Stable Analogue Of Thromboxane A2 Research Articles

Effects of Fatty Acid Metabolites on Adipocytes Britening: Role of Thromboxane A2.

Obesity is a complex disease highly related to diet and lifestyle and is associated with low amount of thermogenic adipocytes. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to fight overweight and associated comorbidities. Recent studies suggest a role for several fatty acids and their metabolites, called lipokines, in the control of thermogenesis. The purpose of this work was to analyze the role of several lipokines in the control of brown/brite adipocyte formation. We used a validated human adipocyte model, human multipotent adipose-derived stem cell model (hMADS). In the absence of rosiglitazone, hMADS cells differentiate into white adipocytes, but convert into brite adipocytes upon rosiglitazone or prostacyclin 2 (PGI2) treatment. Gene expression was quantified using RT-qPCR and protein levels were assessed by Western blotting. We show here that lipokines such as 12,13-diHOME, 12-HEPE, 15dPGJ2 and 15dPGJ3 were not able to induce browning of white hMADS adipocytes. However, both fatty acid esters of hydroxy fatty acids (FAHFAs), 9-PAHPA and 9-PAHSA potentiated brown key marker UCP1 mRNA levels. Interestingly, CTA2, the stable analog of thromboxane A2 (TXA2), but not its inactive metabolite TXB2, inhibited the rosiglitazone and PGI2-induced browning of hMADS adipocytes. These results pinpoint TXA2 as a lipokine inhibiting brown adipocyte formation that is antagonized by PGI2. Our data open new horizons in the development of potential therapies based on the control of thromboxane A2/prostacyclin balance to combat obesity and associated metabolic disorders.

Protein Disulfide Isomerase Negatively Regulates Platelet Function through Modulation of the Thromboxane A2 Receptor

Protein disulfide isomerase (PDI) is an intracellular oxidoreductase that cleaves and forms disulfide bonds in substrate proteins to facilitate their proper folding. However, platelets and endothelium release PDI upon vascular injury. This extracellular PDI serves a critical function in platelet activation and thrombus formation. A PDI antagonist is currently in phase II/III clinical trials for thrombosis prevention. Yet the mechanisms of how PDI enables platelets to respond to the local redox environment remain poorly understood. In particular, the substrates on which PDI acts to modify platelet function have yet to be elucidated.To identify platelet receptors that are controlled by PDI, we evaluated the ability of PDI to modify the effect of different platelet agonists on a platelet aggregation. Recombinant PDI (rPDI) had no effect on platelet aggregation induced by a dose curve of a PAR1 activating peptide, a PAR4 activating peptide, thrombin, collagen, or epinephrine. Unexpectedly, rPDI markedly inhibited aggregation induced by the stable thromboxane A2 analog U46619 (13.8±5.6% of control). In contrast, ERp57, a related thiol isomerase, had little effect and a mutant PDI that lacks the active site cysteines responsible for disulfide bond rearrangement failed to inhibit U46619-induced platelet aggregation, implicating disulfide bond rearrangement. rPDI also blocked U46619-induced dense granule secretion (15.0±5.5% of control), α-granule secretion (24.4±0.1% of control) and α2β3 activation (65.0±0.1% of control). Conversely, inhibition of endogenous PDI augmented U46619-mediated platelet activation. To confirm that PDI was acting upstream in the U46619 activation pathway, we evaluated proximal signaling events. rPDI significantly decreased U46619-induced p38 MAPK phosphorylation in both platelets and HEK cells overexpressing the thromboxane prostanoid (TP) receptor, indicating inhibition of Gα12/13 pathways. In contrast, there was little inhibition of Ca2+ flux, indicating lack of inhibition of Gαq-mediated signaling. These results suggest the presence of a functional disulfide bond within the TP receptor that is sensitive to PDI and required for Gα12/13 signaling.Evaluation of the amino sequence of the TP receptor demonstrates two cysteines, Cys-105 in the extracellular loop I and Cys-183 in the extracellular loop II, that form a disulfide bond and may be modified by redox environment. To determine if the platelet TP receptor is sensitive to modification of redox environment, we evaluated U46619-induced signaling in the presence of GSH and GSSG. Incubation with >0.25 mM GSH reduced U46619-induced platelet aggregation to 11.0±0.6% of control, whereas GSSG did not alter platelet aggregation in response to U46619. These results indicate a functional disulfide bond in the TP receptor that responds to changes in redox environment.Evaluation of additional agonists demonstrated that ADP-induced aggregation was sensitive to rPDI. rPDI did not affect the initial response to 3.5 μM ADP, but completely abrogated the secondary wave of aggregation. This defect in the secondary wave could be overcome by using 5 μM ADP, thus suggesting impaired endogenous ADP release. Indeed, rPDI blocked dense granule release even in response to 5 μM ADP. This result is consistent with the known reliance of dense granule release on TP stimulation and suggests that the negative regulation of PDI on ADP-induced platelet activation is secondary to the inhibition of PDI on the TP receptor.These are the first studies to our knowledge that demonstrate the ability of a thiol isomerase to modulate the function of a G-protein coupled receptor via disulfide bond rearrangement. This mechanism could provide a means to modify signaling through the TP receptor in respond to changes in redox environment. DisclosuresNo relevant conflicts of interest to declare.

Modeling of Acute Pulmonary Arterial Hypertension in Pigs Using a Stable Thromboxane A2 Analogue (U46619): Dose Adjustment and Assessment of Hemodynamic Reactions

U46619, a synthetic analogue of thromboxane A2 was used for modeling acute stable and reversible pulmonary arterial hypertension. Administration of U46619 in high doses led to vascular collapse and inhibition of cardiac function. The doses of U46619 were empirically selected that allow attaining the target level of pulmonary hypertension without systemic hemodynamic disturbances. The possibility of attaining the target level of pulmonary hypertension and reversibility of changes after termination of U46619 infusion make this model attractive for evaluation of the efficiency of different therapeutic methods of treatment of pulmonary hypertension in large animals.

Pulmonary arterial hypertension modeling with synthetic stable thromboxane A2 analogue (U46619) in porcine: dose adjustment and acute hemodynamic reactions

Abstract Introduction Survival in PAH is strongly associated with adaptation of the RV to pressure overload. Purpose To investigate acute hemodynamic reactions during infusion of the stable thromboxane A2 (TXA2) analogue U46619 in a porcine model. Materials and methods The study comprised 9 male Landrace pigs (32.7±3.8 kg) under GA. After heparinization, PAH was induced by continuous and stepwise increasing infusion of U46619 (10 mg/ml; Tocris, USA) according to a pre-specified protocol. The target mPAP was 40 mmHg. Hemodynamics was assessed, cardiac output (CO) was calculated using the Fick equation before and during U46619 infusion. Results U46619 infusion at a previously published rate 0.1, 0.2 μg kg–1 min–1 in pig 1 resulted in rapid hemodynamic deterioration. Two-time reduced U46619 dose infusione resulted in a target mPAP but severe mBP drop in 1, 3, 4 pigs and required cathecholamine support. The U46619 dosage was reduced up to ¼ (0.025, 0.05, 0.075, 0.1, 0.12, 0.15, 0.175 μg kg–1 min–1). In animals on 1/4 dosage U46619 infusion, heart rate (HR), mBP, mPAP, RAP and PVR reliably increased and BP was stable (Table 1). There was a positive correlation between the mPAP and heart rate (HR) on U46619 infusion (r=0.66; t=2.38; p=0.048) and between CO and mBP (r=0.66; t=2.36; p=0.04). Low CO was associated with high PVR (r=−0.98; t=−14.3; p<0,001). Positive correlation between PVR and SVR was revealed (r=0.96; p=0.00002). In 3 pigs with severe mBP drop and PAH right ventricle subendocardial hemorrhage was revealed on autopsy study. Conclusions High dose U46619 infusion was associated with acute RV decompensation due to pressure overload accompanied with systemic BP drop and low CO. Lower dosages of U46619 infusion were characterized by stable target mean PAP. HR acceleration and mean systemic BP are of compensation mechanisms for cardiac output maintenance in PAH. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): RFBR grant 18-315-20050

Biochemical characterization of AeD7L2 and its physiological relevance in blood feeding in the dengue mosquito vector, Aedes aegypti.

Aedes aegypti saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis. Among these salivary proteins are the D7s, which are highly abundant and have been shown to act as scavengers of biogenic amines and eicosanoids. In this work, we performed comparative structural modeling, characterized the binding capabilities, and assessed the physiological functions of the Ae. aegypti salivary protein AeD7L2 compared to the well-characterized AeD7L1. AeD7L1 and AeD7L2 show different binding affinities to several biogenic amines and biolipids involved in host hemostasis. Interestingly, AeD7L2 tightly binds U-46619, the stable analog of thromboxane A2 (KD =69.4nm), which is an important platelet aggregation mediator, while AeD7L1 shows no binding. We tested the ability of these proteins to interfere with the three branches of hemostasis: vasoconstriction, platelet aggregation, and blood coagulation. Pressure myography experiments showed these two proteins reversed isolated resistance artery vasoconstriction induced by either norepinephrine or U-46619. These proteins also inhibited platelet aggregation induced by low doses of collagen or U-46619. However, D7 long proteins did not affect blood coagulation. The different ligand specificity and affinities of AeD7L1 and AeD7L2 matched our experimental observations from studying their effects on vasoconstriction and platelet aggregation, which confirm their role in preventing host hemostasis. This work highlights the complex yet highly specific biological activities of mosquito salivary proteins and serves as another example of the sophisticated biology underlying arthropod blood feeding.

Моделирование острой лёгочной артериальной гипертензии у свиней с помощью синтетического аналога тромбоксана А2 (U46619): подбор дозовых значений и оценка гемодинамических реакций

Моделирование острой лёгочной артериальной гипертензии у свиней с помощью синтетического аналога тромбоксана А2 (U46619): подбор дозовых значений и оценка гемодинамических реакций

Adaptation to acute pulmonary hypertension in pigs.

The extent of right ventricular compensation compared to the left ventricle is restricted and varies among individuals, which makes it difficult to define. While establishing a model of acute pulmonary hypertension in pigs we observed two different kinds of compensation in our animals. Looking deeper into the hemodynamic data we tried to delineate why some animals could compensate and others could not. Pulmonary hypertension (mean pressure 45 mmHg) was induced gradually by infusion of a stable thromboxane A2 analogue U46619 in a porcine model (n = 22). Hemodynamic data (pressure‐volume loops, strain‐analysis of echocardiographic data and coronary flow measurements) were evaluated retrospectively for the short‐term right ventricular compensatory mechanisms and limits (Roehl et al. [2012] Acta Anaesthesiol. Scand., 56:449–58) 10 animals showed stable arterial blood pressures, whereas 12 pigs exhibited a significant drop of 16.4 ± 9.9 mmHg. Cardiac output and heart rate were comparable in both groups. In contrast, right ventricular contractility and coronary flow only rose in the stable group. The unchanging values in the decrease group correlated with an increasing ST‐segment depression and a loss of ventricular synchronism and resulted in a larger septum bulging to the right ventricle. Simultaneously, a reduced left‐ventricular end‐diastolic volume and a missing improvement in contractility in the posterior septal and inferior free wall of the left ventricle have been observed. Our findings suggest that right ventricular compensation during acute pulmonary hypertension is strongly dependent on the individual capability to increase coronary flow. The cause for inter‐individual variability could be the dimension and reactivity of the coronary system.

Relaxation induced by the nitric oxide donor and cyclooxygenase inhibitor NCX2121 in renal hypertensive rat aortas

There is an imbalance between contractile and relaxing endothelial factors in hypertension which induces vascular dysfunction. It involves cyclooxygenase (COX) due to production of vasoconstrictor agonists such as thromboxane A2 (TXA2). In this work we used the NO-NSAID vasodilator compound NCX2121 that has nitric oxide (NO) in its structure and the non-selective COX inhibitor indomethacin. This class of pro-drug has been demonstrated hypotensive effect in animal models of hypertension without alteration in the arterial pressure of normotensive rats. It has been pointed a direct vasodilator effect of NO-NSAIDS which mechanism remains unclear. This study aimed to evaluate the cellular mechanisms involved in the NCX2121-induced relaxation in renal hypertensive two kidney-one clip (2K-1C) rat aorta with intact endothelium and denuded aortas. Our data demonstrated that NO was directly released from the compound NCX2121 in the cytosol of the vascular smooth muscle cells. The relaxation induced by NCX2121 was similar in 2K-1C and normotensive two kidney (2K). It was impaired by the endothelium removal and NO-Synthase (NOS) inhibition in both rat aorta groups. The inhibition of the main NO target, soluble guanylyl-cyclase (sGC) with ODQ, abolished NCX2121-induced relaxation in denuded aorta. The expression of COX-1 and COX-2 as well the produced stable analog of Thromboxane A2 (TXA2) were higher in 2K-1C than in 2K. NCX2121 decreased the augmented production of TXA2 in 2K-1C rat aortas. In intact-endothelium aorta, ODQ impaired the relaxation induced by NCX2121 in 2K but it had no effect in 2K-1C rat aorta. Taken together, our results show that NCX2121-induced relaxation is due to the direct NO release inside the vascular smooth muscle cells and sGC activation in denuded aortas, whereas in intact endothelium aortas, NCX2121-induced relaxation could be due to eNOS activation and inhibition of prostanoids production. In addition, relaxation-induced by NCX2121 is not greater in normotensive than in hypertensive rat aortas because COX activity is higher in 2K-1C than in 2K rat aortas.

The relaxing effect of Poncirus fructus and its flavonoid content on porcine coronary artery.

Coronary artery disease is a common occurrence in human, and causes enormous social cost. Poncirus fructus (PF), the dried immature fruits of Poncirus trifoliata Rafinesquem, is used in the treatment of womb contraction and dyspepsia, as a prokinetic, and in improving blood circulation. This study was performed to investigate the effects of PF and some of its flavonoids components on the coronary from the pig. The arterial ring was suspended by a pair of stainless steel stirrups in an organ bath. The end of the upper stirrup was connected to an isometric force transducer. A dose-dependent induction of relaxation was observed by both water and 70% ethanol extracts of PF in the porcine coronary artery precontracted with U46619 (100 nM), a stable analogue of the potent vasoconstrictor thromboxane A2. The 70% ethanol extract showed more efficacy than the water extract. Pretreatment of the artery with L-NAME (100 µM), a nitric oxide synthase inhibitor, resulted in a significant reduction in the relaxation induced by PF extract. In addition, ODQ (10 µM), a soluble guanylate cyclase inhibitor, also significantly reduced the effects of PF extracts. Hesperidin, a flavonoid present in PF, induced very weak relaxation of the porcine coronary artery at a high concentration (100 µM), while its aglycone, hesperetin, demonstrated a dose-dependent relaxation. In conclusion, PF extracts induced relaxation in the porcine coronary artery, partially through the nitric oxide-cGMP pathway, and the aglycones of flavonoids might be also involved in the relaxation of the same artery.

Up-regulation of Kv7.1 channels in thromboxane A2-induced colonic cancer cell proliferation

Thromboxane A2 (TXA2) is known to stimulate colonic cancer cell proliferation, although the mechanism has not been clarified. In this study, we compared the expression levels of Kv7.1K(+) channels between human colorectal cancer tissue and the accompanying non-tumor mucosa. Kv7.1 proteins were found to be consistently up-regulated in the cancer tissues from different patients. Kv7.1 was also expressed in human colonic cancer cell lines. Treatment of colonic cancer cells with 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of TXA2, significantly increased whole-cell K(+) currents sensitive to chromanol 293B, an inhibitor of Kv7.1 channels, in parallel with an increased expression of Kv7.1 proteins. In contrast, TXB2, an inactive metabolite of TXA2, had no effects on expression level and function of Kv7.1. A TXA2 receptor antagonist (SQ29548) and an inhibitor of cAMP-dependent protein kinase (Rp-8-Br-MB-cAMPS) inhibited STA2-induced increases in both Kv7.1 expression and chromanol 293B-sensitive K(+) currents. Interestingly, STA2-stimulated proliferation of colonic cancer cells was inhibited by chromanol 293B. These results suggest that Kv7.1 channels are involved in the TXA2-induced cancer cell proliferation and that they are up-regulated by the TXA2 receptor-mediated cAMP pathway.

PP041. The underlying mechanisms in reduced action of endothelial nitric oxide in resistance artery in preeclampsia

In preeclampsia (PE), the action of endothelial nitric oxide (eNO) is reduced by in vitro investigation of characteristic changes using vascular strips of resistance artery. We studied underlying mechanisms in the reduced action of it. The vascular strips of omental resistance arteries were obtained from PE and normotensive pregnant women. Informed consent was obtained from each patient. This study was permitted by ethical board. (1) The various isometric tension by NO were measured by bradykinin (BK), sodium nitroprusside (SNP) and the inhibition of 8-pCPT-cGMPVert@during the STA2 (a stable analogue of thromboxane A2)-induced contraction. (2) The [Ca(2+)]i were measured in the endothelium-intact strip loaded cell-permeable form of Fura 2 (Fura 2-AM). (3) The concentrations of cGMP and eNO synthase (eNOS) were measured in endothelial cell intact strips. (1) In PE, endothelium-dependant (by BK) and -independent (by SNP) relaxation of NO in the STA2-induced contractions were significantly smaller, while the action of c GMP was also reduced. (2) BK increased the concentration of [Ca(2+)]i, although the increase was not reduced in PE. (3) The concentrations of cGMP with BK and without BK and eNOS were not reduced in PE. These results revealed that the action of endothelial NO might be reduced not due to decrease in the production of NO in the endothelium but rather to reduced action of cGMP itself.

Optimization of Isolated Perfused/Ventilated Mouse Lung to Study Hypoxic Pulmonary Vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) is a compensatory physiological mechanism in the lung that optimizes the matching of ventilation to perfusion and thereby maximizes gas exchange. Historically, HPV has been primarily studied in isolated perfused/ventilated lungs; however, the results of these studies have varied greatly due to different experimental conditions and species. Therefore, in the present study, we utilized the mouse isolated perfused/ventilated lung model for investigation of the role of extracellular Ca2+ and caveolin-1 and endothelial nitric oxide synthase expression on HPV. We also compared HPV using different perfusate solutions: Physiological salt solution (PSS) with albumin, Ficoll, rat blood, fetal bovine serum (FBS), or Dulbecco's Modified Eagle Medium (DMEM). After stabilization of the pulmonary arterial pressure (PAP), hypoxic (1% O2) and normoxic (21% O2) gases were applied via a ventilator in five-minute intervals to measure HPV. The addition of albumin or Ficoll with PSS did not induce persistent and strong HPV with or without a pretone agent. DMEM with the inclusion of FBS in the perfusate induced strong HPV in the first hypoxic challenge, but the HPV was neither persistent nor repetitive. PSS with rat blood only induced a small increase in HPV amplitude. Persistent and repetitive HPV occurred with PSS with 20% FBS as perfusate. HPV was significantly decreased by the removal of extracellular Ca2+ along with addition of 1 mM EGTA to chelate residual Ca2+ and voltage-dependent Ca2+ channel blocker (nifedipine 1 μM). PAP was also reactive to contractile stimulation by high K+ depolarization and U46619 (a stable analogue of thromboxane A2). In summary, optimal conditions for measuring HPV were established in the isolated perfused/ventilated mouse lung. Using this method, we further confirmed that HPV is dependent on Ca2+ influx.

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.

Aging enhances contraction to thromboxane A2 in aorta from female senescence-accelerated mice

The time-course for aging-associated effects on vascular reactivity to U46619, a stable analogue of thromboxane A(2) (TXA(2)), was studied in aorta from female senescence-accelerated mice-prone (SAMP8), a murine model of accelerated senescence. SAMP8 and senescence-accelerated mice-resistant (SAMR1) were divided into three groups: 3-, 6- and 10-month-old. Contractile curves to U46619 (10(-9) to 10(-6)M) were performed in aortic rings in the absence or in the presence of nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 10(-4)M) and/or cyclooxygenase (COX) inhibitor indomethacin (10(-5)M). Protein and gene expression for COX-1 and COX-2 were determined by immunofluorescence and real-time PCR, respectively. Maximal contraction to U46619 was markedly higher in SAMP8 at all ages. In SAMR1, increases were seen at 10months, while SAMP8 displays augmented contraction at 6months, which was further increased at 10months. L-NAME enhanced U46619 contractions in both 6-month-old groups, although the increase was higher on vessels from SAMR1 at this age. Indomethacin equally increased U46619 contractions in both 3-month-old groups, suggesting the production of vasodilator prostaglandin in young animals. In contrast, at 6 and 10months indomethacin decreased U46619 contractions in both groups, indicating an aging-associated swap to a release of contractile prostanoids in aorta. In conclusion, aging enhances contractile responses to TXA(2) in aorta from female mice by a mechanism involving a decrease of NO production and increased action of contractile prostanoids. This process occurs earlier in SAMP8 mice, establishing these mice as good model to study cardiovascular aging in a convenient and standard time-course.

Red Ginseng Saponin Fraction A Isolated from Korean Red Ginseng by Ultrafiltration on the Porcine Coronary Artery

Red ginseng saponin fraction-A (RGSF-A) contains a high percentage of panaxadiol saponins that were isolated from Korean red ginseng by ultrafiltration. The aim of this study was to elucidate the effects of RGSF-A on the porcine distal left anterior descending (LAD) coronary artery. The relaxant responses to RGSF-A were examined during contractions induced by 100 nM U46619 (9,11-dideoxy-9a,11a-methanoepoxy-prostaglandin F2a), a stable analogue of thromboxane A2. RGSF-A dose-dependently induced biphasic (fast- and slow-) relaxation in the distal LAD coronary artery in the presence of an intact endothelium. The fast-relaxation was quickly achieved in a minute, and then the slow-relaxation was slowly developed and sustained for more than thirty minutes after the administration of RGSF-A. The slow-relaxation had a tendency to be bigger than the fast-relaxation. Fast relaxation induced by RGSF-A was almost blocked by Nω-Nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor. However slow-relaxation induced by RGSF-A was only partially inhibited by L-NAME and ODQ. In the endothelium-removed ring, RGSF-A evoked only slow-relaxation to a certain extent. These data suggest that RGSF-A induced both endothelium dependent fast- and slow-relaxation and endothelium independent slow-relaxation in the porcine distal LAD coronary artery. The endothelium dependent fast-relaxation is mediated by the nitric oxide (NO)-cGMP pathway, and the endothelium dependent slow-relaxation is at least partially mediated by the NO-cGMP pathway. However, the endothelium-independent slow-relaxation remains to be elucidated.

The stable prostacyclin-analogue, iloprost, unlike prostanoids and leukotrienes, potently stimulates cyclic adenosine monophosphate synthesis of primary astroglial cell cultures

The effect of different eicosanoids on adenosine-3', 5'-cyclic-monophosphate (cAMP) accumulation in primary astroglial cell cultures prepared from newborn rat brain was studied. The stable prostacyclin-analogue, iloprost, effectively stimulated cAMP synthesis in a concentration-dependent, saturable manner, the EC50 being about 3 x 10(-8) M. Prostaglandin (PG) E2 was less potent, without reaching plateau even at 10(-5) M. Prostaglandins D2 and F2 alpha, and the stable thromboxane A2-analogue, U 46619, as well as leukotrienes (LT) B4, C4, D4 and E4 were not effective and did not attenuate basal or isoprenaline (10(-8) M)-stimulated astroglial cAMP formation. This is the first indication for the existence of a prostacyclin receptor coupled positively to the adenylate cyclase in astrocytes. Other eicosanoids are unlikely to be involved in receptor-mediated regulation of astroglial cAMP levels.

SENESCENCE AND OVARIECTOMY INCREASE CONTRACTIONS TO THROMBOXANE A2 IN FEMALE MOUSE AORTA: 1D.06

Objective: The present study investigates the effects of ovariectomy on vascular reactivity to U-46619, a stable analog of thromboxane A2 (TXA2) of aorta from senescence-accelerated mice (SAM-P8), a murine model of senescence, and from senescence resistant mice (SAM-R1). Design and Methods: Five month old female SAM-P8 (n = 16) or SAM-R1 (n = 16) were used. Mice were divided in two groups: sham-operated and ovariectomized. Twenty eight days after surgery, mice were sacrificed and vascular rings (4 mm long) from thoracic aorta were mounted for isometric recording of tension in organ bath and concentration response curves for U-46619 (10-9 to 10-6 M) were performed. A segment of thoracic aorta was immediately frozen for protein expression studies. Results: In sham-operated group, TXA2 receptor expression and maximal contraction to U46619 of SAM-P8 aortas were markedly higher than in SAM-R1, suggesting than senescence increases contractile response. Ovariectomy increased contraction to U-46619 in SAM-P8. L-NAME (10-4 M), an inhibitor of nitric oxide (NO) synthase, increased the contractions to U-46619 in SAM-R1 and in SAM-P8 aortas from sham-operated groups but did not change in ovariectomized groups, indicating that estrogens modulate the response to TXA2 through NO pathway. The presence of cyclooxygenase inhibitor indomethacin (10-5 M) decreased the sensitivity to U46619 in aortas from two ovariectomized groups and from sham-operated SAM-P8 group. These results point out that ovariectomy and senescence induce the production of contractile prostaglandins in response to TXA2. Conclusions: The results reveal that senescence enhances expression of TXA2 receptor and contraction to U-46619. Both senescence and ovariectomy change the response to TXA2 by decreasing NO activity and increasing contractile prostaglandins release. These effects were markedly increased in ovariectomized mice with accelerated senescence.

Maxik Channel Beta1 Subunit Interacts and Regulates Thromboxane A2 Receptor Function

MaxiK channel, composed of the pore-forming α (MaxiKα) and regulatory β1 subunits, controls vascular tone via the activation or inhibition of its pore conducting activity. Thromboxane A2 receptor (TPR), a G-protein coupled receptor, induces potent vasoconstriction mediated by its agonist, thromboxane A2. Previously, we demonstrated that U46619, the stable analogue of thromboxane A2 inhibits MaxiK channel in vascular smooth muscle cell contributing to U46619-induced vasoconstriction. In this study, we report that this inhibition is reversed by nanomolar dehydrosoyasaponin I (DHS-I), a pharmacological tool that indicates MaxiKα and β1 association and functional coupling. The reversing effect of DHS-I indicated that the MaxiK channels inhibited by U46619 could be coupled with regulatory β1 subunits. Co-immunoprecipitation and double immunolabeling in co-expressing cells showed that TPR form a complex with β1 on the plasma membrane. To identify the interacting sites in β1 responsible for TPR-β1 complex formation, we prepared serial carboxyl-terminal deletions of β1 and analyzed their interaction properties with TPR in co-immunoprecipitation experiments. β1 lacking amino acids 103-191 reduces the TPR-β1 association by 44 ± 14% (p<0.01), while deletion of residues 73-191 completely reduces the TPR-β1 interaction, suggesting that amino acids 73 to 191 predominantly contribute to the TPR-β1 interaction. To further investigate how β1 regulates TPR-MaxiKα functional coupling, inside-out patch clamp experiments were performed in HEK293T cells expressing TPR + MaxiKα +/- β1 subunit. We found that the β1 subunit reduces U46619-induced MaxiKα inhibition in a dose-dependent manner. In summary, β1 interacts with TPR forming a tripartite complex with MaxiKα and opposes to TPR agonist-induced MaxiK channel inhibition serving as a buffer to vasoconstriction. Thus, in pathological situations like hypertension or aging where β1 expression is compromised the TPR-MaxiK complex would induce severe vasoconstriction. Supported by NIH and AHA.

Arachidonic acid metabolites follow the preferential course of cyclooxygenase pathway for the basal tone in the internal anal sphincter

Present studies determined the roles of the cyclooxygenase (COX) versus the lipoxygenase (LO) pathways in the metabolic pathway of arachidonic acid (AA) in the internal anal sphincter (IAS) tone. Studies were performed in the rat IAS versus the nontonic rectal smooth muscle (RSM). Indomethacin, the dual COX inhibitor, but not nordihydroguaiaretic acid (NDGA), the LO inhibitor, produced a precipitous decrease in the IAS tone. However, when added in the background of indomethacin, NDGA caused significant reversal of the IAS tone. These inhibitors had no significant effect on the RSM. To follow the significance of COX versus LO pathways, we examined the effects of AA and its metabolites. In the IAS, AA caused an increase in the IAS tone (Emax=38.8+/-3.0% and pEC50=3.4+/-0.1). In the RSM, AA was significantly less efficacious and potent (Emax=11.3+/-3.5% and pEC50=2.2+/-0.3). The AA metabolites (via COXs) PGF2alpha and U-46619 (a stable analog of thromboxane A2) produced increases in the IAS tone and force in the RSM. Conversely, AA metabolites (via 5-LO) lipoxin A4, 5-HETE, and leukotriene C4 decreased the IAS tone. Finally, the contractile effects of AA in the IAS were selectively attenuated by the COX-1 but not the COX-2 inhibitor. Collectively, the specific effects of AA and the COX inhibitor, the Western blot and RT-PCR analyses showing specifically higher levels of COX-1, suggest a preferential role of the COX (specifically COX-1) pathway versus the LO in the regulation of the IAS tone.

Ca2+- and Thromboxane-Dependent Distribution of Functional MaxiK Channels in Cultured Astrocytes

Large-conductance, voltage- and Ca2+-activated K+ channels (MaxiK, BK) are broadly expressed ion channels typically observed as a plasma membrane protein in various cell types. In murine astrocyte primary cultures, which are more indicative of in-vivo reactive astrocytes rather than resting astrocytes, our previous results using high-resolution confocal microscopy have revealed the novel finding that MaxiK pore-forming α subunit (MaxiKα) is distributed intracellularly, colocalized along the microtubule network. This MaxiKα association with microtubules was further confirmed by in vitro His-tag pulldown assays, co-immunoprecipitation assays from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine (20mM) or thapsigargin (1μM), resulted in increased MaxiKα labeling at the plasma membrane. More notably U46619, a stable analog of thromboxane A2 (TXA2) which triggers Ca2+-release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiKα surface labeling. We now show using whole-cell patch clamp recordings that U46619 stimulated cells develop a ∼3-fold increase in current amplitude. This data indicates that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. These changes in MaxiKα plasma membrane distribution are effectively blocked by preincubating astrocytes with a cell permeable Ca2+-chelator, BAPTA-AM, or by microtubule disruption prior to stimulation. While microtubules are largely absent in mature astrocytes, our immunohistochemistry results in brain slices show that cortical astrocytes in the developing newborn mouse brain (P1) have a robust expression of microtubules that significantly colocalize with MaxiKα. The results of this study provide the novel insight that suggests Ca2+ released from intracellular stores, may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiKα stores to the plasma membrane of reactive astrocytes. Supported by NIH.