Aims: Bradykinin (BK) stimulates tissue plasminogen activator (t-PA) release from human endothelium. Although BK stimulates both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) release, the role of EDHF in t-PA release remains unexplored. This study sought to determine the mechanisms of BK-stimulated t-PA release in the forearm vasculature of healthy human subjects. Methods: In 33 healthy subjects (age 40.3 ± 1.9 years), forearm blood flow (FBF) and t-PA release were measured at rest and after intra-arterial infusions of BK (400 ng/min) and sodium nitroprusside (3.2 mg/min). Measurements were repeated after intra-arterial infusion of tetraethylammonium chloride (TEA; 1 µmol/min), fluconazole (0.4 µmol·min-1·l-1), and NG-monomethyl-L-arginine (L-NMMA, 8 µmol/min) to block nitric oxide, and their combination in separate studies. Results: BK significantly increased net t-PA release across the forearm (p < 0.0001). Fluconazole attenuated both BK-mediated vasodilation (-23.3 ± 2.7% FBF, p < 0.0001) and t-PA release (from 50.9 ± 9.0 to 21.3 ± 8.9 ng/min/100 ml, p = 0.02). TEA attenuated FBF (-14.7 ± 3.2%, p = 0.002) and abolished BK-stimulated t-PA release (from 22.9 ± 5.7 to -0.8 ± 3.6 ng/min/100 ml, p = 0.0002). L-NMMA attenuated FBF (p < 0.0001), but did not inhibit BK-induced t-PA release (nonsignificant). Conclusion: BK-stimulated t-PA release is partly due to cytochrome P450-derived epoxides and is inhibited by K+Ca channel blockade. Thus, BK stimulates both EDHF-dependent vasodilation and t-PA release.

The endogenous fibrinolytic system is a crucial component of blood flow regulation and protects against intravascular thrombosis. Impairment in endogenous fibrinolysis characterized by inhibition of tissue plasminogen activator (t-PA) release is thought to be an underlying factor in endothelial dysfunction and atherothrombosis [1,2]. The endothelium regulates vasodilator tone and fibrinolytic capacity by releasing vasoactive substances, including nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF) [3,4]. Bradykinin (BK) is an endogenous cardioprotective vasoactive polypeptide that stimulates the release of these molecules and t-PA from the endothelium [5,6,7]. t-PA is synthesized in endothelial cells, stored in small dense granules and released in a dose-dependent manner in response to BK [6,8]. t-PA converts plasminogen to a proteolytic enzyme, plasmin, which digests fibrin-dependent thrombi [9]. Although it is known that BK causes vasodilation via activation of the B2 receptor and subsequent release of NO, prostacyclin, EDHF, and t-PA, the downstream mechanisms governing t-PA release have not been fully elucidated [5,10,11].

There is mounting preclinical evidence to suggest that t-PA release is mediated by EDHF [12,13]. Persistent vasodilation after inhibition of NO and cyclooxygenase can be largely attributed to hyperpolarization by EDHF [3,14]. The hallmark of hyperpolarization is activation of endothelial and/or smooth muscle cell calcium-activated potassium (K+Ca) channels and their inhibition by apamin and charybdotoxin in preclinical studies [3,15]. In human studies, tetraethylammonium chloride (TEA) antagonizes K+Ca channels, thus facilitating characterization of EDHF-dependent responses [16,17]. Endothelium-dependent hyperpolarization may also be partly due to the release of epoxyeicosatrienoic acids (EETs) from cytochrome P450-dependent metabolism of arachidonic acid and subsequent stimulation of K+Ca channels on endothelial cells [18]. The role of EETs as potential EDHFs has been investigated in humans using azole compounds, such as fluconazole, which selectively inhibit epoxidation (EET generation) of arachidonic acid [14,18,19].

Since BK-mediated t-PA release may be crucial in maintaining the endogenous thrombolytic potential, it is important to determine its underlying mechanisms. Our overall goal was to investigate whether EDHF release stimulates t-PA, with the hypothesis that BK stimulates EDHF-mediated t-PA release in the human circulation. We investigated whether activation of the K+Ca channels and/or cytochrome P450-derived EETs mediates t-PA release.

Subjects

Thirty-three healthy subjects aged 21-60 years and free of smoking, hypercholesterolemia, hypertension, diabetes, cardiovascular disease, medication use, or any other systemic disorder participated in the study (ClincalTrials.gov Identifier: NCT00166166; table 1). Written informed consent was obtained and the study was approved by the Emory University Institutional Review Board and the Food and Drug Administration.

Table 1

Subject characteristics

Subject characteristics
Subject characteristics

Experimental Protocols

Subjects were enrolled into 3 separate protocols (described below) performed sequentially. Measurements were performed after an overnight fast in a quiet, temperature-controlled (22-24°C) room. Subjects received 975 mg of oral aspirin 90 min prior to initiation of the study to inhibit prostanoid release [20,21]. An intravenous catheter was placed into a deep antecubital vein for venous sampling and an arterial cannula into the brachial artery for arterial pressure monitoring, drug delivery, and blood sampling.

FBF Measurements

Simultaneous FBF measurements were obtained in both arms with the use of a dual-channel venous occlusion strain gauge plethysmograph (model EC6; DE Hokanson, Bellevue, Wash., USA) as previously described [14]. The mercury-filled silastic strain gauge was placed around the forearm and connected to a plethysmograph calibrated to measure the percent change in volume [14]. An upper arm cuff was inflated to 40 mm Hg to occlude venous outflow and FBF measurements were recorded every 15 s up to eight times, and a mean FBF value (in ml·min-1·100 ml-1) was computed. Forearm vascular resistance was calculated as the mean arterial pressure divided by FBF (expressed as mm Hg/ml·min-1·100 ml-1).

Protocol 1: Effect of K+Ca Channel Activation on BK-Stimulated t-PA Release

FBF was measured after a 30-min rest during saline infusion (total infusion rate 2.5 ml·min-1) and after intra-arterial infusions of 100, 200, and 400 ng/min of BK (Clinalfa, Laufelfingen, Switzerland) given for 8 min each (n = 18). After 30 min, intra-arterial TEA (Sigma-Aldrich, Allentown, Pa., USA, sterilized and tested for pyrogenicity by the Emory Investigational Drug Pharmacy) was infused at 1 µmol/min for 8 min. When given at 0.25-1 mg/min, TEA selectively inhibits K+Ca channels, but reduces FBF with BK only at 1 mg/min (<0.6 µmol/min) [16,22,23,24]. This dose and timing was effective in attenuating FBF and agonist-stimulated vasodilation in previous studies [4,16,25]. While continuing the infusion of TEA, BK infusions were re-administered. Arterial blood pressure and FBF measurements were repeated in the last 2 min of each infusion. Arterial and venous blood was sampled at baseline prior to the infusion of drug and at peak doses of BK.

Protocol 2: Effect of Cytochrome P450 Metabolites to BK-Stimulated t-PA Release

FBF was measured at rest during saline infusion and after intra-arterial infusion of escalating doses of BK (n = 11). Thirty minutes later, fluconazole (Pfizer, New York, N.Y., USA) was infused intra-arterially at 0.4 µmol·min-1·l-1 for 5 min. This dose has been effective in attenuating vasodilation during sustained flow conditions [4,17]. While continuing the infusion of fluconazole, escalating doses of BK were administered. Finally, combined intra-arterial infusions of fluconazole and TEA were given for 8 min and BK infusions were repeated. This protocol allowed to assess the contribution of cytochrome P450 metabolites alone and in combination with K+Ca channel activation to BK-stimulated t-PA release.

Protocol 3: Effect of NO on BK-Stimulated t-PA release

The contribution of NO to BK-stimulated t-PA release was also determined in the presence of K+Ca channel activation (n = 13). FBF was measured at rest and after intra-arterial infusion of BK. Thirty minutes later, TEA (1 µmol/min) was infused for 8 min and followed by repeat infusions of BK. After 30 min, TEA and NG-monomethyl-L-arginine (L-NMMA; Clinalfa) at 8 µmol/min were infused together for 8 min to inhibit EDHF and NO synthesis, followed by repeat administration of BK in escalating doses while continuing infusions of the two antagonists. This dose and timing of L-NMMA were effective in attenuating FBF and agonist-stimulated vasodilation in previous studies [16,25].

Effect of Sodium Nitroprusside on t-PA Release

Finally, in 30 subjects, the comparative contribution of the endothelium-independent vasodilator sodium nitroprusside (SNP) and BK was investigated. FBF was measured during rest and after intra-arterial infusion of escalating doses of BK. Thirty minutes later, SNP (Hospira, Lakeforest, Ill., USA) was infused intra-arterially at 1.6 and 3.2 µg/min.

Blood Sampling and Biochemical Assays

Simultaneous arterial and venous blood samples were obtained from the infused arm before and after peak doses of vasodilator drugs, cooled, centrifuged, and stored in tubes containing 0.5 M citrate buffer (Diapharma, West Chester, Ohio, USA) at -70°C. t-PA antigen levels were determined in duplicate using a two-site enzyme-linked immunosorbent assay (Diapharma) as described previously [26]. Net release or uptake rates at each time point were calculated: net release = (CV - CA) × {FBF × [(101 - hematocrit) / 100]}, where CV and CA represent the concentrations of t-PA in the brachial vein and artery, respectively.

Statistical Analysis

To determine the effects of the interventions on FBF and forearm vascular resistance, changes in the flow and resistance responses between the control, single blockade, and combined blockade were compared using repeated-measures ANOVA. To determine the effects of treatments on net t-PA release, changes in net t-PA release of control, single blockade, and combined blockade were compared using one-way ANOVA with the Tukey post hoc test. Changes in the response were converted to percentage changes of least-square means when presented in the text. Data are presented as means ± SEM in the text. The least-square means and standard errors are presented in the figures. A value of p < 0.05 was considered statistically significant.

Baseline Subject Characteristics

Thirty-three healthy (age 40.3 ± 1.9 years, 64% male) normotensive, nondiabetic, nonobese, nonsmoking subjects with normal serum cholesterol levels were studied (table 1). During intrabrachial drug infusions, no changes in blood pressure or heart rate were observed.

Effect of K+Ca Channel Activation on FBF and BK-Stimulated t-PA Release

BK produced a dose-dependent increase in FBF (p = 0.002) and a decrease in vascular resistance (p = 0.03; fig. 1a, b). BK significantly increased the arteriovenous t-PA concentration gradient and net t-PA release across the forearm (from -0.3 ± 0.5 to 36.9 ± 5.5 ng/min/100 ml, p = 0.002; fig. 1c; table 2). There was no association between net t-PA release and age, sex, body mass index, total cholesterol, and LDL levels.

Table 2

Effect of interventions on the arteriovenous gradient

Effect of interventions on the arteriovenous gradient
Effect of interventions on the arteriovenous gradient
Fig. 1

Contribution of K+Ca channel activation to BK-stimulated vasodilation, and t-PA release. FBF (a) and forearm vascular resistance (b) changes in response to BK (100, 200, and 400 ng/min) alone, and after infusion of TEA (n = 18). Net t-PA release (c) in response to the peak dose of BK (400 ng/min) and after infusion of TEA. Means ± SEM.

Fig. 1

Contribution of K+Ca channel activation to BK-stimulated vasodilation, and t-PA release. FBF (a) and forearm vascular resistance (b) changes in response to BK (100, 200, and 400 ng/min) alone, and after infusion of TEA (n = 18). Net t-PA release (c) in response to the peak dose of BK (400 ng/min) and after infusion of TEA. Means ± SEM.

Close modal

Infusion of TEA decreased resting FBF by 14% (p = 0.0001) and increased vascular resistance by 18% (p = 0.03). Co-administration of TEA with BK attenuated the overall response with a 15% (p = 0.002) lower FBF and a 21% (p = 0.03) higher vascular resistance (fig. 1a, b). TEA infusion effectively abolished t-PA release with BK (from 22.9 ± 5.7 to -0.8 ± 3.6 ng/min/100 ml, p = 0.0002) and the arteriovenous t-PA concentration gradient (table 2), indicating that the effect of BK on endothelial t-PA release is mediated entirely through K+Ca channel activation (fig. 1c).

Effect of Cytochrome P450 Metabolites and K+Ca Channel Activation on FBF and BK-Stimulated t-PA Release

Infusion of fluconazole decreased resting FBF by 19% (p = 0.001) and increased vascular resistance by 27% (p = 0.001). Fluconazole also blunted the overall vasodilator response to BK with a 23% (p < 0.0001) reduction in FBF and a 35% (p < 0.0001) higher vascular resistance (fig. 2a, b). Fluconazole infusion attenuated the t-PA release with BK (from 50.9 ± 9 to 21.3 ± 8.9 ng/min/100 ml, p = 0.02) and the arteriovenous t-PA concentration gradient (table 2), indicating significant contribution of cytochrome P450 metabolites to BK-stimulated t-PA release (fig. 2c). Infusion of TEA after fluconazole reduced FBF by an additional 20% (p < 0.0001, n = 10) and increased vascular resistance by a further 24% (p < 0.0001; fig. 2a, b). In the presence of fluconazole, infusion of TEA tended to attenuate the arteriovenous t-PA gradient (table 2), but the further reduction in net t-PA release did not reach statistical significance (fig. 2c). However, TEA infusion attenuated the t-PA release with BK to a greater extent than fluconazole (85 ± 4 vs. 61 ± 11% reduction, respectively, p = 0.05), indicating that of the potential hyperpolarization mechanisms, K+Ca channel activation is the predominant EDHF signaling pathway mediating BK-stimulated t-PA release.

Fig. 2

Contribution of cytochrome P450 metabolites and K+Ca channel activation to BK-stimulated vasodilation and t-PA release. FBF (a) and forearm vascular resistance (b) changes in response to BK (100, 200, and 400 ng/min) alone, after infusion of fluconazole (FLU), and combined infusions of FLU and TEA (n = 11). Net t-PA release (c) in response to the peak dose of BK (400 ng/min) and after initial infusion of FLU, followed by combined FLU and TEA infusions. Means ± SEM.

Fig. 2

Contribution of cytochrome P450 metabolites and K+Ca channel activation to BK-stimulated vasodilation and t-PA release. FBF (a) and forearm vascular resistance (b) changes in response to BK (100, 200, and 400 ng/min) alone, after infusion of fluconazole (FLU), and combined infusions of FLU and TEA (n = 11). Net t-PA release (c) in response to the peak dose of BK (400 ng/min) and after initial infusion of FLU, followed by combined FLU and TEA infusions. Means ± SEM.

Close modal

Effect of NO and K+Ca Channel Activation on FBF and BK-Stimulated t-PA Release

To assess the comparative contribution of K+Ca channel activation and NO to BK-stimulated t-PA release, individual and combined blockade with TEA and L-NMMA were studied. As seen previously, infusion of TEA attenuated the vasodilator response to BK with a 17% (p < 0.0001) reduction in FBF (fig. 3a). Similarly, t-PA release with BK was reduced from 20.4 ± 6 to 3.5 ± 3 ng/min/100 ml (p = 0.02), indicating a significant contribution of K+Ca channel activation to BK-stimulated t-PA release (fig. 3c). Infusion of L-NMMA after TEA reduced FBF by an additional 21% (p < 0.0001) and increased vascular resistance by a further 42% (p = 0.04; fig. 3a, b). In the presence of TEA, infusion of L-NMMA did not further affect net t-PA release (p = 0.25; fig. 3c; table 2), indicating that BK-stimulated t-PA release is through a K+Ca channel-dependent and NO-synthase-independent pathway.

Fig. 3

Contribution of NO and K+Ca channel activation to BK-stimulated vasodilation and t-PA release. FBF (a) and forearm vascular resistance (b) in response to BK (100, 200, and 400 ng/min) alone, after initial infusion of TEA, and combined infusion of TEA and L-NMMA in healthy (n = 13) subjects. Means ± SEM. Box plot of net t-PA release (c) in response to the peak dose of BK (400 ng/min) and after infusion of TEA, followed by combined TEA and L-NMMA infusions. Minimum and maximum values are depicted by whiskers and the mean by the symbol marker.

Fig. 3

Contribution of NO and K+Ca channel activation to BK-stimulated vasodilation and t-PA release. FBF (a) and forearm vascular resistance (b) in response to BK (100, 200, and 400 ng/min) alone, after initial infusion of TEA, and combined infusion of TEA and L-NMMA in healthy (n = 13) subjects. Means ± SEM. Box plot of net t-PA release (c) in response to the peak dose of BK (400 ng/min) and after infusion of TEA, followed by combined TEA and L-NMMA infusions. Minimum and maximum values are depicted by whiskers and the mean by the symbol marker.

Close modal

Effect of SNP on t-PA Release

Infusions of BK and SNP resulted in similar increases in FBF (p = 0.7) and decreases in FVR (fig. 4a). However, in contrast to BK, the t-PA response (-1.8 ± 2 vs. -0.6 ± 0.4 ng/min/100 ml, nonsignificant) and the arteriovenous t-PA concentration gradient (-0.4 ± 0.3 vs. -0.2 ± 0.3, p = 0.5) remained unchanged with SNP, indicating that SNP does not stimulate endothelial t-PA release (fig. 4b).

Fig. 4

Contribution of BK and SNP to vasodilation and t-PA release. FBF (a), forearm vascular resistance (b), and net t-PA release (c) in response to the peak doses of BK (400 ng/min) and SNP (3.2 µg/min). Means ± SEM. NS = Not statistically significant.

Fig. 4

Contribution of BK and SNP to vasodilation and t-PA release. FBF (a), forearm vascular resistance (b), and net t-PA release (c) in response to the peak doses of BK (400 ng/min) and SNP (3.2 µg/min). Means ± SEM. NS = Not statistically significant.

Close modal

Herein, we demonstrate that although both NO and EDHF contribute to BK-induced vasodilation, t-PA release is solely mediated by EDHF signaling pathways. We confirmed that cytochrome P450-derived EETs partly contribute to BK-stimulated t-PA release and now show that it is completely abolished by inhibition of K+Ca channels, the final site of action for several putative EDHFs [27]. Finally, we demonstrate a lack of contribution of NO to BK-stimulated t-PA release [11].

Endothelial fibrinolytic activity is an endogenous protective mechanism against the development and propagation of arterial thrombi [28]. In the fibrinolytic system, plasminogen is activated to plasmin that degrades fibrin into soluble fibrin degradation products [29]. The endothelium releases t-PA through a calcium- and G-protein-dependent pathway in response to a variety of endogenous mediators, including catecholamines, thrombin, and BK [30,31]. Endogenous BK is produced by activation of the plasma kallikrein-kinin system on endothelial cells and BK B2 receptor blockade inhibits t-PA release [10,11].

In the intact human vasculature, endothelium-dependent hyperpolarization can be mediated by agonists such as BK and by physical stimuli including increases in shear stress that all increase intracellular calcium, causing opening of endothelial K+Ca channels and initiating downstream processes that explain the EDHF phenomena [3,4]. Candidate EDHFs include EETs synthesized from the cytochrome P450-dependent metabolism of arachidonic acid and hydrogen peroxide generated from the degradation of superoxides through various endothelial oxidases such as NADPH oxidase or through superoxide-dismutase-dependent dismutation [32,33]. The availability of TEA, a specific K+Ca channel antagonist, for use in humans has enabled investigations of EDHF activity in the forearm microcirculation [16,17,22]. We now demonstrate that in addition to stimulating vasodilation, BK stimulates t-PA release that is completely abolished by K+Ca channel inhibition [6,34].

Endothelium-derived cytochrome P450 metabolites of arachidonic acid can hyperpolarize membranes primarily by activating the K+Ca channels, although the identity of the specific EETs remains controversial [35,36,37]. Herein, we demonstrate that fluconazole, an inhibitor of cytochrome 2C9, attenuated BK-mediated t-PA release, suggesting that cytochrome P450 metabolites contribute significantly to t-PA release [19], observations that have been confirmed in both preclinical [14,38] and clinical studies in both healthy and hypertensive subjects [27].

Because inhibition of EETs had a lesser effect than inhibition of K+Ca channels on BK-stimulated t-PA release, it appears that other factors that stimulate K+Ca channels also contribute to EDHF activity, which in preclinical studies has been attributed to hydrogen peroxide, gap junctions, or elevations in K+ release from endothelial cells [3,18,39,40,41]. Hydrogen peroxide predominantly contributes to BK-induced human arteriolar vasodilation [42] and to t-PA release from rat hearts [43], but its role in the human circulation in vivo remains to be established. Thus, although hydrogen peroxide activates K+Ca channels and can function as an EDHF, its contribution to vascular homeostasis is complex [44]. It is not only a hyperpolarizing factor, but, depending on the species, blood vessel, and concentration, hydrogen peroxide can also be a signaling molecule and may also cause direct smooth muscle relaxation or depolarization of smooth muscles and vasoconstriction [3].

It may be argued that the BK-mediated t-PA release is at least partly flow mediated. However, with similar increases in blood flow with SNP, we and previous investigators found no increase in t-PA [6,11,27,45,46]. Moreover, infusion of L-NMMA selectively attenuated FBF without affecting the t-PA response to BK.

Limitations

L-NMMA, fluconazole, and TEA are competitive inhibitors and may not completely inhibit NO, cytochrome P450 pathways, and K+Ca channels, respectively, and thus likely underestimate the physiologic importance of these mediators in vivo [47,48]. TEA also acts as a competitive inhibitor of the nicotinic receptor and reduces water permeability of human AQP1 channels; however, at the doses administered, it has been shown to selectively inhibit K+Ca channels [49,50]. It is assumed that TEA effects are reflecting endothelium-dependent hyperpolarization as we cannot measure membrane potentials in this in vivo study. Fluconazole is not a specific inhibitor of cytochrome 2C9; however, we have shown significant inhibition of resting and BK-mediated vasodilation with this inhibitor [4]. Previous studies with a specific 2C9 inhibitor, sulfaphenazole, or with miconazole, have yielded contradictory data [19,51,52,53]. We did not find any ethnic differences in BK-mediated t-PA release even though ethnicity affects the vasodilator response to BK; however, our study was not powered to explore this issue [54]. Our experiments were conducted in the setting of cyclooxygenase inhibition and therefore are unable to explore possible interactions with prostaglandins. Nevertheless, previous studies demonstrated no alteration in BK-mediated t-PA release with cyclooxygenase inhibition [11,55,56].

We demonstrate that BK-stimulated t-PA release is via endothelium-dependent hyperpolarization, partly through cytochrome P450-derived epoxides and ultimately via activation of K+Ca channels. Although BK stimulates both NO and EDHF-dependent vasodilation, BK-mediated t-PA release is purely EDHF dependent. Both endothelium-dependent vasodilation and endothelial fibrinolytic capacity, measured as t-PA release, appear to predict the risk of future cardiovascular events [57,58]. Since abnormalities in the EDHF signaling pathway contribute to both abnormal vasodilation and increased risk of thrombosis, agonists that enhance EDHF activity, such as epoxide hydrolase inhibitors, need to be investigated for their thrombolytic actions.

The study was supported by National Institutes of Health Research Grant RO1 HL79115, and in part by PHS Grant UL1 RR025008 from the Clinical and Translational Science Award Program, and PHS Grant M01 RR00039 from the General Clinical Research Center program, National Institutes of Health, National Center for Research Resources, the British Cardiovascular Society Research Fellowship, National Blood Foundation, NIH NRSA T32 Training Grant, American College of Cardiology Foundation Keating Fellowship, and the American Heart Association Beginning Grant-in-Aid.

The authors have no conflicts of interest to disclose.

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Clinical Trial Registration: http://clinicaltrials.gov/ct2/show/NCT00166166, ClinicalTrials.gov identifier: NCT00166166.

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