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(The FASEB Journal. 2000;14:1411-1422.)
© 2000 FASEB

Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo

ABDULLAH HAJ-YEHIA*, TAHER NASSAR{dagger}, BRUCE S. SACHAIS{ddagger}, ALICE KUO{ddagger}, KHALIL BDEIR{ddagger}, ABU BAKR AL-MEHDI§, ANDREW MAZAR{dagger}{dagger},1, DOUGLAS B. CINES{ddagger},** and ABD AL-ROOF HIGAZI{dagger},{ddagger}2

* School of Pharmacy and the
{dagger} Department of Clinical Biochemistry, Hebrew University-Hadassah Medical Centers, Jerusalem, Israel IL-91120;
{ddagger} Departments of Pathology and Laboratory Medicine,
§ Environmental Medicine and
** Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; and the
{dagger}{dagger} Department of Biology, Angstrom Pharmaceuticals Inc., San Diego California 92121, USA

2Correspondence: University of Pennsylvania, Dept. Pathology and Laboratory Medicine, 513A Stellar-Chance, 422 Curie Blvd., Philadelphia, PA 19104, USA. E-mail: higazi{at}mail.med.upenn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. Å6, a capped peptide corresponding to amino acids 136–143 (KPSSPPEE) of uPA, increased the EC50 of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. Å6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136–143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.—Haj-Yehia, A., Nassar, T., Sachais, B. S., Kuo, A., Bdeir, K., Al-Mehdi, A. B., Mazar, A., Cines, D. B., Higazi, A. A. Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo.


Key Words: smooth muscle cell • intracellular calcium • vascular contractility • rat aorta


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UROKINASE (UPA) IS a serine protease that has been implicated in fibrinolysis (1 2 3 4) , angiogenesis (5 6 7 8) , neointima and aneurysm formation (9 10 11 12 13) , chemotaxis (14) , and wound healing (15) . uPA-/- mice are also more susceptible to lethal pulmonary infection than are wild-type controls (16) . Certain of these activities may require the proteolytic activity of uPA (17 , 18) , whereas others involve intracellular signaling through the urokinase receptor (uPAR) (14 , 19) or additional, as yet undefined receptors (15 , 20 21 22) .

Urokinase is synthesized as a single-chain molecule (scuPA) that expresses little or no intrinsic enzymatic activity (23 24 25) . scuPA is a multidomain protein composed of a carboxyl-terminal protease domain, termed low molecular weight scuPA (LMW-scuPA) and an amino-terminal fragment (ATF). ATF is composed of two subdomains: a growth factor domain (GFD) that binds to uPAR and a single kringle, the function of which is unknown. scuPA can be cleaved by plasmin at the Lys158-Ile159 position to generate an enzymatically active, disulfide-linked two-chain molecule (tcuPA).

Between the ATF and the protease domain is a region designated the ‘connecting peptide’ (amino acids 136–158). Within this region is an eight amino acid sequence (136–143, KPSSPPEE in single-letter code) that can be generated by the combined activities of tcuPA, which cleaves uPA between K135 and K136 (26) , and matrix metalloproteinases, such as matrilysin (MMP-7) and stromelysin (MMP-3), which digest the bond between E143 and L144 (27 , 28) . It has been reported that phosphorylation of Ser-138 or its mutation to glutamic acid abolishes scuPA-induced adhesion and motility of myelomonocytic cells without affecting receptor binding (29 , 30) . However, this conclusion appears to conflict with other studies showing that cell adhesion and motility are mediated by the interaction between uPAR and ATF (31 32 33) .

One potential explanation for these divergent findings is that urokinase can assume more than one conformation, which differ in biological activity. scuPA and tcuPA differ in their intrinsic enzymatic activity (34) , susceptibility to plasminogen activator inhibitors 1 and 2 (35 36 37) and regulation by the urokinase receptor (38 , 39) . Mutations in the connecting peptide may modulate the behavior of each form of uPA in ways that are not evident in the adhesion assay but are operative in vivo.

To address this possibility, we examined the effect of uPA and its fragments on vascular contractility. Vascular reactivity is mediated in part by changes in intracellular Ca2+ concentration in smooth muscle cells (SMC). uPA is known to prime neutrophil production of superoxide by increasing intracellular calcium (40) and to mediate signal transduction in monocytic cells (41 , 42) and vascular SMC (20) , but the effect on calcium flux in this latter cell type has not been described.

The results of the present study indicate that urokinase and its fragments modulate vascular contractility in vitro and in vivo. This activity is mediated by two independent and interdependent epitopes, one residing within the kringle, the other a peptide corresponding to amino acids 136–143. The expression of both epitopes is modulated by conformational changes that occur during the conversion of scuPA to tcuPA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptide synthesis
The capped peptide Å6 (Ac-KPSSPPEE-Am) was synthesized by standard solid-phase methodology using p-methyl-benzhydrylamine resin and L-amino acids protected with t-butyloxycarbonyl (BOC) groups. Removal of the BOC groups was accomplished with 50% trifloroacetic acid in dichloromethane. Coupling was achieved with 1-hydroxybenotriazole and dicyclohexylcarbodiimide. Side-chain protection was with 2-chorobenzyloxycarbonyl for lysine, benzyl for serine, and cyclohexyl for glutamic acid. The amino-terminal lysine was capped by treatment with acetic anhydride. Deprotection and detachment of the completed peptide from the resin were accomplished with anhydrous hydrofluoric acid in the presence of anisole. The peptide was purified on high performance liquid chromatography (HPLC) on a Waters C18 preparative column using 0–40% linear gradient of 1.0% aqueous triethylamine phosphate into CH3CN. The column was washed with 3 column volumes of 1.0% aqueous acetic acid and then eluted with a 0–50% linear gradient of 1.0% aqueous acetic acid into CH3CN. Fractions containing the peptide were reapplied to the column, eluted in the same manner, and lyophilized. The final product was > 99% pure, colorless, and readily soluble in water to > 500 mM. Å7 (Ac-KPSSPPE-Am), Å8 (Ac-PSSPPEE-Am), Å29 (Ac-KPGSPPEE-Am), and the scrambled peptide Å16 (Ac-PSESPEKP-Am), were prepared using similar methodology (see Table 1 ). Uncapped peptides had no effect on vascular function.


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  		Table 1. Urokinase-derived peptides and controls

Contraction response
Male Sprague Dawley rats (250–275 g) were killed by exsanguination. The thoracic aortas were removed with care to avoid damage to the endothelium, dissected free of fat and connective tissue, and cut into transverse rings 5 mm in length (43 44 45 46) . The tissues were kept in an oxygenated (95% O2, 5% CO2) solution of Krebs-Henseliet (KH) buffer (144 mM NaCl, 5.9 mM KCl, 1.6 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM D-glucose). The rings were mounted to record isometric tension in a 10 ml bath containing KH solution under continuous aeration. The rings were equilibrated for 1.5 h at 37°C and maintained under a resting tension of 2 g throughout the experiment. Each aortic ring was then contracted by adding phenylepherine (PE) in stepwise increments (from 10-10 M to 10-5 M). In other experiments, various concentrations of scuPA, tcuPA, or uPA fragments were added 15 min before adding PE. In every experiment, rings exposed to KH buffer alone were analyzed in parallel. Isometric tension was measured with a force displacement transducer and recorded online using a computerized system (ExperimentiaÆ, Budapest, Hungary).

Relaxation response
Separate aortic preparations were used to measure the effect of Å6 on vascular relaxation. After the equilibration period, the aortic rings were contracted with PE at concentrations (3x10-7 M and 6x10-8 M), which induced submaximal contraction (50% and 25%, respectively). After contraction was induced, Å6 was added in stepwise increments (10-9 M to 10-4 M) to the same preparation and the isometric tension was measured as above.

Blood pressure recording
Rats were anesthetized by intraperitoneal (i.p.) administration of ketamine and xylazine (50 and 10 mg/Kg-1, respectively). One cannula was placed in the left carotid artery to record the mean arterial blood pressure and a second was placed in the right jugular vein through which PE (0.1 mg/kg) were administered. Fifteen minutes later, Å6 (0.1 mg), Å6-variants, or buffer were administered in the same manner. The blood pressure was monitored continuously through a transducer placed in the left carotid cannula using the CARDIOSYS computerized system (ExperimentiaÆ).

Conversion of scuPA to tcuPA
In some experiments, two-chain urokinase (tcuPA; gift of American Diagnostica, Greenwich, Conn.) was studied. tcuPA was documented to be free of the isolated amino-terminal fragment of uPA on native gels. In other experiments, scuPA or scuPA variants (see below) (20 µM each) were incubated with plasmin (0.1 µM) for 30 min at 37°C to generate tcuPA. The mixture was added to soluble recombinant human urokinase receptor (suPAR) (see below) bound to CnBr-activated Sepharose (Sigma) for 1 h at 4°C (39) . The beads were washed extensively and the tcuPA was released by adding glycine buffer, pH 3.0. The activity of tcuPA was examined using the chromogenic substrate S-2444 (47) . The completeness of the conversion of scuPA to tcuPA was verified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The preparation was free of plasmin as judged by cleavage of its chromogenic substrate S-2251 (Chromogenics, Molndal, Sweden).

Generation of scuPA{Delta}136–143 and {Delta}kringle-scuPA
A plasmid encoding scuPA{Delta}136–143 was generated using a two-step polymerase chain reaction (PCR). The cDNA encoding full-length scuPA in pcUK176 served as the template. The primers 5'-CGCGGATCCAGCAATGAAC-3' and 5'-TGGCCACACTGAAATTTT AATTTTCCATCTGCGCAGTCAT-3' were used to generate the 438 bp 5'-fragment pUN121 (48) , while 5'-TTAAAATTTCAGTGTGGCCA and 5'-CCAAGCT CGAGGTGCCCG were used to generate the 834 bp 3' fragment. After completion of the second PCR step, the final product was digested with BamHI and XhoI and directionally subcloned into pMT/Bip (Invitrogen, San Diego, Calif.) to yield scuPA{Delta}136–143/pMT/Bip. The recombinant protein was expressed using the Drosophila Expression System (Invitrogen) in Schneider S2 cells according to the manufacturer’s recommendations. scuPA{Delta}136–143/pMT/Bip was purified from S2 medium by affinity chromatography using immobilized rabbit polyclonal anti-human uPAR antibody (ImmunoPure Protein G IgG Plus Orientation Kit, Pierce; Rockford, Ill.). scuPA lacking residues 47–135 ({Delta}k-scuPA) was generated by a two-step PCR procedure in the same manner. The fragment was digested with BglII and XhoI, subcloned into pMT/BiP (Invitrogen), and expressed Schneider 2 cells. {Delta}k-scuPA was purified from the cell supernatant using SP-Sephadex and HPLC chromatography. The protein was characterized by SDS-PAGE under reducing and nonreducing conditions, followed by Western blot analysis with anti-urokinase antibody.

Purification of uPA kringle
tcuPA (7.5 mg/ml) was dialyzed against 0.1 M NaPO4 0.6 M NaCl pH 7.8. Plasmin was added to a final concentration of 1 µM and the mixture was incubated at 37°C for 48 h. Pefablock (1 µM) was added to quench the reaction and the kringle was purified using reverse phase HPLC (RP-HPLC) on a C8 column. Amino-terminal sequencing confirmed the NH2 terminus as starting with Ser-47 of the mature uPA sequence, and the mass of the kringle (determined by MALDI-TOF mass spectrometry) was consistent with a composition corresponding to amino acids 47–135 of uPA (10,138 Da). The kringle was > 95% pure by SDS-PAGE and > 99% pure by analytical C8 RP-HPLC.

Preparation of recombinant suPAR
cDNA encoding the full length of uPAR in uPAR/pGEM (49) was used as a template in a PCR reaction to introduce a stop codon after amino acid residue 277 and to introduce enzyme restriction sites. The PCR product was subcloned into pMT/Bip at the BglII and XhoI sites. The complete sequence was confirmed using automated fluorescence-based sequence analysis (University of Pennsylvania DNA Sequencing Facility). suPAR was expressed using the Drosophila Expression System in Schneider S2 cells as described above and purified by affinity chromatography on scuPA-Sepharose (39) .

Binding of scuPA{Delta}136–143 and {Delta}kringle-scuPA to the urokinase receptor
Binding of wild-type, scuPA{Delta}136–143, and {Delta}kringle-scuPA was measured using a BIA 3000 optical Biosensor (Biacore, AB, Sweden) (50) . This method detects binding interactions in real time by measuring changes in the refractive index (RI) at a biospecific surface, enabling association and dissociation rate constants to be calculated. For these studies, suPAR was coupled to a CM5 research grade sensor chip flow cell (Biacore) via standard amine coupling procedures (51) using N-hydroxysuccinimide/N-ethyl-N'-[3-(dimethylamino) propyl] carbodiimine hydrochloride (Pierce) at a level of 500 response units. The sensor surface was coated with suPAR (10 µg/ml) in 10 µM NaAc buffer, pH 5.0. After immobilization, unreacted groups were blocked with 1 M ethanolamine, pH 8.5. A second flow cell, similarly activated and blocked without immobilization of protein, served as a control. All binding reactions were performed in phosphate-buffered saline (PBS), pH 7.4, 0.005% TWEEN-20. Binding of scuPA (WT, scuPA{Delta}136–143, and {Delta}k-scuPA) was measured at 25°C at a flow rate of 60 µl/min for 2 min, followed by 2 min of dissociation. The bulk shift due to changes in RI was measured using the blank surface and was subtracted from the binding signal at each condition to correct for nonspecific signals. Surfaces were regenerated with 2 x 18 s pulses of 1 M NaCl, pH 3.5, followed by an injection of binding buffer for 1 min to remove this high-salt solution. All injections were performed in a random fashion using the RANDOM command in the automated method. Binding of urokinase was measured over a range of concentrations (12.5–0.2 nM). Data, collected at 2.5 Hz, were fit to a 1:1 Langmuir reaction mechanism using global analysis in the BIA evaluation 3.0 software (Biacore). Secondary plots of the data [ln(|dR/dt|) vs. t] were also performed to unmask any contribution of mass transport to the kinetic data.

Measurement of plasminogen activator activity
The effect of Å6 on the plasminogen activator activity of scuPA/suPAR complexes was performed as described (52) . Briefly, equimolar complexes between scuPA and suPAR were generated at 50-fold final concentration. Purified human fibrinogen was radiolabeled with 125I and resuspended to a specific activity of 30,000 cpm/ml in plasma. Clots were formed from 0.4 ml plasma by adding thrombin (0.4 units/ml final concentration). scuPA/suPAR (10 nM) was incubated in the presence or absence of 50 µM Å6 for 2 h, the clots were washed with PBS, and the release of radiolabeled soluble fibrin degradation products into the lavage solution at 4 h was measured.

Measurement of intracellular calcium
The effect of Å6 on PE-induced intracellular calcium [Ca2+]i in human umbilical vein endothelial cells (HUVEC) was measured (53 , 54) . HUVEC were incubated in media supplemented with 1 µM fluo-3 acetomethyl ester (Fluo-3; Molecular Probes, Eugene, Oreg.) (55) in the presence or absence of 5 or 50 µM Å6 for 30 min at 37°C. The cells were washed three times with Krebs’ ringer bicarbonate solution that contained either 2 mM Ca2+ or no Ca2+ plus 1 mM EGTA (calcium-free medium) to remove unbound dye. One milliliter of Ca2+-containing or Ca2+-free medium was then added to each well. Baseline pictures were taken at 490 nm excitation and 520 nm emission using a Hamamatsu ORCA-100 cooled CCD digital camera; an inverted Nikon-TMD Diaphot epifluorescence microscope. PE (0.1 µM) was then added and Fluo-3 emission was measured at 5 min. Å6 had no effect on dye uptake.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
scuPA and tcuPA have opposing effects on vascular contractility
We examined the effect of urokinase on vascular contractility. To do so, we first studied the effect of scuPA and tcuPA on PE-induced contraction of isolated rat aortic rings. Each form of urokinase contains the identical amino acid sequence, but the molecules differ in their intrinsic enzymatic activity and susceptibility to plasminogen activator inhibitors.

scuPA inhibited PE-induced contraction of the aorta, increasing the EC50 from 29 nM to 182 nM (Fig. 1 ). We then converted scuPA to tcuPA by adding plasmin, which cleaves Lys158-Ile159, generating a molecule composed of two chains that are linked by a single disulfide bond. tcuPA was affinity purified using immobilized soluble uPAR or benzamidine to remove plasmin, with identical results. In direct contrast to scuPA, tcuPA augmented PE-induced aortic contraction, decreasing the EC50 of PE from 29 nM to 4.47 nM (Fig. 1) .



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  		Figure 1. Effect of urokinase on the contraction of rat aortic rings: accumulation curve. Contraction of rat aortic rings was induced by phenylepherine (PE) at the indicted concentrations in the absence ({diamondsuit}) or presence of 10 nM scuPA ({blacktriangleup}) or tcuPA ({blacksquare}). In this and in each figure, the molar concentration of PE is displayed in logarithmic units. The mean ± SD of 6 experiments is shown.

The amino-terminal fragment of uPA promotes vascular contractility
Single- and two-chain full-length uPA may have opposing effects on vascular contractility because they differ in which biologically active epitopes are exposed and/or because intramolecular interactions among the domains differ. To begin to distinguish between these possibilities, experiments were performed using isolated fragments of urokinase.

LMWuPA (amino acids 144–411), which lacks ATF and is catalytically active in tcuPA but nascent in scuPA, had no effect on PE-induced contraction of aortic rings at concentration as high as 20 µM (not shown). Therefore, we turned our attention to the ATF (amino acids 1–135), the portion of the molecule that binds to uPAR. To do so, we first examined the effect of soluble uPAR on uPA-mediated vasoconstriction. suPAR completely abolished the effects of scuPA and tcuPA, as evidenced by a return of the EC50 to 29 nM (not shown). This outcome suggests that uPA-induced aortic contraction is mediated by ATF or a portion of uPA in close enough spatial proximity that it is no longer available when the molecule is bound to its receptor in solution. Isolated ATF was itself procontractile, as evidenced by a decrease in the EC50 for PE to 0.71 nM (Fig. 2 ). Indeed, ATF was sixfold more potent than tcuPA (compare Figs. 1 and 2 ), and its activity was abolished by suPAR as well (Fig. 2) .



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  		Figure 2. Stimulation of PE-mediated vasoconstriction by the amino-terminal fragment of urokinase (ATF). Aortic rings were contracted by sequential addition of increasing concentrations of PE in KH buffer (gray filled square) or KH buffer supplemented with 10 nM ATF ({diamondsuit}) or 10 nM ATF + 10 nM suPAR (*). The mean ± SD of 3 experiments is shown.

The ATF of urokinase is composed of two subdomains: the GFD and the kringle. The GFD is known to mediate the binding of uPA to its receptor. The function of the kringle is unknown. To examine which portion of ATF is responsible for the procontractile effect of tcuPA and ATF, the effect of each fragment on PE-induced vasoconstriction was measured (Fig. 3 ). The kringle did not induce vasoconstriction directly, but markedly augmented the effect of phenylepherine. Addition of 1 nM kringle decreased the EC50 from 29 nM to 0.1 nM, whereas 10 nM GFD did not augment contraction (not shown).



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  		Figure 3. Stimulation of PE-mediated vasoconstriction by the kringle of uPA. Aortic rings were contracted by sequential addition of increasing concentrations of PE in KH buffer (gray X) or KH buffer supplemented with increasing concentrations of uPA kringle: 1 nM (*); 10 nM ({diamondsuit}); 50 nM (gray filled square); 100 nM ({blacktriangleup}). The mean ± SD of 3 experiments is shown.

The connecting peptide of urokinase inhibits vascular contractility
We then performed experiments to identify the portion of scuPA that inhibited PE-induced vasoconstriction. We first examined the effect of Å6, a capped peptide corresponding to amino acids 136–143 (Ac-KPSSPPEE-Am) that lies between LMWuPA and ATF. Å6 increased the EC50 of PE sevenfold to 192 nM. Scrambling the order of the amino acids (Ac-PSESPEKP-Am; designated Å16) completely abolished the inhibitory effect (control, Fig. 4 ). Activity was also lost when the carboxyl-terminal amino acid (E143) (Å7), but not when the amino-terminal amino acid (K136) (Å8), was deleted (Fig. 4) . In accordance with previous findings that mutating Ser-138 to Glu in full-length scuPA abolished its proadhesive effect (29 , 30) , a connecting peptide variant E138 (Å29) had no effect on vascular contractility (not shown).



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  		Figure 4. Effect of Å6 and variants on PE-induced contraction of aortic rings. Aortic rings were contracted by sequential addition of increasing concentrations of PE in KH buffer (control) or KH buffer supplemented with 1 µM wild-type Å6 (WT) or its variants lacking K136 or E143. The mean ± SD of 3 experiments is shown.

We then asked whether Å6 would cause relaxation of precontracted aortic rings. Å6 caused a dose-dependent relaxation of aortic rings pretreated with PE (Fig. 5 ). The tension in rings contracted to 25% of maximum was reduced 70% by 10 µM Å6 (Fig. 5A ) and 40% when rings were first contracted to 50% of maximal tension (Fig. 5B ). The scrambled connecting peptide variant (Å16), a variant lacking E143 (Å7), and a variant S138E (Å29) did not cause vascular relaxation, whereas the Å6 variant lacking the amino-terminal amino acid (K136) (Å8) induced relaxation with the same potency as wild-type Å6 (not shown).



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  		Figure 5. Å6-induced relaxation of precontracted aortic rings. Aortic rings were precontracted with 6 nM PE or 29 nM PE, which induce 25% (A) and 50% (B) of maximal contraction, respectively. Å6 was added at successively higher concentrations and the tension was recorded. The mean ± SD of 3 experiments is shown.

A scuPA variant lacking the connecting peptide lacks vasorelaxation activity
Taken together, these findings indicate that the procontractile effect of tcuPA is mediated by the kringle, an effect that may be overridden in scuPA by a signal involving the connecting peptide. Alternatively, the procontractile epitope may be exposed in tcuPA but not in scuPA. To distinguish between these possibilities, we synthesized a variant scuPA that lacks the connecting peptide (scuPA{Delta}136–143) and studied its vasoactive properties before and after its conversion to a two-chain molecule.

scuPA{Delta}136–143 had no effect on PE-induced aortic contraction (Fig. 6A ). This result demonstrates that the connecting peptide is required to inhibit contraction and that the procontractile elements in ATF are not active in scuPA. We then asked whether the lack of activity of scuPA{Delta}136–143 was due a loss of affinity for uPAR. The binding kinetics of wild-type scuPA and scuPA{Delta}136–143 to suPAR immobilized on a CM5 sensor chip were measured using a BIA 3000 optical biosensor (Fig. 7 ). Binding was kinetically driven as examination of secondary plots failed to reveal any limitation resulting from mass transport. scuPA{Delta}136–143 bound to suPAR with only a small reduction in Kd compared with WT scuPA (Table 2 ), a difference that cannot account for its complete loss of vascular reactivity. Furthermore, scuPA{Delta}136–143 expressed the same plasminogen activator activity as wild-type scuPA in the presence of suPAR (38 , 39) and after cleavage by plasmin to a two-chain molecule (data not shown). In addition, the EC50 for PE-induced contraction in the presence of tcuPA{Delta}136–143 and wild-type tcuPA were almost identical (Fig. 1 and Fig. 6A ). Taken together, this series of experiments supports the hypothesis that the epitope(s) in the kringle that modulate vasoconstriction are exposed only when scuPA is converted to tcuPA.



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  		Figure 6. Effect of uPA{Delta}136–143 and {Delta}k-uPA on PE-induced vasoconstriction. A) Aortic rings were incubated with 10 nM tcuPA{Delta}136–143 ({blacksquare}), 10 nM scuPA{Delta}136–143 ({blacktriangleup}), or PBS control ({diamondsuit}) and then with increasing concentrations of PE as described in the legend to Fig. 1 . The mean ± SD of 3 experiments is shown. B) Aortic rings were incubated with 10 nM {Delta}k-scuPA (x), 10 nM {Delta}k-tcuPA ({blacktriangleup}), or PBS control ({diamondsuit}) and then with increasing concentrations of PE as described in the legend to Fig. 1 . The mean ± SD of 3 experiments is shown.



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  		Figure 7. Binding of scuPA to suPAR measured by surface plasmon resonance. Binding of wild-type scuPA (A), scuPA{Delta}136–143 (B), and {Delta}k-scuPA (C) to suPAR are shown. Urokinase (12.5 nM to 0.2 nM) was then added in twofold increments. An experiment representative of three so performed is shown.


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  		Table 2. Binding of urokinase to urokinase receptora

A tcuPA variant lacking the kringle lacks procontractile activity.
We used a similar approach to ask whether the vasoconstrictive effect of tcuPA is due to the overriding influence of the kringle above that of the connecting peptide or whether there may be a difference in the exposure of two independently functioning domains. Therefore, we examined the effect of a uPA variant lacking the kringle ({Delta}k-uPA) on PE-induced vasoconstriction. {Delta}k-scuPA and WT scuPA bound to suPAR with almost the same Kd (Table 2 and Fig. 7 ). {Delta}k-scuPA also inhibited PE-induced vasoconstriction to the same extent as WT scuPA (Fig. 1 and Fig. 6B ). However, tcuPA lacking the kringle was unable to augment PE-induced vasoconstriction.

Intracellular pathways through which the connecting peptide modulates vascular reactivity
Experiments were then performed to begin to identify the pathway by which the uPA connecting peptide mediates vasorelaxation. First, we asked whether the connecting peptide opposed the contractile activity of PE through a direct effect on the {alpha}-adrenergic receptor or on a postreceptor step. To address this question, we examined the capacity of Å6 to reverse vasoconstriction induced by endothelin-1, which operates through an independent receptor system. Å6 caused a concentration-dependent relaxation of rings contracted by 80 nM endothelin-1, a concentration that induces 50% of maximal contraction (Fig. 8 ). Endothelin-induced constriction was inhibited 40% by 10 µM Å6 (Fig. 8) , whereas the scrambled connecting peptide variant Å16 had no effect (not shown). These data indicate that Å6 does not inhibit PE-induced vasoconstriction by blocking the {alpha}-adrenergic receptor.



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  		Figure 8. Effect of Å6 on vasoconstriction induced by endothelin. Aortic rings were incubated with 80 nM endothelin-1 for 5 min. Å6 was added at successively higher concentrations and the tension was recorded continuously. The tension in rings contracted with endothelin-1 in the presence of scrambled connecting peptide Å16 was unchanged throughout the experiment (not shown). The mean ± SD of 3 experiments is shown.

PE exerts its effect on contraction by increasing intracellular Ca2+, and this effect can be observed in HUVECs used as a model system. The PE-induced rise in intracellular calcium is biphasic, with an initial release from intracellular stores followed by a sustained increase due to influx from the extracellular space (56) . To identify the locus of action of the connecting peptide, PE-mediated Ca2+ influx in calcium-free or calcium-containing media was measured in the presence and absence of Å6. PE induced a 178 ± 14% (n=3; P<0.05 vs. baseline) increase in Calcium Green fluorescence in Ca2+-containing medium, indicative of an increase in intracellular Ca2+, and a 127 ± 11% increase (n=3; P<0.05 vs. baseline) in Ca2+-free medium (Fig. 9A vs. B ). The PE-induced increase in intracellular Ca2+ was reduced 68 ± 11% by 50 µM Å6 (n=3; P<0.05 vs. PE alone) in Ca2+-containing medium and 85 ± 9% (n=3; P<0.05 vs. PE alone) in Ca2+-free medium. The increase in intracellular calcium was reduced 21 ± 6% (n=3; P<0.05 vs. PE alone) by 5 µM Å6 in Ca2+-containing medium and by 45 ± 8% (n=3; P<0.05 vs. PE alone) in Ca2+-free medium.



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  		Figure 9. Effect of Å6 on Ca2+ influx induced by PE. HUVEC were incubated in media containing 1 µM Fluo-3 in the presence or absence of 50 µM Å6 for 30 min at 37°C and cells were washed free of unincorporated dye. A) Baseline emission at 490 nm excitation and 520 nm emission. PE (0.1 µM) was added and Fluo-3 emission was measured 5 min later in the absence (B) or presence (C) of Å6 (designated CP). An experiment representative of three so performed is shown.

The capacity of Å6 to inhibit the contraction of aortic rings in Ca2+-free media was then studied (Fig. 10 ). In the absence of extracellular Ca2+, the EC50 of PE was increased from 25 nM to 1.5 µM, as expected. Addition of Å6 to the media increased the EC50 to > 100 µM, an extent of inhibition even greater than that seen in the presence of extracellular Ca2+ (Fig. 10 vs. Fig. 1 ), whereas the scrambled peptide variant (Å16) had no effect (data not shown). This result indicates that Å6 inhibits SMC contraction by inhibiting the release of intracellular Ca2+, likely from the endoplasmic reticulum.



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  		Figure 10. Contraction of aortic rings in Ca++-free medium. Contraction of aortic rings was induced as described in the legend to Fig. 1 , but in the absence of Ca2+ and in the absence ({diamondsuit}) or presence ({blacksquare}) of 1 µM Å6. The mean ± SD of 3 experiments is shown.

Nitrous oxide (NO) is known to mediate vasorelaxation by decreasing intracellular free Ca2+. To examine whether Å6 works through NO synthesis, the NO synthase inhibitor L-nitro-arg-methyl ester (L-NAME, 1 µM final concentration) was added to the rings (57) . L-NAME did not affect relaxation induced by 10 µM Å6 (not shown). This result indicates that Å6 does not require synthesis of NO to inhibit Ca2+ release from intracellular stores. Prostacyclin also mediates vasorelaxation by decreasing intracellular free Ca2+ (58) . To examine the involvement of the prostaglandin pathway, aortic rings were first contracted with PE. Indomethacin (1 µM final concentration) was added for 10 min and Å6 was then added in progressively higher concentrations. Indomethacin alone had no effect on PE-induced vasoconstriction. However, indomethacin inhibited vasorelaxation induced by Å6 over the entire range of peptide concentrations tested (Fig. 11 ). This experiment indicates that Å6 stimulates the production of prostaglandin derivatives with vasorelaxation activity.



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  		Figure 11. Effect of indomethacin on Å6-induced vasorelaxation. Aortic rings were precontracted with 29 nM PE that induces 50% of maximal contraction. After contraction had reached a plateau, 1 µM indomethacin ({blacksquare}) or control media ({diamondsuit}) was added for 10 min. Å6 was added at successively higher concentrations and a continuous recording of the tension was made. The mean ± SD of 3 experiments is shown. The SDs were too small to be apparent.

Effect of the connecting peptide in vivo.
Last, we examined the effect of the connecting peptide on the blood pressure of rats in vivo. Å6 itself had no effect on ambient blood pressure at concentrations as high as 1 mg, estimated to yield a concentration 10-fold greater than is required to inhibit PE-induced contraction (not shown). However, Å6 markedly attenuated the hypertensive effect of PE. Injection of 0.1 mg of Å6 caused the blood pressure to fall below basal levels in rats injected with PE (Fig. 12A ). In contrast, no effect on blood pressure was observed when the same amount of a connecting peptide variant that lacks E143 (Å7) was injected (Fig. 12B ).



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  		Figure 12. Effect of Å6 in vivo. A) Rats were anesthetized and a blood pressure recorder was placed within the carotid artery. PE (0.1 mg) was injected and the mean arterial blood pressure (MABP) was monitored continuously until a stable baseline was reached (400 s). Saline or Å6 (0.1 mg) was injected and the recording of the mean arterial pressure continued. The data shown are representative of three experiments so performed. B) The protocol described in panel A was used with the exception that a variant lacking E143 was injected.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Signal transduction through the urokinase receptor has been implicated in cell differentiation, adhesion, and migration (31 , 59) . Urokinase has been proposed to induce a conformational change in uPAR that promotes its interaction with vitronectin (52 , 60 , 61) , complements receptor 3 (62) , and modulates ß2-integrin function through intracellular signaling (63 , 64) . However, the determinants in urokinase and within the uPA receptor that promote signal transduction remain incompletely defined. Moreover, auxiliary pathways may exist by which uPA transduces signals independent of uPAR, as suggested by the finding that the concentration of urokinase required to induce mitogenesis in a melanoma cell line was considerably above its Kd for this receptor (21) .

Phosphorylation of Ser-138 or its mutation to Glu abolishes capacity of uPA to promote uPAR-mediated cell adhesion and motility and impairs the motility of uPAR in the cell membrane (13 , 14) . The mechanism by which Ser-138 modulates the function of uPAR with no apparent change in affinity has not been explained. It has been postulated that phosphorylation of Ser-138 impairs a productive interaction with uPAR that is not evident in binding assays or induces novel associations between the ligand and other portions of the receptor that negatively regulate cell adhesion (13 , 14) . These findings imply that all the information required to induce signal transduction is contained within the connecting peptide. It is difficult to reconcile this explanation with the findings of several groups that the amino-terminal fragment of urokinase; indeed, its growth factor subcomponent suffices to induce uPAR-dependent cell adhesion (31 32 33 , 65) .

Our studies reconcile some of these discrepant observations in urokinase-mediated signal transduction by providing novel insights into the nature of intramolecular interactions within the urokinase molecule itself. The finding that single-chain urokinase and two-chain urokinase exert opposing effects on smooth muscle contractility provides not only the first described effect of this plasminogen activator on contractility, but also offers compelling evidence for the existence of more than one functionally active site within the molecule.

One epitope, located within the connecting peptide (amino acids 136–143), inhibits vasoconstriction induced by phenylepherine and endothelin by blocking the release of calcium from intracellular stores as well as its entry from the extracellular space. The effect of the connecting peptide on vasorelaxation is sequence specific and requires both Ser-138 and Glu-143. Further support for the concept that the connecting peptide functions as a biologically active unit derives from its capacity to inhibit cell migration and tumor cell invasiveness (see ref 66 ).

A second epitope, within the kringle of uPA, augments PE-induced vasoconstriction. This procontractile epitope and the epitope within the connecting peptide that promotes vasorelaxation are not exposed simultaneously in either single-chain or two-chain urokinase. Only the connecting peptide is expressed in scuPA. This conclusion is established by several findings. First, a variant of scuPA lacking only these eight amino acids causes neither vasorelaxation nor vasoconstriction. On the other hand, the epitope within the kringle is exposed only in tcuPA. This conclusion is based on two observations. 1) Deleting the kringle did not inhibit the effect of scuPA on vasorelaxation, but the deletion abolished its procontractile activity as a two-chain molecule. 2) Plasmin-mediated activation of scuPA exposes a procontractile epitope within the amino-terminal fragment coincident with the loss of the inhibitory epitope within the connecting peptide. This conclusion derives from the observation that cleavage of the scuPA variant lacking the connecting peptide by plasmin generates a two-chain variant molecule possessing the same procontractile potency as wild-type tcuPA, but does not inhibit PE-induced vasoconstriction as a single-chain molecule.

Our observations may explain why modification of Ser-138 in scuPA by phosphorylation or mutation to Glu abolishes its effect on cell adhesion and motility. The signal-transducing epitope in kringle may not be expressed in scuPA, but is exposed when it is converted to tcuPA or when ATF is released from the remainder of the molecule. In accordance with this hypothesis, both the ATF (67) and the connecting peptide inhibit tumor cell invasiveness (see ref 66 ). In contrast, the finding that ATF and the connecting peptide exert opposing effects on vascular reactivity demonstrates that the two domains are capable of exerting independent effects on signal transduction. This dichotomy is consistent with the finding that cell motility is stimulated by ATF (19) , but is inhibited by the connecting peptide (66) . Thus, our results suggest that the domain in ATF that promotes cell adhesion or other signal-transducing events is not available in scuPA, whereas the connecting peptide is functional. In contrast, the signal-transducing effect of ATF becomes evident when scuPA is converted to tcuPA or the fragment is isolated from the remainder of the molecule.

The mechanism by which uPAR initiates signal transduction—specifically, whether it requires uPAR—was not resolved by this study. Several findings suggest that the vasorelaxation and the procontractile effects of scuPA and tcuPA, respectively, require binding to uPAR. First, the isolated connecting peptide inhibits the binding of scuPA to its receptor, and this inhibition is not competitive (66) . Second, the connecting peptide inhibits the fibrinolytic activity of scuPA/suPAR, which requires complex formation, but not that of tcuPA, which is unaffected by equimolar concentrations of suPAR (38 , 39) . Third, the effects of tcuPA and scuPA on vascular reactivity were abrogated by suPAR. On the other hand, several compelling observations argue against the direct involvement of uPAR. tcuPA and scuPA bind with the same affinity to uPAR but differ in their effects on vasoconstriction. The same reasoning applies to the differences in the behavior of scuPA{Delta}136–143 and {Delta}k-scuPA, both of which bind to uPAR with essentially the same Kd. Moreover, the amino-terminal fragment of human urokinase does not bind to murine uPAR (68) . Whether the isolated connecting peptide, which differs in sequence from its murine analog, shows cross-reactivity is unknown. The observation that the scuPA variants lacking the connecting peptide or the kringle bound normally to human uPAR but lack vasoreactivity, may indicate that vascular cells express additional, uncharacterized binding sites for scuPA that mediate signal transduction (21) . Similar studies in uPAR-/- mice will be required to determine whether the signal generated by the isolated connecting peptide is delivered through uPAR or through a novel receptor.


   ACKNOWLEDGMENTS
 
This work was supported by NIH-HL60169 and a grant from the University of Pennsylvania NCI Core Cancer Pilot Project.


   FOOTNOTES
 
1 Present address: Attenuon, LLC, 10130 Sorrento Valley Rd., Suite B, San Diego, CA 92121, USA.

Received for publication August 23, 1999. Revision received December 3, 1999.
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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