Protease-activated receptors: potential therapeutic targets in irritable bowel syndrome?

Protease-activated receptors (PARs) are a family of four G-protein-coupled receptors (PAR-1 to PAR-4) activated by the proteolytic cleavage of their N-terminal extracellular domain. This activation first involves the recognition of the extracellular domain by proteases, such as thrombin, but also trypsin or tryptase which are particularly abundant in the gastrointestinal tract, both under physiological circumstances and in several digestive diseases. Activation of PARs, particularly of PAR-1 and -2, modulates intestinal functions, such as gastrointestinal motility, visceral nociception, mucosal inflammatory response, and epithelial functions (intestinal secretion and permeability). As these physiological properties have been shown to be altered in various extents and combinations in different clinical presentations of irritable bowel syndrome, PARs appear as putative targets for future therapeutic intervention in these patients.     

Protease-activated receptors: potential therapeutic targets in irritable bowel syndrome?

Protease-activated receptors (PARs) are a family of four G-protein-coupled receptors (PAR-1 to PAR-4) activated by the proteolytic cleavage of their N-terminal extracellular domain. This activation first involves the recognition of the extracellular domain by proteases, such as thrombin, but also trypsin or tryptase which are particularly abundant in the gastrointestinal tract, both under physiological circumstances and in several digestive diseases. Activation of PARs, particularly of PAR-1 and -2, modulates intestinal functions, such as gastrointestinal motility, visceral nociception, mucosal inflammatory response, and epithelial functions (intestinal secretion and permeability). As these physiological properties have been shown to be altered in various extents and combinations in different clinical presentations of irritable bowel syndrome, PARs appear as putative targets for future therapeutic intervention in these patients.     

Review article: proteinase-activated receptors - novel signals for gastrointestinal pathophysiology

Proteinase-activated receptors (PARs) have the common property of being activated by the proteolytic cleavage of their extracellular N-terminal domain. The new NH2-terminus acts as a 'tethered ligand' binding and activating the receptor itself. Four members of this family have been cloned, three of which are activated by thrombin (PAR-1, PAR-3 and PAR-4) while the fourth (PAR-2) is activated by trypsin or mast cell tryptase. In physiological or pathophysiological conditions, the gastrointestinal tract is exposed more than other tissues to proteinases (digestive enzymes, proteinases from pathogens or proteinases from inflammatory cells) that can activate PARs. Since PARs are highly expressed throughout the gastrointestinal tract, the study of the role of PARs in these tissues appears to be particularly important. It has already been shown that PAR-2 activation induces calcium mobilization and eicosanoid production in enterocytes as well as changes in ion transport in jejunal tissue segments. PAR-2 activation also causes calcium mobilization and stimulates amylase release from pancreatic acini. Moreover, both PAR-1 and PAR-2 activation can alter the gastrointestinal motility. In inflammatory or allergic conditions, the proteinases that constitute the major agonists for PARs (thrombin, trypsin and mast cell tryptase) are usually released. The activation of PARs by these proteinases might contribute to the gastrointestinal disorders associated with these pathologies. A complete understanding of the role of PARs in the gastrointestinal tract will require the development of selective receptor antagonists that are not yet available. Nonetheless, the use of PAR agonists has already highlighted new potential functions for proteinases in the gastrointestinal tract, thus the control of PAR activation might represent a promising therapeutic target.     

Physiology of protease-activated receptors (PARs): involvement of PARs in digestive functions

The protease-activated receptor (PAR), a G protein-coupled receptor present on cell surface, mediates cellular actions of extracellular proteases. Proteases cleave the extracellular N-terminal of PAR molecules at a specific site, unmasking and exposing a novel N-terminal, a tethered ligand, that binds to the body of receptor molecules resulting in receptor activation. Amongst four distinct PARs that have been cloned, PARs 1, 3 and 4 are activated by thrombin, but PAR-2 is activated by trypsin or mast cell tryptase. Human platelets express two distinct thrombin receptors, PAR-1 and PAR-4, while murine platelets express PAR-3 and PAR-4. Apart from roles of PARs in platelet activation, PARs are distributed to a number of organs in various species, predicting their physiological importance. We have been evaluating agonists specific for each PAR, using multiple procedures including a HEK cell calcium signal receptor desensitization assay. Using specific agonists that we developed, we found the following: 1) the salivary glands express PAR-2 mRNA and secret saliva in response to PAR-2 activation; 2) pancreatic juice secretion occurs following in vivo PAR-2 activation; 3) PAR-1 and PAR-2 modulate duodenal motility. Collectively, PAR plays various physiological and/or pathophysiological roles, especially in the digestive systems, and could be a novel target for drug development.     

Modulation of visceral pain and inflammation by protease-activated receptors

The gastrointestinal (GI) tract is exposed to a large array of proteases, under both physiological and pathophysiological conditions. The discovery of G protein-coupled receptors activated by proteases, the protease-activated receptors (PARs), has highlighted new signaling functions for proteases in the GI tract, particularly in the domains of inflammation and pain mechanisms. Activation of PARs by selective peptidic agonists in the intestine or the pancreas leads to inflammatory events and changes in visceral nociception, suggesting that PARs could be involved in the modulation of visceral pain and inflammation. PARs are present in most of the cells that are potentially actors in the generation of irritable bowel syndrome (IBS) symptoms. Activation of PARs interferes with several pathophysiological factors that are involved in the generation of IBS symptoms, such as altered motility patterns, inflammatory mediator release, altered epithelial functions (immune, permeability and secretory) and altered visceral nociceptive functions. Although definitive studies using genetically modified animals, and, when available, pharmacological tools, in different IBS and inflammatory models have not yet confirmed a role for PARs in those pathologies, PARs appear as promising targets for therapeutic intervention in visceral pain and inflammation processes.     

Proteinase-activated receptors

Proteinase-activated receptors are a recently described, novel family of seven-transmembrane G-protein-coupled receptors. Rather then being stimulated through ligand receptor occupancy, activation is initiated by cleavage of the N terminus of the receptor by a serine protease resulting in the generation of a new tethered ligand that interacts with the receptor within extracellular loop-2. To date, four proteinase-activated receptors (PARs) have been identified, with distinct N-terminal cleavage sites and tethered ligand pharmacology. In addition to the progress in the generation of PAR-1 antagonists, we describe the role of thrombin in such processes as wound healing and the evidence implicating PAR-1 in vascular disorders and cancer. We also identify advances in the understanding of PAR-1-mediated intracellular signaling and receptor desensitization. The cellular functions, signaling events, and desensitization processes involved in PAR-2 activation are also assessed. However, other major aspects of PAR-2 are highlighted, in particular the ability of several serine protease enzymes, in addition to trypsin, to function as activators of PAR-2. The likely physiological and pathophysiological roles for PAR-2 in skin, intestine, blood vessels, and the peripheral nervous system are considered in the context of PAR-2 activation by multiple serine proteases. The recent discovery of PAR-3 and PAR-4 as additional thrombin-sensitive PARs further highlights the complexity in assessing the effects of thrombin in several different systems, an issue that remains to be fully addressed. These discoveries have also highlighted possible PAR-PAR interactions at both functional and molecular levels. The future identification of other PARs and their modes of activation are an important future direction for this expanding field of study.     

Physiological functions of protease-activated receptor-2

Protease-activated receptors (PARs) are a family of G-protein-coupled-seven-trans-membrane-domain-receptors activated by specific proteases, consisting of four family members. PAR-2, a receptor activated by trypsin, tryptase or coagulation factors VIIa and Xa, is unevenly distributed throughout the mammalian body, modulating multiple physiological functions. In the gastrointestinal tract, PAR-2 is involved in gastric mucosal cytoprotection, smooth muscle motility modulation, salivary and pancreatic exocrine secretion, intestinal ionic transport, etc. In the circulatory system, endothelial PAR-2, upon activation, induces vascular relaxation by mechanisms dependent on nitric oxide or endothelium-derived hyperpolarizing factor (EDHF), resulting in hypotension in vivo. In the respiratory system, PAR-2 appears to play a dual role, being pro- and anti-inflammatory. In the nervous system, PAR-2 present in capsaicin-sensitive sensory neurons participates in processing of pain information. PAR-2 is thus involved in a variety of physiological and pathophysiological functions. PAR-2 is now considered one of the most important molecules as a target for drug development.     

Development of agonists/antagonists for protease-activated receptors (PARs) and the possible therapeutic application to gastrointestinal diseases

Protease-activated receptors (PARs), a family of G-protein-coupled seven-transmembrane-domain receptors, are activated by proteolytic unmasking of the N-terminal cryptic tethered ligand by certain serine proteases. Among four PAR family members cloned to date, PAR-1, PAR-2, and PAR-4 can also be activated through a non-enzymatic mechanism, which is achieved by direct binding of exogenously applied synthetic peptides based on the tethered ligand sequence, known as PARs-activating peptides, to the body of the receptor. Various peptide mimetics have been synthesized as agonists for PARs with improved potency, selectivity, and stability. Some peptide mimetics and/or nonpeptide compounds have also been developed as antagonists for PAR-1 and PAR-4. PARs are widely distributed in the mammalian body, especially throughout the alimentary systems, and play various roles in physiological/pathophysiological conditions, i.e., modulation of salivary, gastric, or pancreatic glandular exocrine secretion, gastrointestinal smooth muscle motility, gastric mucosal cytoprotection, suppression/facilitation of visceral pain and inflammation, etc. Thus PARs are now considered novel therapeutic targets, and development of selective agonists and/or antagonists for PARs might provide a novel strategy for the treatment of various diseases that are resistant to current therapeutics.     

Protease-activated receptors: protease signaling in the gastrointestinal tract

Serine proteases from the circulation, inflammatory cells, digestive glands and microorganisms can signal to cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors. Proteases cleave PARs at specific sites to expose tethered ligand domains that bind to and activate the cleaved receptors. Despite this irreversible mechanism of activation, PAR signaling is tightly regulated to prevent the uncontrolled stimulation of cells. Although PARs are found in all organ systems, protease signaling is of particular interest in the gastrointestinal tract, where proteases regulate neurotransmission, secretion, motility, epithelial permeability and intestinal inflammation, and can thus contribute to disease.     

Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1

Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs.Using fura 2 [Ca(2+)](i) measurements, we analyzed three human epithelial cell lines, HBE (human bronchial epithelial), A549 (human pulmonary epithelial) and HEK (human embryonic kidney)-293 cells, which express functional PAR-1 and PAR-2. Human mesotrypsin failed to induce a PAR-mediated Ca(2+) response in human epithelial cells even at high concentrations. In addition, mesotrypsin did not affect the magnitude of PAR activation by subsequently added bovine trypsin. In HBE cells, which like A549 cells express high PAR-2 levels with negligible PAR-1 levels (<11%), half-maximal responses were seen for both cationic and anionic trypsins at about 5 nM. In the epithelial cells, mesotrypsin did not activate PAR-2 or PAR-1, whereas both anionic and cationic trypsins were comparable activators.We also investigated human astrocytoma 1321N1cells, which express PAR-1 and some PAR-3, but no PAR-2. High concentrations (>100 nM) of mesotrypsin produced a relatively weak Ca(2+) signal, apparently through PAR-1 activation. Half-maximal responses were observed at 60 nM mesotrypsin, and at 10-20 nM cationic and anionic trypsins.Using a desensitization assay with PAR-2-AP, we confirmed that both cationic and anionic trypsin isoforms cause [Ca(2+)](i) elevation in HBE cells mainly through PAR-2 activation. Desensitization of PAR-1 with thrombin receptor agonist peptide in 1321N1 cells demonstrated that all three recombinant trypsin isoforms act through PAR-1.Thus, the activity of human cationic and anionic trypsins on PARs was comparable to that of bovine pancreatic trypsin. Mesotrypsin (trypsin IV), in contrast to cationic and anionic trypsin, cannot activate or disable PARs in human epithelial cells, demonstrating that the receptors are no substrates for this isoenzyme. On the other hand, mesotrypsin activates PAR-1 in human astrocytoma cells. This might play a role in protection/degeneration or plasticity processes in the human brain.     

Protease-activated receptor (PAR), a novel family of G protein-coupled seven trans-membrane domain receptors: activation mechanisms and physiological roles

The protease-activated receptor (PAR) belongs to the large superfamily of G-protein-coupled seven trans-membrane domain receptors. The activation of PARs is achieved by proteolytic unmasking of the cryptic N-terminal receptor-activating sequence that binds to the body of the same receptor molecule. PARs-1, -3 and -4 are activated by thrombin, while PAR-2 is activated by trypsin or mast cell tryptase, but not by thrombin. PARs are widely distributed to a variety of tissues and participate in a number of physiological or pathophysiological phenomena such as platelet aggregation, inflammation and cardiovascular, digestive or respiratory functions. Thus, PARs are of physiological importance and also of pharmacological interest as the novel target for drug development.     

Endothelium-dependent and -independent responses to protease-activated receptor-2 (PAR-2) activation in mouse isolated renal arteries

Protease-activated receptors (PARs) are receptors which require proteolytic cleavage to be self-activated by newly exposed N-terminal `tethered ligands'', and hence serve as sensors for protelytic enzymes. While both the thrombin receptor (PAR-1) and PAR-2 (activated by tryptic enzymes) have been shown to mediate endothelium-dependent vasorelaxation, only PAR-1 has been shown to cause direct vascular smooth muscle contraction. In this study, we report that trypsin and the PAR-2 selective peptide ligand SLIGRL-NH₂ not only caused endothelium-dependent relaxation of mouse renal arteries but also direct smooth muscle contraction if endothelial nitric oxide synthase was inhibited or if the endothelium was removed.     

Modulation of capsaicin-evoked visceral pain and referred hyperalgesia by protease-activated receptors 1 and 2

Protease-activated receptors (PARs) 1 and 2 are expressed in capsaicin-sensitive sensory neurons, being anti- and pro-nociceptive, respectively. Given the possible cross talk between PAR-2 and capsaicin receptors, we investigated if PAR-2 activation could facilitate capsaicin-evoked visceral pain and referred hyperalgesia in the mouse and also examined the effect of PAR-1 activation in this model. Intracolonic (i.col.) administration of capsaicin triggered visceral pain-related nociceptive behavior, followed by referred hyperalgesia. The capsaicin-evoked visceral nociception was suppressed by intraperitoneal (i.p.) TFLLR-NH2, a PAR-1-activating peptide, but not FTLLR-NH2, a control peptide, and unaffected by i.col. TFLLR-NH2. SLIGRL-NH2, a PAR-2-activating peptide, but not LRGILS-NH2, a control peptide, administered i.col., facilitated the capsaicin-evoked visceral nociception 6-18 h after administration, while i.p. SLIGRL-NH2 had no effect. The capsaicin-evoked referred hyperalgesia was augmented by i.col. SLIGRL-NH2, but not LRGILS-NH2, 6-18 h after administration, and unaffected by i.p. SLIGRL-NH2, and i.p. or i.col. TFLLR-NH2. Our data suggest that PAR-1 is antinociceptive in processing of visceral pain, whereas PAR-2 expressed in the colonic luminal surface, upon activation, produces delayed sensitization of capsaicin receptors, resulting in facilitation of visceral pain and referred hyperalgesia.     

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.     

NK1 receptor-mediated mechanisms regulate colonic hypersensitivity in the guinea pig

Neurokinin-1 (NK(1)) receptors activated by substance P (SP) are involved in the processing of nociceptive information and are a potential target for therapy of visceral pain. We have evaluated the role of NK(1) receptors using a selective antagonist of NK(1) receptors in two animal models of colorectal hypersensitivity. The behavioral response to colorectal distension was assessed in freely moving guinea pigs by recording visceromotor reflex contractions of the abdominal musculature. Colonic hypersensitivity was induced by intracolonic administration of a chemical irritant (0.6% of acetic acid), or by acute partial restraint stress. Sensitization was characterized by an exaggerated visceromotor response to a low level of colorectal distension (10 mm Hg). In both models of colonic hypersensitivity, oral administration of TAK-637 (0.1-10 mg/kg) normalized visceromotor responses. The intracerebroventricular (10 microg/kg) or intrathecal (10 microg/kg) administration of TAK-637 inhibited colonic hypersensitivity, suggesting an interaction with central NK(1) receptors. In contrast, TAK-637 had no effect on visceromotor responses to colorectal distension at 40 mm Hg in guinea pigs with normosensitive (nonsensitized) colons. In conclusion, central NK(1) receptors play a significant role in colonic hypersensitivity induced by visceral afferent nerve sensitization from gastrointestinal origin or acute psychosomatic stress, but not in the perception of colorectal distension in animals with normosensitive colons.     

Enteric P2X receptors as potential targets for drug treatment of the irritable bowel syndrome

The irritable bowel syndrome (IBS) is a gastrointestinal motility disorder affecting millions of patients. IBS symptoms include diarrhea, constipation and pain. The etiology of IBS is due partly to changes in the function of nerves supplying the gastrointestinal tract, immune system activation and to psychological factors. P2X receptors are multimeric ATP-gated cation channels expressed by neuronal and non-neuronal cells. Sensory nerve endings in the gastrointestinal tract express P2X receptors. ATP released from gastrointestinal cells activates P2X receptors on sensory nerve endings to stimulate motor reflexes and to transmit nociceptive signals. Antagonists acting at P2X receptors on sensory nerves could attenuate abdominal pain in IBS patients. Primary afferent neurons intrinsic to the gut, and enteric motor- and interneurons express P2X receptors. These neurons participate in motor reflexes. Agonists acting at enteric P2X receptors may enhance gastrointestinal propulsion and secretion, and these drugs could be useful for treating constipation-predominant IBS. Antagonists acting at enteric P2X receptors would decrease propulsion and secretion and they might be useful for treating diarrhea-predominant IBS. Current knowledge of P2X receptor distribution and function in the gut of laboratory animals provides a rational basis for further exploration of the therapeutic potential for drugs acting at P2X receptors in IBS patients. However, more information about P2X receptor distribution and function in the human gastrointestinal tract is needed. Data on the distribution and function of P2X receptors on gastrointestinal immune cells would also provide insights into the therapeutic potential of P2X receptor agents in IBS.     

PAR (protease-activated receptor) as a novel target for development of gastric mucosal cytoprotective drugs

Protease-activated receptor-2 (PAR-2), a G protein-coupled seven trans-membrane domain receptor, is distributed throughout the gastrointestinal tract, modulating various functions. In gastric mucosa, PAR-2 present in sensory neurons, upon activation, triggers mucus secretion by stimulating release of CGRP and tachykinins, resulting mucosal cytoprotection. PAR-2 activation also suppresses acid secretion and increase mucosal blood flow, contributing to the protective mechanisms. In contrast, PAR-2 is also present in chief cells, facilitating pepsinogen secretion. PAR-2 would thus appear to be primarily protective in gastric mucosa, but may also have an aggressive property in certain conditions. Finally, functions of PARs other than PAR-2 in gastric mucosa are also discussed.     

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-NH(2) and TFLLR-NH(2) (PAR-1-activating peptides), but not the PAR-1-inactive peptide FSLLR-NH(2), evoked a marked contraction. In contrast, the PAR-2 agonist SLIGRL-NH(2) and the PAR-4 agonist GYPGKF-NH(2) 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-NH(2) further contracted the precontracted preparations with no preceding relaxation. GYPGKF-NH(2), but not the inactive peptide GAPGKF-NH(2), produced marked relaxation. Trypsin or SLIGRL-NH(2) caused no relaxation. The PAR-1-mediated contraction was completely abolished in Ca(2+)-free medium and considerably attenuated by nifedipine (1 microM) and in a low Na(+) medium. The PAR-4-mediated relaxation was resistant to tetrodotoxin (10 microM), apamin (0.1 microM), charybdotoxin (0.1 microM), L-N(G)-nitroarginine methyl ester (100 microM), indomethacin (3 microM), propranolol (5 microM) or adenosine 3', 5'-cyclic monophosphorothioate, 8-bromo, Rp-isomer (30 microM). 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 Ca(2+) channels and subsequent influx of extracellular Ca(2+). 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.     

Physiology and pathophysiology of proteinase-activated receptors (PARs): PARs in the respiratory system: cellular signaling and physiological/pathological roles

Proteinase-activated receptors (PARs), a family of G protein-coupled receptors, are widely distributed in the mammalian body, playing a variety of physiological/pathophysiological roles. In the respiratory systems, PARs, particularly PAR-2 and PAR-1, are expressed in the epithelial and smooth muscle cells. In addition to the G(q/11)-mediated activation of the phospholipase C beta pathway, epithelial PAR activation causes prompt and/or delayed prostanoid formation, leading to airway smooth muscle relaxation and/or modulation of an inflammatory process. PAR-2 present in the epithelium and smooth muscle is considered primarily pro-inflammatory in the respiratory system, although PAR-2 may also be anti-inflammatory under certain conditions. In the lung epithelial cells, PAR-2 can also be activated by exogenous proteinases including house dust mite allergens, in addition to various possible endogenous agonist proteinases. Clinical evidence also suggests possible involvement of PARs, particularly PAR-2, in respiratory diseases. PARs thus appear to play critical roles in the respiratory systems, and the agonists/antagonists for PARs may serve as the novel therapeutic strategy for treatment of certain respiratory diseases including asthma.