Prostaglandin and protein kinase A-dependent modulation of vanilloid receptor function by metabotropic glutamate receptor 5: potential mechanism for thermal hyperalgesia

Abstract

In addition to its role as a CNS neurotransmitter, glutamate has been shown recently to be an important component of the peripheral inflammation response. We demonstrated previously that the group I metabotropic glutamate receptors (mGluRs) mGlu1 and mGlu5 are expressed in the peripheral terminals of sensory neurons and that activation of group I mGluRs in the skin increases thermal sensitivity. In the present study, we provide evidence suggesting that group I mGluRs increase thermal sensitivity by enhancing vanilloid (capsaicin) receptor function. We show that mGlu5 potentiates capsaicin responses in mouse sensory neurons by the phospholipase C pathway but not by activation of protein kinase C. Rather, the effects are mediated by the metabolism of diacylglycerol and the production of prostaglandins via the cyclooxygenase pathway, leading to activation of the cAMP-dependent protein kinase subsequent to prostanoid receptor activation. Behavioral thermal sensitization in mice induced by intraplantar injection of mGlu1/5 agonists was also blocked by inhibitors of protein kinase A and cyclooxygenase, suggesting that a similar signaling pathway operates in vivo. These results demonstrate a novel signaling pathway in sensory neurons and provide a plausible mechanism for the enhancement of thermal sensitivity that occurs with inflammation and after activation of mGluRs on peripheral sensory neuron terminals.

Fig. 1.

Activation of mGlu1/5 enhances VR function in cultured mouse sensory neurons. a, Representative traces of capsaicin (Cap)-induced calcium responses. A 20 nm concentration of capsaicin was delivered twice (bars) with an interapplication interval of 10 min. Application of the mGlu1/5 agonist DHPG (100 μm, 3.5 min) significantly potentiated the second capsaicin response compared with control cells. b, DHPG dose–response curve for the increase in the second capsaicin responses. Values were measured as the ratio of the second capsaicin response to the first response, and the data points represent the means ± SEM from 18 to 33 cells for each point. The inset shows representative second capsaicin responses (normalized to the initial response), demonstrating dose-dependent enhancement of the peak calcium rise. Note that the duration of the response is also dramatically increased by DHPG treatment. c, Representative traces showing whole-cell patch-clamp recordings from cultured DRG neurons. Application of capsaicin (500 nm, 15 sec) induced inward currents that demonstrated desensitization. Application of DHPG (100 μm, 3 min) significantly potentiated the second response compared with control cells. The dashed lines ina and c represent the amplitude of the control response. d, Mean ± SEM response ratio for 11 cells in each condition. e, mGlu1/5 activation does not enhance the KCl-evoked calcium influx. Application of 40 mm KCl induced reproducible calcium transients that were not affected by the application of 100 μm DHPG (n = 36 controls, 33 DHPG). f, Activation of mGlu1/5, PKC, and PKA sensitizes DRG neurons to capsaicin. After PDA, FSK, or DHPG treatment, capsaicin could evoke calcium responses in neurons that initially did not respond to capsaicin. Treatment with 100 μm DHPG, 50 μm forskolin, or 5 μm PDA increased the percentages of cells that responded to a second application of capsaicin in capsaicin-insensitive silent neurons.n = 616 cells from 12 coverslips for control, 592 cells from 12 coverslips for DHPG, 195 cells from 8 coverslips for forskolin, and 305 cells from 8 coverslips for PDA. *p < 0.01; ANOVA.

Fig. 2.

DHPG-induced modulation of capsaicin responses and intracellular calcium levels is mediated by mGlu5. a, Representative traces of DHPG-induced calcium transients in cultured mouse DRG neurons. A 10 μm concentration of MPEP but not 100 μm LY367385 blocked DHPG-induced calcium transients. DHPG is applied for 3.5 min, beginning at the arrows. The percentages of cells responding to DHPG in control conditions or in the presence of MPEP or LY367385 (LY) are shown in b (n = 469 controls, 474 LY367385, and 553 MPEP). c, Representative traces showing the effect of the mGlu5 antagonist MPEP (10 μm) and the mGlu1 antagonist LY367385 (100 μm) on the sensitizing effect of DHPG on capsaicin responses. d, Mean ± SEM data for the experiments shown in c.Asterisks indicate significant differences atp < 0.05 compared with controls.n = 23–39 cells for each condition.

Fig. 3.

mGlu5 modulation of capsaicin (Cap) responses does not involve PKC. a, Representative traces showing the calcium responses to application of capsaicin (20 nm; black bars) and the effect of application of the PKC activator PDA (5 μm; white bars) or the mGlu1/5 agonist DHPG (100 μm;gray bars). Note that both PDA and DHPG enhance capsaicin responses, but only the PDA effects are blocked by the PKC inhibitors RO31−8220 (b) (100 nm) and GF109203X (c) (1 μm).d, Mean ± SEM data for the experiments shown ina–c (n = 22–47 cells for each condition). RO, RO31−8220; GF, GF109203X. *p < 0.05; ANOVA.Asterisks within the bars indicate a significant increase compared with the control response ratio.

Fig. 4.

PKA mediates the mGlu5 modulation of capsaicin receptor function. a, Representative traces showing the calcium responses to application of capsaicin (Cap; 20 nm; black bars) and the effect of application of the adenylyl cyclase activator FSK plus the phosphodiesterase inhibitor IBMX (50 μm each;white bars) or the mGlu1/5 agonist DHPG (100 μm; gray bars). Note that both forskolin and DHPG enhance capsaicin responses, and both are blocked by the PKA inhibitors KT5720 (1 μm; b) and H89 (10 μm; c). d, Means ± SEM data for the experiments shown in a–c(n = 19–47 cells for each condition).KT, KT5720. *p < 0.05; ANOVA.Asterisks within the bars indicate a significant increase compared with the control response ratio.

Fig. 5.

mGlu5 modulation of capsaicin receptors involved the PLC pathway. a, Representative traces showing the inhibition of capsaicin (CAP) responses by U73122 (5 μm), a PLC inhibitor, but not by U73343 (5 μm), a structural analog that does not inhibit PLC.b, U73122 but not U73343 completely inhibits the sensitization of capsaicin responses by DHPG (100 μm).c, Mean ± SEM data for the experiments shown ina and b (n = 20–42 cells for each condition). The dashed line represents the control response ratio for comparison. *p < 0.05; ANOVA. Asterisks within the barsindicate a significant decrease compared with the control response ratio.

Fig. 6.

mGlu5 modulation of capsaicin receptors requires DAG lipase and cyclooxygenase activity. a, Representative traces showing the lack of effect of the COX inhibitor indomethacin (Indo; 1 μm) and the DAG lipase inhibitor RHC-80267 (100 μm) on responses to capsaicin (CAP; 20 nm; black bars) and their desensitization. b, Both indomethacin and RHC-80267 completely block the ability of DHPG (100 μm) to sensitize capsaicin responses. c, Mean ± SEM data for experiments showing block of DHPG-induced potentiation by 1 μm indomethacin, 100 μmibuprofen, or 20 μm RHC-80267 (RHC) (n = 26–39 cells for each condition). Thedashed line represents the control response ratio for comparison. *p < 0.05; ANOVA.

Fig. 7.

mGlu5 modulation of capsaicin receptor function is blocked by a prostanoid receptor antagonist. a, Representative traces showing the sensitization of responses to capsaicin (CAP; 20 nm; black bars) by PGE₂ (250 nm) or DHPG (100 μm). b, Representative traces showing that the effects of PGE₂ or DHPG are completely blocked by the prostanoid receptor antagonist SC-51089 (SC; 10 μm). c, Mean ± SEM data for experiments showing significant sensitization of capsaicin responses by PGE₂ and DHPG and the block of these effects by SC-51089 (n = 22–42 cells for each condition). Thedashed line represents the control response ratio for comparison. *p < 0.05; ANOVA.

Fig. 8.

Blockade of cyclooxygenase or PKA but not PKC prevents DHPG-induced thermal hypersensitivity in mice.a, GF109203X (GF; 2.5 nmol preinjection and coinjection), a PKC inhibitor, fails to block DHPG-induced thermal hypersensitivity but significantly attenuates epinephrine (Epi)-induced thermal hypersensitivity (mean ± SEM; n = 8). b, Rp-cAMPS (10 nmol preinjection) and H89 (2.5 nmol preinjection and coinjection), both PKA inhibitors, completely block DHPG-induced thermal hypersensitivity (mean ± SEM; n = 4). c, The cyclooxygenase inhibitors indomethacin (Indo; 10 nmol preinjection) and aspirin (Asp; 50 nmol preinjection) eliminate DHPG-induced thermal hypersensitivity (mean ± SEM;n = 4 for indomethacin; n = 6 for aspirin). The dashed lines represent the control response for comparison. *p < 0.05; ANOVA followed by post hoc Tukey's comparisons.

Fig. 9.

Model depicting the signal transduction pathways involved in mGlu5 modulation of vanilloid receptor function in sensory neurons. The glutamate is shown being released from the primary afferent terminal, but glutamate may also originate from mast cells or nearby damaged cells. Note that prostaglandins need not be produced in the same cell as the vanilloid receptors that are ultimately modulated.AC, Adenylyl cyclase.