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. 2013 Feb;168(3):761-72.
doi: 10.1111/j.1476-5381.2012.02186.x.

4α-phorbol 12,13-didecanoate activates cultured mouse dorsal root ganglia neurons independently of TRPV4

R Alexander  1 , A Kerby, A A Aubdool, A R Power, S Grover, C Gentry, A D Grant
Affiliations

4α-phorbol 12,13-didecanoate activates cultured mouse dorsal root ganglia neurons independently of TRPV4

R Alexander et al. Br J Pharmacol. 2013 Feb.

Abstract

Background and purpose: The Ca(2+) -permeable cation channel TRPV4 is activated by mechanical disturbance of the cell membrane and is implicated in mechanical hyperalgesia. Nerve growth factor (NGF) is increased during inflammation and causes mechanical hyperalgesia. 4α-phorbol 12,13-didecanoate (4αPDD) has been described as a selective TRPV4 agonist. We investigated NGF-induced hyperalgesia in TRPV4 wild-type (+/+) and knockout (-/-) mice, and the increases in [Ca(2+) ](i) produced by 4αPDD in cultured mouse dorsal root ganglia neurons following exposure to NGF.

Experimental approach: Withdrawal thresholds to heat, von Frey hairs and pressure were measured in mice before and after systemic administration of NGF. Changes in intracellular Ca(2+) concentration were measured by ratiometric imaging with Fura-2 in cultured DRG and trigeminal ganglia (TG) neurons during perfusion of TRPV4 agonists.

Key results: Administration of NGF caused a significant sensitization to heat and von Frey stimuli in TRPV4 +/+ and -/- mice, but only TRPV4 +/+ mice showed sensitization to noxious pressure. 4αPDD stimulated a dose-dependent increase in [Ca(2+) ](i) in neurons from +/+ and -/- mice, with the proportion of responding neurons and magnitude of increase unaffected by the genotype. In contrast, the selective TRPV4 agonist GSK1016790A failed to stimulate an increase in intracellular Ca(2+) in cultured neurons. Responses to 4αPDD were unaffected by pretreatment with NGF.

Conclusions and implications: TRPV4 contributes to mechanosensation in vivo, but there is little evidence for functional TRPV4 in cultured DRG and TG neurons. We conclude that 4αPDD activates these neurons independently of TRPV4, so it is not appropriate to refer to 4αPDD as a selective TRPV4 agonist.

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Figures

Figure 1
Figure 1
(A) Expression of TRPV4 in dorsal root ganglia lysate. Lysates (30 μg of total protein) from both TRPV4 +/+ and –/– mice showed expression of βIII-tubulin (50 kDa, left column). Only lysates from TRPV4 +/+ mice showed the double band corresponding to TRPV4 (98 and 104 kDa, right column). (B) Immunostaining of cultured dorsal root ganglion neurons from TRPV4 +/+ and –/– mice with rabbit anti-TRPV4 (1:200) and goat anti-rabbit-Alexa488 (1:1000) revealed no difference in staining intensity between +/+, –/– and +/+ (no primary antibody) cells. Images are representative of data obtained from n = 3 independent experiments.
Figure 2
Figure 2
Effect of systemic NGF (1 μg·g−1; i.p.) treatment on thermal and mechanical nociceptive thresholds in TRPV4 +/+ and –/– mice. (A). Latency of withdrawal to radiant heat, (B) 50% withdrawal threshold to application of von Frey hairs and (C) withdrawal pressure were measured at baseline and 1 and 24 h following NGF administration. *P < 0.05 compared with baseline, n = 10.
Figure 3
Figure 3
The effect of hypotonic challenge (264, 234 and 206 mOsm) on [Ca2+]i in cultured mouse dorsal root ganglion neurons from TRPV4 +/+ and –/– mice. Example response profiles of (A) TRPV4 +/+ and (B). TRPV4 –/– neurons to 206 mOsm buffer, 1 μM capsaicin (C) and 50 mM KCl (K). (C) The % of total neurons responding and (D) mean response of individual responding neurons relative to the response to KCl are also shown. n = 6–9 coverslips, with a total of at least 200 neurons from three mice of each genotype for each data point. *P < 0.05 compared with 264 mOsm, one-way anova followed by Dunnett's test.
Figure 4
Figure 4
The effect of 4αPDD (1–10 μM) on [Ca2+]i in cultured mouse dorsal root ganglion neurons from TRPV4 +/+ and –/– mice. Example response profiles of (A) TRPV4 +/+ and (B) TRPV4 –/– neurons to 10 μM 4αPDD, 1 μM capsaicin (C) and 50 mM KCl (K). (C) The % of total neurons responding and (D) mean response of individual responding neurons relative to the response to KCl are also shown. n = 6–9 coverslips, with a total of at least 200 neurons from three mice of each genotype for each data point. *P < 0.05 compared with 1 μM 4αPDD, one-way anova followed by Dunnett's test.
Figure 5
Figure 5
The effect of GSK1016790A (100 nM–1 μM) on [Ca2+]i in cultured mouse dorsal root ganglion neurons from TRPV4 +/+ mice. (A) The % of total neurons responding and (B) mean response of individual responding neurons relative to the response to KCl are shown. n = 8–9 coverslips, with a total of at least 300 neurons from three mice for each data point. (C) GSK1016790A (5 nM–500 nM) caused a dose-dependent increase in [Ca2+]i in HaCaT keratinocytes. (D) The TRPV4 antagonist HC067047 caused a dose-dependent inhibition of the increase in [Ca2+]i caused by application of 100 nM GSK1016790A to HaCaT keratinocytes.
Figure 6
Figure 6
(A) The % of cultured thoracic DRG and trigeminal ganglion neurons responding to GSK1016790A (1 μM) or 4αPDD (10 μM) with an increase in [Ca2+]i and (B) mean response of individual responding neurons relative to the response to KCl. n = 8–11 coverslips, with a total of at least 250 (thoracic) or 180 (TG) neurons from three mice of each genotype for each data point. (C) The % of total neurons responding to 234 mOsm buffer, 4αPDD (3 μM), AITC (100 μM) and capsaicin (1 μM) on [Ca2+]i in cultured mouse dorsal root ganglion neurons from TRPV4 +/+, TRPV1 –/– and TRPA1 –/– mice. n = 5–6 coverslips, with a total of at least 100 neurons from two mice for each data point.
Figure 7
Figure 7
The effect of pre-treatment with NGF (100 ng·mL−1; 24 h) on responses to hypotonic buffer (264 mOsm and 234 mOsm) or 4αPDD (1 and 3 μM) in cultured mouse dorsal root ganglion neurons from TRPV4 +/+ (WT) and TRPV4 –/– (KO) mice. (A and C) The % of total neurons responding and (B and D) mean response of individual responding neurons relative to the response to KCl are shown. n = 8–9 coverslips, with a total of at least 200 neurons from three mice for each data point.
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