Protease Activated Receptors 1 and 4 Sensitize TRPV1 in Nociceptive Neurones

Abstract

Protease-activated receptors (PAR1-4) are activated by proteases released by cell damage or blood clotting, and are known to be involved in promoting pain and hyperalgesia. Previous studies have shown that PAR2 receptors enhance activation of TRPV1 but the role of other PARs is less clear. In this paper we investigate the expression and function of the PAR1, 3 and 4 thrombin-activated receptors in sensory neurones. Immunocytochemistry and in situ hybridization show that PAR1 and PAR4 are expressed in 10 - 15% of neurons, distributed across all size classes. Thrombin or a specific PAR1 or PAR4 activating peptide (PAR1/4-AP) caused functional effects characteristic of activation of the PLCβ/PKC pathway: intracellular calcium release, sensitisation of TRPV1, and translocation of the epsilon isoform of PKC (PKCε) to the neuronal cell membrane. Sensitisation of TRPV1 was significantly reduced by PKC inhibitors. Neurons responding to thrombin or PAR1-AP were either small nociceptive neurones of the peptidergic subclass, or larger neurones which expressed markers for myelinated fibres. Sequential application of PAR1-AP and PAR4-AP showed that PAR4 is expressed in a subset of the PAR1-expressing neurons. Calcium responses to PAR2-AP were by contrast seen in a distinct population of small IB4+ nociceptive neurones. PAR3 appears to be non-functional in sensory neurones. In a skin-nerve preparation the release of the neuropeptide CGRP by heat was potentiated by PAR1-AP. Culture with nerve growth factor (NGF) increased the proportion of thrombin-responsive neurons in the IB4- population, while glial-derived neurotropic factor (GDNF) and neurturin upregulated the proportion of thrombin-responsive neurons in the IB4+ population. We conclude that PAR1 and PAR4 are functionally expressed in large myelinated fibre neurons, and are also expressed in small nociceptors of the peptidergic subclass, where they are able to potentiate TRPV1 activity.

Mapping Human Protease-activated Receptor 4 (PAR4) Homodimer Interface to Transmembrane Helix 4

“…However, the details appear to be tissue-specific. For example, in dorsal root ganglia, PAR4 sensitized TRPV1, resulting in the release of calcitonin gene-related peptide (25). In other studies, PAR4 sensitization was independent of TRPV1 (24).…”

“…Inhibiting PAR4 elicited cell survival pathways through PI3K/ Akt, ERK1/2, nitric-oxide synthase, and K ATP channels in a adenosine receptor-mediated pathway (23). In addition to roles in cardiovascular tissues, PAR4 has been implicated in nociception (24,25). However, the details appear to be tissue-specific.…”

“…The sustained signaling from PAR4 may be required for stable clot formation (21,22). In addition to its role in activation of platelets by thrombin, there is increasing evidence of roles for PAR4 in other physiological processes including nociception, colon cancer cell proliferation, cardiac response to ischemia/reperfusion injury, and diabetic arterial plaques (23)(24)(25)(26).…”

The G Protein–Coupled Receptor–Transient Receptor Potential Channel Axis: Molecular Insights for Targeting Disorders of Sensation and Inflammation

“…Proteases cleave specific sites within the extracellular N-terminal domains of PAR 1 , PAR 2 , and PAR 4 to reveal tethered ligand domains that bind to and activate cleaved receptors. PAR 1 , PAR 2 , and PAR 4 are expressed by nociceptive neurons (Vellani et al, 2010), and PAR 2 in particular can sensitize or excite sensory neurons to promote neurogenic inflammation, pain, and itch (Ramachandran et al, 2012). PARs couple to the major Ga subunits (Ga q/11 , Ga s , Ga i/o , Ga 12/13 ) to stimulate PLC and phospholipase A 2 (PLA 2 ) and activate PKA, PKC, and protein kinase D (atypical PKCm).…”

TRPV1 Antagonists as Analgesic Agents

“…In addition, TRPV1 is receptive to pro-inflammatory agents such as prostaglandins, bradykinin, adenosine triphosphate (ATP), 5-hydroxytryptamine, protease activate receptors (PAR) 1, 2 and 4, nerve growth factor (NGF) and tumor necrosis factor alpha (TNF-alpha) [50] that cause allosteric modification of the channel protein, either directly or indirectly, such that the probability of channel opening by heat, protons and capsaicin is enhanced [9,40,43,[51][52][53][54] Fig. (1).…”