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P A Cahill

Publications and source records attributed to P A Cahill.

At least 19 recordsLinked to original sources
PubMed2005

An assessment of the utility of the yeast GreenScreen assay in pharmaceutical screening.

In this paper we describe an initial reproducibility study of 12 proprietary compounds followed by the assessment of 51 marketed pharmaceuticals and, lastly, a summary of the data so far from 2698 proprietary compounds from the Johnson & Johnson (J&J) compound library, in the yeast GreenScreen assay (GSA). In this assay, a reporter system in the yeast cells employs the DNA damage inducible promoter of the RAD54 gene, fused to the extremely stable green fluorescent protein (GFP). The assay proved to be very robust, the Excel templates provided by Gentronix with the assay interfaced well with in-house J&J systems with little adaptation, the assay was very rapid to perform and used very little compound. The results confirm previous work which suggests that the yeast GSA detects different classes of genotoxic compounds to the Ames assay and as a result can help screen out important genotoxic compounds at the pre-regulatory test phase that are missed by Ames-test-based screens alone. A combination of SAR evaluation of genotoxicity plus an Ames-test-based screen and the GSA provides a powerful pre-regulatory test battery to aid in the selection of successful drug candidates.

Biological Assay↗
PubMed2004

The GreenScreen genotoxicity assay: a screening validation programme.

A yeast (Saccharomyces cerevisiae) DNA repair reporter assay termed the GreenScreen assay (GSA) is described. This is a novel, cost-effective genotoxicity screen, developed to provide a pre-regulatory screening assay for use by the pharmaceutical industry and in other applications where significant numbers of compounds need to be tested. It provides a higher throughput and a lower compound consumption than existing eukaryotic genotoxicity assays and is sensitive to a broad spectrum of mutagens and, importantly, clastogens. We describe a simple, robust assay protocol and a validation study. The end-point of the test reflects the typically eukaryotic chromosomes and DNA metabolizing enzymes of yeast. The capacity for metabolic activation (MA) in yeast is limited compared with the mammalian liver or its extracts, but the assay does detect a subset of compounds that would require MA in existing genotoxicity tests. The GSA detects a different spectrum of compounds to bacterial genotoxicity assays and thus, together with an in silico structure-activity relationship (SAR) screen, and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests.

DNA Helicases↗
PubMed2003

Novel roles of neuropeptide processing enzymes: EC3.4.24.15 in the neurome.

Neuropeptide processing metalloenzymes, such as angiotensin converting enzyme, neprilysin, endothelin converting enzyme, neurolysin, and EC3.4.24.15 (EP24.15), are central to the formation and degradation of bioactive peptides. We present EP24.15 as a paradigm for novel functions ascribed to these enzymes in the neurome. Although the neurome typically encompasses proteomes of the brain and central nervous system, exciting new roles of these neuropeptidases have been demonstrated in other organ systems. We discuss the involvement of EP24.15 with clinical sequelae involving the use of gonadotropin-releasing hormone (GnRH; LHRH) analogs that act as enzyme inhibitors, in vascular physiology (blood pressure regulation), and in the hematologic system (immune surveillance). Hemodynamic forces, such as cyclic strain and shear stress, on vascular cells, induce an increase in EP24.15 transcription, suggesting that neuropeptidase-mediated hydrolysis of pressor/depressor peptides is likely regulated by changes in hemodynamic force and blood pressure. Lastly, EP24.15 regulates surface expression of major histocompatibility complex Class I proteins in vivo, suggesting that EP24.15 may play an important role in maintenance of immune privilege in sites of increased endogenous expression. In these extraneural systems, regulation of both neuropeptide and other peptide substrates by neuropeptidases indicates that the influence of these enzymes may be more global than was anticipated previously, and suggests that their attributed role as neuropeptidases underestimates their physiologic actions in the neural system.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Animals↗
PubMed2000

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).

Animals↗
PubMed1999

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.

Actins↗
PubMed1999

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.

Blood Circulation↗
PubMed1999

Nonanticoagulant heparin prevents coronary endothelial dysfunction after brief ischemia-reperfusion injury in the dog.

BACKGROUND: Coronary endothelial dysfunction after brief ischemia-reperfusion (IR) remains a clinical problem. We investigated the role of heparin and N-acetylheparin, a nonanticoagulant heparin derivative, in modulating coronary endothelial function after IR injury, with an emphasis on defining the role of the nitric oxide (NO)-cGMP pathway in the heparin-mediated effect. METHODS AND RESULTS: Male mongrel dogs were surgically instrumented, and the effects of both bovine heparin and N-acetylheparin on coronary endothelial vasomotor function, expressed as percent change from baseline flow after acetylcholine challenge, were studied after 15 minutes of regional ischemia of the left anterior descending artery (LAD) followed by 120 minutes of reperfusion. In dogs treated with placebo (saline), coronary vasomotor function was significantly (P</=0.03) decreased after 15 and 30 minutes of reperfusion (65+/-12% and 73+/-12%) compared with preischemia (103+/-6%). In contrast, the vasodilatory response to the endothelium-independent vasodilator sodium nitroprusside was maintained during reperfusion. Preischemic administration of both bovine heparin and N-acetylheparin (6.0 mg/kg IV) preserved coronary endothelial function throughout reperfusion. In a parallel group of dogs, nitrate/nitrite (NOx) and cGMP levels in the LAD were measured after treatment and during 15-minute reperfusion. Preischemic administration of N-acetylheparin caused a significant increase in basal NOx and cGMP levels compared with saline controls. Pretreatment with N-acetylheparin also caused a significant increase in NOx and cGMP levels in the LAD after 15 minutes of reperfusion compared with IR alone. CONCLUSIONS: These results suggest that heparin preserves coronary endothelial function after brief IR injury by a mechanism independent of its anticoagulant activity and that the effect of heparin may be mediated in part by activation of the NO-cGMP pathway.

Animals↗
PubMed1999

Altered Gq/G11 guanine nucleotide regulatory protein expression in a rat model of hepatocellular carcinoma: role in mitogenesis.

Guanine nucleotide regulatory proteins (G-proteins) represent an important transmembrane pathway whereby extra-cellular signals are transduced to intracellular signaling pathways. The mitogen-activated protein kinase (MAPK) cascade has been identified as a key factor in transducing numerous mitogenic stimuli. MAPK activity is regulated via numerous receptor types, including those linked to Gq/G11-proteins, which regulate phospholipase-C activity. We hypothesized that alterations in a Gq/G11-PLC pathway may contribute to the enhanced cellular mitogenesis characteristic of hepatocellular carcinoma (HCC), possibly via a MAPK-dependent pathway. By using an in vivo model of HCC we investigated changes in Gq/G11-protein expression in tumorigenic tissue versus adjacent, non-neoplastic liver. In addition we addressed the role of Gq/G11-proteins in the regulation of MAPK-linked mitogenesis by using rat hepatic tumorigenic cells (H4IIE) and isolated hepatocytes in culture. Western blot analysis showed significant increases in Gqalpha and G11alpha expression in tumorigenic liver versus normal liver specimens, an effect that was augmented in cultured H4IIE cells versus isolated cultured hepatocytes. Furthermore, phosphoinositol specific phospholipase-C (PLC) activity was significantly increased in HCC versus normal liver. A specific PLC inhibitor (Et-18-OCH3) caused a dose-dependent decrease in serum stimulated DNA synthesis in both cultured H4IIE cells and isolated rat hepatocytes, the H4IIE cell line showing greater sensitivity to Et-18-OCH3. In addition, serum-stimulated MAPK activity was significantly enhanced in H4IIE versus cultured hepatocytes. Moreover, treatment with Et-18-OCH3 significantly attenuated serum stimulated MAPK activity in both cultured hepatocytes and H4IIE cells. Furthermore, U73122 (Gqalpha-PLC specific uncoupler) and GP2A (Gqalpha specific inhibitor) mirrored the effects of those observed for Et-18-OCH3 whereas PD98059 (specific MEK inhibitor) completely abolished serum-stimulated DNA synthesis in tumorigenic H4IIE cells. We conclude that HCC is associated with enhanced Gq/G11-PLC expression/activity as compared with normal liver. Furthermore, a PLC-linked MAPK cascade plays a significant role in the progression of the enhanced mitogenesis characteristic of HCC.

Animals↗
PubMed1999

Enhanced Gi-protein-mediated mitogenesis following chronic ethanol exposure in a rat model of experimental hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is associated with increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the effects of chronic ethanol exposure on the expression and function of adenylyl cyclase (AC)-linked G-proteins (Gs and Gi) and growth in experimental HCC. G-protein expression and function was determined by immunoblot in the hepatic tumorigenic H4IIE cell line and isolated cultured hepatocytes in the absence or presence of ethanol (5-100 mmol/L). Chronic exposure (24 hours) to ethanol dose-dependently increased Gialpha1/2 expression in the H4IIE cell line, but not in cultured hepatocytes. Gsalpha-protein expression remained unchanged in both H4IIE cells and cultured hepatocytes following ethanol treatment. In addition, ethanol directly activated a Gi-protein, because pertussis toxin (PTx)-catalyzed, adenosine diphosphate (ADP)-dependent ribosylation of Gialpha substrates decreased following ethanol treatment. The increased functional activity of Gialpha1/2-protein expression was confirmed by demonstrating that ethanol dose-dependently inhibited basal and stimulated AC activity in H4IIE cells, while not significantly altering basal AC activity in isolated cultured hepatocytes. Furthermore, while ethanol had no significant effect on basal mitogenesis in H4IIE cells or hepatocytes, increased mitogenesis caused by direct Gialpha-protein stimulation (mastoparan M7; 10-5,000 nmol/L) was further enhanced in the presence of ethanol, an effect that was completely blocked following Gi-protein inhibition (PTx; 100 ng/mL). In contrast, activation of Gi-proteins using M7 failed to alter cellular mitogenesis in isolated cultured hepatocytes, whether in the absence or presence of ethanol. Finally, analysis of mitogen-activated protein kinase (MAPK) activity demonstrated that chronic ethanol treatment further enhanced Gi-protein-stimulated MAPK activity in hepatic tumorigenic cells. In conclusion, these data demonstrate that ethanol enhances cellular mitogenesis in experimental HCC as a result of, at least in part, a Gi-MAPK-dependent pathway. Furthermore, this effect may be caused by ethanol's direct up-regulation of the expression and activity of Gi-proteins in HCC.

Adenosine Diphosphate Ribose↗
PubMed1999

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.

Animals↗
PubMed1999

Inhibitory guanine nucleotide regulatory protein activation of mitogen-activated protein kinase in experimental hepatocellular carcinoma in vitro.

OBJECTIVE: Hepatocellular carcinoma (HCC) is associated with altered expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the interaction between Gi-proteins and the extracellular regulated kinase (ERK) component of the mitogen activated protein kinase (MAPK) cascade in regulating mitogenesis in an experimental model of HCC. DESIGN: Pharmacological agents which selectively interact with specific target proteins involved in signal transduction through a Gi-MAPK pathway have recently become available. These agents in combination with scientific assays allow us to address the role of individual components of this cascade in the regulation of mitogenesis in HCC. METHODS: These studies were performed using rat hepatic tumorigenic cells (H4IIE) and isolated cultured hepatocytes in vitro in conjunction with pharmacological agents which interact with Gi-protein or MAPK components of intracellular signalling. RESULTS: Direct activation of Gi-proteins with mastoparan M7 (M7) significantly increased nuclear thymidine incorporation in hepatic tumorigenic H4IIE cells in a dose-dependent manner (10-1000 nM, n = 4, P < 0.05), an effect that was abolished by treatment with either pertussis toxin (PTx) or the selective mitogen-activated ERK-regulated kinase (MEK) inhibitor PD098059. In contrast, M7 inhibited nuclear thymidine incorporation in serum-stimulated isolated hepatocytes. ERK2 activity was then determined as the ability of immunoprecipitated ERK2 proteins to phosphorylate the ERK substrate myelin basic protein. These studies demonstrated a time- and dose-dependent increase in ERK2 activity in H4IIE cells following Gi-protein activation with M7, a maximal response being attained at 20 min. In contrast, M7 failed to significantly alter ERK2 activity in isolated cultured hepatocytes at any of the doses or time points assayed (10-5000 nM, 10-120 min). Gi-stimulated ERK activation was completely blocked in tumorigenic cells following treatment with PTx. CONCLUSIONS: These data demonstrate for the first time a Gi-linked MAPK cascade in experimental HCC, activation of which stimulates cellular mitogenesis.

Animals↗
PubMed1999

The role of cAMP-MAPK signalling in the regulation of human hepatocellular carcinoma growth in vitro.

OBJECTIVE: We have previously identified that primary human hepatocellular carcinoma (HCC) is associated with altered guanine nucleotide regulatory protein (G-protein) expression concomitant with decreased adenylyl cyclase (AC) and increased mitogen activated protein kinase (MAPK) activity in vivo. This study aims to address the potential link between Gs protein regulation of AC activity/ cyclic adenosine monophosphate (cAMP) production and the subsequent downstream regulation of MAPK activity and mitogenesis. DESIGN: Pharmacological agents which selectively interact with specific target proteins involved in signal transduction via the Gs-AC-cAMP-MAPK signalling pathway were employed in cultured human HCC cell lines in these studies. These agents allow us to address the role of individual components of these pathways in the regulation of mitogenesis in HCC. METHODS: These studies utilized three distinct human HCC cell lines (HepG2, Hep3B and SKHep) in the absence and presence of agents that alter AC-cAMP dependent signalling. De novo DNA synthesis was determined as a marker of altered cellular proliferation, and MAPK activity was determined as the ability to catalyse myelin basic protein (MBP) phosphorylation. RESULTS: 8-Bromo-cAMP (8-Br-cAMP; a cell-permeable cAMP analogue) and forskolin (AC activator) dose-dependently decreased thymidine incorporation in all three cell lines. In addition, serum-stimulated [3H] thymidine incorporation was significantly decreased in HepG2, Hep3B and SKHep cell lines following treatment with either 8-Br-cAMP or forskolin. By contrast, MDL12330A (MDL; irreversible AC inhibitor) enhanced thymidine incorporation in all three cell lines. Treatment with either 8-Br-cAMP or forskolin significantly decreased serum-stimulated MAPK activity. CONCLUSIONS: These data suggest that cAMP acts as an anti-mitogenic agent in these hepatic tumorigenic cell lines in vitro such that inhibition of AC activity promotes MAPK activity and cellular mitogenesis in HCC.

Carcinoma, Hepatocellular↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1998

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.

Animals↗