Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization

Abstract

Although the PI3K (phosphatidylinositol 3-kinase) pathway typically regulates cell growth and survival, increasing evidence indicates the involvement of this pathway in neural plasticity. It is unknown whether the PI3K pathway can mediate pain hypersensitivity. Intradermal injection of capsaicin and NGF produce heat hyperalgesia by activating their respective TRPV1 (transient receptor potential vanilloid receptor-1) and TrkA receptors on nociceptor sensory nerve terminals. We examined the activation of PI3K in primary sensory DRG neurons by these inflammatory agents and the contribution of PI3K activation to inflammatory pain. We further investigated the correlation between the PI3K and the ERK (extracellular signal-regulated protein kinase) pathway. Capsaicin and NGF induce phosphorylation of the PI3K downstream target AKT (protein kinase B), which is blocked by the PI3K inhibitors LY294002 and wortmannin, indicative of the activation of PI3K by both agents. ERK activation by capsaicin and NGF was also blocked by PI3K inhibitors. Similarly, intradermal capsaicin in rats activated PI3K and ERK in C-fiber DRG neurons and epidermal nerve fibers. Injection of PI3K or MEK (ERK kinase) inhibitors into the hindpaw attenuated capsaicin- and NGF-evoked heat hyperalgesia but did not change basal heat sensitivity. Furthermore, PI3K, but not ERK, inhibition blocked early induction of hyperalgesia. In acutely dissociated DRG neurons, the capsaicin-induced TRPV1 current was strikingly potentiated by NGF, and this potentiation was completely blocked by PI3K inhibitors and primarily suppressed by MEK inhibitors. Therefore, PI3K induces heat hyperalgesia, possibly by regulating TRPV1 activity, in an ERK-dependent manner. The PI3K pathway also appears to play a role that is distinct from ERK by regulating the early onset of inflammatory pain.

Figure 3.

a-d, PI3K and ERK are required for the induction and expression of capsaicin-induced heat hyperalgesia without affecting basal sensitivity. a, b, Pretreatment, Intradermal injection of PI3K inhibitors (LY294002, 1 and 10 μg; wortmannin, 0.2 μg) or MEK inhibitors (PD98059, 1 and 10 μg; U0126, 1 μg) before capsaicin injection prevents heat hyperalgesia in a dose-dependent manner. ^*p < 0.01; ⁺p < 0.05, compared with vehicle control (n = 6). c, Posttreatment, Intradermal injection of LY294002 (10 μg) or PD98059 (10 μg), 15 min after capsaicin injection, reverses heat hyperalgesia. ^*p < 0.01, compared with control (n = 6). d, LY294002 (10 μg) or PD98059 (10 μg) has no effect on basal heat sensitivity (n = 6). p > 0.05. Heat sensitivity was measured by PWL and plotted as a proportion of preinjection baseline. The basal PWL (in seconds) in a, b, c, and d is 15.4 ± 0.6 (n = 24), 16.4 ± 0.5 (n = 24), 14.4 ± 0.5 (n = 18), 17.0 ± 0.8 (n = 12), respectively (mean ± SEM). Cont, Control; Ly, LY294002; PD, PD98059; Wort, wortmannin.

Figure 1.

a-h, Capsaicin induces activation of AKT and ERK in cultured DRG neurons. a-c″, Bath application of capsaicin induces a rapid phosphorylation, within 2 min, of AKT at Thr308 (pAKT-T) and ERK. pAKT-T and pERK are colocalized in the same DRG neurons (b, b′', as indicated by arrows), and the capsaicin-induced phosphorylation of AKT-T (a, b) and ERK (a′, b′) is abolished by the PI3K inhibitor LY294002 (100 μm) (c, c′). a′, b′, and c′ are the same fields as a, b, and c, respectively, showing double staining. a″, b″, and c″ are images with high contrast to show all of the cells in each field. Scale bar, 50 μm. The DRG cultures were grown for 24 hr and stimulated with 3 μm capsaicin for 2 min. d, Time course of capsaicin (3 μm)-induced pAKT-T and pERK levels, as measured by the intensity of immunofluorescence. At all time points, pAKT-T and pERK levels are significantly higher (p < 0.01) than control (Cont) levels (n = 4). e, Concentration-dependent inhibition of capsaicin (Cap)-evoked pAKT-T induction by LY249002 and wortmannin. ^*p < 0.01, compared with control; ⁺p < 0.01, compared with capsaicin group (n = 4). f, Inhibition of capsaicin-evoked pERK induction by LY294002 (LY) and wortmannin (Wort). ^*p < 0.01, compared with control; ⁺p < 0.01, compared with capsaicin group (n = 4). g, Western blot reveals that the capsaicin-induced increase in pAKT-T and pERK levels is blocked by LY294002 (100 μm). Total AKT levels were used as loading controls. h, Capsaicin-induced pAKT-T and pERK is suppressed by EGTA (4 mm). ^*p < 0.01, compared with control; ⁺p < 0.01, compared with capsaicin group (n = 4). The medium (containing 1.8 mm Ca²⁺) was incubated with 4 mm EGTA for 30 min in the culture incubator. The control medium, with or without capsaicin (3 μm), and EGTA-containing medium with capsaicin were added to DRG cultures for 2 min.

Figure 2.

a-i, Intradermal capsaicin induces pAKT-T and pERK in the DRG and hindpaw skin. a, b, Capsaicin increases pAKT-T levels in the L5 DRG. c, Quantification of the percentage of all pAKT-T-positive neuronal profiles in each section. ^*p < 0.01, compared with vehicle control (n = 4). d, e, Double immunofluorescence indicates that capsaicin-induced pAKT-T is rarely colocalized with NF-200, a marker for myelinated A-fibers (d), but primarily colocalized with the TRPV1 capsaicin receptor. f-i, More pAKT-T- and pERK-positive nerve fibers are found in the capsaicin-stimulated epidermis of the hindpaw skin (g, i) than in controls (f, h). The same settings of contrast and brightness were used in control and capsaicin-stimulated images. Arrows indicate labeled single nerve fibers in the epidermis. Arrowheads show transected large nerve bundles in the border of the epidermis and dermis. The animals were fixed by perfusion 5 min after the capsaicin injection. Scale bars, 50 μm.

Figure 4.

a-j, NGF induces activation of AKT and ERK in cultured DRG neurons. a-f′, Bath application of NGF (100 ng/ml) to DRG cultures results in phosphorylation of AKT at Ser473 (pAKT-S; a, b) and ERK (d, e). The induction of pAKT-S (c) and pERK (f) is abolished by LY294002 (100 μm; LY). a′-f′ are images of corresponding to a-f with high contrast to show all of the cells in each field. Scale bar, 50 μm. The DRG cultures were grown for 24 hr and stimulated with NGF for 10 min. g, NGF does not change AKT phosphorylation at Thr308 (pAKT-T). h, i, Intensity of pAKT-S and pERK immunofluorescence. NGF-evoked pAKT-S (h) and pERK (i) induction is effectively blocked by LY294002 (100 μm). ^*p < 0.01, compared with control; ⁺p < 0.01, compared with NGF group (n = 4). j, Western blot confirms that NGF-induced increase in pERK levels is blocked by LY294002 (100 μm). Total AKT levels were used as loading controls. Cont, Control.

Figure 5.

PI3K and ERK are required for NGF-induced heat hyperalgesia. Intradermal injection of LY294002 (10 μg; LY) or PD98059 (10 μg; PD), 5 or 20 min before NGF injection, respectively, inhibits NGF-induced heat hyperalgesia at the time points indicated. ^*p < 0.01, compared with control (n = 6). Heat sensitivity was measured by PWL and plotted as a proportion of preinjection baseline. The basal PWL is 16.6 ± 1.0 sec (n = 18; mean ± SEM). Cont, Control.

Figure 6.

a-e, PI3K and ERK are required for NGF-induced potentiation of capsaicin-induced current in DRG neurons. a, Desensitization (tachyphylaxis) of I_Cap during multiple bath applications of capsaicin (Cap). Standard bath solution and BAPTA-based pipette solutions were used. The cell was held at -60 mV. Interval between capsaicin (40 nm) applications was 10 min. b, Long-lasting NGF-evoked potentiation of I_Cap. In the absence of NGF, the second I_Cap (170 pA) was slightly smaller than the first I_Cap (200 pA). However, after exposure to 100 ng/ml NGF for 10 min, I_Cap increased by 14-fold to ∼2400 pA. Inset, Enhanced view of NGF-evoked augmentation of I_Cap. Note the continued potentiation (4th I_Cap) 10 min after removal of NGF from the bath. c, NGF failed to induce any potentiation of I_Cap in a neuron pretreated (>10 min) with the PI3K inhibitor LY294002 (20 μm). After washout of LY294002, NGF markedly potentiated I_Cap. d, Attenuation of NGF-induced potentiation by pretreatment with the MEK inhibitor PD98059 (20 μm). Washout of PD98059 was followed by a large NGF-induced potentiation. e, Summary of NGF effect on I_Cap under various conditions. NGF evoked strong potentiation in 15 cells; the other five cells exhibited desensitization. Because of the clear distinction between these two categories of responses, data were averaged separately. All cells that were included tested positive for NGF potentiation (see c, d) after washout of inhibitors. ^*p < 0.05. LY, LY294002; PD, PD98059.