Thromboxane A2 Analogue U46619 Research Articles

Role of neuronal nitric oxide synthase in regulating retinal blood flow during flicker-induced hyperemia in cats.

To investigate how neuronal nitric oxide synthase (nNOS) contributes to regulation of the retinal circulation during rest and flicker stimulation in cats. Using laser Doppler velocimetry, we measured the vessel diameter and blood velocity simultaneously and calculated the retinal blood flow (RBF) in feline first-order retinal arterioles. After intravitreal injections of Nω-Nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, and Nω-propyl-L-arginine (L-NPA), a selective nNOS inhibitor, we continuously monitored the retinal circulation without any perturbations for 2 hours. We then examined the changes in the RBF in response to 16-Hz flicker stimuli for 3 minutes at 2 hours after intravitreal injection of phosphate-buffered saline (PBS) as a control, L-NAME, L-NPA, and thromboxane A2 (TXA2) analogue U46619 as a basal tone-adjusted control. After intravitreal injection of L-NAME and L-NPA, the baseline RBF decreased gradually in a dose-dependent manner. In the PBS group, the RBF increased gradually and reached a maximal level after 2 to 3 minutes of flicker stimuli. After 3 minutes of 16-Hz flicker stimuli, the RBF increased by 53.5% ± 3.4% compared with baseline. In the L-NAME and L-NPA groups, the increases in RBF during flicker stimulation were attenuated significantly compared with the PBS group. In the TXA2 group, the reduction in the flicker-induced increase in RBF was comparable to that in the PBS group. The current results suggested that increased RBF in response to flicker stimulation may be mediated by nitric oxide (NO) production via nNOS activation.

Pulmonary vascular and airway responses to systemic vasoconstrictors in anesthetized BALB/c mice.

There is no systematic study in which the effects of vasoactive substances were investigated on pulmonary vascular resistance (PVR) in in vivo mouse by directly measuring cardiac output and the inflow and outflow pressures in the pulmonary circulation. We determined the responses of PVR, total peripheral resistance (TPR), and airway pressure (AWP) to angiotensin II, endothelin-1, vasopressin, phenylephrine, and thromboxane A2 analog U46619 in anesthetized BALB/c mice. Pulmonary arterial pressure, left atrial pressure (LAP), and aortic blood flow were measured. TPR increased dose-dependently in response to consecutive administration of all vasoconstrictors except vasopressin which reduced TPR at the highest dose of 100 nmol/kg. At high doses of vasoconstrictors, pulmonary arterial pressure and AWP increased due to increased LAP, as demonstrated by the separate LAP elevation experiments. When LAP transiently increased at high doses, PVR did not increase but decreased. Nonetheless, enodothelin-1, angiotensin II, and U46619 increased PVR. Vasopressin at 100 nmol/kg increased AWP without LAP elevation. In conclusion, the high doses of the vasoconstrictors studied here exert indirectly a transient pulmonary vasodilatory and AWP increasing actions due to pulmonary congestion evoked by strong systemic vasoconstriction. Nevertheless, enodothelin-1, angiotensin II, and U46619 cause pulmonary vasoconstriction, and vasopressin constricts airway in anesthetized BALB/c mice.

Hydrogen Sulfide Plays a Key Role in the Inhibitory Neurotransmission to the Pig Intravesical Ureter

According to previous observations nitric oxide (NO), as well as an unknown nature mediator are involved in the inhibitory neurotransmission to the intravesical ureter. This study investigates the hydrogen sulfide (H2S) role in the neurogenic relaxation of the pig intravesical ureter. We have performed western blot and immunohistochemistry to study the expression of the H2S synthesis enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), measurement of enzymatic production of H2S and myographic studies for isometric force recording. Immunohistochemical assays showed a high CSE expression in the intravesical ureter muscular layer, as well as a strong CSE-immunoreactivity within nerve fibres distributed along smooth muscle bundles. CBS expression, however, was not consistently observed. On ureteral strips precontracted with thromboxane A2 analogue U46619, electrical field stimulation (EFS) and the H2S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked frequency- and concentration-dependent relaxations. CSE inhibition with DL-propargylglycine (PPG) reduced EFS-elicited responses and a combined blockade of both CSE and NO synthase (NOS) with, respectively, PPG and NG-nitro-L-arginine (L-NOARG), greatly reduced such relaxations. Endogenous H2S production rate was reduced by PPG, rescued by addition of GYY4137 and was not changed by L-NOARG. EFS and GYY4137 relaxations were also reduced by capsaicin-sensitive primary afferents (CSPA) desensitization with capsaicin and blockade of ATP-dependent K+ (KATP) channels, transient receptor potential A1 (TRPA1), transient receptor potential vanilloid 1 (TRPV1), vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide (VIP/PACAP) and calcitonin gene-related peptide (CGRP) receptors with glibenclamide, HC030031, AMG9810, PACAP6–38 and CGRP8–37, respectively. These results suggest that H2S, synthesized by CSE, is involved in the inhibitory neurotransmission to the pig intravesical ureter, through an NO-independent pathway, producing smooth muscle relaxation via KATP channel activation. H2S also promotes the release of inhibitory neuropeptides, as PACAP 38 and/or CGRP from CSPA through TRPA1, TRPV1 and related ion channel activation.

Endocannabinoids control platelet activation and limit aggregate formation under flow.

BackgroundThe endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function.ObjectivesOur aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo.Methods and ResultsWe studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa.ConclusionsEndocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function may prove beneficial in the search for new antithrombotic therapies.

MC-002 exhibits positive effects against platelets aggregation and endothelial dysfunction through thromboxane A 2 inhibition

Thromboxane A2 (TXA2) induces platelet aggregation and vasoconstriction, and agents that inhibit TXA2 production or interaction with receptors may exert potential application in stroke therapy. To illustrate the platelet aggregation antagonistic and endothelial protective effect of (E) - 3 - (3 - methoxy - 4 - ((3, 5, 6 - trimethylpyrazin - 2 - yl) methoxy) phenyl) sodium acrylate (MC-002) through TXA2 inhibition and underline mechanisms. Platelets aggregation and thoracic aorta ring contraction of rabbits were induced by U46619. Human umbilical vein endothelial cells (HUVECs) were further applied to explore the protective effect of MC-002 on endothelium when exposed to tumor necrosis factor - α (TNF-α). MTT method was used to assess cell damage, and ELISA analysis was exerted to estimate nitrogen monoxide (NO), endothelin-1 (ET-1), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) releasing. Fluorescence spectrophotometry was conducted to determine intracellular calcium concentration ([Ca(2+)]i), and western blotting method was applied to evaluate the protein expressions of intracellular adhesion molecule-1 (ICAM-1), P-selectin and nuclear factor-kappa B (NF-κB). TXA2 analog U46619 mediated obvious platelet aggregation and vasoconstriction. MC-002 inhibited platelet aggregation through administration in vivo and incubation with platelet in vitro, and relaxed aorta ring in endothelium dependent manner. MC-002 alleviated cell damage, [Ca(2+)]i overload, ET-1 overexcretion and TXB2 activation, but improved NO availability reduction in HUVECs treated with TNF-α. Furthermore, MC-002 downregulated ICAM-1, P-selectin and NF-κB overexpression induced by TNF-α. In conclusion, MC-002 exerted antiplatelet aggregation effect through TXA2 inhibition and relieved inflammatory injury of endothelial cells through NF-κB signal pathway.

Potential for intranasal drug delivery to alter cerebrospinal fluid outflow via the nasal turbinate lymphatics

BackgroundCerebrospinal fluid absorption (CSF) at the cribriform plate is mediated by direct extracranial connections to the lymphatic system. Given the accessibility of these pharmacologically responsive vessels we hypothesized that the rate of CSF outflow can be modulated via the intranasal delivery of drugs known to affect lymphatic contractile activity.FindingsFluid was infused into the lateral ventricle of anesthetized sheep and inflow rate and CSF pressure measured during intranasal administration of pharmacological agents. CSF absorption was calculated at steady-state CSF pressures. The ability of a pharmacological agent to alter CSF absorption was related to the steady-state intracranial pressure (ICP), the concentration and the class of pharmacological agent delivered. An increase in drug concentration correlated with an increase in CSF absorption at high ICP (45 cm H2O, r = 0.42, p = 0.0058). Specifically, the delivery of NG-monomethyl L-arginine (L-NMMA) significantly increased CSF absorption by 2.29 fold over no treatment (2.29 ± 0.34 mL/min), while the thromboxane A2 analogue U46619 resulted in a 2.44 fold increase in CSF absorption over no treatment (2.44 ± 0.55 mL/min). Saline delivery did not significantly increase CSF absorption (0.88 ± 0.097 mL/min). A trend of increased CSF absorption upon noradrenaline delivery was observed: however, this did not reach statistical significance. Increasing drug concentrations inversely correlated with CSF outflow resistance across all drug classes (r = -0.26, p = 0.046).ConclusionsThe administration of nebulized pharmacological agents intranasally has the potential to provide an alternate method to non-invasively modulate CSF absorption and outflow resistance.

P2X1-initiated p38 signalling enhances thromboxane A2-induced platelet secretion and aggregation

ATP released by activated platelets can serve as a positive feedback machinery to amplify platelet responses by activating P2X1 receptors. It has, however, not been defined how P2X1 activities influence thromboxane A2 (TXA2)-stimulated platelet functional responses. Our aim was to elaborate the molecular mechanisms of P2X1 engagements in TXA2-induced platelet secretion and aggregation. P2X1 inhibition by 1 µM NF449 inhibited platelet P-selectin expression induced by a low concentration of the TXA2 analogue U46619 (0.3 µM) (32.0 ± 2.0% vs 43.4 ± 3.0%; n=5; p<0.05). p38 inhibition by SB203580, but not ERK inhibition by U0126, elicited a similar inhibition by NF499. The combination of NF449 and SB203580 provided, however, no additive effects. U46619-induced platelet aggregation was similarly decreased by NF449 and SB203580 alone or in combination, and by P2X1 pre-desensitisation with α,β-Me-ATP. U46619 caused rapid and reversible P2X1-dependent p38 phosphorylation. However, the P2X1-p38 pathway mainly enhanced mild platelet activation by U46619, because α,β-Me-ATP supplementation or p38 blockade had no effect on intense platelet activation induced by a higher concentration of U46619 (3 µM). In conclusion, P2X1 activation, via p38 signalling, potentiates platelet activation initiated by low doses of U46619. Hence, the P2X1-induced p38 signalling promotes more robust platelet activation in response to mild platelet stimuli.

The Role Of Intraplatelet Reactive Oxygen Species In The Regulation Of Platelet Glycoprotein Ibα Ectodomain Shedding

BackgroundGlycoprotein (GP) Ibα contains binding sites for von Willebrand factor (VWF), α-thrombin, P-selectin, and Mac-1 at the extracellular N-terminal 282 residues. Particularly, the interaction of GPIbα with VWF exposed at the injured vessel wall initiates platelet adhesion, and simultaneously triggers intracellular signaling leading to integrin activation and platelet thrombus formation. Therefore, GPIbα ectodomain shedding, which down-regulates the surface expression of the functional receptor and results in the generation of glycocalicin (GC), a soluble N-terminal fragment of GPIbα, has important implications for thrombosis and hemostasis. Stimulations of platelet with either physiological or chemical compounds result in GPIbα ectodomain shedding in vitro and in vivo. The generation of ROS has been observed in platelets stimulated with physiological or chemical compounds, such as collagen, thrombin, and thromboxane A2 analog U46619. In this paper, we plan to clarify the relationship between physiological stimulation-induced ROS generation and GPIbα shedding and the regulatory mechanism of ADAM17-mediated GPIbα shedding. MethodsWashed platelets (3×108/ml) were pre-incubated with or without N-acetylcysteine (NAC) (10 mM), dithiothreitol (DTT) (3 mM), or vehicle control (DMSO) at RT for 15 minutes, and then were incubated with A23187 (5 μM), thrombin (1 U/ml), collagen (5 ug/ml), or dibucaine (1 mM) at 37 °C (or at RT for dibucaine) for different time. Western blot and flow cytometry was used to evaluate the GPIbα ectodomain shedding. ROS levels in platelets were examined using ROS assay kit according to the manufacture's instruction. Results and conclusionsCollagen, thrombin, and calcium ionophore A23187 induced ROS generation, and simultaneously incurred GPIbα ectodomain shedding. ROS scavengers NAC and DTT abolished not only collagen, thrombin, and A23187 induced ROS production, but also GPIbα ectodomain shedding. Interestingly, a recognized calpain activator, dibucaine, induced both ROS production and GPIbα shedding, which were also obviously reduced by NAC and DTT. Furthermore, calpain inhibitors calpain inhibitor I and carbobenzoxy-valinyl-phenylalaninal (MDL), obviously reduced dibucaine-induced ROS production, and inhibited ROS generations induced by A23187 and thrombin. These data indicate that ROS, regulated by calpain, plays a key role in collagen, thrombin, and A23187 induced GPIbα ectodomain shedding. These findings will help to understand the negative-regulatory mechanisms of platelet function, and may suggest a novel strategy to design new class of anti-platelet drug. Disclosures:No relevant conflicts of interest to declare.

Long-lasting physiological antagonism of calcitonin gene-related peptide towards endothelin-1 in rat mesenteric arteries and human coronary arteries

Endothelin-1 causes long-lasting contraction via endothelin type A receptor (ETAR) in isolated rat mesenteric arteries (RMA) that cannot be readily terminated by removing the agonist, or by adding the ETAR antagonist BQ123 or the NO donor sodium nitroprusside. It could be terminated by adding calcitonin-gene related peptide (CGRP), most likely because CGRP causes ET-1/ETAR dissociation. Here we investigated this phenomenon in human coronary microarteries (HCMA). We simultaneously verified the effects of CGRP in RMA and HCMA towards other vasoconstrictors, i.e., the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U46619 (9,11-dideoxy-11α,9α-epoxy-methano-prostaglandin F2α) and KCl. Long-lasting contraction (remaining after washing away the agonist) was observed for ET-1 in RMA, but not HCMA. Constrictions to phenylephrine, U46619 or KCl did not last upon washing. When added on top of ET-1-initiated contraction in RMA, CGRP effectively counteracted vasoconstriction, i.e., it caused full relaxation. Inhibitory effects of CGRP were also observed when briefly exposing RMA and HCMA to CGRP 1h before the addition of ET-1. Similar inhibitory effects of transient CGRP pre-incubation were seen towards phenylephrine, U46619 or KCl in RMA and HCMA. In conclusion, our data imply that CGRP, like ET-1, causes long-lasting effects that remain apparent up to 1h after its removal from the organ bath. Thus, in addition to the reported dissociation of ET-1/ETAR complexes, CGRP causes long-lasting non-selective arterial smooth muscle relaxation that may add to the neuropeptide being a physiological antagonist of arterial effects of ET-1. Long-lasting, washout-resistant ET-1/ETAR interaction does not occur in HCMAs.

Altered expression and activation of mitogen-activated protein kinases in diabetic heart during cardioplegic arrest and cardiopulmonary bypass

We investigated whether mitogen-activated protein kinases (MAPKs) are changed in the hearts of patients with diabetes after cardioplegia and cardiopulmonary bypass (CP/CPB) operations. Biopsies from the right atrial appendage were harvested pre- and post-CP/CPB from nondiabetic (ND) patients (n = 8, hemoglobin A1c (HbA1c) = 5.4 ± 0.12); patients with controlled diabetes (CDM) (n = 8, HbA1c = 6.5 ± 0.15); and patients with uncontrolled diabetes (UDM) (n = 8, HbA1c = 9.6 ± 0.3) undergoing coronary artery bypass grafting. The expression and/or activation of the p38-MAPK, ERK1/2, JNK, and MKP-1 in the right-atrial tissues were analyzed by Western blotting. The vasomotor function of coronary arterioles was measured by videomicroscopy. The post-CP/CPB levels of total p38-MAPK were decreased in the 3 groups as compared with their pre-CP/CPB levels (P < .05). There were increases in phospho-p38-MAPK, phospho-ERK1/2, and MKP-1 in UDM patients as compared with ND and CDM patients at baseline (P < .05). Compared to pre-CP/CPB, the post-CP/CPB levels of phospho-p38-MAPK decreased in the UDM group but were unaltered in the ND and CDM groups; however, the post-CP/CPB levels of phospho-p38-MAPK still remained greater than the post-CP/CPB levels of the other 2 groups. Post-CP/CPB levels of phospho-ERK1/2 were increased in the ND and CDM groups but were decreased in the UDM group compared to their pre-CP/CPB levels, respectively (P < .05). There were no significant differences in phospho-JNK in 3 groups at baseline. Post-CP/CPB levels of phospho-JNK, however, were increased in the 3 groups and were more pronounced in the myocardium of the UDM group (P < .05). After CP/CPB, the protein levels of MKP-1 were unchanged in the 3 groups when compared with their pre-CP/CPB levels. Post-CP/CPB levels of MKP-1, however, remained greater in the UDM group than in the ND and CDM groups. The post-CP/CPB contractile responses to the thromboxane A2 analog U46619 were significantly impaired in all 3 groups compared with pre-CP/CPB contractile responses. These impairments were more pronounced in the UDM group. Uncontrolled diabetes is associated with changes in expression of and activation of MAPKs and vasomotor dysfunction in the setting of CP/CPB.

Signaling pathways involved in the H2O2-induced vasoconstriction of rat coronary arteries

Hydrogen peroxide (H2O2) is an endogenous endothelium-derived hyperpolarizing factor released by flow and involved in the regulation of coronary blood flow. Because opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, the aim of this study was to assess whether H2O2 may act as a coronary vasoconstrictor and if so to determine the underlying signaling mechanisms. Intramyocardial arteries from male Wistar rats were mounted on microvascular myographs for simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and tension. On coronary arteries precontracted with the thromboxane A2 (TxA2) analogue U46619, H2O2 (1–300μM) elicited further moderate contractions in the proximal arterial segments and relaxed the more distal coronary branches, the contractions being markedly augmented in arteries depolarized by raising extracellular K+. H2O2-elicited vasoconstriction on K+30-precontracted coronary arteries was blunted by catalase and significantly reduced by endothelial cell removal and by inhibitors of cyclooxygenase (COX) and of the TxA2 receptor (TP). H2O2 (50μM) increased by about 10-fold basal superoxide anion (O2−) production in coronary arteries measured by lucigenin-enhanced chemiluminescence, and H2O2-elicited contractions were reduced by the superoxide dismutase mimetic tempol and by NADPH oxidase inhibition. Furthermore, blockade of the ERK and p38 mitogen-activated protein (MAP) kinases significantly reduced the contractions elicited by high and low concentrations of peroxide, respectively, whereas Rho kinase inhibition nearly abolished these responses. H2O2 (50μM) elicited simultaneous and similar sustained increases in [Ca2+]i and tension that were blunted by blockade of voltage-dependent L-type channels, but resistant to the nonselective Ca2+ channel blocker 2-aminoethoxydiphenyl borate. Moreover, endothelial cell removal reduced the increases in [Ca2+]i and contraction elicited by peroxide. The present data demonstrate that H2O2 is an endothelium-dependent vasoconstrictor in rat coronary arteries that activates smooth muscle Ca2+ entry through L-type and non-L-type channels and various intracellular signaling pathways including the release of a COX-derived TP agonist, stimulation of the MAP and Rho kinase pathways, and production of NADPH oxidase-derived superoxide.

Pharmacodynamic effects of EV-077: results of an in vitro pilot investigation in healthy volunteers

Antiplatelet agents are important for the treatment of patients with atherosclerotic disease manifestations. Antiplatelet agents act by inhibiting platelet activation and aggregation processes, which are mediated by several activating factors including thromboxane A2 (TxA2), adenosine diphosphate (ADP), and thrombin. In particular, TxA2 induced platelet activation is a pivotal pharmacological target as supported by the undisputable benefit of aspirin in secondary prevention of ischemic events [1, 2]. TxA2 is a metabolite of arachidonic acid (AA) deriving from the cyclooxygenase (COX)-1 enzyme, which transforms AA into prostaglandin H2 (PGH2), which is ultimately metabolized into TxA2 by thromboxane synthase (TS) [3]. Low dose of aspirin mainly inhibits COX-1 and subsequently TxA2 production. However, up to one-third of aspirin treated patients aspirin show persistent TxA2 production, which in turn exposes these subjects to an increased risk of atherothrombotic events [4]. These findings underscore the need to identify new TxA2 modulators. TxA2 receptor (TP receptor) antagonists, TS inhibitors, and dual TS inhibitors and TP receptor antagonists have been recently developed [3, 5]. Such antagonists and inhibitors have also beneficial effects on TP receptor mediated vascular inflammation and oxidative stress, thus on the progression and development of atherosclerotic lesions [6]. EV-077 is a novel dual TS inhibitor and TP receptor antagonist in early phases of clinical investigation. Limited data is currently available on the pharmacodynamic effects of this agent on human platelets [7]. In this pilot investigation we describe the pharmacodynamics effects on human platelets of escalating in vitro concentrations of EV-077. Ten healthy volunteers aged 35.1 ± 3.6 years old were recruited for this study. None of the subjects were taking any type of medication, including over the counter and herbal supplements. Venous blood was collected by antecubital puncture, in hirudin (25 lg/mL) and sodium citrate (0.150 M) containing tubes. Plasma citrate concentration was adjusted according to the haematocrit and the resulting free plasma citrate were adjusted to a final plasma concentration of 20 mmol/L as previously described [8]. Whole-blood hirudin-tubes were incubated with escalating concentrations of EV-077 (vehicle, 1, 10, 30, 100, and 300 nM) for 10 min at 37 8C. EV-077 was provided by Evolva SA (Reinach, Switzerland). Platelet-rich plasma (PRP) was obtained by centrifugation of whole-blood citrated tubes at 1179 g for 10 min. After measuring the platelet count, PRP was adjusted to 250,000/lL ± 10 %. This PRP was incubated with vehicle or 100 nM of EV-077 for 10 min at 37 C. Platelet aggregation was determined by impedance aggregometry using Multiplate Analyzer (MEA) (Dynabyte Medical, Munich, Germany) for whole-blood samples stimulated with AA (1 mM) (Dynabite Medical, Munich, Germany) and the TxA2 analog U-46619 (7 lM) (Calbiochem, Merck KGaA, Darmstadt, Germany), as well as with an agonist indirectly related to the TxA2 pathway, collagen (3 lg/mL) (Dynabite Medical, Munich, Germany). Lighttransmission platelet aggregometry (LTA) was performed using PRP by the turbidimetric method in a two-channel aggregometer (Chrono-Log 490 Model, Chrono-Log Corp., A. Tello-Montoliu F. Rollini B. Desai G. Pasqualino R. Patel D. J. Angiolillo (&) University of Florida College of Medicine-Jacksonville, 655 West 8th Street, Jacksonville, FL 32209, USA e-mail: dominick.angiolillo@jax.ufl.edu

Xenon is not superior to isoflurane on cardiovascular function during experimental acute pulmonary hypertension

Acute right ventricular afterload increase is a known perioperative challenge for the anaesthetic regime especially for patients with a compromised right ventricle. The accused negative inotropic action of volatile anaesthetics, with the exception of xenon, might be crucial for the adaptation of the right ventricle. Reversible pulmonary hypertension (mean pressure 40 mmHg) was induced by an infusion of the stable thromboxane A(2) analog U46619 in a porcine model (n = 35). The effects of 70 vol% xenon and 0.9 vol% isoflurane on biventricular function were studied by conductance catheter technique. Inflammation and myocardial injury was quantified using serum probes [tumour necrosis factor α (TNFα), interleukin 6 (IL-6), troponin] and myocardial tissue [B natriuretic peptide (BNP), TNFα, activated caspase 3] by enzyme-linked immunosorbance assays and reverse-transcription polymerase chain reaction. After wash in of xenon global haemodynamic parameters remained stable whereas isoflurane caused a systemic vasodilation. This led to a significant decrease in mean arterial pressure in the isoflurane group whereas cardiac output remained stable. Both substances did not alter the biventricular contractility nor did they induce changes in preload for both ventricles. Xenon led to an additional increase in right ventricular afterload, whereas isoflurane reduced pulmonary vascular resistance. No effects on systemic inflammatory response and myocardial injury were found, whereas higher apoptosis rate and expression of BNP and IL-6 was determined in the right ventricle. These results do not support the idea that xenon is more beneficial than isoflurane in right ventricular failure during pulmonary hypertension. Isoflurane did not compromise systolic ventricular function during acute PHT it only led to vasodilation in contrast to xenon.

Tetrahydrobiopterin Is Present in High Quantity in Human Milk and Has a Vasorelaxing Effect on Newborn Rat Mesenteric Arteries

Breast milk reduces the incidence of necrotizing enterocolitis (NEC). BH4 is a cofactor for endothelial NOS (eNOS). Reduced BH4 levels, or its oxidation to dihydrobiopterin (BH2), uncouple eNOS resulting in formation of reactive oxygen species (ROS) that have been implicated in the pathogenesis of NEC. We evaluated colostrum and mature breast milk, as well as infant formula, BH4 and BH2 content. In addition, we tested the BH4 effect on the newborn rat mesenteric arterial vascular tone. BH4 and BH2 content increased 3-fold in mature breast milk, when compared with colostrum (p < 0.01), without a change in their ratio. Infant formula had a negligible BH4 content and lower biopterins ratio, when compared with breast milk. eNOS is the predominant synthase isoform in newborn rat mesenteric arteries. In the presence of BH4, mesenteric arteries contracted less to thromboxane A₂ analog U46619 (p < 0.01) and this effect was abolished following eNOS inhibition. BH4 (10⁻⁶ M) vasorelaxed the newborn rat mesenteric arteries. We conclude that when compared with infant formula, breast milk has a high BH4 content that increases as breastfeeding continues. Given its mesenteric arterial vasorelaxing effect, BH4 may play an important role in the reduced NEC incidence among breast fed infants.

Impaired Contractile Response Of Human Peripheral Arterioles To TXA-2 After Cardiopulmonary Bypass

Introduction: Cardiopulmonary bypass (CPB) is often associated with peripheral vasomotor dysfunction. This is thought to lead to decreased vascular tone, hypotension, and an increased need for vasopressor support. We investigated the contractile response to the vasoconstrictor thromboxane A2 (TXA-2), the blockade of TXA-2 receptors and the inhibition of phospholipase-C (PLC) and phospholipase-A2 (PLA-2) in the human peripheral microvasculature pre- and post- CPB. We also examined the effect of CPB on the gene expression of skeletal muscle TXA-2 and TXA-2 synthase. Methods: Chest wall skeletal muscle tissue was harvested pre- and post-CPB from patients undergoing coronary artery bypass grafting. Skeletal muscle arterioles (90-180 micrometer in diameter) were dissected from the harvested tissue and placed in an organ bath containing Krebs solution. Constractile response of the peripheral arterioles to the TXA-2 analog U46619, with and without the highly selective TXA-2 receptor antagonist, SQ 29548, the specific PLC inhibitor U73122 or the selective PLA-2 inhibitor, quinacrine was assessed by videomicroscopy. TXA-2 receptor and TXA-2 synthase gene expression were examined using microarray analysis. Results: The post-CPB contractile response of peripheral arterioles to TXA-2 analog U46619 (10−6M) was significantly impaired compared with pre-CPB (28.5±2% vs. 42.7±3.6 %, P=0.002). The presence of TXA-2 antagonist SQ 29548 (10−6 M) prevented the contractile response to U46619. Pretreatment with the PLC inhibitor U73122 (10−5M) significantly inhibited the U46619-induced contractile response as well. Addition of the PLA-2 inhibitor,quinacrine (10−6M) failed to affect U46619-induced contraction. Microarray analysis showed no significant changes in the gene expression of TXA-2 receptor and TXA-2 synthase. Conclusions: CPB decreases the contractile response of human peripheral arterioles to TXA-2 analog U46619. The contractile response to the TXA-2 analog U46619 is via activation of TXA-2 receptors and PLC, but not of PLA-2. These results provide novel mechanisms for TXA-2-induced contraction and signaling in peripheral vasomotor dysfunction after CPB and cardiac surgery.

Role of arachidonic acid-derived metabolites in the control of pulmonary arterial pressure and hypoxic pulmonary vasoconstriction in rats

BackgroundThe roles of arachidonic acid (AA) metabolites in hypoxia-induced pulmonary vasoconstriction (HPV), a critical physiological mechanism that prevents ventilation/perfusion mismatch, are still incompletely understood. MethodsPulmonary arterial pressure was measured in ventilated/perfused rat lungs. Isometric tones of rat intralobar pulmonary arteries were also measured, using a myograph. ResultsHypoxia (Po2, 3%)-induced pulmonary arterial pressure increases (ΔPAPhypox) were stable with blood-mixed perfusate, but decayed spontaneously. ΔPAPhypox was inhibited by 29%, 16%, and 28% by the thromboxane A2 (TXA2) antagonist SQ-29548, the 5-lipoxygenase inhibitor, MK886, and the leukotriene D4 antagonist, LY-171883, respectively. The prostacyclin synthase inhibitor tranylcypromine augmented ΔPAPhypox by 5%, whereas inhibition of cytochrome P450 did not affect ΔPAPhypox. Consistently, the TXA2 analogue U46619 increased ΔPAPhypox whereas prostacyclin abolished ΔPAPhypox. However, leukotriene D4 had no direct effect on ΔPAPhypox. In the isolated pulmonary arteries, pretreatment with U46619 was essential to demonstrate hypoxia-induced contraction. ConclusionsThe above results suggest that TXA2 and cysteinyl leukotrienes, other than leukotriene D4, are endogenous factors that facilitate HPV in rats. The indispensable role of TXA2-induced pretone in the HPV of isolated pulmonary arteries indicates that the signal from thromboxane receptors might be a critical component of oxygen sensation mechanisms.

Human Platelets Express Functional α7-Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors, especially α7 (nAChRα7), form Ca(2+) channels and are expressed on a variety of neuronal and nonneuronal cells. Also, megakaryocytic cells have been shown to contain components of a nonneuronal cholinergic system, including acetylcholine and acetylcholine esterase. However, the corresponding nAChRs and their role in platelet function have not been demonstrated until now. Our previous platelet transcriptome data indicated the presence of nAChR gene transcripts. Here, we present evidence that human platelets and megakaryocytic precursor cells express nAChRα7 subunits, as revealed by mRNA and protein expression. The subunits form functional Ca(2+) channels, as demonstrated by Ca(2+) entry in platelets induced by the nAChRα7-selective agonist PNU-282987. PNU-282987 also enhanced fibrinogen receptor activation induced by classical platelet agonists (the thromboxane A(2) analog U46619 and ADP). Furthermore, agonist-induced platelet aggregation was significantly inhibited by the nAChRα7-selective antagonists α-bungarotoxin and methyllycaconitine. Ca(2+) influx via nAChRα7 channels represents a novel pathway for human platelets with significant impact on platelet function. Because platelets were suggested to contain acetylcholine, we conclude that on activation, stored acetylcholine is released, which activates nAChRα7 channels and thereby contributes to maintaining intracellular Ca(2+) levels and supporting platelet activation.

Beneficial Effect of Propofol on Arterial Adenosine Triphosphate-sensitive K+Channel Function Impaired by Thromboxane

It is not known whether thromboxane A2 impairs adenosine triphosphate (ATP)-sensitive K channel function via increased production of superoxide in blood vessels and whether propofol as a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor restores this modification. Rat aortas without endothelium were used for isometric force recording, measurements of membrane potential, and superoxide production and Western immunoblotting. Vasorelaxation to an ATP-sensitive K channel opener levcromakalim was obtained during contraction to phenylephrine (3 x 10(-7) M) or a thromboxane A2 analogue U46619 (3 x 10(-7) M). In some experiments, aortas were incubated with an ATP-sensitive K channel antagonist glibenclamide, a superoxide inhibitor Tiron, a nonselective NADPH oxidase inhibitor apocynin, a hydrogen peroxide scavenger catalase, a xanthine oxidase inhibitor allopurinol, a thromboxane receptor antagonist SQ29548 or propofol (3 x 10(-7) to 3 x 10(-6) M). Levcromakalim-induced vasorelaxation was abolished by glibenclamide in rings contracted with either vasoconstrictor agent. Tiron, apocynin, and propofol, but not catalase, augmented the vasodilator response as well as the hyperpolarization by levcromakalim in aortas contracted with U46619. Tiron, apocynin, SQ29548, and propofol, but not allopurinol, similarly reduced in situ levels of superoxide within aortic vascular smooth muscle exposed to U46619. Protein expression of a NADPH oxidase subunit p47phox increased in these arteries, and this augmentation was abolished by propofol. Thromboxane receptor activation induces vascular oxidative stress via NADPH oxidase, resulting in the impairment of ATP-sensitive K channel function. Propofol reduces this stress via inhibition of a NADPH oxidase subunit p47phox and, therefore, restores ATP-sensitive K channel function.

Roles and Mechanisms of Human Immunodeficiency Virus Protease Inhibitor Ritonavir and Other Anti-Human Immunodeficiency Virus Drugs in Endothelial Dysfunction of Porcine Pulmonary Arteries and Human Pulmonary Artery Endothelial Cells

The objective of this study was to determine the effects of highly active antiretroviral therapy (HAART) drugs on pulmonary endothelial function. Porcine pulmonary arteries or human pulmonary arterial endothelial cells (HPAECs) were incubated with eight HAART drugs [ritonavir, indinavir, lopinavir, zidovudine (AZT), abacavir, stavudine, didanosine (ddI), and lamivudine] individually or in combination [three HAART drugs (3-plex; indinavir, stavudine, and ddI)] at their clinical plasma concentrations for 24 hours. Endothelium-dependent vasorelaxation in response to bradykinin was reduced significantly by the ritonavir in a concentration-dependent manner. Five other HAART drugs (indinavir, lamivudine, abacavir, AZT, and ddI) and the 3-plex significantly also impaired endothelium-dependent vasorelaxation in response to bradykinin. Five HAART drugs (ritonavir, indinavir, lamivudine, abacavir, and AZT) significantly decreased endothelial nitric oxide synthase (eNOS) expression and increased superoxide anion levels in both vessels and HPAECs. Furthermore, both ritonavir and AZT substantially activated ERK2 in HPAECs. Additionally, the antioxidants ginsenoside Rb1 and ginkgolide A effectively reversed HAART drug-induced vasomotor dysfunction and eNOS down-regulation. Inhibition of ERK1/2 also partially blocked ritonavir- and AZT-induced down-regulation of eNOS and vasomotor dysfunction. Thus, HAART drugs significantly impair endothelial functions of porcine pulmonary arteries and HPAECs, which may be mediated by eNOS down-regulation, oxidative stress, and ERK1/2 activation. These findings suggest that HAART drugs may contribute to the high incidence of pulmonary artery hypertension in human immunodeficiency virus-infected patients.

Vascular effects of estrone and diethylstilbestrol in porcine coronary arteries

To explore the effects of different estrogens on vascular function, we compared the vasorelaxant effects of 17beta-estradiol, 17alpha-estradiol, estrone, and the synthetic estrogen diethylstilbestrol (DES) on porcine coronary arterial segments. Porcine coronary arterial rings were contracted with the stable thromboxane A2 analogue U46619 (3 x 10(-8) M), and direct relaxation was examined by the addition of increasing concentrations of 17beta-estradiol, 17alpha-estradiol, estrone, or DES (10(-9) to 10(-4) M). Modulation of agonist-induced contraction and relaxation was studied in coronary arterial rings incubated for 20 minutes with DES or estrone (10(-10)-10(-6) M) with 17beta-estradiol (10(-9) M) as comparison. Direct relaxation of arterial rings potentiated by these estrogens was recorded with a rank order potency of DES > 17beta-estradiol > estrone > 17alpha-estradiol. 17beta-Estradiol potentiated relaxation responses to sodium nitroprusside and levcromakalim but not bradykinin or A23187 while reducing contractions to 5-hydroxytryptamine and U46619. DES and estrone, both at 10(-6) M, mimicked the 17beta-estradiol-potentiated sodium nitroprusside and levcromakalim relaxation responses. Additionally, the inhibitory effects of 17beta-estradiol (10(-9) M) on 5-hydroxytryptamine- and U46619-induced contractions were partially reproducible by DES (10(-6) M) and estrone (10(-6) M). Although DES is the most potent among the tested estrogenic compounds in eliciting relaxation, 17beta-estradiol is more effective than estrone and DES at enhancing endothelium-independent relaxation and reducing vascular contraction in porcine coronary arteries.