nitroarginine and seryl-leucyl-isoleucyl-glycyl--arginyl-leucinamide

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is proteolytically activated by certain endogenous proteases, such as trypsin, tryptase, and factor Xa. PAR2 can also be activated by synthetic peptides if their sequence mimics the tethered ligand exposed after receptor cleavage. Although it is known that PAR2 modulates vascular reactivity, it is unclear whether at the chronic stage of type 2 diabetes there are alterations in PAR2-mediated vascular responses. We investigated this issue by exposing mesenteric artery rings to PAR2-activating peptide (PAR2-AP; SLIGRL-NH(2)), the arteries used being obtained from later-stage (32-40-week-old) type 2 diabetic Goto-Kakizaki (GK) rats. The PAR2-AP-induced relaxation was enhanced in GK rats (vs. age-matched Wistar rats), whereas the ACh-induced relaxation was weaker in GK than in Wistar rats. In both groups, the PAR2-AP-induced relaxation was largely blocked by endothelial denudation or by N(G)-nitro-L-arginine [nitric oxide (NO) synthase inhibitor] treatment, but it was unaffected by indomethacin (cyclooxygenase inhibitor) treatment. Both the NO production induced by PAR2-AP and the PAR2 protein expression were significantly increased in mesenteric arteries from GK rats (vs. Wistar rats). These data are the first to indicate that the PAR2-AP-induced endothelium-dependent relaxation is enhanced in mesenteric arteries isolated from type 2 diabetic GK rats at the chronic stage, and they further suggest that the enhancement may be due to an increased expression of PAR2 receptors in this artery.

Topics: Acetylcholine; Animals; Cyclooxygenase Inhibitors; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Endothelium, Vascular; Indomethacin; Male; Mesenteric Arteries; Nitrates; Nitric Oxide; Nitrites; Nitroarginine; Oligopeptides; Phenylephrine; Rats; Rats, Inbred Strains; Rats, Wistar; Receptor, PAR-2; Vasoconstriction; Vasodilation

Proteinase-activated receptors (PARs) are a novel class of G protein-coupled receptors that respond to signals through endogenous proteinases. PAR activation involves enzymatic cleavage of the extracellular NH(2)-terminal domain and unmasking of a new NH(2) terminus, which serves as an anchored ligand to activate the receptor. At least four PAR subtypes have been identified. In the present study, we used the in vitro perfused hydronephrotic rat kidney to examine the effects of activating PAR-2 on the afferent arteriole. The synthetic peptide SLIGRL-NH(2), which corresponds to the exposed ligand sequence and selectively activates PAR-2, did not alter basal afferent arteriolar diameter but caused a concentration-dependent vasodilation (3-30 microM) of arterioles preconstricted by angiotensin II (0.1 nM). A modified peptide sequence (LSIGRL-NH(2), inactive at PAR-2) had no effect. This vasodilation was characterized by an initial transient component followed by a smaller sustained response. A similar pattern of vasodilation was seen when SLIGRL-NH(2) was administered to isolated perfused normal rat kidney. The sustained component of the PAR-2-induced afferent arteriolar vasodilation was eliminated by nitric oxide (NO) synthase inhibition (100 microM nitro-L-arginine methyl ester). In contrast, the transient vasodilation persisted under these conditions. This transient response was not observed when afferent arterioles were preconstricted with elevated KCl, suggesting involvement of an endothelium-derived hyperpolarizing factor. Finally, RT-PCR revealed the presence of PAR-2 mRNA in isolated afferent arterioles. These findings indicate that PAR-2 is expressed in the afferent arteriole and that its activation elicits afferent arteriolar vasodilation by NO-dependent and NO-independent mechanisms.

Topics: Acetylcholine; Angiotensin II; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arterioles; Enzyme Inhibitors; Hydronephrosis; Ibuprofen; Kidney; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oligopeptides; Potassium Chloride; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Receptors, Thrombin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vasodilation

Protease-activated receptor (PAR)-1 and PAR-2 are expressed on gastrointestinal smooth muscle, but knowledge of their functionality is limited. The aim of this study was to determine if PAR-1 and PAR-2 mediate gastrointestinal smooth muscle relaxation and to clarify the underlying mechanisms.. Responses to PAR activation using the serine proteases thrombin and trypsin and the peptide agonists for PAR-1 and PAR-2, SFLLRN-NH2 and SLIGRL-NH2, respectively, were investigated in submaximally contracted longitudinal strips of mouse gastric fundus and guinea pig taenia coli.. In mouse gastric fundus, both thrombin and trypsin caused relaxations followed by contractions. SFLLRN-NH2 and SLIGRL-NH2 caused similar biphasic responses, the relaxation components of which were eliminated by apamin or ryanodine. For SFLLRN-NH2, apamin and ryanodine revealed contractions. Nifedipine inhibited both relaxations and contractions to each peptide. In guinea-pig taenia coli, thrombin but not trypsin caused relaxation, whereas SFLLRN-NH2 and SLIGRL-NH2 caused concentration-dependent relaxations that were eliminated by apamin but were unaffected by ryanodine.. The mouse gastric fundus and guinea pig taenia coli contain functional PAR-1 and PAR-2 that mediate relaxations via ryanodine-sensitive and -insensitive activation of small-conductance, Ca2+-activated K+ channels. We propose that smooth muscle PARs act as sensors for inflammatory signals in gut and respond by inhibiting gut motility during peritoneal infections or tissue damage.

Topics: Acetylcholine; Animals; Apamin; Calcium Channel Blockers; Colon; Gastric Fundus; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Muscle Relaxation; Muscle, Smooth; Nitroarginine; Oligopeptides; Peptide Fragments; Receptor, PAR-1; Receptor, PAR-2; Receptors, Thrombin; Stomach; Tetrodotoxin; Thrombin

Protease-activated receptor-2 (PAR-2) can be activated after proteolysis of the amino terminal of the receptor by trypsin or by synthetic peptides with a sequence corresponding to the endogenous tethered ligand exposed by trypsin (eg, SLIGRL-NH(2)). PAR-2 mediates nitric oxide (NO)-dependent dilatation in cerebral arteries, but it is unknown whether PAR-2 function is altered in cardiovascular diseases. Since hypertension selectively impairs NO-mediated cerebral vasodilatation in response to acetylcholine and bradykinin, we sought to determine whether PAR-2-mediated vasodilatation is similarly adversely affected by this disease state.. We studied basilar artery responses in Wistar-Kyoto rats (WKY) (normotensive) and spontaneously hypertensive rats (SHR) in vivo (cranial window preparation) and in vitro (isolated arterial rings). The vasodilator effects of acetylcholine, sodium nitroprusside, and activators of PAR-2 and protease-activated receptor-1 (PAR-1) were compared in WKY versus SHR. Immunohistochemical localization of PAR-2 was also assessed in the basilar artery.. Increases in basilar artery diameter in response to acetylcholine were 65% to 85% smaller in SHR versus WKY, whereas responses to sodium nitroprusside were not different. In contrast to acetylcholine, vasodilatation in vivo to SLIGRL-NH(2) was largely preserved in SHR, and SLIGRL-NH(2) was approximately 3-fold more potent in causing vasorelaxation in SHR versus WKY in vitro. In both strains, responses to SLIGRL-NH(2) were abolished by N(G)-nitro-L-arginine, an inhibitor of NO synthesis. Activators of PAR-1 had little or no effect on the rat basilar artery. PAR-2-like immunoreactivity was observed in both the endothelial and smooth muscle cells of the basilar artery in both strains of rat.. These data indicate that NO-mediated vasodilatation to PAR-2 activation is selectively preserved or augmented in SHR and may suggest protective roles for PAR-2 in the cerebral circulation during chronic hypertension.

Topics: Acetylcholine; Animals; Cerebral Arteries; Chronic Disease; Enzyme Inhibitors; Hypertension; Male; Nitroarginine; Nitroprusside; Oligopeptides; Peptide Fragments; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, PAR-1; Receptor, PAR-2; Receptors, Thrombin; Vasodilation

1 The localization of protease-activated receptor-2 (PAR2) and the effects of PAR2 activators were investigated in the mouse isolated ureter in order to test the hypothesis that PAR2 activation may initiate neuropeptide release from sensory nerve fibres and hence contribute to inflammation. 2 PAR2 was localized by fluorescence immunohistochemistry to both the smooth muscle and epithelium of the ureter. Macrophage-like cells in the adventitia of the ureter were also PAR2-immunoreactive. PAR2-immunoreactivity was not observed in mast cells or nerve fibres. 3 In circular muscle preparations of the ureter in which continuous rhythmic beating was induced by KCl (20 mM) and the thromboxane A2 mimetic U46619 (0.3 microM), trypsin (0.3 U ml-1) reduced beat frequency to 84.6+/-2.0% of control rates. The PAR2-selective peptide agonist SLIGRL-NH2 concentration-dependently (0.1-3.0 microM) slowed beat frequency to a maximum of 72.7+/-2.0%. 4 Histamine (1-300 microM) was more efficacious than SLIGRL-NH2 in inhibiting ureter beat frequency in a concentration-dependent manner to a maximum (at 300 microM) of 7.9+/-2.5% of the control rate. 5 Pretreatment of preparations with capsaicin (10 microM for 30 min) markedly attenuated the inhibitory effect of histamine, but not that of SLIGRL-NH2, indicating a role for sensory nerves in the inhibitory effect of histamine only. 6 The inhibitory effect of SLIGRL-NH2 on ureter beat frequency was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM) or the cyclo-oxygenase inhibitor, indomethacin (3 microM). 7 In conclusion, PAR2 activation causes inhibition of beating in the mouse ureter that is not mediated by axon reflex release of inhibitory neuropeptides. This inhibitory effect of PAR2 appears to be mediated directly on smooth muscle cells, although the contribution of non-NO, non-prostanoid epithelium-derived factors cannot be ruled out.

Topics: Animals; Capsaicin; Histamine; Immunohistochemistry; In Vitro Techniques; Indomethacin; Male; Mast Cells; Mice; Mice, Inbred BALB C; Neurons, Afferent; Nitroarginine; Oligopeptides; Receptor, PAR-2; Receptors, Thrombin; Trypsin; Ureter