Cannabinoid WIN 55,212-2 Regulates TRPV1 Phosphorylation in Sensory Neurons

Abstract

Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr 144 and Thr 370 were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.Cannabinoids have been shown to exert anti-inflammatory and anti-hyperalgesic effects via peripheral site(s) of action in several pain models (1-5). These effects are thought to be mediated by cannabinoid type 1 (CB1) 4 and/or 2 (CB2) receptor activation, both peripherally and centrally (4 -7). Cannabinoids could exert their effects by acting on CB1/CB2 receptors located on sensory neurons and/or other peripheral cells influencing sensory neuronal function (8). However, there is a Ͻ5-10% co-localization of metabotropic CB1/CB2 receptors with nociceptive neuronal markers such as TRPV1 (transient receptor potential subtype V1) and calcitonin gene-related peptide in trigeminal and dorsal root ganglion neurons (9 -11), suggesting that cannabinoids could act on nociceptors through non-CB1/CB2 receptor mechanism(s). Certain cannabinoids have been shown to activate channels such as TRPV1, including arachidonyl-2-chloroethylamide (ACEA) (12), N-arachidonoyldopamine (13), and anandamide (14), as well as TRPA1 (transient receptor potential subtype A1), including ⌬ 9 -tetrahydrocannabinol (15). In addition, the synthetic cannabinoid R(ϩ)-WIN 55,212-2 (WIN) has demonstrated non-CB1/CB2 receptor activities in trigeminal ganglia (11). The results from these studies suggest that cannabinoids may activate calcium channel function similar to non-cannabinoid transient receptor potential agonists, including the...

The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine (NADA) and Synthetic Cannabinoid WIN55,212-2 Abate the Inflammatory Activation of Human Endothelial Cells

“…Many of these, including PPAR-␣, PPAR-␥, and calcineurin, are expressed in ECs and can themselves modulate endothelial inflammatory outputs (48,53,54,(117)(118)(119). In fact, WIN55,212-2 has been reported to reduce inflammation in murine brain ECs through PPAR-␥, independent of CB 1 R and CB 2 R, which is consistent with our observations using combined CB 1 R antagonist and CB 2 R inverse agonist (29,117,118).…”

“…As mentioned previously, WIN55,212-2 has high affinity for both CB 1 R and CB 2 R and is reported to inhibit TRPV1 (1,51,53,54). We first analyzed the effects of the CB 1 R antagonist CP945598 and CB 2 R inverse agonist SR144528 together (0.1, 1, and 10 M) on the LPS-induced up-regulation of IL-6 and IL-8 in HMVEC-lung in the presence and absence of WIN55,212-2.…”

“…Abn-CBD, abnormal cannabidiol; NAGly, N-arachidonoylglycine; ND, no data (reported in good faith); ACEA, arachidonoyl-2'-chloroethylamide; CAP, capsaicin; CBD, cannabidiol. (54), calcineurin (53,54), PPAR-␥ (29), and PPAR-␣ (114).…”

“…Although WIN55,212-2 is a strong agonist of both CB 1 R and CB 2 R, NADA displays approximately a 40-fold selectivity for CB 1 R over CB 2 R (1,33,51). Moreover, the TRPV1 ion channel is activated by NADA but is inhibited by WIN55,212-2 (34,(52)(53)(54). Therefore, we sought to determine the expression repertoire of the four G protein-associated CBRs, and TRPV1, in different human primary endothelial cell niches.…”

TRP Channels in Visceral Pain

“…For example, 5-HT enhances sensitivity to heat in colonic DRG neurones via TRPV1 (Sugiura et al, 2004), so that they are activated at normal body temperature. Cannabinoids modulate TRPV1 either directly by a direct interaction with the eicosanoid mediator anandamide with the channel itself, or by coupling through intracellular pathways after acting on G-protein coupled cannabinoid receptors [65][66][67]. The protease receptor PAR2 sensitizes TRPV1 through specific intracellular pathways [65].…”