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

Abstract

The biological activities of the proteinase‐activated receptor number 2 (PAR‐2)‐derived peptides, SLIGRL (PP6) SLIGRL‐NH 2 (PP6‐NH 2) and SLIGR‐NH 2 (PP5‐NH 2) 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‐NH 2 (TP5‐NH 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. In the mouse and rat gastric preparations, the PAR‐2‐derived polypeptides, PP6, PP6‐HN 2 and PP5‐NH 2 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‐NH 2. The cumulative exposure of the rat LM tissue to PP6‐NH 2 led to a desensitization of the contractile response to this polypeptide, but not to TP5‐NH 2 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. In the rat gastric preparation, the potencies of the PAR‐2‐activating peptides were lower than the potency of TP5‐NH 2 (potency order: TP5‐NH 2 > > PP6‐NH 2 ≥ PP6 > PP5‐NH 2); PP6 was a partial agonist in this preparation. The contractile actions of PP6 and PP6‐NH 2 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. 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, N ω‐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‐NH 2. The relaxation response to PP6‐NH 2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. In the rat aortic preparation, the potencies of PP6, PP6‐NH 2 and PP5‐NH 2 were greater than those of the thrombin receptor activating peptide, TP5‐NH 2 (potency order: PP6‐NH 2 ≥ PP6 > PP5‐NH2 > TP5‐NH 2). 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. 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.