Capsaicin Up-Regulates Protease-Activated Receptor-4 mRNA and Protein in Primary Cultured Dorsal Root Ganglion Neurons

Abstract

Previous study has shown that there is a functional link between the transient receptor potential vanilloid type 1 (TRPV1) receptor and protease-activated receptor-4 (PAR4) in modulation of inflammation and pain. Capsaicin activation of TRPV1 is involved in enhancement of the expression of TRPV1 in mRNA and protein in dorsal root ganglion (DRG) in vivo. Whether capsaicin could influence expression of PAR4 in primary sensory neurons remains unknown. In the present study, expression of PAR4 in cultured rat DRG neurons was observed using immunofluorescence, real-time PCR and Western blots to examine whether increases in PAR4 mRNA and protein levels are induced by capsaicin treatment with or without pre-treatment of forskolin, a cyclic AMP/protein kinase A (cAMP/PKA) activator or PKA inhibitor fragment 14-22 (PKI14-22), a PKA inhibitor. Capsaicin treatment of cultured DRG neurons significantly increased the expression of PAR4 in mRNA and protein levels. The percentage of PAR4-, TRPV1-immunoreactive neurons and their co-localization in cultured DRG neurons increased significantly in the presence of capsaicin as compared with that in the absence of capsaicin. Compared with capsaicin-only group, pre-incubation with forskolin strongly enhanced the capsaicin-induced increase of PAR4 in mRNA and protein levels. Consistent with the involvement of PKA in the modulation of PAR4 expression, this evoked expression both at mRNA and protein levels was significantly inhibited after PKA was inhibited by pre-incubation with PKI14-22. Taken together, these results provide evidence that TRPV1 activation significantly increases the expression of PAR4 mRNA and protein levels in primary cultures of DRG neurons after capsaicin incubation. Effects of capsaicin on PAR4 expression appear to be mediated by cAMP/PKA signal pathways in DRG neurons.

New insights into protease-activated receptor 4 signaling pathways in the pathogenesis of inflammation and neuropathic pain: a literature review

“…Accumulating evidence demonstrates that nociceptor activation and inflammation evoke a rapid change in the levels of CGRP mRNA and protein in DRG neurons, [26][27][28] and CGRP expression in these neurons contributes to the regulation of neurogenic inflammation and pain. [29][30][31][32] In a previous study, the PAR4 agonist did not induce a calcium signal in DRG neurons; it reduced the calcium signal of DRG neurons in response to KCl, 16 suggesting that PAR4 activation could inhibit the nociceptive signal in DRG neurons. Furthermore, in an in vivo experiment, the PAR4 agonist was able to increase the nociceptive threshold to thermal and mechanical stimuli, and to reduce thermal and mechanical inflammatory hyperalgesia and allodynia.…”

“…[65][66][67][68] TRPV1 is widely distributed in primary nociceptive afferent neurons, and its expression extensively overlaps with PAR4 immunoreactivity in cultured primary sensory neurons. 31,33,69 PAR4 is expressed in both large neurons with myelinated fiber and small nociceptors of the peptidergic subclass, which is able to potentiate TRPV1 activity and promotes the heat-dependent release of the proinflammatory neuropeptide CGRP. 31 Capsaicin-activated TRPV1 is involved in noxious responses and induction of TRPV1 and CGRP mRNA and protein expression in DRG neurons.…”

“…31,33,69 PAR4 is expressed in both large neurons with myelinated fiber and small nociceptors of the peptidergic subclass, which is able to potentiate TRPV1 activity and promotes the heat-dependent release of the proinflammatory neuropeptide CGRP. 31 Capsaicin-activated TRPV1 is involved in noxious responses and induction of TRPV1 and CGRP mRNA and protein expression in DRG neurons. 28 Thrombin or PAR4-AP causes activation of the PLCb/PKC pathway: intracellular calcium release, sensitization of TRPV1, and translocation of the epsilon isoform of PKC (PKCe) to the neuronal cell membrane.…”

“…101,102 PAR4 activation induced by the PAR4-AP AYPGKF-NH 2 is associated with an increase in CGRP expression in DRG neurons and indicates that PAR4 activation evokes a remarkable increase in the number of CGRP-labeled neurons and CGRP mRNA and protein levels in DRG neurons. 31,32 There is a close correlation between CGRP mRNA and protein levels; therefore, it seems more likely that the effect of PAR4 on CGRP expression results from a change in transcription. 31 Several studies have shown that these rapid changes in CGRP mRNA and protein levels were evoked by nociceptor activation and inflammation.…”

“…31,32 There is a close correlation between CGRP mRNA and protein levels; therefore, it seems more likely that the effect of PAR4 on CGRP expression results from a change in transcription. 31 Several studies have shown that these rapid changes in CGRP mRNA and protein levels were evoked by nociceptor activation and inflammation. 26,28 PAR4-AP induces the upregulation of CGRP expression in primary sensory neurons via activation of the ERK1/2 signaling pathway.…”