal serines to expose a ‘tethered ligand’ that binds residues in its own second extracellular domain to initiate signal transduction (Ossovskaya & Bunnett, 2004). PAR-2 has become the current favourite as a promising target in anti-inflammatory/chronic pain drug discovery programmes because it is expressed in sensory nociceptive neurones, its activation releases peptides (SP/CGRP) from afferent nerve endings in the dorsal horn and results in both thermal and mechanical hyperalgesia, and it can be activated by tryptase, which is secreted by mast cells onto adjacent neurones under inflammatory conditions or neuronal damage (Noorbakhsh et al. 2003; Ossovskaya & Bunnett, 2004). Because it is the only PAR not activated by thrombin, selective blockade of this receptor could be therapeutically useful in pain management without antithrombotic side-effects. How does PAR-2 signalling lead to thermal and mechanical hyperalgesia? A global network of research groups, headed by Nigel Bunnett, have presented a set of tour de force studies showing that PAR-2 activates multiple second messenger pathways to sensitize TRPV1 and TRPV4 receptors on nociceptive neurones and this, in turn, releases nociceptive peptides (SP and CGRP) onto centrally projecting nociceptors that can account for TRPV1-dependent thermal and TRPV4-dependent mechanical hyperalgesia (Amadesi et al. 2006; Grant et al. 2007). TRPV1 and TRPV4 receptors are calcium-permeable cationic ion channels activated by multiple sensory stimuli; of relevance here is that TRPV1 can be gated by noxious heat (> 42°C) and capsaicin, and TRPV4 by warm temperature (> 27°C), hypotonicity and shear stress (Nilius et al. 2004). Amadesi et al. (2006) found that PAR-2 colocalized with protein kinase (PK) Ce, and PKA in a subset of TRPV1-containing dorsal root neurones, that PAR-2 agonists activated these pathways, increased TRPV1-mediated calcium influx and whole-cell currents and caused thermal hyperalgesia in mice. Moreover, inhibition of PKCe and PKA prevented the PAR-2 sensitization of TRPV1 calcium transients and currents as well as PAR-2-induced thermal hyperalgesia in mice. Grant et al. (2007) found that PAR-2 also colocalized with TRPV4, SP and CGRP in dorsal root ganglia, that PAR-2 agonists enhanced TRPV4-induced calcium transients and whole-cell currents and that inhibition of phospholipase Cβ, PKA, PKC and PKD prevented the PAR-2-induced sensitization of TRPV4-induced responses. They also found that activation of TRPV4 stimulated the release of SP and CGRP from dorsal horn neurones and this release was potentiated by PAR-2 activation, similar to their previously demonstrated PAR-2 enhancement of TRPV1 release of SP and CGRP (Amadesi et al. 2004). Intraplantar injection of PAR-2 agonists caused mechanical hyperalgesia as well as sensitized pain responses to TRPV4 activation in wild-type but not in TRPV4-deleted mice. These findings provided the basis for a model outlined by Grant et al. (2007) to account for protease-induced mechanical hyperalgesia, a model that can also apply to protease-induced thermal hyperalgesia. Proteases are released in response to inflammatory stimuli where they can activate PAR-2 on nociceptive afferent nerve endings in peripheral tissue. PAR-2 couples to activation of multiple second messenger kinases (PKA, PKC, PKD) in these nerve endings to sensitize TRPV1 and TRPV4 receptors. Sensitized TRPV1 receptors now respond to non-noxious temperature while sensitized TRPV4 receptors show enhanced response to mechanical stimuli; in both cases their increased gating results in increased release of SP and CGRP from dorsal horn terminals with consequent increased transmission centrally. In terms of pathological pain states, prevention of TRPV1 and TRPV4 receptor sensitization is a more attractive therapeutic aim than inhibition of TRPV receptors per se. In this regard, it is of interest to compare results from a recent study by Alessandri-Haber et al. (2006) who found similar mechanisms of TRPV4-dependent sensitization and mechanical hyperalgesia in response to a cocktail of inflammatory mediators (bradykinin, SP, 5-HT, histamine and prostaglandin), none of which alone was sufficient to engage TRPV4-dependent hyperalgesia. PAR-2 antagonists may well pack more punch against neuropathic or inflammatory pain.