Urinary free cortisol: an intermediate phenotype and a potential genetic marker for a salt-resistant subset of essential hypertension.

CONTEXT: Emerging evidence suggests a role for cortisol in essential hypertension, and preliminary reports indicate that urinary free cortisol (UFC) may be an intermediate phenotype. OBJECTIVES: The objectives of this study were: 1) confirm bimodality of UFC, 2) assess whether UFC variations aggregate in hypertensive families, and 3) compare low-mode and high-mode UFC groups for distinguishing features. SUBJECTS/SETTING: Subjects included 390 hypertensives and 166 normotensives from the general community. DESIGN/INTERVENTIONS: Subjects had blood pressure and laboratory measurements on high- and low-salt diets. Familial aggregation was evaluated in 250 hypertensive siblings from 117 families. RESULTS: Hypertensives had higher UFC than normotensives (P<0.001) and bimodal distribution of UFC (P<0.0001). Analyses were controlled for gender and dietary sodium, which are confounding determinants of UFC. Mean low-mode UFC (33.8+/-10.6 microg per 24 h) was similar to that of normotensives. The high mode, comprising 31.3% of hypertensives, had less change in mean arterial pressure between diets than the low mode (P=0.01) without any other significant differences. Observed proportions of concordance and discordance for UFC mode differed significantly from that expected (P<0.001). Observed concordance for the high mode was twice that expected, whereas for the low mode, it was similar to that expected by chance. Family membership explained a significant proportion of variance in UFC classification (P=0.027). UFC mode of one sibling was a significant predictor of the UFC mode of the other sibling [odds ratio 6.6, 95% confidence interval (2.4-18.0), P<0.001]. CONCLUSION: High-mode UFC is an intermediate phenotype of hypertension associated with salt resistance and a strong familial component supporting heritability.

Urinary prostaglandin F2alpha 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 F(2alpha) (PGF(2alpha)) is present in human urine in significant concentrations and is presumed to be derived from COX activity. We determined, however, that levels of putative PGF(2alpha) 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 PGF(2alpha) 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 PGF(2alpha) and its enantiomer, ent-PGF(2alpha), 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 PGF(2alpha) in human urine is derived from the free radical-initiated peroxidation of arachidonate independent of COX and is composed of PGF(2alpha) and its enantiomer, although the latter compound is approximately 2-fold more abundant. Thus, quantification of urinary PGF(2alpha) 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 PGF(2alpha) in humans is derived from the IsoP pathway has implications regarding PG formation and inhibition in vivo.

Bradykinin B(2) receptor does not contribute to blood pressure lowering during AT(1) receptor blockade.

This study tested the hypothesis that endogenous bradykinin contributes to the effects of angiotensin AT(1) receptor blockade in humans. The effect of the bradykinin B(2) receptor antagonist d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Tic-Oic-Arg (HOE-140) (18 microg/kg/h i.v. for 6 h) on hemodynamic and endocrine responses to acute and chronic (1-month) treatment with valsartan (160 mg/day) was determined in 13 normotensive and 12 hypertensive salt-deplete subjects. Acute valsartan increased plasma renin activity (PRA) from 5.3 +/- 9.9 to 15.6 +/- 19.8 ng of angiotensin (Ang) I/ml/h (P < 0.001) and decreased aldosterone from 18.3 +/- 10.5 to 12.0 +/- 9.6 ng/dl (P < 0.001). Chronic valsartan significantly increased baseline PRA (10.5 +/- 15.5 ng of Ang I/ml/h; P = 0.004) but did not affect baseline angiotensin-converting enzyme activity or aldosterone. HOE-140 tended to increase the PRA response to valsartan, and it attenuated the decrease in aldosterone following chronic valsartan (P = 0.03). Acute valsartan decreased mean arterial pressure 12.7 +/- 6.9% (from 100.2 +/- 8.4 to 87.5 +/- 9.8 mm Hg in hypertensives and from 82.4 +/- 8.6 to 70.3 +/- 8.4 mm Hg in normotensives). HOE-140 did not affect the blood pressure response to either acute (effect of valsartan, P < 0.001; effect of HOE-140, P = 0.98) or chronic (valsartan, P = 0.01; HOE-140, P = 0.84) valsartan. Plasma cGMP was increased significantly during chronic valsartan (P = 0.048) through a bradykinin receptor-independent mechanism (effect of HOE-140, P = 0.13). Both acute (P < 0.001) and chronic (P < 0.001) valsartan increased heart rate. HOE-140 augmented the heart rate response to chronic valsartan (P = 0.04). These data suggest that endogenous bradykinin does not contribute significantly to the blood pressure-lowering effect of valsartan through its B(2) receptor.

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 mug/kg per hour for 10 hours followed by 0.9 mug/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/m(2); 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 F(2)-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 F(2)-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 F(2)-isoprostanes, is mineralocorticoid receptor independent and may be pressor dependent.

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, alpha- and gamma-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 alpha-and gamma-thrombin-induced platelet aggregation (P < 0.01 versus baseline). Bradykinin 1-5 inhibited gamma-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.

Bradykinin B2 receptor knockout mice are protected from thrombosis by increased nitric oxide and prostacyclin.

Bradykinin (BK) liberates nitric oxide, prostacyclin, and tissue plasminogen activator from endothelial cells. We hypothesized that BK B2 receptor knockout (KO) mice (BKB2R(-/-)) have increased thrombosis risk. Paradoxically, the BKB2R(-/-) mice have long bleeding times and delayed carotid artery thrombosis, 78 +/- 6.7 minutes, versus 31 +/- 2.7 minutes in controls. The mechanism(s) for thrombosis protection was sought. In BKB2R(-/-) plasma coagulation, fibrinolysis and anticoagulant proteins are normal except for an increased prekallikrein and decreased factor XI. BKB2R(-/-) mice have elevated BK 1-5 (160 +/- 75 fmol/mL, vs 44 +/- 29 fmol/mL in controls) and angiotensin II (182 +/- 41 pg/mL, vs 49 +/- 7 pg/mL in controls). Ramipril treatment shortens vessel occlusion time. BKB2R(-/-) mice have elevated plasma 6-keto-PGF1alpha (666 +/- 232 ng/mL, vs 23 +/- 5.3 ng/mL in controls) and serum nitrate (61 +/- 5.3 microM, vs 24 +/- 1.8 microM in controls). Treatment with L-NAME (NG-mono-methyl-L-arginine ester) or nimesulide shortens the thrombosis time. BKB2R(-/-) mice have increased angiotensin receptor 2 (AT2R) mRNA and protein expression. Treatment with an AT2R antagonist, PD123 319, normalizes the thrombosis time and nitrate and 6-keto-PGF1alpha. The long bleeding times in BKB2R(-/-) mice also correct with L-NAME and nimesulide therapy. In BKB2R(-/-) mice, angiotensin II binding to an overexpressed AT2R promotes thromboprotection by elevating nitric oxide and prostacyclin. These investigations indicate a pathway for thrombosis risk reduction via the plasma kallikrein/kinin and renin angiotensin systems.

A pilot study indicating that bradykinin B2 receptor antagonism attenuates protamine-related hypotension after cardiopulmonary bypass.

BACKGROUND: The administration of protamine to patients who received heparin during cardiopulmonary bypass (CPB) induces hypotension. Protamine inhibits the carboxypeptidase N-mediated degradation of bradykinin, a peptide that causes vasodilation and tissue-type plasminogen activator (t-PA) release. This study tests the primary hypothesis that blocking the bradykinin B(2) receptor would attenuate protamine-related hypotension. METHODS: We conducted a prospective, double-blind, randomized study in 16 adult male patients undergoing elective cardiac surgery requiring CPB and taking an angiotensin-converting enzyme (ACE) inhibitor preoperatively, because ACE inhibition increases bradykinin concentrations during CPB. Subjects were randomized to receive either saline solution (N = 8) or the bradykinin B(2) receptor antagonist HOE 140 (100 mug/kg, N = 8) before the administration of protamine. Mean arterial pressure (MAP) and t-PA activity were measured intraoperatively and before and after protamine administration. RESULTS: Protamine administration caused a significant increase in bradykinin concentrations in the saline solution group (from 6.0 +/- 1.3 to 10.0 +/- 1.6 fmol/mL, P = .043), as well as the HOE 140 group (from 6.5 +/- 1.8 to 14.3 +/- 4.6 fmol/mL, P = .042). Protamine significantly decreased MAP in the saline solution group (from 69.8 +/- 4.4 mm Hg to a mean individual nadir of 56.1 +/- 2.6 mm Hg, P = .031), but bradykinin receptor antagonism blunted this effect (from 74.3 +/- 3.7 mm Hg to a mean individual nadir of 69.6 +/- 1.2 mm Hg in the HOE 140 group, P = .545). Hence, during protamine infusion, MAP was significantly lower in the saline solution group compared with the HOE 140 group (P = .002). t-PA activity decreased significantly during administration of HOE 140 (from 3.59 +/- 0.31 to 1.67 +/- 0.42 IU/mL, P = .001) but not during saline solution (from 2.12 +/- 0.48 to 1.44 +/- 0.36 IU/mL, P = .214). Similarly, t-PA activity decreased significantly during protamine administration in the HOE 140 group (from 1.67 +/- 0.42 to 0.77 +/- 0.26 IU/mL, P = .038) but not in the saline solution group (from 1.44 +/- 0.36 to 0.99 +/- 0.26 IU/mL, P = .132). CONCLUSION: Increased bradykinin contributes to protamine-related hypotension through its B(2) receptor in ACE inhibitor-treated patients.

Angiotensin-converting enzyme inhibition increases basal vascular tissue plasminogen activator release in women but not in men.

OBJECTIVE: Angiotensin-converting enzyme inhibition (ACEI) increases vascular tissue plasminogen activator (t-PA) release through endogenous bradykinin (BK). We tested the hypothesis that gender influences the effect of ACEI on t-PA release. METHODS AND RESULTS: We measured the effect of intra-arterial enalaprilat (0.33 microg/min per 100 mL forearm volume) on forearm blood flow (FBF) and net t-PA release before and during BK (25 to 400 ng/min) and methacholine (3.2 to 12.8 microg/min) in premenopausal women, postmenopausal women not using hormone replacement, young men, and older men. Baseline net t-PA release was similar among groups. Enalaprilat increased basal t-PA release in premenopausal (from 0.9+/-1.0 to 5.1+/-1.7 ng/min per 100 mL, P=0.023) and postmenopausal women (from -3.9+/-2.2 to 3.9+/-1.1 ng/min per 100 mL, P=0.010) but not in young or older men (P=0.028 men versus women). Enalaprilat potentiated the effect of exogenous BK on FBF similarly in all groups. However, during enalaprilat, BK-stimulated t-PA release was greatest in premenopausal women (339.9+/-86.4 ng/min per 100 mL at the 100 ng/min dose, P<0.05 versus any other group), intermediate in postmenopausal women (243.8+/-51.1 ng/min per 100 mL, P<0.05 versus either male group), and least in young (111.9+/-19.2 ng/min/100 mL) and older men (103.4+/-27.6 ng/min/100 mL). CONCLUSIONS: ACEI enhances basal t-PA release in women, independent of menopausal status, but not in men. During ACEI, both gender and menopausal status affect BK stimulated t-PA release.

Quantification of the major urinary metabolite of PGE2 by a liquid chromatographic/mass spectrometric assay: determination of cyclooxygenase-specific PGE2 synthesis in healthy humans and those with lung cancer.

Prostaglandin (PG)E2 is a major cyclooxygenase (COX) product that is important in human physiology and pathophysiology. Quantification of systemic PG production in humans is best assessed by measuring excreted urinary metabolites. Accurate and easy-to-perform assays to quantify the major urinary metabolite of PGE2, 11alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), do not exist. We now report the development of a robust and facile method to measure urinary PGE-M excretion in humans using stable isotope dilution techniques employing liquid chromatography/tandem mass spectrometry (LC/MS/MS). Concentrations of the metabolite in urine from healthy humans are nearly twofold greater in men than in women (10.4+/-1.5 vs. 6.0+/-0.7 ng/mg creatinine). Levels of PGE-M in healthy humans are suppressed significantly not only by the nonselective COX inhibitor ibuprofen but also by the COX-2 selective inhibitor rofecoxib, suggesting that the majority of PGE2 formed in vivo is derived from COX-2. Increased COX-2 expression and increased PGE2 production are associated with malignancy. Levels of PGE-M were found to be greatly increased in humans with unresectable non-small cell cancer of the lung, and this increase is dramatically reduced by administration of the COX-2 inhibitor celecoxib, implying that COX-2 contributes significantly to the overproduction of PGE2. In summary, quantification of PGE-M using LC/MS/MS provides a facile and accurate method to assess PGE2 formation in human physiological and pathophysiological processes.

Angiotensin-converting enzyme inhibition and smoking potentiate the kinin response to cardiopulmonary bypass.

BACKGROUND: This study tested the hypothesis that angiotensin-converting enzyme (ACE) inhibitors potentiate activation of the kallikrein-kinin system during cardiopulmonary bypass (CPB). METHODS: The effects of CPB on concentrations of bradykinin and its metabolite bradykinin 1-5 (BK1-5) were determined in 31 patients taking an ACE inhibitor who were randomized to continue ACE inhibitors until coronary artery bypass surgery (ACE inhibitor group, N = 19) or to discontinue them 48 hours before surgery (no ACE inhibitor group, N = 12). Arterial and venous blood was sampled before CPB, at 30 minutes of CPB, at 60 minutes of CPB, after separation from CPB, and on postoperative day 1. RESULTS: Arterial bradykinin ( P < .001 [from 22.4 +/- 24.1 fmol/mL to 86.2 +/- 98.7 fmol/mL in the no ACE inhibitor group]) and arterial ( P < .001) and venous ( P = .016) BK1-5 concentrations increased significantly during CPB. Arterial bradykinin concentrations were significantly higher ( P = .017), whereas BK1-5 concentrations ( P = .024) and the molar ratio of BK1-5/bradykinin ( P = .008) were significantly lower in the ACE inhibitor group compared with the no ACE inhibitor group. In addition, arterial bradykinin concentrations were significantly increased in smokers compared with nonsmokers ( P = .015), when we controlled for the ACE inhibitor group. There was no effect of smoking on ACE activity ( P = .597 overall). There was a significant inverse correlation between arterial bradykinin and mean arterial pressure ( r 2 = 0.2137, P = .010) and a significant correlation between arterial bradykinin and tissue-type plasminogen activator antigen concentrations ( r 2 = 0.174, P = .022) during CPB. Tissue-type plasminogen activator antigen was significantly higher in the ACE inhibitor group than in the no ACE inhibitor group (18.0 +/- 7.8 ng/mL versus 12.4 +/- 4.5 ng/mL, P = .016) but not in smokers compared with nonsmokers ( P = .451). CONCLUSION: Preoperative ACE inhibitors and smoking potentiate the kinin response to CPB and may contribute to the hemodynamic and fibrinolytic response observed during CPB.

Uric acid and the state of the intrarenal renin-angiotensin system in humans.

BACKGROUND: Experimental hyperuricemia is marked by an activated intrarenal renin-angiotensin system (RAS). The renal vascular response to exogenous angiotensin II (Ang II) provides an indirect measure of intrarenal RAS activity. We tested the hypothesis that the serum uric acid concentration predicts the renal vascular response to Ang II. METHODS: A total of 249 subjects in high sodium balance had the renal plasma flow (RPF) response to Ang II measured. Para-aminohippuric acid (PAH) clearance was used to estimate RPF. Multivariable regression analysis determined if the serum uric acid concentration independently predicts the RPF response to Ang II. Variables considered included age, gender, race, body mass index (BMI), hypertension status, blood pressure, basal RPF, creatinine clearance, serum insulin, serum glucose, serum high-density lipoprotein (HDL), serum triglycerides, and plasma renin activity (PRA). RESULTS: Uric acid concentration negatively correlated with the RPF response to Ang II (r=-0.37, P < 0.001). In univariate analysis, age, BMI, hypertension, triglycerides, and blood pressure were negatively associated, and basal RPF, HDL, and female gender were positively associated with the RPF response to Ang II. In multivariable analysis, serum uric acid concentration independently predicted the RPF response to Ang II (beta=-5.3, P < 0.001). CONCLUSION: Serum uric acid independently predicted blunted renal vascular responsiveness to Ang II, consistent with results from experimental hyperuricemia showing an activated intrarenal RAS. This could be due to a direct effect of uric acid or reflect a more fundamental renal process. These data may have relevance to the association of uric acid with risk for hypertension and nephropathy.

Thyroid function and blood pressure homeostasis in euthyroid subjects.

Overt and subclinical hypothyroidism are associated with increased systemic vascular resistance and hypertension. We examined the relationship between thyroid function and blood pressure homeostasis in euthyroid individuals. A total of 284 subjects (68% hypertensive) consumed high- (200 mmol) and low- (10 mmol) sodium diets, and their blood pressure responses were assessed as percentage change in the mean arterial pressure (MAP). p-Aminohippuric acid clearance was used to estimate effective renal plasma flow. Renal vascular resistance (RVR) was calculated as MAP divided by effective renal plasma flow. Serum free T(4) index (FTI) was lower (P < 0.0001) and TSH was higher (P = 0.046) in hypertensive compared with normotensive subjects independent of other baseline characteristics. FTI (beta = -1.51, P < 0.0001), baseline MAP, and race independently predicted MAP salt sensitivity. The FTI relationship with salt sensitivity adjusted for baseline MAP and race was similar among normotensive (beta = -1.42, P = 0.008) and hypertensive subjects (beta = -1.66, P = 0.0001). FTI correlated negatively with high- (P = 0.0001) and low- (P = 0.008) salt RVR, whereas TSH correlated positively with high- (P = 0.016) and low- (P = 0.012) salt RVR independent of age, gender, race, and body mass index. We have found that FTI is lower and TSH is higher in hypertensive compared with normotensive euthyroid subjects and that FTI independently predicts blood pressure salt sensitivity. These data show that the influence of thyroid function on blood pressure homeostasis extends into euthyroid range and likely reflects the action of thyroid hormone on peripheral vasculature.

The preparation and characterization of novel peptide antagonists to thrombin and factor VIIa and activation of protease-activated receptor 1.

Thrombin and protease-activated receptor 1 (PAR1) activation antagonists were prepared based upon the peptide RPPGF, the angiotensin-converting enzyme breakdown product of bradykinin. A library of 72 peptides consisting of d and/or synthetic amino acids was designed with various substitutions in positions 1 to 5 in Arg-Pro-Pro-Gly-Phe (RPPGF). Two compounds, rOicPGF (TH146) and betaAK2K-4(rOicPGF) (MAP4-TH146), were characterized further. TH146 or MAP4-TH146 completely inhibits threshold gamma-thrombin-induced platelet aggregation at a concentration of 142 +/- 0.05 or 19 +/- 0.06 microM, respectively. TH146 completely inhibits threshold alpha-thrombin-induced washed platelet aggregation at 444 +/- 0.04 microM. TH146 or MAP4-TH146 blocks 2 nM alpha-thrombin-induced fibroblast calcium mobilization with an IC(50) value of 110 or 18 microM, respectively. Furthermore, significant prolongation of the activated partial thromboplastin time, prothrombin time, or thrombin clotting time occurs at 31, 62, or 7.8 microM TH146 and 0.4, 6.25, or 1.56 microM MAP4-TH146, respectively. TH146 and MAP4-TH146 inhibit both alpha-thrombin with a K(i) value of 97 and 49 microM, respectively, and factor VIIa with a K(i) value of 44 and 5 microM, respectively. Both TH146 and MAP4-TH146 specifically bind to the exodomain of recombinant PAR1. MAP4-TH146 (200 microM) completely blocks thrombocytin, a PAR1-activating snake venom protease, without inhibiting the enzyme's active site. TH146 inhibits gamma-thrombin-induced aggregation of mouse platelets, prolongs mouse bleeding times, and delays the time to mouse carotid artery thrombosis. TH146 and MAP4-TH146 inhibit human and mouse platelet aggregation and mouse thrombosis. Analogs of RPPGF are model compounds to develop PAR1 activation antagonists as well as direct inhibitors to thrombin and factor VIIa.

Loss of sodium modulation of plasma kinins in human hypertension.

We studied the effect of salt intake and hypertension on the systemic kallikrein-kinin system (KKS), as measured by bradykinin (BK) 1-5, a stable circulating bradykinin metabolite, and the tissue KKS, as measured by urinary kallikrein excretion. Venous BK 1-5, urinary kallikrein, and components of the renin-angiotensin-aldosterone system were measured in 35 normotensive and 19 hypertensive subjects who were maintained on a high (200 mmol/day) or low (10 mmol/day) salt diet. Salt restriction decreased mean arterial pressure (MAP) (P < 0.001 overall) and the plasma angiotensin-converting enzyme (P = 0.017) and increased plasma renin activity (P < 0.001) and serum aldosterone (P < 0.001). There was an interactive effect of salt intake and hypertension on plasma BK 1-5 (P = 0.043), with BK 1-5 significantly lower during low compared with high salt intake in normotensive (24.7 +/- 2.6 versus 34.9 +/- 5.6 fmol/ml, P = 0.002) but not hypertensive subjects (30.6 +/- 4.6 versus 27.5 +/- 2.8 fmol/ml, P = 0.335). In normotensives, the change in plasma BK 1-5 from high to low salt intake correlated with the change in MAP (r = 0.533, P = 0.004). Urinary kallikrein was higher during low compared with high salt intake (P < 0.001) in both groups. There was no effect of salt intake on urinary BK 1-5. In summary, the systemic and renal KKSs act in tandem to modulate the response to salt intake. The systemic system is activated during high salt intake and counterbalances increased vascular response to pressors. With sodium restriction, the renal system is activated and counterbalances the increased sodium-retaining state induced by activation of the renin-angiotensin-aldosterone system. With hypertension, these modulating effects are diminished or lost, supporting a role for both systems in the development/maintenance of hypertension.

Angiotensin-converting enzyme inhibition alters the fibrinolytic response to cardiopulmonary bypass.

BACKGROUND: Increased plasminogen activator inhibitor-1 (PAI-1) concentrations after coronary artery bypass grafting (CABG) are associated with increased risk of vein graft occlusion. Because angiotensin II stimulates PAI-1 expression, we tested the hypothesis that preoperative angiotensin-converting enzyme (ACE) inhibition decreases PAI-1 expression after CABG. METHODS AND RESULTS: We measured the effects of cardiopulmonary bypass (CPB) on PAI-1 antigen and tissue-type plasminogen activator (tPA) antigen and activity in 31 patients taking an ACE inhibitor (ACEI) who were randomized to continue ACEI until the morning of surgery (ACEI group, n=19) or to discontinue it 48 hours before surgery (No-ACEI group, n=12). Arterial blood samples were taken at baseline before CPB, twice during CPB, after separation from CPB, and on postoperative day 1 (POD1). CPB caused an early decrease in PAI-1 antigen, followed by an increase in PAI-1 antigen on POD1 (P<0.001 for effect of time). ACE inhibition attenuated the increase in PAI-1 antigen such that both PAI-1 antigen on POD1 (P=0.013) and the change in PAI-1 antigen from baseline to POD1 (P=0.009) were higher in the No-ACEI group (from 17.0+/-5.0 to 48.7+/-8.8 ng/mL) versus the ACEI group (from 19.9+/-3.4 to 33.1+/-6.2 ng/mL). There was no significant difference between the 2 groups in intraoperative tPA activity (P=0.259); however, the increase in tPA activity was significantly greater in the ACEI group than in the No-ACEI group (P=0.030). CONCLUSIONS: Preoperative ACEI attenuates the increase in PAI-1 after CABG, suggesting a role for ACE inhibition in reducing the risk of acute graft thrombosis.

Acute angiotensin II increases plasma F2-isoprostanes in salt-replete human hypertensives.

Angiotensin (Ang) II induces oxidative stress in vitro and in animal models of hypertension. We tested the hypothesis that Ang II increases oxidative stress in human hypertension, as assessed by plasma F2-isoprostane concentrations. Plasma F2-isoprostanes, hemodynamic and endocrine parameters were measured at baseline and following a 55 min infusion of 3 ng/kg/min Ang II in 13 normotensive and 13 hypertensive volunteers ingesting a high- (200 mmol/d) or low- (10 mmol/d) sodium diet. Mean arterial pressure (MAP) and body mass index were higher in hypertensive subjects. Ang II infusion increased MAP (p<.001) and plasma aldosterone concentrations (p<.001) and decreased plasma renin activity (p<.001) and renal plasma flow (p<.001) to a similar extent in both groups. Plasma F2-isoprostane concentrations were similar at baseline. There was no effect of Ang II on F2-isoprostane concentrations during low-salt intake in either group (normotensive 51.7 +/- 7.1 to 53.7 +/- 6.5 pg/ml and hypertensive 52.2 +/- 8.2 to 56.2 +/- 10.0 pg/ml; mean +/- SE). During high-salt intake, Ang II increased F2-isoprostane concentrations in the hypertensive group (52.3 +/- 7.2 to 63.2 +/- 10.4 pg/ml, p=0.010) but not in the normotensive group (54.2 +/- 4.4 to 58.9 +/- 6.6 pg/ml, p=0.83). Acute Ang II infusion increases oxidative stress in vivo in hypertensive humans. The renin-angiotensin system may contribute to oxidative stress in human cardiovascular disease.

Effect of combined AT1 receptor and aldosterone receptor antagonism on plasminogen activator inhibitor-1.

Aldosterone enhances angiotensin II (Ang II)-induced plasminogen activator inhibitor (PAI)-1 expression in vitro. This study tested the hypothesis that angiotensin II type 1 (AT(1)) and aldosterone receptor antagonism interact to decrease PAI-1 in humans. Effects of candesartan (16 mg/d), spironolactone (25 mg/d), or combined candesartan/spironolactone on mean arterial pressure (MAP), endocrine, and fibrinolytic variables were measured in 18 normotensive subjects [age 33.7 yr (95% confidence interval 29.3, 38.0), body mass index 26.6 (24.7, 28.4) kg/m(2)] in whom the renin-angiotensin-aldosterone system was activated by furosemide (20 mg/d). Candesartan [83.3 mm Hg (78.9, 87.7)], but not spironolactone [89.4 mm Hg (85.4, 93.5)], decreased MAP, compared with baseline [92.2 mm Hg (88.9, 95.5), P < 0.001] and furosemide alone [89.1 mm Hg (85.7, 92.4), P = 0.002]. Coadministration of spironolactone with candesartan did not further decrease MAP. Candesartan dramatically increased Ang II [177.9 pg/ml (113.3, 242.6)], compared with baseline [34.8 pg/ml (29.3, 40.4), P = 0.002] and furosemide alone [40.6 pg/ml (29.7, 51.5), P = 0.003]. Spironolactone increased Ang II [51.5 pg/ml (41.3, 61.7), P = 0.014 vs. baseline, P = 0.004 vs. candesartan]. There was no additive effect of candesartan and spironolactone on Ang II [197.6 pg/ml (134.2, 261.0)]. Aldosterone was lower during candesartan [8.9 ng/dl (7.3, 10.6), P = 0.007] than during furosemide alone [14.1 ng/dl (10.9, 17.3), P = 0.007], spironolactone [18.7 ng/dl (14.5, 22.9), P = 0.002], or combined candesartan/spironolactone [13.9 ng/dl (11.8, 15.9), P = 0.006]. Furosemide increased PAI-1 antigen [27.8 ng/ml (20.6, 35.0), P = 0.002 vs. 19.3 ng/ml (13.4, 25.2) baseline], even in the presence of candesartan [27.2 ng/ml (16.5, 37.8), P = 0.042 vs. baseline] or spironolactone [27.3 ng/ml (17.9, 36.8), P = 0.015 vs. baseline]. However, coadministration of AT(1) and aldosterone receptor antagonists prevented the furosemide-induced increase in PAI-1 [19.2 ng/ml (9.8, 28.6), P = 0.974 vs. baseline, P < 0.05 vs. candesartan, spironolactone or furosemide alone]. This study evidences an interactive effect of endogenous Ang II and aldosterone on PAI-1 production in humans.

Human colorectal cancer cells efficiently conjugate the cyclopentenone prostaglandin, prostaglandin J(2), 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.

Formation of highly reactive A-ring and J-ring isoprostane-like compounds (A4/J4-neuroprostanes) in vivo from docosahexaenoic acid.

Free radical-initiated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. Docosahexaenoic acid is the most abundant unsaturated fatty acid in the central nervous system. We have shown previously that this 22-carbon fatty acid can yield, upon oxidation, isoprostane-like compounds termed neuroprostanes, with E/D-type prostane rings (E(4)/D(4)-neuroprostanes). Eicosanoids with E/D-type prostane rings are unstable and dehydrate to cyclopentenone-containing compounds possessing A-type and J-type prostane rings, respectively. We thus explored whether cyclopentenone neuroprostanes (A(4)/J(4)-neuroprostanes) are formed from the dehydration of E(4)/D(4)-neuroprostanes. Indeed, oxidation of docosahexaenoic acid in vitro increased levels of putative A(4)/J(4)-neuroprostanes 64-fold from 88 +/- 43 to 5463 +/- 2579 ng/mg docosahexaenoic acid. Chemical approaches and liquid chromatography/electrospray ionization tandem mass spectrometry definitively identified them as A(4)/J(4)-neuroprostanes. We subsequently showed these compounds are formed in significant amounts from a biological source, rat brain synaptosomes. A(4)/J(4)-neuroprostanes increased 13-fold, from a basal level of 89 +/- 72 ng/mg protein to 1187 +/- 217 ng/mg (n = 4), upon oxidation. We also detected these compounds in very large amounts in fresh brain tissue from rats at levels of 97 +/- 25 ng/g brain tissue (n = 3) and from humans at levels of 98 +/- 26 ng/g brain tissue (n = 5), quantities that are nearly an order of magnitude higher than other classes of neuroprostanes. Because of the fact that A(4)/J(4)-neuroprostanes contain highly reactive cyclopentenone ring structures, it would be predicted that they readily undergo Michael addition with glutathione and adduct covalently to proteins. Indeed, incubation of A(4)/J(4)-neuroprostanes in vitro with excess glutathione resulted in the formation of large amounts of adducts. Thus, these studies have identified novel, highly reactive A/J-ring isoprostane-like compounds that are derived from docosahexaenoic acid in vivo.

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 microg/min; N=20), or methacholine (3.2, 6.4, 12.8 microg/min; N=20), and sodium nitroprusside (0.8, 1.6, and 3.2 microg/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 x min(-1) x 100 mL(-1)), blacks (increase in FBF from 2.8+/-0.3 to 15.2+/-1.9 mL x 100 mL(-1) x 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 x min(-1) x 100 mL(-1) in blacks; from 0.04+/-0.9 to 65.9+/-13.6 ng x min(-1) x 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.