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CELLULAR AND MOLECULAR

Derivatized 2-Furoyl-LIGRLO-amide, a Versatile and Selective Probe for Proteinase-Activated Receptor 2: Binding and Visualization

Inflammation Research Network, Department of Pharmacology & Therapeutics and Department of Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada (M.D.H., B.R., E.H., S.H., N.V., M.S., R.R.); INSERM U563, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France (N.V.); and Université Toulouse III Paul Sabatier, Toulouse, France (N.V.)

Received for publication January 16, 2008
Accepted May 12, 2008.

	Abstract

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The proteinase-activated receptor-2 (PAR₂)-activating peptide with an N-terminal furoyl group modification, 2-furoyl-LIGRLO-NH₂ (2fLI), was derivatized via its free ornithine amino group to yield [³H]propionyl-2fLI and Alexa Fluor 594-2fLI that were used as receptor probes for ligand binding assays and receptor visualization both for cultured cells in vitro and for colonic epithelial cells in vivo. The binding of the radiolabeled and fluorescent PAR₂ probes was shown to be present in PAR₂-transfected Kirsten normal rat kidney cells, but not in vector-alone-transfected cells, and was abolished by pretreatment of cells with saturating concentrations of receptor-selective PAR₂ peptide agonists such as SLIGRL-NH₂ and the parent agonist 2fLI but not by reverse-sequence peptides such as 2-furoyl-OLRGIL-NH₂ that cannot activate PAR₂. The relative orders of potencies for a series of PAR₂ peptide agonists to compete for the binding of [³H]propionyl-2fLI (2fLI >> SLIGRL-NH₂ trans-cinnamoyl-LIGRLO-NH₂ > SLIGKV-NH₂ > SLIGKT-NH₂) mirrored qualitatively their relative potencies for PAR₂-mediated calcium signaling in the same cells or for vasorelaxation in a rat aorta vascular assay. In the vascular assay, the potency of Alexa Fluor 594-2fLI was the same as 2fLI. We conclude that ornithine-derivatized 2fLI peptides are conveniently synthesized PAR₂ probes that will be of value for future studies of receptor binding and visualization.

Despite the expanding interest in the pathophysiology of PAR₂, it has not been possible to assess its abundance and location in live cells because of the lack of reliable and conveniently synthesized radiolabeled and fluorescent receptor binding probes. In our previous work, we developed a [³H]propionyl derivative of the receptor-selective PAR₂-activating peptide, trans-cinnamoyl (tc)-LIGRLO-NH₂ (Al-Ani et al., 1999), that was adequate for a ligand binding assay but not suitable as a fluorescently labeled probe for receptor visualization because of its relatively low receptor affinity. Furthermore, our work revealed that, unlike SLIGRL-NH₂, the trans-cinnamoyl peptide was able to activate receptors in addition to PAR₂ and was therefore not optimal as a receptor probe. Therefore, we developed and evaluated in depth the receptor selectivity of a higher potency PAR₂ agonist, 2-furoyl-LIGRLO-NH₂ (2fLI), that like the trans-cinnamoyl derivative was designed to have a C-terminal ornithine that can be readily derivatized via its side-chain amino group (McGuire et al., 2004). Comparable compounds lacking the C-terminal ornithine (2-furoyl-LIGKV-NH₂ and 2-furoyl-LIGRL-NH₂) were also studied by others (Ferrell et al., 2003; Kawabata et al., 2004). From previous work by us and by others evaluating the structure-activity relationships for PAR₁- and PAR₂-activating peptides (Bernatowicz et al., 1996; Hollenberg et al., 1997; Maryanoff et al., 2001), we knew that only the first five amino acids of the PAR-activating peptides are critical for receptor activation and that C-terminal substitutions are readily introduced into peptides that retain full PAR₂-activating activity. Given that result, we hypothesized that the 2fLI peptide derivatized on the side chain amino group of its ornithine would be a good receptor probe for both ligand binding and receptor visualization studies. Therefore, we synthesized ornithine-substituted [³H]propionyl-2fLI and Alexa Fluor 594-2fLI and tested their suitability as PAR₂ probes. The equivalence of the biological activity of Alexa Fluor 594-2fLI with that of 2fLI was tested in a vascular endothelium-dependent PAR₂-activated relaxation bioassay. These derivatives were then used 1) for a cultured cell binding assay and 2) for visualizing PAR₂ by fluorescence microscopy using cultured KNRK cells that were transfected or not with rat PAR₂ (Al-Ani et al., 1999) and by fluorescence microscopy of fixed colon tissue that had been exposed in vivo to Alexa Fluor 594-2fLI.

	Materials and Methods

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Peptides and Other Reagents
All peptides were synthesized as carboxy amides (>95% purity, assessed by HPLC and mass spectrometry) by Dr. Denis McMaster (Peptide Core Facility at University of Calgary, Calgary, AB, Canada). Unless indicated otherwise, all remaining chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Peptides were dissolved in buffer, pH 7.4, containing 25 mM HEPES. [³H]Propionic anhydride was from Moravek Biochemicals (Brea, CA; 110 Ci/mmol); Alexa Fluor 594 was from Invitrogen (Carlsbad, CA); and dinonyl and dibutyl phthalate were from -Fluka (St. Louis, MO).

KNRK Cells
Kirsten virus-transformed normal rat kidney cells (American Type Culture Collection, Manassas, VA) were transfected with either vector alone or with a pcDNA3 vector containing rat PAR₂ to yield vector-alone or PAR₂-expressing KNRK cell lines that have been described previously and used by us for ligand binding and calcium signaling assays (Al-Ani et al., 1999, 2004).

Cell Growth and Harvesting for Binding and Visualization Studies
Cells were routinely grown at 37°C in 75-cm² plastic T-flasks (NUNC; VWR Scientific, Mississauga, ON, Canada) using 10 ml of Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% bovine fetal calf serum under an atmosphere of 5% CO₂ in air. Cells to be used for the binding assays were grown to approximately 80% confluence and were passaged without the use of trypsin by resuspension in calcium-free isotonic saline EDTA-containing cell dissociation buffer (Invitrogen). For routine binding studies, cells harvested from two to three T-flasks were lifted from the flasks as above, pooled, and resuspended (approximately 10⁷ cells/ml; cell counts by hemacytometer) in binding buffer for the assay (isotonic, 115 mM NaCl buffered at pH 7.4 with 25 mM HEPES and supplemented with 0.1% w/v bovine serum albumin). Cells to be used for fluorescence microscopy were similarly resuspended from the T-flasks, reseeded in Multiwell glass bottom Petri dishes (MatTek Corporation, Ashland, MI), and grown to approximately 60% confluence before use.

Synthesis of [³H]Propionyl- and Alexa Fluor-Derivatized 2-Furoyl-LIGRLO-NH₂
Because the only free amino group available for derivatization of the PAR₂-activating peptide is on the ornithine residue, it was possible to use a simple acylation procedure with either ³H-labeled propionic anhydride (N-succinimidyl propionate-2,3-³H, 110 C_i/mmol; Moravek Biochemicals) or Alexa Fluor 594-succinimide ester (Invitrogen). To prepare the radiolabeled ligand binding and fluorescence microscopy probes, 2-furoyl-LIGRL[N(³H)propionyl]-O-NH₂ and 2-furoyl-LIGRL[N-(Alexa Fluor 594)-O]-NH₂ (Alexa Fluor 594-2fLI), the parent peptide (2-furoyl-LIGRLO-NH₂, 600 nmol in 0.5 ml total volume of sodium borate buffer, pH 8.5), was reacted with acylating reagent (1.2 µmol Alexa Fluor 594; 1 nmol [³H]propionate) for 1 h at room temperature. The reaction scheme for the synthesis of Alexa Fluor 594-2fLI was as shown in Scheme 1. The acylation reactions were terminated by the addition of HPLC solvent (0.1 ml, 0.1% v/v trifluoroacetic acid in water), and the reaction mixture (200-µl aliquots) was immediately subjected to fractionation by HPLC to isolate the derivatized peptides. The [³H]propionyl 2fLI derivative was readily separated from unreacted peptide by HPLC (elution with a 15–40% v/v acetonitrile gradient in 0.1% v/v trifluoroacetic acid over 30 min). The specific activity of radiolabeled [³H]propionyl-2-fLI was approximately 110 Ci/mmol. The Alexa Fluor 594-derivatized peptide was eluted as two isomeric peaks (pooled for use) that were cleanly separated from unreacted peptide, which represented only 10% of the initial peptide used in the reaction (relative HPLC peak heights). The reverse-PAR₂ sequence fluorescent probe, 2-furoyl-(N-Alexa Fluor 594)OLRGIL-NH₂ (Alexa Fluor 594-2fOL), which would not be able to interact with the receptor, was prepared by the same procedure as for Alexa Fluor 594-2-fLI. The purity of the HPLC-isolated Alexa Fluor 594-2fLI and its reverse sequence peptide were verified by mass spectrometry and amino acid analysis.

View larger version (14K): [in this window] [in a new window]   Scheme 1. Synthesis of Alexa Fluor 594-2fLI.

Monitoring Ligand Binding and Binding-Competition by Fluorescence Microscopy
Measurements with Cultured Cells. To visualize PAR₂ by fluorescence microscopy in a cultured cell system, KNRK cells transfected or not with rat PAR₂ were grown to approximately 60% confluence as monolayers on Multiwell glass bottom 35-mm diameter Petri dishes (P35G-0-14-C; MatTek Corporation). In keeping with the binding study, cells were first washed free of growth medium and incubated for 2 min on ice or at room temperature, with or without an excess of nonfluorescent binding probe (20–50 µM in a total volume of 2 ml), at which point Alexa Fluor 594-2fLI (50–500 nM) was then added. The binding reaction was allowed to proceed for 60 min either on ice or at room temperature, and unbound fluorescence was removed by a rapid wash (2–3 x 1 ml) followed by visualization of bound Alexa Fluor 594-2fLI.

Bound fluorescence was visualized with an Olympus IX70 inverted microscope with a 10x (tissue) or 20x (KNRK cells) objective coupled to a CCD camera (Qimaging, Surrey, BC, Canada) and Volocity image capture software (Improvision, Waltham, MA). Fluorescence was activated at a wavelength of 555 nm, with emission recorded at 617 nm. Fluorescence observed in the absence of unlabeled PAR₂ competitor was compared with the fluorescence in the presence of competitor. Measurements were done with five or more independently grown cell monolayers. The amount of fluorescent probe bound was documented both by observation of individual cells and by morphometric analysis of the visualized cells. The relative fluorescence observed in the absence or presence of increasing amounts of unlabeled PAR₂ peptide competitor was quantified by integrating the total fluorescence signal yield (NIH ImageJ software) from randomly selected microscopic fields (each approximately 100 x 100 µm area) containing up to a total of 100 or more cells. The binding-competition curves obtained from these fluorographic morphometric measurements were constructed by plotting the ratio of fluorescence observed in the presence of increasing amounts of nonfluorescent PAR₂ peptide competitor relative to the signal observed in the absence of competitor.

Visualizing PAR₂ in Colonic Tissues in Vivo. Although the vascular assays were done with rat-derived tissues to match data obtained with the rat PAR₂-expressing KNRK cells, a murine in vivo model, rather than the rat, was employed to minimize the amount of reagents required for the experiments. Male C57Bl6 mice (6–8 weeks) were purchased from Charles River Laboratories (Montreal, QC, Canada). All procedures were approved by Institutional Animal Care Committee (University of Calgary). The Alexa Fluor 594-2fLI probe (10 µg in 0.1 ml of 10% v/v ethanol/10% v/v Tween 80 in 0.9% NaCl) and its reverse peptide sequence fluorescent analog were administered intracolonically through a catheter inserted at a distance of approximately 4 to 5 cm from the anus to three groups of C57Bl6 male mice, as described previously (Nguyen et al., 2003). The receptor probe was in solution either with or without a 10-fold excess (100 µg) of nonlabeled 2-fLI. One hour after the intracolonic administration of the fluorescent receptor probe or its reverse-sequence fluorescent control peptide, the animals were lightly anesthetized with sodium pentobarbital (approximately 100 mg/kg i.p.) and then immediately perfused via the heart with 5 ml of saline, followed by 5 ml of 10% formalin for fixation of tissues in vivo. Upon fixation by cardiac perfusion, animals were sacrificed, and colonic tissue at the site of peptide administration was harvested (within 5 min) and fixed further overnight in 10% formalin. Fixed tissues were prepared for microscopic analysis by directly embedding in paraffin or were placed in 20% w/v sucrose for another 24 h at 4°C before embedding in ornithine carbamyl transferase compound (OCT; Miles, Elkhart, IN). OCT-embedded tissues were then cryosectioned at 10 µm, washed in isotonic phosphate-buffered saline and mounted in Prolong mounting medium (Invitrogen) for microscopic analysis. Paraffin-embedded tissues were sectioned at 5 µm, deparaffinized by two 5-min washes in xylene, and were mounted directly with Cytoseal xylene based mounting media (Richard-Allan Scientific, Kalamazoo, MI) to observe the fluorescence signal or were stained with hematoxylin and eosin as per standard protocols on a Fisher Histomatic Slide Stainer (Thermo Fisher Scientific, Waltham, MA) before mounting with Cytoseal. CY-3 fluorescence in the sections was monitored as outlined above with an excitation wavelength of 555 nm and an emission wavelength recorded at 617 nm. For morphometric analysis of the fluorescence present at specific tissue locations (e.g., over the luminal epithelial layer), the fluorescence yield (arbitrary units) was measured over equal tissue areas, and the integrated values were compared. The background fluorescence yield for the biologically inactive Alexa Fluor 594-2fOL was considered as "nonspecific" labeling. The value for the Alexa Fluor 594-2fOL fluorescence was subtracted from the fluorescence observed for 594-2fLI (in the absence or presence of competing nonfluorescent 2fLI) to calculate the "specific" receptor-related fluorescence.

Rat Aorta Relaxation Assay
The endothelium-dependent rat aorta relaxation assay that reflects the activation of endothelial PAR₂ was done using tissue derived from male Sprague-Dawley albino rats (200–250 g) essentially as described previously (Al-Ani et al., 1995; Hollenberg et al., 1997; McGuire et al., 2004). In brief, segments of aorta rings were isolated from animals that had been killed by decapitation and were mounted in an organ bath (4-ml plastic cuvette). 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 mM CaCl₂ 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 4-ml plastic cuvettes containing the standard gassed physiological solution. Tension was measured using Statham or Grass force transducers. Tissues were contracted by exposure to phenylephrine (1 µM), and the relaxant response to PAR₂-activating peptides was measured as a percentage (%Ach) of the relaxation caused in the same tissue by 10 µM acetylcholine.

Calcium Signaling Measurements
The calcium signaling assay that monitors PAR₂ activation in PAR₂-transfected KNRK cells was done essentially as described previously for cultured HEK and PAR-expressing KNRK cells. (Al-Ani et al., 1999; Kawabata et al., 1999; Compton et al., 2000). Cells harvested in an isotonic EDTA-containing dissociation medium without the use of trypsin were incubated in a solution of serum-free Dulbecco's modified minimal essential medium that contained 0.25 mM sulfinpyrazone and 22 µM Fluo-3 acetoxymethyl ester (Invitrogen) for 25 min at room temperature. Cells were then resuspended in a buffered solution, pH 7.4, that contained the 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 using an Aminco Bowman Series 2 Luminescence spectrometer (Thermo Spectronic, Madison, WI). Cell suspensions (2 ml) in 4-ml cuvettes were mixed continuously with a magnetic stirrer and maintained at 25°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] ).

	Results

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Optimization and Validation of the Binding Assay. Preliminary work (not shown) established that, at room temperature, binding equilibrium was reached at approximately 1 h and that equilibration took approximately 2 h at 4°C. Binding was linear with respect to cell number in the range of 10⁵ to 10⁸ cells/ml. Thus, the routine binding-competition experiment used approximately 10⁷ cells/ml with an equilibration time of 1 to 1.5 h at room temperature and 2 to 2.5 h at 4°C for fluorescent ligand binding and for routine binding-competition studies with radiolabeled 2fLI. The binding curve for increasing concentrations of [³H]propionyl-2fLI was done at 4°C, with an equilibration time of 2 h. Table 1 shows, for a representative experiment, the binding of radiolabeled [³H]propionyl-2fLI in the absence and presence of an excess (100–200 µM) of either unlabeled SLIGRL-NH₂ or unlabeled 2-furoyl-LIGRLO-NH₂. Although these two PAR₂-selective agonists were able to compete for the binding of radiolabeled probe, the reverse-sequence-peptide that could not activate PAR₂ (2-furoyl-OLRGIL-NH₂) (McGuire et al., 2004) did not compete for binding. Furthermore, the binding of radioligand by cells that had been transfected with vector alone (KNRK-pcDNA3, 1770 ± 80 CPM) (Table 1, fourth line, experiment 1) was comparable to and even lower than the nonspecific binding level detected in the rat PAR₂-expressing KNRK cells incubated with radiolabeled ligand probe in the presence of an excess of unlabeled PAR₂-activating peptide (KNRK-PAR₂ + SLIGRL-NH₂, 2760 ± 90 cpm) (Table 1, second line, experiment 1). Thus, the binding proved to be both peptide- and receptor-specific, and the degree of nonspecific (presumably nonreceptor) binding was of a sufficiently low magnitude to enable further studies. Binding competition studies done at either room temperature or at 4°C yielded comparable results; therefore, routine binding competition experiments were conducted at room temperature to optimize the convenience of the assay.

Binding-Competition Curves for Unlabeled Ligands and Binding Curve for Radiolabeled Binding Probe. The ability of several PAR₂-activating peptides to compete for the binding of radiolabeled 2fLI is illustrated in Fig. 1, in which the relative IC₅₀s for binding competition (Table 2) revealed a relative affinity order of 2-furoyl-LIGRLO-NH₂ >> SLIGRL-NH₂ trans-cinnamoyl-LIGRLO-NH₂ > SLIGKV-NH₂ > SLIGKT-NH₂ TFLLR-NH₂; LSIGRL-NH₂ and 2-furoyl-OLRGIL-NH₂ were not active. Quantitatively, the relative affinities of these peptides (relative IC_50s for radioligand binding: R_rIC50), normalized to the binding of the most widely used PAR₂-activating peptide, SLIGRL-NH₂, set equal to 1.0 were 0.03:1.0:1.0:5.7:8.6 (Table 2). A value for the R_rIC50 greater than 1.0 denotes a peptide with an affinity lower than that of SLIGRL-NH₂. Qualitatively, this order of binding affinities and the lack of binding competition by the two scrambled peptide analogs are exactly in keeping with the relative potencies of the PAR₂-APs and the lack of activity of the control peptides in the PAR₂ calcium signaling assays (Al-Ani et al., 1999; Kawabata et al., 1999) and vascular assays (see below and McGuire et al., 2004) that we had previously used to evaluate the same peptides.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Binding competition curves for PAR-activating peptides. Triplicate samples of PAR2-expressing KNRK cells were incubated with 3H-labeled 2fLI either in the absence or presence of increasing concentrations of unlabeled peptides, as described under Materials and Methods, and the "net" receptor binding of radioligand was calculated by subtracting, from the total radioactivity bound, the nonspecific binding measured in the presence of an excess (100–200 µM) of unlabeled SLIGRL-NH2 or 2-furoyl-LIGRLO-NH2. The binding was expressed as a percentage (% Max) of the maximal net binding observed in an individual experiment. The data points in each curve were obtained from a minimum of three independent experiments, wherein each data point was measured in triplicate. For clarity, symbols are shown without the S.E.M. (e.g., see Fig. 2) calculated for each point: , 2-furoyl-LIGRLO-NH2; , SLIGRL-NH2; , tc-LIGRLO-NH2; , SLIGKV-NH2; , SLIGKT-NH2; , 2-furoyl-OLRGIL-NH2; , TFLLR-NH2; and , LRGILS-NH2.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Vascular response to PAR-activating peptides and Alexa Fluor 594-2fLI. The relaxant response (relative to that caused by 10 µM acetylcholine: %Ach) of an endothelium-intact rat aorta preparation was measured as outlined under Materials and Methods for increasing concentrations of Alexa Fluor 594-2-furoyl-LIGRLO-NH2 and of the PAR2-activating peptides, as indicated by the inset legend.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Competition for Alexa Fluor 594-fLIGRLO-NH2 binding by PAR2-APs. The competition by increasing concentrations of nonfluorescent PAR-APs for the binding of fluorescently labeled Alexa Fluor 594-2-furoyl-LIGRLO-NH2 (Fluo-fLI) was measured as outlined under Materials and Methods. The binding was expressed as a percentage (%Max) of the maximal fluorescence pixels monitored, corrected for background fluorescence observed in the presence of an excess of nonfluorescent 2-furoyl-LIGRLO-NH2.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Binding of increasing concentrations of [3H]propionyl-2fLI to PAR2 expressed in KNRK cells. PAR2-expressing KNRK cells were incubated with increasing concentrations of radiolabeled 2-furoyl-LIGRLO-NH2 in the absence or presence of an excess (100–200 µM) of unlabeled peptide, and the specific binding of radioligand was measured at each concentration, as outlined under Materials and Methods. Both the "net" receptor binding () and the nonspecific binding () are shown. The S.E.M. shown for the points at the highest concentration of [3H]propionyl-2fLI are representative of the variability seen in all binding experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Calcium signaling by 2fLI in HEK cells: lack of antagonism by 2fOL. The increase in intracellular calcium in Fluo-3-loaded PAR2-expressing KNRK cells was monitored in response to 10 µM 2-furoyl-LIGRLO-NH2 either after (left-hand trace) or before (right-hand trace) exposing the cells to an excess (200 µM) of the reverse-sequence peptide, 2-furoyl-OLRGIL-NH2. The scale for time and fluorescence signal E530 is shown by the inset.

Visualization of PAR₂ in Receptor-Transfected Cells: Receptor Specificity. As shown in Fig. 5, A and C, incubation of cell monolayers with Alexa Fluor 594-2fLI (50–500 nM) on ice permitted a visualization of the receptor with the same membrane-localized distribution (Fig. 5A, white arrowheads) that we have seen previously in fixed cells by an immunohistochemical approach using our B5 anti-PAR₂ antiserum (Al-Ani et al., 1999). It is interesting that when the same experiment was done at room temperature, the fluorescently labeled peptide was observed to internalize into an apparently lysosomal and perinuclear location (Fig. 5B, white arrowheads; data not shown). These results indicated that receptor internalization could be visualized with the Alexa Fluor 594-2fLI probe. However, given that the primary aim of our study was to validate the fluorescent PAR-activating peptide as a ligand probe for PAR₂ and not to evaluate receptor dynamics, the precise location of the internalized receptor was not studied in detail. The receptor was visualized only in PAR₂-expressing cells and was not labeled either in cells that had been transfected with vector alone (Fig. 5D) or in monolayers that had been pretreated with an excess concentration (20 µM) of the PAR₂-selective peptide agonist SLIGRL-NH₂ (Fig. 5F) or 2fLI (data not shown). It is significant that the partial reverse sequence PAR₂ peptide, LSIGRL-NH₂ (20 µM), that cannot activate PAR₂ failed to reduce the binding of Alexa Fluor 594-2fLI (Fig. 5E). Likewise, the reverse sequence PAR₂-inactive peptide, 2-furoyl-OLRGIL-NH₂, did not compete for the binding of the fluorescently labeled probe (Fig. 6).

View larger version (75K): [in this window] [in a new window]   Fig. 5. Visualizing PAR2 in KNRK cells with Alexa Fluor 594-2fLI. KNRK cells either transfected with the pcDNA3 vector (D) or expressing PAR2 (A–C, E, and F) were pretreated or not with an excess of unlabeled peptide (F) or its reverse-sequence PAR-inactive analog (E) and then incubated with Alexa Fluor 594-2-furoyl-LIGRLO-NH2 as outlined under Materials and Methods at either room temperature (B) or on ice (A, C, D–F) for 1 h. After washing monolayers free from the incubation medium, CY-3 fluorescence representing bound Alexa Fluor 594-2-furoyl-LIGRLO-NH2 was visualized as outlined under Materials and Methods. The top two panels compare binding observed simultaneously either on ice (A) or at room temperature (B). C to F show data from a single experiment done with cells cooled on ice.

Binding-Competition Curves for Unlabeled Ligands Measured Using Alexa Fluor 594-2fLI Fluorescence. To establish further the receptor selectivity of the Alexa Fluor 594-2fLI, we evaluated the relative potencies of the parent peptide, 2fLI and SLIGRL-NH₂, for competing with the binding of the Alexa Fluor 594 derivative using a fluorescence readout. Binding competition was quantified by measuring the reduction in fluorescence caused by increasing concentrations of nonfluorescent peptides, as outlined under Materials and Methods. As shown by the binding-competition curves in Fig. 6 and the summarized data in Table 2, the relative IC₅₀s for the ability of the unlabeled peptides to reduce the fluorescence signal qualitatively reflected accurately 1) their relative ability to compete (or not) for the binding of [³H]2fLI and 2) their relative biological activities in the vascular relaxation assay (Fig. 4). In KNRK cells that were transfected with empty vector, the very low fluorescence signal that could be observed was not competed for by the nonfluorescent peptide (Fig. 6, open circles at top) and was thus considered to represent a low level of nonspecific binding. Thus, the receptor specificity of binding of Alexa Fluor 594-2fLI was established unequivocally with the use of the PAR₂-nonexpressing cells and by the binding-competition data (competition with 2fLI and SLIGRL-NH₂ but not by LSIGRL-NH₂ or 2-furoyl-OLRGIL-NH₂).

Visualizing PAR₂ in Vivo. Having validated the Alexa Fluor 594-2fLI receptor probe in the cultured KNRK cells expressing PAR₂, we next turned to its use to visualize the receptor in vivo. One hour after its intracolonic administration (a time in keeping with the beginning of the acute inflammatory response of the colon to a PAR₂-activating peptide) (Cenac et al., 2002), the fluorescence signal yielded by the colonic administration of Alexa Fluor 594-2fLI was found primarily in epithelial cells at the villus tips (Fig. 7, top right panel, Alexa-2fLI, white arrowheads). The fluorescent derivative of the reverse peptide sequence (Alexa Fluor 594-2fOL) in general yielded only a minimal signal (Fig. 7, right middle panel, Alexa-2fOL). However, in the paraffin-embedded specimen, a fluorescence signal was observed for the reverse-sequence peptide (2fOL) in the muscular layer. This signal was not observed in the OCT-processed sections (data not shown) and was presumed to represent nonspecific uptake of the fluorescent probe. The fluorescence signal of Alexa Fluor 594-2fLI at the villus tips was clearly diminished when the fluorescent receptor probe was administered along with a 10-fold molar excess of nonfluorescent 2fLI (Fig. 7, bottom right panel, Competition). Morphometric analysis of the fluorescence signal in the area of the epithelial cells (as done for the Alexa Fluor 594-labeled KNRK cells) showed a reduction of approximately 60% in the signal when the 10-fold excess of unlabeled peptide was added along with the fluorescent probe (Fig. 7, histograms, bottom panel, Competition). Because of the marked inflammatory effects of 2fLI and its derivatives in the mouse colon at 1 h (data not shown), we were not able to use a larger excess (e.g., 100-fold) of the nonfluorescent 2fLI. This reduction in fluorescence in the presence of unlabeled 2fLI shown for a representative experiment in Fig. 7 (Fig. 7, histogram on right) ranged from approximately a 40 to 80% competition by the unlabeled peptide (data not shown). Only a very low fluorescence signal in the epithelial cell area was observed for the reverse-sequence receptor-inactive probe, Alexa Fluor 594-2fOL (Fig. 7, middle histogram, bottom panel). The tissues visualized by the hematoxylin-eosin staining procedure (Fig. 7, left panels) show the location of the fluorescence in relation to the intestinal lumen and in relation to the different intestinal cells.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Visualizing PAR2 in mouse colon tissue in vivo. Top and middle panels, Alexa Fluor 594-2-furoyl-LIGRLO-NH2 (Alexa-2fLI, top) or the Alexa Fluor 594-substituted PAR-inactive reverse sequence peptide, Alexa Fluor 594-2-furoyl OLRGIL-NH2 (Alexa-2fOL, middle), was administered intracolonically either with (bottom panels, Competition) or without (top two panels) an excess of nonfluorescent peptide. After 1 h, the animals were killed, and tissue was harvested for fixation, paraffin embedding, and fluorescence microscopy as outlined under Materials and Methods. The hematoxylin and eosin-stained sections of each tissue are shown on the left, for which the fluorescence photographs are shown on the right. The location of the lumen is visible, as is the scale for 50 µm (solid line, 50 µm). The arrowheads denote the areas of PAR2-associated fluorescence similar to those used for the morphometric analysis of binding competition by nonfluorescent peptide (histograms). Bottom histograms, the fluorescence signal observed over equal area regions of the villus tip (e.g., arrowheads) was quantified by morphometric analysis for the three panels on the right, as described under Materials and Methods. The integrated fluorescence in arbitrary units is shown in the histograms.

	Discussion

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Receptor Selectivity and Utility of Derivatized 2-Furoyl-LIGRLO-NH₂ for Monitoring PAR₂. The goal of our study was to validate the derivatized PAR₂ ligand, 2-furoyl-LIGRLO-NH₂, as a receptor probe. The main finding of our study was that the N-acylated, ornithine-substituted derivatives of this PAR₂ agonist are potent and selective ligand probes for monitoring the receptor by ligand binding and fluorescence microscopy approaches. Both as a ligand for binding studies and as a fluorescent receptor probe, the ornithine-substituted analogs of 2fLI displayed the ligand specificity expected for PAR₂: 1) in terms of the competition for binding of the ornithine-substituted derivatives by recognized PAR₂-activating peptides, with an order of affinities (IC₅₀s) of 2-furoyl-LIGRLO-NH₂ >> SLIGRL-NH₂ trans-cinnamoyl-LIGRLO-NH₂ > SLIGKV-NH₂ > SLIGKT-NH₂ TFLLR-NH₂ that was the same as that for the EC₅₀s for activation of PAR₂ in either a calcium signaling or aorta relaxation assay; 2) the lack of binding of the derivatized 2-fLI analogs to KNRK cells that do not express the receptor, in comparison with receptor-expressing cells; 3) the lack of binding competition by the PAR-inactive scrambled sequences, LSIGRL-NH₂ and 2-furoyl-OLRGIL-NH₂; 4) a lack of fluorescence labeling of PAR₂-expressing cells or tissues exposed to the reverse-sequence biologically inactive Alexa Fluor 594-2fOL; and 5) the reduced fluorescence signal from the cultured PAR₂-expressing KNRK cells or the in vivo colon tissues exposed to the active fluorescent receptor probe combined with an excess of nonfluorescent 2-fLI that would compete for receptor binding. It is noteworthy that the Alexa Fluor 594-2fLI derivative demonstrated a biological potency in the aorta relaxation assay that was the same as that of unsubstituted 2fLI and that the derivative was able to compete effectively for the binding of radiolabeled 2fLI to the PAR₂-expressing KNRK cells. These data demonstrated that the ornithine side chain can be substituted with a relatively large substituent without affecting the biological activity or receptor binding activity of the peptide sequence. Furthermore, qualitatively, the relative potencies (IC₅₀s) of 2fLI and SLIGRL-NH₂ for reducing the binding of the fluorescent receptor probe were exactly in agreement with their relative potencies in the vascular and [³H]2fLI binding assays. Thus, we anticipate that this fluorescent PAR₂ probe will prove to be of value for visualizing the receptor in intact tissues in a variety of settings and to follow the dynamics and locations of receptor internalization.

It can be pointed out that, quantitatively, the relative IC_50s of the peptide agonists in the radioligand binding assay, normalized to the IC₅₀ of SLIGRL-NH₂ = 1.0 (2-furoyl-LIGRLO-NH₂: SLIGRL-NH₂:trans-cinnamoyl-LIGRLO-NH₂:SLIGKV-NH₂: SLIGKT-NH₂:: 0.03:1.0:1.0:5.7:8.6) (Table 2, R_rIC50 values) did not match exactly the relative EC_50s of the same peptides in the vascular bioassay (Table 2, R_bEC50 values), normalized to the EC₅₀ of SLIGRL-NH₂ = 1.0 (0.2:1.0: 1.0:1.9:6.7). This quantitative difference in the relative values (Table 2, R_rIC50 versus R_bEC50 values) between the binding IC₅₀s and bioassay EC₅₀s points to differences in the relative peptide intrinsic activities/efficacies, as defined by Stephenson (1956) and Ariens et al. (1957), in the bioassay. The distinct intrinsic activities/efficacies would not be reflected by the radioligand binding assay. These potential differences between the agonist peptides in terms of their bioassay efficacies merit study in the future.

It is clear that the apical aspect of the intestinal colonic villus epithelial cells was labeled in vivo, as we have found previously using an anti-receptor antibody probe (Kong et al., 1997; Cenac et al., 2002; Nguyen et al., 2003). Future studies could investigate the detailed expression of PAR₂ with this fluorescent probe at different time points after its intraluminal administration and correlate these observations with the appearance of pathophysiological features, such as altered smooth muscle contractility, enteric nervous system activation, increased intestinal permeability, or generation of hypersensitivity (Cenac et al., 2007). Whereas the effects of administering PAR₂-activating peptides in vivo have been studied in a variety of different organs (including the intestine, the lungs, the heart, the liver, and the musculoskeletal system), one important question still remains unanswered in those in vivo models. Which cell types are the target for PAR₂ activation and the resulting pathophysiological consequences? The use of the probe we have developed in the present study should be a valuable tool to answer this question.

Although the Alexa Fluor 594-2fLI or [³H]2fLI probes might in principle bind to non-PAR₂ sites, the "net" receptor signal (either binding or fluorescence) calculated by subtracting from the signal detected in the absence of SLIGRL-NH₂ or 2-fLI, the signal observed in the presence of a large molar excess of a nonlabeled PAR₂ agonist, can be taken to reflect accurately the presence of PAR₂ in tissues or cells of interest (Fig. 7, histogram). Our new data complement and support a study that appeared upon completion of our work (Kanke et al., 2006), demonstrating the utility of a ³H-labeled derivative of 2-furoyl-LIGRL-NH₂ to serve as a ligand binding reagent for PAR₂. Our data indicate that the affinity and selectivity of the ornithine-substituted derivatives (both tritiated and labeled with Alexa Fluor 594) are essentially equivalent to those of ³H-labeled 2-furoyl-LIGRL-NH₂. In that previous study, the radioligand was synthesized by the catalytic introduction of ³H into the leucine side chains of dehydro-Leu-2-furoyl-LIGRL-NH₂. Our data indicate that the introduction of a substituent on the side-chain amino group of the ornithine in 2-fLI does not alter the receptor selectivity of the ligand and yields a receptor probe equivalent to [³H]2-furoyl-LIGRL-NH₂ via a more versatile synthetic route that will be more accessible to most laboratories than the tritium gas-palladium catalysis procedure. Our curve-fitting analysis of the binding isotherm (Fig. 2) reveals a plateau in binding in the middle of the curve (between approximately 50 and 120 nM: Fig. 2), indicating a lower capacity binding site with a somewhat higher affinity (approximately 50 nM) than the one previously reported (120 nM) (Kanke et al., 2006). The higher affinity site we report here was not observed by Kanke et al., (2006). Our finding of curvilinearity of the Scatchard plot at the extremes of the concentration range that we used was not explored further. However, to us, the curvilinearity sounds a cautionary note in accepting the Scatchard plot analysis uncritically, without a more in-depth evaluation than was warranted by our study aimed principally at validating the receptor probe. We chose to show the direct binding isotherm deconstructed by simple curve fitting as evidence for the two-site binding curve to emphasize its nature but not to overinterpret the data as possibly done using a detailed mathematical analysis. Because binding at both relatively low (i.e., below 100 nM) and high (i.e., in the micromolar range) concentrations of radioligand were competed for by the unlabeled ligand (i.e., ligand-specific), the data may well reflect the multiple affinity states that a G-protein-coupled receptor can exhibit, depending on its effector interactions (Cuatrecasas and Hollenberg, 1976; DeLean et al., 1978). We suggest that other ornithine-derivatized analogs of 2fLI (e.g., biotinylated or affinity column-coupled) will display a receptor affinity and selectivity comparable to the two derivatives we describe here as "proof of principle". Thus, labeling the peptide with an iodinated acylating reagent, according to the procedure described by Bolton and Hunter (1973) could in principle yield a PAR₂ probe suitable for examining the higher affinity binding site that we have observed in more depth. Furthermore, the ornithine-derivatized peptide could provide for the synthesis of useful receptor affinity columns. Given the binding of the PAR₂-activating peptides to receptor sites that are in some ways distinct from those that bind the tethered ligand (Al-Ani et al., 2004), we predict that the 2fLI probes we describe here will interact both with the nascent and proteolytically activated form of PAR₂. Our continuing work is directed to testing this hypothesis.

Conclusion. The ornithine-derivatized PAR₂-activating peptides that we describe provide a number of advantages over the ligand binding probe prepared by catalytic substitution of dehydro-leucine with tritium. The ornithine-substituted PAR₂ probes are not only more readily synthesized than those prepared by catalytic tritium substitution but our synthesis platform is quite versatile in providing for the preparation of a variety of different receptor probes, two of which we illustrate in this series of experiments. This principle can be used for the development for other receptor probes, for example, to study chemokines receptors like CXCR3 (Vergote et al., 2006). It is clear that the PAR₂-targeted reagents with ornithine substitutions yield ligands that are PAR₂-specific and of sufficiently high affinity to be used in ligand binding and receptor visualization studies. Our data show that the receptor binding of such ornithine-substituted ligands to PAR₂ can be assessed by the usual binding competition paradigms that have been generalized for measurements of receptor binding in other systems (Hollenberg and Cuatrecasas, 1976). We suggest that the reagents and synthesis platform we describe will be of value, not only for the design of PAR₂-targeted reagents but also for the synthesis of comparable receptor probes for PARs 1 and 4. It is our intent to use such reagents for the further study of the molecular pharmacology and physiology of PAR₂ in both cultured cell and intact tissue settings in vivo.

	Acknowledgements

 
We are grateful to Kevin Chapman for assistance with the in vivo evaluation of the binding of Alexa Fluor 594-2fLI, to John (Zhenguo) Yu for help with the vascular bioassay measurements, and to Mahmoud El-Daly for assistance with graphics for the preparation of Alexa Fluor 594-2fLI. The fluorescence measurements for the Alexa Fluor 594-derivatized peptides would not have been possible without the advice and assistance of Dr. Giuseppina (Pina) Colarusso under the auspices of the Canadian Institutes of Health Research (CIHR)-sponsored Group on Inflammatory Disease, with funding for the imaging equipment coming from the Canadian Foundation for Innovation and the Alberta Heritage Foundation for Medical Research.

	Footnotes

 
These studies were supported in large part by term grants from the Canadian Institutes of Health Research (CIRH) (to M.D.H. and N.V.) and by a grant from the Crohn's and Colitis Foundation of Canada (to N.V.), with supplementary support from the National Institutes of Health Grant 1R01MN07568301A1 (to M.D.H.) and the Foundation Bettencourt-Schueller through an INSERM-Avenir program (to N.V.). S.H. was the recipient of an Alberta Heritage Foundation for Medical Research scholarship and a Canadian Association of Gastroenterology scholarship. R.R. is currently supported by a Canadian Association of Gastroenterology/CIHR/Ortho-Jensen Postdoctoral fellowship.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.136432.

ABBREVIATIONS: PAR, proteinase-activated receptor; Alexa Fluor 594, pyranol[3,4-g:5,6-g']diquinolin-13-ium, 6-[2-carboxy-4(or 5)[[(2,5-dioxo-1-pyrrolidinyl)oxycarbonyl]phenyl]-1,2,2,10,10,11-hexamethyl-4,8-bis(sulfomethyl)-succinimidyl ester; Alexa Fluor 594-2fLI, 2-furoyl-LIGRL(N-Alexa Fluor 594)-O-NH₂; Alexa Fluor 594-2fOL, reverse-sequence receptor-inactive 2-furoyl-(N-Alexa Fluor 594)OLRGIL-NH₂; 2fLI, 2-furoyl-LIGRLO-NH₂; 2fO or 2fOL, 2-furoyl-OLRGIL-NH₂; [³H]propionyl-2fLI, 2-furoyl-LIGRL(N-[³H]propionyl)-O-NH₂; PAR₂-AP, PAR₂-activating peptide; KNRK, Kirsten normal rat kidney; IC₅₀, concentration of binding competitor for which specific ligand binding is inhibited by 50%; R_rIC50, R_aIC50, and R_bIC50, relative IC₅₀s for radioligand binding, Alexa-594 2fLI binding, and vascular bioassay, respectively, normalized to the value for SLIGRL-NH₂ = 1.0; HPLC, high-performance liquid chromatography; OCT, ornithine carbamyl transferase; HEK, human embryonic kidney; A23187 [GenBank] , calcimycin; tc, trans-cinnamoyl.

Address correspondence to: Dr. Morley D. Hollenberg, Department of Pharmacology and Therapeutics, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1. E-mail: mhollenb{at}ucalgary.ca

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