Shear stress-induced nitric oxide release triggers the liver regeneration cascade.

The trigger of the liver regeneration cascade is currently unknown and has been the subject of debate. We hypothesize that, following 2/3 partial hepatectomy (PHX), an increase in the blood flow-to-liver mass ratio results in shear stress-induced nitric oxide (NO) release, which triggers the liver regeneration cascade. Portal venous pressure (PVP), reflecting shear stress in the liver, increased to the same extent following PHX and selective portal vein branch ligation (PVL), a hemodynamic model of PHX, suggesting similar amounts of shear stress in both models. Two indices of the initiation of the liver regeneration cascade were used: proliferative factor (PF) activity in blood 4 h after PHX or PVL and hepatic c-fos mRNA expression 15 min. after PHX or PVL. PF activity and c-fos mRNA expression were increased to similar extents after PHX and PVL, suggesting a similar stimulus in both models. PF activity and c-fos mRNA expression were inhibited by administration of the nitric oxide synthase antagonist, l-NAME, and the NO donor, SIN-1, reversed the inhibition in both models. These results provide support for the hypothesis that a hemodynamic change results in increased shear stress in the liver causing generation of NO, which then triggers the liver regeneration cascade.     

Nitric oxide-deficiency regulates hepatic heme oxygenase-1.

Nitric oxide plays a crucial role in the maintenance of liver function and integrity. During stress, the inducible heme oxygenase-1 protein and its reaction products, including carbon monoxide, also exert potent hepatoprotective effects. We investigated a potential relationship between endogenous nitric oxide synthesis and the hepatic regulation of heme oxygenase-1. Inhibition of nitric oxide synthesis in vivo by injection of l-NAME led to a dose-dependent induction of heme oxygenase-1 mRNA, protein and activity in the rat liver, whereas did not affect the expression of other heat shock proteins. The effect of l-NAME was demonstrated by hemodynamic changes within the liver circulation as measured by ultrasonic flow probes. Inhibition of nitric oxide synthase led to a decline in hepatic arterial and portal venous blood flow, and subsequently caused liver cell damage. In contrast, the combined administration of l-NAME and the nitric oxide-independent intestinal vasodilator dihydralazine completely restored portal venous flow, abolished the liver cell damage, and prevented the upregulation of heme oxygenase-1, despite inhibition of nitric oxide production. In conclusion, nitric oxide deficiency upregulates hepatic heme oxygenase-1, which is reversible by maintaining hepatic blood flow. This interdependence has important implications for the development of therapeutic strategies aimed at modulating the activity of these hepatoprotective mediator systems.     

Vasoactive factors and hemodynamic mechanisms in the pathophysiology of portal hypertension in cirrhosis

Portal hypertension is primarily caused by the increase in resistance to portal outflow and secondly by an increase in splanchnic blood flow, which worsens and maintains the increased portal pressure. Increased portal inflow plays a role in the hyperdynamic circulatory syndrome, a characteristic feature of portal hypertensive patients. Almost all the known vasoactive systems/substances are activated in portal hypertension, but most authors stress the pathogenetic role of endothelial factors, such as COX-derivatives, nitric oxide, carbon monoxide. Endothelial dysfunction is differentially involved in different vascular beds and consists in alteration in response both to vasodilators and to vasoconstrictors. Understanding the pathogenesis of portal hypertension could be of great utility in preventing and curing the complications of portal hypertension, such as esophageal varices, hepatic encephalopathy, ascites.     

Intermittent selective clamping improves rat liver regeneration by attenuating oxidative and endoplasmic reticulum stress

Intermittent clamping of the portal trial is an effective method to avoid excessive blood loss during hepatic resection, but this procedure may cause ischemic damage to liver. Intermittent selective clamping of the lobes to be resected may represent a good alternative as it exposes the remnant liver only to the reperfusion stress. We compared the effect of intermittent total or selective clamping on hepatocellular injury and liver regeneration. Entire hepatic lobes or only lobes to be resected were subjected twice to 10 min of ischemia followed by 5 min of reperfusion before hepatectomy. We provided evidence that the effect of intermittent clamping can be damaging or beneficial depending to its mode of application. Although transaminase levels were similar in all groups, intermittent total clamping impaired liver regeneration and increased apoptosis. In contrast, intermittent selective clamping improved liver protein secretion and hepatocyte proliferation when compared with standard hepatectomy. This beneficial effect was linked to better adenosine-5′-triphosphate (ATP) recovery, nitric oxide production, antioxidant activities and endoplasmic reticulum adaptation leading to limit mitochondrial damage and apoptosis. Interestingly, transient and early chaperone inductions resulted in a controlled activation of the unfolded protein response concomitantly to endothelial nitric oxide synthase, extracellular signal-regulated kinase-1/2 (ERK1/2) and p38 MAPK activation that favors liver regeneration. Endoplasmic reticulum stress is a central target through which intermittent selective clamping exerts its cytoprotective effect and improves liver regeneration. This procedure could be applied as a powerful protective modality in the field of living donor liver transplantation and liver surgery.     

Molecular metabolic fingerprinting approach to investigate the effects of borneol on metabolic alterations in the liver of nitric oxide deficient hypertensive rats

Hypertension is one of the major risk factor that underlie a wide range of cardiovascular irregularities which causes functional and metabolic alterations in vascular system and major organs. Nitric oxide is the central regulator of the vascular system and its deficiency leads to increased blood pressure and metabolic alterations in liver. Fourier transform infrared spectroscopy (FTIR) is a vibrational spectroscopic technique that uses infrared radiation to vibrate molecular bonds with in the sample that absorbs it and different samples contain diverse configurations of molecular bonds. Both wavenumber and area of the vibrational spectra can be used to explore the qualitative and quantitative constituent of macromolecules. In this study, we intended to evaluate the protective role of borneol, a natural terpene on liver metabolism in a nitric oxide deficient model of hypertension through interpretation of FTIR spectral information. Results demonstrate that FTIR can successfully indicate the molecular changes that occur in all groups. The over all findings demonstrate that in nitric oxide deficient animal model of hypertension, the liver metabolic program is altered through increasing the structural modification in proteins and triglycerides, and quantitative alteration in proteins, lipids, and glycogen. All the above mentioned modifications were protected by borneol in liver and showed its ability to exert a novel defensive action on hepatic metabolism.     

基于转录组测序的扎龙湿地野大麦耐盐性分析

通过转录组测序技术对扎龙湿地野大麦幼苗在200 mmol/L NaCl胁迫2 d后的叶片进行分析。对测序结果进行De novo拼接后,将差异表达基因在GO、KEGG数据库中进行比对注释。结果表明:NaCl胁迫2 d后,对照组与处理组分别获得Unigene序列61038个和46754个,检测到差异表达基因25465个,差异基因GO功能注释到3个大类的55个功能组。差异表达基因被注释到135个Pathway上,直观地显示出NaCl胁迫下野大麦幼苗体内发生调节及改变的代谢过程和信号通路,其中主要涉及光合作用、脯氨酸代谢、叶绿素代谢等途径。通过对野大麦幼苗叶片转录组分析,发掘相关耐盐基因,有助于培育野大麦及其近缘作物新品种,并对揭示植物耐盐性分子机制及相关代谢途径具有重要意义。     

Nitric oxide mediates inactivation of glutathione S-transferase in suspension culture of Taxus cuspidata during shear stress

The importance of nitric oxide (NO) in regulating plant cell responses to environmental stresses is becoming evident. Here the possible role of NO in suspension cultures of Taxus cuspidata under shear stress was investigated in a Couette-type shear reactor. It was found that shear stress with 190 s(-1) caused NO generation in 8 h. NO formation can be inhibited by N-nitro-L-arginine, a nitric oxide synthase inhibitor. Moreover, the activity of glutathione S-transferase (GST), a principal enzyme responsible for detoxification, decreased during shear stress. This inactivation partially recovered when NOS inhibitor or NO scavenger was added into cell cultures during shear stress. Treatment with reactive nitrogen species (RNS) also caused inactivation of GST in cells. The results indicate that NO plays a crucial role in GST inactivation in Taxus cuspidata cells under shear stress.     

Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.     

Immunocytochemical study of the hepatic innervation in the rat after partial hepatectomy

Summary The autonomic nervous system in rats has been assessed by means of indirect immunofluorescence using monospecific antibodies to neuron-specific enolase, neurofilaments, glial fibrillary acidic protein and S-100 protein (10 days after partial (70%) hepatectomy). Different groups of rats were studied:group A: 70% resection and normal dual blood supply (n=5);group B: 70% resection with only portal blood to the liver remnant (n=5);group C: 70% resection with only arterial blood to the liver remnant (n=5);group D: sham operated controls (n=5).All rats of groups A and D showed normal liver/body weight ratios after 10 days in contrast to groups B and C where liver weights were 50–60% of the preresection weight. In group A the regeneration process was histologically normal and associated with a remarkable increase of autonomic innervation patterns in the portal triad. In contrast, livers of animals in groups B and C showed under the light microscope features of hepatocyte degeneration associated with a decreased autonomic innervation compared to the controls. The changes are identical in groups B and C, and are therefore irrespective of the type of blood deprivation (arterial or portal).These results support the importance of dual blood supply for an optimal regenerative response in liver remnants after liver resection. We suggest that the autonomic nerve supply of the portal triad plays at least an important permissive role in liver regeneration.     

Inter-organ communication between intestine and liver in vivo and in vitro

The maintenance of body homeostrasis requires a finely tuned system of interorgan communication. The intimate metabolic interrelation between intestine and liver is characterized by the unique anatomic position of both tissues using the portal vein as a private channel with the pancreas in optimal position to modulate hepatic metabolism.Gut-derived peptides (such as glucagon-like peptide-1) appear to be involved in the process of liver regeneration by regulating the release of pancreatic hormones (e.g. insulin). Extensive bowel resection or functional exclusion of small intestine may lead to severe liver dysfunction and even cirrhosis, which may be due to the lack of some intestine-derived and as yet unknown factor(s). Here a close cooperation between small intestinal mucosa and hepatocytes is demonstrated leading to the concept of a metabolic gut-liver unit. This metabolic interaction forms a wide spectrum ranging from the secretion of peptide hormones to changes in (portal-venous) substrate availability or hepatocyte cell volume. Further investigation and identification of the mechanisms of such regulatory processes may be facilitated by combined perfusion of isolated rat intestine and liver. Using this in vitro approach we could demonstrate the presence of metabolic interorgan communication between isolated perfused tissues independent of plasma borne hormones or extrinsic neural control.     

Regulation of nitric oxide and bcl-2 expression by shear stress in human osteoarthritic chondrocytes in vitro

Onset and progression of cartilage degeneration is associated with shear stress occurring in diarthrodial joints subjected to inappropriate loading. This study tested the hypothesis that shear stress induced nitric oxide is associated with altered expression of regulatory onco-proteins, bcl-2, and Fas (APO-1/CD95) and apoptosis in primary human osteoarthritic chondrocyte cultures. Shear stress induced membrane phosphatidylserine and nucleosomal degradation were taken as evidence of chondrocyte apoptosis. Application of shear stress upregulated nitric oxide in a dose-dependent manner and was associated with increases in membrane phosphatidylserine and nucleosomal degradation. Increasing levels of shear stress decreased expression of the anti-apoptotic factor, bcl-2, from 44 to 10 U/ml. Addition of the nitric oxide antagonists, L-N(5)-(1-iminoethyl) ornithine and Nomega-nitro-L-arginine methyl ester (L-NAME), reduced shear stress induced nucleosomal degradation by 62% and 74%, respectively. Inhibition of shear stress induced nitric oxide release by L-NAME coincided with a 2.7-fold increase of bcl-2, when compared to chondrocytes exposed to shear stress in the absence of L-NAME. These data suggest that shear stress induced nitric oxide is associated with changes in apoptotic regulatory factors that alter chondrocyte metabolism and may contribute to joint degeneration.     

Coordinated regulation of endothelial calcium signaling and shear stress-induced nitric oxide production by PKCβ and PKCη

BackgroundProtein Kinase C (PKC) is a promiscuous serine/threonine kinase regulating vasodilatory responses in vascular endothelial cells. Calcium-dependent PKCbeta (PKCβ) and calcium-independent PKCeta (PKCη) have both been implicated in the regulation and dysfunction of endothelial responses to shear stress and agonists.ObjectiveWe hypothesized that PKCβ and PKCη differentially modulate shear stress-induced nitric oxide (NO) production by regulating the transduced calcium signals and the resultant eNOS activation. As such, this study sought to characterize the contribution of PKCη and PKCβ in regulating calcium signaling and endothelial nitric oxide synthase (eNOS) activation after exposure of endothelial cells to ATP or shear stress.MethodsBovine aortic endothelial cells were stimulated in vitro under pharmacological inhibition of PKCβ with LY333531 or PKCη targeting with a pseudosubstrate inhibitor. The participation of PKC isozymes in calcium flux, eNOS phosphorylation and NO production was assessed following stimulation with ATP or shear stress.ResultsPKCη proved to be a robust regulator of agonist- and shear stress-induced eNOS activation, modulating calcium fluxes and tuning eNOS activity by multi-site phosphorylation. PKCβ showed modest influence in this pathway, promoting eNOS activation basally and in response to shear stress. Both PKC isozymes contributed to the constitutive and induced phosphorylation of eNOS. The observed PKC signaling architecture is intricate, recruiting Src to mediate a portion of PKCη's control on calcium entry and eNOS phosphorylation. Elucidation of the importance of PKCη in this pathway was tempered by evidence of a single stimulus producing concurrent phosphorylation at ser1179 and thr497 which are antagonistic to eNOS activity.ConclusionsWe have, for the first time, shown in a single species in vitro that shear stress- and ATP-stimulated NO production are differentially regulated by classical and novel PKCs. This study furthers our understanding of the PKC isozyme interplay that optimizes NO production. These considerations will inform the ongoing design of drugs for the treatment of PKC-sensitive cardiovascular pathologies.     

Endothelial function and coronary artery disease

The endothelium produces a number of vasodilator and vasoconstrictor substances that not only regulate vasomotor tone, but also the recruitment and activity of inflammatory cells and the propensity towards thrombosis. Endothelial vasomotor function is a convenient way to assess these other functions, and is related to the long-term risk of cardiovascular disease. Lipids (particularly low density lipoprotein cholesterol) and oxidant stress play a major role in impairing these functions, by reducing the bioavailability of nitric oxide and activating pro-inflammatory signalling pathways such as nuclear factor kappa B. Biomechanical forces on the endothelium, including low shear stress from disturbed blood flow, also activate the endothelium increasing vasomotor dysfunction and promoting inflammation by upregulating pro-atherogenic genes. In contrast, normal laminar shear stress promotes the expression of genes that may protect against atherosclerosis. The sub-cellular structure of endothelial cells includes caveolae that play an integral part in regulating the activity of endothelial nitric oxide synthase. Low density lipoprotein cholesterol and oxidant stress impair caveolae structure and function and adversely affect endothelial function. Lipid-independent pathways of endothelial cell activation are increasingly recognized, and may provide new therapeutic targets. Endothelial vasoconstrictors, such as endothelin, antagonize endothelium-derived vasodilators and contribute to endothelial dysfunction. Some but not all studies have linked certain genetic polymorphisms of the nitric oxide synthase enzyme to vascular disease and impaired endothelial function. Such genetic heterogeneity may nonetheless offer new insights into the variability of endothelial function.     

Chemical signaling under abiotic stress environment in plants

Many chemicals are critical for plant growth and development and play an important role in integrating various stress signals and controlling downstream stress responses by modulating gene expression machinery and regulating various transporters/pumps and biochemical reactions. These chemicals include calcium (Ca²⁺), cyclic nucleotides, polyphosphoinositides, nitric oxide (NO), sugars, abscisic acid (ABA), jasmonates (JA), salicylic acid (SA) and polyamines. Ca²⁺ is one of the very important ubiquitous second messengers in signal transduction pathways and usually its concentration increases in response to the stimuli including stress signals. Many Ca²⁺ sensors detect the Ca²⁺ signals and direct them to downstream signaling pathways by binding and activating diverse targets. cAMP or cGMP protects the cell with ion toxicity. Phosphoinositides are known to be involved both in transmission of signal across the plasma membrane and in intracellular signaling. NO activates various defense genes and acts as a developmental regulator in plants. Sugars affect the expression of many genes involved in photosynthesis, glycolysis, nitrogen metabolism, sucrose and starch metabolism, defense mechanisms and cell cycle regulation. ABA, JA, SA and polyamines are also involved in many stress responses. Cross-talk between these chemical signaling pathways is very common in plant responses to abiotic and bitotic factors. In this article we have described the role of these chemicals in initiating signaling under stress conditions mainly the abiotic stress.Key words: ABA, abiotic stress, Ca²⁺ binding proteins, calcium signaling, cyclic nucleotides, nitric oxide, phosphoinositides signaling, signal transduction, sugar signaling     

Effects of chronic renal failure on caveolin-1, guanylate cyclase and AKT protein expression

Chronic renal failure (CRF) has been documented to cause oxidative stress and alter nitric oxide (NO) metabolism. However, the effect of CRF on proteins related to NO bioactivity has not been investigated. The present study was designed to test the hypothesis that CRF would induce changes in caveolin-1 (Cav-1), soluble guanylate cyclase (sGC) and Akt, three proteins important in regulating NO synthase (NOS) functionality. Male Sprague-Dawley rats were randomized to CRF via 5/6 nephrectomy or sham-operated control groups. After 6 weeks, body weight, blood pressure, creatinine clearance, plasma creatinine, urinary cyclic guanosine monophosphate (cGMP) and immunodetectable levels of Cav-1, sGC and Akt were determined in the renal, aorta, heart and liver tissues from both groups. CRF resulted in marked decreases in body weight and creatinine clearance, and elevation of blood pressure and plasma creatinine. An apparent upregulation of sGC protein abundance in renal tissue was noted, with no change in aorta, heart and liver. This was accompanied by a reduction in urinary cGMP levels, indicative of sGC dysfunction. Cav-1 protein abundance was increased in aortic, liver and renal tissues. In contrast, CRF depressed Akt abundance in aorta, heart and liver tissues. These data document that CRF is characterized by alteration in the abundance of proteins regulating NO function in hepatic, vascular, cardiac and renal tissues, and a decrease in cGMP, which contributes to hypertension and changes in NO bioactivity previously noted in this model.     

A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension

Nitric oxide (NO) production by endothelial cell nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. We sought to understand the mechanism underlying defective eNOS function. Phosphorylation of the serine-threonine kinase Akt, which activates eNOS, was substantially reduced in sinusoidal endothelial cells from injured livers. Overexpression of Akt in vivo restored phosphorylation of Akt and production of NO and reduced portal pressure in portal hypertensive rats. We found that Akt physically interacts with G-protein-coupled receptor kinase-2 (GRK2), and that this interaction inhibits Akt activity. Furthermore, GRK2 expression increased in sinusoidal endothelial cells from portal hypertensive rats and knockdown of GRK2 restored Akt phosphorylation and NO production, and normalized portal pressure. Finally, after liver injury, GRK2-deficient mice developed less severe portal hypertension than control mice. Thus, an important mechanism underlying impaired activity of eNOS in injured sinusoidal endothelial cells is defective phosphorylation of Akt caused by overexpression of GRK2 after injury.     

Hypo- and Hyperglycemia Impair Endothelial Cell Actin Alignment and Nitric Oxide Synthase Activation in Response to Shear Stress

Uncontrolled blood glucose in people with diabetes correlates with endothelial cell dysfunction, which contributes to accelerated atherosclerosis and subsequent myocardial infarction, stroke, and peripheral vascular disease. In vitro, both low and high glucose induce endothelial cell dysfunction; however the effect of altered glucose on endothelial cell fluid flow response has not been studied. This is critical to understanding diabetic cardiovascular disease, since endothelial cell cytoskeletal alignment and nitric oxide release in response to shear stress from flowing blood are atheroprotective. In this study, porcine aortic endothelial cells were cultured in 1, 5.55, and 33 mM D-glucose medium (low, normal, and high glucose) and exposed to 20 dynes/cm² shear stress for up to 24 hours in a parallel plate flow chamber. Actin alignment and endothelial nitric oxide synthase phosphorylation increased with shear stress for cells in normal glucose, but not cells in low and high glucose. Both low and high glucose elevated protein kinase C (PKC) levels; however PKC blockade only restored actin alignment in high glucose cells. Cells in low glucose instead released vascular endothelial growth factor (VEGF), which translocated β-catenin away from the cell membrane and disabled the mechanosensory complex. Blocking VEGF in low glucose restored cell actin alignment in response to shear stress. These data suggest that low and high glucose alter endothelial cell alignment and nitric oxide production in response to shear stress through different mechanisms.     

Inbreeding by environmental interactions affect gene expression in Drosophila melanogaster

Genomewide gene expression patterns were investigated in inbred and noninbred Drosophila melanogaster lines under benign and stressful (high temperature) environmental conditions in a highly replicated experiment using Affymetrix gene chips. We found that both heat-shock protein and metabolism genes are strongly affected by temperature stress and that genes involved in metabolism are differentially expressed in inbred compared with noninbred lines, and that this effect is accentuated after heat stress exposure. Furthermore we show that inbreeding and temperature stress cause increased between-line variance in gene expression patterns. We conclude that inbreeding and environmental stress both independently and synergistically affect gene expression patterns. Interactions between inbreeding and the environment are often observed at the phenotypic level and our results reveal some of the genes that are involved at the individual gene level. Our observation of several metabolism genes being differentially expressed in inbred lines and more so after exposure to temperature stress, together with lower fitness in the investigated inbred lines, supports the hypothesis that superiority of heterozygous individuals partly derives from increased metabolic efficiency.     

Modulation of pulmonary endothelial endothelin B receptor expression and signaling: implications for experimental hepatopulmonary syndrome

The hepatopulmonary syndrome (HPS) results from intrapulmonary vasodilation in the setting of cirrhosis and portal hypertension. In experimental HPS, pulmonary endothelial endothelin B (ET(B)) receptor overexpression and increased circulating endothelin-1 (ET-1) contribute to vasodilation through enhanced endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. In both experimental cirrhosis and prehepatic portal hypertension, ET(B) receptor overexpression correlates with increased vascular shear stress, a known modulator of ET(B) receptor expression. We investigated the mechanisms of pulmonary endothelial ET(B) receptor-mediated eNOS activation by ET-1 in vitro and in vivo. The effect of shear stress on ET(B) receptor expression was assessed in rat pulmonary microvascular endothelial cells (RPMVECs). The consequences of ET(B) receptor overexpression on ET-1-dependent ET(B) receptor-mediated eNOS activation were evaluated in RPMVECs and in prehepatic portal hypertensive animals exposed to exogenous ET-1. Laminar shear stress increased ET(B) receptor expression in RPMVECs without altering mRNA stability. Both shear-mediated and targeted overexpression of the ET(B) receptor enhanced ET-1-mediated ET(B) receptor-dependent eNOS activation in RPMVECs through Ca(2+)-mediated signaling pathways and independent of Akt activation. In prehepatic portal hypertensive animals relative to control, ET-1 administration also activated eNOS independent of Akt activation and triggered HPS. These findings support that increased pulmonary microvascular endothelial ET(B) receptor expression modulates ET-1-mediated eNOS activation, independent of Akt, and contributes to the development of HPS.