Bahjat Al-Ani

Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue.

1 The biological activities of the proteinase‐activated receptor number 2 (PAR‐2)‐derived peptides, SLIGRL (PP6) SLIGRL‐NH2 (PP6‐NH2) and SLIGR‐NH2 (PP5‐NH2) were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR‐2‐derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR‐NH2 (TP5‐NH2). 2 From a neonatal rat intestinal cDNA library, and from intestinal and kidney‐derived cDNA, the coding region of the rat PAR‐2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR‐2 receptor; and this information, along with a reverse‐transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR‐2 and thrombin receptor mRNA in both tissues. 3 In the mouse and rat gastric preparations, the PAR‐2‐derived polypeptides, PP6, PP6‐HN2 and PP5‐NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml−1; 10 nM) and that were equivalent to contractions caused by TP5‐NH2. 4 The cumulative exposure of the rat LM tissue to PP6‐NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5‐NH2 and vice versa, so as to indicate a lack of cross‐desensitization between the receptors responsive to the PAR‐2 and thrombin receptor‐derived peptides. 5 In the rat gastric preparation, the potencies of the PAR‐2‐activating peptides were lower than the potency of TP5‐NH2 (potency order: TP5‐NH2 > > PP6‐NH2 ≥ PP6 > PP5‐NH2); PP6 was a partial agonist in this preparation. 6 The contractile actions of PP6 and PP6‐NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo‐oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7 In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR‐2‐derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, Nω‐nitro‐L‐arginine‐methyl ester (L‐NAME) but not by D‐NAME; in an endothelium‐free preparation, which possessed mRNA for both the PAR‐2 and thrombin receptors, the PAR‐2‐activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5‐NH2. The relaxation response to PP6‐NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8 In the rat aortic preparation, the potencies of PP6, PP6‐NH2 and PP5‐NH2 were greater than those of the thrombin receptor activating peptide, TP5‐NH2 (potency order: PP6‐NH2 ≥ PP6 > PP5‐NH2 > TP5‐NH2). 9 In the rat aortic preparation, the relaxant actions of the PAR‐2‐derived peptides were mimicked by trypsin, at concentrations (0.5‐1 u ml−1; 1–2 nM) lower than those that can activate the thrombin receptor. 10 The bioassay data obtained with the PAR‐2 peptides and with trypsin, along with the molecular cloning/RT‐PCR analysis, point to the presence of functional PAR‐2 receptors that can activate distinct responses in the gastric and vascular smooth muscle preparations. These responses were comparable to those resulting from thrombin receptor activation in the same tissues, so as to suggest that the receptor for the PAR‐2‐activating peptides may play a physiological role as far reaching as the one proposed for the thrombin receptor.

Thrombin and mast cell tryptase regulate guinea‐pig myenteric neurons through proteinase‐activated receptors‐1 and −2

1 Proteases regulate cells by cleaving proteinase‐activated receptors (PARs). Thrombin and trypsin cleave PAR‐1 and PAR‐2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR‐1 and PAR‐2. 2 We detected immunoreactive PAR‐1 and PAR‐2 in > 60 % of neurons from the myenteric plexus of guinea‐pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR‐1 and PAR‐2. We confirmed expression of PAR‐1 and PAR‐2 in the myenteric plexus by RT‐PCR using primers based on sequences of cloned guinea‐pig receptors. 3 Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR‐1 and PAR‐2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR‐1 agonists responded to PAR‐2 agonists, and > 90 % of PAR‐1 and PAR‐2 responsive neurons responded to ATP. 4 These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR‐1 and PAR‐2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.

Endothelium‐dependent contractile actions of proteinase‐activated receptor‐2‐activating peptides in human umbilical vein: release of a contracting factor via a novel receptor

1 The contractile actions of the proteinase‐activated receptor‐2‐activating peptides (PAR2APs), SLIGRL‐NH2 (SL‐NH2), SLIGKV‐NH2 (KV‐NH2), trans‐cinnamoyl‐LIGRLO‐NH2 (tc‐NH2), and the PAR1‐AP, TFLLR‐NH2 (TF‐NH2) as well as trypsin and thrombin were studied in endothelium‐denuded and intact human umbilical vein (HUV) ring preparations. 2 In HUV rings with, but not without an intact endothelium, PAR2APs caused a concentration‐dependent contractile response, whereas LSIGRL‐NH2 trypsin and PAR1APs were inactive. The contractile response was not affected by the endothelin ETA receptor antagonist, BQ123, the cyclooxygenase inhibitor, indomethacin, the leukotriene synthesis inhibitor, MK886, or the epoxygenase/P450 inhibitor, SKF‐525A. Other pharmacological antagonists (prazosin, Losartan®) were similarly inactive. 3 The order of potencies of the PAR2APs to cause a contraction in the endothelium‐intact preparation was: SL‐NH2>>KV‐NH2tc‐NH2. 4 Using an endothelium‐free rat aorta ring as a reporter tissue, surrounded with endothelium‐intact HUV as a donor tissue in a ‘sandwich assay,’ we also monitored the ability of SL‐NH2, TF‐NH2, trypsin and thrombin to release either contractile (EDCF) or relaxant (EDRF) factors. 5 In the ‘sandwich assay’ done in the presence of L‐NAME (0.1 mm), the endothelium‐intact HUV tissue (but not endothelium‐denuded HUV) released a contractile factor (EDCF) in response to SL‐NH2 (50 μm) but not to trypsin or LSIGRL‐NH2. The SL‐NH2‐mediated release/action of the EDCF was not affected by BQ123, indomethacin, MK886 or SKF‐525A. 6 In the ‘sandwich assay’, trypsin (4–10 nm), SL‐NH2, KV‐NH2 and tc‐NH2 caused the release of a relaxant activity (EDRF) from the endothelium‐intact (but not the denuded) HUV preparation. The release of EDRF was blocked by 0.1 mm ωnitro‐L‐arginine‐methylester (L‐NAME). Neither thrombin (10 u ml−1, 100 nm) nor TF‐NH2 (50 μm) were active in this EDRF‐release assay. 7 The relative potencies of the PAR2 agonists for causing the release of EDRF in the HUV sandwich assay were: trypsin>>SL‐NH2>>tc‐NH2>KV‐NH2. This order of potencies differed from the one observed for the same agonists in the HUV contraction assay (above) and in an intracellular calcium signalling assay, conducted with cloned human PAR2 that was expressed in cultured rat kidney KNRK cells: trypsin>>SL‐NH2=tc‐NH2>KV‐NH2. 8 We conclude that PAR2APs (but not PAR1APs) via a receptor distinct from PAR2, can cause a contractile response in endothelium‐intact HUV tissue via the release of a diffusable EDCF, that is different from previously recognized smooth muscle agonists (e.g. prostanoid metabolites, endothelin, noradrenaline, angiotensin‐II, acetylcholine).

Proteinase activated receptor 2: role of extracellular loop 2 for ligand-mediated activation

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. SLIGEL‐NH2 exhibited low potency (1u2003:u2003200 relative to SLIGRL‐NH2) in wild‐type PAR2. Its activity was increased 5 fold in PAR2PRR, but it was inactive in PAR2NET. 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. 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. We conclude that the acidic EL‐2 tripeptide, PEE, in PAR2 plays an important role in governing agonist activity. 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. 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.

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.

Contractile actions of proteinase-activated receptor-derived polypeptides in guinea-pig gastric and lung parenchymal strips: evidence for distinct receptor systems

We have measured the contractile activities and relative potencies (EC50s) of six thrombin PAR1 receptor‐derived receptor‐activating peptides (PAR‐APs): AparafluroFRChaCit‐y‐NH2 (Cit‐NH2); SFLLRNP(P7); SFLLRNP‐NH2 (P7‐NH2); SFLLR (P5); SFLLR‐NH2 (P5‐NH2); TFLLR‐NH2 (TF‐NH2) and a PAR2 receptor activating peptide [SLIGRL‐NH2 (SL‐NH2)] (a) in a guinea‐pig lung peripheral parenchymal strip preparation and (b) in a gastric longitudinal smooth muscle preparation. The relative potencies of the PAR‐APs in the lung preparation (Cit‐NH2≅TF‐NH2≅P5‐NH2>P7≅P5≅P7‐NH2; SL‐NH2 not active) differed appreciably from their relative potencies in the gastric preparation: Cit‐NH2≅TF‐NH2≅P7‐NH2≅P5‐NH2>P7≅percnt;SL‐NH2. The contractile actions of the PAR1‐selective peptide, TF‐NH2 in the gastric preparation were entirely dependent on extracellular calcium and were blocked by tyrosine kinase inhibitors (genistein, tyrphostin 47/AG213, PP1) and by the cyclooxygenase inhibitor, indomethacin, whereas in the lung preparation, the PAR1‐mediated contractile response was only partially dependent on extracellular calcium and was refractory to the actions of either tyrosine kinase inhibitors or indomethacin. Partial sequencing of the PAR cDNAs detected by RTu2003–u2003PCR both in whole lung and in the peripheral parenchymal strip bioassay tissue demonstrated the presence of both PAR1 and PAR2 mRNA; the expression of PAR2 was detected by immunohistochemistry. The data point to the presence of distinct receptor systems for the PAR1‐APs in guinea‐pig lung parenchymal and gastric smooth muscle and indicate that PAR2 does not regulate contractile activity in peripheral parenchymal guinea‐pig lung tissue