Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice

Abstract

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

Fig. 1.

Localization of transient receptor potential (TRP) channels TRPV4-LI and TRPA1-LI in dorsal root ganglia (DRG) neurons innervating the pancreas, where LI indicates TRPA1-like immunoreactivity. The retrograde tracer 1,1′-dioctadecyl-3,3,3′,3-tetramethyl-indocarbocyanine perchlorate (DiI) was injected into the pancreas. After 10–15 days, TRPV4-LI (A) and TRPA1-LI (B) were localized with DiI in DRG (T₈–T₁₀) by immunofluorescence. Antibody specificities were evaluated by preincubation of diluted antibodies with immunizing peptides, as shown in C (TRPV4, 25 μM peptide; TRPA1, 10 μM peptide). Arrows indicate DiI-labeled neurons expressing TRPV4-LI or TRPA1-LI, and arrowheads indicate neurons expressing TRPV4-LI or TRPA1-LI but not DiI. Scale bar = 10 μm.

Fig. 2.

Localization of TRPV4-LI and TRPA1-LI in pancreatic nerve fibers. TRPV4-LI (A) and TRPA1-LI (B) were localized in pancreatic nerve fibers. Arrows indicate TRP-expressing nerve fibers. Right: controls of pancreas sections stained with preadsorbed antibodies, as in Fig. 1. Scale bar = 20 μm.

Fig. 3.

TRP channel-mediated calcium signaling in DRG neurons innervating the pancreas. DiI or Alexa Fluor 594-conjugated choleratoxin B (Alexa-CTB) was injected into the pancreas. After 10–15 days (DiI) or 4 days (Alexa-CTB), DRG (T₈–T₁₀) were isolated and cultured. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in individual neurons challenged with 4α-phorbol 12,13 didecanoate (4αPDD; A), mustard oil (MO; B), 15-deoxy-Δ^12,14-prostaglandin J₂ (15dPGJ₂; C), or 4-hydroxy-2-nonenol (HNE; D). Left: changes in 340/380 ratio, which is proportional to [Ca²⁺]_i, in individual DiI-labeled neurons from trpv4- (trpv4-wt; A) and trpv1-wild-type mice (trpa1-wt; B–D). Traces are from individual neurons indicated by arrows (middle) (A, C, and D are fluorescence images; B is merged fluorescence and phase image). Right: maximal increases over basal values in 340/380 ratio (Δ340/380) in neurons from trpv4-wt and trpv4-ko mice (A) and trpa1-wt and trpa1-ko mice (B–D). *P < 0.05.

Fig. 4.

Effects of TRPV4 agonists on pancreatic inflammation. 4αPDD or vehicle control (VC) (A) or hypotonic (Hypo) or isotonic (Iso) NaCl (B) were injected into the pancreatic duct of trpv4-wt and trpv4-ko mice. After 2.5 h, serum amylase and pancreatic myeloperoxidase (MPO) and histology severity score (HSS) were measured. 4αPDD did not cause detectable pancreatitis compared with vehicle in trpv4-wt and trpv4-ko mice. Hypotonic NaCl did not affect serum amylase or pancreatic MPO but did increase pancreatic HSS, which was not different in trpv4-wt and trpv4-ko mice.

Fig. 5.

Effects of TRPV4 agonists on c-Fos expression in spinal neurons. 4αPDD or vehicle control (A and C) or hypotonic or isotonic NaCl (B) were injected into the pancreatic duct of trpv4-wt and trpv4-ko mice. After 2.5 h, c-Fos-LI was localized in the dorsal horn of the spinal cord (T₆–T₁₂) and the number of c-Fos-LI-positive nuclei per field was determined. 4αPDD and hypotonic NaCl increased c-Fos-LI in the nucleus of neurons in superficial laminae (arrows) of T₈–T₁₀ of trpv4-wt but not trpv4-ko mice. There was no increase in c-Fos-LI in T₆ or T₁₂. *,#P < 0.05. Scale bar = 100 μm.

Fig. 6.

Effects of TRPA1 agonists on pancreatic inflammation. MO (A), 15dPGJ₂ (B), HNE (C), or vehicle control were injected into the pancreatic duct of trpa1-wt and trpa1-ko mice. After 2.5 h, serum amylase and pancreatic MPO and HSS were measured. All agonists induced pancreatic inflammation in trpa1-wt mice, and inflammation was diminished in trpa1-ko mice. *,#P < 0.05.

Fig. 7.

Effects of TRPA1 agonists on pancreatic histology. MO, HNE, or vehicle control were injected into the pancreatic duct of trpa1-wt (top) and trpa1-ko (bottom) mice. After 2.5 h, tissue was collected for histology. In trpa1-wt mice, MO and HNE induced edema, cellular infiltration, formation of vacuoles, and necrosis, which were diminished in trpa1-ko mice. Scale bar = 50 μm.

Fig. 8.

Effects of TRPA1 agonists on fos expression in spinal neurons. MO (A), 15dPGJ₂ (dPGJ₂; B), HNE (C and D), or vehicle control were injected into the pancreatic duct of trpa1-wt and trpa1-ko mice. After 2.5 h, c-Fos-LI was localized in the dorsal horn of the spinal cord and the number of c-Fos-LI-positive nuclei per field was determined. MO, 15dPGJ₂, and HNE increased c-Fos-LI in the nucleus of neurons in superficial laminae (arrows) of T₈–T₁₀ of trpa1-wt but not trpa1-ko mice. There was no increase in fos expression in T₆ or T₁₂. *,#P < 0.05. Scale bar = 100 μm.

Fig. 9.

Cerulein-induced pancreatitis. The trpv4-wt or trpv4-ko mice (A and C) and trpa1-wt or trpa1-ko mice (B and C) were treated with cerulein (CE) or vehicle control. After 12 h, serum amylase and pancreatic MPO and HSS were measured. In trpv4-wt and trpa1-wt mice, cerulein increased serum amylase, pancreatic MPO, and HSS, which was characterized by edema, cellular infiltration, formation of vacuoles, and necrosis. Inflammation was similar in trpv4-ko mice but was diminished in trpa1-ko mice. *,#P < 0.05. Scale bar = 50 μm.

Fig. 10.

Cerulein-induced fos expression in spinal neurons. The trpv4-wt or trpv4-ko mice (A) and trpa1-wt or trpa1-ko mice (B) were treated with cerulein or vehicle control. After 12 h, c-Fos-LI was localized in the dorsal horn of the spinal cord and the number of c-Fos-LI-positive nuclei per field was determined. In trpv4-wt and trpa1-wt mice, cerulein increased c-Fos-LI in the nucleus of neurons in superficial laminae (arrows) of T₆–T₁₂. c-Fos-LI was diminished at all levels in trpv4-ko and trpa1-ko mice. *,#P < 0.05. Scale bar = 100 μm.

Fig. 11.

Cerulein-induced pain behavior. The trpv4-wt or trpv4-ko mice (A) and trpa1-wt or trpa1-ko mice (B) were treated with cerulein or vehicle control. After 12 h, pain behavior was scored. In trpv4-wt and trpa1-wt mice, cerulein increased behaviors that are characteristic of visceral pain. Pain-related behavior was diminished in trpv4-ko and trpa1-ko mice. *,#P < 0.05.

Fig. 12.

Mechanisms of pancreatitis pain. Proinflammatory agents generated during pancreatitis can directly activate TRPV4 (1) and TRPA1 (2) expressed by primary spinal afferent neurons. Factors that activate TRPV4 include elevated intraductal pressures, altered tonicity of edema fluid, and arachidonic acid (AA) metabolites such as 5′,6′-epoxyeicosatrienoic acid (EET). Factors that activate TRPA1 include HNE, formed by peroxidation of membrane lipids in response to reactive oxygen species (ROS), and cyclopentenone prostaglandin metabolites. Proinflammatory agents, notably trypsins that activate PAR₂, also sensitize TRPV4 and TRPA1 (3) to enhance responsiveness of sensory nerves to TRP agonists. Activated TRPA1 stimulates release of substance P (SP) and calcitonin gene-related peptide (CGRP) in the pancreas, where SP activates the neurokinin 1 receptor (NK₁R) and CGRP activates the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), causing neurogenic inflammation (4). Activated TRPV4 and TRPA1 induce central transmission of painful stimuli with SP and CGRP release in the spinal cord and activation of spinal nociceptive neurons (5).