Nitric Oxide

Nitric oxide (NO) is produced by many cells in the body; however, its production by vascular endothelium is particularly important in the regulation of  blood flow. Because of its importance in vascular function, abnormal production of NO, as occurs in different disease states, can adversely affect blood flow and other vascular functions.

NO Biosynthesis

NO is produced from the amino acid L-arginine by the enzymatic action of nitric oxide synthase (NOS). There are two endothelial forms of NOS: constitutive NOS (cNOS; type III) and inducible NOS (iNOS; type II). Co-factors for NOS include oxygen, NADPH, tetrahydrobiopterin and flavin adenine nucleotides. In addition to endothelial NOS, there is a neural NOS (nNOS; type I) that serves as a transmitter in the brain and in different nerves of the peripheral nervous system, such as non-adrenergic, non-cholinergic (NANC) autonomic nerves that innervate penile erectile tissues and other specialized tissues in the body to produce vasodilation. Under normal, basal conditions in blood vessels, NO is continually being produced by cNOS. The activity of cNOS is calcium and calmodulin dependent. There are two basic pathways for the stimulation of cNOS, both of which involve release of calcium ions from subsarcolemmal storage sites. First, shearing forces acting on the vascular endothelium generated by blood flow causes a release of calcium and subsequent cNOS activation. Therefore, increases in blood flow stimulate NO formation (flow-dependent NO formation). Second, endothelial receptors for a variety of ligands stimulate calcium release and subsequent NO production (receptor-stimulated NO formation). Included are receptors for acetylcholine, bradykinin, substance-P, adenosine, and many others vasoactive substances. In the late 1970s, Dr. Robert Furchgott observed that acetylcholine released a substance that produced vascular relaxation, but only when the endothelium was intact. This observation opened this field of research and eventually led to his receiving a Nobel prize. Initially, Furchgott called this substance endothelium-derived relaxing factor (EDRF), but by the mid-1980 he and others identified this substance as being NO.
The other isoform of endothelial NOS is iNOS. It differs, in part, from cNOS in that its activation is calcium independent. Under normal, basal conditions, the activity of iNOS is very low. The activity of iNOS is stimulated during inflammation by bacterial endotoxins (e.g., lipopolysaccharide) and cytokines such as tumor necrosis factor (TNF) and interleukins. During inflammation, the amount of NO produced by iNOS may be a 1,000-fold greater than that produced by cNOS.

Intracellular Mechanisms

When NO forms, it has a half-life of only a few seconds, in large part because superoxide anion has a high affinity for NO (both molecules have an unpaired electron making them highly reactive). Therefore, superoxide anion reduces NO bioavailability. NO also avidly binds to the heme moiety of hemoglobin (in red blood cells) and the heme moiety of the enzyme guanylyl cyclase, which is found in vascular smooth muscle cells and most other cells of the body. Therefore, when NO is formed by vascular endothelium, it rapidly diffuses into the blood where it binds to hemoglobin and subsequently broken down. It also diffuses into the vascular smooth muscle cells adjacent to the endothelium where it binds to and activates guanylyl cyclase. This enzyme catalyzes the dephosphorylation of GTP to cGMP, which serves as a second messenger for many important cellular functions, particularly for signalling smooth muscle relaxation.
Cyclic GMP induces smooth muscle relaxation by multiple mechanisms including

1. increased intracellular cGMP, which inhibits calcium entry into the cell, and decreases intracellular calcium concentrations
2. activates K⁺ channels, which leads to hyperpolarization and relaxation
3. stimulates a cGMP-dependent protein kinase that activates myosin light chain phosphatase, the enzyme that dephosphorylates myosin light chains, which leads to smooth muscle relaxation.

Because of the central role of cGMP in NO-mediated vasodilation, drugs (e.g., Viagra®) that inhibit the breakdown of cGMP (cGMP-dependent phosphodiesterase inhibitors) are used to enhance NO-mediated vasodilation, particularly in penile erectile tissue in the treatment of erectile dysfunction. Increased cGMP also has an important anti-platelet, anti-aggregatory effect.

Vascular actions of NO include the following:

• Direct vasodilation (flow dependent and receptor mediated)
• Indirect vasodilation by inhibiting vasoconstrictor influences (e.g., inhibits angiotensin II and sympathetic vasoconstriction)
• Anti-thrombotic effect - inhibits platelet adhesion to the vascular endothelium
• Anti-inflammatory effect - inhibits leukocyte adhesion to vascular endothelium; scavenges superoxide anion
• Anti-proliferative effect - inhibits smooth muscle hyperplasia

Because of the above actions of NO, when its production is impaired or its bioavailability is reduced, the following can result:

• Vasoconstriction (e.g., coronary vasospasm, elevated systemic vascular resistance, hypertension)
• Thrombosis due to platelet aggregation and adhesion to vascular endothelium
• Inflammation due to upregulation of leukocyte and endothelial adhesion molecules
• Vascular hypertrophy and stenosis

Diseases or Conditions Associated with Abnormal NO Production and Bioavailability

• Hypertension
• Obesity
• Dyslipidemias (particularly hypercholesterolemia and hypertriglyceridemia)
• Diabetes (both type I and II)
• Heart failure
• Atherosclerosis
• Aging
• Cigarette smoking