Urokinase plasminogen activator regulates pulmonary arterial contractility and vascular permeability in mice

Abstract

The concentration of urokinase plasminogen activator (uPA) is elevated in pathological settings such as acute lung injury, where pulmonary arterial contractility and permeability are disrupted. uPA limits the accretion of fibrin after injury. Here we investigated whether uPA also regulates pulmonary arterial contractility and permeability. Contractility was measured using isolated pulmonary arterial rings. Pulmonary blood flow was measured in vivo by Doppler and pulmonary vascular permeability, according to the extravasation of Evans blue. Our data show that uPA regulates the in vitro pulmonary arterial contractility induced by phenylephrine in a dose-dependent manner through two receptor-dependent pathways, and regulates vascular contractility and permeability in vivo. Physiological concentrations of uPA (≤1 nM) stimulate the contractility of pulmonary arterial rings induced by phenylephrine through the low-density lipoprotein receptor-related protein receptor. The procontractile effect of uPA is independent of its catalytic activity. At pathophysiological concentrations, uPA (20 nM) inhibits contractility and increases vascular permeability. The inhibition of vascular contractility and increase of vascular permeability is mediated through a two-step process that involves docking to N-methyl-d-aspartate receptor-1 (NMDA-R1) on pulmonary vascular smooth muscle cells, and requires catalytic activity. Peptides that specifically inhibit the docking of uPA to NMDA-R, or the uPA variant with a mutated receptor docking site, abolished both the effects of uPA on vascular contractility and permeability, without affecting its catalytic activity. These data show that uPA, at concentrations found under pathological conditions, reduces pulmonary arterial contractility and increases permeability though the activation of NMDA-R1. The selective inhibition of NMDAR-1 activation by uPA can be accomplished without a loss of fibrinolytic activity.

Figure 1.

Effect of urokinase-type plasminogen activator (uPA) on the contraction of arterial rings. (A) Effect of uPA on the contraction of pulmonary arterial rings. Contraction of isolated pulmonary arterial rings was induced by phenylephrine (PE) at the indicated concentrations in the absence (solid squares) or presence of 1 nM (open squares) or 20 (open triangles) nM uPA. (B) Effect of uPA on the contraction of isolated aortic rings. Contraction of isolated aortic rings was induced by PE at the indicted concentrations in the absence (solid squares) or presence of 1 nM (open squares) or 20 nM (open triangles) uPA. The mean ± SD of four experiments is shown.

Figure 2.

Involvement of LRP and uPA catalytic activity in uPA-induced alterations of pulmonary arterial contractility. (A) Contraction of isolated pulmonary arterial rings was induced by increasing concentrations of PE from 10⁻¹⁰ M to 10⁻⁵ M (for more details, see Methods in the online supplement) in the absence (Control) or presence of 1 nM uPA, alone or together with recombinant receptor-associated protein (rRAP), anti–low-density lipoprotein–related receptor antibody (anti LRP Ab), or irrelevant IgG (IR IgG). (B) Contraction of isolated pulmonary arterial rings was induced by increasing concentrations of PE (as in A) in the absence (Control) or presence of 20 nM uPA, alone or together with rRAP or anti-LRP antibody (anti LRP Ab). (C) Contraction of isolated pulmonary arterial rings was induced by PE at the indicted concentrations in the absence (open squares) or presence of 1 (open triangles) or 20 nM (solid squares) of a catalytically inactive variant of uPa containing a single mutation within the active site (uPA-S³⁵⁶A). The mean ± SD of three experiments is shown. *Statistical significance between columns 1 and 2; ^#statistical significance between column 1 and the other columns.

Figure 3.

Involvement of N-methyl-d-aspartate receptors (NMDA-Rs) in the contraction of pulmonary arterial rings. Contraction of isolated pulmonary arterial rings was induced by PE in the absence (Control) or presence of 20 nM uPA, alone or together with plasminogen activator inhibitor–1 (PAI-1)–derived peptide (uPA + Pep, 1 μM), NMDA-R antagonist MK-801 (100 nM), or the NMDA-R agonist glutamate (Glut, 150 μM). The mean ± SD of three experiments is shown. *Statistical significance between columns 1 and 2; ^#statistical significance between column 1 and the other columns.

Figure 4.

Effect of uPA and NMDA-Rs on pulmonary vascular permeability. Lung permeability, as measured by the extravasation of intravenously administered Evans blue into the bronchoalveolar lavage, was determined after intravenous injection of saline (Control), wild-type (WT) uPA (uPA, 1 mg/kg), catalytically inactive uPA (uPA S356A), PAI-1 derived peptide (Pep), uPA plus PAI-1–derived peptide (uPA + Pep, 1μM), the NMDA-R antagonist MK-801, (or uPA plus MK-801. The mean ± SD of three experiments is shown. OD = optical density. *Statistical significance between columns 1 and 2; ^#statistical significance between column 1 and the other columns.

Figure 5.

Interaction of uPA and NMDA-R1 in pulmonary arteries. (A) uPA binds to NMDA-R from the pulmonary artery. Homogenates of pulmonary arterial rings isolated from uPA^−/− mice were preincubated with the indicated concentrations of uPA at 4°C, and precipitated with an antibody against uPA, followed by immunoblotting with an antibody against the NR1 subunit of NMDA-R1. The results of an experiment representative of three independent analyses are shown. (B) The docking sites in uPA mediate its interactions with NMDA-R. As in A, homogenates of pulmonary arteries rings isolated from uPA^−/− mice were preincubated with WT uPA (40 nM) (uPA), WT uPA with PAI-1 derived peptide EEIIMD (EEIIMD, 1 μM), or the uPA variant that lacks a functional docking site (ΔDSuPA) at 4°C, precipitated with an antibody against uPA, followed by immunoblotting with an antibody against the NR1 subunit of NMDA-R1. The results of an experiment representative of three independent analyses are shown. (C) uPA cleaves the NR1 subunit of NMDA-R1. Pulmonary arterial rings isolated from uPA^−/− mice were incubated in buffer alone or buffer containing 20 nM WT uPA (uPA), WT uPA with PAI-1–derived peptide EEIIMD (1 μM), or the uPA variant that lacks a functional docking site (ΔDSuPA) for 120 minutes at 37°C. Tissue homogenates were analyzed by SDS-PAGE and Western blotting, using an antibody against the NR1 subunit of NMDA-R1. The results of an experiment representative of three independent analyses are shown.

Figure 6.

The uPA variant that lacks a functional docking site (ΔDSuPA) fails to affect the contractility or permeability of the pulmonary vasculature. (A) The contraction of pulmonary arterial rings was induced by PE in the absence (Control) or presence of 20 nM WT uPA or ΔDSuPA. The mean ± SD of three experiments is shown. (B) Lung permeability was measured as in Figure 5 after intravenous injection of saline (Control), WT uPA (uPA, 1 mg/kg), or ΔDSuPA. The mean ± SD of three experiments is shown. EC = effective concentration; OD = optical density.