Role of cyclooxygenase isoforms in prostacyclin biosynthesis and murine prehepatic portal hypertension.

Portal hypertension (PHT) is a common complication of liver cirrhosis and significantly increases morbidity and mortality. Abrogation of PHT using NSAIDs has demonstrated that prostacyclin (PGI(2)), a direct downstream metabolic product of cyclooxygenase (COX) activity, is an important mediator in the development of experimental and clinical PHT. However, the role of COX isoforms in PGI(2) biosynthesis and PHT is not fully understood. Prehepatic PHT was induced by portal vein ligation (PVL) in wild-type, COX-1(-/-), and COX-2(-/-) mice treated with and without COX-2 (NS398) or COX-1 (SC560) inhibitors. Hemodynamic measurements and PGI(2) biosynthesis were determined 1-7 days after PVL or sham surgery. Gene deletion or pharmacological inhibition of COX-1 or COX-2 attenuated but did not ameliorate PGI(2) biosynthesis after PVL or prevent PHT. In contrast, treatment of COX-1(-/-) mice with NS398 or COX-2(-/-) mice with SC560 restricted PGI(2) biosynthesis and abrogated the development of PHT following PVL. In conclusion, either COX-1 or COX-2 can mediate elevated PGI(2) biosynthesis and the development of experimental prehepatic PHT. Consequently, PGI(2) rather then COX-selective drugs are indicated in the treatment of PHT. Identification of additional target sites downstream of COX may benefit the >27,000 patients whom die annually from cirrhosis in the United States alone.

Endothelial dysfunction in cirrhosis and portal hypertension.

Portal hypertension (PHT) is a common clinical syndrome associated with chronic liver diseases; it is characterized by a pathological increase in portal pressure. Pharmacotherapy for PHT is aimed at reducing both intrahepatic vascular tone and elevated splanchnic blood flow. Due to the altered hemodynamic profile in PHT, dramatic changes in mechanical forces, both pressure and flow, may play a pivotal role in controlling endothelial and vascular smooth muscle cell signaling, structure, and function in cirrhotics. Nitric oxide, prostacyclin, endothelial-derived contracting factors, and endothelial-derived hyperpolarizing factor are powerful vasoactive substances released from the endothelium in response to both humoral and mechanical stimuli that can profoundly affect both the function and structure of the underlying vascular smooth muscle. This review will examine the contributory role of hormonal- and mechanical force-induced changes in endothelial function and signaling and the consequence of these changes on the structural and functional response of the underlying vascular smooth muscle. It will focus on the pivotal role of hormonal and mechanical force-induced endothelial release of vasoactive substances in dictating the reactivity of the underlying vascular smooth muscle, i.e., whether hyporeactive or hyperreactive, and will examine the extent to which these substances may exert a protective and/or detrimental influence on the structure of the underlying vascular smooth muscle in both a normal hemodynamic environment and following hemodynamic perturbations typical of PHT and cirrhosis. Finally, it will discuss the intracellular processes that regulate the release/expression of these vasoactive substances and that control the transformation of this normally protective cell to one that may promote the development of vasculopathy in PHT.

Endothelial cells inhibit flow-induced smooth muscle cell migration: role of plasminogen activator inhibitor-1.

BACKGROUND: The endothelium may play a pivotal role in hemodynamic force-induced vascular remodeling. We investigated the role of endothelial cell (EC) plasminogen activator inhibitor-1 (PAI-1) in modulating flow-induced smooth muscle cell (SMC) migration. METHODS AND RESULTS: Human SMCs cocultured with or without human ECs were exposed to static (0 mL/min) or flow (26 mL/min; shear stress 23 dyne/cm(2)) conditions for 24 hours in a perfused capillary culture system. SMC migration was then assessed with a Transwell migration assay. In the absence but not in the presence of ECs, pulsatile flow significantly increased the migration of SMCs (264+/-26%) compared with SMCs under static conditions, concomitant with a 3- and 4-fold increase in PAI-1 mRNA and protein, respectively, in cocultured ECs. In the presence of PAI-1-/- ECs, flow increased wild-type SMC migration (226+/-25%), an effect that was reversed by exogenous PAI-1. To determine whether the antimigratory activity of PAI-1 was dependent primarily on inhibition of PAs or its association with vitronectin, experiments were conducted with PAI-1R (a mutant PAI-1 that binds to vitronectin but does not inhibit PA) and PAI-1K (a mutant that inhibits PA but has reduced affinity for vitronectin). PAI-1R inhibited both basal and flow-induced migration, whereas PAI-1K inhibited flow-induced migration in the absence of any effect on baseline migration. CONCLUSIONS: Flow-induced EC PAI-1 inhibits flow-induced SMC migration in vitro. EC PAI-1 expression may be one of the predominant mechanisms responsible for controlling the process of vascular remodeling.

Potential mechanisms for cardiovascular protective effect of ethanol.

Epidemiological studies demonstrate a significant protective effect of moderate alcohol consumption on the incidence of cardiovascular diseases which accounts for the majority of deaths in the Western world. In this review, possible mechanisms to explain the cardioprotective effect of ethanol are discussed. While the prevailing theory supported by a number of clinical and animal studies indicates that the ability of ethanol to elevate serum high-density lipoprotein (HDL) cholesterol levels is an important mechanism in ameliorating cardiovascular disease, other mechanisms whereby ethanol could exert its beneficial effect have been proposed. Namely, its ability to affect platelet function and endothelial cell and vascular smooth muscle cell function (In this review, the terms alcohol and ethanol are used interchangeably).

Phenotype dictates the growth response of vascular smooth muscle cells to pulse pressure in vitro.

The objective of this study was to determine the effect of phenotype on pulse pressure-induced signaling and growth of vascular smooth muscle cells in vitro. Using a perfused transcapillary culture system, cells were exposed to increases in pulsatile flow and hence pulse pressure and maintained for 72 h before cells were harvested. Cell proliferation was determined by cell number, DNA synthesis, and proliferating cell nuclear antigen expression. Mitogen-activated protein kinase (MAPK) levels were determined by immunoblot and kinase activity by phosphorylation of myelin basic protein. Cell phenotype was determined by immunoblot and immunocytofluorescence using antisera specific for the differentiation markers alpha-actin, myosin, calponin, osteopontin, and phospholamban. In cells that highly expressed these differentiation markers, there was a significant increase in cell growth in response to chronic increases in pulse pressure without a significant change in MAPK activity in these cells. In contrast, in cells that weakly expressed SMC differentiation markers, there was a significant decrease in cell growth concomitant with a significant decrease in MAPK signaling in these cells. We conclude that SMC phenotype dictates the growth response of SMC to mechanical force in vitro.

Ethanol inhibits basal and flow-induced vascular smooth muscle cell migration in vitro.

BACKGROUND: Alcohol consumption protects against coronary heart disease by as yet unclear mechanisms. The aim of this study was to determine the effect of ethanol on vascular smooth muscle cell (SMC) migration which plays an important role in the pathogenesis of atherosclerosis. MATERIALS AND METHODS: Cultures of human SMC under static (no flow) or pulsatile flow conditions (perfused transcapillary culture system) were pretreated in the absence or presence of ethanol (EtOH) whereupon their random migration (chemokinesis) was assessed by Transwell assay. RESULTS: Ethanol pretreatment (24 h) dose dependently inhibited migration of HuSMC from static cultures with a maximal inhibition of 60.8 +/- 4.4% observed at 40-80 mM, in the absence of any effect on cell adhesion or cell viability as assessed by trypan blue exclusion. In HuSMC exposed to pulsatile flow (0.3 to 25 ml/min, 24 h), there was a flow-dependent increase in migration ranging from a 1.3 +/- 0.16- to 2.67 +/- 0.26-fold increase, compared to static cells, concomitant with a significant increase in urokinase-type plasminogen activator (uPA) mRNA levels. Ethanol pretreatment (20-80 mM, 24 h) dose dependently inhibited the flow-induced increase in SMC migration but did not affect uPA mRNA expression. CONCLUSIONS: The inhibitory effect of ethanol on basal and flow-stimulated SMC migration may be relevant to its cardiovascular effects in vivo.

Ethanol enhances basal and flow-stimulated nitric oxide synthase activity in vitro by activating an inhibitory guanine nucleotide binding protein.

The aim of this study was to determine the effect of ethanol on endothelial nitric oxide synthase (eNOS), the enzyme responsible for the production of the important vasoactive agent nitric oxide. The effect of ethanol (0.8-160 mM) on both basal and flow-stimulated eNOS activity was determined using cultured bovine aortic endothelial cells (EC). In "static" EC ethanol dose-dependently increased basal eNOS activity with a maximum response (approximately 2.0-fold increase) achieved at 40 mM in the absence of any effect on cell viability or nitric oxide synthase protein expression. Pertussis toxin (PTX) pretreatment significantly inhibited the ethanol-induced increase in basal eNOS activity. EC exposed to steady laminar flow exhibited a flow- and time-dependent increase in eNOS activity. Ethanol significantly enhanced the laminar flow-induced eNOS response from 0.62 +/- 0.1 to 1.06 +/- 0. 06 pmol [14C]citrulline/mg/min, a response that was inhibited by PTX. PTX-catalyzed ribosylation of Gialpha substrates, an index of G-protein functional activity, was increased in laminar flow-exposed EC compared with static controls and was further enhanced by ethanol treatment. Likewise, EC exposed to low ( approximately 0.5 dynes/cm2) and high ( approximately 12 dynes/cm2) pulsatile flow demonstrated increased eNOS activity, an effect that was associated with increased PTX-catalyzed ribosylation of Gialpha substrates. Ethanol enhanced the low flow response in a PTX-sensitive manner. These data demonstrate a stimulatory effect of ethanol on basal and flow-stimulated eNOS activity, mediated in part by a mechanism involving a PTX-sensitive G protein.

Sustained pulsatile flow regulates endothelial nitric oxide synthase and cyclooxygenase expression in co-cultured vascular endothelial and smooth muscle cells.

This study addresses the effect of sustained increased pulsatile flow on nitric oxide synthase (NOS) and cyclooxygenase (Cox) expression and activity in co-cultured endothelial cells (EC) and vascular smooth muscle cells (SMC). Using a perfused transcapillary co-culture system which permits the chronic exposure of cultured EC and SMC to physiological shear stresses, co-cultures were exposed to step-wise increases in flow up to: (i) 2 ml/min (low flow: 0.5 dyn/cm2): or (ii) 44 ml/min (high flow: 15 dyn/cm2) and maintained for 72 h before SMC and EC were harvested separately. There was no NOS activity or protein expression in co-cultured SMC under flow conditions. There was a significant increase in eNOS activity in co-cultured EC under high flow conditions, compared to low flow, which correlated with an increase in eNOS expression and mRNA levels. The flow-induced increase in eNOS activity was potentiated by indomethacin treatment, suggesting a modulatory role for a cyclooxygenase product. Prostacyclin levels in co-culture perfusate were significantly elevated under high flow conditions. While both co-cultured EC and SMC expressed cyclooxygenase (Cox-I and Cox-II), they were differentially regulated by pulsatile flow, EC Cox-I and Cox-II protein expression were both decreased. Indomethacin treatment increased the expression of both Cox-I and Cox-II in co-cultured SMC under high flow conditions. We conclude that sustained increases in pulsatile flow maintain elevated eNOS and Cox protein expression and activity in EC while decreasing Cox expression in co-cultured SMC. These data suggest that regulation of these pathways may contribute to flow-induced vascular remodeling in vivo.

Effect of pulse pressure on vascular smooth muscle cell migration: the role of urokinase and matrix metalloproteinase.

Plasminogen activator (PA) expression plays an important role in smooth muscle cell (SMC) migration and may therefore contribute to mechanical force-induced arterialization of vein grafts. The aim of this study was to determine whether pulse pressure due to pulsatile flow modulates SMC migration via urokinase (u-PA)-dependent mechanisms. Using a perfused transcapillary culture system, human umbilical vein SMC were exposed to pulse pressures (0-56 mmHg), in the absence or presence of human umbilical vein endothelial cells (EC) by varying pulsatile flow rates (0 ml/min to 25 ml/min). SMC cultured in the absence of EC increased their migration following exposure to increased pulse pressure (248+/-14%). Both u-PA and matrix metallo-proteinase 1 (MMP-1) expression was significantly elevated in SMC exposed to pressure as compared to static controls. The role of proteases in the pulse pressure-induced enhancement of SMC migration was confirmed following pretreatment with aprotinin, an anti u-PA antibody and metalloproteinase inhibitors (181+/-14% for aprotinin vs. 256+/-25% for control, 108+/-4% for anti-u-PA antibody vs. 233+/-17% for non-immune IgG, and 114+/-9% for BB-94, 105+/-7% for BB-3103 vs. 222+/-5% for control). Using SMC derived from u-PA gene knock-out mice, the SMC migratory response to increased pulse pressure was completely inhibited despite a significant increase in MMP expression in these cells. These results suggest that pulse pressure due to pulsatile flow induces SMC migration in vitro via u-PA and MMP-dependent mechanisms. Moreover, u-PA gene deletion results in blunting of pressure-induced SMC migration despite the endogenous upregulation of metalloproteinase. Modulation of u-PA expression by pressure may thus represent an important mechanism whereby hemodynamic forces regulate smooth muscle cell migration.

Increased expression of endothelin receptors in the vasculature of portal hypertensive rats: role in splanchnic hemodynamics.

Portal hypertension (PHT) is characterized by increased portal pressure caused in part by a reduction in mesenteric vascular resistance. The aim of this study was to evaluate the role of endothelin (ET) and specific ET receptors in maintaining the vasculopathy of PHT. PHT was created in Sprague-Dawley rats by a partial portal vein ligation. Control animals were sham-operated. ET receptor expression was determined in the superior mesenteric artery of sham and PHT rats by in situ autoradiography, radioligand binding analysis, and reverse-transcription polymerase chain reactions (RT-PCR). The pressor response to ET-1 was determined in vitro using isolated vascular rings and in vivo by measuring mean arterial pressure, splanchnic blood flow, and portal venous pressure following treatment with ET and selective ET receptor antagonists. The pressor response to ET in vitro was significantly enhanced in PHT concomitant with increased ET-A and ET-B receptor expression. There was a significant increase in the peak pressor response to ET (10 microg/kg intravenously) in portal hypertensive rats without any significant change in plasma ET-1 levels. There was no significant difference in the peak splanchnic blood flow or portal venous pressure response following ET-A receptor blockade with JKC-301 infusion (200 microg/kg intravenously). In contrast, ET-B receptor blockade with IRL-1038 (200 microg/kg intravenously) preferentially decreased splanchnic blood flow and portal venous pressure in portal hypertensive rats. These data suggest that enhanced ET-B receptor expression in portal hypertensive vessels contributes to the maintenance of elevated portal pressure in these animals.

Flow-mediated regulation of G-protein expression in cocultured vascular smooth muscle and endothelial cells.

G-proteins have been implicated in the transduction of a number of flow-induced responses. We determined whether flow can modulate vascular endothelial or smooth muscle cell G-protein signaling. By use of a perfused transcapillary coculture system that permits the chronic exposure of cultured endothelial cells (ECs) and smooth muscle cells (SMCs) to physiological shear stresses, cocultures were exposed to stepwise increases in flow up to (1) 2 mL/min (low flow: 0.5 dyne/cm2), or (2) 44 mL/min (high flow: 15 dyne/cm2) and maintained for 72 hours before SMCs and ECs were harvested separately. Using Western blot analysis, EC Gi alpha3 expression was significantly increased (41+/-2.9%) by high-flow conditions compared with low-flow. The changes in G-protein expression were associated with a significant increase in endothelial nitric oxide synthase (eNOS) activity, elevated prostacyclin levels in the perfusing media, increased pertussis toxin-catalyzed ADP ribosylation of Gi alpha substrates, and enhanced agonist-stimulated GTPase activity in cocultured ECs. In contrast, high flow induced a significant decrease in Gi alpha1-2 expression (57 5%) in SMCs cocultured with ECs, an effect that was endothelium dependent, inhibited by indomethacin, and correlated with a decrease in pertussis toxin-catalyzed ADP ribosylation of Gi alpha substrates, reduced agonist-stimulated GTPase activity, and enhanced basal and G-protein-stimulated adenylyl cyclase activity. These data demonstrate that flow mediates selective changes in EC and SMC G-protein expression concomitant with changes in G-protein functionality and cellular signaling capacity. Moreover, flow-induced changes in SMC G-protein signaling capacity are endothelium dependent and require a cyclooxygenase product. G-protein modulation may thus represent an important mechanism whereby hemodynamic forces regulate vessel wall function.

Enhanced cyclooxygenase-1 expression within the superior mesenteric artery of portal hypertensive rats: role in the hyperdynamic circulation.

Portal hypertension (PHT) is characterized by splanchnic hyperemia due to enhanced production of vasodilator substances. Enhanced vasodilation and increased splanchnic blood flow contribute to the elevated portal pressure characteristic of PHT. The aim of this study was to determine whether cyclooxygenase (Cox) expression is altered in PHT vessels and whether chronic inhibition of this enzyme impacts on splanchnic blood flow in PHT. PHT was created in Sprague-Dawley rats by a partial portal vein ligation. Control animals were sham operated. Plasma 6-keto-PGF1alpha (prostaglandin F1alpha) levels were significantly elevated in PHT after 2 days as compared with sham and remained elevated up to day 15. Treatment with indomethacin (2 mg/kg i.p. daily for 15 days) resulted in a significant decrease in 6-keto-PGF1alpha levels, which was concomitant with a significant decrease in superior mesenteric artery blood flow (Qsma) after 15 days in PHT rats. Cox-I expression was differentially enhanced in the PHT superior mesenteric artery and thoracic aorta during the development and progression of PHT. In contrast, Cox-II messenger RNA (mRNA) and protein expression was not detected in either of these vessels throughout the development of PHT. These data suggest that PHT is associated with enhanced Cox-I expression within the splanchnic vasculature concomitant with elevated plasma prostacyclin levels and a significant pressor response to indomethacin in PHT animals. We conclude that enhanced Cox-I expression results in increased prostacyclin levels that partially contribute to the maintenance of the hyperemia typical of PHT.

Non-anticoagulant heparin increases endothelial nitric oxide synthase activity: role of inhibitory guanine nucleotide proteins.

Heparin, which is widely used clinically, has recently been shown to have specific properties affecting the vascular endothelium. We hypothesized that heparin stimulates endothelial nitric oxide synthase (eNOS) activity by a mechanism independent of its anticoagulant properties and dependent on an inhibitory guanine nucleotide regulatory protein (Gi). We determined the effect of both heparin and N-acetyl heparin (Non-Hep), a heparin derivative without anticoagulant properties, on eNOS activity in cultured bovine aortic endothelial cells and on endothelium-dependent relaxation in isolated vascular rings. The eNOS activity was determined by measuring both citrulline and nitric oxide (NO) metabolite formation. Heparin and Non-Hep dose-dependently increased basal eNOS activity (ED50 1.0 microgram/ml or 0.15 U/ml), an effect that was significantly inhibited by pertussis toxin (100 ng/ml), a Gi-protein inhibitor. Agonist-stimulated (acetylcholine, 10 microM) eNOS activity was potentiated following pre-treatment with both heparin and Non-Hep and reversed by pertussis toxin. Heparin and Non-Hep induced a dose-dependent relaxation in preconstricted thoracic aortic rings, an effect that was significantly inhibited by pertussis toxin, endothelial inactivation (following treatment with sodium deoxycholate) and NG-nitro-L-arginine-methyl ester (L-NAME). We conclude that heparin and non-anticoagulant heparin induce endothelium-dependent relaxation following activation of eNOS by a mechanism involving a Gi-protein. Administration of heparin derivatives without anticoagulant properties may have therapeutic implications for the preservation of eNOS in conditions characterized by endothelial dysfunction.

Ethanol inhibits mitogen activated protein kinase activity and growth of vascular smooth muscle cells in vitro.

The aim of this study was to determine the effect of ethanol on vascular smooth muscle cell proliferation and mitogen activated protein kinase (MAPK) signaling. Rat aortic smooth muscle cell growth in vitro was determined by measuring cell counts and [3H]thymidine incorporation. MAPK signaling was determined by assessing MEK (also referred to as MAPK kinase) activity by measuring phosphorylated extracellular signal-regulated kinase (pp44ERK - 1 and pp42ERK - 2) expression, and ERK activity by measuring ERK-2-dependent phosphorylation of myelin basic protein (MBP). In quiesced smooth muscle cells, ethanol treatment (24 h) inhibited serum-stimulated mitogenesis in a dose-dependent manner, (IC50 = 60 mM), in the absence of any effect on smooth muscle cell viability. In addition, ethanol treatment caused a significant shift to the right in the smooth muscle cell growth curve, extending the population doubling time from approximately 48 h (control) to approximately 70 h (ethanol). Acute (15 min) ethanol treatment reduced serum-stimulated pp44ERK - 1 and pp42ERK - 2 expression in a dose dependent fashion; 24.5+/-1.5% and 77.6+/-3.2% inhibition for 20 mM and 160 mM ethanol, respectively. Furthermore, there was a significant dose-dependent decrease in ERK2 activity in ethanol treated smooth muscle cells as compared to control smooth muscle cells. These data demonstrate an inhibitory effect of ethanol on smooth muscle cell proliferation and MAPK signalling in vitro. It is tempting to speculate that these actions of ethanol may contribute to its cardiovascular effects in vivo.

Potential paracrine role of the pericardium in the regulation of cardiac function.

OBJECTIVE: Both coronary and endocardial endothelium regulate cardiac contractile function via paracrine pathways. We investigated whether pericardial fluid (PF) and pericardial mesothelial cells (PMC) could exert a similar paracrine action. METHODS: Both PF and PMC were extracted from sheep pericardial space. Endothelin-1, prostaglandins and atrial natriuretic factor were measured in PF in vivo. In the other hand, PMC were grown on T-75 flasks and microcarrier beads to investigate endothelin-1, nitric oxide and prostaglandin pathways in vitro. In addition, effects of PF and PMC effluent were tested on adult rat cardiac myocyte contraction in vitro. RESULTS: In vitro, cultured PMC expressed endothelin-1 mRNA but not the endothelial nitric oxide synthase III, and released endothelin-1 and prostaglandins. Both PF and cultured PMC superfusate induced a potent, rapidly reversible decrease in the shortening of isolated rat cardiac myocytes. This effect was not associated with changes in intracellular calcium. In vivo, prostaglandins, atrial natriuretic factor and endothelin were present in PF. A greater concentration of atrial natriuretic factor was present in PF than in serum, suggesting molecular diffusion from the myocardium to PF. Preliminary results show that the instillation of vasoactive agents into the pericardial space of dogs rapidly alter coronary and systemic vascular tone, consistent with a molecular diffusion of these substances from PF into the myocardium and circulation. CONCLUSIONS: In addition to its mechanical role, the pericardium may contribute to the integration and the regulation of cardiovascular function via a paracrine mechanism.

Enhanced G-protein-induced relaxation in portal hypertensive rats: role of nitric oxide.

Portal hypertension (PHT) is characterized by splanchnic hyperemia due to a reduction in mesenteric vascular resistance. The reasons for the decreased resistance include an increased responsiveness to a vasodilator substance. Because the activation of an inhibitory guanine nucleotide regulatory (Gi) protein can result in endothelium-dependent relaxation, we tested the hypothesis that exaggerated Gi-protein induced relaxation via a nitric oxide (NO)-dependent pathway partly reflects the enhanced Gi-protein expression in PHT vessels. PHT was created in Sprague-Dawley rats by a partial portal-vein ligation. Control animals were sham operated. Using isolated vascular rings in the absence or presence of an intact endothelium, N(G)-nitro-L-arginine methyl ester (L-NAME), and pertussis toxin, dose response relationships for sodium fluoride (NaF; range, 0.1-4 mmol/L), a Gi protein activator, were determined in a cumulative manner. Gi-protein expression was determined by Western blotting. NaF caused a dose-dependent relaxation in both sham and portal hypertensive pre-contracted vessels, an effect that was significantly inhibited by pertussis toxin, endothelial denudation, and L-NAME. Concentrations of NaF greater than 4 mmol/L caused contractions, an effect that was unaffected by L-NAME. The NaF-induced relaxation response was significantly greater in PHT vessels as compared with sham concomitant with increased Gi-protein expression in PHT vessels. These data suggest that the enhanced endothelial Gi-protein-induced relaxation in PHT vessels may partly reflect enhanced expression of Gi-proteins in PHT vessels and may, thus, represent an important mechanism for exaggerated NO-dependent relaxation in the PHT vasculature.

Flow-mediated regulation of endothelin receptors in cocultured vascular smooth muscle cells: an endothelium-dependent effect.

The objective of this study was to determine the effect of pulsatile flow on endothelin (ET) receptor expression in vascular smooth muscle cells (VSMC) cocultured with endothelial cells (EC). Using a perfused transcapillary coculture system which permits the chronic exposure of cultured EC and VSMC to physiological shear stresses, cocultures were exposed to stepwise increases in flow up to (1) 2 ml/min (low flow: 0.5 dyn/cm2) or (2) 44 ml/min (high flow: 15 dyn/cm2) and maintained for 72 h before SMC and EC were harvested separately. There was a significant increase in [125I]-ET-1 binding in cocultured VSMC exposed to high flow as compared to low flow (Bmax: 75 +/- 22 vs. 152 +/- 10 fmol [125I]ET-1 bound/mg protein) in the absence of any change in the affinity (KD) of ET-1 for its receptor. ET-1 peptide mRNA levels were significantly decreased in EC exposed to high flow. The increase in [125I]ET-1 binding was associated with an increase in ET-A and ET-B receptor mRNA levels and was EC dependent as [125I]ET-1 binding in monocultured VSMC was the same, regardless of flow conditions. However, the amount of [125I]ET-1 binding on VSMC cultured in the absence of EC was significantly greater than that on cocultured VSMC. High flow caused a significant increase in endothelial nitric oxide synthase (NOS) activity in EC and prostacyclin levels in the perfusing medium. Flow-mediated upregulation of ET receptors was diminished by treatment with NG-nitro-L-arginine-methyl ester, a NOS inhibitor, whereas indomethacin, a cyclooxygenase inhibitor, had no significant effect. Collectively, these data suggest that flow-induced changes in ET receptor expression in VSMC are endothelium dependent and are in part mediated by nitric oxide. Modulation of ET receptor expression by EC may thus represent an important mechanism whereby hemodynamic forces regulate vessel wall function.

Increased endothelial nitric oxide synthase activity in the hyperemic vessels of portal hypertensive rats.

BACKGROUND/AIM: Portal hypertension is characterized by splanchnic hyperemia due to a reduction in mesenteric vascular resistance. Mediators of this hyperemia include nitric oxide. This is based on several reports indicating a marked splanchnic hyporesponsiveness in portal hypertension to vasoconstrictor stimuli both in vitro and in vivo, and a subsequent reversal using specific inhibitors of nitric oxide synthase. The objective of this study was to determine firstly whether the functional activity and/or expression of nitric oxide synthase is altered in portal hypertensive vasculature and secondly which isoenzyme form was responsible for the preferential response to nitric oxide blockade in these animals. METHODS: We compared nitric oxide synthase functional activity in the hyperemic vasculature of sham and portal hypertensive rats (following partial portal vein ligation). Nitric oxide synthase activities were determined by measuring the conversion of L-arginine to citrulline using ion-exchange chromatography and the amount of immunodetectable nitric oxide synthase in sham and portal hypertensive vessels was determined by Western blot. RESULTS: Ca(2+)-dependent nitric oxide synthase activity was significantly elevated (p < 0.05) in portal hypertensive particulate fractions from the superior mesenteric artery, thoracic aorta and portal vein. Vascular tissue cGMP levels and plasma nitrite levels were both significantly elevated in portal hypertension. Immunodetection with specific antisera raised against the inducible nitric oxide synthase demonstrated a lack of induction within the hyperemic vasculature. Immunodetection with antisera against endothelial nitric oxide synthase showed a significant increase in portal hypertensive portal vein only. These results demonstrate enhanced calcium-dependent nitric oxide synthase activity in portal hypertension hyperemic vessels concurrent with elevated tissue cGMP levels. CONCLUSION: We conclude that enhanced endothelial nitric oxide synthesis may in part contribute to the hyperdynamic circulation of portal hypertension.

Regulation of endothelin receptors by nitric oxide in cultured rat vascular smooth muscle cells.

Two important mediators of endothelium-dependent regulation of vascular smooth muscle tone and proliferation are nitric oxide (NO) and endothelin (ET-1). An imbalance between NO and ET-1 may contribute to the alterations in vascular tone characteristic of cardiovascular disease. The objective of this study was to determine whether NO regulates ET receptors in cultured rat superior mesenteric artery vascular smooth muscle cells (RVSMC). Chronic treatment of quiescent RVSMC with any one of three chemically dissimilar NO-generating drugs, S-nitroso-N-acetyl penicillamine (SNAP), sodium nitroprusside (SNP), and isosorbide dinitrate (ISDN) produced a significant dose- and time-dependent increase in the number of ET-A receptors, while concomitantly increasing the affinity of ET-1 for this receptor. This effect was mimicked by both 8-bromo-cGMP and 8-bromo-cAMP. The requirement of both protein and RNA synthesis and activation of a cAMP-dependent protein kinase (A-kinase) was demonstrated following inhibition of this regulation by cycloheximide, actinomycin D and KT5720 (a specific A-kinase inhibitor), respectively. In addition, the cytokine interleukin 1 beta (IL-1 beta) which induced NOS activity with subsequent NO synthesis in vascular smooth muscle, also caused a similar upregulation of ET receptors. This effect was attenuated in the presence of the specific NOS inhibitor, L-NAME. To assess the possible functional consequences of this NO-mediated upregulation, the effect of SNAP pretreatment on isolated vessel reactivity was determined. In both superior mesenteric artery and thoracic aorta rings, SNAP pretreatment caused a significant increase in the maximal force of contraction to ET-1. Collectively, these data suggest that NO regulates ET-A receptors in vitro through a cGMP-dependent mechanism via activation of the cAMP-dependent protein kinase. We conclude that a similar interaction between NO and ET-1 may be operational in vivo.