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Background and Purpose: Up-regulation of proteinase-activated receptor-2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor., Experimental Approach: We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), trypsin and the PAR2 activating peptide, 2-furoyl(2f)-LIGKV-OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen-activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence., Key Results: The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR-3, also increased following TNFalpha treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation., Conclusions and Implications: These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.

We have previously reported that the contractile response to thrombin and trypsin was enhanced in the pregnant rat myometrium. We herein determined whether or not sex hormones contribute to this enhancement and the expression of protease-activated receptors (PARs). The nonpregnant rats received daily injections of either 17beta-estradiol or progesterone, and then the contractile response of the myometrium was examined ex vivo. Treatment with either 17beta-estradiol or progesterone had almost no significant enhancing effect on the high K(+)- or oxytocin-induced contraction. On the other hand, both 17beta-estradiol and progesterone dose-dependently enhanced the contractile response to trypsin. A maximal enhancement was obtained at 25 and 40 mg kg weight(-1) day(-1) for 17beta-estradiol and progesterone, respectively. The extent of the enhancement of the trypsin-induced contraction seen in the sex hormone-treated rats in the present study was comparable to that reported in the pregnant rats. However, the contractile response to thrombin and PAR1/PAR2-AP, SFLLRNP was not enhanced either by progesterone or 17beta-estradiol. PAR2-AP and PAR4-AP failed to induce contraction under any conditions. PAR1 mRNA was scarcely detected in the control myometrium by an RT-PCR analysis, while it slightly increased only in the progesterone-treated rats. Neither PAR2 nor PAR4 mRNA was detected. We thus conclude that the responsiveness to trypsin, but not thrombin, is controlled by sex hormones. A novel type of receptor, other than PAR1, PAR2 or PAR4, is suggested to mediate the trypsin-induced contraction as in the case of the pregnant rat myometrium.

1. We evaluated the ability of a number of peptides based on the tethered ligand sequences of human, rat and murine proteinase-activated receptor-4 (PAR(4)), to serve as receptor-activating probes or antagonists for bioassays carried out in vitro and for in vivo models of inflammation. 2. In a rat PAR(4)-dependent platelet aggregation assay, the relative potencies of the active sequences (AYPGKF-NH(2)>GYPGKF-NH(2)>GYPGFK-NH(2)>GFPGKP-NH(2)) were consistent with an activation of PAR(4). 3. In the aggregation assay, the reverse or partial reverse-sequence peptides (VQGPYG-NH(2), YAPGKF-NH(2) and FKGPYA-NH(2)) were inactive, while trans-cinnamoyl (Tc)-YPGKF-NH(2), Tc-APGKF-NH(2) and N-palmitoyl-SGRRYGHALR-NH(2) (pepducin P4pal-10) were antagonists. 4. However, in an endothelium-dependent NO-mediated rat aorta (RA) relaxation assay and in a gastric longitudinal muscle (LM) contraction assay, these antagonist peptides were agonists as were most other peptides, with distinct orders of potencies that differed for both the RA and LM assays and from the platelet assay. 5. We conclude that PAR(4)-derived tethered ligand peptide agonists can act at receptors other than PAR(4) and that a judicious choice of ligands is required to probe for PAR(4) function in bioassay systems and in particular for in vivo models. 6. By selecting from these peptides the ones most reliably reflecting PAR(4) activation (AYPGKF-NH(2) as a standard agonist; YAPGKF-NH(2) as a PAR(4)-inactive standard), we were able to establish an inflammatory role for the PAR(4)-activating peptides acting via a non-neurogenic mechanism in a rat paw oedema model.

1. Human mast cell tryptase appears to display considerable variation in activating proteinase-activated receptor 2 (PAR(2)). We found tryptase to be an inefficient activator of wild-type rat-PAR(2) (wt-rPAR(2)) and therefore decided to explore the factors that may influence tryptase activation of PAR(2). 2. Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt-rPAR(2), tryptase was as efficient as trypsin in releasing the receptor-activating sequence (SLIGRL.). However, in the presence of either human-PAR(2) or wt-r PAR(2) expressing cells, tryptase could only activate PAR(2) by releasing SLIGRL from the P20 peptide, suggesting that PAR(2) expressed on the cells was protected from tryptase activation. 3. Three approaches were employed to test the hypothesis that PAR(2) receptor glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR(2) expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase-mediated PAR(2) activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase-mediated PAR(2) activation. (c) Wt-rPAR(2) devoid of the N-terminal glycosylation sequon (PAR(2)T25(-)), but not rPAR(2) devoid of the glycosylation sequon located on extracellular loop-2 (PAR(2)T224A), was selectively and substantially (>30 fold) more sensitive to tryptase compared with the wt-rPAR(2). 4. Immunocytochemistry using antisera that specifically recognized the N-terminal precleavage sequence of PAR(2) demonstrated that tryptase released the precleavage domain from PAR(2)T25(-) but not from wt-rPAR(2). 5. Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR(2)T25(-). 6. Our results indicate that glycosylation of PAR(2) and heparin-inhibition of PAR(2) activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.

1. The mechanism of trypsin-induced contraction in the rat myometrium was investigated using front-surface fluorimetry on fura-PE3-loaded strips. The expression of protease-activated receptors (PARs) in the rat myometrium was determined by reverse transcription polymerase chain reaction (RT - PCR). 2. In non-pregnant rats, 10 microM trypsin developed a force of up to 30.5 +/- 5.1% of that obtained during the 40 mM K(+)-depolarization-induced contraction. In pregnant rats, the maximal level of the cytosolic Ca(2+) concentration and tension obtained with 3 microM trypsin was 143.2 +/- 6.0% and 63.2 +/- 7.9%, respectively. The depletion of the extracellular Ca2+ abolished the trypsin-induced contraction. 3. Trypsin-induced contraction was abolished by the pre-treatment of a serine protease inhibitor. PAR1-activating peptide (PAR1-AP) caused a potent contraction of the myometrium, while neither PAR2-AP nor PAR4-AP induced any contraction. 4. RT - PCR analysis detected the expression of PAR1 mRNA. However, neither PAR2 nor PAR4 mRNA was detected in the rat myometrium. 5. Once the strips were stimulated with thrombin, the subsequent application of thrombin failed to induce any contraction, while trypsin induced a contraction similar to that observed without the pre-stimulation with thrombin. Once the strip was stimulated with trypsin, neither trypsin nor thrombin induced any contraction. The response to PAR1-AP remained after the pre-stimulation with thrombin and trypsin. 6. In conclusion, PAR1 was the only known receptor for trypsin expressed in the rat myometrium, but it was suggested to be cleaved and inactivated by trypsin. Trypsin was thus suggested to contract the rat myometrium via a novel type of PAR, which might be upregulated during pregnancy.

1. This study investigates, whether in addition to the protease-activated receptor-1 (PAR-1), PAR-4 is present in vascular smooth muscle cells (SMC) of the human saphenous vein and whether this receptor is functionally active. PAR-1 and PAR-4 are stimulated by thrombin and by the synthetic peptides SFLLRN and GYPGQV, respectively. 2. mRNAs for both, PAR-1 and PAR-4, were detected in the SMC by using reverse transcriptase polymerase chain reaction (RT - PCR). 3. Treatment of the SMC with GYPGQV (200 microM) resulted in a transient increase in free intracellular calcium. This calcium signal was completely abolished after a preceding challenge with thrombin (10 nM), indicating homologous receptor desensitization. 4. Stimulation of the SMC with 10 nM thrombin or 200 microM SFLLRN caused a time-dependent activation of the extracellular signal-regulated kinases-1/2 (ERK-1/2) with a maximum at 5 min. In contrast, 100 nM thrombin as well as 200 microM of GYPGQV induced a prolonged phosphorylation of ERK-1/2 with a maximum at 60 min. These data suggest that PAR-1 and PAR-4 are activated by thrombin at distinct concentrations and with distinct kinetics. 5. GYPGQV stimulated [(3)H]-thymidine incorporation in SMC. At 500 microM, the peptide increased DNA synthesis 2.5 fold above controls. A comparable mitogenic effect was obtained after stimulation of the SMC by 10 nM thrombin or 100 microM SFLLRN, respectively. 6. These data indicate that a functionally active PAR-4 is present in SMC and, in addition to PAR-1, might contribute to thrombin-induced mitogenesis.

Thrombin causes various cellular events by activating protease-activated receptors (PARs). Here, we showed, for the first time, that thrombin induced myometrial contraction. To determine the mechanism of thrombin-induced myometrial contraction, we simultaneously measured intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension of fura-PE3-loaded rat myometrium using front-surface fluorimetry. The expression of thrombin receptor mRNA in the rat myometrium were determined by reverse transcription-polymerase chain reaction analysis (RT - PCR analysis). Thrombin (0.01 - 3 u ml(-1)) caused dose-dependent increase in [Ca(2+)](i) and tension in the rat myometrium, and this effect was greatly enhanced in the pregnant myometrium. PAR1-activating peptide mimicked the effects of thrombin. In Ca(2+)-free PSS, thrombin induced no increase in [Ca(2+)](i) and tension in the pregnant myometrium. Both diltiazem (10 microM) and SK-F 96365 (10 microM) significantly inhibited the thrombin-induced elevations of [Ca(2+)](i) and tension, and their effects were additive. RT - PCR analysis revealed an approximately 10 fold increase in the level of thrombin receptor mRNA in the pregnant myometrium compared to that obtained in the non-pregnant myometrium. In conclusion, the contractile response to thrombin was greatly enhanced in the pregnant myometrium, mainly due to the up-regulation of thrombin receptor. We propose that initiation of a post-parturitional myometrial contraction is one of the most important physiological roles of thrombin receptor.

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.

1. Rat proteinase-activated receptor-2 (PAR2) variants were stably expressed in rat KNRK cells: (a) wild-type (wt) - PAR2; (b) PAR2PRR, with the extracellular loop 2 (EL-2) sequence P231E232E233mutated to PRR and (c) PAR2NET, with the EL-2 sequence, PEEV changed to NETL. Cell lines were evaluated for their sensitivity (calcium signalling) towards trypsin and the receptor-activating peptides, SLIGRL-NH2, SLIGEL-NH2, trans-cinnamoyl(tc)-LIGRLO-NH2, and SFLLR-NH2. 2. SLIGEL-NH2 exhibited low potency (1 : 200 relative to SLIGRL-NH2) in wild-type PAR2. Its activity was increased 5 fold in PAR2PRR, but it was inactive in PAR2NET. 3. In PAR2PRR, the potencies of SLIGRL-NH2, tc-LIGRLO-NH2, and SFLLR-NH2 were decreased by 80 - 100 fold. But, the potency of trypsin was decreased by only 7 fold. 4. In PAR2NET, highly homologous in EL-2 with proteinase-activated receptor-1 (PAR1), the potency of the PAR1-derived peptide, SFLLR-NH2, was reduced by 100 fold compared with wt-PAR2, whereas the potency of the PAR2-derived AP, SLIGRL-NH2 was reduced 10 fold. In contrast, the potency of trypsin in PAR2NET was almost the same as in wt-PAR2. 5. We conclude that the acidic EL-2 tripeptide, PEE, in PAR2 plays an important role in governing agonist activity. 6. The data obtained with the PEEV-->NETL mutation suggested: (a) that SLIGRL-NH2 and SFLLR-NH2 interact in a distinct manner with PAR2 and (b) that SFLLR-NH2 may interact differently with PAR2 than it does with PAR1. 7 The differential reductions in the potencies of SLIGRL-NH2, compared with trypsin in the PAR2PRR and PAR2NET cell lines point to differences between the interactions of the trypsin-revealed tethered ligand and the free receptor-activating peptide with PAR2.

1. We studied the structure-activity profiles of four thrombin receptor-derived polypeptides (TRPs) (P5, SFLLR; P5-NH2, SFLLR-NH2; P7, SFLLRNP; P7-NH2, SFLLRN) in contractile human placental artery (PA), umbilical artery (UA) and umbilical vein (UV) preparations and in a human platelet aggregation assay. 2. The contractile actions of the TRPs in the two arterial preparations were endothelium-independent, whereas in the UV tissue a contractile response was observed only in an endothelium-denuded preparation; no endothelium-mediated relaxation responses were observed in any of the vascular preparations. 3. In the three vascular preparations, the contractile responses required extracellular calcium and were attenuated by the tyrosine kinase inhibitor, genistein. 4. The relative contractile orders of potencies of the TRPs in the three vascular preparations were distinct from each other (PA: P7-NH2 > P7 > P5-NH2 > P5; UA: P7-NH2 > or = P5-NH2 approximately = P7 > > P5; UV: P5-NH2 > > P7-NH2 = P7 > > P5) and these were in turn distinct from the potency order observed in the platelet aggregation assay (P5-NH2 > or = P7-NH2 > P7 > > P5). 5. Despite the markedly dissimilar TRP potency orders in the placental artery and umbilical vein preparations, the cDNA sequences for the thrombin receptor obtained by polymerase chain reaction cloning of cDNA from the two tissue sources were identical. 6. We conclude that the four tissues studied possess functionally distinct thrombin receptor systems that interact in a distinct way with agonist peptides. In view of the identity of the thrombin receptor cDNA in the two tissues displaying the most dissimilar structure-activity profiles, we suggest that in different tissues, differences in post-translational receptor processing or differences in receptor-effector coupling interactions may result in unique thrombin receptor systems that can display distinct structure-activity profiles.