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Cardiovascular Research 2004 61(4):683-692; doi:10.1016/j.cardiores.2003.11.030
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Distinct roles for protease-activated receptors 1 and 2 in vasomotor modulation in rat superior mesenteric artery

Atsufumi Kawabata*,a, Satoko Kuboa, Yumiko Nakayaa, Tsuyoshi Ishikia, Ryotaro Kurodaa, Fumiko Sekiguchia, Naoyuki Kawaoa and Hiroyuki Nishikawab

aDivision of Physiology and Pathophysiology, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowake, Higashi-Osaka 577-8502, Japan
bResearch and Development Center, Fuso Pharmaceutical Industries Ltd., Osaka 536-8523, Japan

* Corresponding author. Tel.: +81-6-6721-2332x3863; fax: +81-6-6730-1394. kawabata{at}phar.kindai.ac.jp

Received 24 July 2003; revised 12 November 2003; accepted 25 November 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Protease-activated receptors (PARs) 1 and 2 are expressed in various blood vessels including rat aorta, modulating vascular tone. We investigated the roles of PAR-1 and PAR-2 in vasomotor modulation in rat superior mesenteric artery. Methods and results: Effects of the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-amide) and the PAR-1-activating peptide Thr-Phe-Leu-Leu-Arg-amide (TFLLR-amide) on isometric tension were examined in isolated rat superior mesenteric artery or aorta. Both SLIGRL-amide and TFLLR-amide caused relaxation in the precontracted rat aortic rings. The latter peptide, but not the former, produced contraction in the resting rings. NG-nitro-L-arginine methyl ester (L-NAME), but not apamin/charybdotoxin known to block the endothelium-derived hyperpolarizing factor (EDHF) pathway, abolished the relaxation and facilitated the contraction. In the precontracted rat superior mesenteric artery, SLIGRL-amide, but not TFLLR-amide, elicited endothelium-dependent relaxation, which was only partially inhibited by L-NAME with and without indomethacin. The residual relaxation was abolished by apamin/charybdotoxin. Carbenoxolone, a gap junction inhibitor, significantly attenuated the SLIGRL-amide-evoked, EDHF-dependent relaxation, although neither 17-octadecynoic acid, a P450 epoxygenase inhibitor, nor catalase, a hydrogen peroxide scavenger, revealed inhibitory effects. The residual response resistant to carbenoxolone was unaffected by ouabain/BaCl2. In the resting artery, TFLLR-amide, but not SLIGRL-amide, produced only slight contraction, which was dramatically facilitated by combination of L-NAME and apamin/charybdotoxin or by removal of the endothelium. Conclusions: Our data suggest that, in rat superior mesenteric artery, endothelial PAR-2, upon activation, causes relaxation via both NO and EDHF pathways, and that activation of muscular PAR-1 exhibits potential contractile activity that is largely masked by NO and EDHFs pathways triggered by endothelial PAR-1. Gap junctions might be involved in the EDHF mechanisms in this artery.

KEYWORDS Arteries; Nitric oxide; Proteases; Receptors; Vasoconstriction/dilation

This article is referred to in the Editorial by T. Traupe and M. Barton (pages 645–647) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Protease-activated receptors (PARs), a family of G-protein-coupled-seven-trans-membrane-domain receptors, mediate a variety of cellular functions of certain serine proteases [1]. Among four PAR family members, PAR-1, PAR-3 and PAR-4 are specifically activated by thrombin [2–5], whereas PAR-2 is a receptor for trypsin, mast cell tryptase and coagulation factors VIIa and Xa [1,6]. Activation of PARs is triggered by proteolytic unmasking of the cryptic tethered ligand present in the N-terminal extracellular domain. Exogenously applied synthetic peptides as short as five to six amino acids based on the tethered ligand sequence of PAR-1, PAR-2 or PAR-4 [i.e., Ser-Phe-Leu-Leu-Arg (SFLLR), Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) and Gly-Tyr-Pro-Gly-Gln-Val (GYPGQV) derived from human PAR-1, PAR-2 and PAR-4, respectively], but not PAR-3, are capable of causing specific, non-enzymatic activation of the corresponding receptor. PAR-1 and PAR-2 are expressed in a variety of organs/tissues/cells [1]. Multiple functions of PAR-1 and PAR-2 have been well described in the gastrointestinal [7–10], respiratory [11,12], neuronal [13–15] and also circulatory systems [16].

In vivo activation of PAR-1 or PAR-2 by receptor-activating peptides causes hypotension in anesthetized rats or mice [17–19]. In vascular tissues, both PAR-1 and PAR-2 are expressed abundantly in the endothelial cells, modulating vasomotor activity in various isolated blood vessels. In the precontracted rat aortic ring, activation of endothelial PAR-1 and PAR-2 elicits production of nitric oxide (NO), which activates soluble guanylate cyclase, resulting in relaxation responses [20–22]. Agonists for PAR-2, but not PAR-1, cause endothelial NO-dependent relaxation in rat basilar artery [23]. There are species differences in the effects of PAR-1 and PAR-2 agonists on the coronary arteries. Both the PAR-1 and PAR-2 agonists produce vasorelaxation in porcine coronary artery in a manner dependent on both endothelial NO and endothelium-derived hyperpolarizing factor (EDHF) [24], whereas the agonist for PAR-1, but not PAR-2, elicits relaxation in human coronary artery predominantly via NO production [25]. In rat coronary artery, PAR-2 activation causes vasodilation by a mechanism dependent on EDHF and capsaicin-sensitive sensory neurons, but not NO [26]. In rat renal artery, PAR-2, but not PAR-1, triggers vasodilation through both NO-dependent and -independent pathways [27]. Our most recent study has shown that the PAR-2 agonist induces relaxation in rat gastric arterial rings in vitro and increases in gastric mucosal blood flow in anesthetized rats in vivo, and that EDHF plays a predominant role in the induction of the in vitro and in vivo effects of the PAR-2 agonist [19]. In mouse mesenteric artery, the PAR-2 agonist causes EDHF-dependent vasodilation [28], although no information is available about the effect of the PAR-1 agonist. In rat perfused mesenteric artery, endothelial PAR-1 might mediate some of NO-dependent relaxation caused by thrombin [29]. In contrast, PAR-1 is also present in the smooth muscle layer of rat aorta, and, upon activation, is capable of producing contractile responses in the rat aortic ring [20]. The PAR-1 agonist also causes renal vasoconstriction in isolated perfused rat kidney [27]. Nevertheless, little information is available with respect to contractile activity of the PAR-1 agonist in other blood vessels. Collectively, the vascular actions of agonists for PAR-1 and PAR-2 greatly differ with distinct blood vessels and/or species, implying multiplicity of the roles for PAR-1 and PAR-2 in vasomotor modulation. Physiological significance of these complicated roles these receptors play is poorly understood.

In the present study, we investigated the roles for PAR-1 and PAR-2 in modulation of vasomotor activity in rat superior mesenteric artery in vitro. Here, we show that, in this artery, endothelial PAR-2, upon activation, stimulates both the NO and EDHF pathways, producing relaxation, and that PAR-1 present in the smooth muscle, when activated by agonist stimuli, causes contraction that is suppressed and veiled by NO and EDHF produced by concomitant activation of endothelial PAR-1.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Tissue preparation and isometric tension recording
Male Wistar rats (7–8 weeks old) were obtained from Japan SLC. (Shizuoka, Japan), and used with approval from the Kinki University School of Pharmaceutical Science's Committee for the Care and Use of Laboratory Animals, which conforms with the NIH guidelines. The animal was sacrificed by decapitation under urethane (1.5 g/kg, i.p.) anesthesia, and the superior mesenteric artery and/or thoracic aorta were removed. The ring segments of the superior mesenteric artery (0.5 mm in diameter, 3 mm length) and aorta (about 2 mm in diameter, and 4 mm length) were prepared in an ice-cooled Krebs–Henseleit solution (composition in mM: NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25; KH2PO4, 1.2; glucose, 10). In some experiments, the arterial rings were stripped of the endothelium by gently rubbing the inner surface with a cotton string. The ring preparations were mounted between two triangle wire hooks and suspended in organ baths containing 2 ml of Krebs–Henseleit solution maintained at 37 °C and bubbled with 95% O2/5% CO2 to keep the pH at 7.4. The segment was allowed to equilibrate for 1 h under a resting tension of 10 mN, and isometric tension was recorded through a force-displacement transducer (UL-10GR, Minebea, Japan). The integrity of the ring segment was monitored a few times by measuring the contractile response to a high K+ (50 mM)-containing Krebs-Henseleit solution in which the corresponding molar equivalent of NaCl was removed. The contractile responses are expressed as a percentage (% KCl) of the contraction induced by 50 mM K+, and the relaxation responses are represented as a percentage (% papaverine) of the relaxation to 100 µM papaverine. In experiments to determine the effects of Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-amide) on the relaxation responses to subsequent application of acetylcholine and papaverine, complete relaxation was achieved by replacement of the bath medium with Ca2+-free Krebs-Henseleit solution containing 10 mM EGTA, and the relaxation responses are expressed as a percentage of the 10 mM EGTA-evoked relaxation.

2.2 Experimental protocol
2.2.1 Aorta
After observing the contractile responses to 50 mM KCl, the aortic ring was precontracted with 1 µM phenylephrine, and the PAR-2-activating peptide SLIGRL-amide, PAR-1-activating peptide Thr-Phe-Leu-Leu-Arg-amide (TFLLR-amide) and acetylcholine at supramaximal concentrations, 100, 100 and 10 µM, respectively, was applied to the tissue bath. NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, at 100 µM [30] was added to the bath 5 min before addition of phenylephrine. Apamin, an inhibitor of small-conductance, Ca2+-activated K+ channels, and charybdotoxin, an inhibitor of large- and intermediate-conductance, Ca2+-activated K+ channels, when co-administered, are known to block the EDHF pathway [19,31]. To evaluate involvement of the EDHF pathway, therefore, apamin at 0.1 µM plus charybdotoxin at 0.1 µM [32] was applied 5 min before phenylephrine. In contraction experiments, SLIGRL-amide or TFLLR-amide at 100 µM was added to the tissue bath in the absence or presence of L-NAME or apamin plus charybdotoxin that was applied 5 min before the peptide challenge.

2.2.2 Superior mesenteric artery
The arterial rings with or without the endothelium were used. Relaxant effects of SLIGRL-amide (applied cumulatively), TFLLR-amide at 0.1–100 µM (applied non-cumulatively), trypsin at 0.3–30 nM (applied cumulatively) or acetylcholine at 10 µM were tested in the rings precontracted with 1 µM phenylephrine. L-NAME and apamin/charybdotoxin were applied as described above, and indomethacin, an inhibitor of cyclo-oxygenase, at 10 µM [32] was added to the tissue bath 5 min before phenylephrine. To clarify the EDHF pathways mediating the relaxation caused by PAR-2 activation in the superior mesenteric artery, we first evaluated possible involvement of three candidates for EDHF, including (1) epoxyeicosatrienoic acid and its analogues (EETs), P450 epoxygenase metabolites of arachidonic acid [33,34], (2) hydrogen peroxide [35,36], and (3) gap junction [37–40]. 17-Octadecynoic acid, an inhibitor of P450 isozymes including CYP2C11 that is expressed in rat blood vessels, at 10 µM [34], catalase, a scavenger of hydrogen peroxide, at 6250 U/ml [36], or carbenoxolone and 18{alpha}-glycyrrhetinic acid, inhibitors of gap junction, at 300 and 100 µM, respectively [36,39], were applied 5 min before addition of phenylephrine, in the presence of L-NAME at 100 µM and indomethacin at 10 µM. Furthermore, ouabain at 0.5 mM plus BaCl2 at 30 µM, known to inhibit the Na+/K+-pump and the inward rectifying K+ channels, respectively, responsible for the relaxant effect of K+ as an EDHF candidate [32,41], was applied 5 min before phenylephrine, in the presence of L-NAME at 100 µM, indomethacin at 10 µM and carbenoxolone at 300 µM. In contraction experiments, TFLLR-amide or SLIGRL-amide at 100 µM was added to the tissue bath without precontraction, and L-NAME at 100 µM and/or apamin at 0.1 µM plus charybdotoxin at 0.1 µM were applied 5 min before the peptide challenge. To obtain the concentration-response relationship in the presence of L-NAME, apamin, and charybdotoxin, effect of TFLLR-amide at a single concentration of 3–100 µM, was tested in a non-cumulative manner. In the experiments to examine involvement of endogenous superoxide in the PAR-1-mediated contraction, superoxide dismutase (SOD) at 200 U/ml was applied to the tissues 5 min before TFLLR-amide at 100 µM.

2.3 Peptides and other chemicals employed
SLIGRL-amide and TFLLR-amide were synthesized by a standard solid phase synthesis procedures and purified by high-performance liquid chromatography (HPLC), and the concentration, purity and composition of the peptides were determined by mass spectrometry and quantitative amino acid analysis. L-NAME hydrochloride, apamin, 17-octadecynoic acid, catalase, carbenoxolone, SOD, 18{alpha}-glycyrrhetinic acid and ouabain were purchased from Sigma (St. Louis, USA), and charybdotoxin was from Peptide Institute (Minoh, Japan). Indomethacin and papaverine were obtained from Wako (Osaka, Japan), and phenylephrine hydrochloride and acetylcholine chloride were from ACROS (New Jersey, USA) and Tokyo Kasei Kogyo (Tokyo, Japan), respectively. Indomethacin was dissolved in 5 mM Na2CO3, immediately before the use, and 18{alpha}-glycyrrhetinic acid, 17-octadecynoic acid and ouabain were dissolved in 100%, 80% and 50% DMSO, respectively. All other chemicals were dissolved in distilled water.

2.4 Statistics
Data are represented as mean±S.E.M. Statistical analysis was performed by the Tukey's test for multiple comparisons or Student's t-test for two-group comparisons. Differences between the concentration–response curves were analyzed statistically by Bonferroni's test. Significance was set at a P<0.05 level.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Characterization of the relaxation and contractile responses to PAR-2- and PAR-1-activating peptides in rat aortic rings
In the endothelium-intact aortic preparation precontracted with phenylephrine, the PAR-2-activating peptide SLIGRL-amide at 100 µM produced potent relaxation, the magnitude being even greater than that caused by 10 µM acetylcholine. The PAR-1-activating peptide TFLLR-amide at 100 µM produced small relaxation response (Fig. 1a). These relaxation responses caused by SLIGRL-amide, TFLLR-amide or acetylcholine were abolished by pretreatment with L-NAME at 100 µM, but not apamin at 0.1 µM in combination with charybdotoxin at 0.1 µM (Fig. 1a), indicating involvement of NO, but not the EDHF pathways. TFLLR-amide, but not SLIGRL-amide, at 100 µM caused contractile response in the aortic ring (Fig. 1b). This contraction caused by PAR-1 activation was facilitated by pretreatment with L-NAME, but not apamin in combination with charybdotoxin (Fig. 1b), indicating that the contraction caused by the PAR-1 agonist was suppressed by concomitant increase in endothelial NO production, but not by the EDHF pathways.


Figure 1
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  		Fig. 1 Relaxant (a) and contractile (b) activity of the PAR-2-activating peptide SLIGRL-amide and the PAR-1-activating peptide TFLLR-amide in rat aortic rings. Relaxation responses were examined in the aorta precontracted with 1 µM phenylephrine. L-NAME at 100 µM or apamin (Apa) at 0.1 µM in combination with charybdotoxin (ChTX) at 0.1 µM was added to the bath 5 min before phenylephrine (a) or peptides (b). Data show the mean with S.E.M. from five to six experiments. ns, not significant.

 
3.2 Relaxation responses to PAR-2- and PAR-1-activating peptides in rat superior mesenteric arterial rings
In the endothelium-intact arterial ring precontracted with phenylephrine, the PAR-2 agonist SLIGRL-amide, applied cumulatively at 0.1–100 µM, caused potent relaxation responses in a concentration-dependent manner (Figs. 2a and 3a)Go, whereas the PAR-1 agonist TFLLR-amide at the same concentration range had no relaxant effects (Fig. 2a). The relaxation response to SLIGRL-amide was not observed in the endothelium-denuded preparations (Fig. 2a). Of note is that application of SLIGRL-amide did not affect the relaxation responses to subsequent acetylcholine or papaverine in the precontracted arterial rings (Fig. 2b).


Figure 2
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  		Fig. 2 Relaxant activities of SLIGRL-amide and TFLLR-amide in rat superior mesenteric artery with or without the endothelium. (a) SLIGRL-amide or TFLLR-amide was applied to the endothelium-intact (Endo+) or -free (Endo–) arterial rings precontracted with 1 µM phenylephrine. The relaxation response is presented as a percentage of the relaxation caused by papaverine at 100 µM. (b) Lack of effect of SLIGRL-amide on the relaxation caused by subsequent acetylcholine and papaverine in the endothelium-intact arterial rings precontracted phenylephrine. The relaxation response is presented as a percentage of the relaxation caused by depletion of extra- and intracellular Ca2+ with 10 mM EGTA. ns, not significant. Data show the mean with S.E.M. from four to seven experiments.

 

Figure 3
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  		Fig. 3 Typical recordings of the SLIGRL-amide-evoked relaxation in rat superior mesenteric artery. SLIGRL-amide was cumulatively applied to the arterial rings precontracted with 1 µM phenylephrine (PE) (a). The relaxant effects of SLIGRL-amide were also tested in the presence of L-NAME plus indomethacin (Indo) (b) or L-NAME plus indomethacin in combination with apamin (Apa) plus charybdotoxin (ChTX) (c).

 
L-NAME at 100 µM partially inhibited the SLIGRL-amide-evoked relaxation in the superior mesenteric artery (Fig. 4), and indomethacin at 10 µM, when applied in addition to L-NAME, exhibited no additional inhibition (Figs. 3b and 4)Go. In the presence of L-NAME and indomethacin, charybdotoxin at 0.1 µM produced additional attenuation of the relaxant effect of SLIGRL-amide, whereas apamin at 0.1 µM had no such effect (Fig. 4). Combined application of apamin and charybdotoxin abolished the residual responses to SLIGRL-amide in the presence of L-NAME and indomethacin (Figs. 3c and 4)Go, indicating involvement of the EDHF pathway.


Figure 4
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  		Fig. 4 Inhibitory effects of L-NAME, indomethacin and apamin/charybdotoxin on the SLIGRL-amide-evoked relaxation in rat superior mesenteric artery. SLIGRL-amide was cumulatively applied to the arterial rings precontracted with 1 µM phenylephrine. L-NAME at 100 µM, indomethacin (Indo) at 10 µM, apamin (Apa) at 0.1 µM and charybdotoxin (ChTX) at 0.1 µM were added to the tissue bath 5 min before phenylephrine. Data show the mean with S.E.M. from seven to eight experiments.

 
3.3 Relaxation responses to trypsin, an endogenous PAR-2 activator, in rat superior mesenteric arterial rings
In the endothelium-intact arterial ring precontracted with phenylephrine, the endogenous PAR-2 activator trypsin, applied cumulatively at 0.3–30 U/ml, caused potent relaxation responses in a concentration-dependent manner (Fig. 5). As seen in the relaxation responses to SLIGRL-amide, the trypsin-evoked relaxation was partially inhibited by L-NAME, and the residual response was not altered by indomethacin, but abolished by additional application of apamin plus charybdotoxin (Fig. 5).


Figure 5
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  		Fig. 5 Relaxant effect of trypsin, an endogenous PAR-2 activator, and its characterization in rat superior mesenteric artery. Trypsin was cumulatively applied to the arterial rings precontracted with 1 µM phenylephrine. L-NAME at 100 µM, indomethacin (Indo) at 10 µM and apamin (Apa) at 0.1 µM plus charybdotoxin (ChTX) at 0.1 µM were added to the tissue bath 5 min before phenylephrine. Data show the mean with S.E.M. from four experiments.

 
3.4 Evaluation of involvement of candidates for EDHFs in the relaxation responses to the PAR-2 agonist in the presence of L-NAME and indomethacin
To identify the EDHF pathways involved in the PAR-2-triggered endothelium-dependent relaxation in rat superior mesenteric artery, the effect of the currently known three candidates for EDHF, EETs, hydrogen peroxide and gap junction, were evaluated. 17-Octadecynoic acid, an inhibitor of P450 epoxygenase that produces EETs from arachidonic acid, when applied at 10 µM failed to alter the concentration-dependent curve for the SLIGRL-amide-evoked relaxation responses in the presence of L-NAME and indomethacin (Fig. 6a). Catalase, a scavenger of hydrogen peroxide, at 6250 U/ml also had no effect on the relaxation responses to SLIGRL-amide (Fig. 6b). In contrast, carbenoxolone, an inhibitor of gap junction, significantly attenuated the SLIGRL-amide-induced relaxation in the presence of L-NAME and indomethacin in the superior mesenteric artery (Fig. 6c). Carbenoxolone also suppressed the acetylcholine (10 µM)-induced relaxation by approximately 50%, an magnitude of the inhibition being equivalent to that of the relaxation induced by SLIGRL-amide at 100 µM, a maximal concentration (Fig. 6c,d). Similarly, 18{alpha}-glycyrrhetinic acid, another inhibitor of gap junction, at 100 µM also significantly suppressed the SLIGRL-amide-evoked relaxation (data not shown). The residual relaxation responses to SLIGRL-amide in the presence of carbenoxolone in addition to L-NAME and indomethacin was unaffected by ouabain at 0.5 mM plus BaCl2 at 30 µM, known to abolish the relaxant effect of K+ as an EDHF (Fig. 6c), indicating lack of involvement of K+.


Figure 6
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  		Fig. 6 Effects of 17-octadecynoic acid, catalase, carbenoxolone or ouabain/BaCl2 on the EDHF-dependent relaxation caused by SLIGRL-amide-evoked (a–c) or acetylcholine (d), in rat superior mesenteric artery. SLIGRL-amide and acetylcholine were applied to the arterial rings precontracted with 1 µM phenylephrine in the presence of L-NAME at 100 µM and indomethacin at 10 µM. 17-Octadecynoic acid (17-Oct) at 10 µM (a), catalase at 6250 U ml–1 (b), carbenoxolone (Carb) at 300 µM (c, d) and ouabain at 0.5 mM plus BaCl2 at 30 µM (c) were added to the tissue bath 5 min before phenylephrine. Data show the mean with S.E.M. from five to nine experiments. ns, not significant.

 
3.5 Contractile activities of agonists for PAR-1 and PAR-2 in rat superior mesenteric artery
The PAR-1-activating peptide TFLLR-amide at 100 µM caused only slight contractile responses in rat superior mesenteric arterial rings (Figs. 7a and 8a)Go. This contractile activity of TFLLR-amide was slightly augmented by pretreatment with L-NAME at 100 µM or apamin at 0.1 µM plus charybdotoxin at 0.1 µM (Figs. 7b,c and 8a)Go. Combined application of L-NAME and apamin plus charybdotoxin dramatically enhanced the contractile activity of TFLLR-amide at 100 µM (Figs. 7d and 8a)Go. In the presence of L-NAME in combination with apamin plus charybdotoxin, TFLLR-amide at 3–100 µM produced potent contractile responses in a concentration-dependent manner (Fig. 8b). In contrast, the PAR-2-activating peptide SLIGRL-amide exhibited no contractile activity even in the presence of L-NAME, apamin and charybdotoxin (Figs. 7e and 8a)Go. Similar contractile responses to TFLLR-amide at 100 µM were observed even in the presence of SOD at 200 U/ml (Fig. 9). In the endothelium-denuded arterial rings, TFLLR-amide at 100 µM, but not SLIGRL-amide at 100 µM, produced potent contractile responses (Fig. 10).


Figure 7
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  		Fig. 7 Typical recordings of contractile responses to TFLLR-amide or SLIGRL-amide in rat superior mesenteric artery. TFLLR-amide was applied to the arterial rings in the absence (a) or presence of L-NAME (b), apamin/charybdotoxin (ChTX) (c) or L-NAME/apamin/charybdotoxin (d), and SLIGRL-amide was also applied in the presence of L-NAME/apamin/charybdotoxin (e).

 

Figure 8
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  		Fig. 8 Facilitation by L-NAME and/or apamin plus charybdotoxin of the contractile activity of the TFLLR-amide in rat superior mesenteric artery. (a) TFLLR-amide at 100 µM or SLIGRL-amide at 100 µM was applied to the arterial rings in the absence or presence of L-NAME at 100 µM and/or apamin (Apa) at 0.1 µM plus charybdotoxin (ChTX) at 0.1 µM. (b) TFLLR-amide at distinct concentrations was applied non-cumulatively to the tissue in the presence of L-NAME, apamin and charybdotoxin. Data show the mean with S.E.M. from seven to eight experiments.

 

Figure 9
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  		Fig. 9 Lack of effect of SOD on the contractile activity of the TFLLR-amide in rat superior mesenteric artery. In the presence or absence of SOD at 200 U/ml, the contractile activity of TFLLR-amide at 100 µM and its facilitation by L-NAME at 100 µM and/or apamin (Apa) at 0.1 µM plus charybdotoxin (ChTX) at 0.1 µM were evaluated. ns, not significant.

 

Figure 10
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  		Fig. 10 Contractile activities of TFLLR-amide and SLIGRL-amide in rat superior mesenteric artery with or without the endothelium. TFLLR-amide at 100 µM (a) or SLIGRL-amide at 100 µM (b) was applied to the endothelium-intact (Endo+) or -free (Endo–) arterial rings. Data show the mean with S.E.M. from seven to nine experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study demonstrates that the PAR-2 agonists including SLIGRL-amide and trypsin cause endothelium-dependent relaxation via both the NO and EDHF pathways in rat superior mesenteric artery, and that the EDHF pathway triggered by PAR-2 in this artery involves gap junction, but not EETs, hydrogen peroxide or K+. We also found that the contractile activity of the PAR-1 agonist is unveiled by inhibition of both the NO and EDHF pathways or by removal of the endothelium in the superior mesenteric artery, although the PAR-1 agonist itself is incapable of producing relaxation. Thus, there is a perfect balance between the relaxing and contractile effects of PAR-1 stimulation. Taken together, our data show that functional PAR-2 is present only in the endothelium and, upon activation, causes relaxation by stimulating both NO and EDHF pathways in rat superior mesenteric artery, and that PAR-1 is expressed in both the endothelium and smooth muscle, and activation of smooth muscle PAR-1 exhibits potential contractile activity that is largely veiled by NO and EDHF produced or stimulated by activation of endothelial PAR-1 (Fig. 11).


Figure 11
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  		Fig. 11 Scheme for possible vasomotor modulation by PAR-2 and PAR-1 in rat superior mesenteric artery. Arrows show the pathway triggered by PAR-2, while broken arrows show the pathway triggered by PAR-1.

 
Our present findings obtained in rat aortic rings support previous evidence that endothelial NO predominantly mediates the vasorelaxation caused by either PAR-2 or PAR-1 agonist in the precontracted aorta [20,21,30], and partially blocks the contractile response to smooth muscle PAR-1 activation in the resting aorta [20]. Our data further confirmed lack of involvement of the EDHF pathway in vasomotor modulation by PAR-2 and PAR-1 in rat aorta. In mouse mesenteric arteriole preparations, PAR-2 agonists also cause relaxation responses through both the NO and EDHF pathways, and K+ appears to play a role as one of EDHF candidates [28]. Our results in rat superior mesenteric arterial rings also indicate involvement of both NO and EDHF in the relaxant activity of the PAR-2 agonist, and suggest that the EDHF pathway is, in part, mediated by gap junction, but not EETs, hydrogen peroxide or K+. Additional unknown factors might be also involved in the residual relaxation responses resistant to the two distinct gap junction inhibitors in the presence of L-NAME and indomethacin. Apart from minor inconsistency, these characteristics are in agreement with the evidence that gap junctions, but not K+ or EETs, play a major role as the EDHF mechanism in rat mesenteric artery [34,39,42]. The discrepancy concerning EDHFs in the mouse and rat arteries might be just due to species difference. Nonetheless, our data from the relaxation experiments should be discussed with certain reservations, because some of the inhibitors employed themselves greatly altered the magnitude of the pre-contraction caused by phenylephrine; e.g., the evoked pre-contraction (%KCl) was 56.4±0.5 in the control, and 122.2±1.0 and 137.4±1.2 in the presence of L-NAME/indomethacin and L-NAME/indomethacin/apamin/charybdotoxin, respectively.

The PAR-1 agonist failed to cause vasorelaxation in rat superior mesenteric artery, although it slightly relaxed the rat aortic ring, as reported previously [20,43,44]. Nevertheless, endothelial PAR-1, upon activation, appears to stimulate the NO and EDHF pathways, an effect being overcome by the contractile activity due to concomitant activation of smooth muscle PAR-1. In other words, the contractile activity caused by activation of smooth muscle PAR-1 is strongly suppressed by concomitant activation of endothelial PAR-1, and unveiled by inhibition of the NO and EDHF pathways. PAR-1 would thus appear to play a potential dual role in vasomotor modulation in rat superior mesenteric artery, although the apparent contractile and relaxant activities of the PAR-1 agonist are negligible. It has been shown that thrombin, an activator for PAR-1, PAR-3 and PAR-4, induces the release of superoxide in addition to NO [45,46]. Since the bioactivity of NO could be regulated by both production of NO and its breakdown by superoxide [45,46], it is likely that endogenous superoxide could modulate the PAR-1-mediated contractile responses in the superior mesenteric artery. However, our data from experiments using SOD imply that superoxide, generated following PAR-1 activation, if any, does not play a critical role in modulation of the PAR-1-mediated vascular contraction. The physiological significance of these complicated roles of PAR-1, a thrombin receptor, in vascular modulation is still open to question. It is likely that (1) the vasorelaxation by intravascular thrombin via endothelial PAR-1 may function to maintain blood flow, when prothrombin is converted into thrombin, a coagulation factor, in the vascular lumen, and that (2) the vasoconstriction by extravascular thrombin via smooth muscle PAR-1 may support the hemostatic mechanisms, when thrombin exuded from blood vessels during hemorrhage.

Apart from the underlying mechanisms, PAR-2 appears to be present in the endothelium of most arteries in distinct species, and, upon activation, cause relaxation. In contrast, there is a large variation in roles of PAR-1 in modulation of vascular tone between different species and between different arteries. The contractile activity of PAR-1 activation can be often seen in large arteries including the aorta [20,43,44], whereas the PAR-1-mediated vasorelaxation may be relatively clearly observed in small arteries, as detected by a microperfusion assay [29] and a tissue blood flow measurement [47]. This might suggest certain physiological meanings, although the detailed analysis remains to be achieved.

In conclusion, endothelial PAR-2 and PAR-1 in rat superior mesenteric artery function to suppress vasomotor activity, while smooth muscle PAR-1, upon activation, produces vasoconstriction. Both NO and EDHFs including gap junction are considered to be involved in the vasomotor modulation in this artery.


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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