Hypersensitivity Induced by Activation of Spinal Cord PAR2 Receptors Is Partially Mediated by TRPV1 Receptors

Abstract

Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) receptors in the peripheral nerve endings are implicated in the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states. Both PAR2 and TRPV1 receptors are co-expressed in nociceptive dorsal root ganglion (DRG) neurons on their peripheral endings and also on presynaptic endings in the spinal cord dorsal horn. However, the modulation of nociceptive synaptic transmission in the superficial dorsal horn after activation of PAR2 and their functional coupling with TRPV1 is not clear. To investigate the role of spinal PAR2 activation on nociceptive modulation, intrathecal drug application was used in behavioural experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) were performed on superficial dorsal horn neurons in acute rat spinal cord slices. Intrathecal application of PAR2 activating peptide SLIGKV-NH2 induced thermal hyperalgesia, which was prevented by pretreatment with TRPV1 antagonist SB 366791 and was reduced by protein kinases inhibitor staurosporine. Patch-clamp experiments revealed robust decrease of mEPSC frequency (62.8 ± 4.9%), increase of sEPSC frequency (127.0 ± 5.9%) and eEPSC amplitude (126.9 ± 12.0%) in dorsal horn neurons after acute SLIGKV-NH2 application. All these EPSC changes, induced by PAR2 activation, were prevented by SB 366791 and staurosporine pretreatment. Our results demonstrate an important role of spinal PAR2 receptors in modulation of nociceptive transmission in the spinal cord dorsal horn at least partially mediated by activation of presynaptic TRPV1 receptors. The functional coupling between the PAR2 and TRPV1 receptors on the central branches of DRG neurons may be important especially during different pathological states when it may enhance pain perception.

Fig 1. Activation of spinal PAR2 induced thermal hyperalgesia.

(A) Intrathecal administration of PAR2 activating peptide SLIGKV-NH₂ (8 μg, 10 μl, n = 7) decreased the PWLs to radiant heat stimulation for several hours after the treatment. An inactive reverse peptide VKGILS-NH₂ (8 μg, 10 μl, n = 6) did not change the thermal threshold. TRPV1 antagonist SB 366791 (0.43 μg, 15 μl, n = 6) pre-treatment prevented SLIGKV-NH₂ induced decrease of PWLs. Staurosporine (0.014 μg, 15 μl, n = 7) pre-treatment also partially blocked the PWL decrease induced by SLIGKV-NH₂. White arrowhead: application of SB 366791 or staurosporine. Black arrowhead: application of SLIGKV-NH₂ or VKGILS-NH₂ (B) Paw withdrawal threshold to mechanical stimulation with von Frey filament was not significantly affected by any of the intrathecal treatments. Statistical differences between groups with various treatments were identified using Two-way ANOVA followed by multiple comparisons Student Newman Keuls test (*p < 0.05, **p < 0.01, ***p < 0.001 versus inactive peptide VKGILS-NH₂; ^#p < 0.05, ^###p < 0.001 versus SB 366791 + SLIGKV-NH₂).

Fig 2. Activation of PAR2 decreased the frequency of mEPSCs.

(A) Application of SLIGKV-NH₂ (100 μM, 4 min) lowered the frequency of mEPSC as is documented in the recording from one superficial dorsal horn neuron in acute spinal cord slice. (B) Cumulative amplitude analysis of mEPSCs under control conditions and during application of SLIGKV-NH₂ (100 μM, 4 min, n = 17) did not show statistically significant difference. (C) Application of SLIGKV-NH₂ (100 μM, 4 min) decreased the mEPSC frequency (n = 17; ***p < 0.001) compared to the pretreatment period (100%). Co-application of TRPV1 antagonist SB 366791 (10 μM, 4 min, n = 8) or staurosporine (250 nM, 4 min, n = 10) prevented the inhibitory effect of SLIGKV-NH₂ (100 μM) treatment and the mean mEPSC values were statistically different compared to the application of SLIGKV-NH₂ alone (^#p < 0.05, ^##p < 0.01).

Fig 3. PAR2 activation increased the frequency of sEPSCs.

(A) Application of SLIGKV-NH₂ (100 μM, 4 min) increased the sEPSC frequency as documented in recording from one superficial dorsal horn neuron. (B) The amplitude of the sEPSCs did not change during SLIGKV-NH₂ application (100 μM, 4 min, n = 17). (C) Application of SLIGKV-NH₂ (100 μM, 4 min) increased the sEPSC frequency compared to the pre-treatment values set as 100% (n = 17; ***p < 0.001). Application of TRPV1 antagonist SB 366791 (10 μM, 4 min, n = 10) or staurosporine (250 nM, 4 min, n = 9) prevented the excitatory effect of SLIGKV-NH₂ treatment and the mean sEPSC frequency values were statistically different from the application of SLIGKV-NH₂ alone (^#p < 0.05).

Fig 4. Activation of PAR2 increased the amplitude of EPSCs evoked by dorsal root stimulation.

(A) Application of SLIGKV-NH₂ (100 μM, 4 min) increased the amplitude of the evoked EPSC. (B) The increase of eEPSCs amplitude during the SLIGKV-NH₂ (100 μM, 4 min) application was statistically significant compared to pre-treatment values (n = 17, *p < 0.05). Application of SB 366791 (10 μM, 4 min, n = 10) or staurosporine (250 nM, 4 min, n = 9) prevented the SLIGKV-NH₂ induced eEPSC amplitude increase. The mean eEPSC frequency of SB 366791 and SLIGKV-NH₂ co-application was statistically different from the application of SLIGKV-NH₂ alone (^#p < 0.05).