The plasminogen activator system modulates sympathetic nerve function

Abstract

Sympathetic neurons synthesize and release tissue plasminogen activator (t-PA). We investigated whether t-PA modulates sympathetic activity. t-PA inhibition markedly reduced contraction of the guinea pig vas deferens to electrical field stimulation (EFS) and norepinephrine (NE) exocytosis from cardiac synaptosomes. Recombinant t-PA (rt-PA) induced exocytotic and carrier-mediated NE release from cardiac synaptosomes and cultured neuroblastoma cells; this was a plasmin-independent effect but was potentiated by a fibrinogen cleavage product. Notably, hearts from t-PA-null mice released much less NE upon EFS than their wild-type (WT) controls (i.e., a 76.5% decrease; P<0.01), whereas hearts from plasminogen activator inhibitor-1 (PAI-1)-null mice released much more NE (i.e., a 275% increase; P<0.05). Furthermore, vasa deferentia from t-PA-null mice were hyporesponsive to EFS (P<0.0001) but were normalized by the addition of rt-PA. In contrast, vasa from PAI-1-null mice were much more responsive (P<0.05). Coronary NE overflow from hearts subjected to ischemia/reperfusion was much smaller in t-PA-null than in WT control mice (P<0.01). Furthermore, reperfusion arrhythmias were significantly reduced (P<0.05) in t-PA-null hearts. Thus, t-PA enhances NE release from sympathetic nerves and contributes to cardiac arrhythmias in ischemia/reperfusion. Because the risk of arrhythmias and sudden cardiac death is increased in hyperadrenergic conditions, targeting the NE-releasing effect of t-PA may have valuable therapeutic potential.

Figure 1.

t-PA inhibition attenuates sympathetic responses in the vas deferens and decreases NE exocytosis in cardiac synaptosomes. (A and B) Frequency response curves for the contractile responses of the isolated guinea pig (GP) vas deferens to electrical field stimulation (EFS; 0–64 Hz, supramaximal voltage; stimulation duration of 15 s at 5-min intervals, with pulses lasting 1 ms each). Typically, the contractile response of the vas deferens to EFS consists of two phases: an initial spike (A; purinergic) followed by a plateau (B; adrenergic). Peak response amplitudes (means ± SE [error bars]; n = 4) are expressed as percentages of the response to 40 mM K⁺. Responses were recorded either in the absence or presence of 1 and 10 μM tPA_stop. (C, top) Release of endogenous NE from guinea pig heart synaptosomes by depolarization with 3–100 mM K⁺. Points are mean increases in NE release above basal level (± SE; n = 10; EC₅₀ = 32.8 mM). (bottom) Concentration response curves for the inhibition of NE exocytosis (elicited by depolarization with 100 mM K⁺) by t-PA_stop or rPAI-1. Equieffective inhibitory concentrations of t-PA_stop and rPAI-1 were predetermined in a photometric assay of t-PA activity inhibition (IC₅₀, 1.2 μM tPA_stop and 9.2 nM rPAI-1; not depicted). Points (means ± SE; n = 4) are expressed as the percent inhibition of NE release by 100 mM K⁺.

Figure 2.

rt-PA elicits NE release in guinea pig heart synaptosomes and neuroblastoma cells: potentiation by fibrinogen. (A and C) Concentration response curves for the NE-releasing effects of rt-PA in guinea pig heart synaptosomes (endogenous NE) and human SH-SY5Y neuroblastoma cells ([³H]NE). Points are means (± SE [error bars]; n = 8–14) of percent increases in NE release above baseline. (B and D) Coincubation of rt-PA with 200 μg/ml of the fibrinogen digest CNBr-F significantly potentiates the NE-releasing effect of 1 (B) and 0.3 μg/ml (D) rt-PA in guinea pig heart synaptosomes and SH-SY5Y neuroblastoma cells. Bars are means (± SE; n = 4–8) of increases in NE release above baseline. *, P < 0.05 versus baseline; **, P < 0.01 versus baseline.

Figure 3.

Mechanisms of rt-PA–induced NE release in guinea pig heart synaptosomes. (A) Concentration response curves for the NE-releasing effect of rt-PA and plasmin (each at 0.1–10 μg/ml) in guinea pig (GP) heart synaptosomes. As opposed to rt-PA, plasmin did not elicit NE release. Points are means (± SE [error bars]; n = 10 for r-tPA and n = 4 for plasmin). (B) Effects of various inhibitors on the NE-releasing effect of 10 μg/ml r-tPA. Preincubation with the inhibitors reduced the rt-PA–induced NE release from guinea pig heart synaptosomes. 100 nM Ω-conotoxin (ω-CTX), 10 μM BAPTA-AM, 300 nM desipramine (DMI), 30 μM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and 1 μM cariporide (HOE642) each significantly inhibited rt-PA–induced NE release. In contrast, 0.2 μM α₂-antiplasmin (α₂-AP) did not affect the NE-releasing effect of rt-PA. Bars are means (± SE; n = 3–12) of percent increases in NE release above basal level. *, P < 0.05 versus rt-PA alone.

Figure 4.

Vas deferens contraction and NE exocytosis in the heart are both attenuated in t-PA–null mice, potentiated in mice with PAI-1 gene deletion, and unaffected in plasminogen-deficient mice. (A and B) Frequency response curves for the contractile responses of the isolated mouse vas deferens to electrical field stimulation (EFS; 0–64 Hz, supramaximal voltage; every 1 ms for 15 s). Peak response amplitudes of both purinergic and adrenergic phases (means ± SE [error bars]; n = 6–9) are expressed as percentages of the response to 80 mM K⁺. Vasa deferentia isolated from t-PA^−/− mice developed markedly less tension in response to EFS than vasa from WT control mice. In contrast, vasa from PAI-1^−/− mice developed more tension than vasa from WT mice. Vasa from plasminogen^−/− mice developed the same tension as vasa from WT mice. (C) Coronary NE overflow from isolated mouse hearts in response to EFS (0–9 Hz; 5 V for a duration of 60 s, with pulses of 2 ms each). Hearts were perfused with buffer containing 0.1 μM desipramine, 0.1 μM rauwolscine, 1 μM atropine, and 10 μM hydrocortisone. NE overflow was significantly smaller in hearts from t-PA^−/− mice than in hearts from WT mice, whereas it was significantly greater in PAI-1^−/− hearts. Points are means (± SE; n = 6–9) of x-fold increases in NE overflow above basal levels. (D) K⁺-induced NE exocytosis in mouse heart synaptosomes. Points are means (± SE; n = 12–16) of increases in NE release above basal levels. Synaptosomes isolated from hearts of t-PA^−/− mice released significantly less NE in response to K⁺ than synaptosomes from WT hearts. In contrast, synaptosomes from PAI-1^−/− hearts released greater amounts of NE than synaptosomes from WT hearts, whereas NE exocytosis in synaptosomes from plasminogen^−/− mice was not different from that of synaptosomes from WT hearts.

Figure 5.

The administration of rt-PA restores the contractile response of vasa deferentia isolated from mice lacking t-PA. (A) Contractile responses (both purinergic and adrenergic) of vasa deferentia isolated from WT mice to EFS (30 Hz, supramaximal voltage; duration of 15 s at 5-min intervals, with pulses of 1 ms) either in the absence or presence of 0.6 and 1 μg/ml rt-PA (15-min incubation). The 0.6-μg/ml concentration was ineffective, whereas at 1 μg/ml, rt-PA potentiated both the purinergic and adrenergic responses by ∼70 and ∼50%, respectively (*, P < 0.05). Bars represent the mean contractile response amplitude, which is expressed as a percentage of the response to 80 mM K⁺ (± SE [error bars]; n = 4–8). (B) Frequency response curves for the contractile response of vasa deferentia to EFS (0–32 Hz, supramaximal voltage; for 1 ms every 15 s). Vasa were isolated from t-PA^−/− mice and from their WT controls. Incubation of vasa from t-PA^−/− mice with rt-PA (at the subthreshold concentration of 0.6 μg/ml for 15 min) restored the depressed contractile response to EFS to the same magnitude as in vasa from control mice. Points are means (± SE; n = 8 and 4 for WT and t-PA^−/−, respectively) of maximal contractile amplitudes expressed as percentages of the response to 80 mM K⁺. Arrows indicate the upward shifts elicited by the administration of rt-PA in vasa deferentia isolated from animals deprived of t-PA.

Figure 6.

t-PA potentiates sympathetic responses by an action at prejunctional sites. (A) Noncumulative concentration response curves for the contractile response of mouse vas deferens to the administration of exogenous ATP. Vasa deferentia were isolated from t-PA^−/−, PAI-1^−/−, and their WT control mice. The maximum contractile response for each increment in ATP concentration occurred within 30 s; ATP was quickly washed out thereafter to prevent receptor desensitization. The following higher ATP concentration was added after 30 min of reequilibration. (B) Cumulative concentration response curves for the contractile response of mouse vas deferens to the administration of exogenous NE. Vasa deferentia were isolated from t-PA^−/−, PAI-1^−/−, and their WT control mice. The finding that the concentration response curves obtained from vasa deferentia of gene-deleted mice were superimposable on the curves obtained from their WT controls indicates that the lack of t-PA or PAI-1 does not influence postsynaptic responses at sympathetic junctions and that the action of t-PA on sympathetic neurons is limited to presynaptic sites. (A and B) Points are means (± SE [error bars]; n = 7–10) of maximal contractile responses to ATP (A) and NE (B) expressed as percentages of the response to 80 mM K⁺.

Figure 7.

t-PA promotes NE release and associated arrhythmias in myocardial ischemia/reperfusion. (A) Coronary NE overflow before ischemia (basal) and during 10-min reperfusion in hearts isolated from t-PA^−/−and PAI-1^−/− mice and their WT controls. Global stop-flow ischemia was applied for 30 min after an initial stabilization period of 30 min. NE overflow was individually adjusted for coronary flow and heart weight. Points are means (± SE [error bars]; n = 9–11). Asterisks indicate significant differences from 5-min reperfusion levels in t-PA^−/− hearts (*, P < 0.05). (B) Analysis of ventricular arrhythmias during reperfusion of the same hearts as in A. The incidence of high-grade ventricular arrhythmias (i.e., ventricular tachycardia [VT] and ventricular fibrillation [VF]) is expressed as percentages of the total number of hearts used in each of the three groups (± SE). The duration of VT and VF represents the cumulative duration of arrhythmia during the 30-min reperfusion. The occurrence of premature ventricular contractions (PVCs) is expressed as the total number (n) of PVCs counted during the 30-min reperfusion. Bars are means (± SE; n = 9–11). Asterisks indicate significant differences from WT and PAI-1^−/− hearts (incidence of VT/VF) and from WT and t-PA^−/− hearts (number of PVCs).