Thromboxane Receptor Agonist U46619 Research Articles

Amitriptyline inhibits bronchoconstriction and directly promotes dilatation of the airways

IntroductionThe standard therapy for bronchial asthma consists of combinations of acute (short-acting ß2-sympathomimetics) and, depending on the severity of disease, additional long-term treatment (including inhaled glucocorticoids, long-acting ß2-sympathomimetics, anticholinergics, anti-IL-4R antibodies). The antidepressant amitriptyline has been identified as a relevant down-regulator of immunological TH2-phenotype in asthma, acting—at least partially—through inhibition of acid sphingomyelinase (ASM), an enzyme involved in sphingolipid metabolism. Here, we investigated the non-immunological role of amitriptyline on acute bronchoconstriction, a main feature of airway hyperresponsiveness in asthmatic disease.MethodsAfter stimulation of precision cut lung slices (PCLS) from mice (wildtype and ASM-knockout), rats, guinea pigs and human lungs with mediators of bronchoconstriction (endogenous and exogenous acetylcholine, methacholine, serotonin, endothelin, histamine, thromboxane-receptor agonist U46619 and leukotriene LTD4, airway area was monitored in the absence of or with rising concentrations of amitriptyline. Airway dilatation was also investigated in rat PCLS by prior contraction induced by methacholine. As bronchodilators for maximal relaxation, we used IBMX (PDE inhibitor) and salbutamol (ß2-adrenergic agonist) and compared these effects with the impact of amitriptyline treatment. Isolated perfused lungs (IPL) of wildtype mice were treated with amitriptyline, administered via the vascular system (perfusate) or intratracheally as an inhalation. To this end, amitriptyline was nebulized via pariboy in-vivo and mice were ventilated with the flexiVent setup immediately after inhalation of amitriptyline with monitoring of lung function.ResultsOur results show amitriptyline to be a potential inhibitor of bronchoconstriction, induced by exogenous or endogenous (EFS) acetylcholine, serotonin and histamine, in PCLS from various species. The effects of endothelin, thromboxane and leukotrienes could not be blocked. In acute bronchoconstriction, amitriptyline seems to act ASM-independent, because ASM-deficiency (Smdp1−/−) did not change the effect of acetylcholine on airway contraction. Systemic as well as inhaled amitriptyline ameliorated the resistance of IPL after acetylcholine provocation. With the flexiVent setup, we demonstrated that the acetylcholine-induced rise in central and tissue resistance was much more marked in untreated animals than in amitriptyline-treated ones. Additionally, we provide clear evidence that amitriptyline dilatates pre-contracted airways as effectively as a combination of typical bronchodilators such as IBMX and salbutamol.ConclusionAmitriptyline is a drug of high potential, which inhibits acute bronchoconstriction and induces bronchodilatation in pre-contracted airways. It could be one of the first therapeutic agents in asthmatic disease to have powerful effects on the TH2-allergic phenotype and on acute airway hyperresponsiveness with bronchoconstriction, especially when inhaled.

Epoxyeicosatrienoic Acid and Prostanoid Crosstalk at the Receptor and Intracellular Signaling Levels to Maintain Vascular Tone.

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by the cytochrome P450-(CYP450)-mediated epoxygenation of arachidonic acid. EETs have numerous biological effects on the vascular system, but aspects including their species specificity make their effects on vascular tone controversial. CYP450 enzymes require the 450-reductase (POR) for their activity. We set out to determine the contribution of endothelial CYP450 to murine vascular function using isolated aortic ring preparations from tamoxifen-inducible endothelial cell-specific POR knockout mice (ecPOR−/−). Constrictor responses to phenylephrine were similar between control (CTR) and ecPOR−/− mice. Contrastingly, sensitivity to the thromboxane receptor agonist U46619 and prostaglandin E2 (PGE2) was increased following the deletion of POR. Ex vivo incubation with a non-hydrolyzable EET (14,15-EE-8(Z)-E, EEZE) reversed the increased sensitivity to U46619 to the levels of CTR. EETs had no effect on vascular tone in phenylephrine-preconstricted vessels, but dilated vessels contracted with U46619 or PGE2. As U46619 acts through RhoA-dependent kinase, this system was analyzed. The deletion of POR affected the expression of genes in this pathway and the inhibition of Rho-GTPase with SAR407899 decreased sensitivity to U46619. These data suggest that EET and prostanoid crosstalk at the receptor level and that lack of EET production sensitizes vessels to vasoconstriction via the induction of the Rho kinase system.

Inhibition of neurogenic contractions in renal arteries and of cholinergic contractions in coronary arteries by the presumed inhibitor of ADP-ribosylation factor 6, NAV2729

NAV2729 is a presumed inhibitor of the monomeric GTPase ADP ribosylation factor 6 (ARF6) and inhibits smooth muscle contraction outside the cardiovascular system. Its effects on vascular smooth muscle contraction or a possible role of ARF6 in vasocontraction have not yet been examined. Here, we report effects of NAV2729 on neurogenic and agonist-induced contractions in renal interlobar and coronary arteries. Contractions of pig interlobar and coronary arteries were induced in an organ bath by agonists or by electric field stimulation (EFS). Owing to divergent characteristics of both vessel types, EFS-induced contractions were only examined in interlobar arteries, and contractions by agonists acting on muscarinic receptors only in coronary arteries. NAV2729 inhibited frequency-dependent EFS-induced contractions of interlobar arteries. The degree of inhibition was similar using 5 µM and 10 µM NAV2729. Inhibition of EFS-induced contractions was resistant to a nitric oxide synthase inhibitor and to diclofenac. The neurogenic and adrenergic character of EFS-induced contractions was confirmed by inhibition by tetrodotoxin and prazosin. In coronary arteries, NAV2729 (5 µM) inhibited concentration-dependent contractions induced by carbachol and methacholine. Contractions induced by α1-adrenergic agonists, endothelin-1, the thromboxane receptor agonist U46619, or serotonin remained unchanged by NAV2729 in both vessel types. NAV2729 inhibits neurogenic contractions in interlobar arteries and contractions induced by cholinergic agonists in coronary arteries. In both vessel types, NAV2729 does not inhibit contractions induced by receptor agonists other than those acting on muscarinic receptors. Addressing effects in other vessels and in other smooth muscle–rich organs merits further attention.

Effect of mitochondrial complex III inhibitors on the regulation of vascular tone in porcine coronary artery

In order to gain insight into the regulation of vascular tone by mitochondria, the effects of mitochondrial complex III inhibitors on contractile responses in porcine isolated coronary arteries were investigated. Segments of porcine coronary arteries were set up for isometric tension recording and concentration response curves to contractile agents were carried out in the absence or presence of the complex III inhibitors antimycin A or myxothiazol. Activity of AMP kinase was determined by measuring changes in phosphorylation of AMP kinase at Thr172.Pre-incubation with 10 μM antimycin A (Qi site inhibitor), or myxothiazol (Qo site inhibitor) led to inhibition of the contraction to the thromboxane receptor agonist U46619. Similar effects were seen on contractile responses to extracellular calcium, and the L-type calcium channel opener BAY K 8644, suggesting that both antimycin A and myxothiazol inhibit calcium-dependent contractions. The inhibitory effect of antimycin A was still seen in the absence of extracellular calcium, indicating an additional effect on a calcium independent pathway. The AMP kinase inhibitor dorsomorphin (10 μM) prevented the inhibitory of antimycin A but not myxothiazol. Furthermore, antimycin A increased the phosphorylation of AMP kinase, indicating an increase in activity, suggesting that antimycin A also acts through this pathway.These data indicate that inhibition of complex III attenuates contractile responses through inhibition of calcium influx. However, inhibition of the Qi site can also inhibit the contractile response through activation of AMP kinase.

Prospective Study Reveals Increased Platelet Function Associated with Multiple Myeloma and Its Treatment

Background: Multiple Myeloma (MM) is a rare incurable bone marrow cancer characterised by a malignant proliferation of plasma cells. MM is usually preceded by a premalignant and benign Monoclonal Gammopathy of Undetermined Significance (MGUS). The incidence of arterial and venous thrombosis in MM is substantially higher than in the normal population, however the cause of this increased thrombosis risk and the impact of MM on platelet function is unclear. Treatments for both newly diagnosed and relapsed/refractory patients with MM include Immunomodulatory drugs (IMiDs) such as thalidomide/lenalidomide-based combinations. These treatments improve considerably patient outcomes, however iMiD treatment also increases the risk of thrombotic complications in these patients. Aims: In this prospective study we explored the impact of MM and its treatment on platelet function. Methods: High throughput functional analysis was performed using platelets from normal healthy controls (n=31) and patients with MGUS (n=18), smouldering multiple myeloma (SMM, n= 20), and MM (26). The MM group was further divided into 3 treatment cohorts; (1) no treatment, (2) treatment with proteasome inhibitor (PI) and dexamethasone (Dex), and (3) treatment with PI, Dex, immunomodulatory drug (iMiD) and direct oral anticoagulant. Platelet aggregation and activation (fibrinogen binding and P-selectin exposure) were measured in response to a concentration range of agonists including ADP, the thrombin receptor agonist TRAP-6, collagen, collagen-related peptide (CRP), a thromboxane receptor agonist U46619 and epinephrine. Cereblon protein was detected in platelet protein extracts by immunoblot analysis. Results: Consistent with previous reports, modestly increased VWF and factor VIII levels were detected in MM patients, but no additional differences in coagulation parameters were detected in patient groups compared to normal healthy controls (other than expected due to anticoagulant usage). Platelet aggregation in response to each agonist was increased significantly in the MM patient group compared to the normal healthy controls, suggesting that platelet reactivity is elevated in MM patients through a common mechanism that is shared by different activation pathways or the involvement of multiple mechanisms. P-selectin exposure on platelets from MM patients was not significantly different from normal healthy donors, indicating that enhanced platelet reactivity in MM is specifically through modulation of integrin αIIbβ3 activation, fibrinogen binding and therefore enhanced aggregation. The effects of treatment on platelet function in patients on iMiD vs. non iMiD treatment were assessed. In the iMiD treatment group, patient platelets aggregated in response to lower concentrations of ADP, collagen, epinephrine and CRP in samples taken post-treatment compared to those taken before and during treatment. This demonstrates an increased sensitivity to platelet activation in these patients induced by treatment. Immunoblot analysis revealed that platelets contain cereblon, a therapeutic target of lenalidomide. The potential direct effects of iMiDs on platelets in vitro was therefore explored. Lenalidomide treatment (10mM) increased the ability of platelets to aggregate in response to low concentrations of each agonist tested when compared to normal controls. Conclusions: Platelet reactivity is increased in multiple myeloma and increased further upon iMiD treatment. The presence of the key therapeutic target for iMiDs in platelets and the ability of lenalidomide to modulate platelet function directly, reveals new avenues for investigation to determine the underlying mechanism of action. Disclosures Laffan: CSL: Consultancy; Pfizer: Consultancy; Sobi: Consultancy; Roche: Consultancy; LFB: Consultancy; Shire: Consultancy; Octapharma: Consultancy; Bayer: Speakers Bureau; Roche-Chugai: Speakers Bureau; Takeda: Speakers Bureau; Leo-Pharma: Speakers Bureau; Pfizer: Speakers Bureau. Shapiro:Bayer: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Chugai/Roche: Consultancy, Speakers Bureau; Shire/Takeda: Consultancy, Speakers Bureau. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ramasamy:Takeda: Research Funding; Janssen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Amgen: Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sanofi: Honoraria; Oncopeptides: Honoraria; Takeda: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Bristol Myers Squibb: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers squibb: Membership on an entity's Board of Directors or advisory committees. Gibbins:Bristol Myers Squibb: Research Funding; Arena Pharmaceuticals: Research Funding.

Loss‐ And Gain of Function Mutations in the Type 1 IP3 Receptor (IP3R1) Modify Agonist‐Evoked Vasoactive Responses in Cerebral and Skeletal Muscle Resistance Arteries From Adult Mice

Calcium mobilization within vascular smooth muscle and endothelial cells of small resistance arteries plays a critical role in agonist‐evoked vasoconstriction and dilation, respectively. Despite the recognized contribution of IP3‐mediated intracellular Ca2+ release for agonist‐evoked vasoactive responses, it has been difficult to study this process acutely in native arteries, due to the lack of selective pharmacological agents or experimental strategies to target the predominant IP3R1. In humans, heterozygous mutations in the IP3R1 are known to produce either loss‐of‐function (LOF) or gain‐of‐function phenotypes for Ca2+ release that impact cerebellar activity (e.g. ataxia), yet it remains unclear how these global mutations affect the cardiovascular system. To facilitate such investigation, we have engineered novel genetic knock‐in mice harbouring a single amino acid mutation at position Asp2594 in the transmembrane S6 helix of the endogenous IP3R1 Ca2+ release channel that confers either a LOF (Asp2594Ala) or GOF (Asp2594Lys) phenotype, respectively. In permeabilized HEK293 cells expressing these mutant receptors, the LOF mutation shifted IP3‐mediated ER Ca2+ release ~2‐fold to the right, whereas the GOF mutation produced a 2.3‐fold leftward shift compared with wild‐type (WT) IP3R1. In endothelium‐denuded cerebral arteries pressurized to 10 mmHg, the sensitivity of serotonin‐evoked vasoconstriction was reduced in vessels from heterozygous LOF mice compared with WT, whereas agonist responsiveness was enhanced in arteries from heterozygous GOF mice. Vasoconstriction in response to the thromboxane receptor agonist U46619 was modestly enhanced by the GOF mutation, but largely unaffected in arteries from LOF mice. In cannulated cremaster skeletal muscle arteries from LOF and GOF mice pressurized to 70 mmHg, the sensitivity of endothelium‐dependent, acetylcholine‐mediated inhibition of myogenic tone was right shifted (3.7‐fold) and left shifted (2‐fold), respectively, compared with vessels from WT. Comparable changes in functional sensitivity were observed in response to the endothelium‐dependent vasodilator bradykinin. In contrast, dilation evoked by the NO donor sodium nitroprusside was not different among the three genotypes. Collectively, these data demonstrate that genetic modification of IP3R1‐mediated Ca2+ mobilization in small resistance arteries functionally impacts agonist‐evoked vasoactive responses. These observations highlight the importance of IP3R1 activity in the vascular wall, and further implicate vascular impairment as a contributing factor to the neurological dysfunction observed in patients with LOF and GOF IP3R1 mutations.Support or Funding InformationThis work was funded by the following research grants from the Canadian Institutes of Health Research: MOP‐97988 (WCC), MOP‐142467 (APB) and PJT‐155963 (WC, WCC and APB).

LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Regulates Smooth Muscle Contractility by Modulating Ca2+ Signaling and Expression of Cytoskeleton-Related Proteins.

Objective- Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor-related protein 1) maintains vessel wall integrity, and smLRP1-/- mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results- Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca2+ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1-/- mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1-/- mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1-/- aortic rings and cultured vascular smooth muscle cells. Conclusions- These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.

Anticonvulsant-like effect of thromboxane receptor agonist U-46619 against pentylenetetrazol-induced seizures

Increasing evidence suggests that prostanoid receptors and their ligands may constitute valuable tools for development of new antiepileptic drugs. Thromboxane A2 (TXA2) is a major eicosanoid in cardiovascular homeostasis. TXA2 exerts its action through the specific G protein-coupled TXA2 receptor (TP). In addition to its crucial role in the cardiovascular system, TXA2 and TPs play a role in the brain. Nevertheless, previously identified roles have been limited to cell protection of neurotoxicity, and the role of TPs on seizure activity was not investigated. Here we evaluated the effect of potent and selective TP agonist U-46619 on seizures induced by pentylenetetrazol (PTZ). Adult C57BL/6 mice received increasing doses of U-46619 (0, 30, 100 or 300 μg/kg). After 30 min we measured the latencies to myoclonic and generalized seizures induced by PTZ (60 mg/kg). We found that U-46619 increased the latency to PTZ-induced myoclonic jerks and tonic-clonic seizures. Moreover, U-46619 increased the immunocontent of phosphorylated Ser657 at protein kinase C (PKC) alpha subunit, indicating PKC activation in the hippocampus and cerebral cortex. Levels of TPs were not altered by the agonist. Administration of a TP antagonist, SQ 29,548, did not alter seizures and did not blunt the anticonvulsant-like effect of the agonist. In summary, we showed that a potent and selective TP agonist, U-46619, increased seizure latency in mice. Activation of PKC signaling pathways may underlie the anticonvulsant-like effect. Further investigation is needed to understand the potential of TPs in seizure treatment.

Alpha1 -adrenergic stimulation selectively enhances endothelium-mediated vasodilation in rat cremaster arteries.

We have systematically investigated how vascular smooth muscle α 1‐adrenoceptor activation impacts endothelium‐mediated vasodilation in isolated, myogenically active, rat cremaster muscle 1A arteries. Cannulated cremaster arteries were pressurized intraluminally to 70 mmHg to induce myogenic tone, and exposed to vasoactive agents via bath superfusion at 34°C. Smooth muscle membrane potential was measured via sharp microelectrode recordings in pressurized, myogenic arteries. The α 1‐adrenergic agonist phenylephrine (25–100 nmol/L) produced further constriction of myogenic arteries, but did not alter the vasorelaxant responses to acetylcholine (0.3 μmol/L), SKA‐31 (an activator of endothelial Ca2+‐dependent K+ channels) (3 μmol/L) or sodium nitroprusside (10 μmol/L). Exposure to 0.25–1 μmol/L phenylephrine or 1 μmol/L norepinephrine generated more robust constrictions, and also enhanced the vasodilations evoked by acetylcholine and SKA‐31, but not by sodium nitroprusside. In contrast, the thromboxane receptor agonist U46619 (250 nmol/L) dampened responses to all three vasodilators. Phenylephrine exposure depolarized myogenic arteries, and mimicking this effect with 4‐aminopyridine (1 mmol/L) was sufficient to augment the SKA‐31‐evoked vasodilation. Inhibition of L‐type Ca2+ channels by 1 μmol/L nifedipine decreased myogenic tone, phenylephrine‐induced constriction and prevented α 1‐adrenergic enhancement of endothelium‐evoked vasodilation; these latter deficits were overcome by exposure to 3 and 10 μmol/L phenylephrine. Mechanistically, augmentation of ACh‐evoked dilation by phenylephrine was dampened by eNOS inhibition and abolished by blockade of endothelial KCa channels. Collectively, these data suggest that increasing α 1‐adrenoceptor activation beyond a threshold level augments endothelium‐evoked vasodilation, likely by triggering transcellular signaling between smooth muscle and the endothelium. Physiologically, this negative feedback process may serve as a “brake” to limit the extent of vasoconstriction in the skeletal microcirculation evoked by the elevated sympathetic tone.

P2Y12 antibody inhibits platelet activity and protects against thrombogenesis

Given that platelet hyperactivity is known to give rise to thrombotic disorders, new and/or novel antiplatelet therapies are constantly being developed to add to, or to complement the current arsenal of agents. To this end, adenosine diphosphate (ADP) is an important platelet activator that acts by binding to the G-protein coupled P2Y1 and P2Y12 receptors. Although the contribution of the P2Y12 receptor to the genesis of thrombosis is well established, the parenteral arsenal of drugs targeting this receptor in clinical use is limited to cangrelor. In this study, we investigated the potential antiplatelet activity of an antibody targeting the ligand-binding domain of the P2Y12 receptor (abbreviated P2Y12Ab). Our in vitro studies revealed that the P2Y12Ab could effectively inhibit aggregation induced by ADP, as well as that triggered by the thromboxane receptor agonist U46619. Additionally, using FACS analysis, we observed reduced P-selectin, phosphatidylserine exposure and integrin activation in the presence of P2Y12Ab. As for its in vivo effects, the P2Y12Ab also demonstrated protection against thrombus formation; albeit this was accompanied with a bleeding diathesis (longer bleeding time). Notably, this inhibitory profile is consistent with that observed with oral anti-P2Y12 agents. Collectively, our findings demonstrate that the P2Y12Ab functionally blocks platelet activity in vitro and in vivo, and support the notion that it can be purposed as a parenteral antiplatelet agent, to be used in conjunction with and/or as a complement to current antiplatelet therapies.

Human uterine and placental arteries exhibit tissue-specific acute responses to 17 -estradiol and estrogen-receptor-specific agonists

The discrete regulation of vascular tone in the human uterine and placental circulations is a key determinant of appropriate uteroplacental blood perfusion and pregnancy success. Humoral factors such as estrogen, which increases in the placenta and maternal circulation throughout human pregnancy, may regulate these vascular beds as studies of animal arteries have shown that 17β-estradiol, or agonists of estrogen receptors (ER), can exert acute vasodilatory actions. The aim of this study was to compare how acute exposure to ER-specific agonists, and 17β-estradiol, altered human placental and uterine arterial tone in vitro. Uterine and placental arteries were isolated from biopsies obtained from women with uncomplicated pregnancy delivering a singleton infant at term. Vessels were mounted on a wire myograph, exposed to the thromboxane receptor agonist U46619 (10−6 M), and then incubated with incremental doses (5 min, 0.03–30 µM) of either 17β-estradiol or agonists specific for the ERs ERα (PPT), ERβ (DPN) or the G-protein-coupled estrogen receptor GPER-1 (G1). ERα and ERβ mRNA expression was assessed. 17β-estradiol, PPT and DPN each relaxed myometrial arteries (P < 0.05) in a manner that was partly endothelium-dependent. In contrast, 17β-estradiol or DPN relaxed placental arteries (maximum relaxation to 42 ± 1.1 or 47.6 ± 6.53% of preconstriction, respectively) to a lesser extent than myometrial arteries (to 0.03 ± 0.03 or 8.0 ± 1.0%) and in an endothelial-independent manner whereas PPT was without effect. G1 exposure did not inhibit the constriction of myometrial nor placenta arteries. mRNA expression of ERα and ERβ was greater in myometrial arteries than placental arteries. ER-specific agonists, and 17β-estradiol, differentially modulate the tone of uterine versus placental arteries highlighting that estrogen may regulate human uteroplacental blood flow in a tissue-specific manner.

Induction of Regulator of G-Protein Signaling 2 Expression by Long-Acting β2-Adrenoceptor Agonists and Glucocorticoids in Human Airway Epithelial Cells

In asthma and chronic obstructive pulmonary disease (COPD) multiple mediators act on Gαq-linked G-protein-coupled receptors (GPCRs) to cause bronchoconstriction. However, acting on the airway epithelium, such mediators may also elicit inflammatory responses. In human bronchial epithelial BEAS-2B cells (bronchial epithelium + adenovirus 12-SV40 hybrid), regulator of G-protein signaling (RGS) 2 mRNA and protein were synergistically induced in response to combinations of long-acting β2-adrenoceptor agonist (LABA) (salmeterol, formoterol) plus glucocorticoid (dexamethasone, fluticasone propionate, budesonide). Equivalent responses occurred in primary human bronchial epithelial cells. Concentrations of glucocorticoid plus LABA required to induce RGS2 expression in BEAS-2B cells were consistent with the levels achieved therapeutically in the lungs. As RGS2 is a GTPase-activating protein that switches off Gαq, intracellular free calcium ([Ca(2+)]i) flux was used as a surrogate of responses induced by histamine, methacholine, and the thromboxane receptor agonist U46619 [(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid]. This was significantly attenuated by salmeterol plus dexamethasone pretreatment, or RGS2 overexpression, and the protective effect of salmeterol plus dexamethasone was abolished by RGS2 RNA silencing. Although methacholine and U46619 induced interleukin-8 (IL-8) release and this was inhibited by RGS2 overexpression, the repression of U46619-induced IL-8 release by salmeterol plus dexamethasone was unaffected by RGS2 knockdown. Given a role for Gαq-mediated pathways in inducing IL-8 release, we propose that RGS2 acts redundantly with other effector processes to repress IL-8 expression. Thus, RGS2 expression is a novel effector mechanism in the airway epithelium that is induced by glucocorticoid/LABA combinations. This could contribute to the efficacy of glucocorticoid/LABA combinations in asthma and COPD.

The bitter taste receptor (TAS2R) agonists denatonium and chloroquine display distinct patterns of relaxation of the guinea pig trachea

Activation of taste receptors (TAS2Rs) by bitter taste agonists has been reported to cause bronchodilation. The aim of this study was to extend the information on the effects of bitter taste agonists on responses induced by different contractile mediators in a standard airway physiology preparation. Isometric responses were assessed in guinea pig trachea (GPT). TAS2R agonists were administered either to segments precontracted with different agonists for contraction or given before challenge with the different contractile stimuli, including antigen in tissues from ovalbumin-sensitized animals. TAS2R mRNA expression on GPT epithelium and smooth muscle was measured with real-time PCR. Denatonium, chloroquine, thiamine, and noscapine induced concentration-dependent relaxations (R(max): 98.3 ± 1.6, 100.0 ± 0.0, 100.0 ± 0.0, and 52.3 ± 1.1% of maximum, respectively, in the presence of indomethacin) in segments precontracted with carbachol. The receptors for denatonium (TAS2R4, TAS2R10) and chloroquine (TAS2R3, TAS2R10) were expressed in GPT. Whereas denatonium selectively inhibited contractions induced by carbachol, chloroquine uniformly inhibited contractions evoked by prostaglandin E(2), the thromboxane receptor agonist U-46619, leukotriene D(4), histamine, and antigen. The effects of denatonium, but not those of chloroquine, were partly inhibited by blockers of the large Ca(2+)-activated K(+) channels and decreased by an increase of the level of precontraction. In conclusion, TAS2R agonists mediated strong relaxations and substantial inhibition of contractions in GPT. Chloroquine and denatonium had distinct patterns of activity, indicating different signaling mechanisms. The findings reinforce the hypothesis that TAS2Rs are potential targets for the development of a new class of more efficacious agonists for bronchodilation.

Dynamin-Related Protein-1 Controls Fusion Pore Dynamics During Platelet Granule Secretion and Thrombus Formation In Vivo

Abstract 361Platelet granule secretion serves a central role in hemostasis and thrombosis. During platelet secretion, fusion of granule membranes with those of the plasma membrane results in the release of granule contents. Recently electrochemical techniques using single-cell amperometry have shown that platelet membrane fusion results in the formation of a fusion pore. The fusion pore subsequently expands to enable the complete extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore formation and expansion are not known. To discover novel components of the platelet secretory machinery, we tested >300,000 compounds in a forward chemical genetic screen designed to identify inhibitors of dense granule secretion. A compound, ML160, was found that blocked dense granule release with an IC50 of approximately 0.5 μM. ML160 was also identified in an unrelated high throughput screen designed to detect inhibitors of dynamin-related protein-1 (Drp-1). Although best known as mediators of membrane fission, dynamins also contribute to granule exocytosis by controlling fusion pore expansion. Immunoblot analysis of platelet pellets and supernatants confirmed the presence of Drp-1 in platelets and demonstrated nearly equal distribution between platelet membranes and cytosol. mDivi-1, a well-characterized small molecule inhibitor of Drp-1 that acts outside of the GTP binding site, blocked PAR1-mediated platelet dense granule and α-granule release with an IC50 of approximately 20 μM. mDivi-1 also inhibited granule release induced by the thromboxane receptor agonist U46619, PMA, or Ca2+ ionophore, indicating that Drp-1 acts distally in the secretory pathway. To assess whether Drp-1 functions in platelet fusion pore dynamics, we tested the effect of mDivi-1 on the release of dense granules from rabbit platelets using single-cell amperometry. This technique monitors the release of serotonin from single granules in real-time with sub-millisecond temporal resolution. mDivi-1 exposure (10 μM) retarded each release event, resulting in a prolonged spike width of 23.00 ± 1.702 msec compared to the control value of 14.71 ± 1.194 msec. Although this concentration of mDivi-1 did not change the overall percentage of the fusion pore events or the amount of serotonin released through the fusion pore, it showed a distinct effect on the transition from stable fusion pore to maximal fusion pore dilation (% foot= 17.46 ± 1.809%, 9.464 ± 2.014% for control and mDivi-1 conditions, respectively). Evaluation of fluorescein-dextran incorporation into activated platelets by fluorescence microscopy enabled visualization of fusion pore dynamics and confirmed the effect of mDivi-1 on fusion pore expansion. To assess whether Drp-1 participates in platelet function in vivo, we determined the effect of mDivi-1 on thrombus formation following laser-induced injury of mouse cremaster arterioles. mDivi-1 inhibited platelet accumulation at the site of vascular injury by 74%. In contrast, mDivi-1 had no significant effect on fibrin formation under the same conditions. These results identify Drp-1 in platelets, demonstrate a role for Drp-1 in fusion pore dynamics, and indicate that pharmacological regulation of platelet fusion pore expansion can be used to control thrombus formation in vivo. Disclosures:No relevant conflicts of interest to declare.

(+/-)-Govadine and (+/-)-THP, two tetrahydroprotoberberine alkaloids, as selective alpha 1-adrenoceptor antagonists in vascular smooth muscle cells.

(+/-)-Govadine and (+/-)-THP ((+/-)-2,3,10,11-tetrahydroxytetrahydroprotoberberine HBr) have been shown to inhibit noradrenaline-induced contraction of rat thoracic aortae. The pharmacological activity of the compounds was determined in thoracic aortae and cardiac tissue isolated from the rat and in trachea isolated from the guinea-pig to determine the selectivity of the compounds towards different types of receptor. (+/-)-Govadine and (+/-)-THP were found to be alpha 1-adrenoceptor blocking agents in rat thoracic aorta as revealed by their competitive antagonism of vasoconstriction induced by noradrenaline (pA2 = 6.57 +/- 0.07 and 5.93 +/- 0.06, respectively) or phenylephrine (pA2 = 6.74 +/- 0.08 and 6.06 +/- 0.10, respectively). Removal of endothelium did not affect the antagonistic potencies of (+/-)-govadine (pA2 = 6.83 +/- 0.09) and (+/-)-THP (pA2 = 6.25 +/- 0.06) on phenylephrine-induced vasoconstriction. They were more potent than yohimbine (pA2 = 6.05 +/- 0.05), but less so than phentolamine (pA2 = 7.54 +/- 0.11) and prazosin (pA2 = 9.27 +/- 0.12). (+/-)-Govadine and (+/-)-THP, furthermore, inhibited [3H]inositol monophosphate formation caused by noradrenaline (3 microM) in rat thoracic aorta. (+/-)-Govadine and (+/-)-THP were also alpha 2-adrenoceptor blocking agents with pA2 values 5.50 +/- 0.13 and 5.41 +/- 0.11, respectively. A high concentration of (+/-)-govadine (30 microM) or (+/-)-THP (30 microM) did not, however, affect the contraction induced by the thromboxane receptor agonist U46619, prostaglandin F2 alpha (PGF2 alpha), 5-hydroxytryptamine (5-HT), angiotensin II, endothelin or high K+ in rat aorta denuded of endothelium. Neither the cyclic AMP nor cyclic GMP content of rat thoracic aorta was, furthermore, changed by (+/-)-govadine or (+/-)-THP. Contraction of guinea-pig trachea caused by carbachol, histamine, leukotriene C4 or neurokinin A was not affected by (+/-)-govadine or (+/-)-THP. (+/-)-Govadine or (+/-)-THP also did not block beta 1- or beta 2-adrenoceptor-mediated responses induced by isoprenaline in rat right atria and guinea-pig trachea. It is concluded that (+/-)-govadine and (+/-)-THP are selective alpha 1-adrenoceptor antagonists in vascular smooth muscle.

Abstract 5299: Release of Sphingosine-1-Phosphate From Platelets Requires Thromboxane Synthesis and Thromboxane Receptor Activation

The sphingosine-derived lipid signaling molecule sphingosine-1-phosphate (S1P) is an important mediator of vascular homeostasis. S1P signaling has recently been shown to be a downstream signaling component of the receptors for thrombin, the protease-activated receptors (PARs). The present study investigates the requirement of thromboxane formation for the release of S1P induced by the prototypic thrombin receptor PAR1 from human platelets and the consequences for human monocyte and endothelial cell migration. S1P was detected by thin layer chromatography in [3H]sphingosine-labeled platelets and by mass spectrometry. Release of S1P was stimulated about 5 fold (0.3±0.1 vs. 1.6±0.5 nmol/1×106 platelets) by the selective PAR1-activating-peptide (PAR1-AP, 100 μ M). Acetylsalicylic acid (ASA) or the reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500 mg single dose) attenuated S1P release from platelets in healthy volunteers ex vivo which was paralleled by inhibition of TX formation. S1P release was increased by the thromboxane receptor (TXR) agonist U46619, and inhibited by the TXR antagonist ramatroban. In platelets from TXR-deficient mice, thrombin-induced release of S1P was attenuated and not affected by ASA. Supernatants from PAR1-AP-stimulated platelets increased human umbilical vein endothelial cell (HUVECs) migration and the chemotactic capacity of human peripheral blood monocytes (2- and 3-fold, respectively). These effects were inhibited when platelets were pretreated with ASA. S1P receptor-specific antagonists revealed that the chemotactic responses in HUVECs were mediated by the S1P receptor-1 (S1P1) and by S1P1 and S1P3 in monocytes. These data suggest that S1P release from platelets after thrombin receptor activation requires TX synthesis and TXR activation. This novel pathway likely contributes to the regulation of vascular homeostasis during platelet activation and inflammatory processes. Inhibition of S1P may be involved in the anti-inflammatory actions of ASA in vivo, for example by affecting recruitment of endothelial cells and monocytes to sites of vascular injury.

Glybenclamide Exhibits Thromboxane Receptor‐Dependent Antiplatelet Effects

BackgroundProper platelet function is vital as they are the main cellular agents of hemostasis. Conversely, inappropriate activation of platelets is featured in occlusive disease such myocardial infarction, and stroke. While the thromboxane A2 pathway of platelet activation has been identified as an attractive target for pharmacological intervention, no thromboxane receptor (TPR) antagonists are available for clinical use. Given the structural similarity between the antidiabetic sulfonylurea agent Glybenclamide (Glyb) and the TPR antagonist SQ29548, we hypothesize that Glyb exhibits antiplatelet effects via interaction with TPR.ObjectiveCharacterize the potential antiplatelet capacity of Glyb.MethodsIsolate platelets from healthy donors who denied taking medication in the last 10 days and then investigate the effects of Glyb on platelet aggregation induced by the TPR agonist U46619, the thromboxane A2 precursor arachidonic acid (AA), ADP, as well as the protease‐activated receptor peptide (PAR4) using optical aggregometry.ResultsGlyb was found to dose‐dependently inhibit U46619/AA‐ (i.e., TPR‐) mediated platelet aggregation, but had no detectable effects on that triggered by ADP or PAR4.ConclusionOur findings suggest a therapeutic role for Glyb in thrombotic diseases via blockade of TPRs, and may lay the foundation for the design of therapeutic agents for selective targeting of TPR.

No influence of the VAMP8 rs1010 single nucleotide polymorphism on platelet functions in vitro

No influence of the VAMP8 rs1010 single nucleotide polymorphism on platelet functions <i>in vitro</i>

Allergic lung inflammation induces pulmonary vascular hyperresponsiveness

Pulmonary arterial vasoconstriction is an important early component of pulmonary hypertension. Inflammatory mechanisms play a prominent role in the pathogenesis of pulmonary hypertension. The present authors investigated the potential role of acute allergic lung inflammation for alterations in pulmonary haemodynamics. BALB/c mice were intraperitoneally sensitised to ovalbumin and challenged by ovalbumin inhalation. Subsequently, lungs were ventilated and perfused ex vivo, and pulmonary arterial pressure (P(pa)) was continuously monitored. Isolated perfused lungs of allergen-sensitised and -challenged mice showed five-fold enhanced P(pa) responses to serotonin, which is reported to be a significant contributor to pulmonary hypertension in humans. This increase in P(pa) was abolished by the serotonin receptor-2A antagonist ketanserin, but not the serotonin receptor-1B antagonist GR127935. Intracellular signalling to serotonin involved phosphatidylcholine-specific phospholipase C and protein kinase C, as well as Rho-kinase, as assessed by employing the specific inhibitors D609, bisindolylmaleimide and Y27632, respectively. In addition to serotonin, impressively enhanced P(pa) increases in allergic lungs were also evoked by the thromboxane receptor agonist U46619, angiotensin II and endothelin-1. In conclusion, allergic lung inflammation was accompanied by impressive pulmonary vascular hyperresponsiveness. These results suggest a possible role for allergic inflammation in the development of pulmonary arterial hypertension.

Involvement of peptide histidine isoleucine in non-adrenergic non-cholinergic relaxation of the rat gastric fundus induced by high-frequency neuronal firing.

The role of peptide histidine isoleucine (PHI) as a neurotransmitter of the inhibitory motor neurones, the physiological role of PHI and vasoactive intestinal polypeptide (VIP) in the non-adrenergic non-cholinergic (NANC) relaxation and the relative amounts of VIP- and PHI-like immunoreactivity (LI) co-released by neuronal activation were investigated in the rat gastric fundus. Longitudinal muscle strips from the rat gastric fundus precontracted by the thromboxane receptor agonist U46619 (0.1 micro M) were studied in organ baths under conditions of muscarinic receptor blockade by atropine (1 micro M) and adrenergic neurone blockade by guanethidine (5 micro M) ("NANC conditions"). Concentration-response curves were plotted for both amplitude and area under the curve (AUC) of the relaxant responses induced by VIP (0.3 nM-0.3 micro M), PHI (0.3 nM-1 micro M) and peptide histidine valine [PHV(1-42); 0.3 nM-1 micro M]. All three peptides were more potent in the curve based on amplitude than in that based on the AUC of relaxation. In addition, VIP was 5.3 and 7 times more potent than PHI and PHV(1-42), respectively, in producing relaxation expressed as amplitude, and 2.7 and 2.8 times, respectively, in producing relaxation expressed as AUC. PHI and PHV(1-42) behaved as partial agonists with respect to VIP in producing relaxation expressed as AUC. Electrical field stimulation (EFS; 120 mA, 1 ms, 4-32 Hz, pulse trains of 2 min) evoked frequency-dependent relaxant responses. Alpha-chymotrypsin (1 u/ml or 3 u/ml), an anti-VIP serum (1:100 or 1:50) and an anti-PHI serum (1:25), slightly reduced the amplitude, but greatly inhibited the AUC of the NANC relaxation induced by EFS (13 Hz) [approximately 72%, 47% and 28% less than that seen in time controls or with normal rabbit serum (1:100 or 1:25), respectively]. EFS (8-32 Hz) caused significant, frequency-dependent increases in the outflow of VIP- and PHI-LI from the strips. The EFS-induced release of VIP-LI was approximately 20% of the PHI-LI release. These findings indicate that PHI is involved in EFS-induced NANC relaxation of the rat gastric fundus, the major physiological role of VIP and PHI is the maintenance of smooth muscle relaxation and VIP is co-released in equimolar amounts mainly with PHI.