Thromboxane A2 Mimetic U46619 Research Articles

PERIVASCULAR ADIPOSE TISSUE ALTERS CORONARY ARTERIAL SMOOTH MUSCLE AND ENDOTHELIAL FUNCTION

This study tested the hypothesis that perivascular adipose tissue alters the reactivity of coronary vascular smooth muscle and impairs endothelial function. Experiments were conducted in isolated canine coronary arteries and arterioles with and without perivascular adipose tissue. Isometric tension recordings demonstrated that adding adipose tissue to cleaned arteries elicited contractions (active tension 3.95 ± 0.62 g) that were reversed by inhibition of L-type Ca2+ channels with nicardipine (30 μM). Arteries with intact adipose tissue displayed increased contractions to the thromboxane A2 mimetic U46619 and K+ (~ 25% increase). Adipose tissue attenuated endothelial-dependent vasodilation to bradykinin (1 nM – 30 nM), but not acetylcholine. Inhibition of nitric oxide synthase with L-NAME (300 μM) shifted bradykinin-mediated relaxations to the right and eliminated the effect of adipose tissue. Perivascular adipose tissue attenuated bradykinin-induced NO production assessed with amperometric electrodes. There was no difference in dihydroethidium staining of arteries with and without adipose tissue and tempol (10 μM) did not improve endothelial reactivity. In contrast to conduit arteries, adipose tissue had no effect on the resting diameter or bradykinin-induced vasodilation of pressurized coronary arterioles. In conclusion, perivascular adipose tissue contracts coronary arterial smooth muscle via a voltage-dependent mechanism and selectively impairs endothelial-dependent dilation by diminishing NO production. Support HL67804, HL20605.

Properties of the Human Umbilical Vein as a Living Scaffold for a Tissue-Engineered Vessel Graft

Umbilical cords are usually discarded after delivery, even though they contain a set of functional vessels. We investigated whether the human umbilical vein (HUV) is suitable as a storable scaffold for the tissue engineering of small-caliber vessel grafts. Isolated HUVs were cryopreserved by freezing or vitrification. The reaction of the vessels to vasoactive compounds and the mechanical properties were determined in an organ bath. Mitochondrial metabolism, release of antithrombotic compounds, and platelet adhesion were measured on the luminal vessel surface. Seeding with endothelial cells was tested on denuded HUVs. The vessels showed a weak response to norepinephrine but were readily contracted by serotonin and by the thromboxane A2 mimetic U46619. Endothelium-dependent vasorelaxation was weak, reaching significance only for histamine. However, the vessels relaxed to sodium nitroprusside, and to acetylcholine if sandwiched with human saphenous vein. Cryopreservation did not change the mechanical properties in the relevant tension range. Vasoconstriction to potassium chloride and serotonin were reduced after freezing (22.9+/-7.6%, 27.7+/-10.2%) and after vitrification (2.6+/-5.8%, 4.3+/-7.1%). The mitochondrial metabolism was also attenuated after freezing (57.9+/-25.9%) and after vitrification (21.7+/-6.7%). Prostacyclin release was elevated after both cryopreservation procedures (4.0-fold, 3.9-fold), whereas there was no significant change in the adhesion of platelets. Denuded HUVs could readily be seeded with isolated endothelial cells before and after freezing. We conclude that HUV is suitable as a storable living scaffold with antithrombogenic properties.

Antiplatelet activity of [5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl]guanidine (KR-32570), a novel sodium/hydrogen exchanger-1 and its mechanism of action

The antiplatelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen (10 microg/mL), thrombin (0.05 U/mL), arachidonic acid (100 microM), a thromboxane (TX) A2 mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2, 1 microM) and a Ca2+ ATPase inhibitor thapsigargin (0.5 microM) (IC50 values: 13.8 +/- 1.8, 26.3 +/- 1.2, 8.5 +/- 0.9, 4.3 +/- 1.7 and 49.8 +/- 1.4 microM, respectively). KR-32570 inhibited the collagen-induced liberation of [3H]arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at 50 microM. The TXA2 synthase assay showed that KR-32570 also inhibited the conversion of the substrate PGH2 to TXB2 at all concentrations. Furthermore, KR-32570 significantly inhibited the [Ca2+]i mobilization induced by collagen at 50 microM, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen (10 microg/mL)-induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, TXA2 synthase, the mobilization of cytosolic Ca2+ and NHE-1.

Effect of Hypoxia-Reoxygenation on Endothelial Function in Porcine Cardiac Microveins

The cardiac venous system possesses up to 30% of total coronary vascular resistance and the effect of hypoxia-reoxygenation (H-R) and St Thomas (ST) cardioplegic solution on the vein is unknown. We investigated the effects of H-R, with or without ST, on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in porcine cardiac microveins under clinically relevant temperatures. The microveins (diameter 200 to 450 microM) mounted in a myograph were subjected to hypoxia (Po2 < 5 mm Hg) for 30 minutes in Krebs solution (n = 8) or for 60 minutes in Krebs (n = 8) or in ST at 37 degrees C (n = 8) or 4 degrees C (n = 8), followed by 30-minute reoxygenation. The microvein was precontracted with thromboxane A2 mimetic U46619 (-7 log M) and the EDHF-mediated relaxation was induced by bradykinin (-10 to -6 log M) in the presence of indomethacin, NG-nitro-L-arginine, and oxyhemoglobin before and after H-R. The maximal EDHF-mediated relaxation was significantly reduced after 30-minute hypoxia (38.7 +/- 2.0% vs 61.1 +/- 2.3%, n = 8, p < 0.001) or 60-minute hypoxia in either Krebs or ST at 37 degrees C (Krebs: 27.8 +/- 1.2% vs 56.6 +/- 2.5%, n = 8, p < 0.001; ST: 23.8 +/- 4.1% vs 57.1 +/- 1.5%, n = 8, p < 0.001). The relaxation was significantly less after prolonged H-R in Krebs (p < 0.001). Incubation in Krebs or ST at 4 degrees C also reduced the EDHF-mediated relaxation (Krebs: 25.3 +/- 3.3%, n = 8, p < 0.001; ST: 29.1 +/- 4.4%, n = 8, p < 0.001) and there were no significant differences between Krebs and ST regarding the relaxation at either 37 degrees C or 4 degrees C (p > 0.05). We conclude that (1) H-R impairs EDHF-mediated relaxation in the coronary microveins with more severe injury during prolonged H-R and (2) ST does not provide protection to the EDHF-mediated relaxation impaired by H-R at either 37 degrees C or 4 degrees C.

Thromboxane A2-induced arrhythmias in the anesthetized rabbit

Experiments were conducted in the anesthetized rabbit to investigate mechanisms for arrhythmias that occur after left atrial injection of the thromboxane A(2) (TxA(2)) mimetic U-46619. Arrhythmias were primarily of ventricular origin, dose dependent in frequency, and TxA(2) receptor mediated. The response was receptor specific since arrhythmias were absent after pretreatment with a specific TxA(2) receptor antagonist (SQ-29548) and did not occur in response to another prostaglandin, PGF(2alpha). Alterations in coronary blood flow were unlikely the cause of these arrhythmias because coronary blood flow (as measured with fluorescent microspheres) was unchanged after U-46619, and there were no observable changes in the ECG-ST segment. In addition, arrhythmias did not occur after administration of another vasoconstrictor (phenylephrine). The potential involvement of autonomic cardiac efferent nerves in these arrhythmias was also investigated because TxA(2) has been shown to stimulate peripheral nerves. Pretreatment of animals with the beta-adrenergic receptor antagonist propranolol did not reduce the frequency of these arrhythmias. Pretreatment with atropine or bilateral vagotomy resulted in an increased frequency of arrhythmias, suggesting that parasympathetic nerves may actually inhibit the arrhythmogenic activity of TxA(2). These experiments demonstrate that left atrial injection of U-46619 elicits arrhythmias via a mechanism independent of a significant reduction in coronary blood flow or activation of the autonomic nervous system. It is possible that TxA(2) may have a direct effect on the electrical activity of the heart in vivo, which provides significant implications for cardiac events where TxA(2) is increased, e.g., after myocardial ischemia or administration of cyclooxygenase-2 inhibitors.

A Novel Inherited Platelet Function Disorder Involving the Thromboxane A2 Pathway of Platelet Activation.

Thromboxane A2 (TXA2) is a short-lived metabolite of the fatty acid arachidonate that is formed upon platelet activation; it stimulates platelet responses including shape change, aggregation and secretion of granule contents. The TXA2 pathway of platelet activation is important clinically as demonstrated by the beneficial effect of aspirin, that inhibits TXA2 formation, in preventing arterial thrombotic events. TXA2 elicits its stimulatory responses on platelets by binding to the G protein-coupled thromboxane prostanoid (TP) receptor (approximately 55 kD). Of the α and β isoforms of the TP receptor that have been identified, TPα appears to be the predominant form expressed in platelets, utilizing Gq and G12 in signaling. There have been reports of inherited platelet function disorders involving the TXA2 pathway of platelet activation, and the best characterized is a single amino acid substitution (Arg60 → Leu) in the first cytoplasmic loop of the TPα receptor in a dominantly-inherited bleeding disorder characterized by a defective platelet response to TXA2. Here, we describe a pediatric patient with an inherited platelet defect in the TXA2 pathway that does not appear to involve a mutation in the TPα receptor. The 10 year-old patient and his mother (but not his father) have significant bleeding histories involving recurrent epistaxis (patient) and severe menorrhagia with chronic iron deficiency (mother). Platelet counts of both son and mother are in the normal range. Consistent findings with platelet aggregation testing were prolonged lag phases with collagen, decreased (mother) to minimal (patient) responses with arachidonate and shape change only with the stable TXA2 mimetic U46619. PFA-100 closure times were prolonged with the aspirin-sensitive collagen/epinephrine cartridge: >271 sec (patient), 222 sec (mother) (upper limit of normal: 163 sec for children, 142 sec for adults), but normal with the collagen/ADP cartridge: 97 sec (patient), 85 sec (mother) (upper limit of normal: 111 sec for children and adults). Dense granule counts, determined by whole mount electron microscopy, were slightly lower than normal for both the patient (2.3/platelet) and his mother (2.8/platelet) (normal range: 3–7/platelet). Western blotting utilizing the polyclonal P2 rabbit anti-human TP receptor antibody showed the presence of an approximately 55 kD band in both subjects. Fresh platelets from the patient's mother were available for flow cytometric analysis with the P2 antibody; mean fluorescence intensity of P2 binding for the patient's mother's platelets was 67.6 compared with 60.4 for platelets from a healthy control. Analysis of the TPα receptor gene of the patient indicated a heterozygous synonomous substitution C795T in Ile265 of the sixth transmembrane domain; this substitution was not present in the mother. Taken together, these results suggest a dominantly-inherited platelet function disorder involving the TXA2 pathway of platelet activation. The presence of a normal TPα receptor gene and normal levels of the TPα protein indicate the likelihood of a downstream TXA2-signaling defect.

Newborn lamb coronary artery reactivity is programmed by early gestation dexamethasone before the onset of systemic hypertension

Exposure of the early gestation ovine fetus to exogenous glucocorticoids induces organ-specific alterations in postnatal cardiovascular physiology. To determine whether early gestation corticosteroid exposure alters coronary reactivity before the development of systemic hypertension, dexamethasone (0.28 mg x kg(-1) x day(-1)) was administered to pregnant ewes by intravenous infusion over 48 h beginning at 27 days gestation (term, 145 days). Vascular responsiveness was assessed in endothelium-intact coronary arteries isolated from 1-wk-old steroid-exposed and age-matched control lambs (N = 6). Calcium imaging was performed in fura 2-loaded primary cultures of vascular smooth muscle cells (VSMC) from the harvested coronary arteries. Early gestation steroid exposure did not significantly alter mean arterial blood pressure or coronary reactivity to KCl, thromboxane A(2) mimetic U-46619, or ANG II. Steroid exposure significantly increased coronary artery vasoconstriction to acetylcholine and endothelin-1. Vasodilatation to adenosine, but not nitroprusside or forskolin, was significantly attenuated following early gestation steroid exposure. Endothelin-1 or U-46619 stimulation resulted in a comparable increase in intracellular calcium concentration ([Ca(2+)](i)) in coronary VSMC isolated from either dexamethasone-treated or control animals. However, the ANG II- or KCl-mediated increase in [Ca(2+)](i) in control VSMC was significantly attenuated in VSMC harvested from dexamethasone-treated lambs. Coronary expression of muscle voltage-gated l-type calcium channel alpha-1 subunit protein was not significantly altered by steroid exposure, whereas endothelial nitric oxide synthase expression was attenuated. These findings demonstrate that early gestation glucocorticoid exposure elicits primary alterations in coronary responsiveness before the development of systemic hypertension. Glucocorticoid-induced alterations in coronary physiology may provide a mechanistic link between an adverse intrauterine environment and later cardiovascular disease.

Angiotensin-(1-7) Enhances Anti-Aggregatory Effects of the Nitric Oxide Donor Sodium Nitroprusside

Patients with ischemic heart disease have platelets that are resistant to the anti-aggregatory effects of nitric oxide (NO) donors. This NO resistance is associated with increased whole blood superoxide radical (O2-) content. Angiotensin II (Ang II) has been shown to augment O2- formation. Recent studies have demonstrated that angiotensin-(1-7) [Ang-(1-7)] has opposite actions to those of Ang II in the vasculature. This study compares the effects of Ang-(1-7) and Ang II on platelet aggregation and platelet responsiveness to the NO donor sodium nitroprusside (SNP). Platelet aggregation was induced by the thromboxane A2 mimetic U46619 (1-5 micromol/L), and the inhibitory effects of SNP (10 micromol/L) on the rate and extent of aggregation were quantified. Ang II did not induce aggregation, but 10-100 nmol/L Ang II potentiated U46619-induced aggregation by 21+/-6% in the absence and by 26+/-9% in the presence of SNP (P<0.01 for both), in blood samples from 8 normal subjects. By contrast, Ang-(1-7) alone did not affect platelet aggregation, but 10-100 nmol/L Ang-(1-7) potentiated the anti-aggregatory effects of SNP in blood samples from both normal subjects (n=17) and patients with acute coronary syndromes (n=17). This effect of Ang-(1-7) was bimodal, and at higher concentrations of Ang-(1-7), potentiation was abolished. The maximum incremental effects of Ang-(1-7) on inhibition of aggregation were 25+/-4% and 28+/-5%, for rate and extent of aggregation respectively (P<0.01 for both), corresponding to a 2.3-fold potentiation of the anti-aggregatory effect of SNP. Platelets from patients were resistant to the anti-aggregatory effect of SNP, but potentiation of SNP effects by Ang-(1-7) was similar for patients and normal subjects. Thus, Ang-(1-7) potentiates the anti-aggregatory effects of NO donor, and may therefore counteract platelet NO resistance that accompanies cardiovascular disease.

3 Developmental Contractile Response in the Chicken Embryo Ductus Arteriosus in Vitro

Background and aim: During prenatal life the chicken embryo presents a pattern of circulation similar to mammalians. Despite the abundant information about ontogeny of vascular reactivity in mammalian ductus arteriosus (DA), this has not been characterized in the chicken embryo. The aim of the present work was to study the response to vasoconstrictors of the chicken embryo DA between 0.7 and 0.9 incubation. Methods: Isolated right DA segments from chick embryos at day 15 and day 19 of the 21-d incubation were mounted in a myograph for isometric tension recording, bubbled with 0%, 5% or 95% O2/5% CO2. The contractile responses induced by KCl (31.25 mM-125 mM), the thromboxane A2 mimetic U46619 (10 nM-0.1 microM), endothelin-1 (ET-1, 10 nM- 10 microM), and the adrenergic agonist noradrenaline (NA, 0.3 nM- 10 mM) were tested. Response to oxygen was tested in a separate protocol. Sympathetic neuroeffector mechanisms were studied using electrical field stimulation (EFS, 0.25 16 Hz, 2 ms, 85 mA). Density of sympathetic innervation was evaluated using glyoxylic acid staining. Results: Chicken embryo DA responded to depolarizing high-K+ solution with a tonic contraction that increased with age. Also the contractile responses to NA, U46619 and ET-1 augmented. Depletion of calcium abolished the contractile response to K+ and NA, whereas the response to ET-1 decreased 77% and to U46619 decreased 20%. EFS did not evoke any response and catecholamine-containing perivascular nerves were not observed at any age. No clear response to oxygen in either 15 or 19 days embryos was observed. A contractile response to oxygen was only observed in 21 days externally pipped embryos. Conclusions: The DA of the chicken embryo responded, in vitro, to receptor-dependent and -independent contraction. The responses to contractile agonists augmented with age. The response to oxygen was only present at a very late stage of incubation.

404 Relaxant Effects of the Soluble Guanylate Cyclase Activator and NO Sensitizer YC-1 in Piglet Pulmonary Arteries

Background: The indazole derivative YC-1 has been characterized as an nitric oxide (NO)-independent and heme dependent soluble guanylate cyclase (sGC) activator, which also sensitizes sGC to NO. Objective: To examine the effects of YC-1on vascular relaxation in newborn and 2 week-old piglet pulmonary arteries. The effect of YC-1 on the relaxation induced by exogenous NO was also analyzed. Design/Methods: Isolated rings from third generation pulmonary arteries (outer diameter 500-1000 microm) and small distal pulmonary arteries (outer diameter 150–200 microm) were mounted in organ chambers for isometric tension recording. Arteries were precontracted with the thromboxane A2 mimetic U46619 (0.1 microM) Results: YC-1 induced a concentration-dependent relaxation that was significantly greater in 2 week-old pulmonary arteries and was abolished by the sGC inhibitor ODQ (10 microM). YC-1 induced relaxation was similar in conduit pulmonary arteries and arterioles. In the 2-week-old pulmonary arteries, the response to YC-1 was significantly reduced when the endothelium was removed or after incubation with the NO synthase inhibitor L-NAME (0.1 mM). YC-1 (3 microM) augmented NO-induced relaxation in 2-week-old but not in neonatal pulmonary arteries. Conclusions: YC-1 induced pulmonary vascular relaxation in conduit and resistance pulmonary arteries and these effects increased with postnatal age. In the 2-week-old pulmonary arteries and besides its direct activator of sGC, YC-1 produced endothelium-dependent relaxation and synergized with exogenous NO.

Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2+ entry and Ca2+ sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855, but not Thr-697.

The signal transduction pathway whereby the TxA2 (thromboxane A2) mimetic U-46619 activates vascular smooth muscle contraction was investigated in de-endothelialized rat caudal artery. U-46619-evoked contraction was inhibited by the TP receptor (TxA2 receptor) antagonist SQ-29548, the ROK (Rho-associated kinase) inhibitors Y-27632 and H-1152, the MLCK (myosin light-chain kinase) inhibitors ML-7, ML-9 and wortmannin, the voltagegated Ca2+-channel blocker nicardipine, and removal of extracellular Ca2+; the protein kinase C inhibitor GF109203x had no effect. U-46619 elicited Ca2+ sensitization in a-toxin-permeabilized tissue. U-46619 induced activation of the small GTPase RhoA, consistent with the involvement of ROK. Two downstream targets of ROK were investigated: CPI-17 [protein kinase C-potentiated inhibitory protein for PP1 (protein phosphatase type 1) of 17 kDa], a myosin light-chain phosphatase inhibitor, was not phosphorylated at the functional site (Thr-38); phosphorylation of MYPT1 (myosin-targeting subunit of myosin light-chain phosphatase) was significantly increased at Thr-855, but not Thr-697. U-46619-evoked contraction correlated with phosphorylation of the 20 kDa light chains of myosin. We conclude that: (i) U-46619 induces contraction via activation of the Ca2+/calmodulin/MLCK pathway and of the RhoA/ROK pathway; (ii) Thr-855 of MYPT1 is phosphorylated by ROK at rest and in response to U-46619 stimulation; (iii) Thr-697 of MYPT1 is phosphorylated by a kinase other than ROK under resting conditions, and is not increased in response to U-46619 treatment; and (iv) neither ROK nor protein kinase C phosphorylates CPI-17 in this vascular smooth muscle in response to U-46619.

Cellular mechanisms of thromboxane A2-mediated contraction in pulmonary veins

Our objectives were to identify the relative contributions of [Ca2+]i and myofilament Ca2+ sensitivity in the pulmonary venous smooth muscle (PVSM) contractile response to the thromboxane A2 mimetic U-46619 and to assess the roles of PKC, tyrosine kinases (TK), and Rho-kinase (ROK) in that response. We tested the hypothesis that U-46619-induced contraction in PVSM is mediated by both increases in [Ca2+]i and myofilament Ca2+ sensitivity and that the PKC, TK, and ROK signaling pathways are involved. Isometric tension was measured in isolated endothelium-denuded (E-) canine pulmonary venous (PV) rings. In addition, [Ca2+]i and tension were simultaneously measured in fura-2-loaded E- PVSM strips. U-46619 (0.1 nM-1 microM) caused dose-dependent (P < 0.001) contraction in PV rings. U-46619 contraction was attenuated by inhibitors of L-type voltage-operated Ca2+ channels (nifedipine, P < 0.001), inositol 1,4,5-trisphosphate-mediated Ca2+ release (2-aminoethoxydiphenylborate, P < 0.001), PKC (bisindolylmaleimide I, P < 0.001), TK (tyrphostin A-47, P = 0.014), and ROK (Y-27632, P = 0.008). In PV strips, U-46619 contraction was associated with increases in [Ca2+]i and myofilament Ca2+ sensitivity. Both Ca2+ influx and release mediated the early transient increase in [Ca2+]i, whereas the late sustained increase in [Ca2+]i only involved Ca2+ influx. Inhibition of both PKC and ROK (P = 0.006 and P = 0.002, respectively), but not TK, attenuated the U-46619-induced increase in myofilament Ca2+ sensitivity. These results suggest that U-46619 contraction is mediated by Ca2+ influx, Ca2+ release, and increased myofilament Ca2+ sensitivity. The PKC, TK, and ROK signaling pathways are involved in U-46619 contraction.

Inhibition of endogenous nitric oxide in the heart enhances matrix metalloproteinase‐2 release

Matrix metalloproteinase (MMP) activity is upregulated in pathologies such as atherosclerosis during which endogenous nitric oxide (NO) biosynthesis is reduced. Diminished levels of NO, an antioxidant species, may result in higher oxidative stress. Oxidants are capable of activating MMPs from their zymogen forms. We examined whether basal biosynthesis of NO in the coronary circulation regulates MMP-2 activity. In isolated rat hearts perfused with Krebs-Henseleit buffer at a constant flow of 10 ml min(-1), we measured the release of MMP-2 into the coronary effluent by gelatin zymography. The main gelatinolytic activity of 72-kDa corresponds to MMP-2. Infusion of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) concentration dependently increased coronary perfusion pressure (CPP) (by 48+/-11 mmHg with 100 microM) and enhanced the release of the 72-kDa MMP-2 in the effluent. Coinfusion of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1 microM) with L-NAME abolished both the increase in CPP and the enhanced MMP-2 release. The thromboxane A2 mimetic U46619 increased CPP to the same extent as L-NAME without increasing 72-kDa activity in the effluent, suggesting that MMP-2 release is not caused simply by enhanced perfusion pressure. Infusion of either L-NAME or U46619 did not significantly enhance LDH release. L-NAME infusion concentration dependently increased the level of lipid hydroperoxides in homogenates prepared from the perfused hearts. Coinfusion of SNAP prevented this increase. These data reveal another cytoprotective mechanism of endogenous NO biosynthesis in the heart, the inhibition of MMP-2 release.

Testosterone treatment increases thromboxane function in rat cerebral arteries

We previously showed that testosterone, administered in vivo, increases the tone of cerebral arteries. A possible underlying mechanism is increased vasoconstriction through the thromboxane A2 (TxA2) pathway. Therefore, we investigated the effect of chronic testosterone treatment (4 wk) on TxA2 synthase levels and the contribution of TxA2 to vascular tone in rat middle cerebral arteries (MCAs). Using immunofluorescence and confocal microscopy, we demonstrated that TxA2 synthase is present in MCA segments in both smooth muscle and endothelial layers. Using Western blot analysis, we found that TxA2 synthase protein levels are higher in cerebral vessel homogenates from testosterone-treated orchiectomized (ORX + T) rats compared with orchiectomized (ORX) control animals. Functional consequences of changes in cerebrovascular TxA2 synthase were determined using cannulated, pressurized MCA segments in vitro. Constrictor responses to the TxA2 mimetic U-46619 were not different between the ORX + T and ORX groups. However, dilator responses to either the selective TxA2 synthase inhibitor furegrelate or the TxA2-endoperoxide receptor (TP) antagonist SQ-29548 were greater in the ORX + T compared with ORX group. In endothelium-denuded arteries, the dilation to furegrelate was attenuated in both the ORX and ORX + T groups, and the difference between the groups was abolished. These data suggest that chronic testosterone treatment enhances TxA2-mediated tone in rat cerebral arteries by increasing endothelial TxA2 synthesis without altering the TP receptors mediating constriction. The effect of in vivo testosterone on cerebrovascular TxA2 synthase, observed here after chronic hormone administration, may contribute to the risk of vasospasm and thrombosis related to cerebrovascular disease.

U-46619 but not serotonin increases endocannabinoid content in middle cerebral artery: evidence for functional relevance

Cerebral vascular smooth muscle cells express the CB(1) cannabinoid receptor, and CB(1) receptor agonists produce vasodilation of cerebral arteries. The purpose of this study was to determine whether vasoconstriction of rat middle cerebral artery (MCA) results in the local formation of endocannabinoids (eCBs), which, via activation of CB(1) receptors, oppose the vasoconstriction in a feedback manner. The thromboxane A(2) (TXA(2)) mimetic U-46619 significantly increased N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol (2-AG) content of isolated MCA, whereas 5-hydroxytrypamine (5-HT) decreased AEA and 2-AG content. If eCBs play a feedback role in the regulation of MCA tone, then CB(1) receptor antagonists should enhance the constriction of MCA produced by U-46619 but not 5-HT. U-46619 caused concentration-dependent constrictions of endothelium-denuded MCA. Two CB(1) receptor antagonists SR-141716 and AM-251 decreased the EC(50) value for U-46619 to constrict endothelium-denuded MCA without affecting the maximal effect. A low concentration of CB(1) receptor agonist Win-55212-2 (30 nM) produced vasodilation of MCAs constricted with low but not saturating concentrations of U-46619. SR-141716 had no effect on the 5-HT concentration-contraction relationship. These data suggest that TXA(2) receptor activation increases MCA eCB content, which, via activation of CB(1) receptors, reduces the constriction produced by moderate concentrations of the TXA(2) agonist. Although 5-HT-induced vasoconstriction is reduced by exogenous CB(1) receptor agonist, activation of 5-HT receptors does not increase eCB content. These results suggest that MCA production of eCBs is not regulated by constriction per se but likely via a signaling pathway that is specific for TXA(2) receptors and not 5-HT receptors.

Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humour outflow facility

Cellular contraction and relaxation and integrity of the actin cytoskeleton in trabecular meshwork (TM) tissue have been thought to influence aqueous humour outflow. However, the cellular pathways that regulate these events in TM cells are not well understood. In this study, we investigated physiological agonist-mediated regulation of myosin light chain (MLC) phosphorylation in the TM, and correlated such effects with alterations in aqueous outflow facility, since MLC phosphorylation is a critical biochemical determinant of cellular contraction in TM cells. Treatment of serum starved human TM cells with endothelin-1 (0·1 μ m), thromboxane A2 mimetic U-46619 (1·0 μ m), or angiotensin II (1 μ m), all of which are agonists of G-protein coupled receptors, triggered activation of MLC phosphorylation, as determined by urea/glycerol-based Western blot analysis. Agonist-stimulated increase in MLC phosphorylation was associated with activation of Rho GTPase in TM cells, as determined in pull-down assays. In contrast, treatment of human TM cells with a novel Rho-kinase inhibitor H-1152 (0·1–2 μ m), in the presence of serum reduced basal MLC phosphorylation. H-1152 also increased aqueous outflow facility significantly in a dose-dependent fashion, in perfusion studies with cadaver porcine eyes. This effect of H-1152 on outflow facility was associated with decreased MLC phosphorylation in TM tissue of drug-perfused eyes. Collectively, this study identifies potential physiological regulators of MLC phosphorylation in human TM cells and demonstrates the significance of Rho/Rho-kinase pathway-mediated MLC phosphorylation in modulation of aqueous outflow facility through TM.

Cytotoxicity of the E2-isoprostane 15-E2t-IsoP on oligodendrocyte progenitors

Oxidant stress plays a significant role in the pathogenesis of periventricular leukomalacia (PVL). Isoprostanes (IsoPs) are bioactive products of lipid peroxidation abundantly generated during hypoxic–ischemic injuries. Because loss of oligodendrocytes (OLs) occurs early in PVL, we hypothesized that IsoPs could induce progenitor OL death. 15-E2t-IsoP but not 15-F2t-IsoP elicited a concentration-dependent death of progenitor OLs by oncosis and not by apoptosis, but exerted minimal effects on mature OLs. 15-E2t-IsoP-induced cytotoxicity could not be explained by its conversion into cyclopentenones, because PGA2 was hardly cytotoxic. On the other hand, thromboxane A2 (TxA2) synthase inhibitor CGS12970 and cyclooxygenase inhibitor ibuprofen attenuated 15-E2t-IsoP-induced cytotoxicity. Susceptibility of progenitor OLs was independent of TxA2 receptor (TP) expression, which was far less in progenitor than in mature OLs. However, TxA2 synthase was detected in precursor but not in mature OLs, and TxA2 mimetic U46619 induced hydroperoxides generation and progenitor OL death. The glutathione synthesis enhancer N-acetylcysteine prevented 15-E2t-IsoP-induced progenitor cell death. Depletion of glutathione in mature OLs with buthionine sulfoximine rendered them susceptible to cytotoxicity of 15-E2t-IsoP. These novel data implicate 15-E2t-IsoP as a product of oxidative stress that may contribute in the genesis of PVL.

Chronic in ovo hypoxia decreases pulmonary arterial contractile reactivity and induces biventricular cardiac enlargement in the chicken embryo.

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic (15% O(2)) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 +/- 0.5 vs. 26.6 +/- 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane A(2) mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [pD(2) (= -log EC(50)): 6.51 +/- 0.1 vs. 7.05 +/- 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy.

Cyclopiazonic acid decreases spontaneous transient depolarizations in guinea pig mesenteric lymphatic vessels in endothelium-dependent and -independent manners.

Guinea pig mesenteric lymphatic vessels exhibit vasomotion through a pacemaker mechanism that involves intracellular Ca(2+) release and resultant spontaneous transient depolarizations (STDs) of the smooth muscle membrane potential. This study presents a detailed characterization of the effects of cyclopiazonic acid (CPA) on this pacemaker activity. Microelectrode recordings from smooth muscle in vessel segments revealed that application of CPA (1-10 microM) caused a hyperpolarization accompanied by a decrease in the frequency and amplitude of STDs. The CPA-induced hyperpolarization was abolished after destruction of the endothelium and in the presence of N(G)-nitro-L-arginine (100 microM) or 1H-[1,2,4]oxadiazolol-[4,3-a]quinoxaline-1-one (10 microM), which suggests a contribution of endothelium-derived nitric oxide (EDNO) in this response. In the absence of EDNO-induced effects, CPA decreased the frequency and amplitude of STDs recorded before and in the presence of the thromboxane A(2) mimetic U-46619, norepinephrine, or thimerosal. CPA abolished U-46619-induced vasomotion as determined by measurement of constriction-associated intracellular Ca2+ concentration using the ratiometric Ca2+ indicator fura-2. The endothelial actions of CPA were compared with those of ACh, which is known to cause EDNO release in this preparation. Although CPA and ACh both increased endothelial intracellular Ca2+ concentration and depolarized the membrane potential, the kinetics of action for both parameters were markedly slower for CPA than ACh. These results suggest that CPA first hyperpolarizes the lymphatic smooth muscle and decreases STD frequency and amplitude through endothelial release of EDNO, and second, consistent with the action of CPA to inhibit sarcoplasmic reticulum Ca2+-ATPase and deplete Ca2+ stores, it further reduces STD activity. Inhibition of the lymphatic smooth muscle pacemaker mechanism is thought to abolish agonist-induced vasomotion.

Pharmacologically distinct intracellular calcium pools regulate tonic and oscillatory responses in porcine thoracic duct.

The present study investigated the mechanisms by which the thromboxane A2 mimetic U46619 can elicit phasic and tonic contractions in the pig thoracic duct, whereas other agonists like 5-hydroxytryptamine (5-HT) produce tonic contractions only. Tonic contractions in response to either agonist were abolished by the l-type voltage-operated calcium channel (VOCC) inhibitor nifedipine, the store-operated calcium channel inhibitor SKF 96365, the calcium-sensitive chloride channel (ClCa) inhibitor niflumic acid, and by removal of extracellular Cl-. Superimposed phasic responses to U46619 were abolished by only nifedipine. Inhibitors of K+ channels did not prevent phasic contractions to U46619. The IP3 receptor antagonist 2-APB attenuated tonic contractions only, whereas ryanodine and removal of extracellular Na+ selectively abolished phasic contractions to U46619. Therefore, selective initiation of phasic contractions by U46619 appears to depend on intracellular Ca2+ from a ryanodine-sensitive store that causes depolarization via Na+/Ca2+ exchange, whereas tonic contractions to U46619 and 5-HT are mediated primarily by release of IP3-mobilized intracellular Ca2+ that subsequently causes ClCa opening, membrane depolarization, and Ca2+ entry via l-type VOCC.