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, 166 (4), 1433-43

Nociceptive and Pro-Inflammatory Effects of Dimethylallyl Pyrophosphate via TRPV4 Activation

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Nociceptive and Pro-Inflammatory Effects of Dimethylallyl Pyrophosphate via TRPV4 Activation

S Bang et al. Br J Pharmacol.

Abstract

Background and purpose: Sensory neuronal and epidermal transient receptor potential ion channels (TRPs) serve an important role as pain sensor molecules. While many natural and synthetic ligands for sensory TRPs have been identified, little is known about the endogenous activator for TRPV4. Recently, we reported that endogenous metabolites produced by the mevalonate pathway regulate the activities of sensory neuronal TRPs. Here, we show that dimethylallyl pyrophosphate (DMAPP), a substance produced by the same pathway is an activator of TRPV4.

Experimental approach: We examined the effects of DMAPP on sensory TRPs using Ca²⁺ imaging and whole-cell electrophysiology experiments with a heterologous expression system (HEK293T cells transfected with individual TRP channels), cultured sensory neurons and keratinocytes. We then evaluated nociceptive behavioural and inflammatory changes upon DMAPP administration in mice in vivo.

Key results: In the HEK cell heterologous expression system, cultured sensory neurons and keratinocytes, µM concentrations of DMAPP activated TRPV4. Agonistic and antagonistic potencies of DMAPP for other sensory TRP channels were examined and activation of TRPV3 by camphor was found to be inhibited by DMAPP. In vivo assays, intraplantar injection of DMAPP acutely elicited nociceptive flinches that were prevented by pretreatment with TRPV4 blockers, indicating that DMAPP is a novel pain-producing molecule through TRPV4 activation. Further, DMAPP induced acute inflammation and noxious mechanical hypersensitivities in a TRPV4-dependent manner.

Conclusions and implications: Overall, we found a novel sensory TRP acting metabolite and suggest that its use may help to elucidate the physiological role of TRPV4 in nociception and associated inflammation.

Figures

Figure 1
Figure 1
TRPV4 is activated by DMAPP. (A) 10 µM DMAPP elevated intracellular Ca2+ levels in TRPV4-transfected HEK293T cells in the Fluo-3 Ca2+ imaging experiments (n= 47). A non-specific TRP blocker RR blocked these intracellular Ca2+ elevations in the same cells. Responses during Ca2+ imaging experiments are displayed as means ± SEM. (B) Left: current–voltage curves obtained from 10 µM DMAPP-evoked or 4αPDD-evoked increase in the outwardly rectifying current of TRPV4-HEK cells in the whole-cell voltage clamp study (n= 6 and n= 5 respectively). Right: the DMAPP current was inhibited by co-application of a TRPV4-specific blocker, RN-1734 (30 µM; n= 5). Buffer indicates the curve without drug. (C) Summary of the intracellular Ca2+ elevation, assessed by Fluo-3 Ca2+ imaging, observed in cells transfected with TRPV4 or other sensory TRP channels after treatment with 10 µM DMAPP. DMAPP elevated intracellular Ca2+ levels only in the TRPV4-transfected HEK cells. DMAPP did not increase intracellular Ca2+ in untransfected cells (data not shown). More than 33 cells were used to test each TRP for sensory activity (n= 33–108). The threshold for intracellular Ca2+ increase was defined as 20% increase above the basal Ca2+ level (Story et al., 2003; Ryu et al., 2010). (D) Dose–response curve for DMAPP on TRPV4, assessed as increase in Ca2+ levels by Fluo-3 Ca2+ imaging. The curve was fitted by the Hill equation (EC50= 2.5 µM and Hill coefficient = 2.0). Symbols represent mean values of the responses of Ca2+ influx to different concentrations of DMAPP (n= 21–38 for each point).
Figure 2
Figure 2
DMAPP inhibits TRPV3 activation. (A) DMAPP 30 µM suppressed the increase in intracellular Ca2+ induced by 4 mM camphor in hTRPV3-transfected HEK293T cells (n= 36) in the Fluo-3 Ca2+ imaging experiments. (B) Current–voltage curves obtained from 4 mM camphor-evoked outwardly rectifying current increases in the whole-cell voltage clamp study using the TRPV3-HEK cells (n= 5). The camphor-evoked current was inhibited by co-application with DMAPP (30 µM) (n= 5). (C) Summary of the suppression of agonist-induced increase in intracellular Ca2+ by DMAPP in cells transfected with TRPV3 or other sensory TRP channels (assessed by Fluo-3 Ca2+ imaging). Of the HEK cells transfected with TRP channels, DMAPP only reduced the agonist-induced increase in intracellular Ca2+ in the TRPV3-expressing HEK cells (agonists used: 0.1 µM capsaicin for TRPV1; 100 µM probenecid for TRPV2; 4 mM camphor for TRPV3; 3 µM 4α-PDD for TRPV4; 300 µM menthol for TRPM8; 300 µM cinnamaldehyde for TRPA1). More than 48 cells were used for the tests of each TRP activity (n= 28–76). (D) Dose–response curve for inhibitory effect of DMAPP on TRPV3, as assessed by Fluo-3 Ca2+ imaging. The curve was fitted by Hill equation (EC50= 10.4 µM and Hill coefficient = 3.3). Symbols represent mean values of responses of Ca2+ influx to TRPV3 activation by 4 mM camphor in the presence of different DMAPP concentrations (n= 19–31 for each point).
Figure 3
Figure 3
DMAPP activates TRPV4 and inhibits TRPV3 in native cells. (A) DMAPP 10 µM elevated intracellular Ca2+ levels in primarily cultured mouse DRG neurons (n= 8) in the Fura-2 Ca2+ imaging experiments. 4α-PDD also induced responses in the same cells. The finding that high KCl (60 mM) induced a response (via activation of voltage-gated channels) indicates that the cultured neurons retain their excitability (upper). Treatments with RR or RN-1734 blocked the increase in intracellular Ca2+ induced by DMAPP (middle and lower). (B) Current–voltage curves for the increases in outwardly rectifying current evoked by 10 µM DMAPP or 4αPDD in the whole-cell voltage clamp experiments with the cultured DRG neurons (n= 5 for both). The humps on the curves at ∼0 mV were caused by activation of voltage-gated channels by the depolarizing ramp, indicating that the cultured neurons retained their excitability under our culture conditions (Bessac et al., 2008). (C) DMAPP 10 µM elevated intracellular Ca2+ levels in human epidermal keratinocytes in the Fura-2 Ca2+ imaging experiments (n= 67). 4αPDD also induced a response in the same cells. (D) Current–voltage curves obtained for the increases in the outwardly rectifying current evoked by 10 µM DMAPP in the whole-cell voltage clamp experiments using keratinocytes (n= 5). (E) DMAPP 30 µM blocked the increase in intracellular Ca2+ induced by 4 mM camphor in human epidermal keratinocytes (n= 51) in the Ca2+ imaging experiments. (F) Inhibitory effects of 30 µM DMAPP on outwardly rectifying current responses to camphor in the whole-cell voltage clamp study using keratinocytes (n= 5).
Figure 4
Figure 4
DMAPP induces acute nociception and lowers the mechanical threshold. (A) Summary of the time course of the flinching behaviours in mice injected intraplantarly with 3 mM DMAPP or 3 mM 4αPDD (in 10 µL vehicle) for a 10 min period immediately after the injection. The hindpaws for the test were primed with 100 ng PGE2 5 min before the DMAPP injection (n= 5). The animals primed with PGE2 without the DMAPP injection or injected with DMAPP without PGE2 priming showed no flinching responses for 30 min (data not shown). Also when vehicle alone was injected into the primed hind paw, no flinch occurred in the mice for 10 min (data not shown). Mice pretreated with RR, injected into hind paw, showed no flinches from either DMAPP injected or non-injected hind paws (n= 5). (B) Summary of the total number of flinch responses from (A). The mean values of the accumulated flinch responses during the recording period (10 min) are displayed. (C) Summary of the changes in the mechanical thresholds induced by intraplantar DMAPP administration from von Frey tests. The average decreased ratio of the von Frey thresholds induced by DMAPP was 55.7 ± 6.6% (n= 6) and for 4αPDD was 37.2 ± 7.4% (n= 5). Immediately after intradermal pretreatment of the hind paw with RN-1734 (10 mM in 10 µL) or with HC067047 (1 mM in 10 µL) these threshold decreases were reversed (n= 6 for RN-1734; n= 5 for HC067047). (D) Summary of the changes in the mechanical thresholds induced by intraplantar DMAPP from Randall–Selitto tests. The average decreased ratio of the mechanical threshold induced by intraplantar administration of DMAPP (3 mM in 10 µL) was 64.2 ± 1.6% (n= 6). Immediately after intradermal pretreatment of the hind paw with RN-1734 (10 mM in 10 µL) or with HC067047 (1 mM in 10 µL) the threshold decreases induced by DMAPP were reversed (n= 5 for RN-1734; n= 5 for HC067047). (E) Summary of changes in heat thresholds from Hargreaves tests induced by intraplantar DMAPP (n= 5). Injection of vehicle alone did not affect von Frey or Randall–Selitto thresholds (data not shown). Hypo, hypotonic deionized water; PDD, 4αPDD; ND, no significance detected.
Figure 5
Figure 5
DMAPP induces acute inflammation. (A) Summary of the changes in ipsilateral paw diameter of mice injected intraplantarly with 3 mM DMAPP, 3 mM 4αPDD, 10 mM RN-1734 or 1 mM HC067047 in 10 µL vehicle. Five animals were tested for each drug. (B) Summary of the changes in ipsilateral paw diameter of mice injected intraplantarly with 3 mM DMAPP or 3 mM 4αPDD in 10 µL vehicle when 10 mM RN-1734 were additionally injected 1 h after the DMAPP or 4αPDD injection. Five animals were tested for each drug treatment. (C) Summary of the changes in ipsilateral paw diameter of mice injected intraplantarly with 3 mM DMAPP or 3 mM 4αPDD in 10 µL vehicle when 10 mM RN-1734 were injected before the DMAPP or 4αPDD injection. Five animals were tested for each drug treatment. (D) Tissue MPO activities of the animals in (B). (E) Tissue MPO activities of the animals in (C). Student's t-test was used to analyse results for (A)–(C) and anova followed by Bonferroni post hoc test for (D) and (E).

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