nitric oxide

nitric oxide Two strands of research in the late 1970s led to a revolution in biology. Robert Furchgott was unravelling a paradox concerning the well-known neurotransmitter acetylcholine: when injected intravenously it lowers blood pressure, showing that the body's blood vessels are relaxing, but when applied directly to blood vessels in isolation, outside the body, acetylcholine normally makes them contract. Furchgott showed that the response of blood vessels depended critically on the innermost layer of cells lining the vessel — the endothelium. When the endothelium was present, acetylcholine relaxed blood vessels, but when it was removed they contracted. Acetylcholine was shown to cause the release of a diffusible factor from endothelium, and it was this endothelium-derived relaxing factor (EDRF) — its nature then unknown — which relaxed the blood vessels.

Meanwhile, Ferid Murad was investigating how blood vessels are relaxed by the nitrovasodilators (e.g. glyceryl trinitrate, amyl nitrite) used in the treatment of angina. He found that these compounds increase the levels of cyclic GMP (known to promote relaxation) inside the smooth muscle cells of the vessels, probably by generating the substance nitric oxide (more correctly called nitrogen monoxide; NO).

It was soon shown that EDRF also increases the levels of cyclic GMP in smooth muscle, and the two lines of research converged when it was found that various agents, including haemoglobin and the dye methylene blue, inhibit the action both of EDRF and of the nitrovasodilators. By 1987, Furchgott and Louis Ignarro were suggesting that EDRF and NO were one and the same. This was confirmed by Salvador Moncada, who showed that NO is synthesized from the dietary amino acid L-arginine.

It then became evident that the endothelium of blood vessels was not the only site where NO is made. The enzyme nitric oxide synthase (NOS), which uses L-arginine, oxygen, and cofactors to make NO, exists in three forms: neuronal NOS (nNOS), from nerve cells; inducible NOS (iNOS), in inflammatory and other cells when the body's defence mechanisms are activated; and endothelial NOS (eNOS). nNOS and eNOS are ‘constitutive’ enzymes — that is, they are always present in their cells — and they are activated by increases in the intracellular concentration of calcium ions. iNOS is produced in many cells in response to bacterial infection and other circumstances (such as rheumatoid arthritis) when the immune system is activated; it is not regulated by calcium.

Nitric oxide is a tiny molecule. It is a gas, but dissolves in the fluids in and around cells. It is a free radical (sometimes denoted •NO to show that it has an unpaired electron, which makes it react very readily with other molecules). In particular, NO reacts with the radical anion, superoxide (•O^2-), to form peroxynitrite (ONOO^-), which can damage cellular DNA and hence cause cell death. Thus, excess NO can cause damage to cells. In some situations this is valuable: for instance, cells of the immune system use NO to kill invading bacteria. However, it can also be dangerous. After a stroke, for example, the nerve cells that have been deprived of oxygen fail to control their intracellular calcium; this rises and triggers a massive synthesis of NO by nNOS activation, leading to peroxynitrite production and the death of surrounding neurons. Peroxynitrite also oxidizes low density lipoproteins (LDL), which carry cholesterol in the blood, and oxidized LDL promotes atherosclerosis of blood vessels.

In septicaemia (blood poisoning), bacteria circulate in large numbers in the blood, and, in response, much NO is produced by many types of cells, including the muscle cells of the blood vessels this causes the blood vessels to relax. This leads to a severe fall in blood pressure, septic shock, which is difficult to reverse with agents such as adrenaline and dopamine, which normally increase blood pressure by constricting blood vessels. Septicaemia causes many deaths each year, and inhibition of NO synthesis appeared to be a promising treatment. But early attempts using inhibitors of NOS were largely unsuccessful. However, these were not selective for the relevant type (iNOS) and simultaneous inhibition of eNOS was deleterious to organ function. The increase in iNOS activity in inflammation can be inhibited by glucocorticoids (e.g. prednisolone, corticosterone), which explains part of their anti-inflammatory action, but they have no effect on the level of enzyme once it has been made. Thus the search is on for selective iNOS inhibitors which will leave nNOS and eNOS uninhibited.

NO has a good side and a bad side. While excess NO in septicaemia, inflammation, and stroke causes severe damage, localized production of small quantities of NO is essential for normal body function. Endothelial NOS maintains blood vessel function, and inhibition of this enzyme increases blood pressure. Indeed, hypertension is associated with decreased effectiveness of endothelial NO mechanisms. In blood vessels NO also regulates multiplication of muscle cells. Coronary artery bypass surgery sometimes fails because of narrowing of the newly replaced vessels; this is caused by thickening of their walls through muscle cell proliferation — thought to be due to decreased local production of NO as a result of endothelial damage during the surgery.

NO acts as a neurotransmitter in nerves involved in controlling a number of ‘vegetative’ functions, including operation of the sphincters in the gut and erection of the penis. Viagra (Sildenafil) works by prolonging the actions of NO released from penile nerves. In the brain, NO could even be involved in the formation of memory. ‘Long-term potentiation’ — the increase in strength of synapses in the hippocampus, as a result of strong stimulation which is thought to underlie certain forms of memory — is blocked by drugs that inhibit NO synthesis.

This small inorganic molecule, which was probably best known to the general public as a pollutant in car exhausts, became the biological molecule of the 1990s. Its importance was recognized by the award of a Nobel Prize to Furchgott, Ignarro, and Murad in 1998 ‘for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system’.

C. Robin Hiley

See also blood vessels.