Timothy J. McCabe

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

Domain Mapping Studies Reveal That the M Domain of hsp90 Serves as a Molecular Scaffold to Regulate Akt-Dependent Phosphorylation of Endothelial Nitric Oxide Synthase and NO Release

Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation.

Reconstitution of an Endothelial Nitric-oxide Synthase (eNOS), hsp90, and Caveolin-1 Complex in Vitro EVIDENCE THAT hsp90 FACILITATES CALMODULIN STIMULATED DISPLACEMENT OF eNOS FROM CAVEOLIN-1

The activity of endothelial nitric-oxide synthase (eNOS) is regulated by its subcellular localization, phosphorylation and through its interaction with different proteins. The association of eNOS with caveolin-1 (Cav) is believed to maintain eNOS in an inactive state; however, increased association of eNOS to heat shock protein 90 (hsp90) is observed following activation. In this study, we investigate the relationship between caveolin and hsp90 as opposing regulatory proteins on eNOS function. Immunoprecipitation of Cav-1 from bovine lung microvascular endothelial cells shows that eNOS and hsp90 are present in the Cav-1 complex. eNOS and hsp90 from the lysate also interact with exogenous glutathione S-transferase-linked caveolin-1 (GST-Cav), and the addition of calcium-activated calmodulin (CaM) to the GST-Cav complex partially inhibited the association of eNOS and hsp90. Purified eNOS associates with GST-Cav specifically through the caveolin-scaffolding domain (residues 82–101); however, the addition of CaM slightly, but nonstatistically, reduces eNOS binding to GST-Cav. When hsp90 is present in the binding reaction, the addition of increasing concentrations of CaM significantly displaces eNOS and hsp90 from GST-Cav. eNOS enzymatic activity is also less sensitive to inhibition by the caveolin scaffolding peptide (residues 82–101) when eNOS is prebound to hsp90. Collectively, our results show that the actions of CaM on eNOS dissociation from caveolin are facilitated in the presence of hsp90.

Direct Interaction between Endothelial Nitric-oxide Synthase and Dynamin-2 IMPLICATIONS FOR NITRIC-OXIDE SYNTHASE FUNCTION

Endothelial nitric-oxide synthase (eNOS) is regulated in part through specific protein interactions. Dynamin-2 is a large GTPase residing within similar membrane compartments as eNOS. Here we show that dynamin-2 binds directly with eNOS thereby augmenting eNOS activity. Double label confocal immunofluorescence demonstrates colocalization of eNOS and dynamin in both Clone 9 cells cotransfected with green fluorescent protein-dynamin and eNOS, as well as in bovine aortic endothelial cells (BAEC) expressing both proteins endogenously, predominantly in a Golgi membrane distribution. Immunoprecipitation of eNOS from BAEC lysate coprecipitates dynamin and, conversely, immunoprecipitation of dynamin coprecipitates eNOS. Additionally, the calcium ionophore, A23187, a reagent that promotes nitric oxide release, enhances coprecipitation of dynamin with eNOS in BAEC, suggesting the interaction between the proteins can be regulated by intracellular signals. In vitro studies demonstrate that glutathione S-transferase (GST)-dynamin-2 quantitatively precipitates both purified recombinant eNOS protein as well as in vitro transcribed 35S-labeled eNOS from solution indicating a direct interaction between the proteins in vitro. Scatchard analysis of binding studies demonstrates an equilibrium dissociation constant (K d ) of 27.6 nm. Incubation of purified recombinant eNOS protein with GST-dynamin-2 significantly increases eNOS activity as does overexpression of dynamin-2 in ECV 304 cells stably transfected with eNOS-green fluorescent protein. These studies demonstrate a direct protein-protein interaction between eNOS and dynamin-2, thereby identifying a new NOS-associated protein and providing a novel function for dynamin. These events may have relevance for eNOS regulation and trafficking within vascular endothelium.

Erratum: Regulation of endothelium-derived nitric oxide production by the protein kinase Akt (Nature (1999) 399 (597-601))

This corrects the article DOI: 21218

Relationships between NADPH diaphorase staining and neuronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues

Abstract The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent enzymes – neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450 reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue. However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver, sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea, zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms.

Sparing of mdx extraocular muscles from dystrophic pathology is not attributable to normalized concentration or distribution of neuronal nitric oxide synthase

Previous findings have led to speculations that decreased concentration of nNOS (neuronal nitric oxide synthase) may underlie some aspects of the pathophysiology of dystrophic muscle. We have tested whether the sparing of extraocular muscles (EOM) in muscular dystrophy is attributable to the presence of normal nNOS concentration and distribution in these muscles. Measurements of total nNOS concentration in control muscle showed that total nNOS comprises approximately 0.05% of total muscle protein, indicating a molar stoichiometry of approximately 60 and 20 to total dystrophin and syntrophin, respectively. Thus, most muscle nNOS is either not associated with the dystrophin complex, or binds to yet unidentified sites in the complex. nNOS concentration was at least two-fold greater in C57 EOM and tibialis anterior (TA) compared with mdx samples. No significant differences in nNOS concentration in EOM versus TA in either mdx or C57 mice were observed, nNOS was concentrated at the sarcolemma of all C57 samples, while mdx nNOS displayed a cytosolic distribution, except in fibers that reverted to express dystrophin. These data show that mdx EOM are spared by a mechanism other than normalized concentration and location of nNOS.

Structure-Function Studies and Physiological Roles of Eicosanoids Metabolized by Cytochrome P450 ω-Hydroxylases

The cytochrome P450-mediated oxygenation of many fatty acids and eicosanoids occurs in a variety of tissues. The cytochromes P450 responsible for these activities belong to a closely related gene family, the first member of which was purified and characterized from clofibrate-treated rats by Tamburini, et al. (1) and cloned and sequenced by Hardwick, et al (2). In the latest listing of members of the cytochrome P450 gene superfamily by Nebert, et al. (3), there are now 9 members of the CYP4A gene subfamily which includes those cytochromes P450 catalyzing the ω-hydroxylation of fatty acids and eicosanoids from different organs and species. Since the mid-1970’s, the interest of Masters’ laboratory has centered on fatty acid and prostaglandin hydroxylations catalyzed by pig and rabbit kidney and lung microsomes. Following the observation by Powell and Solomon (4) and Powell (5) that the ω-hydroxylation of prostaglandins and thromboxanes was induced in maternal rabbit lung over 100-fold during pregnancy, Williams, et al. (6) purified a cytochrome P450 prostaglandin w-hydroxylase from this source. Simultaneously, Yamamoto, et al. (7) isolated a similar or identical protein from the lungs of progesterone-treated rabbits and, later, Matsubara, et al (8) cloned and sequenced the cDNA for their protein. There is strong evidence that the high level of induction of this enzyme is regulated at the transcriptional level, since the gestational age dependence of enzyme activity parallels the levels of protein (Western blots) and in vitro-translatable mRNA (9). Northern blotting techniques have also revealed an increase in mRNA levels as a result of pregnancy in female rabbits or progesterone treatment in male rabbits (8,10). Recently, attention has been directed toward the possible physiological significance of these enzymes (11–15) especially in the regulation of hemodynamics. The experiments to be described have been designed to ascertain the structure-function relationships of the various members of the CYP4A gene subfamily by correlating the various substrate specificities with manipulation of their amino acid sequences and expression in a transient mammalian cell expression system, i.e., African green monkey kidney cells (COS-1). The regulatory aspects of these many and varied, but closely related, enzymes are being studied in whole animals by pretreatment with steroid hormones, some of which have been shown previously to be inducers of various members of this subfamily. The physiological roles of these various eicosanoid-metabolizing cytochromes P450 are being probed utilizing a combination of biochemical approaches, including the use of broad spectrum and specific inhibitors of cytochrome P450 systems in microdissected renal arcuate arteries.