Evidence that PAR-1 and PAR-2 mediate prostanoid-dependent contraction in isolated guinea-pig gallbladder

We have investigated the ability of protease-activated receptor-1 (PAR-1), PAR-2, PAR-3 and PAR-4 agonists to induce contractile responses in isolated guinea-pig gallbladder. Thrombin, trypsin, mouse PAR-1 activating (SFLLRN-NH(2)) peptide, and mouse PAR-2 activating (SLIGRL-NH(2)) and human PAR-2 activating (SLIGKV-NH(2)) peptides produced a concentration-dependent contractile response. Mouse PAR-4 activating (GYPGKF-NH(2)) peptide, the mouse PAR-1 reverse (NRLLFS-NH(2)) peptide, the mouse PAR-2 reverse (LRGILS-NH(2)) and human PAR-2 reverse (VKGILS-NH(2)) peptides caused negligible contractile responses at the highest concentrations tested. An additive effect was observed following the contractile response induced by either trypsin or thrombin, with the addition of a different PAR agonist (SFLLRN-NH(2) and SLIGRL-NH(2), respectively). Desensitization to PAR-2 activating peptide attenuated the response to trypsin but failed to attenuate the response to PAR-1 agonists, and conversely desensitization to PAR-1 attenuated the response to thrombin but failed to alter contractile responses to PAR-2 agonists. The contractile responses produced by thrombin, trypsin, SFLLRN-NH(2) and SLIGRL-NH(2) were markedly reduced in the presence of the cyclo-oxygenase inhibitor, indomethacin, whilst the small contractile response produced by NRLLFS-NH(2) and LRGILS-NH(2) were insensitive to indomethacin. The contractile responses to thrombin, trypsin, SFLLRN-NH(2) and SLIGRL-NH(2) were unaffected by the presence of: the non-selective muscarinic antagonist, atropine; the nitric oxide synthase inhibitor, L-NAME; the sodium channel blocker, tetrodotoxin; the combination of selective tachykinin NK(1) and NK(2) receptor antagonists, (S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl) piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2] octane chloride (SR140333) and (S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino-2-(3, 4-dichlorophenyl)-butyl] benzamide (SR48968), respectively. The results indicate that PAR-1 and PAR-2 activation causes contractile responses in the guinea-pig gallbladder, an effect that is mediated principally by prostanoid release, and is independent of neural mechanisms.     

Role of PGE(2) in protease-activated receptor-1, -2 and -4 mediated relaxation in the mouse isolated trachea

1. The potential mediator role of the prostanoid PGE(2) in airway smooth muscle relaxations induced by peptidic and proteolytic activators of PAR-1, PAR-2, PAR-3 and PAR-4 was investigated in carbachol-precontracted mouse isolated tracheal segments. 2. The tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced smooth muscle relaxation that was abolished by the non-selective cyclo-oxygenase (COX) inhibitor, indomethacin. The relative order for mean peak relaxation was SLIGRL-NH(2)>GYPGKF-NH(2) approximately amp; SFFLRN-NH(2)>SFNGGP-NH(2). 3. SFFLRN-NH(2), SLIGRL-NH(2) and GYPGKF-NH(2), but not SFNGGP-NH(2), induced significant PGE(2) release that was abolished by indomethacin. Like that for relaxation, the relative order for mean PGE(2) release was SLIGRL-NH(2)>GYPGKF-NH(2)>SFFLRN-NH(2)>SFNGGP-NH(2). 4. In dose-response studies, SLIGRL-NH(2) induced concentration-dependent increases in PGE(2) release (EC(50)=20.4 microM) and smooth muscle relaxation (EC(50)=15.8 microM). 5. The selective COX-2 inhibitor, nimesulide, but not the COX-1 inhibitor valeryl salicylate, significantly attenuated SLIGRL-NH(2)-induced smooth muscle relaxation and PGE(2) release. 6. Exogenously applied PGE(2) induced potent smooth muscle relaxation (EC(50)=60.3 nM) that was inhibited by the mixed DP/EP(1)/EP(2) prostanoid receptor antagonist, AH6809. SLIGRL-NH(2)-induced relaxation was also significantly inhibited by AH6809. 7. In summary, the results of this study strongly suggest that PAR-mediated relaxation in murine tracheal smooth muscle is dependent on the generation of the spasmolytic prostanoid, PGE(2). PAR-stimulated PGE(2) release appears to be generated preferentially by COX-2 rather than COX-1, and induces relaxation via activation of the EP(2) receptor.     

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.     

屋尘螨变应原对人肥大细胞脱颗粒的影响

目的：阐明屋尘螨变应原Derp1对人肥大细胞HMC-1释放类胰蛋白酶水平的影响。方法：流式细胞法测定HMC-1细胞表面蛋白酶激活受体2（PAR2）表达水平;将Derp1、SLIGRL-NH2（PAR2激活剂）、LRGILS-NH2（PAR2对照肽）及Derp1+ FSLLY（ PAR2拮抗剂）分别处理HMC-1细胞,用ELISA检测药物刺激后类胰蛋白酶水平;采用钙绿实验检测PAR2生物学功能。结果：肥大细胞表面表达PAR2受体;Derp1单独作用于HMC-1可明显引起HMC-1细胞内钙流释放及类胰蛋白酶的释放,类胰蛋白酶释放水平明显高于SLIGRL-NH2及正常对照;将Derp1与FSLLY共同作用于HMC-1时,可部分抑制HMC-1细胞内的钙流释放及类胰蛋白酶的释放。结论：屋尘螨变应原Derp1可以通过激活人肥大细胞HMC-1表面PAR2受体引起肥大细胞脱颗粒释放类胰蛋白酶。     

Characterization of protease-activated receptors in rat peritoneal mast cells

Activation of protease-activated receptor (PAR)-1 or PAR-2 elicits inflammation most probably via mast cell degranulation in vivo. The present study aimed at characterizing PARs in rat peritoneal mast cells (PMC). Messenger RNA for PAR-1, but not for PAR-2, was detected in PMC. Thrombin, the PAR-1 agonist SFLLR-NH2 or the PAR-2 agonist SLIGRL-NH2 failed to induce histamine release from PMC. Surprisingly, the PAR-2-inactive control peptide LSIGRL-NH2 triggered histamine release from PMC. Thus, PAR-1, but not PAR-2, are expressed in PMC, whereas neither PAR-1 nor PAR-2 are considered to be involved in degranulation of PMC. LSIGRL-NH2 does not appear to be appropriate as a control peptide for PAR-2 in inflammation studies.     

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.     

Mast cell tryptase stimulates DLD-1 carcinoma through prostaglandin- and MAP kinase-dependent manners

We found that striptease-positive mast cells were abundant in the invasive front of human colon adenocarcinoma by examining 30 cases. Because tryptase has been suggested to be the agonist proteinase for protease-activated receptor-2 (PAR-2), we investigated the effects of stimulation of PAR-2 by tryptase on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line. PAR-2 stimulation by tryptase induced the increase in [Ca(2+)](i), which was desensitized by the prior application of PAR-2 activating peptide (AP). The proliferative responses of DLD-1 to tryptase and PAR-2 AP were associated with the phosphorylation of MEK and MAP kinase. Inhibition of MEK by PD98059 completely inhibited the proliferation-enhancing effects of tryptase and PAR-2 AP as well as phosphorylation of MAP kinase. Moreover, tryptase and PAR-2 AP stimulated the production of prostaglandin E2 and the inhibition of prostaglandin synthesis by indomethacin or NS398 resulted in the complete inhibition of the proliferative responses to tryptase and PAR-2 AP. Furthermore, the tryptase-stimulated proliferation of DLD-1 was concentration-dependently inhibited by nafamostat mesilate, a specific inhibitor of tryptase. These results as a whole indicated that tryptase has proliferative effects on DLD-1 through cyclooxygenase- and MAP kinase-dependent manners acting on PAR-2 by its proteolytic activity.     

Modulation of airway smooth muscle tone by protease activated receptor-1,-2,-3 and -4 in trachea isolated from influenza A virus-infected mice

Relaxant and contractile effects of the tethered ligand domain sequences of murine PAR-1, PAR-2, PAR-3 and PAR-4, and of the proteases thrombin and trypsin were examined in mouse isolated tracheal preparations. The epithelium- and cyclo-oxygenase-dependence of these effects and the potential modulatory effects of respiratory tract viral infection were also investigated. In carbachol-contracted preparations, trypsin, thrombin, and the tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced transient, relaxant responses that were abolished by the cyclo-oxygenase inhibitor indomethacin. Repeated administration of SFFLRN-NH(2), SLIGRL-NH(2) or GYPGKF-NH(2) (30 microM) was associated with markedly diminished relaxation responses (homologous desensitization), although there was no evidence of cross-desensitization between these peptides. The tethered ligand domain sequences for PAR-1 and PAR-4 induced a rapid, transient contractile response that preceded the relaxant response. Contractions were not inhibited by indomethacin and were not induced by either thrombin or trypsin. Influenza A virus infection did not significantly affect the responses induced by either the proteases or peptides. Furthermore, epithelial disruption caused by mechanical rubbing had no significant effect on responses to these PAR activators in preparations from either virus- or sham-infected mice. In summary, the proteases trypsin and thrombin, and peptide activators of PAR-1, PAR-2 and PAR-4 induced relaxant responses of mouse isolated tracheal smooth muscle preparations, which were mediated by a prostanoid, probably PGE(2). Interestingly, PAR-mediated relaxations were not significantly diminished following acute damage to the epithelium caused by mechanical rubbing and/or the respiratory tract viral pathogen, influenza A. British Journal of Pharmacology (2000) 129, 63 - 70.     

Proteinase-activated receptor-1 (PAR-1) activation contracts the isolated human renal artery in vitro

1. The in vitro motor function of protease-activated recepter-1 (PAR-1), PAR-2 and PAR-4 and the presence by immunohistochemistry of PAR-1 in the human renal artery have been investigated. 2. Thrombin and the human PAR-1 (SFLLRN-NH(2)) activating peptide, but not the PAR-1 reverse peptide (NRLLFS-NH(2)), contracted both endothelial-intact and endothelial-denuded human renal artery strips, whereas no relaxation was observed either in strips non-precontracted or precontracted with phenylephrine. Maximum contraction by thrombin or SFLLRN-NH(2) was about 60% of phenylephrine. However, thrombin was approximately 1000-fold more potent than SFLLRN-NH(2). 3. PAR-1 desensitisation, using repeated applications of SFLLRN-NH(2), almost completely blocked the response to thrombin. The contractile effect produced by thrombin and SFLLRN-NH(2) was not affected by nitric oxide synthase inhibition, but was significantly reduced by cyclooxygenase blockade. 4. Trypsin, the PAR-2 (SLIGKV-NH(2) and SLIGRL-NH(2)) and PAR-4 (GYPGQV-NH(2) and AYPGKF-NH(2)) activating peptides did not produce any significant contraction or relaxation. 5. In agreement with the motor function data immunohistochemistry showed specific staining patterns for PAR-1 in the human renal artery. 6. Combined, these studies would suggest a possible role for PAR-1 in renal vascular homeostasis.     

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

1. 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. 2. Intraperitoneal administration of the PAR-2-activating peptide SLIGRL-NH(2), but not the inactive control LSIGRL-NH(2), at 1 - 5 micromol kg(-1), in combination with the aminopeptidase inhibitor amastatin at 2.5 micromol kg(-1), facilitated gastrointestinal transit in a dose-dependent manner. The human PAR-1-derived peptide SFLLR-NH(2) and the specific PAR-1 agonist TFLLR-NH(2), but not the inactive control FSLLR-NH(2), at 2.5 - 10 micromol kg(-1), in combination with amastatin, also promoted gastrointestinal transit. 3. The Ca2+-activated, small conductance K+ channel inhibitor apamin at 0.01 micromol kg(-1) significantly potentiated the actions of SLIGRL-NH(2) and TFLLR-NH(2) at subeffective doses. 4. The increased gastrointestinal transit exerted by either SLIGRL-NH(2) at 5 micromol kg(-1) or TFLLR-NH(2) at 10 micromol kg(-1) was completely abolished by the L-type Ca2+ channel inhibitor verapamil at 61.6 micromol kg(-1). In contrast, the tyrosine kinase inhibitor genistein at 18.5 micromol kg(-1) failed to modify the effects of the agonists for PAR-2 or PAR-1. 5. 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.     

Impairment of PAR-2-mediated relaxation system in colonic smooth muscle after intestinal inflammation

Protease-activated receptor (PAR)-2 plays important roles in intestinal inflammatory responses. Changes in PAR-2-mediated smooth muscle function may contribute pathophysiologically to the intestinal motility disorders often observed in inflammatory bowel disease (IBD).Stimulation of PAR-2 by trypsin-induced relaxation of carbachol- and KCl-induced contractions in normal rat colonic smooth muscle was completely resolved by tissue pretreatment with apamin, but not by pretreatment with l-NMMA or a cocktail of neuronal blockers (tetrodotoxin, hexamethonium and propranolol).In colon inflamed by dextran sodium sulphate (DSS), trypsin-induced inhibitory effects were significantly reduced. Relaxation induced by SLIGRL-NH(2), a selective PAR-2-activating peptide, was also reduced in DSS-treated rat colon. However, inhibitory effects of 1-ethylbenzimidazolin-2-one, an activator of small conductance Ca(2+)-activated K(+) channel, were unaffected.Expression of PAR-2 mRNA in colonic muscularis externa was significantly lower in DSS-treated rats than in control rats.These results suggest that the PAR-2 mediated relaxation system in colonic smooth muscle is suppressed in this experimental colitis rat model, and may contribute to motility disorders in IBD.     

Protease-activated receptor-2-mediated contraction in the rat urinary bladder: the role of urinary bladder mucosa

The role of protease-activated receptor-2 (PAR-2) in the regulation of the rat urinary bladder contractility was investigated. Both trypsin and PAR-2 activating peptide (SLIGRL-NH(2)) produced a concentration-dependent contractile response in the urinary bladder preparations. These contractions were abolished by removal of the urinary bladder mucosa and were significantly reduced by indomethacin (10 microM). These results suggest that activation of PAR-2 stimulates release of prostaglandins from mucosal layer and thereby contracts rat urinary bladder smooth muscles.     

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

1 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. 2 Thrombin at 0.03-0.1 microM and the PAR-1-activating peptide SFLLR-NH2 at 3-100 microM or TFLLR-NH2 at 10-50 microM produced a dual action, relaxation followed by contraction of the duodenal longitudinal muscle. The PAR-2-activating peptide SLIGRL-NH2 at 10-100 microM elicited only small contraction. Trypsin at 0.08 microM induced small contraction, or relaxation followed by contraction, depending on preparations. The PAR-4-activating peptide GYPGKF-NH2 at 1000 microM exhibited no effect. 3 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 microM), the protein kinase C inhibitor GF109203X (1 microM) and the tyrosine kinase inhibitor genistein (15 microM), but were resistant to indomethacin (3 microM) and tetrodotoxin (1-10 microM). 4 The relaxation of the preparations exerted by TFLLR-NH2 was unaffected by indomethacin (3 microM), propranolol (5 microM), NG-nitro-L-arginine methyl ester (100 microM) and tetrodotoxin (1-10 microM). This relaxation was resistant to either GF109203X (1 microM) or genistein (15 microM), but was, remarkably, attenuated by combined application of these two kinase inhibitors. 5 Apamin (0.1 microM), an inhibitor of calcium-activated, small-conductance potassium channels, but not charybdotoxin (0.1 microM), completely abolished the PAR-1-mediated duodenal relaxation, and significantly enhanced the PAR-1-mediated contraction. 6 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.     

Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist

A growing body of evidence suggests an important contribution of the cellular actions of thrombin to thrombosis and restenosis following angioplasty. Recently we reported on SCH 79797 (N3-cyclopropyl-7-?[4-(1-methylethyl)phenyl]methyl?-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine) and its analogs as new potent, nonpeptide thrombin receptor antagonists. This study further characterizes the biochemical and pharmacological actions of pyrroloquinazoline inhibitors of protease activated receptor-1 (PAR-1) in human platelets and coronary artery smooth muscle cells (hCASMC). SCH 79797 and its N-methyl analog (SCH 203099) inhibited binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-[(3)H]Tyr-NH(2)) to PAR-1 with IC(50) values of 70 and 45 nM, respectively. SCH 79797 inhibited [(3)H]haTRAP binding in a competitive manner. SCH 79797 and SCH 203099 inhibited alpha-thrombin- and haTRAP-induced aggregation of human platelets, but did not inhibit human platelet aggregation induced by the tethered ligand agonist for protease-activated receptor-4 (PAR-4), gamma-thrombin, ADP, or collagen. SCH 203099 inhibited surface expression of P-selectin induced by haTRAP and thrombin, and it did not increase P-selectin expression or prevent thrombin cleavage of the receptor. Thrombin and TFLLRNPNDK-NH(2) (TK), a PAR-1-selective agonist, produced transient increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in hCASMC. This increase in [Ca(2+)](i) was inhibited effectively by SCH 79797. However, the Ca(2+) transients induced by SLIGKV-NH(2,) a PAR-2-selective agonist, were not inhibited by SCH 79797. Thrombin- and TK-stimulated [(3)H]thymidine incorporation also was inhibited completely by SCH 79797. The results of this study demonstrate that SCH 79797 and SCH 203099 are potent, selective antagonists of PAR-1 in human platelets and hCASMC. These data also suggest that the thrombin stimulation of Ca(2+) transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1.     

Protease-activated receptor-2-mediated contraction of urinary bladder is enhanced in cyclophosphamide-treated rats

Protease-activated receptor-2 (PAR-2) is activated by serine proteases, such as trypsin and mast cell tryptase. Recently, we have shown that activators of PAR-2 contract the rat urinary bladder mainly by stimulating release of prostaglandins (PGs) from the mucosal layer. In the present study, we investigated how the PAR-2-mediated responses are altered in rats with cyclophosphamide (CYP)-induced cystitis. The contractile responses to trypsin and PAR-2 activating peptide (PAR-2 AP; SLIGRL-NH₂) in the urinary bladders were augmented by treatment of rats with CYP. The contractile effects of these PAR-2 activators on the smooth muscles of the urinary bladder were also potentiated after induction of cystitis by CYP. On the other hand, CYP-induced cystitis significantly attenuated contractions produced by PGE₂ in the smooth muscles of the urinary bladder. The PAR-2-mediated contractions were significantly prevented by indomethacin or NS-398, an inhibitor of cyclooxygenase-2. Both trypsin and PAR-2 AP increased the release of PGE₂ from the urinary bladder mucosa and smooth muscle. CYP-induced cystitis enhanced the PAR-2 activators-induced PGE₂ releases from the urinary mucosa without affecting those from the smooth muscle of the urinary bladder. The PGE₂ releases were prevented by indomethacin or NS-398. The mRNAs for PAR-2 in the urinary bladder mucosa and smooth muscle preparations were not altered in CYP-induced cystitis. These results suggest that PAR-2-mediated responses were enhanced in bladders from CYP-treated rats. The enhancement of PAR-2-mediated contraction might be ascribed to the increased production of PGs and the altered sensitivity of smooth muscle to PAR-2 activators.     

Activation of human tonsil and skin mast cells by agonists of proteinase activated receptor-2

Aim: To investigate the effects of the agonists of proteinase activated receptor (PAR)-2,and histamine on degranulation of human mast cells. Methods: Human mast cells were enzymatically dispersed from tonsil and skin tissues. The dispersed cells were then cultured with various stimuli, and tryptase and histamine levels in cell supernatants collected from challenge tubes were measured. Results:PAR-2 agonist peptide SLIGKV provoked a dose-dependent release of histamine from skin mast cells. It also induced tryptase release from tonsil mast cells, tcLIGRLO appeared less potent than SLIGKV in induction of release of histamine and tryptase. Trypsin was able to induce a “bell” shape increase in tryptase release from tonsil mast cells. It was also able to induce a dose-dependent release of histamine from both tonsil and skin mast cells. The actions of trypsin on mast cells were inhibited by soy bean trypsin inhibitor (SBTI) or α1-antitrypsin (α1-AT).Time course study revealed that both stimulated tryptase or histamine release initiated within 10s and reached their peak release between 4 and 6min. Pretreatment of cells with metabolic inhibitors or pertussis toxin reduced the ability of mast cells to release tryptase or histamine. Conclusion: It was demonstrated that the in vitro tryptase release properties of human tonsil and skin mast cells suggested a novel type of mast cell heterogeneity. The activation of mast cells by PAR-2 agonists indicated a self-amplification mechanism of mast cell degranulation.     

Dual effect mediated by protease-activated receptors on the mechanical activity of rat colon

1. The present study examined the mechanical effects of agonist enzymes and receptor-activating peptides for protease-activated receptor (PAR)-1 and PAR-2 on longitudinal and circular muscle of rat isolated colonic segments in the attempt to clarify the PAR functional role in intestinal motility. 2. The responses to PAR-1 and PAR-2 activation were examined in vitro by recording simultaneously the changes of endoluminal pressure (index of circular muscle activity) and of isometric tension (index of longitudinal muscle activity). 3. Both PAR-1 agonists, thrombin (0.1 nM - 3 microM) and SFLLRN-NH2 (1 nM - 3 microM), and PAR-2 agonists, trypsin (0.1 nM - 10 microM) and SLIGRL-NH2 (1 nM - 10 microM), induced different effects in the two muscular layers: a reduction of the spontaneous contractions in the circular muscle and a contractile effect or biphasic, relaxation followed by contraction, depending on the concentration, in the longitudinal muscle. 4. The inhibitory effects were greatly reduced or abolished by apamin (0.1 microM) indicating that they mainly occur via activation of Ca2+-dependent small conductance, K+-channels. 5. The responses to PAR-1 and PAR-2 were unaffected by tetrodotoxin (1 microM) or indomethacin (1 microM) suggesting that are independent by products of cyclooxygenase or by neural action potentials. 6. These findings indicate that both PAR-1 and PAR-2 are functionally expressed in rat colon. PARs mediate changes of the mechanical activity of longitudinal and circular muscle which might explain the alterations of colonic motility observed during inflammatory conditions.     

The mechanism of LTE4-induced histamine hyperresponsiveness in guinea-pig tracheal and human bronchial smooth muscle, in vitro.

1. Preincubation of guinea-pig tracheal smooth muscle with leukotriene E4 (LTE4) in vitro increased its subsequent responsiveness to histamine. 2. LTE4 pretreatment of guinea-pig tracheal strips did not affect the subsequent responsiveness to either the contractile agents, carbachol and KCl, or to the relaxant beta-adrenoceptor agonist, isoprenaline. 3. LTE4-induced airway histamine hyperresponsiveness was blocked by indomethacin (5 microM), GR32191 (3 microM), atropine (1 microM) and tetrodotoxin (1 microM). 4. U46619, a stable thromboxane A2-analogue, at a non-contractile concentration of 4 nM, increased tracheal smooth muscle sensitivity to histamine. 5. Both LTE4 and U46619 pretreatment increased the contractile response of tracheal smooth muscle to electrical field stimulation. 6. Preincubation of human bronchial spirals with LTE4 in vitro increased its subsequent responsiveness to histamine. 7. LTE4-induced histamine hyperresponsiveness of human bronchus was inhibited by GR32191 (3 microM) and atropine (1 microM). 8. It is proposed that LTE4 induces guinea-pig airway smooth muscle hyperresponsiveness to histamine via a facilitation of cholinergic neurotransmission, which is dependent upon the secondary generation of prostanoid mediator(s) acting on TP-receptors situated on cholinergic nerve terminals. In addition, it is suggested that LTE4 may induce histamine hyperresponsiveness of human bronchus in vitro by a similar mechanism as to that seen in guinea-pig central airway smooth muscle.