Thromboxane A2 Analogue U46619 Research Articles

Genetic Evidence for a Predominant Role of PI3Kβ In ITAM— and Integrin-Mediated Signaling in Platelets

Phosphoinositide 3-kinases (PI3K) have been implicated in platelet activation downstream of G protein-coupled receptors (GPCRs), ITAM-bearing receptors, as well as integrins. Among the multiple PI3K isoforms expressed in platelets, PI3Kγ and PI3Kβ are considered to be involved in GPCRs-mediated signaling leading to Rap1b activation and cell aggregation. However, a definitive genetic analysis of the relative contribution of PI3Kβ versus PI3Kγ in the different contexts of platelet activation has not been provided yet. PI3Kγ knockout mice have been widely investigated, but PI3Kγ has been recognized to mediate also kinase-independent effects. Moreover, deletion of p110β is embryonic lethal, and thus PI3Kβ knockout platelets have not been previously available for analysis. Knockin mice carrying a kinase dead form of either PI3Kβ or PI3Kγ have been recently generated by introducing K833R mutation in p110γ, or K805R mutation in p110β, respectively (Patrucco et al, Cell 2004: 118,375; Ciraolo et al, 2008, in press). In this study, we have compared platelet activation in mice homozygous for the kinase dead forms of PI3Kβ (PI3KβKD) or PI3Kγ (PI3KγKD). Platelet count and tail bleeding time were not altered in either PI3KγKD or PI3KβKD mice. Platelet aggregation induced by the thromboxane A2 analogue U46619, which stimulates GPCRs, was only slightly reduced in PI3KγKD platelets, and was not further inhibited by treatment with the selective PI3Kβ inhibitor TGX221. Accordingly, catalytic inactivation of PI3Kβ caused only a modest reduction of platelet aggregation induced by U46619. Similar results were also obtained upon stimulation of GPCRs for ADP or thrombin. ADP- and U46619-induced activation of the small GTPase Rap1b, a key regulator of platelet aggregation, was only reduced in PI3KγKD, but was almost completely suppressed in PI3KβKD, pointing to a major role for this isoform in the GPCRs-mediated regulation of the GTPase. Rap1b was normally activated upon stimulation of PI3KγKD platelets with the GPVI ligand convulxin (CVX), both in the absence and presence of the ADP scavenger apyrase. Moreover, CVX-induced aggregation of PI3KγKD platelets was only slightly reduced. By contrast, PI3KβKD platelets completely failed to aggregate in response to a wide range of concentrations of CVX, and accumulation of active GTP-bound Rap1b was almost undetectable. Accordingly, the PI3Kβ inhibitor TGX221 completely suppressed both Rap1b activation and aggregation induced by GPVI stimulation in either wild type or PI3KγKD platelets, both in the presence and absence of apyrase. PI3KγKD platelets adhered normally to immobilize type I collagen or fibrinogen, and displayed only a small defect of spreading on fibrinogen. Similarly, adhesion to collagen was comparable in PI3KβKD and wild type platelets. However, PI3KβKD revealed a severely impaired adhesion to fibrinogen. In addition, adherent cells completely failed to spread, and virtually no lamellipodia-forming platelets were observed. Altogether these results indicate both PI3Kγ and PI3Kβ are both involved in Rap1b activation and platelet aggregation upon stimulation of GPCRs, but provide the first genetic evidence that PI3Kβ, rather than PI3Kγ, plays an essential role in platelet activation downstream of the ITAM-bearing receptor GPVI, as well as in integrin αIIbβ3 outside-in signaling.

Role of nitric oxide and prostanoids in the adrenergic contraction of goat cerebral arteries after ischemia‐reperfusion

To analyze the role of nitric oxide and prostanoids in the adrenergic cerebrovascular response after ischemia‐reperfusion (I‐R), left middle cerebral artery (MCA) was occluded for 120 min followed by 60 min of reperfusion in anesthetized goats. After this, the animals were sacrificed, the brain was removed and 3 mm‐long arterial rings were taken from branches of the left (I‐R) and right (control) MCA, and prepared for isometric tension recording in organ baths. Noradrenaline (NA) (10−8 – 10−4 M, in the presence of beta‐adrenoceptor blockade) produced concentration‐dependent contractions, and the maximal contraction but not the sensitivity was higher in I‐R than in control arteries. The inhibitor of nitric oxide synthesis L‐NAME (10−4 M) increased the contraction to NA similarly in control and I‐R arteries, while the cyclooxygenase inhibitor meclofenamate (10−5 M) augmented the maximal response to NA in control but not in I‐R arteries. The relaxation induced by ADP (10−8–10−5 M) in the arteries precontracted with the thromboxane A2 analogue U46619 was similar in control and I‐R arteries. Therefore, ischemia‐reperfusion may increase the adrenergic cerebrovascular reactivity due, at least in part, to reduced formation of prostacyclin; the modulatory role of NO in this reactivity may be preserved after I‐R. (Supported, in part, by FMMA and MEyC)

Growth-related oncogene-α induces endothelial dysfunction through oxidative stress and downregulation of eNOS in porcine coronary arteries

Growth-related oncogene-alpha (GRO-alpha) is a member of the CXC chemokine family, which is involved in the inflammatory process including atherosclerosis. We hypothesized that GRO-alpha may affect endothelial functions in both porcine coronary arteries and human coronary artery endothelial cells (HCAECs). Vasomotor function was analyzed in response to thromboxane A2 analog U-46619 for contraction, bradykinin for endothelium-dependent vasorelaxation, and sodium nitroprusside (SNP) for endothelium-independent vasorelaxation. In response to 10(-6) M bradykinin, GRO-alpha (50 and 100 ng/ml) significantly reduced endothelium-dependent vasorelaxation by 34.73 and 48.8%, respectively, compared with controls (P < 0.05). There were no changes in response to U-46619 or SNP between treated and control groups. With the lucigenin-enhanced chemiluminescence assay, superoxide anion production in GRO-alpha-treated vessels (50 and 100 ng/ml) was significantly increased by 50 and 86%, respectively, compared with controls (P < 0.05). With real-time PCR analysis, endothelial nitric oxide synthase (eNOS) mRNA levels in porcine coronary arteries and HCAECs after GRO-alpha treatment were significantly decreased compared with controls (P < 0.05). The eNOS protein levels by both immunohistochemistry and Western blot analyses were also decreased in GRO-alpha-treated vessels. Antioxidant seleno-l-methionine and anti-GRO-alpha antibody effectively blocked these effects of GRO-alpha on both porcine coronary arteries and HCAECs. In addition, GRO-alpha immunoreactivity was substantially increased in the atherosclerotic regions compared with nonatherosclerotic regions in human coronary arteries. Thus GRO-alpha impairs endothelium-dependent vasorelaxation in porcine coronary arteries through a mechanism of overproduction of superoxide anion and downregulation of eNOS. GRO-alpha may contribute to human coronary artery disease.

Transglutaminase-dependent RhoA Activation and Depletion by Serotonin in Vascular Smooth Muscle Cells

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension.

Role of murine integrin α2β1 in thrombus stabilization and embolization: Contribution of thromboxane A2

Platelets stably interact with collagen via glycoprotein (GP)VI and alpha2beta1integrin. With alpha2-null mice, we investigated the role of alpha2beta1 in thrombus formation and stability in vivo and in vitro. Using a FeCl(3)-induced thrombosis model, in arteries from alpha2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects, while the thromboxane A(2) analogue U46619 was borderline effective in suppressing the embolisation in alpha2-null mice. In vitro, perfusion of alpha2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from alpha2-null mice. We conclude that integrin alpha2beta1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A(2) release. The increased embolization in alpha2-null mice may argue against the use of alpha2beta1 integrin inhibitors for antithrombotic therapy.

Food Intake Enhances Thromboxane Receptor–Mediated Platelet Activation in Type 2 Diabetic Patients but Not in Healthy Subjects

Diabetes is associated with platelet hyperactivity (1,2), increases in circulating activated platelets and platelet-leukocyte aggregates (PLAs), and platelet hyperreactivity to in vitro stimulation (2,3). Thromboxane-mediated platelet activation is of special interest since diabetes is associated with increased platelet-dependent thromboxane A2 (TxA2) production (2), and diabetic patients appear to benefit less from prevention with aspirin than nondiabetic patients (4,5). Insufficient insulin production and/or insulin resistance in diabetes leads to hyperglycemia, especially after food intake. Accumulating evidence indicates that postprandial hyperglycemia contributes to diabetes cardiovascular complications and may even predict complications more closely than fasting glucose levels (6). We recently reported (7) the effects of food intake and oral antidiabetes treatment on platelet function in type 2 diabetic patients. We found that food intake augmented platelet activation stimulated by ADP but not by thrombin (7). Responses to the TxA2 analog U46619 were also studied but have not been reported. Healthy control subjects were only studied in the fasting state in the original study design (7). We therefore recalled them to test if the meal causes similar enhancement of platelet activation in subjects with normal blood glucose regulation, and we currently focus on the responsiveness to U46619. Fifteen patients with fairly well-controlled type 2 diabetes (mean ± SE age 53 ± 6 years, 11 male and 4 female, BMI 28.1 ± 3.8 kg/m2, A1C 6.8 ± 1.7% [reference <5.2%]). The study originally recruited 15 matched healthy control subjects, but only 10 (age 51 …

Chronic estrogen treatment reduces vaso-constrictor responses in insulin resistant rats

Previous experiments have shown that chronic estrogen treatment via subcutaneous implants prevented insulin-induced blood pressure elevation and increased insulin sensitivity in ovariectomized female rats. In vitro vascular studies were performed using isolated mesenteric arteries to determine the effect of chronic estrogen and insulin treatments on vascular responses to vasoconstrictor agents. Female Wistar rats were assigned to the following groups: sham-operated, sham-operated plus insulin, sham-operated plus insulin plus estrogen, ovariectomized, ovariectomized plus insulin, and ovariectomized plus insulin plus estrogen. Chronic insulin and estrogen treatments were initiated with subcutaneous placement of insulin implants (2 U/d) and 17beta-estradiol implants (0.5 mg/pellet, 60 day release) at the back of the neck. After 8 weeks of treatment, mesenteric arteries were isolated for assessment of constrictor responses to norepinephrine and the thromboxane A2 analogue U46619 in the presence or absence of the endothelium. The results show that chronic estrogen treatment attenuated the vascular constrictor responses to norepinephrine and U46619 only in endothelium intact vessels. Incubation with insulin did not significantly affect norepinephrine-induced vascular smooth muscle contraction. The study provides evidence that the mechanism by which estrogen prevents insulin-induced blood pressure elevation in insulin-treated ovariectomized rats is by influencing endothelium-derived vasoactive factors such as thromboxane A2.

Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets

Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.

Effects of endothelin receptor blockade on hypervasoreactivity in streptozotocin-diabetic rats: vessel-specific involvement of thromboxane A2

Increased vasoconstrictor response to norepinephrine (NE) and endothelin (ET)-1 in arteries from diabetic animals is ameliorated by chronic endothelin receptor blockade with bosentan and was absent in endothelium-denuded arteries, suggesting the involvement of ET-1 and an endothelium-derived contracting factor such as thromboxane A2 (TxA2). To examine this possibility, we determined the effects of acute blockade of ET receptors or inhibition of TxA2 synthesis on the vascular function of superior mesenteric arteries (SMA) and renal arteries (RA) isolated from nondiabetic and 11-week streptozotocin (STZ) diabetic rats chronically treated with either bosentan or vehicle. Both in vitro incubation with bosentan and a selective ETA receptor blocker, BQ123, eradicated the increase in NE contractile responses in diabetic SMA. Additionally, in vitro incubation with the thromboxane synthase inhibitor, dazmegrel, abrogated the exaggerated NE and ET-1 contractile responses in diabetic SMA. Conversely, in RA, no significant acute effect of bosentan, BQ123, nor dazmegrel on vascular responses to NE was observed. Dazmegrel incubation attenuated the maximum contractile responses to ET-1 in diabetic RA; however, these responses in diabetic RA remained significantly greater than those of other groups. Diabetic RA but not SMA exhibited an enhanced contractile response to the TxA2 analogue U46619, which was corrected by chronic bosentan treatment. Immunohistochemical analyses in diabetic SMA revealed an increase in ETA receptor level that was normalized by chronic bosentan treatment. These data indicate that an interaction between ET-1 and TxA2 may be involved in mediating the exaggerated vasoconstrictor responses in diabetic arteries. Furthermore, the underlying mechanisms appear to be vessel specific.

COX-2, Another Important Player in the Nitric Oxide–Endothelin Cross-Talk

See related article, pages 1439–1445 Traditionally, the role of the endothelium was thought to be primarily that of a selective barrier to the diffusion of macromolecules from the vessel lumen to the interstitial space. During the past 20 years, numerous additional roles for the endothelium have been defined such as regulation of vascular tone, modulation of inflammation, promotion as well as inhibition of vascular growth, and modulation of platelet aggregation and coagulation. Endothelial dysfunction is a characteristic feature of patients with cardiovascular risk factors such as hypercholesterolemia, hypertension, diabetes mellitus, and chronic smoking. More recent studies indicate that it may predict long-term atherosclerotic disease progression as well as cardiovascular event rate.1 There is a growing body of evidence that decreased endothelial bioavailability of nitric oxide (NO·) in particular attributable to increased production of reactive oxygen species, such as superoxide (O2·−), leads to an activation of the renin–angiotensin system,2,3 increased formation of cyclooxygenase (COX)-dependent vasoconstrictors,4 but also to increased expression of the most potent endogenous vasoconstrictor endothelin-1 (ET-1).5–9 Of the 4 active endothelins (ET-1 to ET-4) ET-1 is the predominant isoform in the cardiovascular system. ET-1 exerts its major cardiovascular effects through activation of 2 distinct G protein–coupled receptors, the ETA and ETB receptors. ETA receptors are found exclusively in smooth muscle cells. Endothelin-1 promotes vasoconstriction, mitogenesis, and thrombosis predominantly via binding to the ETA receptor. ETB receptors are localized to some extent in smooth muscle cells, but also in endothelial cells. Activation of ETB receptors has been demonstrated to cause the release of NO· and prostacyclin (PGI2).10,11 In normal states with p reserved vascular (endothelial) NO …

Exaggerated vasomotor response to ANG II in rats with fetal programming of hypertension associated with exposure to a low-protein diet during gestation

The renin-angiotensin system plays a key role in the initiation and maintenance of elevated blood pressure associated with altered intrauterine milieu. The current studies were undertaken to verify whether vascular response to ANG II is increased in adult offspring of low-protein fed dams (LP) compared with control (CTRL) and if so, to examine underlying mechanism(s). ANG II-induced contraction of carotid rings was increased in LP (E(max), the maximum asymptote of the curve, relative to maximal response to KCl 80 mM: 230 +/- 3% LP vs. 201 +/- 2% CTRL, P < 0.05). In both groups, contraction to ANG II was mediated solely by AT1R. Responses to thromboxane A2 analog U-46619 and to KCl 80 mM under step increases in tension were similar between groups. Endothelium depletion enhanced contraction to ANG II in both groups, more so in LP. Blockade of endothelin formation had no effect on response to ANG II, and ANG-(1-7) did not elicit vasomotor response in either group. Superoxide dismutase (SOD) analog Tempol normalized LP without modifying CTRL response to ANG II. Basal levels of superoxide (aortic segments, lucigenin-enhanced chemiluminescence and fluorescent dye hydroethidine) were higher in LP. ANG II further increased superoxide production in LP only, and this was inhibited by coincubation with diphenylene iodonium or apocynin (inhibitor of NADPH oxidase complex). AT1R expression in carotid arteries was increased in LP, whereas SOD expression was unchanged. In conclusion, vasoconstriction to ANG II is exaggerated in this model of developmental programming of hypertension, secondary to enhanced vascular production of superoxide anion by NADPH oxidase with concomitant increase of AT1R expression.

Increased Expression of Cyclooxygenase-2 Mediates Enhanced Contraction to Endothelin ET A Receptor Stimulation in Endothelial Nitric Oxide Synthase Knockout Mice

The aim of this study was to determine whether prolonged loss of NO activity, in endothelial NO synthase knockout (eNOS(-/-)) mice, influences endothelin (ET) ETA receptor-mediated smooth muscle contraction and, if so, to define the underlying mechanism(s). In isolated endothelium-denuded abdominal aortas, contractions to the selective ETA receptor agonist ET-1(1-31) were significantly increased in aortas from eNOS(-/-) compared with wild-type (WT) mice. In contrast, contractions to the alpha1-adrenergic agonist phenylephrine or the thromboxane (TX) A2 analog U-46619 were similar between eNOS(-/-) and WT mice. Immunofluorescent and Western blot analysis demonstrated that the aortic expression of ETA receptors was decreased in eNOS(-/-) compared with WT mice. Contractions evoked by ET-1(1-31), but not phenylephrine, were reduced by inhibition of cyclooxygenase-2 (COX-2) (indomethacin or celecoxib) or of TXA2/prostaglandin H2 receptors (SQ-29548). After COX inhibition, contractions to ET-1(1-31) were no longer increased and were actually decreased in eNOS(-/-) compared with WT aortas. Western blot analysis revealed that endothelium-denuded abdominal aortas express COX-2, but not COX-1, and that expression of COX-2 was significantly increased in eNOS(-/-) compared with WT mice. Contractions to the COX substrate arachidonic acid were also increased in eNOS(-/-) aortas. Furthermore, ET-1(1-31) but not phenylephrine stimulated production of the TXA2 metabolite TXB2, which was increased in eNOS(-/-) compared with WT aortas. Therefore, COX-2 plays a crucial and selective role in ETA-mediated smooth muscle contraction. Furthermore, COX-2 expression is increased in eNOS(-/-) mice, which overcomes a reduced expression of ETA receptors and enables a selective increase in contraction to ETA receptor stimulation.

Bronchial Muscle Peristaltic Activity in the Fetal Rat

Aside for the potential for tonic contraction, the airway smooth muscle exhibits intermittent phasic rhythmic activity that may contribute to lung growth during fetal life. Therefore, we examined 4th generation rat 18-22 d gestation fetal, 4-6 d of age newborn and adult bronchial ring from Sprague Dawley rats to compare differences in smooth muscle function. We hypothesized that phasic contractions were greatest before birth. Bronchial muscle spontaneous rhythmic contractions were greatest in the fetus and absent in the adult. In response to KCl stimulation, the fetal bronchial smooth muscle only developed tonic force that was 3.5 +/- 0.6 and lower than measured in the newborn 9.0 +/- 0.3 and adult 13.7 +/- 1.4 mN/mm2. The thromboxane A2 analogue U46619 induced tonic and phasic muscle contractions and the amplitude and frequency of the phasic contractions were greater in the fetus as compared with the adult and increased with gestational age. The U46619-induced rhythmic contractions were abrogated by ryanodine, thapsigargin and reduction of extracellular Na+, suggesting intracellular Ca2+ dependence and involvement of the Na+/Ca2+ exchanger. The inward rectifier K+ blocker BaCl2 induced phasic contractions in unstimulated fetal, but not adult bronchial muscle of the same amplitude and frequency as for the spontaneous and U46619-induced ones. We conclude that the airway smooth muscle phasic activity is greatest in the fetus and tends to disappear post-natally with age suggesting an in utero role during lung development.

Neutrophil antimicrobial peptide α-defensin causes endothelial dysfunction in porcine coronary arteries

Defensins are cysteine-rich cationic polypeptides released from neutrophils that exhibit powerful antimicrobial activities. Because inflammation, including neutrophil infiltration and release of defensins, may play an important role in atherosclerosis and other vascular diseases, we determined whether alpha-defensin could cause endothelial dysfunction, a major initial event of atherosclerosis, in porcine coronary arteries. Porcine coronary arteries were sliced into 5-mm rings and treated with different concentrations of human recombinant alpha-defensin for 24 hours. Vasomotor reactivity was studied by using a myograph system. Levels of superoxide anion were detected by the lucigenin-enhanced chemiluminescence method. Endothelial nitric oxide synthase (eNOS) messenger RNA (mRNA) and protein levels were determined by real-time polymerase chain reaction and immunohistochemistry analysis, respectively. Endothelium-dependent relaxation in response to bradykinin was significantly reduced by 40% for the rings treated with 1500 nM of alpha-defensin compared with controls (P< .05). Vessel contractility in response to the thromboxane A2 analogue U46619 and endothelium-independent relaxation in response to sodium nitroprusside were not affected with defensin treatment. In addition, the superoxide anion level at the endothelial layer of porcine coronary artery rings was significantly increased by 80% in the defensin-treated (1500 nM) vessels compared with controls (P< .05). Furthermore, the eNOS mRNA levels in endothelial cells isolated from the cultured rings treated with defensin (1500 nM) were significantly decreased by 27% compared with controls (P< .05). Immunoreactivity of eNOS in the defensin-treated vessel rings was also substantially reduced. Defensin reduces the endothelium-dependent vasorelaxation. This effect is associated with increased superoxide radical production and decreased eNOS expression in porcine coronary arteries. Inflammation is an important mechanism of atherosclerosis and other vascular diseases. The roles and interactions of biomediators released from inflammatory cells are not fully understood, however. This study provides new information about effects and potential molecular mechanisms of a major neutrophil releasing factor, alpha-defensin, on endothelial dysfunction of porcine coronary arteries. Thus, targeting alpha-defensin and its associated molecular mechanisms may become a new strategy to prevent vascular diseases.

Regulation of Protein Kinase C by the Platelet P2Y12 ADP Receptor.

The Gi-coupled P2Y12 receptor plays a crucial role in platelet activation induced by ADP, and is also required for the action of a number of agonists whose receptors are couple to Gq, such as thromboxane A2. Stimulation of P2Y12 leads to the inhibition of adenylyl cyclase through the Gai subunit, and to the activation of phosphatidylinositol 3-kinase (PI-3K) and Rap1b through the bg subunit. Pharmacological inhibition of P2Y12 receptor results in the prevention of platelet aggregation induced by a number of agonists, including ADP and the the thromboxane A2 analogue U46619. In this study, we report that P2Y12 receptor is absolutely necessary for the stimulation of protein kinase C (PKC) and for PKC-directed protein phosphorylation. By using 32P-labeled human platelets, as well as by immunoblotting with anti-phospho(Ser) PKC substrates antibody, we analyzed the phosphorylation of pleckstrin, the main platelet substrate for PKC. As expected, we found that ADP-induced pleckstrin phosphorylation was totally suppressed by antagonists of the Gq-coupled P2Y1 receptor, but, surprisingly, we found that it was also completely prevented upon blockade of the P2Y12 receptor by AR-C69931MX or by 2-MeSAMP. Although we failed to directly detect any production of IP3 in ADP-stimulated platelets, we observed that ADP-induced Ca2+ release from intracellular stores was not affected by P2Y12 receptor antagonists. Moreover, AR-C69931MX or 2-MeSAMP strongly impaired pleckstrin phosphorylation induced by U46619, without affecting intracellular Ca2+ mobilization. These results indicated that blockade of the P2Y12 receptor did not interfere with phospholipase C stimulation, and that the reduced activation of PKC is probably not the consequence of a reduced production of diacylglycerol. Experiments performed with specific cell permeable inhibitors demonstrated that, in ADP-treated platelets, neither inhibition of adenylyl cyclase nor activation of PI-3K downstream P2Y12 receptor stimulation was responsible for PKC regulation. Subthreshold concentrations of PMA were able to restore ADP- or U46619-induced pleckstrin phosphorylation in the presence of P2Y12 antagonists, but were unable to restore platelet aggregation. Moreover, direct inhibition of PKC by Ro31-8220 prevented ADP-induced pleckstrin phosphorylation, but aggregation occurred normally. These results indicate that although PKC-directed protein phosphorylation is targeted by the P2Y12 receptor, it is not responsible for the essential contribution of this receptor to platelet aggregation. By contrast, we found that inhibition of platelet diacylglycerol kinase by R59949 completely reverted the inhibitory effect of AR-C69931MX or 2-MeSAMP on ADP- and U46619-induced activation of PKC, and completely restored platelet aggregation. These results suggest that diacylglycerol kinase may be regulated downstream the Gi-coupled P2Y12 receptor, and may be an important new regulator of platelet aggregation.

A New Role for FcγRIIA in the Potentiation of Human Platelet Activation Induced by Weak Stimulation.

The low affinity receptor for immunoglobulin G, FcγRIIA, is expressed in human platelets, mediates heparin-associated thrombocytopenia, and participates in platelet activation induced by von Willebrand factor. Activation of FcγRIIA occurs upon clustering of the receptor induced by immunocomplexes, and consists in the phosphorylation of two tyrosine residues within the ITAM, typically promoted by an associated Src kinase. The phosphorylated receptor acts as a docking site for SH2 domain-containing signaling proteins, including the tyrosine kinase Syk. This event initiates an intracellular tyrosine kinase-based signaling cascade that eventually leads to phosphorylation and activation of phospholipase C (PLC) γ2, and elicits cellular responses. To date, very little is known on the possible involvement of FcγRIIA in platelet activation induced by soluble agonists. We have found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in Src-kinase-mediated tyrosine phosphorylation of FcγRIIA. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcγRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane A2 analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcγRIIA. We also found that the anti-FcγRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. Thrombin- and U46619-induced tyrosine phosphorylation of Syk and PLCγ2, which represent substrates typically involved in FcγRIIA-mediated signaling, was clearly reduced by incubation with anti-FcγRIIA antibody IV.3. Morever, we were able to demonstrated that platelet stimulation by thrombin induced the association of FcγRIIA with Syk. Signaling through immunoreceptor typically takes places in characteristic membrane microdomains called lipid rafts. Upon stimulation with thrombin, FcγRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcγRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcγRIIA, and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcγRIIA can be activated in human platelets downstream G-protein-coupled receptors, and initiates a tyrosine kinase-based signaling pathway that significantly contributes to platelet activation and aggregation in response to weak stimulation.

Inhibition of phosphodiesterase 1 augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs with acute pulmonary hypertension

Phosphodiesterase 1 (PDE1) modulates vascular tone and the development of tolerance to nitric oxide (NO)-releasing drugs in the systemic circulation. Any role of PDE1 in the pulmonary circulation remains largely uncertain. We measured the expression of genes encoding PDE1 isozymes in the pulmonary vasculature and examined whether or not selective inhibition of PDE1 by vinpocetine attenuates pulmonary hypertension and augments the pulmonary vasodilator response to inhaled NO in lambs. Using RT-PCR, we detected PDE1A, PDE1B, and PDE1C mRNAs in pulmonary arteries and veins isolated from healthy lambs. In 13 lambs, the thromboxane A(2) analog U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mmHg. Four animals received an intravenous infusion of vinpocetine at incremental doses of 0.3, 1, and 3 mg.kg(-1).h(-1). In nine lambs, inhaled NO was administered in a random order at 2, 5, 10, and 20 ppm before and after an intravenous infusion of 1 mg.kg(-1).h(-1) vinpocetine. Administration of vinpocetine did not alter pulmonary and systemic hemodynamics or transpulmonary cGMP or cAMP release. Inhaled NO selectively reduced mean pulmonary arterial pressure, pulmonary capillary pressure, and pulmonary vascular resistance index, while increasing transpulmonary cGMP release. The addition of vinpocetine enhanced pulmonary vasodilation and transpulmonary cGMP release induced by NO breathing without causing systemic vasodilation but did not prolong the duration of pulmonary vasodilation after NO inhalation was discontinued. Our findings demonstrate that selective inhibition of PDE1 augments the therapeutic efficacy of inhaled NO in an ovine model of acute chemically induced pulmonary hypertension.

Stimulatory Roles of Nitric-oxide Synthase 3 and Guanylyl Cyclase in Platelet Activation

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.

Estrogen potentiates vasopressin-induced contraction of female rat aorta by enhancing cyclooxygenase-2 and thromboxane function

To determine the roles of estrogen and constrictor prostanoids in vasopressin (VP)-induced contraction of female rat aorta, vascular reactivity to VP was determined in thoracic aortas of intact, ovariectomized, and ovariectomized + estrogen-replaced female rats in the presence of indomethacin (Indo), NS-398, SQ-29,548, or vehicle control. The effects of estrogen on vascular reactivity to the thromboxane A(2) analog U-46619 were also examined. Maximal contractile response to VP in intact female rats (5,567 +/- 276 mg/mg of aortic ring wt) was markedly attenuated by ovariectomy (2,485 +/- 394 mg; P < 0.001) and restored by estrogen replacement with 17beta-estradiol (5,059 +/- 194 mg; P > 0.1). Indo and NS-398 significantly attenuated maximal responses to VP in intact female rats to a similar extent [3,176 +/- 179 (P < 0.0001) and 3,258 +/- 152 mg (P < 0.0001), respectively]. Ovariectomy abolished and estrogen replacement restored the inhibitory effects of Indo, NS-398, and SQ-29,548. Contractile responses of rat aorta to U-46619 were significantly greater (P < 0.0001) in females (5,040 +/- 238 mg) than in males (3,679 +/- 96 mg). Ovariectomy markedly attenuated (3,923 +/- 84 mg; P < 0.01) and estrogen replacement restored (5,024 +/- 155 mg; P > 0.1) responses to U-46619 in female aortas. These data reveal that estrogen is an important regulator of the contractile responses of female rat aorta to VP, which appears to potentiate both cyclooxygenase-2 and constrictor prostanoid function in the vascular wall.

Ginsenoside Rb1 blocks homocysteine-induced endothelial dysfunction in porcine coronary arteries

Homocysteine (Hcy) is an independent risk factor for atherosclerosis. This study investigates the effects of ginsenoside Rb1, a major constituent of ginseng, on Hcy-induced endothelial dysfunction and molecular changes in porcine coronary arteries. The coronary arteries were harvested from pig hearts and cut into 5-mm ring segments, which were then divided into six groups, including control, Hcy alone (50 microM), low-dose (1 microM) or high-dose (10 microM) Rb1 alone, and Hcy plus low-dose or high-dose Rb1. After 24-hour incubation, the rings were analyzed for vasomotor function in response to thromboxane A2 analog U46619, bradykinin, and sodium nitroprusside (SNP), respectively. In addition, superoxide anion was assessed by lucigenin-enhanced chemiluminescence analysis. Endothelial nitric oxide synthase (eNOS) was studied using real-time polymerase chain reaction and Western blot. Endothelium-dependent relaxation (bradykinin) was significantly reduced in rings treated with Hcy alone as compared with the control (49.80% vs 71.77%, n = 8, P < .05), whereas neither high-dose nor low-dose Rb1 alone affected the endothelium-dependent relaxation. The low-dose Rb1-Hcy combined group had a partially improved endothelium-dependent relaxation (54.44%), whereas the high-dose Rb1-Hcy combined group showed a complete recovery of endothelium-dependent relaxation (72.89%). There was no substantial difference in maximal contraction induced by U46619 or endothelium-independent relaxation by SNP among all groups (P > .05). Furthermore, superoxide anion was markedly increased by 137% in the Hcy-treated group as compared with the control, but there were no statistically significant changes from the control in all other groups (P > .05). Lastly, eNOS mRNA and protein levels were substantially reduced in the Hcy-treated group, but not in the Rb1-Hcy combined groups. This is the first study to show that ginsenoside Rb1 can effectively block Hcy-induced endothelial dysfunction and superoxide anion production as well as eNOS downregulation in porcine coronary arteries. This study suggests that ginseng and its active constituents may have potential clinical applications in controlling Hcy-associated vascular injuries. Homocysteine (Hcy) is an independent risk factor for atherosclerosis and other vascular lesions. It causes endothelial dysfunction and oxidative stress. Ginseng compounds have effects of vasorelaxation and antioxidation. The purpose of this study was to determine the effect of ginsenoside Rb1, a major constituent of ginseng, on Hcy-induced endothelial dysfunction and molecular changes in porcine coronary arteries. Our results showed that ginsenoside Rb1 can effectively block Hcy-induced dysfunction of endothelium-dependent vasorelaxation as well as superoxide anion production and eNOS downregulation. This study suggests that ginseng compounds may have potential clinical applications in controlling Hcy-associated vascular diseases and other vascular lesions.