Introduction

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Proteinase-activated receptors (PARs) are members of the heptahelical family of G-protein-coupled receptors that contain seven putative transmembrane domains (Coughlin, 2001; Macfarlane et al., 2001; Hollenberg and Compton, 2002). Their activation can result in an increased intracellular calcium concentration [Ca²⁺]_i (Rasmussen et al., 1991; Nystedt et al., 1994, 1995; Molino et al., 1997). Proteolysis of the extracellular N terminus of PARs by serine proteases, including thrombin and/or trypsin, reveal a characteristic tethered ligand peptide sequence that is believed to interact with the extracellular loop 2 of the receptor to initiate receptor activation (Vu et al., 1991; Coughlin et al., 1992). Specific tethered ligand sequences have been identified for each of the four PAR family members (PAR1, PAR2, PAR3, and PAR4). Synthetic peptides based on the N terminus proteolytically revealed that tethered ligand sequences have been shown to be specific agonists for PAR1, PAR2, and PAR4 (Nystedt et al., 1994; Blackhart et al., 1996; Hollenberg et al., 1997; Kahn et al., 1998; Xu et al., 1998; Kawabata et al., 1999; Hollenberg and Saifeddine, 2001). Structure-activity relationships have also led to the synthesis of chemically modified peptides that either are equipotent to the revealed tethered ligand sequence [e.g., trans-cinnamoyl-LIGRLO-NH₂ (tc-LI) for PAR2] or are receptor antagonists (e.g., N-trans-cinnamoyl-p-fluoro-F-p-guanidino-FLR-NH₂ and trans-cinnamoyl-YPGKF-NH₂ for PAR1 and PAR4, respectively) (Bernatowicz et al., 1996; Hollenberg et al., 1997; Hollenberg and Saifeddine, 2001).

PAR2-derived activating peptides (PAR2-APs) cause endothelium-dependent relaxation of blood vessels, principally via activation of the nitric-oxide synthase (NOS) expressed by endothelial cells (NOS3), presumably a consequence of elevated [Ca²⁺]_i in these cells (Saifeddine et al., 1996; Moffatt and Cocks, 1998; Sobey et al., 1999; Cicala, 2002). In addition, a non-NOS/nonsoluble guanylyl cyclase (sGC)/noncyclooxygenases (COX) endothelium-dependent PAR2-mediated relaxation of murine-isolated resistance arteries has also been recently described (McGuire et al., 2002). Both endothelium-independent contraction (mouse renal arteries) (Moffatt and Cocks, 1998) and endothelium-dependent contraction (rat pulmonary artery and human umbilical vein) (Roy et al., 1998) have been reported as vascular responses to Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH₂; SLI-NH₂) in vitro. In the former study, endothelium-independent contraction via activation of PAR2 localized to vascular smooth muscle was demonstrated by agonist cross-desensitization experiments (Moffatt and Cocks, 1998). An earlier study with the isolated aorta and femoral arteries from rats reported the release of endothelin-1 by stimulation with PAR2-AP SLIGRL (Magazine et al., 1996). With respect to hemodynamics in vivo, the duration, but not magnitude, of the blood pressure-lowering effect of SLI-NH₂ in anesthetized wild-type PAR2 mice was decreased by pretreatment with NOS inhibitors but not by nonselective COX inhibitors; the blood pressure-lowering effect was absent in PAR2 (/) mice (Damiano et al., 1999).

In a study by Vergnolle et al. (1998), SLI-NH₂ and tc-LI were found to be equipotent for eliciting the relaxation of precontracted rat aorta and the activation of calcium-signaling in a PAR2-transfected cell line, but the two peptides displayed a significant difference in potency for activation of rat jejunal chloride transport, as measured by short-circuit current in Ussing chambers. The differences in the structure-activity relationships observed for the jejunal chloride transport assay compared with the other two assays (vascular relaxation and calcium signaling) were taken as evidence for the activation of a non-PAR2 receptor in the jejunum by PAR2-APs. Likewise, there is evidence demonstrating non-PAR2-mediated contraction by SLI-NH₂, but not tc-LI, in rat pulmonary artery and human umbilical vein preparations (Roy et al., 1998; Saifeddine et al., 1998).

The goals of our study were 1) to evaluate the vascular actions of tc-LI in murine arterial vessels in vitro and 2) to determine whether those effects could be ascribed to PAR2. Both mesenteric and renal arterial vessels served as controls for the known relaxant and contractile responses of murine PAR2 activation, whereas using the femoral artery extended our knowledge to a tissue in which the response to PAR2 activation was not known.

	Methods and Materials

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Materials. The following peptides were synthesized as carboxy-amides by the Peptide Synthesis Core Facility (University of Calgary, Calgary, AB, Canada) and were purified by preparatory high-pressure liquid chromatography (>95% purity): SLI-NH₂, tc-LI, LRGILS-amide, trans-cinnamoyl-OLRGIL-amide, and AYPGKF-amide. Stock solutions of all peptides and their dilutions were made in 25 mM HEPES buffer, pH 7.4. The concentration of the peptides and purity of all peptide stock solutions were verified by quantitative amino acid analysis and mass spectrometry. Apamin, atropine, BQ123, BQ788, caffeine, charybdotoxin, chlorpheniramine, cyclopiazonic acid, indomethacin, nifedipine, L-N^G-nitroarginine methyl ester hydrochloride (L-NAME), 1-H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), prazosin, and SQ29548 were purchased from Sigma-Aldrich (St. Loius, MO). SR140333 and SR48968 were gifts from SANOFI Research Institute (Montpellier, France) to Dr. Nathalie Vergnolle (University of Calgary). Tetrodotoxin (without citrate) was purchased from Alamone (Jerusalem, Israel).

Animal Sources. Wild-type genetic background male mouse strains (C57BL/6J; Jackson Laboratories, Bar Harbor, ME) were used as control animals and were matched for sizes when experiments were conducted. Protocols for using animals were approved by the Animal Resources Committee at the University of Calgary and were in accordance with the guidelines of the Canadian Council for Animal Care in Research. NOS3 (/) mice were also purchased from Jackson Laboratories. PAR2 (/) mice, derived from C57BL/6 background, were supplied by R.W. Johnson Pharmaceutical Research Institute (Spring House, PA).

Bioassay Preparation. Male mice (8 to 16 weeks of age; 20-40 g) were sacrificed by cervical dislocation and then the intestinal tissues, the kidneys with arteries and veins attached, or the quadriceps skeletal muscles containing femoral vein, artery, and nerve were removed and placed in ice-cooled modified Krebs' buffer. With the aid of a dissection light microscope, the second-order small mesenteric arteries (MA) (arterial segments separated by two branch points from the superior mesenteric artery), renal arteries, and femoral arteries were carefully isolated from surrounding tissue. Modified Krebs-bicarbonate solution was gassed with 95% O₂/5% CO₂ (pH 7.4 at 37°C) and had the following composition: 118 mM NaCl, 4.7 mM KCl, 0.87 mM MgSO₄, 0.86 mM KH₂PO₄, 2.5 mM CaCl₂, 10 mM D-glucose, and 25 mM NaHCO₃. Two to four rings (1- to 2-mm lengths) were cut from isolated blood vessels to be used for isometric tension measurements. Rings were suspended between a micropositioner and force transducer with gold-plated tungsten wires (20-µm diameter) in a Mulvany-Halpern-style organ bath (5 ml volume; 610 Multi-Myograph system; J.P. Trading, Copenhagen, Denmark). Isometric tension was recorded on-line via a serial connection to a computer hard drive at a rate of 1 Hz. Resting tension (1 mN for small mesenteric arteries or 1.5 mN for renal and femoral arteries) was fixed for an initial 1-h equilibration period. Software for data acquisition and analysis (Myodaq 2.01/Myodata 2.02) were designed by J.P. Trading for use with the 610 Multi-Myograph system.

Bioassay Protocols. Tissues were routinely contracted with 60 to 120 mM KCl to determine their viability. Then tissues were submaximally (50-75% of E_Max) precontracted with cirazoline (0.06-0.3 µM for femoral artery, 0.06-1 µM for renal artery, and 0.1-0.3 µM for MA), and the response to either a single concentration (10 µM) or a cumulative concentration range (1 nM to 10 µM) was determined for ACh to assess the responsiveness of the endothelium. Tissues from wild-type animals and PAR2 (/) mice typically responded to ACh with >80% reversal of precontracted tension. In experiments designed to determine the role of the endothelium in the relaxation effects, a stainless steel 220-µm diameter wire was used to rub the interior surface of the rings, thereby removing the endothelium. These tissues were deemed to be endothelium-denuded only if there was no immediate relaxation response to a single application of 10 µM ACh. Equilibration periods between treatments and the incubation of inhibitors with tissues were 20 min each. Relaxation activity was determined by the reversal of a submaximal precontraction induced by the contractile agonist; contractions by either cirazoline or U46619 were 50 to 75% of a maximal response, as determined by their maximal responses to U46619 or cirazoline, whereas contractions by 30 mM KCl were about 30% of the maximal response to 120 mM KCl. Either single concentrations (50 or 100 µM) or cumulative concentration-response relationships were determined for SLI-NH₂, tc-LI, and the reversed-sequence control peptides LRGILS-NH₂ and trancinnamoyl-OLRGIL-NH₂, respectively.

Data Analysis and Statistics. Arterial ring relaxant responses are reported on the graphs as a percentage of the initial tension (% initial tension) generated by the contractile agonists. Arterial ring contraction caused by peptides from the baseline tension were standardized as a percentage of maximal contraction generated by 120 mM KCl. The pD₂ values for relaxation in response to PAR2-APs were determined from individual concentration-response relationships by manual graph interpolation. E_Max values are reported as percent relaxation, whereby 100% relaxation represents a complete reversal of agonist-induced tone to baseline tension. Values represent the means ± S.E. mean (error bars) for 3 to 13 animals with two to four measurements per animal unless otherwise indicated. The comparisons of mean values for pD₂ and E_Max were made using either Student's t tests and one-way analysis of variance calculations (ANOVA) followed by Student-Newman-Keuls post hoc tests (GraphPad Instat 2.01; GraphPad Software, San Diego, CA) or entire data sets by two-way ANOVA followed by Bonferroni post hoc. Differences between means were considered significant if the Student's t test or post hoc tests indicated a p value less than 0.05.

	Results

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SLI-NH₂ and tc-LI Cause PAR2-Dependent Relaxation and PAR2-Independent Contraction of Murine Femoral Arteries Precontracted with either Cirazoline or U46619. Initially, to assess the vascular actions of PAR2-APs, we determined cumulative concentration-response relationships for SLI-NH₂ and tc-LI applied to cirazoline-precontracted femoral arteries from C57BL/6J and PAR2 (/) mice. Both SLI-NH₂ and tc-LI caused a biphasic concentration-dependent response in femoral arteries from C57BL/6J mice that included an initial relaxant effect (0.1 to 10 µM) that was followed by a contractile effect at higher concentrations (Fig. 1, a and b). The E_Max and pD₂ values for SLI-NH₂-induced relaxation of femoral arteries from C67BL/6J (n = 7) mice were 70 ± 5% and 5.8 ± 0.2, respectively. The E_Max and pD₂ values for tc-LI-induced relaxation of femoral arteries from C67BL/6J mice (n = 7) were 78 ± 6% and 5.8 ± 0.1, respectively. In contrast, in the femoral arteries preparations from PAR2 (/) mice, the addition of PAR2-APs failed to cause relaxation but induced a further contractile response of submaximal cirazoline-precontracted tissue (Fig. 1c). The contractile responses to tc-LI (10 to 100 µM) were significantly greater than the contractions elicited by equivalent concentrations of SLI-NH₂ in the cirazoline precontracted femoral arteries of PAR2 (/) mice [Fig. 1c; P < 0.0001; two-way ANOVA followed by (P < 0.01) Bonferroni post hoc].

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effects of SLI-NH2 and tc-LI on femoral arteries from PAR2 (+/+) and PAR2 (/) mice: inhibition of relaxation by inhibitors of NOS/sGC, and the lack of effect of ET-1 (ETA and ETB) receptor antagonists in PAR2 (+/+) mice. C57BL/6J (a and b) or PAR2 (/) (c) mouse arteries were precontracted to 50% of maximal with cirazoline before obtaining cumulative concentration-response relationships for peptides. For several experiments vessels from C57BL/6J were pretreated for 20 min with the indicated inhibitors (10 µM ODQ, 100 µM L-NAME, 1 µM BQ123, and 1 µM BQ788). Numbers within parentheses indicate the number of animals.

Precontraction by 30 mM KCl Inhibits PAR2-Induced Relaxation of Femoral Arteries, but Not Renal Arteries. To determine the effect of vascular smooth muscle membrane depolarization on the relaxant activity of the PAR2-APs (i.e., relaxation mediated via activation of K⁺ channels), femoral arteries from C57BL/6J, NOS3 (/), and PAR2 (/) mice were precontracted by the addition of 30 mM KCl (Fig. 2). Precontraction of femoral arteries by depolarization with 30 mM KCl reduced the relaxation by PAR2-APs in preparations from the PAR2 (+/+) mice (Fig. 2a; p < 0.05, Student's unpaired t test). Neither peptide caused a relaxation response in femoral arteries from either NOS3 (/) or PAR2 (/) mice (Fig. 2, b and c). The contractile responses elicited by tc-LI in femoral arteries from background control (Fig. 2a), NOS3(/) (Fig. 2b), and PAR2 (/) (Fig. 2c) mice were the same (P > 0.05; two-way ANOVA). Similarly, whether complete blockade of vasodilator activity by SLI-NH₂ was caused by NOS/sGC inhibitors (Fig. 1a) or by targeted gene deletions of either NOS3(/) (Figs. 2b) or PAR2 (/) (Figs. 1c and 2c), the contractile responses by SLI-NH₂ were the same (P > 0.05; two-way ANOVA). In contrast with the results obtained with the femoral arterial preparation, the relaxation responses of renal arteries from C57BL/6J mice were not reduced by precontraction of these arteries with 30 mM KCl compared with precontraction with cirazoline (Fig. 3; p > 0.05; Student's unpaired t test). E_Max values for SLI-NH₂-induced relaxation of renal arteries were: cirazoline-treated (n = 3), 31 ± 9% compared with 30 mM KCl-treated (n = 3), 49 ± 12%. E_Max values for tc-LI-induced relaxation were: cirazoline-treated (n = 4), 30 ± 7% compared with 30 mM KCl-treated (n = 3), 43 ± 2%.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Vascular smooth muscle depolarization attenuates SLI-NH2 and tc-LI-induced relaxation. C57BL/6J (a), NOS3 (/) (b) or PAR2 (/) (c) arteries were precontracted to 30 to 50% of maximum with 30 mM KCl before obtaining cumulative concentration-response relationships for peptides. Numbers within parentheses indicate the number of animals.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   SLI-NH2 and tc-LI-induced relaxation of PAR2 (+/+) renal arteries. Arteries were precontracted with either cirazoline (a) or 30 mM KCl (b) before obtaining cumulative concentration-response relationships for peptide-induced changes in tension. Numbers within parentheses indicate the number of animals.

Non-NOS/Non-sGC/Non-COX-Mediated Relaxation of Murine Mesenterics by tc-LI. We have reported that SLIGRL-NH₂ caused the vasodilation of small murine second-order mesenteric arteries via a non-NOS/non-sGC/non-COX mechanism (McGuire et al., 2002), and thus, we determined whether tc-LI had a similar action. Indeed, tc-LI caused a vasodilation that was partly L-NAME/ODQ/indomethacin-insensitive (Fig. 4) and was completely inhibited by additional pretreatment of these tissues with 1 µM apamin plus 0.1 µM charybdotoxin (Fig. 4). The E_Max values were all significantly different than each other (n = 3; p < 0.05; ANOVA followed by Student-Newman-Keuls post hoc). There was no significant difference between the pD₂ values determined for control and L-NAME/ODQ/indomethacin-treated tissues. The pD₂ values and E_Max values for tc-LIGRLO-NH₂-induced relaxation were: control (n = 4), 6.0 ± 0.1 and 90 ± 5% and L-NAME/ODQ/indomethacin-treated (n = 3), 5.9 ± 0.3 and 52 ± 15%. These pD₂ values were virtually identical to the pD₂ values for SLI-NH₂-induced relaxation of MA reported previously (McGuire et al., 2002). The E_Max value for tissues treated with L-NAME + ODQ + indomethacin + apamin + charybdotoxin (n = 3) was 6 ± 4%, and the pD₂ value could not be determined. tc-LI (0.1 to 3 µM) failed to cause the relaxation of 0.1 µM cirazoline precontracted small mesenteric arteries from PAR2 (/) mice, but at concentrations >10 µM, tc-LI further contracted these vessels (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Effects of NOS/sGC/COX inhibition and selective inhibitors of calcium-activated K+ channels, SKCa and BKCa, on tc-LI-induced relaxation of C57BL/6J second-order small mesenteric arteries. Arteries were pretreated for 20 min with inhibitors (100 µM L-NAME, 10 µM ODQ, 10 µM indomethacin, 1 µM apamin, and 0.1 µM charybdotoxin) and then precontracted with 0.1 µM cirazoline before obtaining cumulative concentration-response relationships for tc-LI. Numbers within parentheses indicate the number of animals.

SLI-NH₂ Induces PAR2-Mediated Endothelium-Independent Contraction of Murine Arteries and tc-LI Induces Non-PAR2 Endothelium-Independent Arterial Contraction. To assess the contribution of the vascular smooth muscle expression of PAR2 to renal arterial contraction by SLI-NH₂ and tc-LI, renal arteries from C57BL/6J and PAR2 (/) mice were treated with these agonists. Consistent with a previous report (Moffatt and Cocks, 1998), SLI-NH₂ elicited a contraction response from renal arteries of PAR2 (+/+) mice when these arteries were coincubated with an inhibitor of NOS (300 µM L-NAME) (Fig. 5) but did not contract the nontreated (control) renal arteries. The other PAR2 ligand agonist, tc-LI, contracted renal arteries whether NOS inhibitor was present or not (Fig. 5). tc-LI, however, also caused the contraction of renal arteries from PAR2 (/) mice, whereas SLI-NH₂ did not, even in the presence of NOS inhibitor (Fig. 5). Renal arteries from PAR2 (/) that were precontracted with cirazoline neither relaxed nor produced further contraction when exposed to concentrations 100 µM of SLI-NH₂, but contracted further to tc-LI ( 3 µM) (data not shown). KCl (120 mM) elicited equivalent contractile responses from renal arteries from each of the mouse strains, 4 to 5 mN/1-mm length of tissue.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   SLI-NH2 and tc-LI-induced contraction of mouse renal arteries from baseline tension in the presence or absence of PAR2 expression, with and without simultaneous NOS inhibition. Arteries were pretreated for 20 min with NOS inhibitor (300 µM L-NAME) before application of either peptide (100 µM). Numbers above each bar indicate the number of animals.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effects of SLI-NH2 and tc-LI on murine femoral arteries at baseline tension in the absence or presence of NOS inhibitor, an intact endothelium, or the absence of either NOS3 or PAR2 expression. Arteries were pretreated for 20 min with NOS inhibitor (300 µM L-NAME) as indicated before application of either peptide (100 µM). Numbers above each bar indicated the number of animals.

Intracellular Caffeine-Sensitive Calcium Stores are Necessary for tc-LI-Induced Vascular Contractions. To determine whether an influx of extracellular calcium contributed to the contractile response of femoral arteries by tc-LI, tissues were preincubated in the presence of inhibitors of either voltage-gated calcium channels (nifedipine) or nonspecific cation channels (CdCl₂) or were incubated in calcium-free Kreb's solution. Significantly, all three of these treatments failed to inhibit contractions by 50 µM tc-LI (Fig. 7, a and b; results not shown for CdCl₂; n = 3; p > 0.05; ANOVA). To characterize the intracellular stores of calcium that elicited the contractions of femoral arteries by tc-LI, tissues were pretreated with cyclopiazonic acid (10 µM), to inhibit active uptake of calcium by the sarcoplasmic reticulum, and then exposed to caffeine (10 mM), to deplete caffeine-sensitive stores, before exposure to 50 µM tc-LI. Indeed, the depletion of intracellular caffeine-sensitive calcium stores reduced significantly the contractions caused subsequently by the addition of 50 µM tc-LI (Fig. 7c).

View larger version (9K): [in this window] [in a new window]   Fig. 7.   Roles of extracellular influx and intracellular stores of calcium on tc-LI-induced contractions of mouse femoral artery. To examine the role of extracellular Ca2+ influx, tissues were either repetitively exposed to 50 µM tc-LI (i.e., controls for the lack of tachyphylaxis) (a) or pretreated with voltage-gated calcium channel antagonist before or incubated in Ca2+-free Kreb's solution during exposure to tc-LI (b). To examine the role of intracellular Ca2+ stores (c), tissues were treated with sarcoplasmic reticulum Ca2+-ATPase pump inhibitor and then depleted of caffeine-sensitive Ca2+ stores by repetitive exposures to increasing concentrations of caffeine before treatment with tc-LI. Percent inhibition of tc-LI-induced contractions were (mean ± S.D.): caffeine-treatment (data not illustrated; n = 3), 41 ± 13% inhibition, and cyclopiazonic acid + caffeine (n = 4), 71 ± 11%. P < 0.05 for treated-tissue responses compared with control (untreated tissue responses), ANOVA followed by Student-Newman-Keuls post hoc.

Contraction of Femoral Arteries by tc-LI Does Not Involve Activation of PAR4, Vascular Muscarinic (M₂), Endothelin-1 (ET_A), Histamine (H_1/2), Angiotensin II (AT₁), ₁-Adrenergic, Substance P (NK₁/NK₂), or Thromboxane A₂ (TP) Receptors Nor the activation of COX. A series of known receptor inhibitors were used to test the hypothesis that if any of a number of vascular agonists, including M₂, ET_A, H₁, AT₁, ₁, NK₁/NK₂, or TP receptors, were involved in tc-LI-induced contraction, then receptor antagonists to these vasoactive compounds should block tc-LI-induced contraction. However, pretreatment of femoral arteries with the following receptor antagonists failed to inhibit 50 µM tc-LI-induced contraction of femoral arteries: atropine (5 µM), BQ123 (1 µM), chlorpheniramine (10 µM), Losartan (1 µM), prazosin (1.5 µM), SR140333 (100 nM) plus SR48986 (100 nM), or SQ29548 (10 µM); the nonselective COX inhibitor indomethacin (10 µM) also failed to block contraction (data not shown). In addition, the PAR4-selective agonist AYPGKF-NH₂ that activated PAR4-mediated rat, mouse, and human platelet aggregation (Faruqi et al., 2000; Hollenberg and Saifeddine, 2001; Chung et al., 2002) failed to cause a contractile response of the femoral artery (data not shown).

Reverse-Sequenced Control PAR2-Derived Activating Peptides Fail to Affect Vascular Tone. To determine whether the vascular actions of SLI-NH₂ and tc-LI resulted from nonspecific effects, we chose to determine the effects of the reverse-sequenced peptides and trans-cinnamoic acid. In all tissues from each strain of mouse, neither LRGILS-NH₂ nor trans-cinnamoyl-OLRGIL-NH₂ (100 µM of each) failed to change the tone of either precontracted arteries or arteries at baseline tension whether or not NOS inhibitor was present (data not shown). Furthermore, 100 µM of trans-cinnamoic acid did not affect tone (data not shown).

	Discussion

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The major finding of our study is that we have identified a non-PAR2-mediated vasoconstrictor activity elicited by the PAR2-AP tc-LIGRLO-NH₂. By the use of several different murine blood vessels and the use of mice with selective gene deletions for PAR2 and NOS3, we also demonstrated vessel-selective PAR2-dependent relaxant effects caused by both SLI-NH₂ and tc-LI and confirmed the contractile effects for murine PAR2 activation in vitro. However, tc-LI-mediated vasoconstriction was common to all of the murine arteries studied here. The clearest distinction between PAR2 and the distinct PAR-AP receptor responsible for the action of tc-LI is illustrated in Fig. 6, wherein the femoral arteries from PAR2 (/) mice contracted in response to tc-LI but not to SLI-NH₂. A series of receptor antagonists to known vasoconstrictor substances failed to inhibit tc-LI-induced contraction of murine femoral arteries. Taken together with the failure of the removal of the endothelium to inhibit contractile activity, the data suggest the existence of a receptor with a novel pharmacology that is expressed on murine vascular smooth muscle. In addition, these data indicate that tc-LI may not be a selective ligand for PAR2. Furthermore the data indicate that caffeine-sensitive intracellular calcium stores and not extracellular influx are essential for mediating the contractile effect linked to activation of this novel tc-LI-activated receptor.

The vascular relaxant effects of the PAR2-derived tethered ligand-activating peptide sequences SLI-NH₂ and tc-LI were found to be dependent upon the activation of PAR2 in renal arteries, femoral arteries, and MA. Both SLI-NH₂ and tc-LI, which induced relaxation of murine femoral arteries with identical potencies, were inhibited by pretreatment with a combination of L-NAME + ODQ (Fig. 1) or by precontraction with 30 mM KCl (Fig. 2). KCl (30 mM) abolished the relaxation of femoral arteries by tc-LI at low concentrations, whereas the relaxation by SLI-NH₂ was inhibited partially; yet, both peptide agonists used the same signal transduction pathway for relaxation. Thus, it appears that the vasoconstrictor activity of tc-LI was greater than the vasodilator activity in these precontracted tissues. In femoral arteries from NOS3 (/) mice, PAR2-APs failed to initiate vascular smooth muscle relaxation. These results are consistent with the observations reported previously for the relaxation response of precontracted renal arteries from mice that also demonstrated the prerequisite role of NOS in PAR2-mediated vasodilation in vitro (Moffatt and Cocks, 1998). For comparison, we also demonstrate that tc-LI and SLI-NH₂ had equivalent actions in the C57BL/6J mouse renal artery (Fig. 3). The coincidence of these concentration-relaxation curves for the peptides indicates that endogenous peptidases do not appear to account for the differences seen in the contractile responses by these peptides. Additional pretreatment of femoral arteries with ET-1 (ET_A and ET_B) receptor antagonists failed to have any additional effects, and this result differed from an earlier study that reported an endothelin-1 associated contractile effect in the femoral artery from rats (Magazine et al., 1996).

As previously reported for SLI-NH₂- and trypsin-induced PAR2-mediated effects in MA (McGuire et al., 2002), tc-LI caused an L-NAME/ODQ/COX-insensitive relaxation. As well, the relaxation of MA elicited by tc-LI was completely inhibited by pretreatment with a combination of apamin + charybdotoxin. This result is an indication of the involvement of small-, intermediate-, and large (big)-conductance Ca²⁺-activated K⁺ channels (SK_Ca, IK_Ca, and BK_Ca, respectively), most likely via an endothelium-dependent hyperpolarization, in PAR2-induced vasodilation of resistance arteries. We were not surprised to find that a NOS/sGC mechanism mediated PAR2-dependent relaxation of murine femoral arteries, whereas a non-NOS/non-sGC/non-COX mechanism mediated the relaxation of MA. The importance of endothelium-dependent hyperpolarization to vascular smooth muscle relaxation has been highlighted for resistance rather than conduit arteries, and thus, our data agree with this argument (McGuire et al., 2001).

It has been reported by ourselves and others that high concentrations (>30 µM) of PAR2-APs induce further contraction of precontracted murine arteries (Moffatt and Cocks, 1998; McGuire et al., 2002). These effects appear to be both peptide- and tissue-specific responses. In renal arteries, the further contraction by SLI-NH₂, but not by tc-LI, is dependent upon PAR2 expression, and these results confirmed a previous study (Moffatt and Cocks, 1998). In femoral arteries, however, it is clearly a non-PAR2 mechanism that mediates the added SLI-NH₂-induced contractions because this response is present in PAR2 (/) mice. Interestingly, in the PAR2 (/) preparations, the renal arteries responded in a similar way to the MA, wherein high concentrations of SLI-NH₂ failed to contract MA from PAR2 (/) mice further (McGuire et al., 2002). In support of a study by Moffatt and Cocks (1998), we also found that SLIGRL-NH₂ induced an endothelium-independent, PAR2-dependent contraction of arteries from baseline tension when NOS activity was either inhibited or absent [NOS3 (/) animals]. This PAR2-dependent contractile response to SLI-NH₂ exhibited rapid tachyphylaxis, unlike the PAR2-dependent relaxation responses, and thus, we suggest differential regulation mechanisms for endothelial versus vascular smooth muscle PAR2.

Our results are significantly different from those of previous studies that propose non-PAR2 effects for SLI-NH₂ and/or tc-LI. In the study by Vergnolle et al. (1998), the non-PAR2 effects on intestinal chloride transport were inhibited by indomethacin, but the contractile response to tc-LI in the femoral artery was not indomethacin-sensitive. In the rat and human vascular tissue bioassays, contraction elicited by non-PAR2 actions of SLI-NH₂ was endothelium-dependent (Roy et al., 1998; Saifeddine et al., 1998), whereas in our study both PAR2 and non-PAR2 contractile effects were endothelium-independent. The question of whether the non-PAR2 contractile mechanism of the PAR2-APs in precontracted femoral arteries is the same as the non-PAR2 mechanism of contraction by tc-LI at baseline tension is unanswered. We did not identify a specific receptor inhibitor of the tc-LI-induced contractile effect in femoral arteries; nor was the contractile response due to the activation of PAR4. Thus, these results point to the existence of a receptor for tc-LI with a novel pharmacology. These data indicate that several likely candidate mechanisms such as cyclooxygenase products, NK₁ and NK₂, M₁ and M₂, ₁, H₁, ET_A, AT₁, TP, and PAR4 receptors are not involved. The identification of any inhibitor of this activity would allow us to determine whether the actions at baseline tension and precontraction are the same.

In conclusion, we determined that the vasodilator action of tc-LI in a series of different murine blood vessels was due to PAR2 activation and that PAR2 activation of vascular smooth muscle in murine arteries can also produce contraction. Furthermore, we conclude that tc-LI, in addition to activation of PAR2, is also an agonist for an as yet unidentified receptor that can mediate the vasoconstriction of murine arteries in vitro. Future studies to determine more precisely the signal transduction mechanism of the contraction of murine vascular smooth muscle from femoral arteries, likely to be facilitated by the use of PAR2 (/) mice, should provide a basis for the subsequent isolation and identification of the receptor.