Trolox and 17beta-estradiol protect against amyloid beta-peptide neurotoxicity by a mechanism that involves modulation of the Wnt signaling pathway

Oxidative stress is a key mechanism in amyloid beta-peptide (A beta)-mediated neurotoxicity; therefore, the protective roles of 17beta-estradiol (E2) and antioxidants (Trolox and vitamin C) were assayed on hippocampal neurons. Our results show the following: 1) E2 and Trolox attenuated the neurotoxicity mediated by A beta and H2O2 as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays, quantification of apoptotic cells, and morphological studies of the integrity of the neurite network. 2) Vitamin C failed to protect neurons from A beta toxicity. 3) A beta-mediated endoperoxide production, reported to induce cell damage, was decreased in the presence of E2 and Trolox. 4) Two key Wnt signaling components were affected by E2 and Trolox; in fact, the enzyme glycogen synthase kinase 3beta was inhibited by both E2 and Trolox, and both compounds were able to stabilize cytoplasmic beta-catenin. 5) E2 activated the expression of the Wnt-5a and Wnt-7a ligands, and at the same time, E2, through the alpha-estrogen receptor, was able to prevent the excitotoxic A beta-induced rise in bulk-free Ca2+ as an alternative pathway to increase cell viability. 6) Finally, the Wnt-7a ligand protected against cytoplasmic calcium disturbances induced by A beta treatment. Our results suggest that control of oxidative stress, regulation of cytoplasmic calcium, and activation of Wnt signaling may prevent A beta neurotoxicity.     

Macrophage colony stimulatory factor and interferon-gamma trigger distinct mechanisms for augmentation of beta-amyloid-induced microglia-mediated neurotoxicity

Dysregulated stimulation of microglia, the resident macrophages in the brain, can lead to excessive induction of inflammatory agents and subsequently damage to neurons. Fibrillar beta-amyloid peptide (fA beta), a major component of senile plaques in Alzheimer's disease (AD) brain, is known to induce microglial-mediated neurotoxicity under certain conditions. Microglial 'priming' by macrophage colony stimulatory factor (MCSF) or interferon-gamma (IFN gamma) appears to be required for this fA beta-induced microglia mediated neurotoxicity in vitro. We report here that while both MCSF and IFN gamma induce microglial-mediated fA beta neurotoxicity, their mechanisms of toxicity differ. The enhancement of neurotoxicity by IFN gamma or MCSF is not due to enhanced A beta ingestion by microglia or to the direct effect of proinflammatory cytokine production. The neurotoxicity resulting from IFN gamma/fA beta treatment was blocked by pretreatment with nitric oxide synthase inhibitor L-N-5-(1-iminoethyl) ornithine hydrochloride (L-NIO), consistent with a role for nitric oxide in the IFN gamma-mediated toxicity mechanism. In contrast, no induction of nitric oxide production was detected for microglia treated with MCSF/fA beta. Furthermore, inhibiting the generation of reactive oxygen species (ROS) using the specific NADPH oxidase inhibitor apocynin reversed fA beta/MCSF-induced neurotoxicity while L-NIO had little effect. As MCSF is endogenously expressed within the brain, and both its level and that of the MCSF receptor are dramatically increased in the AD brain, the neurotoxicity resulting from ROS release by fA beta/MCSF coactivated microglia may be a more appropriate model for assessing fA beta-induced microglial-mediated neuropathology in AD.     

Evidence of Trolox and some gallates as synergistic protectors of erythrocytes against peroxyl radicals.

The peroxidation of human erythrocytes induced by peroxyl radical initiator and its inhibition by several gallate esters (e.g., propyl, methyl, ethyl) and Trolox (a more polar analogue of vitamin E) have been studied. The antioxidant activity was determined on erythrocytes against hemolysis generated by a thermal activator, 2,2'-azobis-(2-amidinopropane)dihydrogenchloride. It was found that propyl gallate and its two analogues were more effective than Trolox in preventing cell lysis. However, the combination of gallate esters and Trolox produced a protective effect exceeding the arithmetic sum of their individual contributions. These perceived synergisms occur at more than one level of Trolox at a given level of a gallate ester.     

A Kinetic Analysis of the Mechanism of beta-Amyloid Induced G Protein Activation

beta-Amyloid (A beta) is the primary protein component of senile plaques found in Alzheimer's disease. In an aggregated (amyloid fibril, protofibril, or low molecular weight oligomer) state, A beta has been consistently shown to be toxic to neurons, but the molecular mechanism of this toxicity is poorly understood. We have previously shown that A beta activates a G(i/o) protein, and that inhibition of this specific G protein activation attenuated A beta-induced cell toxicity. In the present study, we use a kinetic analysis to examine the mechanism of A beta-induced G protein activation. Using synthetic A beta(1-40) and phospholipid vesicles containing purified G(0)alpha subunits, we examined the relationship between A beta concentration, G(0)alpha subunit concentration, GTP concentration and rate of GTP hydrolysis experimentally. We found that at low concentrations of A beta (less than 10 microM), A beta increased the rate of GTP hydrolysis over the rate of hydrolysis in the absence of peptide, however, at high concentrations of A beta, significantly decreased rates of GTP hydrolysis were observed. We postulated several molecular level mechanisms for the observed rate behavior, from those mechanisms derived rate equations, and then tested the mechanisms against our experimental rate data. Based on our results, we identified a plausible mechanism for A beta-induced G protein activation which is consistent with available experimental data. This work demonstrates the utility of an engineering approach to examining steps in the mechanism of A beta-induced cell toxicity and could provide insight into our understanding of the mechanism of Alzheimer's disease.     

Cardiorespiratory responses to interleukin-1beta in adult rats: role of nitric oxide, eicosanoids and glucocorticoids

Interleukin-1beta (IL-1beta) receptors are abundantly expressed in brain stem regions involved in respiratory control. We hypothesized that systemic administration of IL-1beta would increase ventilation (V(E )), and that nitric oxide, eicosanoids, and glucocorticoid receptors would modulate IL-1beta-induced cardioventilatory responses. Intravenous injections of three doses (37.5 ng kg(-1), 75 ng kg(-1 ) and 150 ng kg(-1)) of IL-1b induced monophasic increases in (V(E)), heart rate (HR), and blood pressure (BP) which had a distinctly different onset and duration of action compared to IL-1beta-induced body temperature elevations. Pre-treatment with the nitric oxide inhibitor L-NAME was associated with decreased peak V(E) responses, without affecting the latency and duration of IL-1beta. L-NAME also enhanced HR responses while pressor responses were attenuated. Eicosanoid inhibition with indomethacin resulted in markedly attenuated V responses. However, cardiovascular responses to IL-1beta were not modified by indomethacin. In contrast, pre-treatment with dexamethasone, was not associated with any changes in the IL-1beta-induced V(E), HR, or BP responses. We conclude that IL-1beta increases of V(E) are dose-dependent and are not time-locked with the pyrexic response suggesting the possibility that distinct neural pathways may underlie these responses. In addition, nitric oxide and eicosanoid-dependent mechanisms modulate IL-1beta ventilatory effects.     

Glutathione cycle impairment mediates A beta-induced cell toxicity

Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the action of amyloid beta-peptide (A beta). We observed that A beta 25-35 induced an increase in reactive oxygen species (ROS) in NT2 rho+ cells, leading to protein and lipid oxidation. This oxidative status was partially prevented by the antioxidants, vitamin E, reduced glutathione, and by melatonin. However, NT2 rho0 cells (that lack mitochondrial DNA) in the absence of A beta showed an increase in ROS production, lipid and protein oxidation, as compared with parental rho+ cells. Upon A beta 25-35 treatment, in rho+ cells, a decrease in glutathione reductase activity and in GSH levels was observed, whereas glutathione peroxidase activity was shown to be increased. In NT2 rho0 cells, in the absence of A beta, GSH levels were maintained, whereas glutathione reductase and peroxidase activities were increased. The exposure of A beta to rho0 cells did not induce any change in these parameters. We observed that melatonin prevented caspase activation and DNA fragmentation in rho+ cells treated with A beta. Considering the evidence presented, we argue that the glutathione cycle impairment is a key event in A beta-induced cell toxicity.     

Effects of interleukins on plasma arginine vasopressin and oxytocin levels in conscious, freely moving rats.

To elucidate whether interleukins are involved in vasopressin or oxytocin release during cytokine-related stressful conditions, we examined the effects of human interleukin-1 beta and interleukin-6 on plasma vasopressin and oxytocin levels in rats. Interleukin-1 beta administrated intravenously stimulated both the vasopressin and oxytocin secretion in dose-dependent manners. Neither hormone release was observed following interleukin-6 administration. Pretreatment with aspirin significantly attenuated the effects of interleukin-1 beta on both the vasopressin and oxytocin levels. SC-19220, a prostaglandin E2 receptor antagonist, did not affect the interleukin-1 beta-induced increase of plasma oxytocin levels, but almost completely abolished its effect on plasma vasopressin levels. These results suggest that under certain stressful conditions which accompany the stimulation of cytokine production, interleukin-1 is involved in the increase of plasma vasopressin and oxytocin levels and, moreover, different kinds of prostaglandins are suggested to participate in these interleukin-1-induced hormone release.     

A Role for 4-Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, in Disruption of Ion Homeostasis and Neuronal Death Induced by Amyloid β-Peptide

Abstract: Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid β-peptide (Aβ) can promote free radical production, we tested the hypothesis that HNE mediates Aβ25-35-induced disruption of neuronal ion homeostasis and cell death. Aβ induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na⁺,K⁺-ATPase activity and induced an increase of neuronal intracellular free Ca²⁺ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both Aβ and HNE. The antioxidant propyl gallate protected neurons against Aβ toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates Aβ-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.     

The effect of vitamins C and E on biomarkers of oxidative stress depends on baseline level

Oxidative stress is elevated in obesity, and may be a major mechanism for obesity-related diseases. Nonsmokers (n=396) were randomized to 1000 mg/day vitamin C, 800 IU/day vitamin E, or placebo, for 2 months. Treatment effect was examined in multiple regression analyses using an intention-to-treat approach. Vitamin C (P=0.001) and vitamin E (P=0.043) reduced plasma F2-isoprostanes. In the overall sample, changes from baseline were +6.8, -10.6, and -3.9% for placebo, vitamin C, and vitamin E groups, respectively. However, a significant interaction with baseline F2-isoprostane was found. When baseline F2-isoprostane was >50 microg/mL, vitamin C reduced F2-isoprostane by 22% (P=0.01). Vitamin E reduced it by 9.8% (P=0.46). Below that cut point, neither treatment produced further reductions. F2-isoprostane>50 microg/mL was strongly associated with obesity, and was present in 42% of the sample. Change in malondialdehyde concentration was minimal. These findings suggest a role for vitamin C in reducing lipid peroxidation. Future research on effects of vitamins C or E on plasma F2-isoprostane should limit participants to those with baseline levels >50 mug/mL. Further studies are needed to establish whether treatment with vitamins C or E in persons with concentrations above that cut point could slow the development of cardiovascular disease.     

Antioxidants prevent amyloid peptide-induced apoptosis and alteration of calcium homeostasis in cultured cortical neurons

Beta-amyloid ((A)beta) is a peptide of 39-42 amino acids that is the primary component of plaques in Alzheimer's disease (AD). The mechanism by which (A)beta expresses its neurotoxic effects may involve induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels. Cultured cortical cells were utilized to study the alterations in calcium homeostasis underlying the neurotoxic effect of (A)beta. Serum supplement B27 and vitamin E were effective in preventing neuronal death as assessed by lactate dehydrogenase (LDH) release, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and number of apoptotic nuclei. In addition, (A)beta-induced cytosolic free calcium ([Ca2+]i) was blocked by antioxidants vitamin E and U83836E, but not by N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801, or by voltage-gated calcium channel blocker nimodipine. Taken together, the results suggest that NMDA receptor and voltage-gated calcium channels are not involved in (A)beta-induced [Ca2+]i increase. This increase appeared to be the result of extracellular calcium influx by some unknown mechanisms. In addition, antioxidants such as B27 were effective in protecting cultured cortical neurons against (A)beta, and correlated with (A)beta attenuation of early calcium response.     

Oxidant stress is a significant feature of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic cholestatic disorder characterised by an immunological, and often granulomatous, attack on bile ducts leading to fibrosis, cirrhosis, liver failure and death. Animal and human studies suggest that oxidant stress plays a key role in progression of other liver diseases, but no comprehensive investigation has been performed previously in PBC. A wide range of lipid peroxidation and antioxidant markers were measured in the blood and urine of 41 patients with histologically confirmed PBC. Lipid peroxidation markers were significantly elevated [plasma and urinary 8-isoprostane, P<0.001; plasma malondialdehyde (MDA), P=0.007] compared to age- and sex-matched controls. The most striking antioxidant depletion occurred with plasma total glutathione where levels were significantly reduced (30% of controls). Total serum antioxidant levels were decreased (P=0.013) and serum selenium and vitamin A were also lower (both P<0.001); vitamins C and E were normal. Most patients had early disease biochemically and were Child-Pugh grade A. Urinary 8-isoprostane correlated positively with Ludwig stage and markers of hepatic injury and cholestasis. This study clearly demonstrates that oxidant stress, as reflected in a comprehensive spectrum of lipid peroxidation and antioxidant markers, is a significant feature of early-stage PBC.     

Using model membranes for the study of amyloid beta:lipid interactions and neurotoxicity

One of the major tasks in understanding the etiopathogenesis of amyloid beta-induced neurotoxicity of Alzheimer's disease (AD), is in fully capturing the large number of the biochemical processes that influence each other during the course of the disease, in vivo. Model membranes possess, as their main strength, the ability to enable the researcher to manipulate a 'biological' micro-vesicle under a controlled environment. This review narrowly focuses on discussing the exploitation of model membranes for improved understanding of some of the mechanisms governing AD's amyloid beta-induced neurotoxicity. Amyloid beta (Abeta) is cleaved from a membrane-located amyloid precursor protein by membrane-located enzymes. The relative spatial localization of the involved biomolecules within the membrane bilayer is crucial in influencing Abeta production, its aggregation on the membrane surface or insertion into the membrane, and fibril formation: all important processes in causing neurotoxicity. The lipid composition of the bilayer is similarly important. The review also attempts to highlight current and future challenges in using model membranes for studying biochemical processes.     

The role of nerve growth factor in hyperosmolar stress induced apoptosis

Nerve growth factor (NGF) is a neurotrophic factor that plays an important role in the differentiation and growth of neuronal cells. It is also regarded as an inflammatory mediator in non-neuronal tissues under physiological stress conditions. The mechanisms of NGF production and its roles in hyperosmolar stress conditions have not been established. In this study, we show that NGF levels in cultured human corneal epithelial cells (HCECs) were up-regulated during hyperosmolar stress by IL-1beta, but not TNF-alpha. NF-kappaB activity, but not AP-1, increased significantly under hyperosmolar conditions, and NF-kappaB was involved in IL-1beta-induced NGF production. IL-1beta-induced NGF production reduced JNK phosphorylation and HCEC apoptosis. These changes were accompanied by down-regulated Bax and caspase-3, -8, -9 activities. NGF siRNA and the tyrosine kinase inhibitor K252a significantly enhanced Bax up-regulation. Thus, up-regulated NGF under hyperosmolar stress conditions may contribute, at least in part, to reduced HCEC apoptosis. This conclusion suggests that enhanced NGF expression may be beneficial in recovering corneal damage due to chronic hyperosmolar stress.     

Butylated hydroxyanisole specifically inhibits tumor necrosis factor-induced cytotoxicity and growth enhancement.

The effect of commonly used food antioxidants on recombinant tumor necrosis factor alpha (rTNF-alpha)-induced cytotoxicity, growth enhancement and adhesion has been evaluated. Butylated hydroxyanisole (BHA) and 4-hydroxymethyl-2,6-di-t-butylphenol (HBP) were the only two of nine antioxidants that completely inhibited rTNF-alpha-induced cytotoxicity in L929 and WEHI 164 fibrosarcoma cells. Ethoxyquin, propyl gallate and butylated hydroquinone only partially inhibited rTNF-alpha-induced cytotoxicity, while the antioxidants butylated hydroxytoluene (BHT), alpha-tocopherol, ascorbic acid and thiodipropionic acid had minimal effects. The only difference between the molecular structure of the efficient HBP and the non-efficient BHT, is a hydroxymethyl group instead of a hydroxyl group on the phenolic ring. Neither BHA nor BHT inhibited the activation of NF kappa B after 10 or 60 min challenge with rTNF-alpha in L929 cells. BHA also inhibited rTNF-alpha-induced, but not rIL-1 beta-induced growth enhancement in FS-4 fibroblasts. Further, BHA blocked both rTNF-alpha-induced and rIL-1 beta-induced prostaglandin E2 synthesis in FS-4 fibroblasts. BHA inhibited the rTNF-alpha-induced release of arachidonic acid in both FS-4 and L929 cells, suggesting that BHA inhibits cellular phospholipase(s). Neither alpha-tocopherol nor BHA inhibited rTNF-alpha-induced adhesiveness of human endothelial cells. The results indicate that BHA is a specific and potent inhibitor of rTNF-alpha- and rTNF-beta-induced cytotoxicity, as well as of rTNF-alpha-induced growth enhancement.     

Effects of antioxidant vitamin supplements on Helicobacter pylori-induced gastritis in Mongolian gerbils

BACKGROUND: Epidemiological studies show that high intake of food-bound vitamin C and E reduces the risk of gastric cancer. Whether dietary supplementation with antioxidant micronutrients interferes with Helicobacter pylori infection and associated diseases is unclear. The aim of this study was to investigate if dietary vitamin C or E supplementation influences the progression of gastritis, gastric mucosal nitrosative and oxidative protein damage, gastric mucosal lipid peroxidation, or gastric mucosal oxidative DNA damage in H. pylori-infected Mongolian gerbils. MATERIALS AND METHODS: Gerbils were divided into four groups: H. pylori-infected animals fed with vitamin C- or vitamin E-supplemented food, and infected and uninfected animals given standard rodent food. Subgroups of animals were killed at different time-points until 52 weeks postinfection. Concentrations of 3-nitrotyrosine and thiobarbituric acid-reactive substances (TBARS) in the gastric mucosa were determined with an immunodot blot and a fluorometric method, respectively. Mucosal concentrations of carbonyl carbons on proteins and 8-hydroxydeoxyguanosine were determined by enzyme-linked immunosorbent assay. Gastritis was scored semiquantitatively. RESULTS: Vitamin supplements had no effect on the colonization with H. pylori. Vitamin C as well as vitamin E supplements reduced mucosal 3-nitrotyrosine concentrations to normal levels in infected animals. Vitamin E supplements decreased mucosal protein carbonyls and TBARS in short-term gastritis. In addition, vitamin C supplements caused attenuated mucosal oxidative DNA damage and milder mucosal inflammation in short-term gastritis. CONCLUSION: Vitamin C or vitamin E supplementation leads to some short-term protective effects on H. pylori-induced gastritis in Mongolian gerbils. These effects seem to subside over time when the infection persists.     

Gallic acid, a metabolite of the antioxidant propyl gallate, inhibits gap junctional intercellular communication via phosphorylation of connexin 43 and extracellular-signal-regulated kinase1/2 in rat liver epithelial cells

Propyl gallate and its metabolite, gallic acid, are widely used as antioxidants in the food industry, but they have been shown to exhibit liver toxicity and enhance carcinogenesis. In the present study, we investigated the possible undesirable effects of propyl gallate and gallic acid on gap junctional intercellular communication (GJIC), inhibition of which is closely linked to carcinogenesis. Gallic acid and propyl gallate exhibited dose-dependent free-radical-scavenging activities as determined by 1,1-diphenyl-2-picrylhydrazyl- or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-radical-scavenging assays, and the free-radical-scavenging activity of gallic acid was stronger than that of propyl gallate. However, using WB-F344 rat liver epithelial cells, gallic acid inhibited GJIC in a dose-dependent manner, while propyl gallate had no significant effect compared with untreated controls. The gallic-acid-induced inhibition of GJIC was reversible, with a recovery of nearly 65% after 120 min. Gallic acid induced the phosphorylation of connexin 43 (Cx43) and phosphorylation of extracellular-signal-regulated kinase1/2 (ERK1/2). The gallic-acid-induced inhibition of GJIC was attenuated by treatment with mitogen-activated protein kinase kinase inhibitors (U0126 and PD098059). U0126 blocked the gallic-acid-induced phosphorylation of Cx43 and ERK1/2, indicating that the gallic-acid-induced inhibition of GJIC is mediated by phosphorylation of Cx43 via activation of ERK1/2. In addition, gallic-acid-induced inhibition of GJIC was protected by ascorbic acid and quercetin, which might represent a simple example of the different effects of natural antioxidants in carcinogenesis.     

Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.     

8-Isoprostane-induced endothelin-1 production by infant rat pulmonary artery smooth muscle cells is mediated by Rho-kinase

We have reported that 8-isoprostane stimulated the production of endothelin (ET)-1, a potent vasoconstrictor and critical mediator of chronic pulmonary hypertension, by infant rat pulmonary artery smooth muscle cells (PASMCs), through stimulation of the thromboxane A2 receptor. The aim of this study was to examine the contribution of putative downstream intracellular mediators of thromboxane A2 receptor stimulation to this effect. PASMCs from infant rats were treated with calcium ionophore (A23187), 8-isoprostane, or 8-isoprostane together with inhibitors of tyrosine kinase, protein kinase C, phosphatidylinositol 3-kinase, mitogen-activated protein kinases, or Rho-kinases (ROCK). A23187 had no effect on ET-1 production, excluding raised intracellular Ca2+ as a major contributor. Increased ET-1 production induced by 8-isoprostane was significantly attenuated by the ROCK inhibitors Y-27632 and hydroxyfasudil, but not by inhibitors of the other pathways. 8-Isoprostane also increased membrane binding of RhoA, a major determinant of ROCK activity, and ROCK-II expression through the protein kinase C pathway. These data indicate that the RhoA/ROCK pathway mediates increased ET-1 production by PASMCs, which we speculate may at least partly explain the beneficial effects of both antioxidants and ROCK inhibitors in animal models of chronic pulmonary hypertension.