Thromboxane Analogue U46619 Research Articles

P63RhoGEF Couples G αq/11 -Mediated Signaling to Ca 2+ Sensitization of Vascular Smooth Muscle Contractility

In normal and diseased vascular smooth muscle (SM), the RhoA pathway, which is activated by multiple agonists through G protein-coupled receptors (GPCRs), plays a central role in regulating basal tone and peripheral resistance. This occurs through inhibition of myosin light chain phosphatase, leading to increased phosphorylation of the myosin regulatory light chain. Although it is thought that specific agonists and GPCRs may couple to distinct RhoA guanine nucleotide exchange factors (GEFs), thus raising the possibility of selective targeting of specific GEFs for therapeutic use, this notion is largely unexplored for SM contraction. We examine whether p63RhoGEF, known to couple specifically to Gα(q/11) in vitro, is functional in blood vessels as a mediator of RhoA activation and if it is selectively activated by Gα(q/11) coupled agonists. We find that p63RhoGEF is present across SM tissues and demonstrate that silencing of the endogenous p63RhoGEF in mouse portal vein inhibits contractile force induced by endothelin-1 to a greater extent than the predominantly Gα(12/13)-mediated thromboxane analog U46619. This is because endothelin-1 acts on Gα(q/11) as well as Gα(12/13). Introduction of the exogenous isolated pleckstrin-homology (PH) domain of p63RhoGEF (residues 331-580) into permeabilized rabbit portal vein inhibited Ca2+ sensitized force and activation of RhoA, when phenylephrine was used as an agonist. This reinforces the results based on endothelin-1, because phenylephrine is thought to act exclusively through Gα(q/11). We demonstrate that p63RhoGEF selectively couples Gα(q/11) but not Gα(12/13), to RhoA activation in blood vessels and cultured cells and thus mediates the physiologically important Ca2+ sensitization of force induced with Gα(q/11)-coupled agonists. Our results suggest that signaling through p63RhoGEF provides a novel mechanism for selective regulation of blood pressure.

Prostaglandin D2 induces contractions through activation of TP receptors in peripheral lung tissue from the guinea pig

Prostaglandin D(2) (PGD(2)), released through mast cell activation, is used as a non-invasive biomarker in patients with asthma. Since PGD(2) can elicit opposing effects on airway tone via activation of the PGD(2) receptors DP(1) and DP(2) as well as the thromboxane receptor TP, the aim of this study was to characterize the receptors that are activated by PGD(2) in the guinea pig lung parenchyma. PGD(2) and the thromboxane analog U46619 induced concentration-dependent contractions. U46619 was more potent and caused stronger effect than PGD(2). The specific TP receptor antagonist SQ-29548 and the combined TP and DP(2) receptor antagonist BAYu3405 concentration-dependently shifted the curves for both agonists to the right. The DP(1) receptor agonist BW245 induced a weak relaxation at high concentrations, whereas the DP(1) receptor antagonist BWA868C did not affect the PGD(2) induced contractions. The specific DP(2) receptor agonist 13,14-dihydro-15-keto-PGD(2) showed neither contractile nor relaxant effect in the parenchyma. Furthermore, studies in precision-cut lung slices specified that airways as well as pulmonary arteries and veins contracted to both PGD(2) and U46619. When the lung parenchyma from ovalbumin sensitized guinea pigs were exposed to ovalbumin, both thromboxane B(2) and PGD(2) were released. Ovalbumin also induced maximal contractions at similar level as PGD(2) in the parenchyma, which was partly reduced by SQ-29548. These data show that PGD(2) should be recognized as a TP receptor agonist in the peripheral lung inducing contraction on airways, arteries and veins. Therefore, a TP receptor antagonist can be useful in combination treatment of allergic responses in asthma.

T-type voltage-gated calcium channels regulate the tone of mouse efferent arterioles

Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation-contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium-induced constriction. Further, constriction by the thromboxane analogue U46619 was significantly inhibited by mibefradil at a concentration specific for T-type channels. Using PCR, we found that two channel subtypes, Ca(v)3.1 and Ca(v)3.2, were expressed in microdissected efferent arterioles. Ca(v)3.1 was found by immunocytochemistry to be located in mouse efferent arterioles, human pre- and postglomerular vasculature, and Ca(v)3.2 in rat glomerular arterioles. Inhibition of endothelial nitric oxide synthase by L-NAME or its deletion by gene knockout changed the potassium-elicited transient constriction to a sustained response. Low concentrations of nickel, an agent that blocks Ca(v)3.2, had a similar effect. Thus, T-type voltage-gated calcium channels are functionally important for depolarization-induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles.

Vasoconstrictor Potential of Coronary Aspirate From Patients Undergoing Stenting of Saphenous Vein Aortocoronary Bypass Grafts and Its Pharmacological Attenuation

Stent implantation into atherosclerotic plaques releases, apart from particulate debris, soluble substances that contribute to impaired microvascular perfusion. To quantify the release of vasoconstrictors and to determine the efficacy of coronary dilators to attenuate their action. Using a distal protection/aspiration device, coronary arterial blood was retrieved before and during stenting in 22 patients with severe saphenous vein aorto-coronary bypass stenoses. The release of catecholamines, endothelin, serotonin, thromboxane B(2), and tumor necrosis factor (TNF)α was measured. The response of rat mesenteric arteries with intact (+E) and denuded (-E) endothelium to aspirate plasma was normalized to that by KCl. Responses to selective receptor blockade, adenosine, nitroprusside, and verapamil against the aspirate-induced constriction were determined. The coronary arterial plasma withdrawn before stenting induced 21±5% and the aspirate plasma after stenting induced 95±8% of maximum KCl-induced vasoconstriction. Serotonin, thromboxane B(2), and TNFα release into aspirate plasma increased by 1.9±0.2 μmol/L, 25.6±3.1 pg/mL, and 19.7±6.1 pg/mL, respectively, during stenting. The aspirate-induced vasoconstriction was largely antagonized by selective serotonin receptor blockade, with little further antagonism by additional thromboxane receptor blockade. TNFα did not induce constriction per se but potentiated the constriction with serotonin and the thromboxane-analog U-46619 in arteries +E. The concentrations to induce half-maximal vasodilation were comparable for nitroprusside (+E, 3.3×10(-8); -E, 1.9×10(-8) mol/L) and verapamil (+E, 8.3×10(-8); -E, 7.8×10(-8) mol/L), and the vasoconstriction was eventually eliminated. The vasodilator response to adenosine was dependent on functional endothelium and weaker. Serotonin is the main coronary vasoconstrictor after stenting, and thromboxane and TNFα somewhat potentiate the serotonin response. Nitroprusside and verapamil are more potent than adenosine to attenuate the aspirate plasma-induced vasoconstriction, and they are not dependent on functional endothelium.

A Chemical Genetic Analysis of Platelet Activation Identifies An Antithrombotic Allosteric Modulator That Acts through Helix 8 of Par1

AbstractAbstract 483The essential challenge in pharmacologic control of platelet activation during thrombus formation is to achieve inhibition of platelet mediated thrombosis without impairing hemostasis. Inhibition of platelet GPCRs has focused primarily on inhibition of orthosteric binding sites, selecting for compounds that compete with endogenous ligands. However, small molecules that act at non-orthosteric sites can stabilize alternative GPCR conformations and modify GPCR function. Such allosteric modulators demonstrate saturable binding, selective effects on affinity and efficacy, and could provide improved physiologic control of platelet function. However, cell-based screens are required for the identification of such modulators. We screened a 16,000 compound library for inhibitors of platelet activation and identified a family of alkylated quinolines that modified Par1-mediated granule secretion. Activity of these compounds was determined by the length of their alkyl tails such that a two carbon addition to the tail converted a compound that inhibited Par1 activating peptide (SFLLRN)-induced P-selectin expression into a compound that augmented Par1-mediated activation. The most potent inhibitory compound, JF5 (IC50 = 4 μM), blocked Par1 activating peptide-induced GTPase activity and GTP[γ-35S] binding but not GTPγS-induced platelet activation in permeabilized platelets, thus indicating activity at Par1. The compound failed to inhibit platelet activation induced by a Par4 activating peptide, the thromboxane analog U46619, arginine vasopressin, PMA, or Ca2+ ionophore. Studies using cultured cells overexpressing specific GPCRs demonstrated that JF5 inhibited Par1-mediated activation, but failed to inhibit activation through TP, P2Y1 or EP3 receptors. However, JF5 did demonstrate inhibition of the α2A-adrenergic receptor. Radioligand binding studies confirmed non-competitive inhibition of the α2A-adrenergic receptor by JF5. Comparative phenotyping using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to select GPCRs. Evaluation of the amino acid sequence of these receptors demonstrated that sensitive receptors possessed a constrained 8th helix, as defined by a C-terminal palmitoylation site and interactions of the 8th helix with TM7 and the i1 loop. The 8th helix of GPCRs is oriented parallel to the intracellular face of the plasma membrane and is thought to transduce signals to cognate Gα subunits. To assess the hypothesis that the 8th helix contributes to the inhibitory activity of JF5, the compound was evaluated for its ability to inhibit IP3 generation in COS7 cells overexpressing wild-type Par1 or cells expressing a Par1 mutant in which residues 376–386 had been replaced with 3 alanine residues (ΔH8 Par1). JF5 completely inhibited SFLLRN-induced IP3 generation in cells expressing wild-type Par1. Inhibition was reversed in the ΔH8 Par1 mutant, confirming the involvement of the 8th helix in JF5 inhibitory activity. Despite its potent inhibition of Par1-mediated aggregation, secretion, and IP3 generation, JF5 did not inhibit SFLLRN-induced shape change. To evaluate the premise that JF5 inhibited coupling to Gαq but not Gα12/13, we determined the effect of JF5 on SFLLRN-induced Gα12-dependent decreased barrier function by measuring changes in transepithelial resistance (TER) in MDCK cells overexpressing Gα12. JF5 demonstrated no inhibition of SFLLRN-mediated decrease in TER at concentrations up to 200 μM, whereas the orthosteric Par1 inhibitor, Sch79797, completely blocked SFLLRN-induced TER at 1 μM. These results confirmed that JF5 inhibits Par1 coupling to Gαq, but not Gα12. JF was also evaluated in a mouse model of thrombus formation following laser-induced injury of cremaster arterioles. JF5 inhibited thrombus formation with an IC50 of 1 mg/kg and delayed thrombus formation by 2.3-fold. Evaluation of the 8th helix of mouse Par4 demonstrated that, unlike the 8th helix of human Par4, it contained a palmitoylation site and is predicted to have a constrained conformation based on interactions with TM7. These results indicate a role for the 8th helix in conferring sensitivity to small molecules and show that this sensitivity can be exploited to control platelet activation during thrombus formation. JF5 will serve as a useful probe to evaluate the role of the 8th helix in coupling of GPCRs to cognate Gα subunits. Disclosures:No relevant conflicts of interest to declare.

Systems Biology to Predict Platelet Function

AbstractAbstract SCI-38Systems Biology seeks to provide patient-specific prediction of dynamic cellular response to multiple stimuli, critical information toward predicting risk, disease progression, or response to therapy. We deployed two distinct approaches, bottom-up and top-down analyses, to gain insight into platelet signaling.The bottom-up approach required a definition of reaction network and kinetic equations (topology), kinetic parameters, and initial concentrations in order to simulate platelet signaling. We developed a computational platelet model – assembled from 24 peer-reviewed platelet studies to yield 132 measured kinetic rate constants – that accurately predicts resting levels of cytosolic calcium, IP3, diacylglycerol, phosphatidic acid, phosphoinositol, PIP, and PIP2. The model accurately predicts the full transient calcium dynamics in response to increasing levels of ADP. In the first full stochastic simulation of single platelet response to ADP, the model provides an extremely accurate prediction of the statistics of the asynchronous [Ca]i spikes observed in single platelets. Specifically, this is the first work to provide a quantitative molecular explanation of the asynchronous calcium spiking observed in ADP-activated human platelets. We show the asynchronous spiking is a result of the fundamentally stochastic nature of signal transduction in cells as small as human platelets. Specific testable predictions have emerged about the requirement of high SERCA/IP3R ratios in functional platelets, limits on the concentration of calcium in the DTS, and relative potencies of PAR peptides and ADP.For functional phenotyping platelets, a top-down approach linking multiple inputs to functional outputs was used to understand how human platelets integrate diverse signals encountered during thrombosis. We developed a high-throughput platform that measures the human platelet calcium mobilization in response to all pairwise combinations of six major agonists. Agonists tested in this study were: convulxin (CVX; GPVI activator), ADP, the thromboxane analog U46619, PAR1 agonist peptide (SFLLRN), PAR4 agonist peptide (AYPGKF), and PGE2 (activator of IP and EP receptor). The calcium responses to single agonists at 0.1, 1, 10′ EC50 and 135 pairwise combinations trained a neural network (NN) model to predict the entire 6-dimensional platelet response space. The NN model successfully predicted responses to sequential additions and 27 ternary combinations of [ADP], [convulxin], and [SFLLRN] (R=0.881). With 4077 NN simulations spanning the 6-dimensional agonist space, 45 combinations of 4–6 agonists (ranging from synergism to antagonism) were selected and confirmed experimentally (R=0.883), revealing a highly synergistic condition of high U46619/PGE2 ratio, consistent with the risk of COX-2 therapy. Furthermore, pairwise agonist scanning (PAS) provided a direct measurement of 135 synergy values, thus allowing a unique phenotypic scoring of 10 human donors.Patient-specific training of NNs represent a compact and robust approach for prediction of cellular integration of multiple signals in a complex disease milieu. Either bottom-up models or top-down NN models are ideal for incorporation into systems biology simulations of thrombotic pathways under flow conditions. Disclosures:No relevant conflicts of interest to declare.

Thromboxane Antagonism with terutroban in Peripheral Arterial Disease: the TAIPAD study.

Terutroban is a selective prostaglandin endoperoxide (TP) receptor antagonist with antithrombotic, antivasoconstrictive and antiatherosclerotic properties and is currently in development for long-term cardiovascular secondary prevention. TAIPAD is an international, double-blind, randomized controlled study comparing the effects of five dosages of oral terutroban vs. aspirin and placebo on platelet aggregation in peripheral arterial disease (PAD) patients. After 10 day's placebo run-in, included patients (n = 435; ankle-brachial pressure index, 0.7 ± 0.1) were randomly allocated to aspirin 75 mg day(-1), terutroban 1, 2.5, 5, 10 or 30 mg day(-1) or placebo. On day 5, the placebo group was reallocated to one of the terutroban groups for the rest of the study (day 83). Ex vivo platelet aggregation induced by the thromboxane analog U46619 (7 μm) was measured 24 h after dosing, as well as platelet aggregation induced by arachidonic acid (AA), collagen and ADP. Terutroban dose-dependently inhibited U46619-induced platelet aggregation at days 5 and 83. At day 5, the inhibition was significant vs. placebo for all terutroban dosages (P < 0.001). Terutroban (5, 10 and 30 mg day(-1)) was at least as effective as aspirin in inhibiting platelet aggregation induced by arachidonic acid (AA), collagen and adenosine diphosphate (ADP). Terutroban was well tolerated, with a safety profile similar to aspirin. In PAD patients, terutroban dose-dependently inhibited platelet aggregation 24 h after dosing, and was at least as effective as aspirin at 5, 10 and 30 mg day(-1). Terutroban was well tolerated.

Voltage-gated calcium channels are involved in the regulation of calcium oscillations in vascular smooth muscle cells from isolated porcine retinal arterioles

Disturbances in the regulation of retinal arteriolar tone are involved in the pathophysiology of major vision threatening diseases. Therefore, improving the treatment of these diseases requires knowledge about how the contraction of retinal arterioles is controlled. The present study hypothesized that in addition to calcium release from sarcoplasmic reticulum, calcium entry from the extracellular space is also involved in the generation of calcium oscillations as a basis for initiation of contraction in retinal arterioles. Isolated porcine retinal arterioles were mounted in a confocal microvascular myograph and loaded with the calcium sensitive fluorophore Oregon Green allowing simultaneous measurements of oscillations in the intracellular calcium concentration in individual smooth muscle cells and changes in the vascular tone. Recordings were made after pre-contraction with the thromboxane analogue U46619 in a low and a high ambient calcium concentration, and after subsequent blocking of voltage-gated calcium channels using nifedipine or the sarcoplasmic reticulum dependent calcium recruitment using ryanodine, caffeine, thapsigargin and cyclopiazonic acid. U46619 increased the number of oscillations in a low but not in physiological extracellular calcium concentration. In contrast to mesenteric arterioles, blocking of sarcoplasmic reticulum Ca 2+ channels with ryanodine was without effect on both retinal arteriolar tone and calcium oscillations. Conversely, blocking of the sarcoplasmic reticulum Ca 2+ pump with cyclopiazonic acid or thapsigargin significantly reduced calcium oscillations, but not arteriolar tone. At a physiological extracellular calcium concentration, addition of nifedipine, an L-type calcium channel blocker, reduced vascular tone but increased the number of cells displaying calcium oscillations. Fast oscillations in the intracellular calcium concentration in retinal smooth muscle cells depend on calcium transport by both the SR and the sarcolemma. However, both sites of calcium recruitment seem to have unique properties in porcine retinal arterioles as compared to other organs.

Expression and function of phosphodiesterases in nitrofen‐induced congenital diaphragmatic hernia in rats

Congenital diaphragmatic hernia (CDH) is an anomaly associated with pulmonary hypoplasia and pulmonary hypertension (PH). The limited efficacy of current approaches to treat PH in CDH, including inhaled nitric oxide (NO), drives the search for other therapies. Phosphodiesterases (PDEs) degrade cyclic nucleotide second messenger cAMP and cGMP downstream of NO thereby limiting the vasodilatory response to NO. To identify therapeutic targets by cataloguing the expression and function of PDE isoforms in the pulmonary vasculature in nitrofen-induced CDH in fetal rats. Quantitative RT-PCR revealed PDE1-5 and PDE9 mRNA expression in pulmonary arteries (PAs) of control and nitrofen-induced CDH term fetal rats. In this order of potency, the PDE inhibitors Sildenafil (PDE5) > EHNA (PDE2) > Rolipram (PDE4) > Cilostamide (PDE3) all dilated isolated third generation PA after pre-constriction with the thromboxane analog U46619. Hyperoxic pre-incubation of PAs significantly attenuated vasodilatation induced by the PDE5 inhibitor Sildenafil (65% vs. 33%, P < 0.004). CDH PAs dilated significantly less to PDE2 inhibitor EHNA compared to control (51% vs. 72%, P < 0.05). Subsequently PDE2 protein expression was higher in PAs of CDH animals. Most PDE isoforms exist in the PAs of fetal rats and their inhibition causes pulmonary vasodilatation. PDE5 inhibition was the most potent vasodilator, however, there were no differences between groups. PDE5-induced vasodilatation was attenuated by hyperoxic pre-incubation. PDE inhibitors might be considered therapeutic targets in combination with iNO in neonates with CDH.

Treatment With the Leukotriene Inhibitor Montelukast for 10 Days Attenuates Portal Hypertension in Rat Liver Cirrhosis

The mechanisms underlying intrahepatic vasoconstriction are not fully elucidated. Here we investigated the Kupffer cell (KC)-dependent increase in portal pressure by way of actions of vasoconstrictive cysteinyl leukotrienes (Cys-LTs). Liver cirrhosis was induced in rats by bile duct ligation (BDL for 4 weeks; controls: sham-operation) and thioacetamide application (18 weeks). Infusion of leukotriene (LT) C(4) or LTD(4) in isolated perfused livers (20 nM, BDL and sham) demonstrated that LTC(4) is a more relevant vasoconstrictor. In BDL animals the Cys-LT(1) receptor inhibitor montelukast (1 microM) reduced the maximal portal perfusion pressure following LTC(4) or LTD(4) infusion. The infusion of LTC(4) or D(4) in vivo (15 microg/kg b.w.) confirmed LTC(4) as the more relevant vasoconstrictor. Activation of KCs with zymosan (150 microg/mL) in isolated perfused BDL livers increased the portal perfusion pressure markedly, which was attenuated by LT receptor blockade (Ly171883, 20 microM). Cys-LTs in the effluent perfusate increased with KC activation but less with additional blockade of KCs with gadolinium chloride (10 mg/kg body weight, 48 and 24 hours pretreatment). KCs were isolated from normal rat livers and activated with zymosan or lipopolysaccharide at different timepoints. This resulted in an increase in Cys-LT production that was not influenced by preincubation with montelukast (1 microM). Infusion of LTC(4) (20 nM) and the thromboxane analog U46619 (0.1 microM) further enhanced portal pressure, indicating additive effects. Treatment with montelukast for 10 days resulted in an impressive reduction in the basal portal pressure and an attenuation of the KC-dependent increase in portal pressure. Activation of isolated KCs produced Cys-LTs. Infusion of Cys-LTs increased portal pressure and, vice versa, treatment with montelukast reduced portal pressure in rat liver cirrhosis. Therefore, montelukast may be of therapeutic benefit for patients with portal hypertension.

Diagnostic Utility of Platelet Secretion Testing by ATP Release Assays: Results From a Prospective Study of Individuals Referred for Bleeding Disorder Assessments.

Abstract 1314Poster Board I-338 BackgroundPlatelet secretion defects are a common type of inherited platelet disorder. Experts agree that ATP release assays are often helpful to assess for platelet secretion disorders among individuals with suspected bleeding problems. However, the diagnostic utility of ATP release assays has not been established, and it is unclear if the findings correlate with aggregation results. Our goal was to evaluate the incidence and spectrum of platelet release abnormalities in a prospective cohort of individuals referred for testing as part of a bleeding disorder assessment, and describe the diagnostic utility of platelet ATP release and its relationship to aggregation findings. MethodsData were analyzed for 40 healthy controls and 76 patients from a prospective cohort of individuals referred for bleeding disorder assessments. ATP release and light transmission aggregometry (LTA) were assessed by standardized methods, with validated reference intervals (derived by non-parametric analysis), using platelet-rich plasma adjusted to 250 × 109 platelets L−1 with autologous platelet poor plasma. ATP release was assessed using the Chronolog lumiaggregometer, as recommended, and a modified agonist panel (1.6 mM arachidonic acid, 1 uM thromboxane analogue U46619, 100 uM epinephrine, 6 uM epinephrine, 5 ug·mL−1 collagen, 1.25·g mL−1 collagen, 1 U·mL−1 thrombin, and 5 uM ADP). The test sensitivity and specificity for inherited platelet disorders were estimated and the likelihoods of detecting an inherited platelet disorder by ATP release (using different agonists or the agonist panel) were expressed as odds ratios (OR) and 95% confidence intervals (95% CI). Correlations between ATP release triggered by different agonists, and between ATP release and maximal aggregation with the same agonist, were expressed as Pearson correlation coefficients (ρ). ResultsReduced ATP release with one or more agonists was more common among the referred individuals that a hematologist categorized as having a bleeding problem (n = 66; 45% abnormal) than among healthy controls (5% false positives, p < 0.0001). The likelihood of an inherited platelet disorder was high (OR 83, 95% CI 16 - 445) when ATP release was abnormal with one or more agonists, even when LTA results were normal (OR 56, 95% CI 10 - 318). Impaired ATP release with some agonists (e.g. 5 uM ADP and 100 uM epinephrine) could not be detected as the reference intervals did not have a measurable lower limit. Among other agonists, 1 U·mL−1 thrombin had the highest likelihood of detecting impaired ATP release due to an inherited platelet disorder (OR 32 compared to 17- 25 for other agonists).Receiver operator curve analyses indicated that an assessment of ATP release with the full panel, or a more limited panel of 4 agonists, had high specificity and moderate sensitivity for detecting inherited defects in platelet function. The predictive power was improved by considering results as continuous rather than categorical (normal/abnormal) variables, however, this required complex mathematical equations. Most abnormalities in ATP release due to common inherited platelet disorders were detected by a limited number of agonists in the panel. The optimal panel, with minimized false positive and false negative rates (5% and 18% respectively), included: 1 uM thromboxane analogue U46619, 6 uM epinephrine, 5.0 ug·mL−1 collagen, and 1 U·mL−1 thrombin.Among patient subjects and healthy controls, there was a significant correlation (ρ = 0.25 – 0.92; p values < 0.02) between the amount of ATP release induced by most agonists. Maximal aggregation and ATP release responses for each agonist did not correlate significantly among healthy controls, and showed only a weak correlation among patient subjects. ConclusionsLike light transmission aggregometry, ATP release assays have important diagnostic utility for detecting impaired platelet function due to common inherited bleeding disorders. The poor correlation between ATP release and maximal platelet aggregation suggests that these tests are predominantly influenced by different parameters of platelet function. Our data on useful agonists for detecting impaired ATP release may prove helpful to laboratories that wish to optimize testing for common platelet disorders. The knowledge that platelet secretion assays are very helpful for bleeding disorder assessments should be translated into clinical practice. DisclosuresNo relevant conflicts of interest to declare.

Hemodynamic Alterations in the Coronary Circulation of Cardiomyopathic Hamsters: Age and Ang II–dependent Mechanisms

Background Coronary vasospasms have been reported in the early stages of cardiomyopathy in the Syrian cardiomyopathic hamster (CM; BIO-TO2 strain). It has been proposed these alterations could lead to ischemic heart disease and heart failure. However, the cause of these coronary abnormalities has not been established. In this study, we evaluated coronary hemodynamic to assess the role of Ang-II, reactive oxygen species, and nitric oxide (NO) in the development of these alterations in CM of 1, 2, and 6 months of age. Methods and Results Excised hearts from control (CT) and CM were retroperfused with Krebs-Ringer bicarbonate solution (KRB), and coronary resistance (CR) was determined. The experimental protocol involved sequential infusions of the thromboxane analog U46619 (THX, 0.1 μmol/L), bradykinin (BKN, 10 μmol/L), and sodium nitroprusside (SNP, 10 μmol/L). Similar experiments were conducted after treatment of hearts with N ω -nitro-L-arginine methyl ester (L-NAME, 10 μmol/L). Basal CR increased with age, but no significant differences were observed between CT and CM. Reactivity to THX was increased (69%, P < .05) in 2-month-old CM when compared with CT. This effect was observed concomitantly with a significant reduction (53%, P < .05) in BKN-induced relaxation. The reduction in BKN-dependent relaxation was prevented by treatment for 1 month with the antioxidant N-acetylcysteine (1 g·kg·day), or losartan, an Ang II type 1 receptor blocker (10 mg·kg·day). Losartan also prevented the THX-induced increased reactivity in 2-month-old CM. The BKN-induced relaxation occurred through an L-NAME–sensitive pathway that was impaired with age. SNP dilation was preserved in all animal groups. Conclusions Our results strongly implicate vascular renin-angiotensin-system (RAS) and oxidative stress in endothelial dysfunction and increased reactivity in the early stages of cardiomyopathy in CM. These findings could be relevant to understand the etiology of cardiovascular disorders, in particular, in patients with sarcoglycanopathies.

Alpha1‐Adrenoceptor mediated aortic contraction involves transactivation of EGF receptor

Prolonged elevation of plasma catecholamines is a risk factor for vascular diseases such as hypertension and atherosclerosis. Catecholamines induce hypertrophy of vascular smooth muscle through alpha1‐adrenoceptors (α1‐ARs). In cell culture this involves the transactivation of epidermal growth factor receptor (EGFR). We hypothesized EGFR transactivation to be involved also in the α1‐AR mediated aortic contraction. Thoracic aorta was isolated from 12‐14 weeks old male Wistar rats. In vitro aortic contractile responses to cumulative doses of phenylephrine (PE) were investigated in the absence and presence of EGFR antagonists AG1478 and DAPH in intact and endothelium‐denuded rings. Involvement of signal transduction pathways was investigated by using heparin and inhibitors of Src, MMP, ERK1/2 and IP3K. AG1478 concentration dependently decreased PE‐induced contractile responses in both intact (pA2=5,52) and denuded (pA2=5,47) rings, as did DAPH. In contrast, AG1478 did not affect contraction response to the thromboxane analog U46619. Blockade of IP3K by wortmannin abrogated the effect of AG1478 on the PE response, whereas heparin and inhibitors of Src, MMP and ERK1/2 did not affect the AG1478 action. α1‐AR mediated contraction of aortic ring involves transactivation of EGFR, most likely via activation of the IP3K pathway and not by shedding of heparin‐binding EGF. This study was supported by GUIDE.

An Increased Regional Blood Flow Precedes Mesenteric Inflammation in Rats Treated by a Phosphodiesterase 4 Inhibitor

The study was undertaken to assess the hemodynamic effects induced by a single dose of the phosphodiesterase 4 (PDE4) inhibitor, CI-1044, which is known to cause mesenteric vascular alterations in rats. In the present study, an administration of 160 mg/kg of CI-1044 caused perivascular and interstitial inflammation, with infiltrates of admixed neutrophils and macrophages but without evidence of vascular necrosis (ileum, 15/20 rats; duodenum + jejunum, 7/20 rats). Four hours after administration, blood pressure was decreased (- 13%). A fluorescent microsphere technique demonstrated that, in these conditions, cardiac output was doubled (+ 100%) and total peripheral resistance was decreased (- 54%). The largest increases in blood flow were measured in the duodenum (+ 101%), in the jejunum (+ 110%), and in the ileum (+ 192%). Therefore, the mesentery was the most sensitive organ affected by the drug and, within this area, parts with the highest incidence of vascular alteration were those which had shown the highest increase in flow. In addition, isolated precontracted mesenteric resistance arteries dissected from untreated animals were fully relaxed when incubated with increasing concentrations of CI-1044 up to 2.5 x 10(-5)M. At this latter concentration, contractile abilities and sensitivities to the physiological agonist noradrenaline (NA) and to the thromboxane analogue U46619 were significantly attenuated (- 28 and - 27%, respectively). This effect could lead to a decreased response to NA and possibly to other agonists in vivo consistent with the vasodilation observed with the microsphere technique. These data provide evidence that the PDE4 inhibitor CI-1044 induces changes of vascular tone that could lead to histological alterations in the mesenteric area.

Normothermic and hypothermic models for studying the deleterious effects of hypoxia-reoxygenation on EDHF-mediated relaxation in isolated porcine coronary arteries

Introduction The vasomotor response of the coronary artery is altered by hypoxia–reoxygenation (H–R) induced damage. The aim of our study was to compare and evaluate normothermic and hypothermic models which are suitable for future drug studies of vasoprotective action against H–R injury. Methods Porcine coronary arterial rings were isolated and placed in Krebs–Henseleit (K–H) solution. Rings were exposed to normoxic conditions (control group) and two different H–R conditions: the first induced by a 95% N 2–5% CO 2 gas mixture (40- and 60-min hypoxia) in a normothermic protocol, and the second induced by hypothermic (4 °C) hypoxia–reoxygenation in an air-tight beaker filled with K–H solution (24- and 48-hours hypoxia). Reoxygenation was applied by introducing K–H solution aerated with a 95% O 2–5% CO 2 mixture under normothermic (37 °C) conditions. To test the EDHF-mediated relaxation by substance P, rings were first incubated in L-NNA, nitric oxide synthase inhibitor, and indomethacin, cyclooxygenase inhibitor, and then pre-contracted with thromboxane analogue U-46619. Analysis of the maximum relaxation of the arterial rings was performed by one-way ANOVA, followed by Bonferroni's post-test. Results Distal segments of the coronary artery responded faster to contraction induced by U-46619 and were relaxed by substance P to a greater extent than proximal segments. Maximal relaxations of arterial rings induced by a 10 nM solution of substance P were significantly reduced ( p < 0.001) from the values for normoxic rings (81.0 ± 1.0%, n = 30) after 40-min H–R (50.5 ± 5.3%, n = 30), 60-min H–R (32.1 ± 3.5%, n = 30), 24-hours hypothermic H–R (56.0 ± 2.3%, n = 30) and after 48-hours hypothermic H–R (38.5 ± 5.1%, n = 30). Conclusions The model employing 40-min normothermic H–R is as effective as 24-hours hypothermic H–R, and 60-min normothermic H–R as 48-hours hypothermic H–R for studying the deleterious effects of H–R on EDHF-mediated relaxation.

Sevoflurane and the Feto-Placental Vasculature: The Role of Nitric Oxide and Vasoactive Eicosanoids

The effects and mechanisms of action of volatile anesthetics on the feto-placental vasculature are not known. We aimed to quantify the vasoactive effects of sevoflurane and determine the role of nitric oxide (NO) and of vasoactive eicosanoids in mediating these effects in isolated human chorionic plate arterial rings. Quadruplicate ex vivo human chorionic plate arterial rings were used in all studies. Series 1 quantified the vasodilation produced by sevoflurane in rings preconstricted with the thromboxane analog U46619. Series 2A-C examined the role of NO in sevoflurane-mediated vasodilation. In separate experiments, we examined the potential for the nonspecific NO inhibitors, L-NAME, L-nMMA, and the inactive D-NAME, to modulate the vasodilation produced by sevoflurane. Series 2D determined whether sevoflurane altered vascular smooth muscle sensitivity to exogenous NO. Series 3A-D examined the role of vasoactive eicosanoids in sevoflurane-mediated vasodilation. In separate experimental series, we examined whether the nonspecific cyclooxygenase inhibitor, indomethacin, or the 5-lipoxygenase inhibitor, nordihydroguaiaretic acid, modulated sevoflurane-mediated vasodilation. Sevoflurane produced dose-dependent vasodilation of preconstricted chorionic plate arterial rings, with mean ring vasodilation increasing from 15 +/- 7% at 2% sevoflurane to 67 +/- 17% (mean +/- sd) at 8% sevoflurane. Blockade of NO synthase did not attenuate the vasodilator effects of sevoflurane. Sevoflurane did not alter smooth muscle sensitivity to NO. Indomethacin augmented sevoflurane vasodilation at 10(-5) M, but not at 10(-6) M. Conversely, nordihydroguaiaretic acid attenuated sevoflurane-mediated vasodilation at 3 x 10(-6) M but not at 3 x 10(-7) M. Sevoflurane was a vasodilator in the feto-placental vasculature in this in vitro model. Sevoflurane-mediated vasodilation is NO and cyclooxygenase-independent and appears to be mediated in part via a lipoxygenase generated vasodilator eicosanoid.

The Effect of Maternal Catecholamines on the Caliber of Gravid Uterine Microvessels

Changes in maternal catecholamines that accompany the onset of labor analgesia include a decrease in epinephrine (EPI) but no change in norepinephrine (NE). Because EPI exerts predominantly beta-adrenergic, and NE predominantly alpha-adrenergic effects in circulating concentrations, we hypothesized that these changes could lead to uterine arteriole vasoconstriction. Uterine microvessels (73-120 microm internal diameter, n = 18) were harvested from near-term pregnant Sprague-Dawley rats, isolated and studied in a pressurized no-flow state with video microscopy. Drugs were applied extraluminally to the superfusion reservoir and the steady-state vessel diameter recorded. Dose-response curves were constructed for NE with and without the addition of the alpha-adrenergic antagonist prazosin, EPI (after 20%-30% preconstruction with the thromboxane analog U46619) with and without the addition of the beta-adrenergic antagonist propranolol, and NE in the presence of 10(-8) M EPI. Washout experiments modeled the changes in circulating maternal catecholamines observed during onset of analgesia: 10(-8) M EPI and 10(-6.5) M of NE (the EC50) were added simultaneously, then washed with NE only, and then with the beta2-adrenergic agonist terbutaline and NE. The washout protocol was repeated in the presence of propranolol. NE caused dose-dependent vasoconstriction (P < 0.0001), which was blocked by prazosin (P < 0.0001). EPI, added to U46619-preconstricted microvessels, caused vasodilation at lower concentrations and vasoconstriction at higher doses (P < 0.0001). Propranolol converted this response to monophasic dose-dependent vasoconstriction (P < 0.0001). Pretreatment of nonprecontracted vessels with EPI, 10(-8) M, significantly attenuated NE-induced vasoconstriction (P < 0.0001). In washout experiments, removal of EPI with continued presence of NE resulted in vasoconstriction that was reversed by terbutaline. Propranolol blocked the effect of both EPI and terbutaline. The results demonstrate that EPI, in concentrations found in the plasma of laboring women, vasodilates uterine resistance vessels and attenuates NE-induced vasoconstriction. This observation may have implications for changes in uterine blood flow that may accompany the onset of labor analgesia in human parturients, as effective analgesia is accompanied by an acute decrease in circulating EPI levels.

Thromboxane analog U46619 redistributes pulmonary microvascular perfusion in isolated rat lungs, but does not retain 3 μm diameter particles in lung capillaries

U46619 is a potent vasoconstrictor that increases vascular resistance 2–4 fold in isolated, perfused rat lungs. We wondered if any of this constriction occurred in lung capillaries. To test this, we infused 3.0 μm diameter fluorescent latex particles into the pulmonary arteries of isolated rat lungs perfused with U46619. We then measured the percentage of the infused particles that flowed out of the left atrium over 5 minutes. This percentage averaged 14.2±10.0% in vasoconstricted lungs, and 11.5±10.2% in flow‐matched controls (n.s.) (mean flow, 4.9±1.0 ml/min). To further investigate the effects of U46619 vasoconstriction, we imaged trapping patterns of the particles within the lungs and quantified these using dispersion index (DI) analysis. LogDI, a measure of latex particle distribution, averaged 0.81±0.43 in vasoconstricted lungs and 0.54±0.28 in flow‐matched controls (p = 0.03). The larger logDI value in the U‐46619‐perfused lungs demonstrates that the trapped latex particles were more clustered (non‐uniform) than those in the flow‐matched controls. Our results suggest that U46619 redistributes pulmonary microvascular perfusion, but most likely not through constriction of lung capillaries. Supported by a grant from the Department of Veterans Affairs.

Time-Limited Hyporesponsiveness to Inhaled Nitric Oxide and Pulmonary Phosphodiesterase Activity in Endotoxemic Rats

In acute lung injury (ALI) pulmonary hyporesponsiveness to inhaled nitric oxide (iNO) still represents an unresolved clinical challenge. In septic ALI-patients the incidence of hyporesponsiveness to iNO is increased; therefore, endotoxemia appears to play a major role. Experimental data suggest that endotoxemia, e.g., induced by lipopolysaccharides (LPS), contribute to the hyporesponsiveness to iNO. Guanosine 3',5'-cyclic monophosphate (cGMP) is metabolized by phosphodiesterases (PDE). The role of PDE in reduced pulmonary vascular response in experimental endotoxemia is still not known. Here, we hypothesized that PDE activity modulates initial pulmonary responsiveness to iNO in ALI following systemic endotoxin exposure. Rats were treated with LPS or used as controls. Lungs were isolated-perfused 0-36 h after LPS injection and the synthetic thromboxane analogue U46619 was added to increase pulmonary artery pressure by 6-8 mmHg (n = 47). Then, the pulmonary vasodilatory response to 3 doses of iNO (0.4, 4 and 40 ppm) was measured. Furthermore, lungs were prepared as described previously, and 2, 10, and 18 h after LPS the change in pulmonary artery pressure in response to two different inhibitors of PDE, one of which is PDE sensitive (8-Br-cGMP) and one is PDE stable (8-pCPT-cGMP), was determined (n = 43). Serum nitrite/nitrate levels started to increase 4 h after LPS, with a maximum at 18 h. In contrast, decreased pulmonary vasoreactivity in response to iNO developed as early as 2 h later and remained depressed up to 18 h. The pulmonary vasoreactivity to the PDE-sensitive 8-Br-cGMP after LPS-stimulation was lower than that in lungs treated with the PDE-stable 8-pCPT-cGMP. In rats pretreated with LPS, hyporesponsiveness of pulmonary vessels to iNO is time-limited and associated with increased serum nitrite/nitrate levels, and appears to be attributed in part to increased pulmonary PDE activity.

Intravital microscopy of the murine pulmonary microcirculation

Intravital microscopy (IVM) is considered as the gold standard for in vivo investigations of dynamic microvascular regulation. The availability of transgenic and knockout animals has propelled the development of murine IVM models for various organs, but technical approaches to the pulmonary microcirculation are still scarce. In anesthetized and ventilated BALB/c mice, we established a microscopic access to the surface of the right upper lung lobe by surgical excision of a window of 7- to 10-mm diameter from the right thoracic wall. The window was covered by a transparent polyvinylidene membrane and sealed with alpha-cyanoacrylate. Removal of intrathoracic air via a trans-diaphragmal intrapleural catheter coupled the lung surface to the window membrane. IVM preparations were hemodynamically stable for at least 120 min, with mean arterial blood pressure above 70 mmHg, and mean arterial Po(2) and arterial Pco(2) in the range of 90-100 Torr and 30-40 Torr, respectively. Imaged lungs did not show any signs of acute lung injury or edema. Following infusion of FITC dextran, subpleural pulmonary arterioles and venules of up to 50-microm diameter and alveolar capillary networks could be visualized during successive expiratory plateau phases over a period of at least 2 h. Vasoconstrictive responses to hypoxia (11% O(2)) or infusion of the thromboxane analog U-46619 were prominent in medium-sized arterioles (30- to 50-microm diameter), minor in small arterioles <30 microm, and absent in venules. The presented IVM model may constitute a powerful new tool for investigations of pulmonary microvascular responses in mice.