The effects of sympathetic outflow on upregulation of vanilloid receptors TRPV(1) in primary afferent neurons evoked by intradermal capsaicin

Abstract

The vanilloid receptor TRPV(1) is a key nociceptive molecule located in primary afferent nociceptive neurons in dorsal root ganglia (DRG) for initiating neurogenic inflammation and pain. Our recent study demonstrates that up-regulation of TRPV(1) receptors by intradermal injection of capsaicin is modulated by activation of the protein kinase C (PKC) cascade. Neurogenic inflammation and pain resulting from capsaicin injection are sympathetically dependent, responding to norepinephrine, adenosine 5'-triphosphate (ATP) and/or neuropeptide Y released from sympathetic efferents. In a rat model of acute neurogenic inflammatory pain produced by capsaicin injection, we used immunofluorescence and Western blots combined with pharmacology and surgical sympathectomies to analyze whether the capsaicin-evoked up-regulation of TRPV(1) in DRG neurons is affected by sympathetic outflow by way of activating the PKC cascade. Sympathetic denervation reduced significantly the capsaicin-evoked expressions of TRPV(1), calcitonin gene-related peptide and/or phosphorylated PKC and their co-expression. These reductions could be restored by exogenous pretreatment with an analog of ATP, alpha,beta-methylene ATP. Inhibition of PKC with chelerythrine chloride prevented the ATP effect. Consistent results were obtained from experiments in which capsaicin-evoked changes in cutaneous inflammation (vasodilation and edema) were examined after sympathetic denervation, and the effects of the above pharmacological manipulations were evaluated. Our findings suggest that the capsaicin-evoked up-regulation of TRPV(1) receptors in DRG neurons is modulated sympathetically by the action of ATP released from sympathetic efferents to activate the PKC cascade. Thus, this study proposes a potential new mechanism of sympathetic modulation of neurogenic inflammation.

Figure 1

Confocal immunofluorescence images showing single labeling of TRPV₁, p-PKCα and CGRP, and double or triple labeling of these three molecules in L₄ DRG on the side ipsilateral to intradermal injection of CAP at 30 min after intradermal injection of CAP or vehicle in sham-sympathectomized (Sham-symp.+Veh, Sham-symp.+CAP) and sympathectomized (Symp.+Veh, Symp.+CAP) rats. Bar=100 μm.

Figure 2

Grouped data summarizing changes in the number of TRPV₁-, p-PKC (α and β subunits)-, and CGRP-positive neurons (A–H) and in percent TRPV₁ positive neurons with p-PKC double staining, percent TRPV₁ positive neurons with CGRP double staining, and percent TRPV₁-p-PKC positive neurons with CGRP triple staining (I–R) in L_4–5 DRGs after CAP injection in sympathetically intact (Sham-symp.+CAP) and sympathectomized (Symp.+CAP) groups. DRGs were sampled at 30, 60 and 90 min after vehicle or CAP injection. Statistical analysis (*, P<0.05, **, P<0.01; ***, P<0.001) was made of changes due to CAP injection vs. those due to vehicle injection at the same time point in the group having sham-sympathectomy or sympathectomy, and of the CAP-induced effects between the groups of sham-sympathectomy and sympathectomy at the same time point (+, P<0.05; ++, P<0.01; + ++, P<0.001).

Figure 3

Western blot analyses of the evoked changes in the relative density of TRPV₁, p-PKCα and p-PKCε in L₄ and L₅ DRG tissue following CAP injection in sham-sympathectomized and sympathectomized rats at 30, 60 and 90 min after unilateral injection of CAP. Immunoreactivity of TRPV₁, p-PKCα and p-PKCε was normalized to β-actin. A. Representative Western blots for TRPV₁, p-PKCα and p-PKCε to CAP injection at 30, 60 and 90 min after injection in the sham-sympathectomized and sympathectomized rats. B. Grouped data of changes in these molecules. * P<0.05, ** P<0.01, *** P<0.001, compared with vehicle injection at the same time point in the group of sham-sympathectomy or sympathectomy. +, P<0.05, ++ P<0.01, +++ P<0.001, compared with the CAP-induced effects under sham-sympathectomized conditions.

Figure 4

Confocal immunofluorescence images showing the restoration by activation of peripheral P2X (not P2Y) receptors of the reduction in the CAP-evoked single staining for TRPV₁, p-PKC, and CGRP and double or triple staining for these molecules in L₄ DRG on the side ipsilateral to CAP injection after sympathectomy and the effects of PKC inhibition. Data were sampled at 30 min after CAP injection. Symp.+Veh+Veh: CAP-evoked expression in a sympathectomized rat with vehicle pretreatments (used for dissolving the P2X or P2Y agonist and C.C.); Symp.+Veh+α,β-meATP: CAP-evoked expression in a sympathectomized rat without C.C. and with α,β-meATP pretreatments; Symp.+Veh+UTP: CAP-evoked expression in a sympathectomized rat without C.C. and with UTP pretreatments; Symp.+C.C.+α,β-meATP: CAP-evoked expression with C.C. and α,β-meATP pretreatments. Scale bars=100 μm.

Figure 5

Grouped data summarizing the restoration by activation of peripheral P2X (not P2Y) receptors on the reduction in the CAP-evoked single staining for TRPV₁, p-PKC, and CGRP (A–D) and the reduction in percentage TRPV₁ positive neurons with p-PKC double staining (E and F), percentage TRPV₁ positive neurons with CGRP double staining (G), and percentage TRPV₁-p-PKC positive neurons with CGRP triple staining (H and I) in L₄ and L₅ DRGs on the side ipsilateral to CAP injection after sympathectomy and the effects of PKC inhibition. *, **, and ***: P<0.05, P<0.01 and P<0.001, compared to the group of sympathectomy with vehicle pretreatment (Symp.+Veh+Veh). +, ++, and +++: P<0.05, P<0.01 and P<0.001, comparison between the groups of sympathectomy without C.C. and with α,β-meATP pretreatments (Symp.+Veh+α,β-meATP) and of sympathectomy with C.C. and α,β-meATP pretreatments (Symp.+C.C.+α,β-meATP).

Figure 6

Western blot analyses of the restoration by activation of peripheral P2X (not P2Y) receptors on the reduction in the CAP-evoked expressions of TRPV₁, p-PKCα and p-PKCε proteins in L₄ and L₅ DRGs after sympathectomy and the effects of PKC inhibition. A: Representative Western blots for TRPV₁, p-PKCα and p-PKCε in DRGs ipsilateral to CAP injection at 30 min after CAP injection in the sympathectomized rats pretreated with and without α,β-meATP or UTP when PKC was and was not inhibited by chelerythrine chloride. B: Grouped data of showing changes in these molecules. * and ***: P<0.05 and P<0.001, compared to the group with symapthectomy and vehicle pretreatments (Symp.+Veh+Veh). ++ and +++: P<0.01 and P<0.001, comparison between the groups of sympathectomy without C.C. and with α,β-meATP pretreatments (Symp.+Veh+α,β-meATP) and sympathectomy with C.C. and α,β-meATP pretreatments (Symp.+C.C.+ α,β-meATP).

Figure 7

Confocal immunofluorescence images showing changes in the CAP-evoked single labeling of TRPV₁, p-PKCα, and CGRP and double or triple labeling of these molecules in neurons of the L₄ DRG on the side ipsilateral to CAP injection after sympathectomy (E–H, Symp.+Veh) compared to the CAP-evoked changes in the sham-sympathectomized (A–D, Sham-symp.+Veh) and the effects of PKC activation by pretreatment with PDBu (I–L, Symp.+PDBu). Data were sampled at 30 min after CAP injection. Scale bar=100 μm.

Figure 8

Grouped data summarizing changes in the CAP-evoked single labeling for TRPV₁, p-PKC (α and ε subunits), and CGRP (A–D) and changes in the CAP-evoked percentage of TRPV₁ positive neurons with p-PKC double staining (E and F), percentage of TRPV₁ positive neurons with CGRP double staining (G), and percentage of TRPV₁-p-PKC positive neurons with CGRP triple staining (H and I) in L₄ and L₅ DRGs on the side ipsilateral to CAP injection after sympathectomy and the effects of PKC activation by pretreatment with PDBu (Symp.+PDBu). Data were sampled at 30 min after CAP injection. *, **, and ***, P<0.05, P<0.01, and P<0.001, compared with the group having sham-sympathectomy with vehicle pretreatment (Sham-symp.+Veh). +, ++, and +++: P<0.05, P<0.01, and P<0.001, comparison between the groups of sympathectomy with vehicle pretreatment (Symp.+Veh) and of sympathectomy with PDBu pretreatment (Symp.+PDBu).

Figure 9

Western blot analyses of the CAP-evoked changes in the relative density of TRPV₁, p-PKCα and p-PKCε in L₄ and L₅ DRG tissue after sympathectomy and the effects of PKC activation by pretreatment with PDBu. Data were sampled at 30 min after CAP injection. A: Representative Western blots for TRPV₁, p-PKCα and p-PKCε in DRG tissue ipsilateral to CAP injection at 30 min after CAP injection in the sham-sympathectomized and sympathectomized rats with and without PDBu pretreatments. B: Grouped data of changes in these molecules. * and ***: P<0.05 and P<0.001, compared to the group of sham-sympathectomy with vehicle pretreatment. +++: P<0.001, comparison between groups of sympathectomy with vehicle pretreatment and sympathectomy with PDBu pretreatment.

Figure 10

A: Restorative effect of activation of peripheral P2X (not P2Y) receptors on the reduction in the CAP-induced enhancement of vasodilation (left panel) and edema (right panel) due to sympathectomy and the effect of PKC inhibition. Chelerythrine chloride (C.C., a PKC inhibitor) was given 20 min prior to CAP injection. α,β-meATP (a P2X agonist) or UTP (a P2Y agonist) was injected 10 min prior to CAP injection. **: P<0.01, compared to the group of sympathectomy with vehicle pretreatment/without C.C. pretreatment (Symp. +Vehicle+Vehicle). +: P<0.05, compared to the group of sympathectomy with α,β-meATP pretreatment/without C.C. pretreatment (Symp.+Vehicle+α,β-meATP). B: Restorative effect of activation of PKC by intra-arterial injection of PDBu (a PKC activator) on the reduction in the CAP-induced enhancement of vasodilation (left panel) and edema (right panel) due to sympathectomy (Symp.). PDBu was given 10 min prior to CAP injection. **: P<0.01, compared to the group of sympathectomy with vehicle pretreatment (Symp. +Vehicle). +: P<0.05, compared to the group of sham-sympathectomy with vehicle pretreatment (Sham-symp.+Vehicle).