Journal of Pharmacology and Experimental Therapeutics CARDIOVASCULAR

[CARDIOVASCULAR] Niacin-induced "Flush" Involves Release of Prostaglandin D2 from Mast Cells and Serotonin from Platelets: Evidence from Human Cells in Vitro and an Animal Model

2008-11-12

Niacin lowers serum cholesterol, low-density lipoprotein, and triglycerides, and it raises high-density lipoprotein. However, most patients experience cutaneous warmth and vasodilation (flush). Acetylsalicylic acid (ASA) can reduce this flush, presumably by decreasing prostaglandin D₂ (PGD₂) release from macrophages. Here, we show that methylnicotinate induces significant PGD₂ release from human mast cells and serotonin from human platelets. Intradermal injection of methylnicotinate induces rat skin vasodilation and vascular permeability. Niacin increases plasma PGD₂ and serotonin in a rat model of flush. The phenothiazine prochlorperazine, the H₁, serotonin receptor antagonist cyproheptadine, and the specific serotonin receptor-2A antagonist ketanserin inhibit niacin-induced temperature increase by 90% (n = 5, p < 0.05), 90 and 50% (n = 3, p < 0.05), and 85% (n = 6, p = 0.0008), respectively, in this animal model. These results indicate that niacin-induced flush involves both PGD₂ and serotonin, suggesting that drugs other than ASA are required to effectively inhibit niacin-induced flush.

[CARDIOVASCULAR] Improvement of Endothelial Function of the Corpus Cavernosum in Apolipoprotein E Knockout Mice Treated with Irbesartan

Baumhakel, M., Custodis, F., Schlimmer, N., Laufs, U., Bohm, M. — 2008-11-12

Angiotensin receptor blockers enhance endothelial function and are suggested to improve erectile function. The effects and underlying mechanisms of treatment with the angiotensin receptor blocker irbesartan on penile endothelial function in apolipoprotein E (ApoE)^-/- mice were determined. Wild-type (C57/B6) and ApoE^-/- mice were fed with a high-fat, cholesterol-rich diet for 7 weeks and treated with irbesartan (50 mg/kg · day) or hydralazine (250 mg/l). Vital parameters were measured with the tail-cuff method. Endothelial (aortic rings) and erectile function (corpora cavernosa) were assessed by pharmacological stimulation in an organ bath chamber. Oxidative stress and angiotensin receptor expression were determined. Blood pressure was significantly decreased in irbesartan- and hydralazine-treated ApoE^-/- mice (p < 0.05) compared with controls and wild-type mice. Endothelial function of the aorta and corpus cavernosum was significantly impaired in ApoE^-/- mice (p < 0.05) and could be restored by treatment with irbesartan (p < 0.05). Consistently, nitric oxide production of corpora cavernosa was impaired in ApoE^-/- mice (p < 0.01), with a restoration in irbesartan- but not hydralazine-treated mice. Dihydroethidium-stained sections and lipid peroxidase assay revealed a reduction of superoxide production in irbesartan (p < 0.05) compared with hydralazine-treated and control ApoE^-/- mice. In summary, irbesartan improves penile endothelial function in ApoE^-/- mice by reduction of vascular and cavernosal oxidative stress. This result emphasizes the beneficial effect of inhibition of the renin-angiotensin system even in terms of erectile function.

[CARDIOVASCULAR] Novel Peroxisome Proliferator-Activated Receptor {alpha} Agonists Lower Low-Density Lipoprotein and Triglycerides, Raise High-Density Lipoprotein, and Synergistically Increase Cholesterol Excretion with a Liver X Receptor Agonist

2008-11-12

The first generation peroxisome proliferator-activated receptor (PPAR) agonist gemfibrozil reduces the risk of major cardiovascular events; therefore, more potent PPAR agonists for the treatment of cardiovascular diseases have been actively sought. We describe two novel, potent oxybenzylglycine PPAR-selective agonists, BMS-687453 [N-[[3-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]phenyl]methyl]-N-(methoxycarbonyl)-glycine] and BMS-711939 N-[[5-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]-2-fluorophenyl]methyl]-N-(methoxycarbonyl)-glycine], that robustly increase apolipoprotein (Apo) A1 and high-density lipoprotein cholesterol in human ApoA1 transgenic mice and lower low-density lipoprotein-cholesterol and triglycerides in fat-fed hamsters. These compounds have much lower potency against mouse PPAR than human PPAR; therefore, they were tested in PPAR-humanized mice that do not express murine PPAR but express human PPAR selectively in the liver. We developed hepatic gene induction as a novel biomarker for efficacy and demonstrate hepatic gene induction at very low doses of these compounds. BMS-711939 induces fecal cholesterol excretion, which is further increased upon cotreatment with a liver X receptor (LXR) agonist. It is surprising that this synergistic increase upon coadministration is also observed in mice that express PPAR in the liver only. BMS-711939 also prevented the LXR agonist-induced elevation of serum triglycerides. Such PPAR agonists could be attractive candidates to explore for the treatment of cardiovascular diseases, especially in combination with a suitable LXR agonist.

[CARDIOVASCULAR] Pharmacology of Macitentan, an Orally Active Tissue-Targeting Dual Endothelin Receptor Antagonist

2008-11-12

Macitentan, also called Actelion-1 or ACT-064992 [N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-N'-propylaminosulfonamide], is a new dual ET_A/ET_B endothelin (ET) receptor antagonist designed for tissue targeting. Selection of macitentan was based on inhibitory potency on both ET receptors and optimization of physicochemical properties to achieve high affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major and pharmacologically active metabolite, ACT-132577. Macitentan and its metabolite antagonized the specific binding of ET-1 on membranes of cells overexpressing ET_A and ET_B receptors and blunted ET-1-induced calcium mobilization in various natural cell lines, with inhibitory constants within the nanomolar range. In functional assays, macitentan and ACT-132577 inhibited ET-1-induced contractions in isolated endothelium-denuded rat aorta (ET_A receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea (ET_B receptors). In rats with pulmonary hypertension, macitentan prevented both the increase of pulmonary pressure and the right ventricle hypertrophy, and it markedly improved survival. In diabetic rats, chronic administration of macitentan decreased blood pressure and proteinuria and prevented end-organ damage (renal vascular hypertrophy and structural injury). In conclusion, macitentan, by its tissue-targeting properties and dual antagonism of ET receptors, protects against end-organ damage in diabetes and improves survival in pulmonary hypertensive rats. This profile makes macitentan a new agent to treat cardiovascular disorders associated with chronic tissue ET system activation.

[CARDIOVASCULAR] Bivalirudin Decreases NO Bioavailability by Vascular Immobilization of Myeloperoxidase

2008-10-17

Bivalirudin, a direct thrombin inhibitor, has emerged as an important alternative to heparin in patients undergoing percutaneous coronary intervention. However, it remains elusive if potentially adverse extracoagulant properties are responsible for the fact that its favorable effects in clinical studies are mainly driven by a reduction in bleeding events. The aim of the current study was to determine the effects and mechanisms of acute treatment with bivalirudin in comparison to heparin on NO bioavailability, an important factor for the pathogenesis of ischemic events. In particular, we studied the interaction between bivalirudin and myeloperoxidase (MPO), a leukocyte-derived enzyme that consumes endothelial-derived nitric oxide (NO), modifies a variety of biological targets, and thus affects the integrity of the vessel wall. In patients undergoing elective percutaneous coronary intervention, bivalirudin, in contrast to heparin, exhibited a significant decrease in plasma MPO levels (p = 0.03) accompanied by a deterioration of flow-mediated dilation (p = 0.02), a surrogate for endothelial NO bioavailability. In vitro experiments revealed avid binding of bivalirudin to both bovine aortic endothelial cells (BAEC) and MPO. Methylation of bivalirudin carboxyl groups at the carboxyl-terminal end revealed the specific binding site of bivalirudin to MPO. Bivalirudin-facilitated binding of MPO to BAEC resulted also in functional changes in terms of increased NO consumption as well as enhanced MPO-mediated redox modifications. These results illustrate dichotomous extracoagulant properties of heparins and thrombin inhibitors and suggest that bivalirudin acutely impairs endothelial NO bioavailability, thereby underscoring the potentially critical role of MPO as a mediator of vascular function.

[CARDIOVASCULAR] Antiatherosclerotic Effects of a Novel Synthetic Tissue-Selective Steroidal Liver X Receptor Agonist in Low-Density Lipoprotein Receptor-Deficient Mice

2008-10-17

Liver X receptor (LXR) agonists have the potential to treat atherosclerosis based on their ability to enhance reverse cholesterol transport. However, their side effects, such as induction of liver lipogenesis and triglyceridemia, may limit their pharmaceutical development. In contrast to the nonsteroidal LXR agonist N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317), 3, 6, 24-trihydroxy-24, 24-di(trifluoromethyl)-5β-cholane (ATI-829), a novel potent synthetic steroidal LXR agonist, was a poor inducer of sterol regulatory element-binding protein 1c expression in hepatoma HepG2 cells, whereas both compounds increased ABCA1 expression in macrophage THP-1 cells. In male low-density lipoprotein receptor-deficient mice, ATI-829 selectively activated LXR target gene expression in mouse intestines and macrophages but not in the liver. A significant increase in liver triglyceride and plasma triglyceriderich small very low-density lipoprotein (VLDL) was observed in T0901317 but not ATI-829-treated mice. Compared with vehicle-treated mice, atherosclerosis development was significantly inhibited in the innominate artery after treatment with either compound. However, in the aortic root, inhibition of atherosclerosis was only observed in the right (right coronary artery-associated sinus) but not the left coronary-related sinus (left coronary artery-associated sinus; LC) of mice treated with either compound. Lesions in the innominate artery were less complex after treatment with either compound and contained mostly macrophage foam cells. In contrast, LC lesions were more complex and had a large collagen-positive fibrous cap and less macrophage foam cell area after treatment with either compound. The T0901317-induced hypertriglyceridemia was accompanied by an increase in small triglyceride-rich VLDL that may influence LXR agonist-mediated antiatherosclerotic effects at certain vascular sites. ATI-829, by selectively activating LXR in certain tissues without inducing hypertriglyceridemia, is a good candidate for drug development.

[CARDIOVASCULAR] Formation of Hydrogen Peroxide and Reduction of Peroxynitrite via Dismutation of Superoxide at Reperfusion Enhances Myocardial Blood Flow and Oxygen Consumption in Postischemic Mouse Heart

Xu, Y., Liu, B., Zweier, J. L., He, G. — 2008-10-17

Reactive oxygen/nitrogen species suppress myocardial oxygen consumption. In this study, we determined that endogenous hydrogen peroxide through dismutation of superoxide enhances postischemic myocardial blood perfusion and oxygen consumption. Electron paramagnetic resonance oximetry was applied to monitor in vivo tissue Po₂ in mouse heart subjected to regional ischemia reperfusion. Heart rate, arterial blood pressure, blood flow, infarction, and activities of mitochondrial NADH dehydrogenase and cytochrome c oxidase were measured in six groups of wild-type (WT) and endothelial nitricoxide synthase knock-out (eNOS^-/-) mice treated with phosphate-buffered saline (PBS), superoxide dismutase mimetic (SOD_m) M40403 [a manganese(II)-bis(cyclohexylpyridine)-substituted macrocyclic superoxide dismutase mimetic, C₂₁H₃₅Cl₂MnN₅], 10006329 EUK 134 [EUK134, manganese 3-methoxy N,N¹-bis(salicyclidene)ethylenediamine chloride], and SOD_m plus glibenclamide to study the protective effect of hydrogen peroxide via dismutation of superoxide on the activation of sarcolemmal potassium channels. In the PBS group, there was an overshoot of tissue Po₂ after reperfusion. Treatment with SOD_m, EUK134, and SOD_m + glibenclamide protected mitochondrial enzyme activities, reduced infarct size, and suppressed the postischemic hyperoxygenation. In particular, in the SOD_m-treated group, there was a transient peak of tissue Po₂ at 9 min after reperfusion, which was dependent on endogenous hydrogen peroxide but not nitric oxide formation as it appeared in both WT and eNOS^-/- mice. Blood flow and rate pressure product were higher in the SOD_m group than in other groups, which contributed to the transient oxygen peak. Thus, SOD mimetics protected mouse heart from superoxide-induced reperfusion injury. With treatment of different SOD mimetics, it is concluded that endogenous hydrogen peroxide via dismutation of superoxide at reperfusion enhances postischemic myocardial blood perfusion and mitochondrial oxygen consumption, possibly through activation of sarcolemmal ATP-sensitive potassium channels.

[CARDIOVASCULAR] Effect of cGMP on Pharmacomechanical Coupling in the Uterine Artery of Near-Term Pregnant Sheep

Zhang, L., Xiao, D., Hu, X. — 2008-10-17

The present study examined the role of cGMP in the regulation of ₁-adrenoceptor-mediated pharmacomechanical coupling in the uterine artery of near-term pregnant sheep. The cell-permeable cGMP analog 8-bromo-cGMP produced a dose-dependent relaxation of the uterine artery and shifted norepinephrine (NE) dose-response curve to the right with a decreased maximal contraction. Accordingly, 8-bromo-cGMP significantly decreased the potency and the maximal response of NE-induced inositol 1,4,5-trisphosphate (IP₃) synthesis in the uterine artery. In addition, 8-bromo-cGMP significantly reduced the binding affinity of IP₃ to the IP₃ receptor. The density of IP₃ receptors was not affected. Simultaneous measurement of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) and tensions in the same tissue indicated that 8-bromo-cGMP decreased NE-induced contractions by 92% but only blocked 44% [Ca²⁺]_i. In accordance, 8-bromo-cGMP significantly decreased tension generation for a given [Ca²⁺]_i (g/R_f340/380, 24.87 ± 3.43 versus 3.10 ± 0.35). In the absence of extracellular Ca²⁺, NE produced a transient increase in [Ca²⁺]_i and contraction, which were inhibited by 8-bromo-cGMP by 47 and 76%, respectively. In contrast to NE-induced responses, 8-bromo-cGMP had no significant effects on KCl-induced [Ca²⁺]_i and contractions. The results indicate that cGMP suppresses ₁-adrenoceptor-mediated pharmacomechanical coupling in the uterine artery by inhibiting IP₃ synthesis and Ca²⁺ release from intracellular stores, as well as inhibiting the agonist-mediated Ca²⁺ sensitization of myofilaments, which is likely to play an important role in the adaptation of uterine artery contractility during pregnancy.

[CARDIOVASCULAR] Anti-Inflammatory and Anti-Apoptotic Effects of Fumonisin B1, an Inhibitor of Ceramide Synthase, in a Rodent Model of Splanchnic Ischemia and Reperfusion Injury

2008-09-19

Ceramide is a sphingolipid with potent proinflammatory and proapoptotic properties. This study sought to determine whether pharmacological inhibition of ceramide biosynthesis in the intestine attenuates pathophysiological sequelae of shock induced by splanchnic artery occlusion and reperfusion. Ischemia and reperfusion injury was induced in anesthetized rats by clamping both the superior mesenteric artery and the celiac artery for 45 min followed by reperfusion. Within 6 min after reperfusion, animals developed significant systemic hypotension with 100% of the animals dying during the 4-h period of reperfusion. In parallel experiments, animals were necropsied after 60 min of reperfusion, and the ileum was harvested for histological examination and assessment of biochemical changes. Administration of fumonisin B1 (FB1), a competitive and reversible inhibitor of ceramide synthase (3 mg/kg, 15 min before reperfusion), significantly reduced i) the increased ceramide expression as detected by immunohistochemistry; ii) peroxynitrite-mediated protein nitration; iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils following a decrease in intercellular adhesion molecule-1 expression; iv) production of the proinflammatory cytokine tumor necrosis factor-; and v) apoptosis in the ileum. Overall, tissue-protective effects were clearly observed upon histological examination of the ileum. These beneficial events were ultimately linked to decreases in both the development of hypotension and overall mortality. These results implicate ceramide as a key signaling molecule in splanchnic arterial ischemia and reperfusion-induced shock. The broader implications of our results provide a pharmacological rationale for the development of inhibitors of ceramide biosynthesis as novel therapeutics for ischemia and reperfusion-induced shock of several etiologies.

[CARDIOVASCULAR] Dissociation between Superoxide Accumulation and Nitroglycerin-Induced Tolerance

Tsou, P.-S., Addanki, V., Fung, H.-L. — 2008-09-19

We hypothesize that superoxide (O₂^·) accumulation is not a crucial causative factor in inducing nitroglycerin (NTG) tolerance. In LLC-PK1 cells, pre-exposure to NTG resulted in increased O₂^· accumulation and reduced cGMP response to NTG versus vehicle control. O₂^· stimulated by NTG was reduced by oxypurinol (100 µM), a xanthine oxidase inhibitor. Exposure to angiotensin II (Ang II) increased O₂^· but did not reduce cGMP response. The O₂^· scavenger tiron reduced Ang II-induced O₂^· production but did not increase NTG-stimulated cGMP production. Using p47^phox–/– and gp91^phox–/– mice versus their respective wild-type controls (WT), we showed that aorta from mice null of these critical NADPH oxidase subunits exhibited similar vascular tolerance after NTG dosing (20 mg/kg s.c., t.i.d. for 3 days), as indicated by their ex vivo pEC₅₀ and cGMP accumulation upon NTG challenge. In vitro aorta O₂^· production was enhanced by NTG incubation in both p47^phox null and WT mice. Pre-exposure of isolated mice aorta to 100 µM NTG for 1 h resulted in vascular tolerance toward NTG and increased O₂^· accumulation. Oxypurinol (1 mM) reduced O₂^· but did not attenuate vascular tolerance. These results suggest that O₂^· does not initiate either in vitro and in vivo NTG tolerance, and that the p47^phox and gp91^phox subunits of NADPH oxidase are not critically required. Increased O₂^· accumulation may be an effect, rather than an initiating cause, of NTG tolerance.

[CARDIOVASCULAR] Sodium Hydrogen Exchange 1 (NHE-1) Regulates Connexin 43 Expression in Cardiomyocytes via Reverse Mode Sodium Calcium Exchange and c-Jun NH2-Terminal Kinase-Dependent Pathways

Stanbouly, S., Kirshenbaum, L. A., Jones, D. L., Karmazyn, M. — 2008-09-19

Connexin 43, the major connexin isoform in gap junctions of cardiac ventricular myocytes, undergoes changes in distribution and expression in cardiac diseases. The Na⁺-H⁺ exchanger (NHE-1), a key mediator of hypertrophy and heart failure, has been shown to be localized in the cardiomyocyte gap junctional regions; however, whether NHE-1 regulates gap junction proteins in the hypertrophied cardiomyocyte is not known. To address this question, neonatal rat ventricular myocytes were treated with phenylephrine (PE) for 24 h to induce hypertrophy. Increased Cx43 expression observed with PE treatment (132.4 ± 6.3% compared to control; P < 0.05) was further significantly augmented by the specific NHE-1 inhibitor EMD87580 [N-[2-methyl-4,5-bis(methylsulfonyl)-benzoyl]-guanidine hydrochloride] (173.2 ± 8.7% increase compared to control; P < 0.05 versus PE), an effect that was mimicked by another NHE-1 inhibitor cariporide [4-isopropyl-3-(methylsulfonyl)benzoyl-guanidine methanesulfonate]. PE-induced hypertrophy was associated with mitogen-activated protein kinase c-Jun NH₂-terminal kinase (JNK) 1/2 activation, whereas inhibition of JNK1/2 with either SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] or small interfering RNA significantly increased PE-induced up-regulation of Cx43 protein levels. Inhibition of reverse mode Na⁺-Ca²⁺ exchange (NCX) with KB-R7943 [2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate] partially reversed JNK1/2 activation (195.2 ± 21.4 versus 143.7 ± 14.4% with KB-R7943; P < 0.05) and augmented up-regulation of Cx43 protein (121.1 ± 8.3 versus 215.9 ± 25.6% with KB-R7943; P < 0.05) in the presence of PE. Our results demonstrate that NHE-1 negatively regulates Cx43 protein expression in PE-induced cardiomyocyte hypertrophy via a JNK1/2-dependent pathway, which is probably activated by reverse mode NCX activity.

[CARDIOVASCULAR] Simvastatin Inhibits Catecholamine Secretion and Synthesis Induced by Acetylcholine via Blocking Na+ and Ca2+ Influx in Bovine Adrenal Medullary Cells

Matsuda, T., Toyohira, Y., Ueno, S., Tsutsui, M., Yanagihara, N. — 2008-09-19

Simvastatin, an inhibitor of HMG-CoA reductase, is a potent inhibitor of cholesterol biosynthesis and has beneficial effects in the primary and secondary prevention of cardiovascular diseases. In this study, we report the effects of simvastatin on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells used as a model of sympathetic neurons. Simvastatin inhibited catecholamine secretion induced by acetylcholine, an agonist of the nicotinic acetylcholine receptor; by veratridine, an activator of voltage-dependent Na⁺ channels; and by high K⁺, an activator of voltage-dependent Ca²⁺ channels (IC₅₀ = 3.8, 7.8, and 6.1 µM, respectively). Simvastatin also suppressed acetylcholine-induced ²²Na⁺ influx (IC₅₀ = 4.3 µM) and ⁴⁵Ca²⁺ influx (IC₅₀ = 6.1 µM), veratridine-induced ²²Na⁺ influx (IC₅₀ = 6.6 µM) and ⁴⁵Ca²⁺ influx (IC₅₀ = 12 µM), and high K⁺-induced ⁴⁵Ca²⁺ influx (IC₅₀ = 11 µM). The reduction of catecholamine secretion caused by simvastatin was not overcome by increasing the concentration of acetylcholine or by treatment with mevalonate, the first metabolite of HMG-CoA. The inhibitory effect of simvastatin on histamine-induced secretion of catecholamines was observed in the presence of extracellular Ca²⁺, but not in a Ca²⁺-free medium, suggesting that simvastatin does not interfere with histamine receptors nonselectively. Simvastatin also suppressed acetylcholine-induced [¹⁴C]catecholamine synthesis from [¹⁴C]tyrosine as well as tyrosine hydroxylase activity. These findings suggest that simvastatin inhibits catecholamine secretion and synthesis induced by acetylcholine through suppression of Na⁺ and Ca²⁺ influx in the adrenal medulla and probably in the sympathetic neurons.

[CARDIOVASCULAR] Gap Junction Inhibitors Reduce Endothelium-Dependent Contractions in the Aorta of Spontaneously Hypertensive Rats

Tang, E. H. C., Vanhoutte, P. M. — 2008-09-19

Experiments were designed to determine the effect of gap junction inhibitors on endothelium-dependent contractions. Isolated aortic rings of spontaneously hypertensive rats (SHR) were suspended in vitro for isometric force recording. The nonselective gap junction inhibitor, carbenoxolone, reduced endothelium-dependent contractions to acetylcholine and the calcium ionophore A23187 [5-methylamino-2-(2S,3R,5R,8S,9S)-3,5,9-trimethyl-2-(1-oxo-(1H-pyrrol-2-yl)propan-2-yl)-1,7-dioxaspiro-(5,5)undecan-8-yl)methyl)benzooxazole-4-carboxylic acid]. There was no or modest effect of the gap peptides ⁴⁰Gap27, ^37,43Gap27, or ⁴³Gap26 when applied alone on endothelium-dependent contractions. However, the combined treatment with the three gap peptides significantly decreased endothelium-dependent contractions. The combined inhibition of the three connexins was not as effective as carbenoxolone, suggesting the involvement of other connexins in the process of endothelium-dependent contraction. The present study shows the involvement of gap junctions in endothelium-dependent contractions of the SHR aorta, presumably that of the combination of connexins 37, 40, and 43 rather than a single subtype of these proteins. Contractions of the vascular smooth muscle caused by 9,11-dideoxy-11, 9-epoxymethanoprostaglandin F₂ (U46619) and prostacyclin, but not to those of endoperoxides and phenylephrine, were reduced only minimally by carbenoxolone. Thus, if gap junction signaling is involved in the contraction of the vascular smooth muscle to thromboxane-prostanoid receptor agonists, their contribution is small. This suggests that the reduction of endothelium-dependent contractions by carbenoxolone and the gap peptides cannot be attributed to the homocellular gap junctions between vascular smooth muscle, but is more likely to involve the homocellular gap junctions between endothelial cells and/or myoendothelial gap junctions.