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1.
Am J Physiol Cell Physiol ; 304(4): C324-33, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23195071

ABSTRACT

Cardiotonic steroids (CTS) of the strophanthus and digitalis families have opposing effects on long-term blood pressure (BP). This implies hitherto unrecognized divergent signaling pathways for these CTS. Prolonged ouabain treatment upregulates Ca(2+) entry via Na(+)/Ca(2+) exchanger-1 (NCX1) and TRPC6 gene-encoded receptor-operated channels in mesenteric artery smooth muscle cells (ASMCs) in vivo and in vitro. Here, we test the effects of digoxin on Ca(2+) entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 µg·kg(-1)·day(-1) for 3 wk) did not raise BP and had no effect on resting cytolic free Ca(2+) concentration ([Ca(2+)](cyt)) or phenylephrine-induced Ca(2+) signals in isolated ASMCs. Expression of transporters in the α2 Na(+) pump-NCX1-TRPC6 Ca(2+) signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated α2 Na(+) pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ~4.5-fold) were observed in ASMCs from rats treated with ouabain but not digoxin. Moreover, in primary cultured ASMCs from normal rats, treatment with digoxin (100 nM, 72 h) did not upregulate NCX1 and TRPC6 but blocked the ouabain-induced upregulation of these transporters. Pretreatment of ASMCs with the c-Src inhibitor PP2 (1 µM; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analog eliminated the effect of ouabain on NCX1 and TRPC6 expression and ATP-induced Ca(2+) entry. Thus, in contrast to ouabain, the interaction of digoxin with α2 Na(+) pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca(2+) signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP.


Subject(s)
Calcium Signaling/drug effects , Cardiotonic Agents/pharmacology , Digoxin/pharmacology , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , Ouabain/pharmacology , src-Family Kinases/metabolism , Animals , Aorta/cytology , Calcium Channels/metabolism , Cardiotonic Agents/administration & dosage , Cells, Cultured , Digoxin/administration & dosage , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Male , Mesenteric Arteries/cytology , Myocytes, Smooth Muscle/drug effects , Nifedipine/pharmacology , Ouabain/administration & dosage , Phosphorylation , Protein Processing, Post-Translational/drug effects , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Sodium-Calcium Exchanger/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , TRPC Cation Channels/metabolism , src-Family Kinases/antagonists & inhibitors
2.
Am J Hypertens ; 24(2): 241-6, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20966899

ABSTRACT

BACKGROUND: A silent polymorphism (+1166 A/C single-nucleotide polymorphism) localized in the 3'-UTR (untranslated region) of the human angiotensin II type-1 receptor (AT1R) has been associated with hypertension and cardiovascular complications. The +1166 A/C is recognized by a specific microRNA-155 (miR-155), which is base-pairing complementary with the +1166 A-allele but not with the mutant +1166 C allele. Aim of our study was to investigate the interplay between miR-155 and AT1R protein expression. METHODS: Sixty-four subjects were selected for the +1166 A/C from the cohort of hypertensives (n = 573) of the Hypertension and Ambulatory Recording Venetia Study (HARVEST): 25 were homozygous for the 1166 A allele, 20 heterozygous, and 19 homozygous for the 1166 C allele. RESULTS: miR-155 expression was significantly decreased in subjects with CC genotype in comparison to AA and AC genotype. AT1R protein expression was significantly increased in the CC group in comparison to AA and AC (P < 0.01) although AT1R mRNA expression was not significantly different in the three groups. AT1R protein expression was positively correlated with systolic and diastolic blood pressure and negatively correlated with miR-155 expression level. Plasma transforming growth factor-ß1 (TGF-ß1) may have a modulator role in the interplay between miR-155 and AT1R protein expression as it was correlated negatively with miR-155 expression and positively with AT1R protein expression in subjects with CC genotype. CONCLUSION: The interplay between miR-155 expression, +1166C polymorphism, and AT1R protein expression may have a role in the regulation of blood pressure.


Subject(s)
Blood Pressure/genetics , Hypertension/genetics , MicroRNAs/analysis , Polymorphism, Genetic , Receptor, Angiotensin, Type 1/genetics , 3' Untranslated Regions , Adult , Age of Onset , Female , Genetic Predisposition to Disease , Heterozygote , Homozygote , Humans , Hypertension/blood , Hypertension/epidemiology , Hypertension/physiopathology , Italy/epidemiology , Male , Phenotype , Prospective Studies , RNA, Messenger/blood , Receptor, Angiotensin, Type 1/blood , Transforming Growth Factor beta1/blood
3.
J Hypertens ; 28(5): 1104-8, 2010 May.
Article in English | MEDLINE | ID: mdl-20375904

ABSTRACT

OBJECTIVE: RGS2 (regulators of G-protein signalling) is a negative regulator of Galphaq protein signalling, which mediates the action of several vasoconstrictors. Low RGS2 expression increases G-protein-coupled signalling in hypertensive patients. The aim of the present study was to correlate RGS2 expression in peripheral blood mononuclear cells (PBMs) with response to antihypertensive therapy in never-treated patients with essential hypertension. METHODS AND DESIGN: RGS2 expression was measured by real-time quantitative RT-PCR in peripheral blood mononuclear cells (PBMs) from 102 essential hypertensives. The diagnosis of essential hypertension was based on all clinically required tests, including the captopril suppression test. Antihypertensive treatment was given in accordance to international guidelines. End-point of the study was systolic blood pressure (BP) less than 140 mmHg and diastolic BP less than 90 mmHg with three or less different antihypertensive agents, which identified responders to treatment. Resistant hypertension was defined as the failure to control systolic and/or diastolic BP despite at least three different classes of antihypertensive agents, including a diuretic. RESULTS: During follow-up, 85 (83%) patients reached the end point (responders). Resistant hypertensives (n = 17, 17%) were older, had higher baseline BP, plasma aldosterone and aldosterone: renin ratio (ARR) and lower plasma renin activity than patients who reached the end point. RGS2 was negatively correlated to systolic BP at enrollment and significantly lower in PBMs from resistant hypertensives in comparison with patients that reached BP goal. According to logistic regression analysis, high RGS2 expression was predictor of reaching BP goal, whereas high ARR after captopril, age and systolic pressure at enrolment were predictor of resistant hypertension. CONCLUSION: RGS2 expression affects the response to antihypertensive treatment. Reduced RGS2 expression contributes to resistance to antihypertensive agents through poor negative feedback on the effects of aldosterone and of other vasoactive agents.


Subject(s)
Aldosterone/blood , Antihypertensive Agents/therapeutic use , Hypertension/drug therapy , RGS Proteins/genetics , Renin/blood , Adolescent , Adult , Aged , Blood Pressure/drug effects , Cohort Studies , Drug Resistance , Feedback, Physiological , Female , Gene Expression/drug effects , Humans , Hypertension/blood , Hypertension/genetics , Hypertension/physiopathology , Male , Middle Aged , RNA, Messenger/blood , RNA, Messenger/genetics , Young Adult
4.
Diabetes ; 59(4): 1006-15, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20068143

ABSTRACT

OBJECTIVE: Sirtuins (SIRTs) are NAD(+)-dependent deacetylases that regulate metabolism and life span. We used peripheral blood mononuclear cells (PBMCs) to determine ex vivo whether insulin resistance/metabolic syndrome influences SIRTs. We also assessed the potential mechanisms linking metabolic alterations to SIRTs in human monocytes (THP-1) in vitro. RESEARCH DESIGN AND METHODS: SIRT1-SIRT7 gene and protein expression was determined in PBMCs of 54 subjects (41 with normal glucose tolerance and 13 with metabolic syndrome). Insulin sensitivity was assessed by the minimal model analysis. Subclinical atherosclerosis was assessed by carotid intima-media thickness (IMT). In THP-1 cells exposed to high glucose or fatty acids in vitro, we explored SIRT1 expression, p53 acetylation, Jun NH(2)-terminal kinase (JNK) activation, NAD(+) levels, and nicotinamide phosphoribosyltransferase (NAMPT) expression. The effects of SIRT1 induction by resveratrol and of SIRT1 gene silencing were also assessed. RESULTS: In vivo, insulin resistance and metabolic syndrome were associated with low PBMC SIRT1 gene and protein expression. SIRT1 gene expression was negatively correlated with carotid IMT. In THP-1 cells, high glucose and palmitate reduced SIRT1 and NAMPT expression and reduced the levels of intracellular NAD(+) through oxidative stress. No effect was observed in cells exposed to linoleate or insulin. High glucose and palmitate increased p53 acetylation and JNK phosphorylation; these effects were abolished in siRNA SIRT1-treated cells. Glucose- and palmitate-mediated effects on NAMPT and SIRT1 were prevented by resveratrol in vitro. CONCLUSIONS: Insulin resistance and subclinical atherosclerosis are associated with SIRT1 downregulation in monocytes. Glucotoxicity and lypotoxicity play a relevant role in quenching SIRT1 expression.


Subject(s)
Down-Regulation , Insulin Resistance/genetics , Metabolic Syndrome/genetics , Sirtuin 1/genetics , Angiogenesis Inhibitors/pharmacology , Atherosclerosis/genetics , Atherosclerosis/pathology , Carotid Arteries/pathology , Glucose/pharmacology , Glucose Tolerance Test , Humans , Longevity , Metabolic Syndrome/pathology , Monocytes/physiology , Palmitic Acid/pharmacology , Reference Values , Resveratrol , Stilbenes/pharmacology , Tunica Intima/drug effects , Tunica Intima/pathology , Tunica Media/drug effects , Tunica Media/pathology
5.
Am J Physiol Heart Circ Physiol ; 298(1): H263-74, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19897708

ABSTRACT

Prolonged ouabain administration (25 microg kg(-1) day(-1) for 5 wk) induces "ouabain hypertension" (OH) in rats, but the molecular mechanisms by which ouabain elevates blood pressure are unknown. Here, we compared Ca(2+) signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free Ca(2+) concentration ([Ca(2+)](cyt); measured with fura-2) and phenylephrine-induced Ca(2+) transients were augmented in freshly dissociated OH ASMCs. Immunoblots revealed that the expression of the ouabain-sensitive alpha(2)-subunit of Na(+) pumps, but not the predominant, ouabain-resistant alpha(1)-subunit, was increased (2.5-fold vs. NT ASMCs) as was Na(+)/Ca(2+) exchanger-1 (NCX1; 6-fold vs. NT) in OH arteries. Ca(2+) entry, activated by sarcoplasmic reticulum (SR) Ca(2+) store depletion with cyclopiazonic acid (SR Ca(2+)-ATPase inhibitor) or caffeine, was augmented in OH ASMCs. This reflected an augmented expression of 2.5-fold in OH ASMCs of C-type transient receptor potential TRPC1, an essential component of store-operated channels (SOCs); two other components of some SOCs were not expressed (TRPC4) or were not upregulated (TRPC5). Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol [a measure of receptor-operated channel (ROC) activity] was much greater in OH than NT ASMCs. This correlated with a sixfold upregulation of TRPC6 protein, a ROC family member. Importantly, in primary cultured mesenteric ASMCs from normal rats, 72-h treatment with 100 nM ouabain significantly augmented NCX1 and TRPC6 protein expression and increased resting [Ca(2+)](cyt) and ROC activity. SOC activity was also increased. Silencer RNA knockdown of NCX1 markedly downregulated TRPC6 and eliminated the ouabain-induced augmentation; silencer RNA knockdown of TRPC6 did not affect NCX1 expression but greatly attenuated its upregulation by ouabain. Clearly, NCX1 and TRPC6 expression are interrelated. Thus, prolonged ouabain treatment upregulates the Na(+) pump alpha(2)-subunit-NCX1-TRPC6 (ROC) Ca(2+) signaling pathway in arterial myocytes in vitro as well as in vivo. This may explain the augmented myogenic responses and enhanced phenylephrine-induced vasoconstriction in OH arteries (83) as well as the high blood pressure in OH rats.


Subject(s)
Cardiotonic Agents , Hypertension/chemically induced , Hypertension/metabolism , Muscle, Smooth, Vascular/metabolism , Ouabain , Sodium-Calcium Exchanger/biosynthesis , Sodium-Potassium-Exchanging ATPase/biosynthesis , Animals , Blotting, Western , Calcium Channels/metabolism , Fluorescent Dyes , Fura-2 , Homeostasis/physiology , Image Processing, Computer-Assisted , Male , Mesenteric Arteries/metabolism , Mesenteric Arteries/physiology , RNA, Small Interfering , Rats , Rats, Sprague-Dawley , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Sodium-Calcium Exchanger/metabolism , TRPC Cation Channels/biosynthesis , TRPC Cation Channels/genetics , TRPC6 Cation Channel , Up-Regulation
6.
J Hypertens ; 27(8): 1594-601, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19451835

ABSTRACT

OBJECTIVES: The longitudinal relationship between coffee use and hypertension is still controversial. Cytochrome P450 1A2 (CYP1A2) is the main responsible enzyme for the metabolism of caffeine. The aim of the present study was to investigate the effect of coffee intake on the risk of developing hypertension needing antihypertensive treatment in individuals stratified by CYP1A2 genotype. DESIGN: We assessed prospectively 553 young White individuals screened for stage 1 hypertension. Coffee intake was ascertained from regularly administered questionnaires. Incident physician-diagnosed hypertension was the outcome measure. Genotyping of CYP1A2 SNP was performed by real time PCR. RESULTS: During a median follow-up of 8.2 years, 323 individuals developed hypertension. For carriers of the slow *1F allele (59%), hazard ratios of hypertension from multivariable Cox analysis were 1.00 in abstainers (reference), 1.72 (95%CI, 1.21-2.44) in moderate coffee drinkers (P = 0.03), and 3.00 (1.53-5.90) in heavy drinkers (P = 0.001). In contrast, hazard ratios for coffee drinkers with the rapid *1A/*1A genotype were 0.80 (0.52-1.23, P = 0.29) for moderate drinkers and 0.36 (0.14-0.89, P = 0.026) for heavy drinkers. In a two-way ANCOVA, a gene x coffee interactive effect was found on follow-up changes in systolic (P = 0.000) and diastolic (P = 0.007) blood pressure. Urinary epinephrine was higher in coffee drinkers than abstainers but only among individuals with slow *1F allele (P = 0.001). CONCLUSION: These data show that the risk of hypertension associated with coffee intake varies according to CYP1A2 genotype. Carriers of slow *1F allele are at increased risk and should thus abstain from coffee, whereas individuals with *1A/*1A genotype can safely drink coffee.


Subject(s)
Coffee/adverse effects , Cytochrome P-450 CYP1A2/genetics , Hypertension/etiology , Adolescent , Adult , Epinephrine/urine , Follow-Up Studies , Genotype , Humans , Hypertension/genetics , Middle Aged , Polymorphism, Single Nucleotide , Prospective Studies , Risk
7.
Am J Hypertens ; 22(2): 208-14, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19023273

ABSTRACT

BACKGROUND: The aim of the study was to investigate the effect of polymorphism A1166C for AGTR1 and -1332G/A for AGTR2 on the incidence of sustained hypertension (HT) and metabolic syndrome in a cohort of young patients screened for stage 1 HT. METHODS: We assessed 420 white hypertensive subjects never treated for HT and followed up for 7.3 years in the HT and Ambulatory Recording Venetia Study (HARVEST). Incident physician-diagnosed HT, increase in ambulatory blood pressure (BP), and new onset metabolic syndrome were the outcome measures. RESULTS: For AGTR1, 37.2% of the subjects in the group with AA genotype, 47.5% in the group with AC genotype, and 66.7% in the group with CC genotype developed HT during follow-up (P = 0.001). Ambulatory systolic (P = 0.007) and diastolic (P < 0.001) BPs increased largely in the patients with CC genotype than in the rest of the group. New onset metabolic syndrome during follow-up (n = 30, P = 0.008), and the frequency of the metabolic syndrome at the end of follow-up (n = 65, P = 0.002) were also more common among the patients with CC and AC genotype. In a Cox analysis, subjects with CC genotype had an increased risk of developing HT (hazard ratio (HR) 1.6, 95% confidence interval (CI) 1.2-2.0, P = 0.000) and metabolic syndrome (HR 2.8, 1.5-5.2, P = 0.002) than AA subjects. No association was found between the AGTR2 polymorphism and any outcome measure. CONCLUSIONS: The AGTR1 A1166C polymorphism may be considered a genetic marker predisposing to an increase in BP and the development of the metabolic syndrome in subjects screened for stage 1 HT.


Subject(s)
Hypertension/genetics , Metabolic Syndrome/genetics , Receptor, Angiotensin, Type 1/genetics , Adult , Female , Follow-Up Studies , Humans , Male , Polymorphism, Genetic , Receptor, Angiotensin, Type 2/genetics , Regression Analysis , Sex Factors
8.
Hepatology ; 48(6): 1913-23, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19003918

ABSTRACT

Decreased cardiac contractility and beta-adrenergic responsiveness have been observed in cirrhotic cardiomyopathy, but their molecular mechanisms remain unclear. To study beta-adrenergic-stimulated contractility and beta-adrenergic gene expression patterns, 20 Wistar Kyoto rats were treated with carbon tetrachloride to induce cirrhosis and 20 rats were used as controls. Left ventricular contractility was recorded in electrically driven isolated hearts perfused at constant flow with isoproterenol (10(-10) to 10(-6) M). A cardiac gene expression profile was obtained using a microarray for the myocyte adrenergic pathway. The cardiac contractility maximal response to isoproterenol was significantly reduced in cirrhotic rats in comparison to control rats, whereas the half-maximal effective concentration was not different. In cirrhotic rats, cardiac gene expression analysis showed a significant overexpression of G protein alpha-inhibiting subunit 2 (Galpha(i2)), cyclic nucleotide phosphodiesterase (PDE2a), regulator of G-protein signaling 2 (RGS2), and down-expression of adenylate cyclase (Adcy3). These results indicate that overexpression of Galpha(i2), PDE2a, and RGS2 down-regulates the beta-adrenergic signaling pathway, thus contributing to the pathogenesis of cirrhotic cardiomyopathy.


Subject(s)
Cardiomyopathies/genetics , Gene Expression Regulation/physiology , Liver Cirrhosis/complications , Receptors, Adrenergic, beta/genetics , Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Adrenergic beta-Agonists/pharmacology , Animals , Carbon Tetrachloride , Cardiomyopathies/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 2/genetics , Cyclic Nucleotide Phosphodiesterases, Type 2/metabolism , Dose-Response Relationship, Drug , GTP-Binding Protein alpha Subunit, Gi2/genetics , GTP-Binding Protein alpha Subunit, Gi2/metabolism , Gene Expression Regulation/drug effects , Heart Rate/drug effects , Isoproterenol/pharmacology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/metabolism , Male , Myocardial Contraction/drug effects , RGS Proteins/genetics , RGS Proteins/metabolism , Rats , Rats, Inbred WKY , Receptors, Adrenergic, beta/metabolism , Signal Transduction
9.
J Nutr ; 138(9): 1596-601, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18716156

ABSTRACT

We previously documented a clear-cut antihypertensive effect of green teat extract (GTE), which was associated with correction of endothelial dysfunction and prevention of left ventricular hypertrophy in an angiotensin II (Ang II)-dependent model of hypertension, but the molecular mechanisms remain to be defined. As several effects of Ang II involve production of reactive oxygen species (ROS) and activation of 2nd messengers, such as mitogen-activated protein kinase (MAPK) and Akt, we investigated the effect of GTE on these signal transduction pathways in Ang II-treated rats. Rats were treated for 2 wk with Ang II infusion (700 mug.kg(-1).d(-1); n = 6, via osmotic minipumps), Ang II plus GTE (6 g/L) dissolved in the drinking water; n = 6), or vehicle (n = 6) to serve as controls. Blood pressure was monitored by telemetry throughout the study. The activation and expression of NAD(P)H oxidase subunits, protein kinase C isoforms, Src, epidermal growth factor receptor (EGFR), Akt, and MAPK were determined in the heart in vitro through immunoprecipitation and western blot analysis with specific antibodies. NAD(P)H oxidase enzymatic activity was measured by cytochrome c reduction assay. GTE blunted Ang II-induced blood pressure increase and cardiac hypertrophy. In Ang II-treated rats, GTE decreased the expression of the NAD(P)H oxidase subunit gp91(phox) and the translocation of Rac-1, as well as NAD(P)H oxidase enzymatic activity. Furthermore, it specifically reduced Ang II-induced Src, EGFR, and Akt phosphorylation. These results show that GTE blunts Ang II-induced cardiac hypertrophy specifically by regulating ROS production and the Src/EGFR/Akt signaling pathway activated by Ang II.


Subject(s)
Cardiomegaly/drug therapy , ErbB Receptors/metabolism , Oncogene Protein v-akt/metabolism , Reactive Oxygen Species/metabolism , Tea , src-Family Kinases/metabolism , Angiotensin II/toxicity , Animals , Cardiomegaly/chemically induced , Cardiomegaly/metabolism , Enzyme Activation , Isoenzymes , Male , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , NADPH Oxidases/metabolism , Oncogene Protein v-akt/genetics , Protein Kinase C/chemistry , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Subunits , Rats , Rats, Sprague-Dawley , Signal Transduction
10.
J Hypertens ; 26(5): 938-45, 2008 May.
Article in English | MEDLINE | ID: mdl-18398336

ABSTRACT

OBJECTIVE: Regulator of G-protein signaling (RGS)-2 is a regulator of angiotensin II (Ang II) signaling. In Bartter's syndrome/Gitelman's syndrome (BS/GS), we have demonstrated increased RGS-2 levels and blunted Ang II signaling which contribute to their reduced vasomotor tone and remodeling. The present study investigates the effect of silencing RGS-2 in fibroblasts from six BS/GS patients on intracellular Ca2+ (CaI2+) mobilization and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, established Ang II-mediated responses. METHODS: Fibroblasts were RGS-2 silenced by transfecting chemically synthesized small interfering RNA. Silencing efficiency and Ang II-induced ERK 1/2 phosphorylation were evaluated by western blot and Ang II-induced Cai2+ using Fura-2 AM. RESULTS: RGS-2 expression in not silenced BS/GS fibroblasts from patients is increased compared with healthy controls [0.34 +/- 0.02 vs. 0.19 +/- 0.01 densitometric units (d.u.), P = 0.0005]. Silencing RGS-2 in BS/GS patients was achieved to the level of controls. Ang II-induced Cai2+ release and ERK 1/2 phosphorylation were reduced in not silenced cells from BG/GS patients compared with controls (112.16 +/- 13.2 vs. 130.33 +/- 13.64 mmol/l, P = 0.011 and 0.64 +/- 0.08 vs. 0.91 +/- 0.03 mmol/l, P < 0.006, respectively). Silencing RGS-2 in BS/GS patients increased Ang II-induced Cai2+ release and ERK 1/2 phosphorylation in silenced cells compared with not silenced cells [59.3 +/- 10.8 (peak-basal) vs. 40.5 +/- 14.1 nmol/l, P = 0.017 and 0.84 +/- 0.06 vs. 0.64 +/- 0.08 nmol/l, P < 0.03, respectively], whereas they were not different compared with controls (60.1 +/- 4.3 and 0.91 +/- 0.03 nmol/l). Integrating the Cai2+ response over time showed increased Cai2+ area under the curve (AUC) of BS/GS silenced cells compared with that of not silenced cells (P = 0.013). CONCLUSION: This is the first report of silencing RGS-2 effect on Ang II signaling in a human clinical condition of altered vascular tone regulation and remodeling and establishes RGS-2 as a key regulatory element of Ang II signaling in humans.


Subject(s)
Angiotensin II/physiology , Bartter Syndrome/physiopathology , Gitelman Syndrome/physiopathology , Mitogen-Activated Protein Kinase 3/metabolism , RGS Proteins/metabolism , Adult , Calcium/metabolism , Cells, Cultured , Cohort Studies , Female , Fibroblasts/physiology , Gene Silencing , Humans , Male , Middle Aged , Mitogen-Activated Protein Kinase 1/metabolism , Phosphorylation , Signal Transduction
11.
Metabolism ; 57(3): 421-7, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18249218

ABSTRACT

RGS2 is a negative regulator of Galpha protein signaling and promotes adipocyte differentiation. Recently, we described a polymorphism at the C1114G locus with the G allele associated with hypertension in a cross-sectional study. The aim of the present study was to assess whether the RGS2 C1114G is predictive of overweight in young subjects with grade I hypertension. We genotyped at the RGS2 C1114G locus 406 (male, n = 294; female, n = 112) white hypertensive subjects (age, 33 +/- 9 years) never treated for hypertension and at low cardiovascular risk. Median follow-up was 7.85 years. At baseline, male patients carrying the RGS2 1114G allele had higher body mass index (BMI) than patients with CC genotype (26.1 +/- 0.3 vs 25.3 +/- 0.3 kg/m2, P < .05). The frequency of male patients with BMI > or = 25 was similar between the patients with G allele and those with CC genotype (55.1% vs 47.8%, P = not significant). No significant difference between the 2 groups was observed with regard to physical activity, blood pressure, and heart rate. At the end of follow-up, BMI was higher in male patients with G allele compared with patients with CC genotype (26.8 +/- 0.3 vs 25.8 +/- 0.2 kg/m2, P < .01); and the frequency of male patients with BMI >25 kg/m2 was greater in the former (69.0% vs 52.2%, P < .01). According to Cox regression, allele G was a significant predictor of developing overweight or obesity during follow-up. These epidemiologic relations were not significant in female patients. In young male patients with grade I hypertension, RGS2 1114G allele is associated with increased BMI and with greater risk of developing overweight or obesity. The RGS2 1114G allele may be considered a genetic marker that predicts an individual's predisposition to gaining weight.


Subject(s)
Hypertension/genetics , Obesity/genetics , Overweight/genetics , Polymorphism, Genetic/genetics , RGS Proteins/genetics , Weight Gain/genetics , 3' Untranslated Regions/genetics , Adolescent , Adult , Alleles , Cohort Studies , Female , Follow-Up Studies , Gene Frequency , Genotype , Humans , Hypertension/epidemiology , Male , Middle Aged , Multivariate Analysis , Obesity/epidemiology , Overweight/epidemiology , Sex Characteristics
12.
Arterioscler Thromb Vasc Biol ; 27(12): 2627-33, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17916771

ABSTRACT

OBJECTIVE: Hyperglycemia is the main determinant of long-term diabetic complications, mainly through induction of oxidative stress. NAD(P)H oxidase is a major source of glucose-induced oxidative stress. In this study, we tested the hypothesis that rosiglitazone (RSG) is able to quench oxidative stress initiated by high glucose through prevention of NAD(P)H oxidase activation. METHODS AND RESULTS: Intracellular ROS were measured using the fluoroprobe TEMPO-9-AC in HUVECs exposed to control (5 mmol/L) and moderately high (10 mmol/L) glucose concentrations. NAD(P)H oxidase and AMPK activities were determined by Western blot. We found that 10 mmol/L glucose increased significantly ROS production in comparison with 5 mmol/L glucose, and that this effect was completely abolished by RSG. Interestingly, inhibition of AMPK, but not PPARgamma, prevented this effect of RSG. AMPK phosphorylation by RSG was necessary for its ability to hamper NAD(P)H oxidase activation, which was indispensable for glucose-induced oxidative stress. Downstream of AMPK activation, RSG exerts antioxidative effects by inhibiting PKC. CONCLUSIONS: This study demonstrates that RSG activates AMPK which, in turn, prevents hyperactivity of NAD(P)H oxidase induced by high glucose, possibly through PKC inhibition. Therefore, RSG protects endothelial cells against glucose-induced oxidative stress with an AMPK-dependent and a PPARgamma-independent mechanism.


Subject(s)
Antioxidants/pharmacology , Endothelial Cells/drug effects , Glucose/metabolism , Multienzyme Complexes/metabolism , NADPH Oxidases/metabolism , Oxidative Stress/drug effects , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/drug effects , Thiazolidinediones/pharmacology , AMP-Activated Protein Kinases , Animals , Cells, Cultured , Endothelial Cells/enzymology , Endothelial Cells/metabolism , Enzyme Activation , Enzyme Activators/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Male , Membrane Glycoproteins/metabolism , Multienzyme Complexes/genetics , Muscle, Skeletal/drug effects , Muscle, Skeletal/enzymology , Muscle, Skeletal/metabolism , NADPH Oxidase 2 , NADPH Oxidases/antagonists & inhibitors , PPAR gamma/drug effects , PPAR gamma/metabolism , Phosphoproteins/metabolism , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Transport , RNA Interference , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Rosiglitazone , Transfection , rac1 GTP-Binding Protein/metabolism
13.
Cardiovasc Res ; 73(2): 432-8, 2007 Jan 15.
Article in English | MEDLINE | ID: mdl-17123493

ABSTRACT

OBJECTIVE: Cardiovascular risk is increased among HIV-infected patients receiving antiretroviral therapy due to the development of hypertension and metabolic abnormalities. In this study, we investigated the effects of long-term treatment with zidovudine (AZT) and vitamin C, alone and in combination, on blood pressure and on the chain of events linking oxidative stress to cardiac damage in the rat. METHODS: Six adult Wistar Kyoto rats received AZT (1 mg/ml) in the drinking water for 8 months, six vitamin C (10 g/kg of food) and AZT, six vitamin C alone, and six served as controls. RESULTS: AZT increased systolic blood pressure, expression of gp91(phox) and p47(phox) subunits of NAD(P)H oxidase, and protein kinase C (PKC) delta activation and reduced antioxidant power of plasma and cardiac homogenates. AZT also caused morphological alterations in cardiac myocyte mitochondria, indicative of functional damage. All of these effects were prevented by vitamin C. CONCLUSION: Chronic AZT administration increases blood pressure and promotes cardiovascular damage through a NAD(P)H oxidase-dependent mechanism that involves PKC delta. Vitamin C antagonizes these adverse effects of AZT in the cardiovascular system.


Subject(s)
Antimetabolites/pharmacology , Ascorbic Acid/pharmacology , Hypertension/prevention & control , NADPH Oxidases/metabolism , Vitamins/pharmacology , Zidovudine/pharmacology , Animals , Antioxidants/metabolism , Blotting, Western/methods , Enzyme Activation/drug effects , Hypertension/metabolism , Hypertension/pathology , Male , Membrane Glycoproteins/analysis , Membrane Glycoproteins/metabolism , Microscopy, Electron , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/ultrastructure , NADPH Oxidase 2 , NADPH Oxidases/analysis , Nuclear Proteins/analysis , Nuclear Proteins/metabolism , Protein Kinase C/metabolism , Rats , Rats, Inbred WKY , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
14.
Free Radic Biol Med ; 41(3): 473-83, 2006 Aug 01.
Article in English | MEDLINE | ID: mdl-16843828

ABSTRACT

Insulin can generate oxygen free radicals. Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, exert a powerful antioxidant effect. The present study aimed to clarify the mechanisms through which insulin generates free radicals and to assess whether pravastatin modulates such effects. In cultured skin fibroblasts from human volunteers exposed to high insulin concentration, either in the presence or in the absence of pravastatin, insulin induced translocation of the p47(phox) subunit of NAD(P)H oxidase from the cytosol to the membrane and generation of radical oxygen species through a PKC delta-dependent mechanism. The insulin-induced translocation of p47(phox) was PKC delta dependent and attenuated by pravastatin, but independent of the activation of Akt and Rac1. Insulin-induced Akt phosphorylation was increased by pravastatin and ERK1/2 phosphorylation attenuated. The present study demonstrates a novel mechanism by which insulin stimulates the generation of free radicals in human fibroblasts, ex vivo. It involves phosphatidylinositol 3-kinase, PKC delta, and p47(phox) translocation and promotes ERK1/2 phosphorylation. Pravastatin inhibited radical oxygen species production by inhibiting PKC delta. These observations offer a robust explanation for the positive effects of pravastatin treatment in patients with insulin resistance syndrome.


Subject(s)
Insulin/pharmacology , Pravastatin/pharmacology , Protein Kinase C-delta/metabolism , Cells, Cultured , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts , Free Radicals/metabolism , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , NADPH Oxidases/metabolism , Phosphorylation/drug effects , Protein Kinase C-delta/genetics , Protein Transport , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Skin/drug effects , Skin/metabolism , rac1 GTP-Binding Protein/antagonists & inhibitors , rac1 GTP-Binding Protein/metabolism
15.
J Hypertens ; 24(6): 1115-24, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16685212

ABSTRACT

CONTEXT: RGS2 (regulators of G-protein signaling) is a negative regulator of Galphaq protein signaling, which mediates the action of several vasoconstrictors. RGS2-deficient mouse line exhibits a hypertensive phenotype and a prolonged response to vasoconstrictors. OBJECTIVE: To compare RGS2 expression in peripheral blood mononuclear cells (PBMs) and cultured fibroblasts from normotensive subjects and hypertensive patients. METHODS: PBMs were isolated from 100 controls and 150 essential hypertensives. Additionally, fibroblasts were isolated from skin biopsy of 11 normotensives and 12 hypertensives and cultured up to the third passage. Quantitative mRNA and protein RGS2 expression were performed by real-time quantitative reverse transcriptase-polymerase chain reaction and by immunoblotting, respectively. Free Ca measurement was performed in monolayers of 24-h serum-deprived cells, using FURA-2 AM. Phosphorylation of the extracellular signal-regulated kinases ERK1/2 was measured by immunoblotting. Polymorphism (C1114G) in the 3' untranslated region of the RGS2 gene was investigated by direct sequencing and real-time polymerase chain reaction (PCR). RESULTS: RGS2 mRNA expression was significantly lower in PBM and in fibroblasts from hypertensives, in comparison to normotensives. C1114G polymorphism was associated with RGS2 expression, with the lowest values in GG hypertensives. The 1114G allele frequency was increased in hypertensives compared with normotensives. Angiotensin II-stimulated intracellular Ca increase and ERK1/2 phosphorylation were higher in fibroblasts from hypertensive patients compared with control subjects, and in those with the G allele, independently of the blood pressure status. The angiotensin II-stimulated Ca mobilization and ERK1/2 phosphorylation were negatively correlated with RGS2 mRNA expression. CONCLUSION: Low expression of RGS2 contributes to increased G-protein-coupled signaling in hypertensive patients. The allele G is associated with low RGS2 expression and blood pressure increase in humans.


Subject(s)
Angiotensin II/physiology , Calcium/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Hypertension/metabolism , RGS Proteins/metabolism , Adult , Cells, Cultured , Cytosine/physiology , Female , Fibroblasts/metabolism , Guanine/physiology , Humans , Hypertension/genetics , Intracellular Fluid/metabolism , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Phosphorylation , Polymorphism, Single Nucleotide , RGS Proteins/genetics
16.
J Hypertens ; 24(4): 757-66, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16531806

ABSTRACT

OBJECTIVE: The aim of the present study was to determine whether inhibition of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase and of various superoxide generating systems could affect the collagen production, the mRNA and protein expression of collagen types I and III in control and angiotensin II-treated cardiac fibroblasts. METHODS: Cardiac fibroblasts from passage 2 from normal male adult rats were cultured to confluency and incubated in serum-free Dulbecco's modified Eagle's medium for 24 h. The cells were then preincubated with(out) the tested inhibitors for 1 h and then further incubated with(out) angiotensin II (1 micromol/l) for 24 h. Collagen production was measured spectrophotometrically with picrosirius red as dye and with [3H]proline incorporation; collagen type I and III content by enzyme-linked immunosorbent assay and collagen type I and III mRNA expression by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). NAD(P)H-dependent superoxide anion production was assayed as superoxide dismutase-inhibitable cytochrome c reduction. Intracellular formation of reactive oxygen species was assessed with 2',7'-dichlorofluorescein diacetate as fluorescent probe. RESULTS: Angiotensin II stimulated the collagen production, the collagen I and III content and mRNA expression in cardiac fibroblasts, and apocynin, a membrane NAD(P)H oxidase inhibitor, abolished this induction. Rotenone, allopurinol, indomethacin, nordihydroguiaretic acid, ketoconazole and nitro-L-arginine (inhibitors of mitochondrial NAD(P)H oxidase, xanthine oxidase, cyclooxygenase, lipoxygenase, cytochrome P450 oxygenase and nitric oxide synthase, respectively) did not affect the angiotensin II-induced collagen production. Angiotensin II increased the NAD(P)H-dependent superoxide anion production and the intracellular generation of reactive oxygen species in cardiac fibroblasts, and apocynin abrogated this rise. CONCLUSIONS: Our data show that in adult rat cardiac fibroblasts the membrane-associated NAD(P)H oxidase complex is the predominant source of superoxide anion and reactive oxygen species generation in angiotensin II-stimulated adult cardiac fibroblasts. Inhibition of this NAD(P)H oxidase complex with apocynin completely blocked the angiotensin II-stimulated collagen production, and collagen I and III protein and mRNA expression.


Subject(s)
Angiotensin II/pharmacology , Collagen/metabolism , Fibroblasts/drug effects , Myocardium/cytology , Reactive Oxygen Species/metabolism , Acetophenones/pharmacology , Animals , Cells, Cultured , Collagen/genetics , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type III/genetics , Collagen Type III/metabolism , Enzyme Inhibitors/pharmacology , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression/drug effects , Male , Matrix Metalloproteinase 1/metabolism , NADP/metabolism , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Onium Compounds/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Tissue Inhibitor of Metalloproteinase-1/metabolism
18.
J Hypertens ; 23(4): 793-800, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15775784

ABSTRACT

BACKGROUND: Fibroblasts are involved in the remodeling of the heart and of the vasculature associated to arterial hypertension, and an abnormal extracellular signal-regulated kinase 1/2 (ERK1/2) activation by angiotensin II (Ang II) plays a pivotal role in this process. However, the intracellular pathways leading to cell hypertrophy and hyperplasia, as well as to collagen production, are still incompletely known. OBJECTIVE: To investigate the role of superoxide anion (O2) and of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase in Ang II-stimulated ERK1/2 over-activation in fibroblasts from hypertensive patients. METHODS: O2 production was measured in skin fibroblasts from hypertensives (HT, n = 11) and from normotensive controls (NT, n = 10) by electron spin resonance technique. ERK1/2 phosphorylation and p47phox NAD(P)H oxidase subunit translocation were measured by western blot. RESULTS: Ang II (1 micromol/l) induced a larger p47phox subunit translocation and increased intracellular O2 production to a larger extent in HT in comparison to NT and this effect was blocked by apocynin, an inhibitor of the NAD(P)H oxidase. Ang II increased ERK1/2 phosphorylation more in HT than in NT. The Ang II-induced ERK1/2 phosphorylation was inhibited by apocynin in a dose-dependent manner in NT, but not in HT. CONCLUSIONS: The chain of cellular events leading to increased ERK1/2 responsiveness to Ang II in hypertension include an exaggerated response of p47phox, NAD(P)H oxidase and O2, but it is partially resistant to apocynin. Therefore, NAD(P)H-dependent reactive oxygen species (ROS) production is not the only determinant of the exaggerated ERK1/2 responsiveness in fibroblasts of hypertensives (HT).


Subject(s)
Angiotensin II/metabolism , Hypertension/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphoproteins/metabolism , Vasoconstrictor Agents/metabolism , Acetophenones/pharmacology , Adult , Angiotensin II/pharmacology , Antioxidants/pharmacology , Cell Membrane/enzymology , Cells, Cultured , Cytosol/enzymology , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Free Radicals/metabolism , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Middle Aged , NADPH Oxidases , Phosphorylation/drug effects , Reactive Oxygen Species/metabolism , Vasoconstrictor Agents/pharmacology
19.
Biochem Pharmacol ; 68(7): 1331-8, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15345322

ABSTRACT

Callipeltin A is a cyclic depsidecapeptide isolated from the marine sponges Callipelta sp. and Latrunculia sp. that has been previously shown to increase the force of contraction of guinea-pig atria through the inhibition of Na+/Ca2+ exchanger (NCX). We investigated the effect of callipeltin A on guinea-pig aortic rings contracted by procedures that activate NCX in "calcium entry mode". Callipeltin A did not inhibit these contractions. Resting aorta responded to callipeltin A with a remarkable contraction that was concentration-dependent (EC50 0.44microM). This contraction was not inhibited by the calcium channel blocker verapamil and was not mediated by the activation of alpha-adrenergic or endothelin-1 receptors. Pre-incubation of aortic rings with 0.5mM amiloride, an inhibitor of NCX, completely prevented callipeltin A-induced contraction. Furthermore, callipeltin A (EC50 0.51microM) increased Na+ efflux of Na-loaded erythrocytes. 1H and 13C NMR resonances of callipeltin A revealed small but significant changes in the titration with K+ and Na+ salts. It is suggested that the effect of callipeltin A on cardiac and vascular preparations is linked to a Na-ionophore action.


Subject(s)
Depsipeptides , Ionophores/metabolism , Muscle, Smooth, Vascular/drug effects , Peptides, Cyclic/pharmacology , Sodium/metabolism , Vasoconstriction/drug effects , Animals , Aorta , Biological Transport/drug effects , Calcium/metabolism , Guinea Pigs , In Vitro Techniques , Magnetic Resonance Spectroscopy , Muscle, Smooth, Vascular/physiology
20.
Am J Hypertens ; 17(7): 604-10, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15233979

ABSTRACT

BACKGROUND: Insulin resistance, a frequent finding in hypertensive patients, leads to accelerated cardiovascular damage. It has been suggested that a crosstalk between angiotensin II and insulin signaling pathways may provoke insulin resistance, and may contribute to the development of cardiovascular damage. To identify a common pathophysiologic pathway between metabolic disorders and cardiovascular remodeling, we investigated the effect of angiotensin II and insulin on extracellular signal regulated kinases 1 and 2 (ERK1/2), isoforms of mitogen-activated protein kinases (MAPK) involved in cellular proliferation and extracellular matrix deposition. METHODS: Skin fibroblasts from normotensive subjects, insulin sensitive hypertensive subjects, and insulin resistant hypertensive subjects were cultured and used after four passages. The ERK1/2 expression and phosphorylation were measured by Western blot using specific antibodies, respectively anti-ERK1/2 and anti-pERK1/2. Expression of AT1 receptor for angiotensin II was determined by reverse transcriptase-polymerase chain reaction in real time. RESULTS: The ERK1/2 were similarly expressed in skin fibroblasts from all groups; ERK1/2 phosporylation evoked by angiotensin II was significantly higher in fibroblasts from hypertensive patients in comparison to normotensive subjects, but the increase was observed only in insulin resistant hypertensive subjects. The effect of insulin on ERK1/2 phosphorylation was not significantly different in the three groups. Treatment with the combination of insulin and angiotensin II increased ERK1/2 phosphorylation to a greater extent in comparison to the single agonists in normotensive subjects and in insulin sensitive but not in insulin resistant hypertensive subjects. CONCLUSIONS: Angiotensin II stimulated ERK1/2 activation is increased in insulin resistant hypertensive subjects, and it may play a role in the pathogenesis of insulin resistance and accelerated cardiovascular damage.


Subject(s)
Angiotensin II/therapeutic use , Enzyme Activation/drug effects , Hypertension/drug therapy , Hypertension/metabolism , Hypoglycemic Agents/therapeutic use , Insulin/therapeutic use , Mitogen-Activated Protein Kinase 1/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Vasoconstrictor Agents/therapeutic use , Adult , Angiotensin II/administration & dosage , Biomarkers/blood , Blood Pressure/drug effects , Dose-Response Relationship, Drug , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Hypertension/physiopathology , Hypoglycemic Agents/administration & dosage , Insulin/administration & dosage , Insulin Resistance , Italy , Male , Middle Aged , Mitogen-Activated Protein Kinase 3 , Phosphorylation/drug effects , Phosphotransferases/drug effects , Phosphotransferases/metabolism , Receptor, Angiotensin, Type 1/therapeutic use , Time Factors , Treatment Outcome , Vasoconstrictor Agents/administration & dosage
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