The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

AKT/PKB signaling: navigating downstream.

Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: A report of the International Brachial Artery Reactivity Task Force

The programmed cell death that occurs as part of normal mammalian development was first observed nearly a century ago (Collin 1906). It has since been established that approximately half of all neurons in the neuroaxis and >99.9% of the total number of cells generated during the course of a human lifetime go on to die through a process of apoptosis (for review, see Datta and Greenberg 1998; Vaux and Korsmeyer 1999). The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. The purification in the 1950s of the nerve growth factor (NGF), which promotes the survival of sympathetic neurons, set the stage for the discovery that peptide trophic factors promote the survival of a wide variety of cell types in vitro and in vivo (Levi-Montalcini 1987). The profound biological consequences of growth factor (GF) suppression of apoptosis are exemplified by the critical role of target-derived neurotrophins in the survival of neurons and the maintenance of functional neuronal circuits. (Pettmann and Henderson 1998). Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 38-OH kinase (PI3K)/c-Akt kinase cascade. Several targets of the PI3K/c-Akt signaling pathway have been recently identified that may underlie the ability of this regulatory cascade to promote survival. These substrates include two components of the intrinsic cell death machinery, BAD and caspase 9, transcription factors of the forkhead family, and a kinase, IKK, that regulates the NF-kB transcription factor. This article reviews the mechanisms by which survival factors regulate the PI3K/c-Akt cascade, the evidence that activation of the PI3K/c-Akt pathway promotes cell survival, and the current spectrum of c-Akt targets and their roles in mediating c-Akt-dependent cell survival.

/pdf/cellular-survival-a-play-in-three-akts-3wtxbzcnm7.pdf

Cellular survival: a play in three Akts

Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is a fundamental determinant of cardiovascular homesotasis: it regulates systemic blood pressure, vascular remodelling and angiogenesis. Physiologically, the most important stimulus for the continuous formation of NO is the viscous drag (shear stress) generated by the streaming blood on the endothelial layer. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increased NO production. Inhibition of the phosphatidylinositol-3-OH kinase/Akt pathway or mutation of the Akt site on eNOS protein (at serine 1177) attenuates the serine phosphorylation and prevents the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca2+, rendering its activity maximal at sub-physiological concentrations of Ca2+. Thus, phosphorylation of eNOS by Akt represents a novel Ca2+-independent regulatory mechanism for activation of eNOS.

Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation

The endothelial nitric oxide synthase (eNOS), the expression of which is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO) in response to a number of stimuli. The physiologically most important determinants for the continuous generation of NO and thus the regulation of local blood flow are fluid shear stress and pulsatile stretch. Although eNOS activity is coupled to changes in endothelial cell Ca(2+) levels, an increase in Ca(2+) alone is not sufficient to affect enzyme activity because the binding of calmodulin (CaM) and the flow of electrons from the reductase to the oxygenase domain of the enzyme is dependent on protein phosphorylation and dephosphorylation. Two amino acids seem to be particularly important in regulating eNOS activity and these are a serine residue in the reductase domain (Ser(1177)) and a threonine residue (Thr(495)) located within the CaM-binding domain. Simultaneous alterations in the phosphorylation of Ser(1177) and Thr(495) in response to a variety of stimuli are regulated by a number of kinases and phosphatases that continuously associate with and dissociate from the eNOS signaling complex. eNOS associated proteins, such as caveolin, heat shock protein 90, eNOS interacting protein, and possibly also motor proteins provide the scaffold for the formation of the protein complex as well as its intracellular localization.

https://www.physiology.org/doi/pdf/10.1152/ajpregu.00323.2002

Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase

In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.

Cytochrome P450 2C is an EDHF synthase in coronary arteries

https://www.cell.com/trends/pharmacological-sciences/pdf/S0165-6147(02)02050-3.pdf

EDHF: bringing the concepts together.

—The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca2+ by the phosphorylation of Ser1177 but results only in a low nitric oxide (NO) output In the present study, we assessed whether the agonist-induced (Ca2+-dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of Thr495 in the calmodulin (CaM)-binding domain eNOS Thr495 was constitutively phosphorylated in porcine aortic endothelial cells and was rapidly dephosphorylated after bradykinin stimulation In the same cells, bradykinin enhanced the phosphorylation of Ser1177, which was maximal after 5 minutes, and abolished by the CaM-dependent kinase II (CaMKII) inhibitor KN-93 Bradykinin also enhanced the association of CaMKII with eNOS Phosphorylation of Thr495 was attenuated by the protein kinase C (PKC) inhibitor Ro 31-8220 and after PKC downregulation using phorbol 12-myristate 13-acetate The agonist-induced dephosphorylation of Thr495 was completely Ca2+-dependent and inhibited by the PP1 inhibitor calyculin A Little CaM was bound to eNOS immunoprecipitated from unstimulated cells, but the agonist-induced dephosphorylation of Thr495 enhanced the association of CaM Mutation of Thr495 to alanine increased CaM binding to eNOS in the absence of cell stimulation, whereas the corresponding Asp495 mutant bound almost no CaM Accordingly, NO production by the Ala495 mutant was more sensitive to Ca2+/CaM than the aspartate mutant These results suggest that the dual phosphorylation of Ser1177 and Thr495 determines the activity of eNOS in agonist-stimulated endothelial cells Moreover, the dephosphorylation of Thr495 by PP1 precedes the phosphorylation of Ser1177 by CaMKII The full text of this article is available at http://wwwcircresahaorg

Ingrid Fleming

Papers

Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation

Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase

Cytochrome P450 2C is an EDHF synthase in coronary arteries

EDHF: bringing the concepts together.

Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity.