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CARDIOVASCULAR

2-Furoyl-LIGRLO-amide: A Potent and Selective Proteinase-Activated Receptor 2 Agonist

Canadian Institutes of Health Research Group on Regulation of Vascular Contractility, Smooth Muscle Research Group (J.J.M., C.R.T., M.D.H.) and Mucosal Inflammation Research Group (M.S., K.S., M.D.H.), Departments of Pharmacology & Therapeutics and Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada

Received December 17, 2003; accepted February 19, 2004.

	Abstract

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A peptide corresponding to a proteinase-activated receptor 2 (PAR₂)-activating peptide with an N-terminal furoyl group modification, 2-furoyl-LIGRLO-NH₂, was assessed for PAR₂-dependent and -independent biological activities. 2-Furoyl-LIGRLO-NH₂ was equally effective to and 10 to 25 times more potent than SLIGRLNH₂ for increasing intracellular calcium in cultured human and rat PAR₂-expressing cells, respectively. In bioassays of tissue PAR₂ activity, measured as arterial vasodilation and hyperpolarization, 2-furoyl-LIGRLO-NH₂ was 10 to 300 times more potent than SLIGRL-NH₂. Unlike trans-cinnamoyl-LIGRLO-NH₂, 2-furoyl-LI-GRLO-NH₂ did not cause a prominent non-PAR₂-mediated contraction of murine femoral arteries. In conclusion, 2-furoyl-LI-GRLO-NH₂ represents the most potent and selective activator of PAR₂ in biological systems described to date.

Recently, a new compound from a series of PAR₂-activating peptides, 2-furoyl-LIGKV-OH, was found to mimic the actions of the PAR₂-activating peptide SLIGRL-NH₂ but with an extended duration of action in vivo in a mouse model of chronic arthritis (Ferrell et al., 2003). In this mouse model of arthritis, the intra-articular injection of SLIGRL-NH₂ caused knee joint swelling that reached a maximum at 4 h and then declined, whereas 2-furoyl-LIGKV-OH caused swelling that was not only equivalent to that of SLIGRL-NH₂ at 4 h, but reached a maximum at 24 h that almost doubled the maximal swelling caused by SLIGRL-NH₂ and then persisted for at least an additional 48 h thereafter (Ferrell et al., 2003). The N-terminal furoyl-modification of the human PAR₂-activating peptide sequence was in keeping with prior studies using N-terminal acylation to yield PAR antagonists (Bernatowicz et al., 1996) and to protect PAR peptide-based agonists from endogenous aminopeptidases that are expected to decrease their bioavailability (Coller et al., 1993; Vergnolle et al., 1998; Maryanoff et al., 2001). Among the list of selective PAR₂-activating peptides was the compound trans-cinnamoyl-LIGRLO-NH₂, which is a potent and selective PAR₂ agonist like SLIGRL-NH₂ that possesses a carboxy-terminal ornithine. When acylated with [³H]propionate, trans-cinnamoyl-LIGRLO-NH₂ can be used as a radioligand receptor-binding probe (Al Ani et al., 1999). However, our continued experience with the use of the trans-cinnamoyl PAR₂-activating peptide for studies done in vitro and in vivo revealed that it also can stimulate receptors other than PAR₂ in the mouse vasculature (McGuire et al., 2002a). Thus, despite its selective and potent action on PAR₂ in cultured cells, this acylated agonist does not seem to be the best probe for PAR₂ functions in complex biological systems in vitro or in vivo, where it can have other actions (McGuire et al., 2002a). It is interesting to note that the N-acylated PAR₂ peptide is a full agonist, whereas the N-trans-cinnamoyl derivatives of PAR₁- and PAR₄-activating peptides are antagonists for their respective receptors (Bernatowicz et al., 1996; Hollenberg and Saifeddine, 2001).

Given the limited pharmacological profile described for 2-furoyl-LIGKV-OH (Ferrell et al., 2003), we sought to evaluate the action of the comparable peptide 2-furoyl-LIGRLO-NH₂ in more depth to determine whether it might be a more useful compound than trans-cinnamoyl-LIGRLO-NH₂ in assessing the potential effects of activating PAR₂ in complex biological systems. We designed the new PAR₂-activating peptide to be more potent than 2-furoyl-LIGKV-OH by taking advantage of the increase in peptide potency we have previously observed upon substituting an arginine for lysine at position 5 and upon C-terminal amidation (Hollenberg et al., 1996; Al Ani et al., 1999).

	Materials and Methods

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Materials. All peptides were synthesized as carboxy amides (>95% purity, assessed by high-performance liquid chromatography and mass spectrometry) by Dr. Denis McMaster and Tyler Vanderputten (Peptide Core Facility at the University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada). Unless otherwise indicated, all remaining chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Peptides were dissolved in phosphate-buffered saline (pH 7.4) containing 25 mM HEPES.

Animals. Sprague-Dawley male rats (350 g) and C57BL/6 male mice (8-10 weeks old) were supplied by Charles River Canada (Montreal, PQ, Canada). Procedures that involved animals were approved by the Animal Resources Committee at the University of Calgary and were in accordance with the guidelines of the Canadian Council on the Care of Animals in Research.

Global Intracellular Calcium Measurements. The details of the experimental protocol for measuring changes in global intracellular calcium in cells suspended in solution have been described previously (Al Ani et al., 1999; Kawabata et al., 1999; Compton et al., 2000). The experiments described herein employed either human or rat PAR₂-expressing KNRK cells (Al Ani et al., 1999; Compton et al., 2000) or HEK293 cells, which constitutively coexpress human PAR₁ and PAR₂ (Kawabata et al., 1999). Cells harvested without the use of trypsin in an isotonic EDTA-containing dissociation medium were incubated in a solution of -minimal essential medium that contained 10% (v/v) fetal calf serum, 0.25 mM sulfinpyrazone, and 22 µM Fluo-3 acetoxymethyl ester (Molecular Probes, Eugene, OR) for 25 min at room temperature. Cells were then resuspended in a buffered solution (pH 7.4) that contained 150 mM NaCl, 3 mM KCl, 1.5 mM CaCl₂, 10 mM glucose, 20 mM HEPES, and 0.25 mM sulfinpyrazone. Light emission at 530 nm using a 480-nm excitation wavelength was monitored on an AMINCO Bowman Series 2 luminescence spectrometer (Spectronic Unicam, Rochester, NY). Cell suspensions (2 ml) in 4-ml cuvettes were mixed continuously with a magnetic stirrer and maintained at 24°C. The response that resulted from the addition of a test agonist was standardized relative to the peak fluorescence elicited by the addition of calcium ionophore (2 µM A23187 [GenBank] ). In the cross-desensitization experiments, a sufficient time was allowed between the sequential addition of agonists (5-10 min) to enable a complete refilling of the intracellular calcium stores (Kawabata et al., 1999).

Rat Platelet Activation Assay. The platelet aggregation assay used to test for PAR₄ activation of isolated washed rat platelets was carried out as described previously (Hollenberg and Saifeddine, 2001).

Isometric Tension and Membrane Potential Measurements. The details of experiments for measuring the isometric tension of isolated rat aorta, murine femoral, and mesenteric arteries were as described previously (Compton et al., 2002; McGuire et al., 2002a,b, 2003). Briefly, segments of arteries were isolated from animals that had been killed by cervical dislocation. Tissues were continuously maintained in a standard physiological salt solution buffer containing 114 mM NaCl, 4.7 mM KCl, 0.8 mM KH₂PO₄, 1.2 mM MgCl₂, 11 mM D-glucose, 25 mM NaHCO₃, and 2.5 mMCaCl₂ that was bubbled with a 95%/5% O₂/CO₂ gas mixture to maintain the buffer at pH 7.4. Rings of rat aorta were suspended vertically by two metal hooks; the upper hook was connected to an isometric force transducer, and the lower hook was connected to an immovable support in 5 ml of cuvettes containing the standard physiological solution. Two gold-plated tungsten wires (diameter, 0.02 mm) suspended murine arteries horizontally (one connected to a force transducer and the other to a micropositioner) in a Mulvany-style myograph chamber (Danish Myo Technology A/S, Inc., Skejbyparken, Denmark). For electrophysiological studies, a motorized micropositioner was used to place a sharp glass microelectrode filled with 3M KCl (resistance 50-100 M) close enough to impale cells from the adventitial side of an artery that was suspended in a single myograph chamber. Drugs were added directly to the chambers that contained the vessels. Isometric tension was recorded from rat aorta and murine femoral arteries with a standard paper chart recorder and personal computer using MyoDaq/MyoData 2.1 (Danish Myo Technology A/S, Inc.), respectively. Isometric tension and membrane potentials of murine mesenteric arteries were recorded simultaneously from the same artery with a personal computer using Axotape 2.0 software.

Data Analysis. pD₂ is equal to the negative logarithm, base 10, of the effective concentration of peptide that elicits 50% of the maximal observed response (E_max). Unless otherwise stated, data represent the mean ± S.E.M. (error bars on graphs; n, number of separate experiments). Statistical comparison of concentration-response parameters (pD₂ and E_max) between two experimental groups was made using Student's t test for paired data. Comparison of contractile responses by femoral arteries was made by one-way analysis of variance after logarithmic transformation of the data (logarithm, base 10, percentage of 120 mM KCl-induced contraction) and was followed by Neuman-Keuls post hoc test (p < 0.05 was considered statistically significant).

	Results

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Selective and Potent Activation of PAR₂ by 2-Furoyl-LIGRLO-NH₂. To determine whether 2-furoyl-LIGRLO-NH₂ activates PAR₂, KNRK cells that overexpress the native rat PAR₂ or that were transfected with an empty vector were treated with this peptide, and the resulting global intracellular calcium changes were monitored (Fig. 1). In the cells overexpressing rat PAR₂, 2-furoyl-LIGRLO-NH₂ (0.01-10 µM) caused concentration-dependent increases in intracellular calcium (Fig. 2A) and was about 25 times more potent than the receptor-selective PAR₂-activating peptide SLIGRL-NH₂. The pD₂ values for 2-furoyl-LIGRLO-NH₂ (n = 4) and SLIGRL-NH₂ (n = 3) were 7.0 ± 0.1 and 5.6 ± 0.1, respectively. The maximal response to 2-furoyl-LIGRLO-NH₂ (E_max, 88 ± 3% of 2 µM A23187 [GenBank] -induced response) was not different than that elicited by SLIGRL (E_max, 89% ± 2% of 2 µM A23187 [GenBank] -induced response; p > 0.05; Student's paired t test). In the empty pcDNA3 vector-transfected KNRK cells that do possess mRNA for PAR₂, 2-furoyl-LIGRLO-NH₂ (10 µM) raised intracellular calcium only slightly (n = 4; E_max, 14 ± 4%). Likewise, SLIGRL-NH₂ at 100 µM produced a minimal response (n = 4; E_max, 16 ± 3%).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Calcium signaling and ligand cross-desensitization in rat PAR2-expressing KNRK cells. Rat PAR2-expressing KNRK cell suspensions containing intracellular Fluo-3 were exposed twice to PAR2-desensitizing concentrations of either trypsin (Trp) (10 U ml-1; 20 nM) (A) or SLIGRLNH2 (100 µM) (B) and followed by a test concentration of 2-furoyl-LIGRLO-NH2 (2-f-LIGRLO-NH2) (1 µM). Alternatively, cells were first exposed to desensitizing concentrations of 2-furoyl-LIGRLO-NH2 (50 µM) (C) and followed 10 min later by a test concentration of the selective PAR2-activating peptide SLIGRL-NH2 (25 µM). The calcium signals [fluorescence at 530 nm (E530, upward deflection)] generated by the test concentrations of either 2-furoyl-LIGRLO-NH2 (1 µM) or SLIGRL-NH2 (25 µM) without prior desensitization are shown to the right of each tracing (A, B, and C). The scales for time (minutes) and calcium signal (upward deflection in centimeters) are shown by the inset. On average, intracellular calcium concentrations rose from a baseline value of about 30 nM to a maximum value of about 340 nM.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Concentration-effect curves for calcium signaling by 2-furoyl-LIGRLO-NH2 in PAR2-expressing cells. A and B, suspensions of either rat PAR2-expressing KNRK-(A) or HEK293 cells (B) treated with 2-furoyl-LIGRLO-NH2 (n = 4) at different concentrations and the relative global changes in intracellular calcium measured by fluorescence of Fluo-3. Values were standardized as a percentage relative to a maximal intracellular calcium-dependent fluorescence signal caused by the addition of calcium ionophore (2 µM A23187 [GenBank] ).

A homospecific receptor cross-desensitization protocol (Kawabata et al., 1999) was used to unequivocally determine that the 2-furoyl-LIGRLO-NH₂-induced calcium response was mediated through activation of PAR₂. Rat PAR₂-overexpressing KNRK cells were first treated with trypsin (10 U ml^-1; 20 nM) for 5 to 10 min prior to the addition of 2-furoyl-LIGRLO-NH₂. Under these conditions of PAR₂ desensitization by prior trypsin-dependent PAR₂ activation, a calcium signal generated by the subsequent addition of 2-furoyl-LIGRLO-NH₂ was completely absent (Fig. 1A). Similarly, desensitization of KNRK cell PAR₂ by prior exposure of the cells to SLIGRL-NH₂ completely eliminated a subsequent response to 2-furoyl-LIGRL-NH₂ and vice versa (Fig. 1, B and C). The control responses to the agonists without prior desensitization are shown on the right of each tracing. These experiments demonstrated a homologous desensitization of rat PAR₂ by all agonists. Cross-desensitization data that mirrored the results shown for rat PAR₂-expressing KNRK cells in Fig. 1 exactly were obtained for KNRK cells expressing human instead of rat PAR₂.

Since HEK cells constitutively express both human PAR₁ and PAR₂ (Kawabata et al., 1999), it was possible to evaluate the potential action of 2-furoyl-LIGRLO-NH₂ on both receptors concurrently in this cell line. As shown in Fig. 3, a calcium signal was generated in the HEK cells upon activation by the selective PAR-activating peptides TFLLR-NH₂ (PAR₁) and SLIGRL-NH₂ (PAR₂). Like SLIGRL-NH₂ (Kawabata et al., 1999), 2-furoyl-LIGRLO-NH₂ caused concentration-dependent increases in the intracellular calcium concentration of HEK293 cells (Fig. 2B). The pD₂ value for this action of 2-furoyl-LIGRLO-NH₂ (n = 4) was 5.4 ± 0.1. The E_max value for 2-furoyl-LIGRLO-NH₂ was 92 ± 3% of the maximum calcium response generated by 2 µM A21387 [GenBank] . A reverse-sequence peptide synthesized as a control that should not be able to interact with PAR₂, 2-furoyl-OLRGIL-NH₂ (200 µM), did not cause a calcium signal in either PAR₂-expressing KNRK or HEK293 cells.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Calcium signaling and ligand cross-desensitization in human HEK cells coexpressing PAR1 and PAR2. Cross-desensitization protocols like those outlined in the legend to Fig. 1 were done with Fluo-3-containing human HEK cells that coexpress both PAR1 and PAR2. The sequences of the desensitization protocols were as follows: A, desensitization with 2-furoyl-LIGRLO-NH2 (2-f-LIGRLO-NH2) (100 µM) followed by a test concentration of the PAR1-selective agonist TFLLR-NH2 (5 µM); B, desensitization with thrombin (100 nM) followed by a test concentration of 2-furoyl-LIGRLO-NH2 (2.5 µM); C, desensitization with TFLLR-NH2 (100 µM) followed by a test concentration of 2-furoyl-LIGRLO-NH2; D, desensitization with the PAR2-selective agonist SLIGRL-NH2 (100 µM) followed by a test concentration of 2-furoyl-LIGRLO-NH2 (2.5 µM); and E, desensitization with 2-furoyl-LIGRLO-NH2 (100 µM) followed by a test concentration of SLIGRL-NH2 (25 µM). The calcium signals (E530, upward deflection) generated by the various agonists without prior desensitization are shown on the right of each tracing (A-E). The scales for time (minutes) and calcium signal (upward deflection in centimeters) are shown by the inset. On average, intracellular calcium concentrations rose from a baseline value of about 30 nM to a maximum value of about 340 nM.

To determine whether 2-furoyl-LIGRLO-NH₂ might also activate or antagonize PAR₁, the HEK293 cells were exposed to the receptor-selective PAR₁ agonist TFLLR-NH₂ after prior exposure of the cells to desensitizing concentrations of 2-furoyl-LIGRLO-NH₂ (Fig. 3A). A prior desensitization of the HEK293 cells by 2-furoyl-LIGRLO-NH₂ did not significantly diminish the subsequent signal generated by TFLLRNH₂ in the continued presence of 2-furoyl-LIGRLO-NH₂ (Fig. 3A). Thus, the 2-furoyl peptide neither activated nor antagonized PAR₁. Moreover, after the prior desensitization of PAR₁ by thrombin-dependent (10 U ml^-1; 100 nM) activation of PAR₁ (Fig. 3B) or the PAR₁-selective agonist TFLLR-NH₂ (100 µM) (Fig. 3C), 2-furoyl-LIGRLO-NH₂ elicited changes in intracellular calcium in HEK293 cells that were at least 70% (n = 3) of the control value. Under these desensitizing conditions, the calcium responses to the PAR₁-selective agonists were completely absent (Fig. 3, B and C). In keeping with the results obtained with the KNRK cells expressing human PAR₂, prior desensitization of the HEK cells with the PAR₂-selective agonist SLIGRL-NH₂ abrogated the cell response to 2-furoyl-LIGRLO-NH₂ and vice versa (Fig. 3, D and E). In the desensitization protocols illustrated in Fig. 3, the calcium signals generated by the several agonists without prior desensitization are shown on the right of each tracing.

To determine whether 2-furoyl-LIGRLO-NH₂ might activate or inhibit PAR₄, the actions of this peptide in a PAR₄-dependent rat platelet aggregation assay (Hollenberg and Saifeddine, 2001) were assessed. 2-Furoyl-LIGRLO-NH₂ (200 µM) did not cause rat platelet aggregation (n = 3) nor antagonize platelet aggregation triggered by the PAR₄-activating peptide AYPGKF-NH₂ (15 µM).

Vascular Reactivity of Rat Aorta Preparations. Initially, to assess the vascular activity of 2-furoyl-LIGRLO-NH₂, rat aorta rings with intact endothelium were contracted with phenylephrine (1 µM) and then treated with 2-furoyl-LIGRLO-NH₂ to determine its relaxation activity, as previously documented for SLIGRL-NH₂ (Hollenberg et al., 1996). 2-Furoyl-LIGRLO-NH₂ (0.01-10 µM) caused a concentration-dependent relaxation of the rat aorta preparation (Fig. 4A). As illustrated by Fig. 4A, 2-furoyl-LIGRLO-NH₂ was about 10 times more potent than SLIGRL-NH₂ (p < 0.05 compared with the pD₂ value for SLIGRL-NH₂; Student's t test for paired data). The pD₂ and E_max values of 2-furoyl-LIGRLO-NH₂ (n = 3) for relaxation of rat aorta were 6.5 ± 0.1 and 68 ± 4% relaxation, respectively. The pD₂ and E_max values of SLIGRL-NH₂ (n = 3) for relaxation of rat aorta were 5.6 ± 0.1 and 63 ± 5% relaxation, respectively. The reverse-sequence peptide 2-furoyl-OLRGIL-NH₂ (10 µM) did not relax the phenylephrine-contracted rat aorta.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Concentration-response curves for relaxation of rat aorta and murine femoral arteries by 2-furoyl-LIGRLO-NH2. A and B, rings of rat aorta (A) (n = 4) and murine femoral arteries (B) (n = 4) contracted by submaximal concentrations of phenylephrine and cirazoline, respectively, and tissues then exposed to increasing concentrations of either 2-furoyl-LIGRLO-NH2 or SLIGRL-NH2. 100% relaxation represents the complete reversal of the initial contractions by -adrenergic-agonists.

Vascular Reactivity of Murine Femoral Artery Preparations. To assess the relative PAR₂-dependent and PAR₂-independent biological activities of 2-furoyl-LIGRLO-NH₂, the effects of this peptide on vascular reactivity were measured in murine femoral arteries with intact endothelium, and these responses were compared with those triggered by other PAR₂-activating peptides under the same conditions. The PAR₂-dependent activity of 2-furoyl-LIGRLO-NH₂ in murine femoral arteries was compared with SLIGRL-NH₂,as measured by the relaxation of these cirazoline (0.1 µM)-contracted arteries. 2-Furoyl-LIGRLO-NH₂ (0.01-10 µM) caused a concentration-dependent relaxation of murine femoral arteries (Fig. 4B). The pD₂ and E_max values for 2-furoyl-LIGRLO-NH₂ (n = 4) were 7.9 ± 0.3 and 86 ± 6% relaxation, respectively. The pD₂ and E_max values for SLIGRL-NH₂ (n = 4) were 5.5 ± 0.1 and 74 ± 7% relaxation, respectively (Fig. 4B). The reverse-sequence peptide 2-furoyl-OLRGIL-NH₂ (0.1-10 µM; n = 4) did not relax cirazoline-contracted murine femoral arteries.

The PAR₂-independent activity of 2-furoyl-LIGRLO-NH₂ in murine femoral arteries with intact endothelium was compared with that of trans-cinnamoyl-LIGRLO-NH₂, as measured by the contraction of these arteries from baseline tension (Fig. 5) (McGuire et al., 2002a). At a concentration of 50 µM (i.e., 3000 times the pD₂ value for its relaxation activity), 2-furoyl-LIGRLO-NH₂ caused less than 15% of the contraction elicited by an equal concentration of trans-cinnamoyl-LIGRLONH₂. The contraction of arteries treated a second time with 2-furoyl-LIGRLO-NH₂ after a prior exposure to 50 µM 2-furoyl-LIGRLO-NH₂ followed by a tissue wash (homologous desensitization protocol) was not statistically different than the first exposure. However, the contractile responses of tissues treated with 50 µM 2-furoyl-LIGRLO-NH₂ after prior exposure to 50 µM trans-cinnamoyl-LIGRLO-NH₂ and washout (heterologous desensitization protocol) were abolished. The contractile responses of arteries treated with 50 µM trans-cinnamoyl-NH₂ after prior exposure to 50 µM 2-furoyl-LIGRLO-NH₂ and washout (heterologous desensitization protocol) were unaffected. The reverse-sequence peptide 2-furoyl-OLRGIL-NH₂ (50 µM; n = 4) did not contract murine femoral arteries at baseline tension.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Comparison of the PAR2-independent contraction of murine femoral arteries by trans-cinnamoyl-LIGRLO-NH2 to 2-furoyl-LIGRLO-NH2. Rings of murine femoral arteries with intact endothelium at baseline tension conditions were exposed to 50 µM trans-cinnamoyl-LIGRLO-NH2 or 50 µM 2-furoyl-LIGRLO-NH2, and then the contractile reposes were measured. Contractions are expressed as a percentage relative to a maximal contraction caused by 120 mM KCl. For the homologous desensitization protocol, rings of femoral arteries were exposed to 50 µM 2-furoyl-LIGRLO-NH2 after the washout of the prior addition of 50 µM 2-furoyl-LIGRLO-NH2. For heterologous desensitization protocols, 50 µM 2-furoyl-LIGRLO-NH2 or 50 µM trans-cinnamoyl-LIGRLO-NH2 were added after the washout of the prior addition (50 µM) of the other agonist. One-way analysis of variance: F(5,14) = 28.89, p < 0.0001. Neuman-Keuls post hoc test for multiple comparisons: **, p < 0.01 compared with trans-cinnamoyl-LIGRLO-NH2; a, p < 0.01 compared with 2-furoyl-LIGRLO-NH2; NS, p > 0.05, 2-furoyl-LIGRLO-NH2 homologous desensitization protocol compared with 2-furoyl-LIGRLO-NH2 and trans-cinnamoyl-LIGRLO-NH2 after heterologous desensitization protocol compared with trans-cinnamoyl-LIGRLO-NH2 (n = 3 for each group). Error bars represent the standard deviation. The average contraction of femoral arteries (n = 30) by 120 mM KCl was 5 ± 0.4 mN.

Electrophysiological Responses of Murine Small-Caliber Mesenteric Arteries to 2-Furoyl-LIGRLO-NH₂. To assess the electrophysiological activity of 2-furoyl-LIGRLO-NH₂, the membrane potential of murine mesenteric arteries with intact endothelium was measured either at baseline tension or during contraction with cirazoline and then during treatment with 2-furoyl-LIGRLO-NH₂ (0.01, 0.1, and 1 µM). 2-Furoyl-LIGRLO-NH₂ caused a concentration-dependent hyperpolarization of murine mesenteric arteries at baseline tension (Fig. 6A). When murine mesenteric arteries were contracted by cirazoline, 2-furoyl-LIGRLO-NH₂ (0.01, 0.1, 1 µM) caused simultaneous hyperpolarization and relaxation of these arteries (Fig. 6, B and C). The reverse-sequence peptide 2-furoyl-OLRGIL-NH₂ (0.01-1 µM; n = 3) caused neither hyperpolarization nor relaxation of murine mesenteric arteries.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Vascular smooth muscle cell hyperpolarization and relaxation by 2-furoyl-LIGRLO-NH2. Data summarize the change in membrane potentials of vascular smooth muscle cells in murine small-caliber mesenteric arteries at baseline tension (-57 ± 2 mV steady-state membrane potential) (A) or while contracted by cirazoline (-46 ± 1 mV steady-state membrane potential) after the exposure to different concentrations of 2-furoyl-LIGRLO-NH2 (B). The lower bar graph (C) summarizes the relaxation that was measured coincident with the hyperpolarization shown in panel B and is expressed as a percentage of the reversal of cirazoline-induced tone. During some experiments, the intracellular electrodes were pulled out of the cells prior to obtaining stable membrane recordings during relaxation responses of contracted arteries to 2-furoyl-LIGRLO-NH2; thus, the n values for B and C are different.

	Discussion

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Receptor Selectivity and Increased Potency of 2-Furoyl-LIGRLO-NH₂ for PAR₂. The main finding of our study was that the N-acylated peptide 2-furoyl-LIGRLO-NH₂ is a potent and selective activator of PAR₂ that, like the isoserine derivative of the PAR-activating peptide SFLLRN (Coller et al., 1993), would be expected to be resistant to aminopeptidases. Our new data considerably extend the limited pharmacological information published previously about the receptor selectivity of a comparable peptide, 2-furoyl-LIGKV-OH, used for studies in a murine arthritis model in vivo (Ferrell et al., 2003). Prompted by Ferrell et al. (2003), our newly designed PAR₂-activating peptide agonist 2-furoyl-LIGRLO-NH₂ was almost an order of magnitude more potent than SLIGRL-NH₂ for the activation of rat and human PAR₂, as measured by intracellular calcium signaling in cultured PAR₂-expressing cells; it was also almost an order of magnitude more potent than SLIGRL-NH₂ in causing an endothelium-dependent relaxation of rat aorta rings. In this regard, our novel N-acylated PAR₂ agonist, which would provide for the development of a PAR₂ receptor-binding probe (Al Ani et al., 1999), was also more potent than trans-cinnamoyl-LIGRLO-NH₂, which is equipotent with SLIGRL-NH₂ in such assays (Vergnolle et al., 1998; Al Ani et al., 1999). 2-Furoyl-LIGRLO-NH₂ was specific for the activation of PAR₂ compared with the activation of PAR₁ and PAR₄, which were unaffected. The homologous receptor cross-desensitization experiments using the calcium signaling assay with either PAR₂-expressing KNRK cells or HEK293 cells that constitutively express both PAR₁ and PAR₂ confirmed the receptor selectivity of 2-furoyl-LIGRLO-NH₂, as did its lack of activity in the PAR₄-triggered rat platelet aggregation assay. Interestingly, this N-acylated peptide was not an antagonist for PAR₁, as is the trans-cinnamoyl derivative of PAR₁-related peptide sequences (Bernatowicz et al., 1996). The much greater pD₂ value of 2-furoyl-LIGRLO-NH₂ for relaxation of the rat aorta preparation, compared with the pD₂ value of SLIGRL-NH₂, was comparable to the greater relative potency of 2-furoyl-LIGRLO-NH₂ relative to SLIGRL-NH₂ in the calcium signaling assay using PAR₂-expressing KNRK cells. Thus, the increased potency of the 2-furoyl derivative would seem not to depend on the PAR₂-bearing target cell or tissue on which it acts.

We hypothesize that the addition of the more sterically compact furoyl group to the amino terminus of the PAR₂-activating peptide sequence (LIGRLO-NH₂), compared with the bulky N-terminal trans-cinnamoyl group, provides an even more conformationally favorable motif for interacting with the receptor than does the serine of the native tethered ligand-activating peptide SLIGRL-NH₂ under the same conditions. Based on structure-activity data obtained by us and others for PAR₂-activating peptides (Hollenberg et al., 1996; Al Ani et al., 1999; Maryanoff et al., 2001), it was expected that our derivative with arginine in the penultimate C-terminal position and a C-terminal amide would be more potent than the previously described peptide with lysine at that position and without a C-terminal amide (Ferrell et al., 2003). Given our previous structure-activity data for PAR₂-activating peptides (Hollenberg et al., 1996; Al Ani et al., 1999), it is also possible that the additional ornithine residue at the C terminus would confer increased potency of our peptide over the noncarboxyamidated 2-furoyl derivative previously described (2-furoyl-LIGKV-OH) (Ferrell et al., 2003). This assumption would have to be confirmed experimentally in future work that would be facilitated by the synthesis of radiolabeled ligands and the discovery of a PAR₂ antagonist. Notwithstanding, our new derivative is 2 to 3 times more potent in the HEK293 cell calcium signaling assay (pD₂, 5.5) than the potency reported for the action of 2-furoyl-LIGKV-OH on human PAR₂-expressing NCTC 2544 cells (pD₂, 5.08) (Ferrell et al., 2003). A resistance to amino-peptidase activity in the assays done on cells in vitro cannot explain the comparatively high potency of the 2-furoyl derivative, since our previous work has shown that amino-peptidase inhibitors such as amastatin do not influence the potency of nonacylated peptide agonists in the assay (Kawabata et al., 1999). The likely resistance of the 2-furoyl derivative to amino-peptidase action makes it an attractive agonist to use for studies done in vivo, as demonstrated by the use of the agonist 2-furoyl-LIGKV-OH in studies of murine arthritis (Ferrell et al., 2003).

Relative PAR₂ versus Non-PAR₂-Activities of 2-Furoyl-LIGRLO-NH₂ in the Murine Vasculature. It has been demonstrated conclusively that PAR₂ activation is responsible for the SLIGRL-NH₂-induced relaxation of contracted murine femoral arteries (McGuire et al., 2002a), as is the SLIGRL-NH₂-induced blood pressure lowering activity of SLIGRL-NH₂ in mice measured in vivo (Damiano et al., 1999), because both responses are absent in PAR₂-deficient mice. Remarkably, 2-furoyl-LIGRLO-NH₂ was up to 300 times more potent than SLIGRL-NH₂ in the murine-isolated blood vessel assays, wherein the responses are PAR₂-dependent. In terms of its relaxant action, the peptide trans-cinnamoyl-LIGRLO-NH₂ has been observed to be equipotent with SLIGRL-NH₂ (Vergnolle et al., 1998; Al Ani et al., 1999; McGuire et al., 2002a). In contrast with SLIGRL-NH₂, trans-cinnamoyl-LIGRLO-NH₂ causes a PAR₂-independent contraction of murine femoral arteries from baseline tension at concentrations that are about 50 times greater than its pD₂ value for relaxation (McGuire et al., 2002a). In comparison with the trans-cinnamoyl derivative, 2-furoyl-LIGRLO-NH₂ caused only a minor contraction of the murine femoral arteries (about 15% of the trans-cinnamoyl-LIGRLO-NH₂-induced response) at a concentration that was 3000 times greater than the pD₂ value for its PAR₂-mediated response (relaxation). Therefore, its combined increased receptor potency and very low predicted non-PAR₂-dependent activity enhanced the useful range of concentrations for 2-furoyl-LIGRLO-NH₂ for activating PAR₂, at least as assessed in mouse-based assays. This property of our new agonist is important because of the number of transgenic mouse models that have been developed to predict human diseases in which it will be of considerable interest to determine the consequence of PAR₂ activation via the use of PAR₂-activating peptides. Such studies would preclude the use of PAR₂ agonists that lack the potency and selectivity of our newly designed PAR₂ agonist.

2-Furoyl-LIGLRO-NH₂ had the same actions qualitatively as SLIGRL-NH₂ in the murine small-caliber mesenteric arteries and the same increase in relative potency (>100 times) compared with SLIGRL-NH₂ [pD₂ value of 6 in murine mesenteric arteries (McGuire et al., 2002b] as had been found in the murine femoral arteries. Interestingly, the potency of 2-furoyl-LIGRLO-NH₂ relative to SLIGRL-NH₂ in the murine bioassays was even greater than the potency found for the 2-furoyl derivative relative to SLIGRL-NH₂ in the rat and human PAR₂-mediated calcium signaling and rat aorta relaxation assays. The reason for these differences between assays may possibly relate to subtle species differences between the interactions of the 2-furoyl derivative with the distinct mouse, rat, and human receptor sequences. Alternatively, differences in PAR₂ coupling to signal transduction and calcium regulation in native murine endothelial cells versus the coupling to calcium signaling in PAR₂-expressing cell lines may account for differences in the relative potencies of 2-furoyl-LIGRLO-NH₂ to SLIGRL-NH₂ in the different assay preparations. The premise that the differences in relative potencies are due to differences in the sequences of PAR₂ between species is supported by the apparent difference in potency of 2-furoyl-LIGRLO-NH₂ relative to SLIGRLNH₂ for calcium signaling in the rat compared with the human receptor expressed in cultured cells. Furthermore, the PAR₂-dependent activation of mouse endothelial calcium-activated K⁺ channels that elicit the hyperpolarization response in mesenteric arteries (McGuire et al., 2002b; McGuire and Triggle, 2003) and endothelial NO synthase activation in femoral arteries (McGuire et al., 2002b) would also be expected to be more indicative of subcellularly localized changes in calcium than are the gross global changes to intracellular calcium arising from PAR₂ activation in transformed cells.

Conclusion. The peptide 2-furoyl-LIGRLO-NH₂ greatly surpasses the potency and receptor selectivity in vitro of previously described PAR₂-activating peptides. Given the markedly increased activity of this peptide for PAR₂-dependent activities and the enhanced selectivity for activating PAR₂ compared with the triggering of PAR₂-independent effects in more complex biological assays, this compound should prove to be very useful for studies done in intact animals that are aimed at elucidating the potential patho-physiological responses due to PAR₂ when activated in vivo by locally generated proteinases.

	Footnotes

 
These studies were supported by grants from the Canadian Institutes of Health Research (CIHR) (C.R.T., M.D.H.), the Heart and Stroke Foundation of Alberta, Northwest Territories and Nunavut (C.R.T., M.D.H.), the Johnson and Johnson Focused Giving Program (M.D.H.), and a CIHR-Servier Canada/International University Industry Partnership (M.D.H.). J.J.M. was supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada in conjunction with CIHR and AstraZeneca. Measurements of intracellular calcium were made possible by an equipment grant from the Alberta Heritage Foundation for Medical Research.

DOI: 10.1124/jpet.103.064584.

ABBREVIATIONS: PAR, proteinase-activated receptor; KNRK, Kirsten virus-transformed rat kidney; HEK, human embryonic kidney.

Address correspondence to: Dr. John J. McGuire, Cardiovascular Research Group, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B 3V6. E-mail: mcguire{at}mun.ca

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