Thromboxane A2 Analogue U46619 Research Articles

Medroxyprogesterone Acetate and Dihydrotestosterone Induce Coronary Hyperreactivity in Intact Male Rhesus Monkeys

Coronary hyperreactivity (CH), characterized by persistent severe vasoconstrictions in response to vasoconstrictor challenge, is oppositely influenced by progesterone (P) and medroxyprogesterone acetate (MPA) treatment in surgically menopausal primates. In this study we tested whether multiweek MPA or dihydrotestosterone (DHT) exposure induced CH in intact male rhesus monkeys. Coronary angiographic experiments with intracoronary serotonin and the thromboxane A(2) analog U46619 stimulated brief vasoconstriction (for 1-3 min) in large epicardial coronaries in untreated male monkeys. In contrast, MPA- and DHT-treated monkeys displayed long-duration constrictions (>5 min), with significantly greater reductions in the minimal diameters of epicardial coronaries. Immunocytochemistry demonstrated androgen receptors (AR) and P receptors in aorta and coronary arteries, and immunocytochemistry and Western blotting showed AR and P receptors in rhesus coronary vascular muscle cells. In vivo, MPA or DHT increased thromboxane prostanoid (TP) receptor expression in the aorta. In vitro, MPA or DHT increased, whereas P did not change, TP receptor expression in primary coronary vascular muscle cell. This MPA- or DHT-mediated increase in TP receptor expression was attenuated by the AR antagonist flutamide. MPA or DHT induction of CH in intact adult male primates, hypothesized to occur via androgenic up-regulation of vascular muscle TP receptor expression, could predispose to CH-mediated myocardial ischemia.

Ginsenosides block HIV protease inhibitor ritonavir-induced vascular dysfunction of porcine coronary arteries

Human immunodeficiency virus (HIV) protease inhibitor ritonavir (RTV) may induce vascular dysfunction through oxidative stress. Ginsenosides have been shown to have potential benefits on the cardiovascular system through diverse mechanisms, including antioxidative property. The objective of this study was to determine whether ginsenosides could prevent coronary arteries from RTV-induced dysfunction. Porcine coronary artery rings were incubated with RTV and ginsenosides Rb1, Rc, and Re for 24 h. Vasomotor function was recorded by a myograph tension system. In response to the thromboxane A(2) analog U-46619, the contraction of the vessel rings was significantly reduced. When cocultured with Rb1, Rc, and Re, the contractility significantly increased. In response to bradykinin at 10(-5) M, the endothelium-dependent relaxation of vessel rings was significantly reduced by 59% for RTV compared with controls (P < 0.05). When cocultured with Rb1, Rc, and Re, the relaxation significantly increased 100%, 90%, and 134%, respectively, compared with the RTV-alone groups (P > 0.05). In response to sodium nitroprusside, RTV significantly reduced vasorelaxation. In addition, the endothelial nitric oxide synthase (eNOS) mRNA levels were significantly reduced by 78% for RTV group (P < 0.05) by real-time PCR analysis. The eNOS protein levels measured by Western blot analysis and nitrite concentrations measured by Griess assay were also decreased, whereas O(2)(-) production by lucigenin-enhanced chemiluminescence was significantly increased in the RTV-treated group. These effects of RTV were effectively blocked by ginsenosides. Thus HIV protease inhibitor RTV significantly impaired the vasomotor function of porcine coronary arteries. This effect may be mediated by the downregulation of eNOS and overproduction of O(2)(-). These results suggest that ginsenosides can effectively block RTV-induced vascular dysfunction.

Effects of progesterone and estrogen on endothelial dysfunction in porcine coronary arteries 1

The effects of hormone replacement therapy (HRT) on the vascular endothelium have been controversial. In this study, we determined the effects of HRT on endothelium-dependent relaxation in a porcine coronary artery model. Coronary artery rings harvested from female swine were incubated as controls or with estrogen (10(-9), 10(-8), 10(-7) g/L), progesterone (1 x 10(-6), 1 x 10(-5), 5 x 10(-5) g/L), or a combination of the two (10(-8)g/L estrogen, 1 x 10(-5)g/L progesterone). After 24 h in tissue culture, the rings were tested on a myograph system to measure contraction and endothelium-dependent relaxation. Myograph analysis was performed with the thromboxane A2 analogue U46619 for contraction and bradykinin or sodium nitroprusside for relaxation. Nitric oxide synthase (eNOS) levels were determined by immunohistochemistry. Levels of superoxide anion in the progesterone or estrogen treated tissues were assessed by lucigenin-enhanced chemiluminescence analysis. In response to 10(-7)M bradykinin, porcine coronary artery rings treated with 1 x 10(-6), 1 x 10(-5) and 5 x 10(-5) g/L of progesterone showed a significant reduction in endothelium-dependent vasorelaxation by 36%, 45%, and 68%, respectively, as compared to controls (P <0.05). However, rings treated with estrogen showed no significant difference as compared to controls. Furthermore, estrogen treatment with progesterone reversed the effect of progesterone, showing no difference in vessel relaxation as compared to controls. There were no differences in endothelium-independent vasorelaxation (sodium nitroprusside) or in smooth muscle contractility (U46619) between the control and the hormone-treated groups. The eNOS immunoreactivity was reduced in progesterone-treated coronary artery rings. Furthermore, coronary endothelium exposed to progesterone showed a 59% increase in superoxide anion production, while estrogen produced a 67% decrease when compared to controls (P <0.05 for both). This data suggests that the progesterone component of HRT has a detrimental influence on endothelium-dependent relaxation. This effect appears to be related to decreased eNOS levels, as well as increased consumption of NO by superoxide anion in the endothelium of tissues exposed to progesterone. Estrogen can block progesterone-induced endothelial dysfunction and superoxide anion production in the pig coronary artery model.

Curcumin prevents HIV protease inhibitor ritonavir-induced vascular injury and superoxide anion production in porcine coronary arteries

Introduction: HIV protease inhibitor ritonavir (RTV) may induce vascular dysfunction through oxidative stress. The objective of this study was to determine whether a dietary antioxidant curcumin could prevent coronary arteries from RTV-induced injury. Methods: Porcine coronary arteries were incubated with RTV and/or curcumin for 24 hours. Vasomotor function was studied with a myograph tension system in response to thromboxane A2 analogue U46619 (contraction), and bradykinin (endothelium-dependent relaxation). Superoxide anion levels were determined by lucigenin enhanced chemiluminescence. Results: In response to U46619, the control vessels showed a 71% reduction (p < 0.001, t-test). The combination of RTV and curcumin showed a vessel tension similar to controls. In response to bradykinin, the control vessel rings relaxed by 70.80%, while RTV treated vessels only relaxed by 29.25%, which showed a 59% reduction as compared to controls (p < 0.001, t-test). Curcumin plus RTV showed 54.20% of vasorelaxation, respectively. Superoxide anion level in the RTV-treated group was increased by 47%, as compared to controls (P < 0.05, t-test). Curcumin treatment significantly inhibited RTV induced superoxide anion production. Conclusions: HIV protease inhibitor RTV significantly decreases smooth muscle cell contractivity and endothelium-dependent vasorelaxation, but increases superoxide anion production in porcine coronary arteries. Curcumin can effectively inhibit RTV-induced vascular injury and oxidative stress.

Bupivacaine inhibits thromboxane A2-induced vasoconstriction in rat thoracic aorta.

Plasma levels of thromboxane A2 (TXA2), an inflammatory mediator inducing platelet aggregation, bronchoconstriction, and vasoconstriction, are increased in the perioperative period. A major role in the pathogenesis of perioperative thromboembolic and ischemic syndromes is attributed to this prostanoid. Local anesthetics (LA) inhibit signaling of TXA2 receptors expressed in cell models. Therefore, we hypothesized that LA may inhibit vasoconstriction induced by the TXA2 analog U46619 in rat thoracic aorta. Rings (3-mm length) of the rat thoracic aorta were mounted in organ baths and isometric contractile force was measured. Rings, with or without endothelium, were incubated for 60 min in bupivacaine (10(-6) or 10(-5) M) or Krebs-Henseleit solution (control group) and subsequently exposed to cumulative concentrations of U46619 (10(-10) to 10(-6) M). The reversibility of the TXA2-induced vasoconstriction by bupivacaine was also studied. Pretreatment of rings with bupivacaine concentration-dependently diminished TXA2-induced contraction in rat aortic rings. We found no significant differences in relaxing effect of bupivacaine between rings with and without endothelium. Contraction in rings established with U46619 could not be reversed by cumulative concentrations of bupivacaine. Bupivacaine inhibited carbachol-induced vascular relaxation. This study provides experimental evidence that bupivacaine is an endothelium-independent inhibitor of TXA2-induced vasoconstriction of rat thoracic aorta.

Cyclooxygenase involvement in thromboxane-dependent contraction in rat mesenteric resistance arteries.

The influence of cyclooxygenase pathway activation following thromboxane-endoperoxide (TP) receptor stimulation was studied in rat mesenteric resistance arteries (n=6 to 10 per group). We studied isolated, perfused, and pressurized mesenteric resistance arteries (mean internal diameter 214 microm) using an arteriograph, enabling us to study arteries in physiological conditions of flow and pressure. Changes in diameter were continuously recorded, and contractions measured as internal diameter reduction. Release of cyclooxygenase pathway metabolites was also assessed by enzyme immunoassay (EIA) analysis of mesenteric bed perfusions. The thromboxane A2 (TxA2) analog U-46619 (1 micromol/L) induced a significant contraction (108 microm maximal diameter reduction). Inhibition by 3 chemically different cyclooxygenase inhibitors (ie, flurbiprofen, indomethacin, and aspirin) potently reduced the contraction to 27%, 25%, and 6% of control, respectively. The selective cyclooxygenase-1 inhibitor SC-58560 inhibited U-46619 contraction, whereas selective cyclooxygenase-2 inhibition (SC-58236) had no effect. Thromboxane synthase inhibition (furegrelate) did not affect U-46619-induced contraction, but it was reduced by cytosolic phospholipase A2 inhibition. Measurement of cyclooxygenase derivatives produced by the isolated mesenteric bed showed that PGE2 was produced after TxA2-receptor stimulation with U-46619. Exogenous prostaglandin E2 (in the presence of the TxA2 receptor antagonist SQ 29 548) and U-46619 contracted mesenteric arteries with a similar potency (EC50: 0.30 and 0.48 micromol/L, respectively). This study provides the first evidence that TxA2-receptor-dependent contraction in a resistant artery involved cyclooxygenase stimulation and, at least in part, a PGE2 formation. This mechanism of TxA2-dependent contraction in resistant arteries might be of importance in the understanding of diseases affecting resistant arteries and involving TxA2, such as hypertension.

Estrogen blocks homocysteine-induced endothelial dysfunction in porcine coronary arteries 1,2

Background The objective of this study was to examine the effect of estrogen combined with homocysteine on vasomotor function and endothelial integrity in intact porcine coronary arteries. Materials and methods Pig coronary artery rings were incubated with estrogen, homocysteine, or estrogen and homocysteine for 24 h. Myographic analysis was performed with thromboxane A2 analog U46619 for contraction and bradykinin or sodium nitroprusside for relaxation. Endothelial nitric oxide synthase (eNOS) levels were determined by immunohistochemistry. Levels of superoxide anion were assessed by lucigenin-enhanced chemiluminescence analysis. Results Endothelium-dependent vasorelaxation (bradykinin) for the homocysteine alone group was 62% compared with control ( P < 0.05), and endothelium-dependent vasorelaxation for the estrogen alone group was 85% compared with control ( P > 0.05). Endothelium-dependent vasorelaxation for the estrogen-homocysteine combined group was 79% compared with 89% for control ( P > 0.05). There were no differences in endothelium-independent vasorelaxation (sodium nitroprusside) or in smooth muscle contractility (U46619) between all three groups and control. In addition, the eNOS immunoreactivity was declined in the homocysteine group and had no major change in the estrogen or estrogen plus homocysteine-treated group as compared with controls. The superoxide free radical measurement showed a marked increase in the homocysteine group, no major change from controls in the estrogen group, and a much-lessened effect in the combination of estrogen and homocysteine. Conclusions These data demonstrate that combining estrogen with homocysteine significantly blocks the effect of homocysteine on impairing endothelium-dependent vasorelaxation as well as on decreasing eNOS expression and increasing oxidative stress in porcine coronary arteries. This study suggests that estrogen may play a role in preventing homocysteine-mediated endothelial dysfunction and may be of benefit in the hyperhomocysteinemic patient.

Enhanced leukocyte–platelet cross‐talk in Type 1 diabetes mellitus: relationship to microangiopathy

Platelets and leukocytes may influence each others' function, i.e. platelet-leukocyte cross-talk. Diabetes mellitus (DM) is associated with platelet and leukocyte dysfunction. To evaluate platelet-leukocyte cross-talk, and if this might contribute to platelet and leukocyte dysfunction and microangiopathy in DM patients. We evaluated platelet and leukocyte function, and cross-talk between these cells in Type 1 DM patients without (n = 19) and with (n = 20) microangiopathy, and healthy subjects (n = 27), using whole blood flow cytometry. Platelet-leukocyte cross-talk was studied in hirudinized whole blood incubated at 37 degrees C with stirring. Basal single platelet P-selectin and leukocyte CD11b expression were similar in DM patients and healthy subjects, whilst circulating platelet-leukocyte aggregates and plasma elastase levels were elevated in DM patients. The thromboxane A2 analog U46619 (3 x 10(-7) m) induced more marked increases of platelet P-selectin expression and platelet-leukocyte aggregation in DM patients than in healthy subjects. The leukocyte-specific agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10(-7) m) induced more marked CD11b expression in DM patients with microangiopathy, compared with healthy subjects. Platelet-leukocyte cross-talk induced by U46619 (10(-6) m) showed no difference between DM patients and healthy subjects. fMLP (10(-6) m) evoked marked leukocyte activation, which subsequently caused mild platelet P-selectin expression. This leukocyte-platelet cross-talk was more pronounced in DM patients than in healthy subjects. Furthermore, enhanced leukocyte-platelet cross-talk was correlated to platelet hyperreactivity among DM patients with microangiopathy only. Type 1 DM is associated with platelet and leukocyte hyperactivity, and enhanced leukocyte-platelet cross-talk, which may contribute to platelet hyperactivity and the microvascular complications seen in Type 1 DM.

Estrogen prevents homocysteine-induced endothelial dysfunction in porcine arteries

Introduction: Hyperhomocysteinemia is an independent risk factor of atherosclerosis and thrombosis. Estrogen may have a cardioprotective effect by improvement of endothelial functions. Thus, we hypothesized that estrogen might protect endothelium from homocysteine damage. The objective of this study was to examine the effect of estrogen on homocysteine treated porcine coronary arteries. Methods: Pig coronary artery rings were incubated with homocysteine (50 μM), estrogen (10−5M), or homocysteine and estrogen (50 μM/10−5M) for 24 h. Myograph tension analysis was performed with thromboxane A2 analogue U46619 for contraction and bradykinin for endothelium dependent vasorelaxation or sodium nitroprusside for endothelium independent vasorelaxation. Results: There were no differences of maximal contraction in response to U46619 among all groups of porcine coronary artery rings. However, endothelium-dependent vasorelaxation was different. Homocysteine treated vessels showed 62% relaxation in response to 10−5M bradykinin, which was significantly reduced compared to control vessels (88% relaxation) (P<0.05). There was no significant difference of endothelium dependent vasorelaxation between estrogen treated and control groups (P>0.05). Furthermore, combining homocysteine and estrogen showed 75% improvement of endothelium dependent vasorelaxation as compared to homocysteine treatment alone. There was no difference in endothelium-independent vasorelaxation (sodium nitroprusside) between all groups. Conclusion: These data demonstrated that homocysteine significantly reduces endothelium dependent vasorelaxation, and estrogen significantly prevents homocysteine-mediated endothelial dysfunction in porcine coronary arteries. This study suggests that estrogen may be of benefit in the hyperhomocysteinemic patient.

Thromboxane A2-induced inhibition of voltage-gated K+ channels and pulmonary vasoconstriction: role of protein kinase Czeta.

Voltage-gated K+ channels (KV) and thromboxane A2 (TXA2) play critical roles in controlling pulmonary arterial tone under physiological and pathological conditions. We hypothesized that TXA2 might inhibit KV channels, thereby establishing a link between these two major pathogenic pathways in pulmonary hypertension. The TXA2 analogue U46619 inhibited IK(V) (Emax=56.1+/-3.9%, EC50=0.054+/-0.019 micromol/L) and depolarized pulmonary artery smooth muscle cells via activation of TP receptors. In isolated pulmonary arteries, U46619 simultaneously increased intracellular Ca2+ concentration and contractile force, and these effects were inhibited by nifedipine or KCl (60 mmol/L). U46619-induced contractions were not altered by the inhibitors of tyrosine kinase genistein or Rho kinase Y-27632 but were prevented by the nonselective protein kinase C (PKC) inhibitors staurosporine and calphostin C. Furthermore, these responses were sensitive to Gö-6983 but insensitive to bisindolylmaleimide I and Gö-6976. Based on the specificity of these drugs, we suggested a role for an atypical PKC in U46619-induced effects. Thus, treatment with a PKCzeta pseudosubstrate inhibitor markedly prevented the vasoconstriction, the inhibition of IK(V), and the depolarization induced by U46619. Western blots showed a transient translocation of PKCzeta from the cytosolic to the particulate fraction on stimulation with U46619. These results indicate that TXA2 inhibits IK(V), leading to depolarization, activation of L-type Ca2+ channels, and vasoconstriction of rat pulmonary arteries. We propose PKCzeta as a link between TP receptor activation and KV channel inhibition.

Sildenafil prevents change in RhoA expression induced by chronic hypoxia in rat pulmonary artery.

Exposure to chronic hypoxia (CH) induces a sustained pulmonary hypertension associated with structural and functional changes in the pulmonary arterial bed, including alterations of contractile properties. The small G-protein RhoA and its effector Rho kinase play a major role in the sustained rise in tension induced by vasoconstrictors. The aim of this study was to analyze the effect of CH on the RhoA/Rho kinase signaling pathway in the rat pulmonary artery. Maximal contraction of pulmonary artery rings to endothelin-1, noradrenaline, and the thromboxane A2 analog U46619 was markedly decreased in rats exposed to CH (10% O2, 2 weeks). This CH-induced decrease response to agonists was attributable to the abolition of RhoA-mediated Ca2+ sensitization of the contraction. Real-time reverse transcriptase-polymerase chain reaction and Western blot analysis revealed a decrease in RhoA mRNA (79.4+/-6.0%, n=4) and RhoA (81.1+/-8.0%, n=4) expression in the main pulmonary artery from CH rats, whereas RhoA expression was not modified in arterial smooth muscle cells and arteries exposed to hypoxia and high intraluminal pressure, respectively. Treatment of rats with sildenafil (25 mg/kg per day) throughout 2 weeks of exposure to CH prevented CH-induced downregulation of RhoA, reduction of contraction, and pulmonary artery remodeling. These findings indicate that CH-induced downregulation of RhoA expression, leading to the abolition of RhoA/Rho kinase-mediated Ca2+ sensitization of contraction, is responsible for the decreased responses to contracting agonists in the pulmonary artery of CH rats. These alterations are prevented by sildenafil, indicating a major role of the NO/cyclic GMP pathway in CH-induced altered RhoA signaling in the pulmonary artery.

Prostanoid-induced modulation of neuropeptide Y and noradrenaline release from the rat mesenteric bed.

1. A variety of prostanoids were examined for their ability to alter the periarterial nerve stimulation-induced release of noradrenaline (NA) and neuropeptide Y immunoreactive compounds (NPY-ir) from the perfused mesenteric arterial bed of the rat. 2. Periarterial nerve stimulation (16 Hz) increased the overflow of NA, NPY-ir and perfusion pressure. 3. The prostacyclin (PGI2) analogues, carbaPGI2 and cicaprost both produced a concentration-dependent attenuation of the nerve stimulation-induced increase in NA, NPY-ir overflow and perfusion pressure. 4. The prostaglandin (PG) analogue PGE2 attenuated the evoked increase in NPY-ir overflow as well as a modest decrease in NA. 5. PGE1, sulprostone and iloprost attenuated the nerve stimulation-induced increase in NA overflow but not NPY-ir. 6. Neither PGF2alpha nor the thromboxane A2 analogue U46619 altered the evoked increase in NA or NPY-ir overflow. 7. The results support the view that sympathetic co-transmitter release can be differentially modulated by paracrine/autocrine mediators at sympathetic neuroeffector junctions.

Pulmonary endothelial dysfunction after cardiopulmonary bypass in neonatal pigs.

In neonatal pigs cardiopulmonary bypass (CPB) is associated with endothelial dysfunction in isolated large pulmonary arteries. It is, however, of great importance if this functional change extends to the small pulmonary resistance arteries, which are the key regulators of pulmonary flow and pressure. The aim of this study was to assess changes in pulmonary microvascular function after CPB using a clinically relevant pediatric procedure. From three groups of neonatal pigs (CPB-, sham- and control group) pulmonary resistance arteries and systemic resistance arteries (from skeletal muscle) were isolated and mounted as ring preparations in wire myographs. Vessel diameters were less than 500 microm. Concentration-response curves were constructed for norepinephrine (NA), vasopressin (Vp), and the thromboxane A2-analog U46619, while the endothelium-dependent and -independent vasodilator functions were assessed as responses to acetylcholine and nitric oxide (NO). Maximum pulmonary vasodilator response to acetylcholine was attenuated after CPB compared with sham-operated and control animals (P=0.04). NO-induced relaxation, and contractile responses to NA, Vp, and U46619 were not influenced by CPB. In systemic arteries no changes in contractile or relaxant responses were seen after CPB. CPB seems to induce pulmonary endothelial dysfunction in pulmonary but not peripheral resistance arteries in neonatal piglets.

An Analysis of Responses to Levosimendan in the Pulmonary Vascular Bed of the Cat

Calcium-sensitizing drugs, such as levosimendan, are a novel class of drug therapy for heart failure. We investigated the hypothesis that levosimendan is a pulmonary vasodepressor mediated through inhibition of phosphodiesterase, adenosine triphosphate (ATP)-dependent potassium channels, or both. We investigated responses to the calcium sensitizer levosimendan in the pulmonary vascular bed of the cat under conditions of controlled pulmonary blood flow and constant left atrial pressure when lobar arterial pressure was increased to a high steady level with the thromboxane A2 analog U-46619. Under increased-tone conditions, levosimendan caused dose-related decreases in lobar arterial pressure without altering systemic arterial and left atrial pressure. Responses to levosimendan were significantly attenuated, although not completely, after the administration of U-37883A, a vascular selective nonsulfonylurea ATP-sensitive K+-channel-blocking drug. Responses to levosimendan were not significantly different after the administration of the nitric oxide synthase inhibitor l-N5-(1-iminoethyl)-ornithine or the cyclooxygenase inhibitor sodium meclofenamate or when lung ventilation was interrupted. These data show that levosimendan has significant vasodilator activity in the pulmonary vascular bed of the cat. They also suggest that pulmonary vasodilator responses to levosimendan are partially dependent on activation of ATP-sensitive K+ channels and independent of the synthesis of nitric oxide, activation of cyclooxygenase enzyme, or changes in bronchomotor tone in the pulmonary vascular bed of the cat.

The role of inducible nitric oxide synthase in lipopolysaccharide-mediated hyporeactivity to vasoconstrictors differs among isolated rat arteries

We investigated whether organ-specific differences exist in the role of inducible nitric oxide synthase (iNOS) in hyporeactivity to vasoconstrictors following 20 h in vitro exposure of isolated superior mesenteric, renal, hepatic and coronary arteries from the rat to bacterial lipopolysaccharide (LPS). LPS attenuated contraction in response to depolarizing KCl in all arteries. Maximum contractile responses to noradrenaline were attenuated in superior mesenteric and hepatic arteries, and those to the thromboxane A(2) analogue U46619 were attenuated in coronary arteries. LPS shifted the concentration-response curve to noradrenaline in renal arteries to the right. Removal of extracellular L-arginine improved the response to noradrenaline in superior mesenteric and renal arteries only. Addition of the iNOS inhibitor aminoguanidine resulted in full recovery of the responses to noradrenaline in superior mesenteric, renal and hepatic arteries. Contractile responses in coronary arteries did not improve after inhibition of iNOS activity. Therefore the pattern of the LPS-induced changes in vascular reactivity, as well as the contribution of iNOS to impaired vascular constriction, differed among vascular beds. These differences are likely to represent a contributory factor in the sepsis-associated redistribution of cardiac output.

Potentiation of human lung fibroblast chemotaxis by the thromboxane A2 analog U-46619

Fibroblast production of extracellular matrix is crucial not only for normal tissue development and maintenance of tissue structure but also for the repair and remodeling processes after injury. Thromboxane A2 (TXA2) is a potent mediator in inflammatory processes. The aim of this study was to investigate the effect of TXA2 on chemotaxis of human fetal lung (HFL-1) fibroblasts induced by human plasma fibronectin or platelet-derived growth factor BB (PDGF-BB). By using the blindwell chamber technique, the TXA2 agonist U-46619 alone had no chemotactic activity. However, U-46619 (200 nmol/L) stimulated HFL-1 fibroblast chemotaxis to human plasma fibronectin (20 μg/mL; 161.8% ± 13.4%; P <.005) and to PDGF-BB (10 ng/mL; 188.5% ± 7.0%; P <.005). Checkerboard analysis of human plasma fibronectin-directed migration confirmed that the TXA2 agonist increased both chemotaxis and chemokinesis. The stimulatory effect of the TXA2 agonist was concentration dependent and increased with time. Another agent known for stimulating the protein kinase C pathway, phorbol 12-myristate 13-acetate (10−8 mol/L), had a similar effect, stimulating chemotaxis to fibronectin (146.2% ± 8.6%). The stimulatory effect of the TXA2 agonist on HFL-1 fibroblast chemotaxis was inhibited by the synthetic thromboxane receptor antagonist SQ29,548 (10−5 mol/L) and the protein kinase C inhibitor calphostin (10−7 mol/L). In summary, TXA2 appears to stimulate fibroblast chemotaxis to fibronectin and PDGF, perhaps by modulating the rate of fibroblast migration. Such an effect may contribute to regulation of wound healing and the development of fibrotic disorders. (J Lab Clin Med 2002;139:43-9)

Homocysteine Decreases Endothelium-Dependent Vasorelaxation in Porcine Arteries

Background. Although hyperhomocysteinemia has long been recognized as an independent risk factor for vascular disease, the mechanisms of the pathogenesis of homocysteine are largely unknown. The objective of this study was to examine the effect of homocysteine on vasomotor function and endothelial integrity in intact porcine arteries.Methods. Pig coronary artery rings were incubated with homocysteine (10, 50, or 100 μM) for 24 h. Myograph analysis was performed with thromboxane A2 analogue U46619 for contraction and bradykinin or sodium nitroprusside for relaxation. Pig carotid arteries were perfusion-cultured in control and 50 and 100 μM homocysteine treatment groups. The diameter change was analyzed in response to norepinephrine and acetylcholine, respectively. Endothelial morphology and nitric oxide systhase (eNOS) levels were determined by histology analysis.Results. Endothelial-dependent vasorelaxation (bradykinin) was significantly reduced by 52, 87, and 97% in the pig coronary artery rings treated with 10, 50, and 100 μM homocysteine, respectively, compared to controls (P < 0.05). There were no differences in endothelium-independent vasorelaxation (sodium nitroprusside) or in smooth muscle contractility (U46619) between control and homocysteine-treated groups (P > 0.05). Acetylcholine-induced vasorelaxation was also significantly reduced by 44 and 98% in the pig carotid arteries treated with 50 and 100 μM homocysteine, respectively, compared to controls (P < 0.05). Variable degrees of endothelial cell injury, such as morphology change and detachment, were observed, and eNOS immunoreactivity was markedly reduced in both pig coronary and carotid arteries that were treated with high doses of homocysteine.Conclusion. These data demonstrated that homocysteine significantly decreased endothelium-dependent vasorelaxation and eNOS immunoreactivity as well as induced marked endothelial injury in both porcine coronary and carotid arteries. This study suggests that homocysteine-mediated endothelial dysfunction and injury may play important roles in vascular lesion formation in the hyperhomocysteinemic patient.

Contractile hyporesponsiveness of hepatic arteries in humans with cirrhosis: Evidence for a receptor-specific mechanism

Splanchnic vasodilatation and vascular hyporesponsiveness to vasopressors are characteristic features of patients with cirrhosis. Although the vascular response to different vasopressors has been shown to be attenuated in cirrhosis, alterations on the receptor level are discussed controversially. Thus, impaired postreceptor signaling has been postulated. However, so far this has not been studied in human splanchnic vessels. Therefore, we assessed the vascular response of human hepatic arteries after activating the G-protein–dependent signal transduction pathway by stimulation with angiotensin II, the thromboxane A2 analog U46619, or by G-protein activation with NaF/AlCl3. After endothelium denudation, cumulative isometric concentration contraction curves were obtained for hepatic arteries from 32 cirrhotic patients undergoing liver transplantation and from 40 organ donors after stimulation with either angiotensin II (10−11-10−5 mol/L), U46619 (10−10-10−6 mol/L) or AlCl3 (30 μmol/L)/NaF (10−4-3 × 10−2 mol/L). Hepatic arteries from cirrhotic patients were markedly less responsive to angiotensin II (P < .0001) than those from organ donors. Both stimulation of the G-protein phospholipase C pathway via the thromboxane A2 receptor and receptor-independent G-protein stimulation with AlCl3/NaF, induced an intact contractile response. In conclusion, the G-protein–dependent signal transduction system itself is unaltered in cirrhosis. Hence, the cause of the hyporesponsiveness to some vasoconstrictors in cirrhosis appears to be a receptor-specific phenomenon localized upstream from the G-protein level. (HEPATOLOGY 2001;34:884-888.)

Inhibition of cyclooxygenase but not nitric oxide synthase influences effects on the human omental artery of the thromboxane A2 mimetic U46619 and 17β-estradiol

Objective: These experiments were performed to study the influence of endothelial factors on the contractile effect of the thromboxane A2 analog U46619 and on the relaxant action of 17β-estradiol on isolated human omental arteries from nonpregnant women, women with normal pregnancies, and women with preeclampsia. Study Design: Arterial rings (3 mm) with or without endothelium were suspended in organ chambers filled with Krebs buffer, 37°C, aerated with 5% carbon dioxide in air, pH approximately 7.4, for isometric tension recording. Rings were incubated with indomethacin, Nω-nitro-L -arginine, or 17β-estradiol, alone or in combination. The concentration that produced 50% of maximal effect, the area under the curve, and the maximal effect of U46619, normalized with respect to a reference contraction in response to potassium chloride, were compared. Results: Neither indomethacin nor Nω-nitro-L -arginine changed the basal tone of omental artery rings. Neither Nω-nitro-L -arginine nor removal of the endothelium affected either the contractile action of U46619 or the relaxant action of 17β-estradiol. In contrast, indomethacin potentiated the contractile effect of U46619 and abolished the inhibitory effect of 17β-estradiol in rings from all three groups. The effects of U46619 and 17β-estradiol were significantly less in rings from women with normal pregnancy than in those from women with preeclampsia. Tissues from women with preeclampsia demonstrated the highest contractile response to U46619. Conclusion: The inhibitory effect of 17β-estradiol is not due to increased production of endothelial nitric oxide but rather involves inhibitory products of the cyclooxygenase pathway. The effect of indomethacin may result from inhibited production or release of eicosanoids or from sensitization of thromboxane A2 receptors. (Am J Obstet Gynecol 2001;185:182-9.)

Homocysteine reduces smooth muscle [Ca2+]i and constrictor responses of isolated arterioles.

Chronic elevation of plasma homocysteine concentration has been shown to be associated with impaired vascular function. The acute direct effect of homocysteine on the tone and vasoactive responses of arterioles and the possible underlying mechanisms, however, have not yet been elucidated. Thus arterioles were isolated from gracilis muscle of rats (d: approximately 130 microm) and their diameter was measured by videomicroscopy. Homocysteine (10(-6)-10(-4) M) elicited dose-dependent dilation of arterioles (maximum: 44+/-6% at 10(-4) M). The dilation was not affected by the presence of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester or by removal of the endothelium, or the free radical scavenger catalase and superoxide dismutase, or the K+ channel inhibitors glibenclamide, 4-aminopyridine, or tetraethyl ammonium. Incubation of vessels with homocysteine (10(-4) M, 20 min) did not affect dilations to acetylcholine or sodium nitroprusside, whereas it significantly decreased constrictions to norepinephrine (at 10(-6) M; control: 57+/-7%, homocysteine: 21+/-5%) and to the thromboxane A2 analogue U46619 (at 10(-8) M: control: 44+/-3%, homocysteine: 20+/-4%). Homocysteine (10(-4) M), similar to the voltage-operated Ca2+ channel inhibitor nitrendipine (10(-8) M), significantly decreased the arteriolar smooth muscle [Ca2+]i as assessed by changes in the fura-2 ratiometric signal (R(Ca), -6+/-1% and -24+/-3%, respectively). These data suggest that in isolated arterioles homocysteine decreases pressure-induced tone and responses to vasoconstrictor agents, likely by altering Ca2+ signaling of arteriolar smooth muscle.