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Results from prevention trials, including the Alzheimer's Disease Anti-inflammatory Prevention Trial (ADAPT), have fueled discussion about the cardiovascular (CV) risks associated with non-steroidal anti-inflammatory drugs (NSAIDs). We tested the hypotheses that (i) adverse CV events reported among ADAPT participants (aged 70 years and older) are associated with increased ratio of urine 11-dehydrothromboxane B(2) (Tx-M) to 2'3-donor-6-keto-PGF1 (PGI-M) attributable to NSAID treatments; (ii) coincident use of aspirin (ASA) would attenuate NSAID-induced changes in Tx-M/PGI-M ratio; and (iii) use of NSAIDs and/or ASA would not alter urine or plasma concentrations of F(2)-isoprostanes (IsoPs), in vivo biomarkers of free radical damage. We quantified urine Tx-M and PGI-M, and urine and plasma F(2)-IsoPs from 315 ADAPT participants using stable isotope dilution assays with gas chromatography/mass spectrometry, and analyzed these data by randomized drug assignment and self-report compliance as well as ASA use. Adverse CV events were significantly associated with higher urine Tx-M/PGI-M ratio, which seemed to derive mainly from lowered PGI-M. Participants taking ASA alone had reduced urine Tx-M/PGI-M compared to no ASA or NSAID; however, participants taking NSAIDs plus ASA did not have reduced urine Tx-M/PGI-M ratio compared to NSAIDs alone. Neither NSAID nor ASA use altered plasma or urine F(2)-IsoPs. These data suggest a possible mechanism for the increased risk of CV events reported in ADAPT participants assigned to NSAIDs, and suggest that the changes in the Tx-M/PGI-M ratio was not substantively mitigated by coincident use of ASA in individuals 70 years or older.
We examined the effects of endogenous nitric oxide (NO) inhibition on the longitudinal distribution of pulmonary vascular resistance and on arachidonic acid metabolism during endotoxemia in awake sheep. Mean pulmonary artery (Ppa), left atrial (Pla), and systemic artery pressure (Psa) were continuously measured, and cardiac output (CO) was continuously monitored by an implanted ultrasonic flow probe. We advanced a 7-French Swan-Ganz catheter into distal pulmonary artery and measured the pulmonary microwedge pressure (Pmw) with the balloon deflated, allowing calculation of upstream pulmonary vascular resistance (PVRup = [Ppa - Pmw]/CO) and down-stream PVR (PVRdown = [Pmw - Pla]/CO), respectively. In paired studies, endotoxin (1 micro g/kg) was infused over 30 minutes with and without N(omega)-nitro-L-arginine (NLA) treatment. NLA (20 mg/kg) was administered 30 minutes before endotoxin infusion. Endotoxin caused increases in PVRup and PVRdown. Pretreatment with NLA increases PVRup at baseline and enhanced increases in both PVRup and PVRdown during endotoxemia. Plasma level of thromboxane B(2) (TxB(2)) and prostacyclin (6-keto = PGF(1alpha)) significantly increased 1 hour after endotoxin administration (TxB(2), 308.3 +/- 94.8 [SE] to 2163.5 +/- 988.5 pg ml(-1), P <.05; 6-keto=PGF(1alpha), 155.6 +/- 91.4 to 564.9 +/- 131.8 pg ml(-1), P <.05), but the increased levels were similar to those in the NLA-pretreated animals. We conclude that endogenous NO mainly regulates precapillary vascular tone at baseline, and that NO modulated pre- and postcapillary vascular constriction during endotoxemia in sheep. It appears that cyclooxygenase production in response to endotoxin is unaffected by NO and its vascular effects.
Acetaminophen has antipyretic and analgesic properties yet differs from the nonsteroidal antiinflammatory drugs and inhibitors of prostaglandin H synthase (PGHS)-2 by exhibiting little effect on platelets or inflammation. We find parallel selectivity at a cellular level; acetaminophen inhibits PGHS activity with an IC(50) of 4.3 microM in interleukin (IL)-1 alpha-stimulated human umbilical vein endothelial cells, in contrast with an IC(50) of 1,870 microM for the platelet, with 2 microM arachidonic acid as substrate. This difference is not caused by isoform selectivity, because acetaminophen inhibits purified ovine PGHS-1 and murine recombinant PGHS-2 equally. We explored the hypothesis that this difference in cellular responsiveness results from antagonism of the reductant action of acetaminophen on the PGHSs by cellular peroxides. Increasing the peroxide product of the PGHS-cyclooxygenase, prostaglandin G(2) (PGG(2)), by elevating the concentration of either enzyme or substrate reverses the inhibitory action of acetaminophen, as does the addition of PGG(2) itself. 12-Hydroperoxyeicosatetraenoic acid (0.3 microM), a major product of the platelet, completely reverses the action of acetaminophen on PGHS-1. Inhibition of PGHS activity by acetaminophen in human umbilical vein endothelial cells is abrogated by t-butyl hydroperoxide. Together these findings support the hypothesis that the clinical action of acetaminophen is mediated by inhibition of PGHS activity, and that hydroperoxide concentration contributes to its cellular selectivity.
BACKGROUND - Bradykinin stimulates dose-dependent tissue plasminogen activator (tPA) release from human endothelium. Although bradykinin is known to cause vasodilation through B(2) receptor-dependent effects on NO, prostacyclin, and endothelium-derived hyperpolarizing factor production, the mechanism(s) underlying tPA release is unknown.
METHODS AND RESULTS - We measured the effects of intra-arterial bradykinin (100, 200, and 400 ng/min), acetylcholine (15, 30, and 60 microg/min), and nitroprusside (0.8, 1.6, and 3.2 microg/min) on forearm vasodilation and tPA release in healthy volunteers in the presence and absence of (1) the B(2) receptor antagonist HOE 140 (100 microg/kg IV), (2) the NO synthase inhibitor L-N:(G)-monomethyl-L-arginine (L-NMMA, 4 micromol/min intra-arterially), and (3) the cyclooxygenase inhibitor indomethacin (50 mg PO TID). B(2) 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 F(1alpha) (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.
CONCLUSIONS - These data indicate that bradykinin stimulates tPA release from human endothelium through a B(2) 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.
CONTEXT - An imbalance in vasodilating (prostacyclin [PGI2]) and vasoconstricting (thromboxane A2 [TxA2]) eicosanoids may be important in preeclampsia, but prospective data from large studies needed to resolve this issue are lacking. Because most trials using aspirin to reduce TxA2 production have failed to prevent preeclampsia, it is critical to determine whether eicosanoid changes occur before the onset of clinical disease or are secondary to clinical manifestations of preeclampsia.
OBJECTIVE - To determine whether PGI2 or TxA2 changes occur before onset of clinical signs of preeclampsia.
DESIGN, SETTING, AND PARTICIPANTS - Multicenter prospective study from 1992 to 1995 of subjects from the placebo arm of the Calcium for Preeclampsia Prevention Trial. Women who developed preeclampsia (n = 134) were compared with matched normotensive control women (n = 139).
MAIN OUTCOME MEASURES - Excretion of urinary metabolites of PGI2 (PGI-M) and TxA2 (Tx-M) as measured from timed urine collections obtained prospectively before 22 weeks', between 26 and 29 weeks', and at 36 weeks' gestation.
RESULTS - Women who developed preeclampsia had significantly lower PGI-M levels throughout pregnancy, even at 13 to 16 weeks' gestation (long before the onset of clinical disease); their gestational age-adjusted levels were 17% lower than those of controls (95% confidence interval [CI], 6%-27%; P=.005). The Tx-M levels of preeclamptic women were not significantly higher overall (9% higher than those of controls; 95% CI, -3% to 23%; P=.14). The ratio of Tx-M to PGI-M, used to express relative vasoconstricting vs vasodilating effects, was 24% higher (95% CI, 6%-45%) in preeclamptic women throughout pregnancy (P=.007).
CONCLUSIONS - Our results show that reduced PGI2 production, but not increased TxA2 production, occurs many months before clinical onset of preeclampsia. Aspirin trials may have failed because an increase in thromboxane production is not the initial anomaly. Future interventions should make correcting prostacyclin deficiency a major part of the strategy to balance the abnormal vasoconstrictor-vasodilator ratio present in preeclampsia.
Clinical manifestations of mastocytosis are mediated, at least in part, by release of the mast cell mediators histamine and prostaglandin D2. It has been previously reported that in addition to prostaglandin D2, mast cells produce other eicosanoids, including thromboxane. Nonetheless, little information exists regarding the formation of other prostanoids in vivo. The most accurate method to examine the systemic production of eicosanoids in vivo is the quantitation of urinary metabolites. We previously developed a highly accurate assay employing mass spectrometry to measure a major urinary metabolite of thromboxane, 11-dehydro-thromboxane B2, in humans. We utilized this assay to quantitate thromboxane production in 17 patients with histologically proven mastocytosis. We report that thromboxane formation was significantly increased (>2 SD above the mean) in at least one urine sample from 65% of patients studied. Of these, 91% of patients with documented systemic involvement had elevated thromboxane generation. In addition, endogenous formation of thromboxane was highly correlated with the urinary excretion of the major urinary metabolite of prostaglandin D2 (r = 0.98) and Ntau-methylhistamine (r = 0.91), suggesting that the cellular source of increased thromboxane in vivo could be the mastocyte. Enhanced thromboxane formation in patients with this disorder is unlikely to be of platelet origin as other markers of platelet activation, platelet factor 4 and beta-thromboglobulin, were not increased in three patients with marked overproduction of thromboxane. Furthermore, the recovery of 11-dehydro-thromboxane B2 excretion in two patients after the administration of aspirin occurred significantly more rapidly than the recovery of platelet thromboxane generation. These studies, therefore, report that thromboxane production is significantly increased in the majority of patients with mastocytosis that we examined and provide the basis to elucidate the role of this eicosanoid in disorders of mast cell activation.
Angiotensin converting enzyme (ACE) inhibitors block degradation of bradykinin and bradykinin stimulates prostacyclin production. ACE inhibitors are reported to increase prostaglandins. Therefore, we set out to determine 1) the contribution of prostacyclin to the bradykinin-mediated vasodepressor effects of ACE inhibitors, 2) whether ACE inhibitors alter the effect of bradykinin on prostacyclin, and 3) whether the effects of ACE inhibitors on bradykinin and prostaglandins are class effects or dependent on ACE inhibitor structure. To address these questions, we compared the effects of captopril, quinapril and placebo on blood pressure, urinary excretion of 2,3-dinor-6-keto-PGF1 alpha, and the vasodepressor response to i.v. bradykinin in 21 salt-replete normal-to-high renin hypertensive patients. Captopril and quinapril doses were titrated to lower pressure similarly. Captopril, but not quinapril, increased excretion of prostacyclin metabolite (217 +/- 50 vs. 135 +/- 21 pg/mg Cr base line, P < .05). Both ACE inhibitors dramatically, equally potentiated the vasodepressor response to bradykinin; the bradykinin dose required to decrease mean arterial pressure 15 mm Hg or increase pulse 20 bpm was 50-fold lower in ACEI-treated than in placebo-treated subjects (10 +/- 0 and 12.1 +/- 2.1 ng/kg/min in captopril and quinapril groups vs. 567 +/- 109 ng/kg/min in the placebo group; P < .005). ACE inhibition significantly attenuated the prostacyclin response to bradykinin at any given level of hypotensive response. Indomethacin abolished the prostacyclin response to bradykinin but did not alter the vasodepressor response. These data demonstrate that ACE inhibitors potentiate bradykinin-mediated vasodepression through a prostaglandin-independent mechanism. They suggest that although ACE inhibitors increase prostaglandins by increasing bradykinin, ACE inhibitors may attenuate prostaglandin production through a second bradykinin-independent mechanism.
Endogenous prostacyclin production is best assessed by the measurement of its excreted metabolites, of which a major one is 2,3-dinor-6-ketoprostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha). Gas chromatographic-mass spectrometric (GC-MS) assays have been developed for this compound but are cumbersome and time-consuming. We now report a modified assay for the measurement of 2,3-dinor-6-keto-PGF1 alpha employing GC-MS in which sample preparation time is markedly shortened by replacing a number of extraction steps with reversed-phase column extraction and by modifying derivatization procedures. Precision of the assay is +/- 5% and the accuracy is 98%. The lower limit of detection in urine is approximately 15 pg/mg creatinine. Normal urinary levels of this metabolite were found to be 141 +/- 54 pg/mg creatinine (mean +/- S.D.). Urinary excretion of 2,3-dinor-6-keto-PGF1 alpha is markedly altered in situations associated with abnormalities of prostacyclin generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous prostacyclin production and to further explore the role of this compound in human health and disease.
The relationship between renal prostaglandin (PG)I2 biosynthesis and renin release was examined in conscious dogs before and during renal artery constriction. Dogs were chronically instrumented with femoral vein, femoral artery and left renal vein catheters and an inflatable cuff and electromagnetic flow probe were positioned on the left renal artery. After 2 days, mean arterial blood pressure, heart rate, renal blood flow and renal secretion rates of renin and 6-keto-PGF1 alpha were determined before and 10 min after a reduction in renal blood flow. Plasma levels of 6-keto-PGF1 alpha, measured by a gas chromatographic-mass spectrometric assay, were used as an index of PGI2 synthesis. A 38% reduction in renal blood flow did not significantly alter mean arterial blood pressure, heart rate or arterial levels of plasma renin activity or 6-keto-PGF1 alpha. In contrast, renal artery constriction increased renal venous plasma levels of both renin activity and 6-keto-PGF1 alpha by 308% (P less than .002) and 132% (P less than .05), respectively. As a consequence, the renal secretion rate of renin was increased from 80 +/- 40 to 917 +/- 231 ng of angiotensin I . min-1 . hr-1 (P less than .02) and the renal secretion rate of 6-keto-PGF1 alpha was increased from -2.1 +/- 1.1 to 9.0 +/- 3.6 ng/min (P less than .05). In addition, there was a significant correlation between the renal secretion rates of renin and 6-keto-PGF1 alpha (r = 0.688; P less than .013; n = 12). These data indicate a close association between the renal biosynthesis and PGI2 and renin release and are consistent with the concept that PGI2 participates in the release of renin.