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Cardiovascular Research 1998 38(2):485-492; doi:10.1016/S0008-6363(98)00017-0
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Copyright © 1998, European Society of Cardiology

The L-arginine/nitric oxide pathway contributes to the acute release of tissue plasminogen activator in vivo in man

David E Newbya,b,*, Robert A Wrightb, Pamela Dawsonc, Christopher A Ludlamc, Nicholas A Boonb, Keith A.A Foxb and David J Webba

aClinical Pharmacology Unit and Research Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
bDepartment of Cardiology, University of Edinburgh, Royal Infirmary, Lauriston Place, Edinburgh EH3 9YW, UK
cDepartment of Haematology, University of Edinburgh, Royal Infirmary, Lauriston Place, Edinburgh EH3 9YW, UK

* Corresponding author. Tel.: +44 (131) 332-1205; Fax: +44 (131) 343-6017; E-mail: d.e.newby@ed.ac.uk

Received 28 October 1997; accepted 18 December 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Effective endogenous fibrinolysis requires rapid release of endothelial tissue plasminogen activator (t-PA). Using the nitric oxide synthase inhibitor, L-NG-monomethylarginine (L-NMMA), we examined the contribution of endogenous nitric oxide to substance P-induced t-PA release in vivo in man. Methods: Blood flow and plasma fibrinolytic and haemostatic factors were measured in both forearms of 8 healthy male volunteers who received unilateral brachial artery infusions of substance P (2–8 pmol/min) and L-NMMA (1–4 µg/min). Results: Substance P caused dose-dependent increases in blood flow (P<0.001) and plasma t-PA antigen (P=0.04) and activity (P<0.001) concentrations confined to the infused forearm, but had no effect on plasminogen activator inhibitor type 1 (PAI-1) or von Willebrand factor concentrations. In the presence of L-NMMA, substance P again caused significant increases in blood flow (P<0.001) and t-PA antigen (P=0.003) and activity (P<0.001) concentrations but these increases were significantly less than with substance P alone (P<0.001, P=0.05 and P<0.01, respectively). L-NMMA alone significantly reduced blood flow in the infused arm, but had no measurable effect on t-PA or PAI-1 concentrations. Conclusions: The L-arginine/nitric oxide pathway contributes to substance P-induced t-PA release in vivo in man. This provides an important potential mechanism whereby endothelial dysfunction increases the risk of atherothrombosis through a reduction in the acute fibrinolytic capacity.

KEYWORDS Thrombolysis; Endothelial factor; Nitric oxide; Blood flow; Endothelial function


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The endogenous fibrinolytic system can have important clinical effects as exemplified by the observation that in ~30% of patients with an acute myocardial infarction, the infarct-related artery spontaneously reperfuses within 12 h [1–3]. The ability of the endothelium to release tissue plasminogen activator (t-PA) rapidly is crucial if endogenous fibrinolysis within the arterial circulation is to be effective, with thrombus dissolution being much more effective if t-PA is incorporated during, rather than after, thrombus formation [4, 5]. Epidemiological studies in a healthy male population and patients with ischaemic heart disease have shown a relationship between plasma fibrinolytic parameters and future cardiovascular events, such as stroke or myocardial infarction [6–9]. However, the capacity of endothelial cells to release t-PA from intracellular storage pools, and the rapidity with which this can be mobilised, may not be reflected in the basal circulating plasma concentrations of t-PA antigen or activity [10].

Endothelial cell culture techniques have limitations in the investigation of t-PA release and may not be truly representative of the in vivo function of these cells. The amount of t-PA released in culture is small and necessitates prolonged incubation periods and sensitive assays. Moreover, the phenotype of endothelial cells in culture, and the ability to release t-PA, changes with increasing passages. In contrast, under in vivo physiological conditions, the endothelium is arranged within a non-planar three-dimensional vascular bed, has a more favourable volume to surface area ratio, and is exposed to pulsatile blood flow and pressure changes. We have recently described an in vivo model to assess acute t-PA release in man [11]. Using intra-brachial infusions of substance P, we have shown a dose-dependent release of t-PA from the human forearm without causing significant release of von Willebrand factor (vWf) or plasminogen activator inhibitor type 1 (PAI-1). This suggests either a selective action of substance P or the lack of a rapidly translocatable pool of PAI-1 and vWf. However, we have previously used only brief (~10 min) substance P infusions [11]and protracted stimulation may release these factors [12–14].

Substance P causes endothelium dependent vasodilatation [15]which is mediated by the endothelial cell neurokinin type 1 receptor [16]and is, in part, related to the release of nitric oxide [17–19]. However, because t-PA release is not seen with infusions of the nitric oxide donor and vasodilator, sodium nitroprusside [11, 20], an increase in nitric oxide and blood flow together do not release t-PA from the endothelium. Nevertheless, it remains a possibility that the L-arginine/nitric oxide pathway contributes to substance P-induced t-PA release.

Therefore, the aims of the current study were two-fold: first, to ascertain whether prolonged substance P infusion can cause vWf or PAI-1 release; and second, to determine whether nitric oxide synthase inhibition using L-NG-monomethylarginine (L-NMMA) affects basal or substance P-induced t-PA release.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Subjects
Eight healthy men aged between 20 and 33 years participated in three studies which were undertaken with the approval of the local research ethics committee and in accordance with the Declaration of Helsinki. The written informed consent of each subject was obtained before entry into the study. None of the subjects received vasoactive or non-steroidal anti-inflammatory drugs in the week before each phase of the study, and all abstained from alcohol for 24 h, and from food, tobacco and caffeine-containing drinks for at least 5 h, before each study. All studies were performed in a quiet, temperature-controlled room maintained at 23.5–24.5°C.

2.2 Intra-arterial administration and drugs
The brachial artery of the non-dominant arm was cannulated with a 27-standard wire gauge steel needle (Cooper's Needle Works, Birmingham, UK) under 1% lignocaine (Xylocaine; Astra Pharmaceuticals, Kings Langley, UK) local anaesthesia. The cannula was attached to a 16-gauge epidural catheter (Portex, Hythe, UK) and patency maintained by infusion of saline (0.9%: Baxter Healthcare, Thetford, UK) via an IVAC P1000 syringe pump (IVAC, Basingstoke, UK). The total rate of intra-arterial infusions was maintained constant throughout all studies at 1 ml/min. Pharmaceutical-grade substance P (Clinalfa, Läufelfingen, Switzerland) and L-NG-monomethylarginine (L-NMMA; Clinalfa) were administered following dissolution in saline.

2.3 Forearm blood flow and blood pressure
Blood flow was measured in both forearms by venous occlusion plethysmography using mercury-in-silastic strain gauges applied to the widest part of the forearm [21]. During measurement periods, the hands were excluded from the circulation by rapid inflation of the wrist cuffs to a pressure of 220 mmHg using E20 Rapid Cuff Inflators (D.E. Hokanson, Washington, USA). Upper arm cuffs were inflated intermittently to 40 mmHg for 10 s in every 15 s to achieve venous occlusion and obtain plethysmographic recordings. Analogue voltage output from an EC-4 Strain Gauge Plethysmograph (D.E. Hokanson) was processed by a MacLab analogue-to-digital converter and Chart v3.3.8 software (AD Instruments, Castle Hill, Australia) and recorded onto a MacIntosh Classic II computer (Apple Computers, Cupertino, USA). Calibration was achieved using the internal standard of the plethysmograph.

Blood pressure was monitored in the non-infused arm at intervals throughout each study using a semi-automated non-invasive oscillometric sphygmomanometer [22](Takeda UA 751, Takeda Medical, Tokyo, Japan).

2.4 Venous sampling and assays
Venous cannulae (17-gauge) were inserted into large subcutaneous veins of the antecubital fossa in both arms as described previously [23]. Ten ml of blood was withdrawn simultaneously from each arm and collected into acidified buffered citrate (Biopool Stabilyte, Umeå, Sweden; for t-PA assays) and citrate (Monovette, Sarstedt, Nümbrecht, Germany; for PAI-1 assays) tubes, and kept on ice before being centrifuged at 2000 g for 30 min at 4°C. Platelet-free plasma was decanted and stored at –80°C before assay.

Plasma PAI-1 and t-PA antigen concentrations were determined using an enzyme-linked immunosorbent assay (ELISA); Coaliza PAI-1 [24]and Coaliza t-PA [25](Chromogenix AB, Mölndal, Sweden) respectively. Plasma PAI-1 and t-PA activities were determined by a photometric method, Coatest PAI-1 [26]and Coaset t-PA [27](Chromogenix). Intra-assay coefficients of variation were 7.0 and 5.5% for t-PA and PAI-1 antigen, and 4.0 and 2.4% for activity, respectively. Inter-assay coefficients of variability were 4.0, 7.3, 4.0 and 7.6%, respectively. The sensitivities of the assays were 2.5 ng/ml, 0.5 ng/ml, 5 AU/ml and 0.10 IU/ml, respectively. vWf antigen was determined [28]using an ELISA (Dako, Glostrup, Denmark) with a sensitivity of 0.05 IU/ml. The intra-assay and inter-assay coefficients of variability were 5.2 and 7.3%, respectively. Factor VIII:C procoagulant activity was determined using a standard one-stage assay on an ACL-3000+coagulometer (Instrumentation Laboratory, Warrington, UK). Haematocrit was determined by capillary tube centrifugation of blood anticoagulated by ethylene diamine tetraacetic acid and was obtained from the infused forearm at baseline and at 120 min.

2.5 Study design
On 3 separate occasions, at 09.00 h, subjects attended fasted and rested recumbent throughout each study. Strain gauges and cuffs were applied and the brachial artery of the non-dominant arm cannulated. Throughout all protocols, measurements of forearm blood flow were made every 10 min. Saline was infused for the first 30 min to allow time for equilibration and the final blood flow measurement during saline infusion was taken as the basal forearm blood flow. Thereafter, subjects underwent the following protocols, in random order, each separated by at least 1 week: protocol 1, each subject received intra-arterial substance P at 2, 4 and 8 pmol/min, for 10 min at each dose, followed by a continuous infusion of 8 pmol/min for a further 90 min; protocol 2, L-NMMA was co-infused at 4 µmol/min for 10 min before and throughout the same substance P infusion as protocol 1; and protocol 3, subjects received intra-arterial L-NMMA at 1, 2 and 4 µmol/min for 10 min at each dose followed by a continuous infusion of 4 µmol/min for a further 90 min. Venous samples were withdrawn from each arm at baseline and at 10, 20, 30, 50, 80 and 120 min after the start of substance P (for protocols 1 and 2) or L-NMMA infusion (protocol 3).

2.6 Data analysis and statistics
Plethysmographic data were extracted from the Chart data files and forearm blood flows were calculated for individual venous occlusion cuff inflations by use of a template spreadsheet (Excel v4.0; Microsoft, Cambridge, USA). Recordings from the first 60 s after wrist-cuff inflation were not used because of the reflex vasoconstriction this causes [21]. Usually, the last five flow recordings in each 3-min measurement period were calculated and averaged for each arm. Estimated net release of t-PA activity and antigen was defined previously [11]as the product of the infused forearm plasma flow (based on the mean haematocrit, HCt, and the infused forearm blood flow, FBF) and the concentration difference between the infused ([t-PA]inf) and non-infused arms ([t-PA]non-inf).


Formula

Data were examined, where appropriate, by two-way analysis of variance (ANOVA) with repeated measures and two-tailed paired Student's t-test using Excel v4.0 (Microsoft). Tachyphylaxis was assessed by comparing the 30-min (peak) and 120-min (final) values with a two-tailed paired Student's t-test. Area under the curve (AUC) was calculated for the estimated net release of t-PA across the study period. All results are expressed as mean±s.e.m. Statistical significance was taken at the 5% level.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
All subjects were normotensive and there were no significant changes in blood pressure, heart rate or blood flow in the contralateral arm throughout any of the studies (Table 1). Haematocrit decreased slightly in each study (Table 1). Between the 3 protocols, there were no significant differences in the baseline values of blood pressure, heart rate, forearm blood flow, haematocrit or plasma concentrations of t-PA and PAI-1 antigen and activity.


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  		Table 1 Haemodynamics and haematocrit at baseline and completion of the 3 study protocols

 
3.1 Isolated infusions of substance P and L-NMMA
Substance P increased blood flow in the infused arm (P<0.001) in a dose-dependent manner (Fig. 1 and Table 2) reaching a maximum increase of 15.9±1.9 ml/100 ml/min after 10 min at 8 pmol/min. Following prolonged infusion, substance P-induced vasodilatation demonstrated tachyphylaxis and decreased to 12.1±1.3 ml/100 ml/min after 100 min of substance P at 8 pmol/min (P<0.003 vs. 10 min). In comparison to the non-infused arm, substance P caused a dose-dependent increase in venous plasma t-PA activity (P<0.001) and antigen (P<0.04) concentrations of the infused arm which did not undergo significant tachyphylaxis (Fig. 2). Concentrations of plasma PAI-1 activity were also reduced in the infused arm (P=0.04; Fig. 2). In contrast, there were no significant changes in plasma PAI-1 antigen, vWf or factor VIII:C concentrations in either arm (Fig. 2 and Table 2).


Figure 1
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  		Fig. 1 Infused forearm blood flow and estimated net release of t-PA antigen and activity during protocol 1 (substance P alone, {circ}), protocol 2 (substance P and L-NMMA, bullet) and protocol 3 (L-NMMA alone; {blacksquare}). *P<0.001; {dagger}P≤0.05; {ddagger}P=0.09 (ANOVA).

 

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  		Table 2 Blood flow and plasma von Willebrand factor and factor VIII:C activity concentrations in both arms during isolated substance P infusion: protocol 1

 

Figure 2
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  		Fig. 2 Plasma concentrations of t-PA and PAI-1 antigen (solid lines) and activity (dashed lines) in the infused (bullet and {blacksquare}, respectively) and non-infused ({circ} and {square}, respectively) arms in the 3 protocols. *P<0.001; {dagger}P<0.003; {ddagger}P≤0.04 (ANOVA).

 
L-NMMA decreased blood flow in the infused arm (P<0.001) in a dose-dependent manner (Fig. 1 and Table 2) reaching 2.1±0.2 ml/100 ml/min after 100 min at 4 µmol/min. There were no significant changes in the concentrations of plasma t-PA and PAI-1 antigen or activity in either arm during infusion of L-NMMA (Fig. 2).

3.2 Co-infusion of L-NMMA and substance P
In the presence of L-NMMA, substance P increased blood flow in the infused arm (P<0.001) in a dose-dependent manner (Fig. 1) reaching a maximum increase of 13.7±1.7 ml/100 ml/min after 10 min at 8 pmol/min. This response underwent tachyphylaxis and decreased to 9.3±1.4 ml/100 ml/min after 100 min of substance P at 8 pmol/min (P<0.002 vs. 10 min). In comparison, with the non-infused arm, substance P co-infused with L-NMMA caused a dose-dependent increase in plasma t-PA activity (P<0.001) and antigen (P<0.003) concentrations of the infused arm which did not undergo significant tachyphylaxis (Fig. 2). L-NMMA caused a significant attenuation of substance P-induced increases in blood flow (P<0.001) and plasma t-PA activity concentrations (P<0.003) in the infused forearm, but not plasma t-PA antigen.

3.3 Estimated net t-PA production
L-NMMA infused alone had no significant effects on t-PA release: 95% confidence intervals for t-PA antigen and activity release are 0.31 to –0.68 ng/100 ml/min and 0.27 to –0.06 IU/100 ml/min, respectively. Substance P caused dose-dependent increases in the estimated net release of t-PA antigen and activity in the presence or absence of L-NMMA (P<0.001) which did not undergo significant tachyphylaxis. However, the magnitude of the increase in release of both t-PA antigen (P=0.05) and activity (P<0.01) was significantly reduced in the presence of L-NMMA (Fig. 1). L-NMMA reduced the AUC for the substance P-induced release of t-PA antigen and activity by 40 and 46% respectively.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We have shown that intra-brachial substance P infusion increases forearm blood flow and plasma t-PA concentrations for up to 2 h without a demonstrable effect on plasma PAI-1 or vWf concentrations. Although the nitric oxide synthase inhibitor, L-NMMA, significantly reduced forearm blood flow without affecting basal t-PA release, it inhibited the increases in blood flow, plasma t-PA concentrations and t-PA release produced by substance P administration in the forearm. These data suggest that the L-arginine/nitric oxide pathway contributes to substance P-induced t-PA release in vivo in man. In contrast, we [11]and others [20]have shown previously that t-PA release is not seen with the large local increases in nitric oxide delivery and blood flow associated with infusions of the nitric oxide donor, sodium nitroprusside. Taken together, these findings indicate that increases in nitric oxide and blood flow are not sufficient per se to release t-PA but, through the L-arginine/nitric oxide pathway, are able to enhance substance P-induced t-PA release.

The permissive role of intracellular mediators in the mechanism of t-PA release has been described previously. In the rat perfused hindlimb model, increasing intracellular calcium alone is insufficient to cause t-PA release whilst it is essential for bradykinin induced t-PA release [29]. However, the regulation of t-PA release is complex and may involve several signal transduction pathways [30]. This is reflected by the diversity of mediators, such as thrombin, bradykinin and desmopressin, which can release t-PA and increase t-PA activity [12, 13, 31, 32]. One can, therefore, only speculate as to whether our findings extend to the acute t-PA release seen with in situ thrombosis. However, both nitric oxide mediated endothelial dysfunction [33–36]and abnormalities of endogenous fibrinolysis [6–9]have been described in many atherosclerotic diseases and the associated risk factors. Thus, the coupling of acute t-PA release to the L-arginine/nitric oxide pathway provides an important potential mechanism whereby endothelial dysfunction might increase the risk of atherothrombosis through a reduction in the acute fibrinolytic capacity. Our initial findings would suggest that this model could be applied to the assessment of the acute fibrinolytic capacity of patients with endothelial dysfunction such as those with hypercholesterolaemia and a smoking habit [35, 36], and to the examination of the subsequent effect of L-arginine supplementation.

Substance P-induced vasodilatation undergoes tachyphylaxis [37]which may relate to internalisation of the neurokinin type 1 receptor from the endothelial cell surface membrane [38]. It has been suggested from ex vivo animal studies [15, 39]that the residual vasodilatation following the development of tachyphylaxis is almost completely nitric oxide-dependent. In the present study, the degree of inhibition of substance P-induced vasodilatation by L-NMMA was less than we [18]and others [17]have previously described and may reflect the higher potency and doses used in this study. Whilst we have readily demonstrated tachyphylaxis of substance P-induced vasodilatation, the co-infusion of L-NMMA did not affect the development of tachyphylaxis and did not abolish the residual substance P-induced vasodilatation following its development. Thus, in contrast to animal studies, residual vasodilatation after the development of tachyphylaxis does not appear to be predominantly nitric oxide mediated in the human forearm. In addition, we were unable to detect significant tachyphylaxis of substance P-induced increases in plasma t-PA antigen and activity concentrations, suggesting that not all the actions of substance P undergo tachyphylaxis.

The substance P-induced reductions in plasma PAI-1 activity of the infused arm without significant alterations in PAI-1 antigen concentrations are consistent with acute t-PA release in the absence of PAI-1 release [40]. PAI-1 binds to the newly released t-PA to form an inactive PAI-1/t-PA complex, thereby reducing the plasma PAI-1 activity. The trend for PAI-1 antigen concentrations to fall in both arms as the study progressed is consistent with systemic (hepatic) clearance of the PAI-1/t-PA complex [40–42]. However, this trend was also seen with isolated L-NMMA infusion in which there was no significant release of t-PA consistent with a circadian fall of PAI-1 antigen during the morning [43].

Despite reducing forearm blood flow by half, L-NMMA did not significantly affect the constitutive release or plasma concentrations of t-PA and PAI-1 antigen and activity. The 95% confidence intervals indicates that if L-NMMA has an effect on basal t-PA or PAI-1 release then it is rather small. This suggests that the L-arginine/nitric oxide pathway does not play a major role in the basal release of t-PA or PAI-1 in the peripheral vasculature of man.

4.1 Study limitations
Since the derivation of t-PA release is a function of plasma flow, it could be argued that the inhibition by L-NMMA of substance P-induced t-PA release reflects the simultaneous reduction in blood flow. However, the reduction in absolute blood flow was only modest (15–20%) in comparison to the reduction in t-PA release (40–46%) and the plasma t-PA activity concentrations in the infused forearm were also significantly reduced by co-infusion of L-NMMA. The findings of the present study would be strengthened by utilising a control vasoconstrictor and demonstrating a neutral effect on substance P-induced t-PA release. However, standard receptor coupled vasoconstrictors used in forearm studies, such as noradrenaline, vasopressin and angiotensin II, are known to stimulate t-PA and PAI-1 release [12, 14, 32, 44]and would not help in interpreting the influence of L-NMMA on substance P-induced t-PA release.

We have previously been unable to detect an acute local release of either vWf or PAI-1 during 10-min infusions of substance P given at 8-fold higher concentrations [11]. In the present study, substance P did not cause significant vWf or PAI-1 release, despite infusion times of up to 120 min, suggesting that the dissociation of substance P-induced t-PA release from vWf is not a temporal effect. However, this dissociated release does not appear to be unique to substance P since this has also been recently described with local forearm infusions of desmopressin [45]. These findings are, however, limited to the peripheral forearm vascular bed and the endothelium in other tissue beds may respond differently to substance P stimulation. The extension of this model to vascular beds associated with atherosclerosis such as the coronary circulation, will be of crucial relevance in determining the influence of atheroma and endothelial dysfunction on the acute local release of t-PA during thrombotic occlusion and plaque rupture.

In summary, in the forearm vascular bed in vivo, we have shown for the first time that the L-arginine/nitric oxide pathway contributes to substance P-induced t-PA release in man. This coupling of acute t-PA release to the L-arginine/nitric oxide pathway provides an important potential mechanism whereby endothelial dysfunction increases the risk of atherothrombosis through a reduction in the acute fibrinolytic capacity.

Time for primary review 23 days.


   Acknowledgements
 
This work has been supported by a grant from the British Heart Foundation (PG/96149). D.E.N. was the recipient of a British Heart Foundation Junior Research Fellowship (FS/95009).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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