Differential modulation of nitric oxide synthases in aging: therapeutic opportunities

Abstract

Vascular aging is the term that describes the structural and functional disturbances of the vasculature with advancing aging. The molecular mechanisms of aging-associated endothelial dysfunction are complex, but reduced nitric oxide (NO) bioavailability and altered vascular expression and activity of NO synthase (NOS) enzymes have been implicated as major players. Impaired vascular relaxation in aging has been attributed to reduced endothelial NOS (eNOS)-derived NO, while increased inducible NOS (iNOS) expression seems to account for nitrosative stress and disrupted vascular homeostasis. Although eNOS is considered the main source of NO in the vascular endothelium, neuronal NOS (nNOS) also contributes to endothelial cells-derived NO, a mechanism that is reduced in aging. Pharmacological modulation of NO generation and expression/activity of NOS isoforms may represent a therapeutic alternative to prevent the progression of cardiovascular diseases. Accordingly, this review will focus on drugs that modulate NO bioavailability, such as nitrite anions and NO-releasing non-steroidal anti-inflammatory drugs, hormones (dehydroepiandrosterone and estrogen), statins, resveratrol, and folic acid, since they may be useful to treat/to prevent aging-associated vascular dysfunction. The impact of these therapies on life quality in elderly and longevity will be discussed.

Keywords: aging; endothelial dysfunction; folic acid; nitric oxide; nitric oxide synthases; resveratrol; statin; uncoupled eNOS.

Figure 1

Role of nitric oxide synthase (NOS) enzymes in endothelial function and aging-associated endothelial dysfunction. Endothelial (e)-NOS isoform converts l-arginine to nitric oxide (NO). eNOS activity is determined by intracellular calcium concentration [i(Ca²⁺)] and/or its phosphorylation (P) state at different sites (e.g., eNOS is positively and negatively modulated by phosphorylation of serine 1177 (S¹¹⁷⁷) and threonine 494 (T⁴⁹⁴), respectively). In addition, eNOS activity is inhibited by its interaction with caveolin-1 and by O-GlcNAc modification, while eNOS association with heat shock protein 90 (hsp90) favors its activation. In endothelial aging, NO synthesis is compromised because eNOS activity is decreased due to increased expression and interaction with caveolin, reduced expression and association with hsp90, reduced phosphorylation of S¹¹⁷⁷ and increased phosphorylation of T⁴⁹⁴. In addition, reduced availability of l-arginine and tetrahydrobiopterin (BH₄) induces eNOS uncoupling or changes the enzyme to a state that favors superoxide anion $•{\text{O}}_2^{\text{−}}$ generation. In aged endothelial cells, up-regulated iNOS, which may be associated with NF-κB-induced vascular inflammation, produces high levels of NO. NO reacts with cysteine residues of proteins (arginase in this example), forming S-nitrosothiol (-SNO)–arginase, which increases arginase activity and l-arginine consumption. NO directly reacts with $•{\text{O}}_2^{\text{−}}$ produced by uncoupled eNOS, and other vascular sources, generating the harmful reactive nitrogen specie peroxynitrite (ONOO⁻), which contributes to vascular dysfunction. The aging–associated NOS alterations are depicted in red. ACh, acetylcholine; Bk, bradykinin; PI₃K, phosphoinositide-3-kinase.

Figure 2

Drugs that affect NO generation/turnover by modulating NOS enzymes. The drugs that modulate NOS enzymes and, consequently NO production, act by changing three important aspects of NOS enzymes function: activity, expression and transcription: (1) activity; estrogen induces eNOS phosphorylation at (S¹¹⁷⁷) to increase enzyme activity; statins and folic acid may cause eNOS recoupling to increase eNOS activity and NO production; (2) expression; statins and DHEA increase eNOS expression and, consequently, NO generation; (3) transcription; statins and resveratrol act at the transcriptional level of eNOS increasing its mRNA stability. In addition, resveratrol activates the promoter gene of eNOS increasing its transcription. Whereas eNOS enzyme function can be altered in three different ways, nNOS and iNOS function can be modulated at the transcriptional level by some of these drugs. Statins may directly induce nNOS transcription and increase nNOS protein levels. On the other hand, resveratrol indirectly decreases iNOS transcription by inhibiting NF-κB. DHEA, dehydroepiandrosterone; ROS, reactive oxygen species; NF-κB, nuclear factor kappa B; mRNA, messenger RNA.