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Comparative Study
. 2018 Aug 8;38(32):7032-7057.
doi: 10.1523/JNEUROSCI.3542-17.2018. Epub 2018 Jul 5.

Angiotensin II Triggers Peripheral Macrophage-to-Sensory Neuron Redox Crosstalk to Elicit Pain

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Free PMC article
Comparative Study

Angiotensin II Triggers Peripheral Macrophage-to-Sensory Neuron Redox Crosstalk to Elicit Pain

Andrew J Shepherd et al. J Neurosci. .
Free PMC article

Abstract

Injury, inflammation, and nerve damage initiate a wide variety of cellular and molecular processes that culminate in hyperexcitation of sensory nerves, which underlies chronic inflammatory and neuropathic pain. Using behavioral readouts of pain hypersensitivity induced by angiotensin II (Ang II) injection into mouse hindpaws, our study shows that activation of the type 2 Ang II receptor (AT2R) and the cell-damage-sensing ion channel TRPA1 are required for peripheral mechanical pain sensitization induced by Ang II in male and female mice. However, we show that AT2R is not expressed in mouse and human dorsal root ganglia (DRG) sensory neurons. Instead, expression/activation of AT2R on peripheral/skin macrophages (MΦs) constitutes a critical trigger of mouse and human DRG sensory neuron excitation. Ang II-induced peripheral mechanical pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs. Furthermore, AT2R activation in MΦs triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on mouse and human DRG sensory neurons via cysteine modification of the channel. Our study thus identifies a translatable immune cell-to-sensory neuron signaling crosstalk underlying peripheral nociceptor sensitization. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic pain and thus identifies multiple druggable analgesic targets.SIGNIFICANCE STATEMENT Pain is a widespread health problem that is undermanaged by currently available analgesics. Findings from a recent clinical trial on a type II angiotensin II receptor (AT2R) antagonist showed effective analgesia for neuropathic pain. AT2R antagonists have been shown to reduce neuropathy-, inflammation- and bone cancer-associated pain in rodents. We report that activation of AT2R in macrophages (MΦs) that infiltrate the site of injury, but not in sensory neurons, triggers an intercellular redox communication with sensory neurons via activation of the cell damage/pain-sensing ion channel TRPA1. This MΦ-to-sensory neuron crosstalk results in peripheral pain sensitization. Our findings provide an evidence-based mechanism underlying the analgesic action of AT2R antagonists, which could accelerate the development of efficacious non-opioid analgesic drugs for multiple pain conditions.

Keywords: AT2R; TRPA1; angiotensin II; neuroimmune interaction; oxidative stress; pain.

Figures

Figure 1.
Figure 1.
Ang II induces peripheral mechanical pain hypersensitivity in mice, without any sex-specific differences. A, Intraplantar Ang II injection dose dependently induces mechanical hypersensitivity in the ipsilateral hindpaw of C57BL/6 mice. Data are presented as mean ± SEM (n = 8 per group). B, Increasing doses of intraplantar Ang II injection does not influence heat sensitivity in mouse hindpaws. Data are presented as mean ± SEM (n = 8 per group). C, Intrathecal Ang II (100 pmol) does not induce significant mechanical hypersensitivity. Data are presented as mean ± SEM (n = 7 per group). D, E, Both male and female mice develop hindpaw mechanical hypersensitivity of a similar magnitude (D) in response to Ang II injection (intraplantar) without influence on hindpaw heat sensitivity (E). Data are presented as mean ± SEM (n = 7 per group). *p < 0.05, **p < 0.01, ***p < 0.001, and not significant (ns) versus their respective baseline values, as well as individual time points in saline group (AC) or contralateral groups (D, E), two-way ANOVA with Tukey's multiple-comparisons post hoc test.
Figure 2.
Figure 2.
AT2R, but not AT1R, mediates Ang II-induced mechanical pain hypersensitivity in mice. A, Coadministration of losartan, an AT1R antagonist (10 pmol, ipl), does not influence Ang II-induced (100 pmol, ipl) mechanical hypersensitivity in C57BL/6 (B6) mice. Ang II (100 pmol, ipl) induces mechanical hypersensitivity in ipsilateral hindpaws of B6 and B6-Agtr1a-KO mice to a similar magnitude. Data are presented as mean ± SEM (n = 6 for B6-losartan group and n = 7 each for the remaining groups); **p < 0.01 and ***p < 0.001 versus their respective baseline values, as well as individual time points in B6-losartan and B6-Agtr1a-KO-saline groups. B, Coadministration of PD123319, an AT2R antagonist (10 pmol, ipl), completely attenuates Ang II-induced (100 pmol, ipl) mechanical hypersensitivity in ipsilateral hindpaws of B6 mice. Ang II (100 pmol, ipl) induces mechanical hypersensitivity in in ipsilateral hindpaws of B6 and FVB-Agtr2-WT mice to a similar magnitude, which is absent in FVB-Agtr2-KO mice. Data are presented as mean ± SEM (n = 7 each for B6-PD123319, B6-Ang II+PD123319, and FVB-WT-Saline groups and n = 8 each for FVB-WT-Ang II, FVB-Agtr2-KO-Saline, and FVB-Agtr2-KO-Ang II groups). *p < 0.05, **p < 0.01, and ***p < 0.001 versus their respective baseline values, as well as individual time points in saline injection, for FVB-Agtr2-WT mice; not significant (ns) versus FVB-Agtr2-KO-saline and B6-PD123319 groups. C, Ang II injection (100 pmol, ipl) does not influence mouse hindpaw heat sensitivity in experimental conditions shown in A and B. ns, Not significant versus the respective group baseline values, as well as individual time points in saline-injected relevant KO genotype groups. D, E, Bradykinin injection (10 pmol, ipl) into the hindpaws of FVB-Agtr2-KO mice leads to development of mechanical (D) and heat (E) hypersensitivity, suggesting no deficits in induced cutaneous hypersensitivity due to Agtr2 gene deletion. Data are presented as mean ± SEM (n = 7 per group). **p < 0.01 and ***p < 0.001 versus their respective baseline values, as well as individual time points in contralateral groups, two-way ANOVA with Tukey's multiple-comparisons post hoc test.
Figure 3.
Figure 3.
Requirement of TRPA1, but not TRPV1 or TRPV4, for Ang II-induced mechanical pain hypersensitivity in mice. A, Coadministration of AP18, a TRPA1 antagonist (10 nmol, ipl) prevents Ang II-induced (100 pmol, ipl) mechanical hypersensitivity in the ipsilateral hindpaws of B6 mice. Ang II (100 pmol, ipl) elicits mechanical hypersensitivity in B6/129S-Trpa1-WT mice to a similar extent as seen in B6 mice, which is absent in B6/129S-Trpa1-KO mice. Data are presented as mean ± SEM (n = 6 each for B6-AP18 and B6/129-Trpa1-WT-Saline groups, n = 7 for B6/129-Trpa1-WT-Ang II group, n = 8 for B6-Ang II+AP18 group, and n = 9 each for B6/129-Trpa1-KO-Saline and B6/129-Trpa1-KO-Ang II groups). ***p < 0.001 versus their respective baseline values, as well as individual time pointsm in B6/129S-Trpa1-WT-saline group; not significant (ns) versus B6-WT-AP18 and B6/129S-Trpa1-KO-saline groups. B, Ang II (100 pmol, ipl) induces a similar magnitude of mechanical hypersensitivity in the ipsilateral hindpaws of B6, B6-Trpv1-KO, and B6-Trpv4-KO mice. Data are presented as mean ± SEM (n = 7 each for B6-Saline, B6-Ang II, B6-Trpv1-KO-Saline, B6-Trpv4-KO-Saline, and B6-Trpv4-KO-Ang II groups and n = 8 for B6-Trpv1-KO-Ang II group). *p < 0.05, **p < 0.01, and ***p < 0.001 versus their respective baseline values, as well as individual time points for saline injection, in respective mouse genotype groups. C, D, Ang II injection (100 pmol, ipl) does not influence hindpaw heat sensitivity in in the hindpaws of mice used in experimental conditions shown in A and B. ns, Not significant versus the respective group baseline values, as well as individual time points, in saline-injected relevant KO genotype groups, two-way ANOVA with Tukey's multiple-comparisons post hoc test.
Figure 4.
Figure 4.
Ang II has no direct influence on sensory neuron TRPA1- and TRPV1-mediated Ca2+ flux. A, Representative traces (from two individual neurons/species) showing that Ang II (1 μm) does not induce [Ca2+]i elevation in cultured mouse and human DRG sensory neurons in culture. AITC (100 μm) was used for the detection of TRPA1+ neurons. B, Quantification of Ang II-induced (1 μm; 1 h) [Ca2+]i elevation in cultured mouse and human DRG neurons. Peak neuronal [Ca2+]i elevation at 5 min intervals are normalized to baseline Ca2+ levels. AITC (100 μm) and 50 mm KCl (K50) are used to detect TRPA1 responsiveness and neurons, respectively. C, D, Ang II (1 μm) fails to potentiate AITC-induced (50 μm; 15 s; C) and capsaicin-induced (50 nm; 15 s; D) [Ca2+]i elevation, as depicted by representative traces from single neuron/species. Potentiation TRPA1- and TRPV1-induced [Ca2+]i elevation are quantified by calculating the ratio of second versus first AITC/Cap-induced Ca2+ flux in cultured mouse/human DRG neurons. As positive controls, bradykinin (100 nm) significantly potentiates AITC- and capsaicin-evoked Ca2+ flux and PGE2 (10 μm) potentiates capsaicin-induced Ca2+ elevation. Data are presented as mean ± SEM in BD. Numbers shown inside each column in BD indicate the number of DRG neurons in ≥4 culture batches from ≥4 mice/group or ≥4 human DRG culture batches/group. *p < 0.05, **p < 0.01, ***p < 0.001, and not significant (ns) versus respective baseline/vehicle groups, one-way ANOVA with Bonferroni's post hoc test.
Figure 5.
Figure 5.
Ang II has no direct influence on sensory neuron membrane potential and excitability. A, B, Ang II (1 μm; 5 min) fails to influence AP firing to ramp current injection and the membrane potential of cultured mouse (A) and human (B) DRG neurons. Data are presented as mean ± SEM. ns, Not significant versus respective vehicle groups, one-way ANOVA with Bonferroni's post hoc test. C, D, Representative AP firing traces to step current injections and graphs for step current thresholds and AP frequency of cultured mouse (C) and human (D) DRG neurons are also unaffected by Ang II exposure (1 μm, 5 min). Data are presented as mean ± SEM. Numbers shown inside each column or on line graphs in all panels indicate the number of DRG neurons in ≥4 culture batches from ≥4 mice/group or ≥4 human DRG culture batches/group. ns, Not significant versus respective vehicle groups, one-way ANOVA with Bonferroni's post hoc test. AP firing traces for vehicle- and Ang II-treated conditions in all panels are offset for distinct visualization from their respective control traces.
Figure 6.
Figure 6.
AT2R expression is not detected on mouse and human DRG sensory neurons. A, Representative confocal microscopy images of DRG tissue sections from FVB-Agtr2-WT and FVB-Agtr2-KO mice immunostained with routinely used anti-AT2R antibodies (red). Scale bar, 50 μm. No difference in AT2R signal intensity can be seen in the DRG sections from both mouse genotypes. B, Representative agarose gel electrophoresis images of RT-PCR amplification of AT1R and AT2R genes (Agtr1 and Agtr2, respectively) from the total RNA isolated from mouse and human DRGs. Plasmids containing mouse and human AT1R and AT2R cDNAs are used as a positive control. In addition, TRPV1 amplification is used as a positive control for DRG tissue. Numbers on the left denote DNA molecular weight markers (in base pairs). C, Ang II (1 μm; 30 min) does not induce phosphorylation of ERK1/2 and p38 MAPK, indicative of Ang II/AT2R activation, in cultured mouse and human DRG neurons. TNF-α (10 nm) is used as a positive control for induction of ERK1/2 and p38 MAPK phosphorylation. D, Quantification of the extent of ERK1/2 and p38 MAPK phosphorylation levels in mouse and human DRG neurons in response to Ang II exposure (1 μm; 30 min), as shown in representative immunoblots in Figure 3A. TNF-α (10 nm; 30 min) is used as a positive control for induction of ERK1/2 and p38 MAPK phosphorylation. Data are presented as individual experimental replicates, with mean ± SEM marked therein (n = 4 per group). *p < 0.05, **p < 0.01, and not significant (ns) versus respective comparison groups, one-way ANOVA with Tukey's multiple-comparisons post hoc test. E, The AT2R gene (Agtr2) is not expressed in neurons and non-neuronal cells in mouse DRG, as verified by lack of GFP signal (as a surrogate marker) in DRG sections (L2–L5) from Agtr2GFP reporter mice in which the Agtr2 promoter drives GFP expression. Representative confocal microscopy images of DRG sections stained with anti-GFP antibodies, along with CGRP and NF200 antibodies to mark peptidergic and myelinated sensory neurons. DAPI was used as a nuclear stain. Scale bar, 50 μm. F, G, Heat map showing mRNA expression levels (from RNAseq experiments) of RAS genes compared with critical pain-associated genes in human DRG tissue (F). No alteration in the mRNA levels of RAS genes can be observed in DRGs obtained from humans without or with chronic pain conditions (G). Red arrows indicate no reliable mRNA expression levels and green arrow indicates considerable mRNA expression of RAS genes.
Figure 7.
Figure 7.
Ang II induces peripheral MΦ infiltration in mouse hindpaw skin and increased MΦ density in skin biopsies from human patients with diabetic and chemotherapy-induced peripheral neuropathy. A, Representative confocal microscopy images of mouse hindpaw plantar punch tissue sections showing hindpaw Ang II injection (100 pmol, ipl) enhances MΦ (green; Iba1) and neutrophil (red, Ly6g; blue, DAPI) infiltration both 1 and 5 h after injection compared with saline injection. Scale bar, 100 μm. Magnified views of area indicated as red dotted rectangular boxes are shown on the right top corner in each image group. B, Representative confocal microscopy images of human plantar punch tissue sections showing increased MΦ density (Iba1) in human leg/ankle skin biopsies from diabetic neuropathy and chemotherapy-induced peripheral neuropathy patients compared with age-matched healthy controls. This is accompanied by a decrease in the density of nociceptive nerve fibers (PGP9.5) in the skin (bottom row images). Green dotted rectangular boxes on the top left corners in top row images represent magnified views of individual MΦs in indicated areas. Scale bar, 100 μm. C, Density of both MΦs and nociceptive fibers in human skin biopsies are quantified and presented as individual experimental replicates, with mean ± SEM marked therein (n = 2 sections each from n = 8 human subjects per group). *p < 0.05, ###p < 0.001, and not significant (ns) versus healthy control groups, one-way ANOVA with Tukey's multiple-comparisons post hoc test.
Figure 8.
Figure 8.
AT2R is expressed in MΦs. A, Representative agarose gel electrophoresis images of RT-PCR amplification of AT1R and AT2R genes (Agtr1a and Agtr2, respectively) from total RNA isolated from mouse MΦs and PMNs. Plasmids containing mouse Agtr1a and Agtr2 cDNAs are used as positive controls and Ly6g amplification is used for identification/validation of PMNs. Numbers on the left denote DNA molecular weight markers (in base pairs). B, Heat map showing moderate to high expression levels of RAS genes in monocytes/MΦs from RNA expression data deposited in the NCBI-GEO database. GEO mouse datasets GSE47426 (Mauer et al., 2014) and GSE57468 (An et al., 2014) and human datasets GSE10856 (Chang et al., 2008) and GSE20484 (Gleissner et al., 2010) are analyzed for RAS gene mRNA expression in monocytes/MΦs. C, Representative immunoblots depicting nonspecificity of routinely used anti-AT2R antibodies in peritoneal MΦ lysates from FVB-Agtr2-WT and FVB-Agtr2-KO mice. Anti-Iba1 and anti-mortalin antibodies are used as positive controls for MΦs and housekeeping protein, respectively. Numbers on the left denote protein molecular weight markers (in kilodaltons). D, E, Ang II (100 nm; 30 min) induces Erk1/2 phosphorylation in mouse (B6-WT) peritoneal MΦs, but not in PMNs. The AT2R inhibitor PD123319 (1 μm), but not the AT1R inhibitor losartan (1 μm), attenuates Ang II-induced Erk1/2 phosphorylation in MΦs. Ang II-induced Erk1/2 phosphorylation is absent in MΦs from FVB-Agtr2-KO mice, but intact in FVB-Agtr2-WT mice. The selective AT2R activator CGP42112A (100 nm) and TNF-α (10 nm) are used in mouse MΦs as positive controls for AT2R activation/signaling and Erk1/2 phosphorylation, respectively. LPS (10 nm) is used as a positive control for Erk1/2 phosphorylation in mouse PMNs. Mortalin (Grp75) immunoreactivity is used as loading control and the magnitude of ERK1/2 phosphorylation is quantified in E. Data are presented as individual experimental replicates, with mean ± SEM marked therein (n = 3 per group). *p < 0.05, #p < 0.05, and not significant (ns) versus indicated comparison groups, one-way ANOVA with Tukey's multiple-comparisons post hoc test.
Figure 9.
Figure 9.
AT2R is expressed in skin MΦs, but not in the neurons microglia/MΦs, of DRGs after hindpaw Ang II injection. A, Representative confocal microscopy images of Agtr2GFP mouse hindpaw plantar punch tissue sections showing hindpaw Ang II injection (100 pmol, ipl; 5 h after injection) enhances MΦ density (F4/80 and CD68 staining), which overlaps with GFP signal (green), suggesting AT2R expression therein. Scale bar, 50 μm. Images on the bottom rows represent magnified red dotted rectangular boxes in the respective top row images. B, Representative confocal microscopy images of Agtr2GFP mouse DRG (L2–L5) tissue sections showing hindpaw Ang II injection (100 pmol, ipl; 5 h after injection) does not enhance microglia/MΦ density (red, Iba1 staining) in the ipsilateral DRGs, as well as no detectable GFP signal (green, with anti-GFP antibody staining), suggesting no AT2R expression in neurons and microglia/MΦ. Scale bar, 50 μm. C, Representative microscopy images of cultured-dissociated DRGs from Agtr2GFP mice showing no detectable GFP signal (green, with anti-GFP antibody staining), suggesting no AT2R expression in neurons (red, β3-tubulin staining) and microglia/MΦ (red, Iba1 staining). Scale bar, 25 μm. D, Representative microscopy images of cultured peritoneal monocytes/MΦs from Agtr2GFP mice showing detection of GFP signal (green, with anti-GFP antibody staining) in Iba1-stained (red) MΦs, suggesting AT2R expression. Scale bar, 25 μm.
Figure 10.
Figure 10.
Peripheral MΦs are required for Ang II-induced mechanical hypersensitivity on mouse hindpaws. A, Experimental scheme for chemogenetic depletion of skin MΦs in MaFIA mice with the administration of a designer drug (B/B-HmD; 2 mg/kg/d for 5 d). Efficacy of peripheral MΦ depletion in skin, but not DRGs and spinal cord, in these mice were verified with immunostaining. Representative confocal microscopy images of hindpaw plantar punch, DRG (L2–L5) and spinal cord sections from MaFIA mice show depletion of skin MΦs without any alteration in microglia/MΦs in the DRG and spinal cord (red, Iba1 staining) of BB-HmD-treated mice. Scale bar, 50 μm. B, Ang II (100 pmol, ipl) fails to induce mechanical hypersensitivity in the hindpaws of MaFIA mice subjected to chemogenetic depletion of peripheral MΦs (as shown in A). Data are presented as mean ± SEM (n = 6 for Vehicle-Saline group, n = 7 each for Vehicle-Ang II and B/B-HmD-Saline groups, and n = 8 for B/B-HmD-Ang II group). ***p < 0.001 versus vehicle/saline-ipsi group, and ###p < 0.001 versus vehicle/Ang II-ipsi group, two-way ANOVA with Tukey's multiple-comparisons post hoc test. C, Chemogenetic depletion of MΦs and subsequent hindpaw Ang II injection (100 pmol, ipl) in MaFIA mice (B) does not influence hindpaw heat sensitivity. Data are presented as mean ± SEM (n = 7 for B/B-HmD-Saline group and n = 8 for B/B-HmD-Ang II group). D, E, Bradykinin injection (10 nmol, ipl) leads to the development of both mechanical (D) and heat (E) hypersensitivity in MaFIA mice subjected to chemogenetic depletion of peripheral MΦs with B/B-HmD administration (2 mg/kg/d for 5 d). Data are presented as mean ± SEM (n = 7 per group). *p < 0.05 **p < 0.01, and ***p < 0.001 versus respective contralateral groups, two-way ANOVA with Tukey's multiple-comparisons post hoc test.
Figure 11.
Figure 11.
Ang II induces no elevation in Ca2+, but ROS/RNS production in MΦs, in an AT2R-dependent manner. A, Representative traces and quantification of ratiometric Ca2+ imaging of primary C57BL/6J peritoneal MΦs showing no response to the TRPA1 agonist AITC (100 μm, 30 s) before or after application of Ang II (1 μm; 5 min). A terminal pulse of the Ca2+ ionophore ionomycin (Inm; 10 μm) serves as a positive control. Peak Ca2+ signal data (ratio of F340/F380) are plotted on the right column graph with the indicated treatment conditions. In addition, capsaicin (1 μm, 30 s) was used, which failed to induce any Ca2+, before or after Ang II application. B, Representative time-lapse images (left) and quantification traces (right) of cultured mouse peritoneal MΦs showing Ang II-induced (100 nm) increase in ROS/RNS production, as determined by increased intensity of DCFDA redox-sensitive fluorescent dye. Scale bar, 50 μm. C, Ang II and Ang III, but not Ang IV, exposure (15 min) dose dependently induces ROS/RNS production in mouse MΦs. D, Ang II (100 nm; 15 min) induces ROS/RNS production to a similar extent in both male and female mouse MΦs. E, Ang II-induced (100 nm; 15 min) MΦ ROS/RNS production can be attenuated by PD123319 (1 μm) and NAC (3 mm), but not with losartan (1 μm) coapplication. Like Ang II, the AT2R-selective agonist CGP42112A (100 nm; 15 min) also elevates ROS/RNS levels. F, ROS/RNS production in mouse MΦs in response to higher Ang II or Ang II doses (10 μm; 15 min) can be attenuated by PD123319 (1 μm). G, Ang II (100 nm; 15 min) increases ROS/RNS levels in Agtr1a-KO mouse MΦs, which can be attenuated by PD123319 (1 μm) coapplication. Both Ang II and CGP42112A (100 nm each; 15 min) increase ROS/RNS levels in FVB-Agtr2-WT, but not in FVB-Agtr2-KO, mouse MΦs. Furthermore, Ang II and CGP42112A (100 nm each; 15 min) significantly increase ROS/RNS levels in MΦs from B6-Agtr2GFP reporter mice similar to that observed in B6-WT mice. H, DCFDA fluorescence emission in mouse and human DRG neurons is unaffected by Ang II (1 μm; 15 min) and CGP42112A (1 μm; 15 min) exposure, indicating no Ang II-induced ROS/RNS production. Data in all panels are presented as mean ± SEM. Numbers shown inside each column in all panels indicate the number of MΦs and DRG neurons in ≥4 culture batches from ≥4 mice (or human DRG culture batches for H) per group. ***p < 0.001, ###p < 0.001, and not significant (ns) versus respective vehicle (C) and indicated comparison groups (A, B and D, H), one-way ANOVA with Tukey's post hoc test.
Figure 12.
Figure 12.
Ang II induces AT2R-dependent ROS/RNS production in mouse hindpaws and attenuation of Ang II-induced hindpaw mechanical hypersensitivity by ROS/RNS scavenging. A, Hindpaw injection of Ang II (100 pmol; 1 h) increases local ROS/RNS production, as determined by increased L-012 redox-sensitive dye luminescence intensity and quantified on the graph (right). Coinjection of PD123319 (10 pmol) and NAC (30 nmol) completely attenuate Ang II-induced ROS/RNS production. Data are presented as mean ± SEM (n = 5 mice per group). ***p < 0.001, ###p < 0.001, and not significant (ns) versus respective comparison groups, one-way ANOVA with Tukey's multiple-comparisons post hoc test. B, Coadministration of NAC (30 nmol, ipl) completely attenuates Ang II-induced (100 pmol, ipl) hindpaw mechanical hypersensitivity in mice. NAC administration does not influence hindpaw heat sensitivity. Data are presented as mean ± SEM (n = 9 per group). ***p < 0.001 versus Ang II/contra, ###p < 0.001 versus Ang II/ipsi groups, and not significant (ns) versus respective comparison groups, two-way ANOVA with Tukey's multiple-comparisons post hoc test.
Figure 13.
Figure 13.
Ang II-induced MΦ ROS/RNS production trans-activates TRPA1 on mouse DRG neurons. A, Representative traces of Ang II-induced (100 nm, 1 h) [Ca2+]i elevation in mouse DRG neurons observed only upon coculturing with mouse MΦs (both B6-WT mice). TRPA1+ neurons are identified by AITC (100 μm) and 50 mm KCl (K50). Area under the curve (AUC) for Ang II-induced [Ca2+]i elevation is subsequently quantified. B, C, Ang II-induced increases in DRG neuron [Ca2+]i elevation in cocultures can be completely attenuated upon coapplication of NAC (3 mm; B) and the TRPA1 antagonist A967079 (1 μm; C). Ang II (100 nm, 1 h) fails to induce [Ca2+]i elevation in FVB-Agtr2-WT DRG neurons cocultured with FVB-Agtr2-KO MΦs; however, increased [Ca2+]i is conserved in FVB-Agtr2-KO DRG neurons cocultured with FVB-Agtr2-WT MΦs (C). Data are presented as mean ± SEM. ***p < 0.001 and not significant (ns) versus respective comparison groups, one-way ANOVA with Tukey's post hoc test. D, Representative traces and quantification of bradykinin (BK; 100 nm; 5 min)-evoked potentiation of AITC-induced (50 μm) and capsaicin-induced (100 nm) Ca2+ flux in Agtr2-KO DRG neurons. Data are presented as mean ± SEM. *p < 0.05 versus control groups, one-way ANOVA with Tukey's post hoc test. E, Top left, Representative agarose gel electrophoresis images of RT-PCR amplification of AT1R and AT2R genes (Agtr1a and Agtr2) from total RNA isolated from the mouse monocyte-MΦ cell line J774A.1. Gapdh amplification is used as a positive control. Numbers on the left denote DNA molecular weight markers (in base pairs). Bottom left, Western blot images demonstrating increased Erk1/2 phosphorylation in J774A.1 cells treated with Ang II (100 nm; 30 min), the AT2R-selective agonist CGP42112A (100 nm; 30 min), or TNF-α (10 nm; 30 min) as a positive control. The Ang II-mediated increase in p-ERK1/2 is inhibited by coapplication of PD123319 (1 μm), but not losartan (1 μm). Numbers on the left denote protein molecular weight markers (in kilodaltons). Right, J774A.1 cells demonstrate AT2R-dependent increased DCFDA fluorescence, which is inhibited by PD123319 (1 μm) or the antioxidant NAC (3 mm), but not losartan (1 μm). Data are presented as mean ± SEM. ***p < 0.001 versus vehicle, ###p < 0.001 versus Ang II. F, Ang II (100 nm, 1 h) induces increase in DRG neuron [Ca2+]i elevation upon coculturing with J774A.1 MΦ cells. Data in BF are presented as mean ± SEM and numbers shown inside each column in these panels indicate the number of DRG neurons (and MΦs for E) in ≥4 culture batches from ≥4 mice/group. **p < 0.05, ***p < 0.001, ###p < 0.001, and not significant (ns) versus the indicated comparison groups, one-way ANOVA with Tukey's post hoc test.
Figure 14.
Figure 14.
Ang II-induced MΦ ROS/RNS production in U937 human MΦ cells trans-activates TRPA1 on human DRG neurons. A, Top left, Representative agarose gel electrophoresis images of RT-PCR amplification of AT1R and AT2R genes (AGTR1 and AGTR2) from total RNA isolated from the human monocyte-MΦ cell line U937. GAPDH amplification is used as a positive control. Numbers on the left denote DNA molecular weight markers (in base pairs). Bottom left, Western blot images demonstrating increased Erk1/2 phosphorylation in U937 cells treated with Ang II (100 nm; 30 min), the AT2R-selective agonist CGP42112A (100 nm; 30 min), or TNF-α (10 nm; 30 min) as a positive control. The Ang II-mediated increase in p-ERK1/2 is inhibited by coapplication of PD123319 (1 μm), but not losartan (1 μm). Numbers on the left denote protein molecular weight markers (in kilodaltons). Right, U937 cells demonstrate AT2R-dependent increased DCFDA fluorescence, which is inhibited by PD123319 (1 μm) or the antioxidant NAC (3 mm), but not losartan (1 μm). B, Ang II (100 nm, 1 h) induces significant elevation of [Ca2+]i levels in human DRG neurons upon coculturing with the U937 human MΦ cell line, which can be completely attenuated upon coapplication of the AT2R antagonist PD123319 (1 μm) and the TRPA1 antagonist A967079 (1 μm). C, Ang II (100 nm, 1 h) induces a significant elevation in [Ca2+]i levels in HEK293 cells transfected with eGFP+hTRPA1-WT, but not with eGFP alone or eGFP+hTRPA1–3C/S mutant cDNAs, upon coculturing with U937 human MΦ cell line. Data in all panels are presented as mean ± SEM and numbers shown inside each column indicate the number of MΦs (A) and DRG neurons (B, C) in ≥4 human DRG culture batches per group. **p < 0.05, ###p < 0.001, and not significant (ns) versus the indicated comparison groups, one-way ANOVA with Tukey's post hoc test.

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