Cellular signaling and NO production

Abstract

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor is nitric oxide (NO), which is synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Endothelium-dependent relaxations involve both pertussis-toxin-sensitive G(i) (e.g., responses to serotonin, sphingosine 1-phosphate, alpha(2)-adrenergic agonists, and thrombin) and pertussis-toxin-insensitive G(q) (e.g., adenosine diphosphate and bradykinin) coupling proteins. eNOS undergoes a complex pattern of intracellular regulation, including post-translational modifications involving enzyme acylation and phosphorylation. eNOS is reversibly targeted to signal-transducing plasmalemmal caveolae where the enzyme interacts with a number of regulatory proteins, many of which are modified in cardiovascular disease states. The release of nitric oxide by the endothelial cell can be up- (e.g., by estrogens, exercise, and dietary factors) and down-regulated (e.g. oxidative stress, smoking, and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g., diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis-toxin-sensitive pathway for NO release which favors vasospasm, thrombosis, penetration of macrophages, cellular growth, and the inflammatory reaction leading to atherosclerosis. The unraveling of the complex interaction of the pathways regulating the presence and the activity of eNOS will enhance the understanding of the perturbations in endothelium-dependent signaling that are seen in cardiovascular disease states, and may lead to the identification of novel targets for therapeutic intervention.

Fig. 1

Some of the neurohumoral mediators that cause the release of endothelium-derived relaxing factors (EDRF) through activation of specific endothelial receptors (circles). E epinephrine, AA arachidonic acid, Ach acetylcholine, ADP adenosine diphosphate, α alpha adrenergic receptor, AVP arginine vasopressin, B kinin receptor, ET endothelin, endothelin-receptor, H histaminergic receptor, 5-HT serotonin (5-hydroxytryptamine), serotoninergic receptor, M muscarinic receptor, NE norepinephrine, P purinergic receptor, T thrombin receptor, VEGF vascular endothelial growth factor, VP vasopressin receptor [from , with permission]

Fig. 2

Schematic of possible mechanisms by which production of nitric oxide is regulated in endothelial cells. Nitric oxide is produced through enzymatic conversion of L-arginine by nitric oxide synthase (endothelial or type III, eNOS). The transcription of this enzyme is regulated genomically by hormones and growth factors. Stability of eNOS mRNA is modulated by statins and hormones. eNOS enzyme activity requires calcium, calmodulin, nicotinamide adenine dinucleotide phosphate, and 5, 6, 7, 8-tetra-hydrobiopterine (BH₄). Enzyme activity is regulated by complexing to these proteins in microdomains of the endothelial cell. Association with this complex of heat shock protein 90 increases enzyme activity. Stimulation of specific receptors on the endothelial surface (R) complexed with guanine nucleotide regulatory proteins, which are sensitive to pertussis toxin (G_i) or insensitive to pertussis toxin (G_q), activate intracellular pathways that modulate eNOS activity post-translationally through heat shock protein 90 or AKT-phosphorylation. Association of eNOS with caveolin-1 or glycosylation of the enzyme reduces activity. A metabolite of L-arginine, asymmetric dimethyl arginine decreases production of the nitric oxide through competitive binding to eNOS. Thus, this endogenous amine may be a risk factor for the development of cardiovascular disease. + indicates stimulation, − indicates inhibition, ? indicates those pathways in which the regulation is unknown [from , with permission]

Fig. 3

Postulated G-protein-mediated signal transduction processes in a normal, native endothelial cell. Activation of the cell causes the release of nitric oxide (NO), which has important protective effects in the vascular wall. Abbreviations: 5-HT serotonin receptor, B bradykinin receptor, P purinoceptor, G coupling proteins [from , with permission]

Fig. 4

Effects of oxidized low-density lipoproteins in a regenerated endothelial cell, resulting in the reduced release of nitric oxide. Abbreviations: 5-HT serotonin receptor, B bradykinin receptor, P purinoceptor, G coupling proteins [from , with permission]

Fig. 5

Mechanisms of oxyLDL-induced impairment of endothelial NO production. The NO synthase (NOS) uses L-arginine to generate NO. NO production could be attenuated in the presence of oxyLDL by interfering with the supply of L-arginine to the enzyme through endogenous competitive inhibitors such as asymmetrical dimethyl-L-arginine as well as degradation of arginine through arginase. NOS expression and specific activity are decreased by oxyLDL through RhoA and PKC. NO bioavailability is reduced by an oxyLDL-mediated activation of the NADPH oxidase, which leads to superoxide anion (O₂⁻) formation. This process facilitates the generation of peroxynitrite (ONOO⁻), which subsequently oxidizes tetrahydrobiopterin (BH₄) of NOS, leading to NOS uncoupling [3]. Uncoupled NOS itself produces O₂⁻, further promoting the process of BH₄ oxidation [from , with permission]

Fig. 6

Overview of principal eNOS post-translational modifications. Consensus binding sites for calmodulin (CaM), FAD, FMN, NADPH, heme, arginine, and tetrahydrobiopterin (BH₄) are shown along the linear sequence of the eNOS protein. eNOS undergoes dual acylation: co-translational N-myristoylation (“My”) at glycine 2 and reversible post-translational thiopalmitoylation (“Pa”) at cysteines 15 and 26. [The numbering scheme used here reflects the amino acid sequence of bovine eNOS, which has been more extensively characterized from a biochemical standpoint.] eNOS-derived NO promotes S-nitrosylation at cysteines 96 and 101, leading to enzyme inhibition (signified by the red arrow). Phosphorylations at serine 116, threonine 497, and tyrosine 659 are generally associated with eNOS enzyme inhibition (shown in red), and phosphorylations at serines 617, 635, and 1179 and at tyrosines 83 generally increase enzyme activity (shown in green)