Laine J. Murphey

Bradykinin Stimulates Tissue Plasminogen Activator Release From Human Forearm Vasculature Through B2 Receptor–Dependent, NO Synthase–Independent, and Cyclooxygenase-Independent Pathway

BackgroundBradykinin stimulates dose-dependent tissue plasminogen activator (tPA) release from human endothelium. Although bradykinin is known to cause vasodilation through B2 receptor–dependent effects on NO, prostacyclin, and endothelium-derived hyperpolarizing factor production, the mechanism(s) underlying tPA release is unknown. Methods and ResultsWe measured the effects of intra-arterial bradykinin (100, 200, and 400 ng/min), acetylcholine (15, 30, and 60 &mgr;g/min), and nitroprusside (0.8, 1.6, and 3.2 &mgr;g/min) on forearm vasodilation and tPA release in healthy volunteers in the presence and absence of (1) the B2 receptor antagonist HOE 140 (100 &mgr;g/kg IV), (2) the NO synthase inhibitor l-NG-monomethyl-l-arginine (L-NMMA, 4 &mgr;mol/min intra-arterially), and (3) the cyclooxygenase inhibitor indomethacin (50 mg PO TID). B2 receptor antagonism attenuated vasodilator (P =0.004) and tPA (P =0.043) responses to bradykinin, without attenuating the vasodilator response to nitroprusside (P =0.36). L-NMMA decreased basal forearm blood flow (from 2.35±0.31 to 1.73±0.22 mL/min per 100 mL, P =0.01) and blunted the vasodilator response to acetylcholine (P =0.013) and bradykinin (P =0.07, P =0.038 for forearm vascular resistance) but not that to nitroprusside (P =0.47). However, there was no effect of L-NMMA on basal (P =0.7) or bradykinin-stimulated tPA release (P =0.45). Indomethacin decreased urinary excretion of the prostacyclin metabolite 2,3-dinor-6-keto-prostaglandin F1&agr; (P =0.04). The vasodilator response to endothelium-dependent (P =0.019 for bradykinin) and endothelium-independent (P =0.019) vasodilators was enhanced during indomethacin administration. In contrast, there was no effect of indomethacin alone (P =0.99) or indomethacin plus L-NMMA (P =0.36) on bradykinin-stimulated tPA release. ConclusionsThese data indicate that bradykinin stimulates tPA release from human endothelium through a B2 receptor–dependent, NO synthase–independent, and cyclooxygenase-independent pathway. Bradykinin-stimulated tPA release may represent a marker for the endothelial effects of endothelium-derived hyperpolarizing factor.

Angiotensin-converting enzyme insertion/deletion polymorphism modulates the human in vivo metabolism of bradykinin

BACKGROUND Bradykinin is a cardioprotective peptide metabolized by the angiotensin-converting enzyme (ACE). An insertion/deletion (I/D) polymorphism in the ACE gene determines plasma ACE levels. The D allele is associated with cardiovascular disease, which may relate to enhanced angiotensin II production or to increased bradykinin degradation to the inactive metabolite bradykinin 1-5 (BK1-5). Therefore, we determined the effect of the ACE I/D polymorphism on human bradykinin metabolism in vivo. METHODS AND RESULTS Bradykinin (400 ng/min) was infused into the brachial artery of volunteers with ACE I/I, I/D, or D/D genotypes (n=9 each). The bradykinin and BK1-5 levels in forearm venous return were quantified by liquid chromatography-mass spectroscopy. Plasma ACE activity was highest in those with the D/D genotype (36.8+/-6.2 U/mL), intermediate in those with the I/D genotype (25.3+/-3.3 U/mL), and lowest in those with the I/I genotype (20.3+/-2.3 U/mL; P=0.017 for effect of number of D alleles). Bradykinin concentrations were 726+/-242, 469+/-50, and 545+/-104 fmol/mL in I/I, I/D, and D/D subjects, respectively (P>0. 10). Significant correlations existed between the number of D alleles and BK1-5 concentrations (1113+/-290, 1520+/-318, and 1887+/-388 fmol/mL in the I/I, I/D, and D/D groups, respectively; P=0.027) and the ratio of BK1-5 to bradykinin (1.87+/-0.35, 3.09+/-0. 40, and 4.31+/-0.97 in the I/I, I/D, and D/D volunteers, respectively; P=0.010). The venous blood BK1-5:bradykinin ratio correlated with plasma ACE activity (r(2)=0.16, P=0.039), and total kinin concentration correlated with net tissue plasminogen activator release across the forearm (r(2)=0.20, P=0.027). CONCLUSIONS The ACE D allele has a significant effect on the in vivo degradation of bradykinin in humans. The ratio of BK1-5:bradykinin may serve as a marker for tissue ACE activity.

Angiotensin II Induces Interleukin-6 in Humans Through a Mineralocorticoid Receptor–Dependent Mechanism

This study tested the hypothesis that angiotensin promotes oxidative stress and inflammation in humans via aldosterone and the mineralocorticoid receptor. We measured the effect of intravenous aldosterone (0.7 &mgr;g/kg per hour for 10 hours followed by 0.9 &mgr;g/kg per hour for 4 hours) and vehicle in a randomized, double-blind crossover study in 11 sodium-restricted normotensive subjects. Aldosterone increased interleukin (IL)-6 (from 4.7±4.9 to 9.4±7.1 pg/mL; F=4.94; P=0.04) but did not affect blood pressure, serum potassium, or high-sensitivity C-reactive protein. We next conducted a randomized, double-blind, placebo-controlled, crossover study to measure the effect of 3-hour infusion of angiotensin II (2 ng/kg per minute) and norepinephrine (30 ng/kg per minute) on separate days after 2 weeks of placebo or spironolactone (50 mg per day) in 14 salt-replete normotensive subjects. Angiotensin II increased blood pressure (increase in systolic pressure: 13.7±7.5 and 15.2±9.4 mm Hg during placebo and spironolactone, respectively; P<0.001 for angiotensin II) and decreased renal plasma flow (−202±73 and −167±112 mL/min/1.73 kg/m2; P<0.001 for angiotensin II effect) similarly during placebo and spironolactone. Spironolactone enhanced the aldosterone response to angiotensin II (increase of 17.0±10.6 versus 9.0±5.7 ng/dL; P=0.002). Angiotensin II transiently increased free plasma F2-isoprostanes similarly during placebo and spironolactone. Angiotensin II increased serum IL-6 concentrations during placebo (from 1.8±1.1 to 2.4±1.4 pg/mL; F=4.5; P=0.04) but spironolactone prevented this effect (F=6.4; P=0.03 for spironolactone effect). Norepinephrine increased blood pressure and F2-isoprostanes but not aldosterone or IL-6. Aldosterone increases IL-6 in humans. These data suggest that angiotensin II induces IL-6 through a mineralocorticoid receptor–dependent mechanism in humans. In contrast, angiotensin II–induced oxidative stress, as measured by F2-isoprostanes, is mineralocorticoid receptor independent and may be pressor dependent.

Urinary prostaglandin F2α is generated from the isoprostane pathway and not the cyclooxygenase in humans

Prostaglandins (PGs) derived from the enzymatic oxidation of arachidonic acid by the cyclooxygenases (COXs) are potent lipid mediators involved in human physiology and pathophysiology. Structurally similar compounds, the isoprostanes (IsoPs), are generated from the free radical-catalyzed oxidation of arachidonic acid independent of COX. IsoPs exhibit significant bioactivity and play a role in the pathogenesis of diseases associated with oxidant injury. As one of the major PGs, prostaglandin F2α (PGF2α) is present in human urine in significant concentrations and is presumed to be derived from COX activity. We determined, however, that levels of putative PGF2α in urine cannot be suppressed by nonsteroidal anti-inflammatory agents, suggesting that it is generated via another mechanism(s). An important difference between COX-derived PGF2α and the IsoPs is that the former is an optically pure compound, whereas IsoPs are racemic. Utilizing a rodent model of oxidative stress, we now show that significant amounts of compounds identical in all respects to PGF2α and its enantiomer, ent-PGF2α, are formed in equal amounts esterified in tissue phospholipids, suggesting that these compounds are derived via the IsoP pathway. Further, employing liquid chromatography/mass spectrometry, the vast majority of putative PGF2α in human urine is derived from the free radical-initiated peroxidation of arachidonate independent of COX and is composed of PGF2α and its enantiomer, although the latter compound is ∼2-fold more abundant. Thus, quantification of urinary PGF2α actually reflects oxidative stress status as opposed to COX activity. Indeed, levels of this compound are elevated in urine from cigarette smokers and in humans with hypercholesterolemia, two conditions associated with oxidant stress. The elucidation that urinary PGF2α in humans is derived from the IsoP pathway has implications regarding PG formation and inhibition in vivo.

Human colorectal cancer cells efficiently conjugate the cyclopentenone prostaglandin, prostaglandin J2, to glutathione

Cyclopentenone prostaglandins (PGs), particularly those of the J-series, affect proliferation and differentiation in a number of cell lines. J-ring PGs have been shown to be ligands for the peroxisome proliferator-activated receptor (PPAR)-gamma and to modulate NF-kappaB-mediated gene transcription. We have previously reported that large quantities of eicosanoids, including PGJ(2), are produced by the human colorectal cancer cell line HCA-7 while lesser amounts of Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) are formed. In this and other cell lines, cyclopentenone PGs have been shown to increase cell proliferation, but factors that influence their formation and metabolism are poorly understood. Unlike other PGs, cyclopentenone PGs contain alpha,beta-unsaturated carbonyl groups that readily adduct various biomolecules such as glutathione (GSH) in vitro. We now report that in HCA-7 cells, PGJ(2) is largely metabolized by conjugation to GSH. Characterization of the adducts by liquid chromatography (LC)-mass spectrometry (MS) revealed two major metabolites consisting of (1) a novel GSH conjugate in which the carbonyl at C-11 of PGJ(2) is reduced and (2) intact PGJ(2) conjugated to GSH. Approximately 70% of the PGJ(2) added to HCA-7 cells was esterifed to GSH after 2 h of incubation, suggesting this pathway represents the major route of metabolic disposition of PGJ(2) in HCA-7 cells.

Ethnicity Affects Vasodilation, but Not Endothelial Tissue Plasminogen Activator Release, in Response to Bradykinin

Previous studies indicate that the vasodilator response to bradykinin (BK) and other endothelium-dependent and -independent agonists is decreased in black Americans compared with white Americans. The purpose of the present study was to determine the effect of ethnicity on fibrinolytic function in humans. Graded doses of BK (100, 200, and 400 ng/min), acetylcholine (15, 30, and 60 &mgr;g/min; N=20), or methacholine (3.2, 6.4, 12.8 &mgr;g/min; N=20), and sodium nitroprusside (0.8, 1.6, and 3.2 &mgr;g/min) were infused via brachial artery in 19 white and 21 black age-matched normotensive subjects. Forearm blood flow (FBF) was measured by plethysmography, and venous and arterial samples were collected for tissue plasminogen activator (tPA) antigen. Compared with whites (increase in FBF from 3.7±0.5 to 23.9±2.5 mL · min−1 · 100 mL−1), blacks (increase in FBF from 2.8±0.3 to 15.2±1.9 mL · 100 mL−1 · min−1) exhibited a blunted FBF response to BK (P =0.035). Responses to sodium nitroprusside and methacholine or acetylcholine were similarly decreased. In contrast, there was no effect of ethnicity on net tPA antigen release in response to BK (increase from −0.2±0.4 to 67.3±15.2 ng · min−1 · 100 mL−1 in blacks; from 0.04±0.9 to 65.9±13.6 ng · min−1 · 100 mL−1 in whites). Thus, ethnicity significantly influenced the relationship between the flow and tPA release responses to BK (P =0.037). These data suggest that the BK-dependent alterations in vascular fibrinolytic function are preserved in black Americans compared with white Americans.

Angiotensin-(1-7) does not affect vasodilator or TPA responses to bradykinin in human forearm

Abstract—Studies in isolated vessels and rat models of hypertension suggest that angiotensin (Ang)-(1-7) potentiates the vasodilator effect of bradykinin, possibly through ACE inhibition. We therefore tested the hypothesis that Ang-(1-7) potentiates the vasodilator or tissue plasminogen activator (TPA) response to bradykinin in the human forearm vasculature. Graded doses of Ang-(1-7) (10, 100, and 300 pmol/min), bradykinin (47, 94, and 189 pmol/min), and Ang I (1, 10, and 30 pmol/min) were administered through the brachial artery to 8 normotensive subjects in random order. Thirty minutes after initiation of a constant infusion of Ang-(1-7) (100 pmol/min), bradykinin and Ang I infusions were repeated. There were no systemic hemodynamic effects of the agonists. Bradykinin significantly increased forearm blood flow (P <0.001, from 3.8±0.5 to 13.9±3.1 mL/min per 100 mL at 189 pmol/min) and net TPA release (P =0.007, from 1.1±1.0 to 23.6±6.2 ng/min per 100 mL at 189 pmol/min), whereas Ang I caused vasoconstriction (P =0.003, from 3.3±0.4 to 2.5±0.3 mL/min per 100 mL at 30-pmol/min dose). There was no effect of Ang-(1-7) on either forearm blood flow (P =0.62, 3.3±0.4 to 3.5±0.4 mL/min per 100 mL at 300 pmol/min) or TPA release (P =0.52, from 0.7±0.8 to 1.0±0.7 ng/min/100 mL at 300 pmol/min). Moreover, there was no effect of 100 pmol/min Ang-(1-7) on the vasodilator [P =0.46 for Ang-(1-7) effect] or TPA [P =0.82 for Ang-(1-7) effect] response to bradykinin or the vasoconstrictor response to Ang I [P =0.62 for Ang-(1-7) effect]. These data do not support a role of Ang-(1-7), given at supraphysiological doses, in the regulation of human peripheral vascular resistance or fibrinolysis.

Interactive Effect of PAI-1 4G/5G Genotype and Salt Intake on PAI-1 Antigen

Abstract—Activation of the renin-angiotensin-aldosterone system (RAAS) is associated with increased circulating PAI-1 antigen and increased risk of thrombotic cardiovascular events. A 4G/5G polymorphism located 675 bp upstream from the transcription start site of the PAI-1 gene affects PAI-1 antigen concentrations. To test the hypothesis that PAI-1 4G/5G genotype influences the effect of activation of the RAAS on PAI-1 expression, we measured morning PAI-1 antigen concentrations in 76 subjects with essential hypertension during low (10 mmol/d) and high (200 mmol/d) salt intake. Low salt intake was associated with activation of the RAAS as measured by plasma renin activity (2.3±0.2 versus 0.5±0.0 ng angiotensin I · mL−1 · h−1, P <0.001) and aldosterone (529±40 versus 145±12 pmol/L). PAI-1 antigen concentrations were 17.9±2.7, 19.2±2.5, and 27.8±4.0 ng/mL during high salt intake and 19.2±2.7, 21.6±2.9, and 38.9±7.2 ng/mL during low salt intake in the 5G/5G (n=14), 4G/5G (n=40), and 4G/4G (n=22) groups, respectively. There was a significant effect of both salt intake (F=6.0, P =0.017) and PAI-1 4G/5G genotype (F=7.6, P =0.001) on PAI-1 antigen. More importantly, there was a significant interactive effect (F=7.8, P =0.001) of salt intake and PAI-1 4G/5G genotype on PAI-1 antigen. PAI-1 4G/5G genotype influenced the relationship between serum triglycerides and PAI-1 antigen such that the relationship was significant only in 4G homozygotes during either high (R2=0.31, P =0.014) or low (R2=0.37, P =0.006) salt intake. This study identifies an important gene-by-environment interaction that may influence cardiovascular morbidity and the response to pharmacological therapies that interrupt the RAAS.

Bradykinin and Its Metabolite Bradykinin 1-5 Inhibit Thrombin-Induced Platelet Aggregation in Humans

Bradykinin 1-5 is a major stable metabolite of bradykinin, formed by the proteolytic action of angiotensin-converting enzyme. In vitro and animal studies suggest that bradykinin 1-5 possesses biological activity. This study tests the hypothesis that bradykinin 1-5 affects vasodilation, fibrinolysis, or platelet aggregation in humans. Graded doses of bradykinin (47-377 pmol/min) and bradykinin 1-5 (47-18,850 pmol/min) were infused in the brachial artery in random order in 36 healthy subjects. Forearm blood flow (FBF) was measured, and simultaneously obtained venous and arterial plasma samples were analyzed for tissue plasminogen activator (t-PA) antigen. In seven subjects each, α- and γ-thrombin-induced platelet aggregation was measured in platelet-rich plasma obtained from antecubital venous blood at baseline and during peptide infusions. Bradykinin caused dose-dependent increases in FBF and net t-PA release (P < 0.001 for both). Bradykinin 1-5 did not affect FBF (P = 0.13) or net t-PA release (P = 0.46) at concentrations >1500 times physiologic. In contrast, both bradykinin and bradykinin 1-5 inhibited α-and γ-thrombin-induced platelet aggregation (P < 0.01 versus baseline). Bradykinin 1-5 inhibited γ-thrombin-induced platelet aggregation 50% at a calculated dose of 183 ± 3 pmol/min. Neither bradykinin nor bradykinin 1-5 affected thrombin receptor-activating peptide-induced platelet aggregation, consistent with the hypothesis that bradykinin and bradykinin 1-5 inhibit thrombin-induced platelet aggregation by preventing cleavage of the thrombin receptor and liberation of thrombin receptor-activating peptide. This study is the first to demonstrate biological activity of bradykinin 1-5 following in vivo administration to humans. By inhibiting thrombin-induced platelet aggregation without causing vasodilation, bradykinin 1-5 may provide a model for small molecule substrate-selective thrombin inhibitors.

Contribution of bradykinin to the cardioprotective effects of ACE inhibitors

Bradykinin, through its B 2 receptor, stimulates endothelial release of a number of vasodilators, such as nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). Angiotensin-converting enzyme (ACE) inhibitors enhance the effects of local bradykinin by decreasing its degradation and by increasing B 2 receptor sensitivity. Clinical and experimental studies demonstrate that blockade of the B 2 receptor attenuates the antihypertensive, antihypertrophic, and antiatherosclerotic effects of ACE inhibitors. Thus, the evidence strongly supports a role for bradykinin in mediating the cardiovascular benefits of ACE inhibitors.