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CARDIOVASCULAR
Department of Pharmacology, Weill Cornell Medical College, New York, New York (U.S., T.M., R.L.); and Laboratory of Neurobiology and Genetics, Rockefeller University, New York, New York (S.V., J.P.M., S.S.)
Received February 12, 2007; accepted April 10, 2007.
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Abstract |
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; O'Rourke et al., 2005). Both enzymes activate the zymogen plasminogen to plasmin, the principal circulating thrombolytic agent; indeed, functional disruption of the t-PA and u-PA genes in mice impairs clot lysis (Carmeliet et al., 1993b, 1994). The activity of t-PA and u-PA in plasma is regulated by specific inhibitors. Of these, plasminogen activator inhibitor type 1 (PAI-1) is considered the main inhibitor (Potempa et al., 1994). The two plasminogen activators have unique biological properties; t-PA, through its fibrin-binding function, is of primary importance in cell-independent fibrinolysis (Collen, 1999; Yakovlev et al., 2000; Medved and Nieuwenhuizen, 2003), whereas u-PA, through its high-affinity cell surface receptor, participates mainly in cell-mediated fibrinolysis, such as fibrin solubilization in organizing wound fields (Blasi et al., 1986; Ellis et al., 1991).
Prominent u-PA immunoreactivity has been demonstrated in the vas deferens and other urogenital organs (Larsson et al., 1984). Furthermore, u-PA mRNA expression was shown in murine gastrointestinal and urogenital tracts, including vas deferens (Kristensen et al., 1991), where the amounts of u-PA mRNA were as high as those of t-PA (Rickles and Strickland, 1988). Interestingly, the expression of t-PA mRNA in the heart was found to be 5-fold greater than in the vas deferens, whereas much lower t-PA mRNA was expressed in the gastrointestinal tract (Rickles and Strickland, 1988).
Recently, the presence of both t-PA and u-PA has been demonstrated in sensory neurons, and enhanced expression of t-PA, u-PA, PAI-1, and PAI-2 has been reported after neuronal injury (Yamanaka et al., 2005). In addition to its endothelial source, t-PA is synthesized in neural tissue (Peng et al., 1999; Jiang et al., 2002; O'Rourke et al., 2005). A recent report using a transgenic mouse model demonstrated a highly confined expression of t-PA in embryonic cells derived from the neural crest, with most prominent staining in the adrenal medulla and cervical ganglia, the sympathetic chain, axons within the myocardium, and the adventitial surface of several arteries (Hao et al., 2006). Importantly, Pittman and coworkers (Krystosek and Seeds, 1981; Pittman et al., 1989) found that sympathetic neurons are capable of releasing both u-PA and t-PA, a process perhaps involved in neurite outgrowth. Notably, physical and mental stress, electrical sympathetic stimulation, and coronary artery ligation all cause the acute release of t-PA into the circulation (Jern et al., 1994; Björkman et al., 2003; Aspelin et al., 2005).
We recently reported that endogenous t-PA enhances norepinephrine (NE) release from sympathetic nerves, whereas lack of t-PA greatly reduces it (Schaefer et al., 2006). We believe these findings to be relevant in conditions of exaggerated NE release, such as hypertension, myocardial ischemia, and congestive heart failure. Because sympathetic activity is a key player in cardiovascular disease, the aim of the present study was to identify the specific components of the plasminogen-activating system (PAS) (i.e., t-PA, u-PA, PAI-1, and plasminogen) critically involved in the modulation of NE release. In the present study, we find that t-PA is the only PAS component capable of influencing sympathetic tone and that this action is independent of u-PA and plasminogen availability.
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Materials and Methods |
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, 1994). Mice deficient in PAI-1, plgn, t-PA, and u-PA-/- were purchased from Jackson Laboratories (Bar Harbor, ME). All the mice used have been back crossed to C57/BL6 mice for at least nine generations. All studies were performed on male young adult mice at 4 to 5 months of age. After pretreatment with heparin (100 I.U. i.p.), mice were anesthetized with CO2 and killed by cervical dislocation (approved by the Weill Cornell Medical College Institutional Animal Care Committee). Hearts and vasa deferentia were quickly excised and cooled in ice-cold modified Krebs-Henseleit (KH) solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4·7H2O, 24 mM NaHCO3, 1.1 mM KH2PO4, 10 mM glucose, 0.5 mM pyruvic acid, and 2.5 mM CaCl2·2H2O). KH solution was continuously equilibrated with 95% O2 + 5% CO2, as described previously (Schaefer et al., 2006). Vas Deferens. The midportion of the vas deferens was rapidly mounted into vessel chambers containing Krebs-Ringer solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4·7H2O, 25 mM NaHCO3, 1.2 mM KH2PO4, 8.3 mM glucose, 2.5 mM CaCl2·and 2H2O, gassed with 95% O2 + 5% CO2) with one end connected to a force transducer. Dissected vasa were allowed to equilibrate for at least 1 h at a resting tension of 250 mg at 37°C. In general, aerated Krebs-Ringer solution was replaced every 30 min. At the beginning of each experiment, a brief contraction with K+ (80 mM) was induced, and the developed tension was recorded with PowerLab/8SP (ADInstruments, Colorado Springs, CO). After washout and an additional equilibration period of 60 min, sympathetic nerves were stimulated by electrical field stimulation (EFS) at 4 to 32 Hz for 15 s at 5-min intervals with a pulse width of 1 ms and supramaximal voltage. EFS was applied using a stimulator (model S48; Grass Instruments, West Warwick, RI) and a stimulation isolation unit (model SIU5; Grass Instruments). Contractile responses [first phase, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) sensitive, purinergic; second phase, prazosin sensitive, adrenergic] were individually analyzed and expressed as percentage of the response to K+ (80 mM). Drugs were incubated for at least 15 min, and the resultant response to EFS was analyzed and compared with control stimulation. Responses were stable for at least three to four consecutive stimulations. To differentiate between pre- and postsynaptic sites of action, concentration-response curves for ATP (0.1–100 µM), phenylephrine (0.01–100 µM), and NE (0.01–100 µM) were constructed and expressed as percentage of the response to K+.
Cardiac Synaptosomes. After excision of the heart, the aorta was cannulated with an 18-gauge custom-made steel cannula, and the heart was perfused at constant pressure (100 cm of H2O) with KH buffer at 37°C. Two hearts per each mouse type were perfused for 20 min to ensure that blood components were removed from the coronary vasculature. Both hearts were minced together in ice-cold sucrose (0.32 M), and synaptosomes were prepared as described previously (Seyedi et al., 1997). Synaptosomes were depolarized with K+, and the release of NE and ATP was analyzed in the supernatant and related to the pellet protein content (Sesti et al., 2003).
Inhibition of t-PA Activity. The activity of u-PAstop and t-PAstop was determined by their ability to inhibit recombinant t-PA, using a colorimetric substrate for the protease. The assay was performed in 96-well microtiter plates. Recombinant t-PA was diluted in a buffer containing 50 mM Tris-imidazole and 300 mM NaCl, pH 8.4, incubated with various concentrations of both inhibitors for 15 min at room temperature. The kinetics of substrate cleavage (20 µl of Pefachrome t-PA; concentration 2 mM; Pentapharm Ltd., Basel, Switzerland) were monitored spectrophotometrically at 405 nm for 5 min (Schaefer et al., 2006).
NE Assay. Synaptosomal supernatants were assayed for NE by HPLC with electrochemical detection as described previously (Seyedi et al., 1997). The detection limit was 
0.05 pmol.
ATP Assay. ATP levels were measured with a firefly luciferin-luciferase assay-based commercial kit (ATP bioluminescence assay kit HS II; Roche Diagnostics, Indianapolis, IN). Samples (50 µl) of each supernatant were pipetted into appropriate test tubes, placed in a luminometer (model 20/20; Turner Designs, Sunnyvale, CA), and processed by autoinjection of 50 µl of luciferin-luciferase reagent. ATP concentrations were calculated from a calibration curve constructed the same day using ATP standards included in the kit. The amount of ATP was expressed as femtomoles per milligram of synaptosomal protein (Sesti et al., 2003).
t-PA Antigen Assay. A 96-well strip format enzyme-linked immunosorbent assay kit (MTPAKT-TOT) was obtained from LOXO-GmbH (Dossenheim, Germany). The detection limit was below 0.05 pg/µg protein.
Drugs and Chemicals. Adenosine 5'-triphosphate (disodium salt), norepinephrine HCl, prazosin HCl, phenylephrine, PPADS, and 
,
-methylene-ATP (,-MeATP) were obtained from Sigma-Aldrich Co. (St. Louis, MO). Plasminogen, t-PAstop, and u-PAstop were purchased from American Diagnostics, Inc. (Stamford, CT). Pefachrome t-PA (Pefa-5037) was purchased from Pentapharm.
Statistics. Values refer to means ± S.E.M. One-way ANOVA followed by Dunnett's post-test, one-sample Student's t test, and unpaired Student's t test were used as indicated. p < 0.05 was considered significant.
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). The second contractile phase is due to activation of postjunctional 1-adrenoceptors by released NE. A certain degree of overlap exists between both phases, in that ATP partially influences the second phase and NE the first phase (Todorov et al., 1996). Indeed, desensitization of P2X-receptors with a high concentration of ,-Me-ATP (10 µM) or blockade with the P2X-receptor antagonist PPADS (10 µM; data not shown) abolished the initial contractile phase but also slightly attenuated the second phase (Fig. 1B). Conversely, preincubation with the 1-adrenoceptor antagonist prazosin (0.1 µM) attenuated the second phase and slightly the first (Fig. 1C). At frequencies between 4 and 32 Hz, EFS induced a progressive increase in the amplitude of both purinergic and adrenergic phases (Fig. 2, A and B). These responses persisted unabated for at least three to four subsequent stimulation cycles (i.e., time controls; data not shown). Frequency-response curves for vasa deferentia isolated from plgn-/- and u-PA-/- mice were superimposable on the respective curves obtained in vasa from WT mice (Fig. 2). In contrast, the frequency-response curves for the vasa deferentia isolated from PAI-1-/- and t-PA-/- mice were shifted to the left and right of the WT curve, respectively. Indeed, at 4 and 32 Hz, peak contractile responses of vasa deferentia were greater in PAI-1-/- than in WT mice and smaller in t-PA-/- (Fig. 2).
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The contractile response elicited by EFS in the vas deferens from WT mice was not different from that of urokinase-deficient mice (see Fig. 2), suggesting that the PAS-induced modulation of sympathetic activity is independent of u-PA. To confirm this result, we used u-PAstop, a selective u-PA inhibitor (Stürzebecher et al., 1999). Concentration-response curves for the inhibition of rt-PA activity by u-PAstop (30 nM–300 µM) and t-PAstop (3 nM–300 µM) are shown in Fig. 3A. IC50 values were 3.1 ± 0.08 and 0.13 ± 0.02 µM for u-PAstop and t-PAstop, respectively. Having determined the relative inhibitory potency of u-PAstop and t-PAstop, we next assessed the effect of pretreatment with these inhibitors on the response of the WT vas deferens to EFS. At 10 µM (i.e., a concentration at which u-PAstop and t-PAstop inhibited t-PA activity by 80 and 100%, respectively; see Fig. 3A), both purinergic and adrenergic frequency-response curves were markedly shifted downwards by t-PAstop and only slightly, but not significantly, by u-PAstop (Fig. 3B). This indicated that the response of the vas deferens to EFS is independent of u-PA but dependent on t-PA.
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Because the neurogenic response of the isolated vas deferens to EFS is the result of pre- and postjunctional components (Todorov et al., 1996), we questioned whether the potentiating role of t-PA also involves postsynaptic sites. Thus, we investigated the contractile response of the vas deferens to exogenous ATP and NE in WT mice and in mice with various PAS deletions. Concentration-response curves for ATP (0.1–100 µM; Fig. 5A) and NE (0.01–100 µM; Fig. 5B) obtained in vasa from t-PA-/-, PAI-1-/-, plgn-/-, and u-PA-/- mice were superimposable on the curves obtained in vasa from WT mice. Comparable results were obtained with phenylephrine (data not shown). In addition, the amplitude of the contractile response of vasa deferentia to K+ (80 mM) was the same in WT, t-PA-/-, PAI-1-/-, plgn-/-, and u-PA-/- mice (Fig. 5C). Moreover, t-PAstop (10 µM) failed to modify the concentration-response curves for the contractile response of vasa deferentia to exogenous NE in WT, t-PA-/-, and PAI-1-/- mice (Fig. 6). The finding that various PAS deletions and selective inhibition of t-PA with t-PAstop did not influence the response of the vas deferens to stimulation of postjunctional sites suggested that t-PA selectively targets prejunctional sites in its modulation of the neurogenic contractile response and that neither plasminogen nor urokinase is involved in this action of t-PA.
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Exocytosis of NE and ATP from Mouse Heart Nerve Endings Is Enhanced by Tissue Plasminogen Activator and Is Independent of Plasminogen and Urokinase. Because our findings suggested a presynaptic site of t-PA action, we chose the cardiac synaptosomal preparation, an optimal model of sympathetic nerve endings, for the investigation of NE and ATP exocytosis (Seyedi et al., 1997; Sesti et al., 2003), to determine whether the action of t-PA entails a modulation of transmitter release. Upon depolarization with 50 and 100 mM K+, NE release from synaptosomes isolated from WT mouse hearts increased by 15 and 30% over basal level, respectively, and further increased to 40 and 45% in synaptosomes from PAI-1-/- mouse hearts (Fig. 7). In contrast, K+-induced NE release in synaptosomes from t-PA-/- hearts was 6-fold lower than WT. In synaptosomes from hearts of plgn-/- and u-PA-/- mice, NE exocytosis was not different from WT synaptosomes (Fig. 7). K+-induced synaptosomal ATP release increased by 10 and 50% over basal level with 50 and 100 mM K+, respectively (Fig. 8). Notably, the concentration-response curve for the K+-induced ATP release in WT synaptosomes was shifted downwards in the presence of t-PAstop (10 µM; Fig. 8A). Likewise, the concentration-response curve for the K+-induced ATP release in t-PA-/- synaptosomes was shifted downwards compared with the curve in WT synaptosomes (Fig. 8B). Thus, the t-PA-induced potentiation of neurogenic sympathetic responses is associated with an increase in both ATP and NE exocytosis, which is plasminogen- and urokinase-independent.
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Presence of t-PA Antigen in Vas Deferens and Mouse Heart Synaptosomes. t-PA antigen levels in vasa deferentia and cardiac synaptosomes isolated from WT mice were similar to those detected in vasa and synaptosomes from PAI-1-/-, u-PA-/-, and plgn-/- mice (Fig. 9). In contrast, t-PA antigen was undetectable in vasa deferentia and cardiac synaptosomes isolated from t-PA-/- mice. Interestingly, t-PA antigen level was 10-fold higher in cardiac synaptosomes than in whole heart (1.7 pg/mg protein in synaptosomes, see Fig. 9A, versus 0.173 ± 0.04 pg/mg in whole-heart homogenate).
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Discussion |
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1 receptors, respectively (Todorov et al., 1996; Westfall et al., 1996). Efficient control of synaptic transmission requires a rapid mechanism for terminating the actions of neurotransmitters. Amino acids and monoamines are taken back up into neurons by specific high-affinity transporters, whereas ATP is broken down in the extracellular space (Amara and Kuhar, 1993; Zimmermann and Braun, 1999). Quantities of released transmitters determine signaling strength. Typically, EFS of vas deferens elicits a biphasic twitch response. Most ATP is released during the first phase of contraction. Neuronally released ATP elicits excitatory junction potentials, which in turn initiate action potentials, resulting in the phasic component of the neurogenic response (Allcorn et al., 1986). Seconds later, NE is released into the sympathetic junction, leading to the tonic component of contraction. The first phase is blocked by ,-MeATP, and the second phase is blocked by prazosin (see Fig. 1). It is well known that with increasing stimulation frequencies, NE release quantitatively exceeds that of ATP; therefore, particularly during low-stimulation frequencies (
4 Hz), purinergic responses can be easily separated from adrenergic responses (>8 Hz). Numerous presynaptic modulators of the sympathetic nervous system have been described previously (Boehm and Kubista, 2002), but PAS components were not part of the list. We recently demonstrated that PAS plays a major role in sympathetic neurotransmission (Schaefer et al., 2006). In addition, there is increasing evidence that sympathetic neurons synthesize, transport, and release t-PA, thereby contributing significantly to plasmatic fibrinolytic capacity. Prominent t-PA immunostaining is present in sympathetic axons of small arteries, arterioles, and vasa vasorum (Lochner et al., 1998; O'Rourke et al., 2005). Evidence for an axonal source of t-PA activity includes: 1) a decreased blood fibrinolytic activity following guanethidine sympathectomy (Peng et al., 1999), 2) an increased regional blood t-PA activity during in vivo electrical stimulations of sympathetic nerves (Jern et al., 1994; Björkman et al., 2003), 3) much slower t-PA-releasing kinetics from resting cultured endothelial cells than from sympathetic neurons, and 4) very prominent arteriolar t-PA immunostaining in striated muscle arterioles, but not in venules (Huber et al., 2002). In addition, u-PA has been found to be present within the sympathetic nervous system (Pittman et al., 1989).
The aim of this study was to identify which PAS components modulate sympathetic neural activity and, in particular, to determine a possible role of u-PA and plasminogen. Using vasa deferentia isolated from gene-targeted mice, we found that only vasa isolated from t-PA-null mice displayed attenuated purinergic and adrenergic neurogenic responses to EFS. In contrast, responses evoked in vasa from u-PA-/- and plasminogen-/- mice were almost identical to those in WT mice. Thus, the interaction between PAS and sympathetic nervous system seems to be restricted to t-PA and most likely independent of u-PA and plasminogen availability. Indeed, as previously reported (Schaefer et al., 2006), we found an enhanced contractile response to EFS in vasa deferentia from PAI-1-/- mice. This enhanced response is probably related to increased t-PA activity, as evidenced by increased fibrinolytic capacity in PAI-1-/- mice (Carmeliet et al., 1993a, b, 1994).
Moreover, we investigated the effects of t-PAstop and u-PAstop, reversible competitive inhibitors of t-PA and u-PA, respectively, on the response to EFS and found additional evidence that only t-PA is involved in the neurogenic contraction. Indeed, only t-PAstop significantly attenuated the contractile response of vasa deferentia isolated from WT mice. Furthermore, t-PAstop failed to influence the contractile response to EFS of vasa from tPA-/- mice but markedly inhibited the response of vasa from PAI-1-/- mice. In contrast, a similar mild inhibition was observed in vasa from WT, u-PA-/-, and plgn-/- mice. Thus, our data clearly suggest that t-PA positively modulates sympathetic neurotransmission independently of urokinase and plasminogen availability. Although the potentiation of sympathetic neurotransmission could involve pre- and/or postjunctional sites, we found that the contractile response of the vas deferens to the administration of ATP and NE or to depolarization with K+ was the same in WT and every single PAS gene-deleted mouse. Moreover, t-PAstop did not modify the contractile response to NE, not only in WT vasa, but also in vasa from t-PA-/- and PAI-1-/- mice. Thus, t-PA modulates sympathetic neurotransmission in the vas deferens exclusively at prejunctional sites.
Prejunctional modulation of sympathetic neurotransmission by t-PA, as evidenced by the enhancement of both purinergic and adrenergic phases of the twitch response of vas deferens to EFS, could involve an enhanced release of ATP and NE and/or an increase in transmitter bioavailability due to reduced NE reuptake and diminished metabolism of released ATP. However, it is unlikely that t-PA would affect both NE reuptake and ATP metabolism, two totally different mechanisms of signal termination. Furthermore, pretreatment with desipramine, a neuronal NE transport inhibitor, diminished rt-PA-induced NE release (Schaefer et al., 2006), discounting the possibility that t-PA or t-PAstop might reduce NE re-uptake. Thus, it would seem that t-PA acts prejunctionally augmenting transmitter release. Indeed, we found that synaptosomes isolated from t-PA-/- mouse hearts released much less ATP and NE upon K+ depolarization than synaptosomes from WT hearts, whereas synaptosomes from PAI-1-/- hearts released much more NE. Again, this effect was restricted to t-PA because NE exocytosis in cardiac synaptosomes from u-PA-/- and plgn-/- mice was not different from that in WT. In addition, we recently demonstrated that administration of therapeutically relevant concentrations of rt-PA to guinea pig heart synaptosomes and primary cultures of human neuroblastoma SH-SY5Y cells releases NE as a function of its concentration (Schaefer et al., 2006). In agreement with our findings, a recent study demonstrated that the release of dopamine and acetylcholine evoked by depolarization in mouse brain was markedly enhanced by local rt-PA microinjections, whereas it was reduced by PAI-1 administration and in t-PA-/- mice (Ito et al., 2006).
We postulate that the activity level of t-PA will determine the magnitude of NE release. In fact we had found that pretreatment with t-PA stimulators (CNBrF) enhances r-tPA-induced NE release in guinea pig heart synaptosomes and SH-SY5Y cells (Schaefer et al., 2006), whereas the vas deferens response to EFS and NE release from cardiac synaptosomes are both markedly increased in PAI-1-/- mice (see Figs. 2, 4, and 7). Furthermore, we predict that the neuromodulatory function of the PAS, as observed in our models, is dependent upon the actions of t-PA and independent of u-PA and plasminogen. In fact, t-PA levels were below detection limits in vasa deferentia and cardiac synaptosomes of t-PA-/- mice. Moreover, only t-PA gene deletion, but not deletion of u-PA and plasminogen, caused a marked attenuation of neuronal function in the vas deferens and a striking decrease in NE release from cardiac synaptosomes compared with WT controls.
The mechanism of the prejunctional action of t-PA resulting in the potentiation of sympathetic neurotransmission remains speculative. Until now, a specific t-PA receptor has not been discovered, although different binding proteins for t-PA have been described previously (Melchor and Strickland, 2005). A specific receptor or substrate is likely to exist because the effects mediated by rt-PA clearly involve well known mechanisms of exocytotic and nonvesicular NE release (i.e., intracellular Ca2+,Na+/H+ exchanger, and NE transporter), as we previously showed in guinea pig heart synaptosomes (Schaefer et al., 2006). A less likely explanation entails a distinct protein-protein interaction. Multiple possibilities for protein-protein interactions involving different subdomains within the five-domain structure of t-PA have been described previously (Melchor and Strickland, 2005). Future studies using site-directed mutagenesis would be one approach to elucidate a specific t-PA subdomain interacting with the sympathetic nervous system. So far, the serine protease domain seems to be a candidate for the effects of t-PA.
Interestingly, brain and heart contain the highest concentrations of t-PA (Christie et al., 1999); this observation is likely to have important pathophysiological implications. Indeed, we found a 10-fold enrichment in t-PA antigen in cardiac synaptosomes compared with the intact heart. Notably, the vas deferens with its dense sympathetic innervation had t-PA antigen levels similar to those of cardiac synaptosomes. Thus, our observations may be applicable to all sympathetically innervated tissues. Administration of r-tPA for thrombolysis in acute myocardial infarction or stroke is still considered an important treatment option. Inasmuch as vascular patency is the major goal to preserve cardiac function in myocardial infarction, magnification of sympathetic stress with rt-PA thrombolysis carries potential risks. In this regard, we found that NE spillover and ventricular arrhythmias during ischemia/reperfusion were markedly decreased in hearts of t-PA-/- mice compared with WT and PAI-1-/- hearts (Schaefer et al., 2006).
In summary, we have used mouse models with single PAS component deletions (i.e., t-PA-/- or PAI-1-/-) to uncover interactions between plasminogen-activating and sympathetic nervous systems. Extending previous data from our laboratory, we offer novel evidence that the neuromodulatory action of PAS is exclusively related to t-PA and independent of u-PA and plasminogen availability. Safeguarding the thrombolytic activity of t-PA and eliminating its prosympathetic effects should be an important goal in preclinical research.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: t-PA, tissue plasminogen activator; u-PA, urinary-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; NE, norepinephrine; PAS, plasminogen activator system; -/-, knockout; plgn, plasminogen; KH, Krebs-Henseleit; EFS, electrical field stimulation; PPADS, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid; ,-MeATP, ,-methylene-ATP; ANOVA, analysis of variance; WT, wild type.
1 Current affiliation: Medizinische Klinik II, Universitätsklinikum Schleswig Holstein, Campus Lübeck, Germany. 
Address correspondence to: Dr. Roberto Levi, Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10021. E-mail: rlevi{at}med.cornell.edu
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