Roles of Cav3.2 and TRPA1 channels targeted by hydrogen sulfide in pancreatic nociceptive processing in mice with or without acute pancreatitis.

Hydrogen sulfide (H(2)S), formed by multiple enzymes, including cystathionine-γ-lyase (CSE), targets Ca(v)3.2 T-type Ca(2+) channels (T channels) and transient receptor potential ankyrin-1 (TRPA1), facilitating somatic pain. Pancreatitis-related pain also appears to involve activation of T channels by H(2)S formed by the upregulated CSE. Therefore, this study investigates the roles of the Ca(v)3.2 isoform and/or TRPA1 in pancreatic nociception in the absence and presence of pancreatitis. In anesthetized mice, AP18, a TRPA1 inhibitor, abolished the Fos expression in the spinal dorsal horn caused by injection of a TRPA1 agonist into the pancreatic duct. As did mibefradil, a T-channel inhibitor, in our previous report, AP18 prevented the Fos expression following ductal NaHS, an H(2)S donor. In the mice with cerulein-induced acute pancreatitis, the referred hyperalgesia was suppressed by NNC 55-0396 (NNC), a selective T-channel inhibitor; zinc chloride; or ascorbic acid, known to inhibit Ca(v)3.2 selectively among three T-channel isoforms; and knockdown of Ca(v)3.2. In contrast, AP18 and knockdown of TRPA1 had no significant effect on the cerulein-induced referred hyperalgesia, although they significantly potentiated the antihyperalgesic effect of NNC at a subeffective dose. TRPA1 but not Ca(v)3.2 in the dorsal root ganglia was downregulated at a protein level in mice with cerulein-induced pancreatitis. The data indicate that TRPA1 and Ca(v)3.2 mediate the exogenous H(2)S-induced pancreatic nociception in naïve mice and suggest that, in the mice with pancreatitis, Ca(v)3.2 targeted by H(2)S primarily participates in the pancreatic pain, whereas TRPA1 is downregulated and plays a secondary role in pancreatic nociceptive signaling.

Contribution of TRPA1 as a downstream signal of proteinase-activated receptor-2 to pancreatic pain.

We examined if TRPA1, like TRPV1, contributes to pancreatic nociceptor excitation following proteinase-activated receptor-2 (PAR2) stimulation and to pancreatitis-related pain in mice. A PAR2-activating peptide, infused into the pancreatic duct, caused spinal Fos expression, which was prevented by AP18, a TRPA1 inhibitor. Repeated administration of cerulein caused referred hyperalgesia accompanying pancreatitis, which was reversed by SB366791, a TRPV1 inhibitor, but not AP18. AP18, administered in combination with a subeffective dose of SB366791, significantly suppressed the referred hyperalgesia. Our findings suggest that TRPA1, like TRPV1, mediates PAR2-triggered pancreatic nociception and that TRPA1 in collaboration with TRPV1 latently contributes to pancreatitis-related pain.

The proteinase/proteinase-activated receptor-2/transient receptor potential vanilloid-1 cascade impacts pancreatic pain in mice.

AIMS: Proteinase-activated receptor-2 (PAR2) and transient receptor potential vanilloid-1 (TRPV1) are co-localized in the primary afferents, and the trans-activation of TRPV1 by PAR2 activation is involved in processing of somatic pain. Given evidence for contribution of PAR2 to pancreatic pain, the present study aimed at clarifying the involvement of TRPV1 in processing of pancreatic pain by the proteinase/PAR2 pathway in mice. MAIN METHODS: Acute pancreatitis was created by repeated administration of cerulein in conscious mice, and the referred allodynia/hyperalgesia was assessed using von Frey filaments. Injection of PAR2 agonists into the pancreatic duct was achieved in anesthetized mice, and expression of Fos in the spinal cord was determined by immunohistochemistry. KEY FINDINGS: The established referred allodynia/hyperalgesia following cerulein treatment was abolished by post-treatment with nafamostat mesilate, a proteinase inhibitor, and with capsazepine, a TRPV1 antagonist, in mice. Injection of trypsin, an endogenous PAR2 agonist, or SLIGRL-NH(2), a PAR2-activating peptide, into the pancreatic duct caused expression of Fos protein in the spinal superficial layers at T8-T10 levels in the mice. The spinal Fos expression caused by trypsin and by SLIGRL-NH(2) was partially blocked by capsazepine, the former effect abolished by nafamostat mesilate. SIGNIFICANCE: Our data thus suggest that the proteinase/PAR2/TRPV1 cascade might impact pancreatic pain, in addition to somatic pain, and play a role in the maintenance of pancreatitis-related pain in mice.

Phosphorylation of ERK in the spinal dorsal horn following pancreatic pronociceptive stimuli with proteinase-activated receptor-2 agonists and hydrogen sulfide in rats: evidence for involvement of distinct mechanisms.

Noxious stimuli cause prompt phosphorylation of extracellular signal-regulated kinase (ERK) in the spinal dorsal horn that contributes to facilitation of pain sensation and is often used as an immediate marker for excitation of spinal neurons following somatic and colonic nociception. Here we asked whether two distinct pronociceptive stimuli with proteinase-activated receptor-2 (PAR2) agonists and hydrogen sulfide (H(2)S) in the pancreas cause phosphorylation of ERK in the spinal dorsal horn and also examined involvement of their possible downstream signaling molecules, transient receptor potential vanilloid-1 (TRPV1) and T-type Ca(2+) channels, respectively. Capsaicin (a TRPV1 agonist), trypsin (an endogenous PAR2 agonist), SLIGRL-NH(2) (a PAR2-activating peptide), and NaHS (an H(2)S donor) were infused into the pancreatic duct in anesthetized rats, and phosphorylated ERK in the spinal cord was detected by immunohistochemistry. Intraductal administration of capsaicin and trypsin caused prompt phosphorylation of ERK in the superficial layers of T9, but not T5 or T12, spinal dorsal horn. SLIGRL-NH(2) and NaHS, administered in the same manner, also produced ERK phosphorylation in the corresponding spinal regions. Mibefradil, a T-type Ca(2+) channel blocker, abolished the phosphorylation of ERK caused by intraductal NaHS but not SLIGRL-NH(2). In contrast, capsazepine, an inhibitor of TRPV1, suppressed the phosphorylation of ERK caused by intraductal SLIGRL-NH(2) but not NaHS. Our data thus demonstrate that pancreatic pronociceptive stimuli with PAR2 agonists and H(2)S cause ERK phosphorylation in the spinal dorsal horn, through activation of TRPV1 and T-type Ca(2+) channels, respectively, and that those two pronociceptive pathways are independent of each other.

The proteinase inhibitor camostat mesilate suppresses pancreatic pain in rodents.

Camostat mesilate, an orally available proteinase inhibitor, is clinically used for treatment of pancreatitis. Given recent evidence that pancreatic proteinases including trypsin and/or proteinase-activated receptor-2 (PAR2) might be involved in pancreatic pain, we examined if camostat mesilate could suppress spinal Fos expression, a marker for neuronal activation, following specific application of trypsin to the pancreas, and pancreatitis-related referred allodynia. Trypsin, administered into the pancreatic duct, caused delayed expression of Fos proteins in the superficial layer of the bilateral T8 and T9 spinal dorsal horns in rats. The trypsin-induced spinal Fos expression was completely abolished by oral pre-administration of camostat mesilate at 300 mg/kg. After hourly repeated (6 times in total) administration of caerulein, mice showed typical symptoms of pancreatitis, accompanied by mechanical allodynia in the upper abdomen (i.e., referred hyperalgesia/allodynia), as assessed by use of von Frey filaments. Camostat mesilate at 100-300 mg/kg, given orally twice before the 1st and 4th doses of caerulein, abolished the pancreatitis-related abdominal allodynia, while it partially prevented the inflammatory signs. The same doses of camostat mesilate, when administered once after the final dose of caerulein, also revealed significant anti-allodynic effect. These data suggest that camostat mesilate prevents and/or depresses pancreatitis-induced pain and/or referred hyperalgesia/allodynia, in which proteinases including trypsin would play a critical role.

Suppression by protease-activated receptor-2 activation of gastric acid secretion in rats.

Activation of protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, induces neurally mediated gastric mucus secretion accompanied by mucosal cytoprotection. In the present study, we investigated whether PAR-2 could modulate gastric acid secretion in rats. Messenger RNAs for PAR-2 and PAR-1 were detected in the gastric mucosa and smooth muscle. The PAR-2-activating peptide SLIGRL-NH(2), but not the inactive control peptide, when administered i.v., strongly suppressed gastric acid secretion in response to carbachol, pentagastrin or 2-deoxy-D-glucose in the rats with a pylorus ligation. The PAR-2-mediated suppression of acid secretion was resistant to cyclooxygenase inhibition or ablation of sensory neurons by capsaicin. Our results provide novel evidence that in addition to stimulating neurally mediated mucus secretion, activation of PAR-2 suppresses gastric acid secretion independently of prostanoid production or sensory neurons. These dual actions of PAR-2 would result in gastric mucosal cytoprotection.