Biomedical subjects

Ivan S Yuhanna

Publications and source records attributed to Ivan S Yuhanna.

8 recordsLinked to original sources

PubMed2005

FcgammaRIIB mediates C-reactive protein inhibition of endothelial NO synthase.

C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk and endothelial dysfunction. Whether CRP has direct actions on endothelium and the mechanisms underlying such actions are unknown. Here we show in cultured endothelium that CRP prevents endothelial NO synthase (eNOS) activation by diverse agonists, resulting in the promotion of monocyte adhesion. CRP antagonism of eNOS occurs nongenomically and is attributable to blunted eNOS phosphorylation at Ser1179. Okadaic acid or knockdown of PP2A by short-interference RNA reverses CRP antagonism of eNOS, indicating a key role for the phosphatase. Aggregated IgG, the known ligand for Fcgamma receptors, causes parallel okadaic acid-sensitive loss of eNOS function, FcgammaRIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires FcgammaRIIB. In FcgammaRIIB(+/+) mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in FcgammaRIIB(-/-) mice. Thus FcgammaRIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.

Acetylcholine↗

PubMed2005

Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor-BI mediate HDL-initiated signaling.

The binding of HDL to scavenger receptor-BI (SR-BI) mediates cholesterol movement. HDL also induces multiple cellular signals, which in endothelium occur through SR-BI and converge to activate eNOS. To determine the molecular basis of a signaling event induced by HDL, we examined the proximal mechanisms in HDL activation of eNOS. In endothelial cells, HDL and methyl-beta-cyclodextrin caused comparable eNOS activation, whereas cholesterol-loaded methyl-beta-cyclodextrin had no effect. Phosphatidylcholine-loaded HDL caused greater stimulation than native HDL, and blocking antibody against SR-BI, which prevents cholesterol efflux, prevented eNOS activation. In a reconstitution model in COS-M6 cells, wild-type SR-BI mediated eNOS activation by both HDL and small unilamellar vesicles (SUVs), whereas the SR-BI mutant AVI, which is incapable of efflux to SUV, transmitted signal by only HDL. In addition, eNOS activation by methyl-beta-cyclodextrin was SR-BI dependent. Studies of mutant and chimeric class B scavenger receptors revealed that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains of SR-BI are both necessary for HDL signaling. Furthermore, we demonstrated direct binding of cholesterol to the C-terminal transmembrane domain using a photoactivated derivative of cholesterol. Thus, HDL signaling requires cholesterol binding and efflux and C-terminal domains of SR-BI, and SR-BI serves as a cholesterol sensor on the plasma membrane.

Animals↗

PubMed2004

Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol: role of ERalpha domains with known nuclear functions.

Estradiol stimulates endothelial nitric oxide synthase (eNOS) via the activation of plasma membrane (PM)-associated estrogen receptor (ER) alpha. The process requires Src and erk signaling and eNOS phosphorylation by phosphoinositide 3-kinase (PI3 kinase)-Akt kinase, with Src and PI3 kinase associating with ERalpha upon ligand activation. To delineate the basis of nongenomic eNOS stimulation, the potential roles of ERalpha domains necessary for classical nuclear function were investigated in COS-7 cells. In cross-linking studies, estradiol-17beta (E2) caused PM-associated ERalpha to form dimers. However, eNOS activation by E2 was unaltered for a dimerization-deficient mutant ERalpha (ERalphaL511R). In contrast, ERalpha mutants lacking the nuclear localization signals (NLS), NLS2,3 (ERalphaDelta250-274) or the DNA binding domain (ERalphaDelta185-251), which targeted normally to PM and caveolae/rafts, were incapable of activating eNOS. The loss of NLS2/NLS3 prevented Src and erk activation, and it altered ligand-induced PI3 kinase-ERalpha interaction and prevented eNOS phosphorylation. Loss of the DNA binding domain did not change E2 activation of Src or erk, but ligand-induced PI3 kinase-ERalpha binding and eNOS phosphorylation did not occur. Thus, dimerization is not required for ERalpha coupling to eNOS; however, NLS2/NLS3 plays a role in Src activation, and the DNA binding region is involved in the dynamic interaction between ERalpha and PI3 kinase.

Animals↗

PubMed2003

Hypoxia-induced left ventricular dysfunction in myoglobin-deficient mice.

Myoglobin-deficient mice are viable and have preserved cardiac function due to their ability to mount a complex compensatory response involving increased vascularization and the induction of the hypoxia gene program (hypoxia-inducible factor-1alpha, endothelial PAS, heat shock protein27, etc.). To further define and explore functional roles for myoglobin, we challenged age- and gender-matched wild-type and myoglobin-null mice to chronic hypoxia (10% oxygen for 1 day to 3 wk). We observed a 30% reduction in cardiac systolic function in the myoglobin mutant mice exposed to chronic hypoxia with no changes observed in the wild-type control hearts. The cardiac dysfunction observed in the hypoxic myoglobin-null mice was reversible with reexposure to normoxic conditions and could be prevented with treatment of an inhibitor of nitric oxide (NO) synthases. These results support the conclusion that hypoxia-induced cardiac dysfunction in myoglobin-null mice occurs via a NO-mediated mechanism. Utilizing enzymatic assays for NO synthases and immunohistochemical analyses, we observed a marked induction of inducible NO synthase in the hypoxic myoglobin mutant ventricle compared with the wild-type hypoxic control ventricle. These new data establish that myoglobin is an important cytoplasmic cardiac hemoprotein that functions in regulating NO homeostasis within cardiomyocytes.

Animals↗

PubMed2003

Vasomodulation by skeletal muscle-derived nitric oxide requires alpha-syntrophin-mediated sarcolemmal localization of neuronal Nitric oxide synthase.

Neuronal nitric oxide synthase (nNOS) is abundantly expressed in skeletal muscle where it associates with the dystrophin complex at the sarcolemma by binding to the PDZ domain of alpha-syntrophin. Nitric oxide (NO) produced by skeletal muscle nNOS is proposed to regulate blood flow in exercising muscle by diffusing from the skeletal muscle fibers to the nearby microvessels where it attenuates alpha-adrenergic vasoconstriction. In the present study, we hypothesized that sarcolemmal localization of nNOS is a critical determinant of the vasoregulatory effect of skeletal muscle-derived NO. To test this hypothesis, we performed experiments in alpha-syntrophin null mice and in transgenic mice expressing a mutated alpha-syntrophin lacking the PDZ domain (DeltaPDZ), both of which are characterized by reduced sarcolemmal nNOS. We found that modulation of alpha-adrenergic vasoconstriction was greatly impaired in the contracting muscles of the alpha-syntrophin null mice and transgenic DeltaPDZ mice compared with wild-type mice and transgenic mice expressing full-length alpha-syntrophin. These in vivo mouse studies highlight the functional importance of appropriate membrane targeting of nNOS by the dystrophin-associated protein alpha-syntrophin and may have implications for the development of potential gene therapy strategies to treat muscular dystrophy or other muscle-related diseases.

Animals↗

PubMed2003

Pulmonary NO synthase expression is attenuated in a fetal baboon model of chronic lung disease.

Nitric oxide (NO), produced by NO synthase (NOS), serves multiple functions in the perinatal lung. In fetal baboons, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) are all primarily expressed in proximal respiratory epithelium. In the present study, NOS expression and activity in proximal lung and minute ventilation of NO standard temperature and pressure (VeNO(STP)) were evaluated in a model of chronic lung disease (CLD) in baboons delivered at 125 days (d) of gestation (term = 185 d) and ventilated for 14 d, obtaining control lung samples from fetuses at 125 or 140 d of gestation. In contrast to the normal 73% increase in total NOS activity from 125 to 140 d of gestation, there was an 83% decline with CLD. This was related to marked diminutions in both nNOS and eNOS expression and enzymatic activity. nNOS accounted for the vast majority of enzymatic activity in all groups. The normal 3.3-fold maturational rise in iNOS protein expression was blunted in CLD, yet iNOS activity was elevated in CLD compared with at birth. The contribution of iNOS to total NOS activity was minimal in all groups. VeNO(STP) remained stable in the range of 0.5-1.0 nl x kg(-1) x min(-1) from birth to day 7 of life, and it then rose by 2.5-fold. Thus the baboon model of CLD is characterized by deficiency of the principal pulmonary isoforms, nNOS and eNOS, and enhanced iNOS activity over the first 2 wk of postnatal life. It is postulated that these alterations in NOS expression and activity may contribute to the pathogenesis of CLD.

Animals↗

PubMed2003

High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases.

High density lipoprotein (HDL) activates endothelial nitric-oxide synthase (eNOS), leading to increased production of the antiatherogenic molecule NO. A variety of stimuli regulate eNOS activity through signaling pathways involving Akt kinase and/or mitogen-activated protein (MAP) kinase. In the present study, we investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I. HDL (10-50 microg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. Phosphoinositide 3-kinase (PI3 kinase) inhibition or dominant negative PI3 kinase also blocked the phosphorylation and activation of eNOS by HDL. Studies with genistein and PP2 showed that the nonreceptor tyrosine kinase, Src, is an upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition fully attenuated eNOS stimulation by HDL without affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt did not alter HDL-induced MAP kinase activation. These results indicate that HDL stimulates eNOS through common upstream, Src-mediated signaling, which leads to parallel activation of Akt and MAP kinases and their resultant independent modulation of the enzyme.

Animals↗

PubMed2002

ERbeta has nongenomic action in caveolae.

ERalpha and ERbeta serve classically as transcription factors, and ERalpha also mediates nongenomic responses to E2 such as the activation of endothelial nitric oxide synthase (eNOS). In contrast, the nongenomic capacities of endogenous ERbeta are poorly understood. We evaluated eNOS activation by E2 in cultured endothelial cells that express endogenous ERbeta to determine whether the ERbeta isoform has nongenomic action and to reveal the subcellular locale of that function. A subpopulation of ERbeta was localized to the endothelial cell plasma membrane, overexpression of ERbeta enhanced rapid eNOS stimulation by E2, and the response to endogenous ER activation was inhibited by the ERbeta-selective antagonist RR-tetrahydrochrysene (THC). eNOS activation through ERbeta was reconstituted and shown to occur independent of ERalpha in COS-7 cells, and ERbeta protein in COS-7 was directed to the plasma membrane. THC also blunted E2 activation of eNOS in isolated endothelial cell plasma membranes. Furthermore, ERbeta protein was detected and THC attenuated E2 stimulation of eNOS in isolated endothelial cell caveolae, and functional ERbeta-eNOS coupling was recapitulated in caveolae from transfected COS-7 cells. These findings in the ER-eNOS signaling paradigm indicate that endogenous ERbeta has nongenomic action in caveolae.

Animals↗