Kenzo Kawai

Proteinase-activated receptor-2 (PAR-2): regulation of salivary and pancreatic exocrine secretion in vivo in rats and mice.

Proteinase‐activated receptor‐2 (PAR‐2) is expressed throughout the gastrointestinal tract including the pancreas, and may be involved in digestive functions. The aim of our study was to evaluate a potential role for PAR‐2 in regulating salivary and pancreatic exocrine secretion in vivo. PAR‐2‐activating peptides (PAR‐2‐APs), but not selective PAR‐1‐APs, administered intravenously, increased salivary secretion in the mouse or rat; this effect of the PAR‐2‐APs was unaffected by atropine, phentolamine, propranolol or indomethacin. Secretion (amylase) by rat parotid gland slices in vitro was also stimulated by PAR‐2‐APs and trypsin, but not by activation of other PARs. PAR‐2‐APs, administered to rats in vivo, caused a prompt effect on pancreatic exocrine secretion. PAR‐2 mRNA, known to be present in pancreatic tissue, was also detected in parotid tissue. Our results indicate that in addition to a potential role in regulating cardiovascular and respiratory functions, PAR‐2 may also play a general role in vivo for the direct regulation of glandular exocrine secretion.

Modulation by protease‐activated receptors of the rat duodenal motility in vitro: possible mechanisms underlying the evoked contraction and relaxation

The present study examined effects of agonist enzymes and receptor‐activating peptides for protease‐activated receptors (PARs) on duodenal motility in the rat, and also investigated possible mechanisms underlying the evoked responses. Thrombin at 0.03–0.1 μM and the PAR‐1‐activating peptide SFLLR‐NH2 at 3–100 μM or TFLLR‐NH2 at 10–50 μM produced a dual action, relaxation followed by contraction of the duodenal longitudinal muscle. The PAR‐2‐activating peptide SLIGRL‐NH2 at 10–100 μM elicited only small contraction. Trypsin at 0.08 μM induced small contraction, or relaxation followed by contraction, depending on preparations. The PAR‐4‐activating peptide GYPGKF‐NH2 at 1000 μM exhibited no effect. The contractile responses of the duodenal strips to TFLLR‐NH2 and to SLIGRL‐NH2 were partially attenuated by the L‐type calcium channel blocker nifedipine (1 μM), the protein kinase C inhibitor GF109203X (1 μM) and the tyrosine kinase inhibitor genistein (15 μM), but were resistant to indomethacin (3 μM) and tetrodotoxin (1–10 μM). The relaxation of the preparations exerted by TFLLR‐NH2 was unaffected by indomethacin (3 μM), propranolol (5 μM), NG‐nitro‐L‐arginine methyl ester (100 μM) and tetrodotoxin (1–10 μM). This relaxation was resistant to either GF109203X (1 μM) or genistein (15 μM), but was, remarkably, attenuated by combined application of these two kinase inhibitors. Apamin (0.1 μM), an inhibitor of calcium‐activated, small‐conductance potassium channels, but not charybdotoxin (0.1 μM), completely abolished the PAR‐1‐mediated duodenal relaxation, and significantly enhanced the PAR‐1‐mediated contraction. These findings demonstrate that PAR‐1 plays a dual role, suppression and facilitation of smooth muscle motility in the rat duodenum, while PAR‐2 plays a minor excitatory role in the muscle, and that PAR‐4 is not involved in the duodenal tension modulation. The results also suggest that the contractile responses to PAR‐1 and PAR‐2 activation are mediated, in part, by activation of L‐type calcium channels, protein kinase C and tyrosine kinase, and that the relaxation response to PAR‐1 activation occurs via activation of apamin‐sensitive, but charybdotoxin‐insensitive, potassium channels, in which both protein kinase C and tyrosine kinase might be involved synergistically.

Protease-activated receptor-2 (PAR-2) in the pancreas and parotid gland: Immunolocalization and involvement of nitric oxide in the evoked amylase secretion.

Protease-activated receptor-2, a G protein-coupled receptor activated by serine proteases such as trypsin, tryptase and coagulation factors VIIa and Xa, modulates pancreatic and salivary exocrine secretion. In the present study, we examined the distribution of PAR-2 in the pancreas and parotid gland, and characterized the PAR-2-mediated secretion of amylase by these tissues in vivo. Immunohistochemical analyses using the polyclonal antibody against rat PAR-2 clearly showed abundant expression of PAR-2 in rat pancreatic and parotid acini. The PAR-2 agonist SLIGRL-NH2, administered intraperitoneally (i.p.) at 1-10 micromol/kg and 1.5-15 micromol/kg, in combination with amastatin, an aminopeptidase inhibitor, facilitated in vivo secretion of pancreatic and salivary amylase in a dose-dependent manner, respectively, in the mouse. The PAR-2-mediated secretion of pancreatic amylase was abolished by pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. The secretion of salivary amylase in response to the PAR-2 agonist at a large dose, 15 micromol/kg, but not at a smaller dose, 5 micromol/kg, was partially reduced by L-NAME. Pretreatment with capsaicin for ablation of the sensory neurons did not modify the PAR-2-mediated secretion of pancreatic and salivary amylase in the mouse. In conclusion, our study demonstrates expression of PAR-2 in rat pancreatic acini as well as parotid acini and indicates that nitric oxide participates in the PAR-2-mediated in vivo secretion of pancreatic amylase, and, to a certain extent, of salivary amylase, although capsaicin-sensitive sensory neurons, known to be activated by PAR-2, are not involved in the evoked pancreatic or salivary amylase secretion.

In vivo evidence that protease-activated receptors 1 and 2 modulate gastrointestinal transit in the mouse

Protease‐activated receptors (PARs) 1 and 2 modulate the gastric and intestinal smooth muscle motility in vitro. In the present study, we examined if activation of PAR‐2 and PAR‐1 could alter gastrointestinal transit in mice. Intraperitoneal administration of the PAR‐2‐activating peptide SLIGRL‐NH2, but not the inactive control LSIGRL‐NH2, at 1–5 μmol kg−1, in combination with the aminopeptidase inhibitor amastatin at 2.5 μmol kg−1, facilitated gastrointestinal transit in a dose‐dependent manner. The human PAR‐1‐derived peptide SFLLR‐NH2 and the specific PAR‐1 agonist TFLLR‐NH2, but not the inactive control FSLLR‐NH2, at 2.5–10 μmol kg−1, in combination with amastatin, also promoted gastrointestinal transit. The Ca2+‐activated, small conductance K+ channel inhibitor apamin at 0.01 μmol kg−1 significantly potentiated the actions of SLIGRL‐NH2 and TFLLR‐NH2 at subeffective doses. The increased gastrointestinal transit exerted by either SLIGRL‐NH2 at 5 μmol kg−1 or TFLLR‐NH2 at 10 μmol kg−1 was completely abolished by the L‐type Ca2+ channel inhibitor verapamil at 61.6 μmol kg−1. In contrast, the tyrosine kinase inhibitor genistein at 18.5 μmol kg−1 failed to modify the effects of the agonists for PAR‐2 or PAR‐1. These findings demonstrate that PAR‐1 and PAR‐2 modulate gastrointestinal transit in mice in vivo. Our data also suggest that the PAR‐1‐and PAR‐2‐mediated effects are modulated by apamin‐sensitive K+ channels and are dependent on activation of L‐type Ca2+ channels, but independent of tyrosine kinase. Our study thus provides novel evidence for the physiological and/or pathophysiological roles of PARs 1 and 2 in the digestive systems, most probably during inflammation.

Protease-activated receptor-2 (PAR-2) in the rat gastric mucosa: immunolocalization and facilitation of pepsin/pepsinogen secretion

Agonists of protease‐activated receptor‐2 (PAR‐2) trigger neurally mediated mucus secretion accompanied by mucosal cytoprotection in the stomach. The present study immunolocalized PAR‐2 in the rat gastric mucosa and examined if PAR‐2 could modulate pepsin/pepsinogen secretion in rats. PAR‐2‐like immunoreactivity was abundant in the deep regions of gastric mucosa, especially in chief cells. The PAR‐2 agonist SLIGRL‐NH2, but not the control peptide LSIGRL‐NH2, administered i.v. repeatedly at 0.3 – 1 μmol kg−1, four times in total, significantly facilitated gastric pepsin secretion, although a single dose produced no significant effect. The PAR‐2‐mediated gastric pepsin secretion was resistant to omeprazole, NG‐nitro‐L‐arginine methyl ester (L‐NAME) or atropine, and also to ablation of sensory neurons by capsaicin. Our study thus provides novel evidence that PAR‐2 is localized in mucosal chief cells and facilitates gastric pepsin secretion in the rats, most probably by a direct mechanism.

Dual modulation by thrombin of the motility of rat oesophageal muscularis mucosae via two distinct protease-activated receptors (PARs): a novel role for PAR-4 as opposed to PAR-1

Since protease‐activated receptors (PARs) are distributed throughout the gastrointestinal tract, we investigated the role of PARs in modulation of the motility of the rat oesophageal muscularis mucosae. Thrombin produced contraction of segments of the upper and lower part of the smooth muscle. Trypsin contracted both the muscle preparations only at high concentrations. SFLLR‐NH2 and TFLLR‐NH2 (PAR‐1‐activating peptides), but not the PAR‐1‐inactive peptide FSLLR‐NH2, evoked a marked contraction. In contrast, the PAR‐2 agonist SLIGRL‐NH2 and the PAR‐4 agonist GYPGKF‐NH2 caused no or only a negligible contraction. In oesophageal preparations precontracted with carbachol, thrombin produced a dual action i.e. relaxation followed by contraction. TFLLR‐NH2 further contracted the precontracted preparations with no preceding relaxation. GYPGKF‐NH2, but not the inactive peptide GAPGKF‐NH2, produced marked relaxation. Trypsin or SLIGRL‐NH2 caused no relaxation. The PAR‐1‐mediated contraction was completely abolished in Ca2+‐free medium and considerably attenuated by nifedipine (1 μM) and in a low Na+ medium. The PAR‐4‐mediated relaxation was resistant to tetrodotoxin (10 μM), apamin (0.1 μM), charybdotoxin (0.1 μM), L‐NG‐nitroarginine methyl ester (100 μM), indomethacin (3 μM), propranolol (5 μM) or adenosine 3′,5′‐cyclic monophosphorothioate, 8‐bromo, Rp‐isomer (30 μM). Thus, thrombin plays a dual role in modulating the motility of the oesophageal muscularis mucosae, producing contraction via PAR‐1 and relaxation via PAR‐4. The PAR‐1‐mediated effect appears to occur largely through increased Na+ permeability followed by activation of L‐type Ca2+ channels and subsequent influx of extracellular Ca2+. Our data could provide evidence for a novel role of PAR‐4 as opposed to PAR‐1, although the underlying mechanisms are still open to question.

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.

Involvement of EDHF in the hypotension and increased gastric mucosal blood flow caused by PAR-2 activation in rats

Agonists for protease‐activated receptor‐2 (PAR‐2) cause hypotension and an increase in gastric mucosal blood flow (GMBF) in vivo. We thus studied the mechanisms underlying the circulatory modulation by PAR‐2 activation in vivo, especially with respect to involvement of endothelium‐derived hyperpolarizing factor (EDHF). Arterial blood pressure and GMBF were measured in anesthetized rats in vivo. Vascular relaxation was assessed in the precontracted rat gastric arterial rings in vitro. The PAR‐2‐activating peptide SLIGRL‐NH2 and/or trypsin, administered i.v., produced largely NO‐independent hypotension and increase in GMBF accompanied by decreased gastric mucosal vascular resistance (GMVR) in rats. Combined administration of apamin and charybdotoxin, but not each of them, specifically abolished the hypotension, increased GMBF and decreased GMVR caused by the PAR‐2 agonists. In the isolated rat gastric artery, SLIGRL‐NH2 elicited endothelium‐dependent relaxation even in the presence of an NO synthase inhibitor and indomethacin, which was abolished by apamin plus charybdotoxin. Our data suggest involvement of apamin/charybdotoxin‐sensitive K+ channels in the PAR‐2‐triggered hypotension and increased GMBF, predicting a role of EDHF‐like factors.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

BackgroundProteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population.MethodsEMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells).ResultsStimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor.ConclusionPAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.

Distinct Activity of Peptide Mimetic Intracellular Ligands (Pepducins) for Proteinase‐Activated Receptor‐1 in Multiple Cells/Tissues

Abstract:  Proteinase‐activated receptor‐1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, can be activated not only by PAR1‐activating peptides (PAR1APs) based on the N‐terminal cryptic tethered ligand sequence but also by an N‐palmitoylated (Pal) peptide, Pal‐RCLSSSAVANRSKKSRALF‐amide (P1pal‐19), based on the intracellular loop 3 of PAR1, designated pepducin, in human platelets or PAR1‐transfected cells. The present article evaluated the actions of P1pal‐19 and also the shorter peptide, Pal‐RCLSSSAVANRS‐amide (P1pal‐12), known as a possible PAR1 antagonist, in multiple cells/tissues that naturally express PAR1. P1pal‐19 as well as a PAR1AP, TFLLR‐amide, evoked cytosolic Ca2+ mobilization in cultured human lung epithelial cells (A549) and rat gastric mucosal epithelial cells (RGM1). P1pal‐19 and TFLLR‐amide, but not a PAR2‐activating peptide, SLIGRL‐amide, caused delayed prostaglandin E2 formation in RGM1 cells. P1pal‐19, like TFLLR‐amide, produced endothelial NO‐dependent relaxation in rat aorta and epithelial prostanoid‐dependent relaxation in mouse bronchus. The P1pal‐19‐induced relaxation remained constant even after desensitization of PAR1 with TFLLR‐amide in either tissue. P1pal‐19 failed to mimic the contractile effects of TFLLR‐amide in the endothelium‐denuded preparations of rat aorta or superior mesenteric artery and the rat gastric longitudinal smooth muscle strips. P1pal‐12 partially inhibited the vasorelaxation caused by TFLLR‐amide and P1pal‐19, but not SLIGRL‐amide, in the rat aorta. Our data thus indicate that P1pal‐19 is capable of mimicking the effects of PAR1APs in the endothelial and epithelial, but not smooth muscle, cells/tissues, and suggest that P1pal‐12 may act as a PAR1 antagonist in the vascular endothelium.