Although nitroglycerin had been used for more than 100 years to treat angina, the mechanism of action was unknown until about 1980. Ignarro's group used isolated strips of bovine coronary artery to show that NO produced a transient but marked relaxation of smooth muscle cells. This relaxation could be blocked by hemoglobin and other hemoproteins, since hemoglobin catalyzes the rapid inactivation of NO. Methylene blue, an inhibitor of GC, was shown to block NO relaxation of smooth muscles cells, thus providing the first clue that NO probably causes relaxation by elevating tissue cGMP. This confirmed work by Merhade in other systems.

Platelet aggregation can be inhibited by sodium nitroprusside as shown by Ignarro. This led to the question of whether the antiplatelet effect of nitroprusside could also be mediated by NO and cGMP, as is the vasodilator effect of nitroprusside. They showed that NO produced a concentration-dependent inhibition of platelet aggregation in tests of human platelets aggregated by ADP. Cigarette smoke, which contains fairly large amounts of NO, was also shown by Ignarro to inhibit platelet aggregation and to be a good coronary artery vasodilator.

As a pharmacologist Ignarro was driven to understand the mechanism of action of the drugs related to nitroglycerin, which work by liberating NO. These drugs, whether organic nitrates, nitrate esthers or sodium nitroprusside, work by liberating NO by one or more mechanism in the cell, such as the smooth muscle cell. NO produces vasorelaxation by activating GC which causes elevation of cGMP levels.

Ignarro noted the irony that the simple molecular structure of NO belies its many different pharmacologic and physiologic effects. Importantly, the unpaired electron in the NO structure allows NO be reactive with iron, especially heme iron, in variety of proteins. Although GC activation by NO had been identified, which stimulates the conversion of GTP to cGMP, the mechanism was unknown. Ignarro's group found that purified GC contained heme, therefore it was a hemoprotein. Because of the very high binding affinity of heme for NO, it was clear that GC could bind NO.

Further work showed that heme combined with the catalytic site of GC, causing stearic hindrance, blocking access of the catalytic site to the substrate GTP. When NO reacts with this site it forms a bond with the iron causing breakage of the bond between the iron and the enzyme protein, thus causing exposure of the catalytic site and allowing much greater exposure to the substrate GTP. This accounts for the marked increase in activity caused by NO when it activates GC.

The knowledge that nitroglycerin and NO are very potent relaxants of both arteries and veins, particularly arteries, led to a series of important laboratory work in the 1980s by Ignarro's group. The presence of receptors for exogenous NO and nitroglycerin led to the questions of why those receptors exist, and whether there is endogenous NO or nitroglycerin that functions in the body. However, the difficulty of the experiments in Ignarro's laboratory did not confirm the presence of endogenous NO.

Endothelium-dependent relaxation

Ignarro's group then conducted experiments based on the observation in 1980 that acetylcholine and bradykinin and other substances cause endothelium-dependent vasodilation by first generating endothelium-derived relaxing factor (EDRF). But, the chemical basis of EDRF was unknown.

Ignarro therefore conducted studies to determine whether endothelium-dependent relaxation involves the actions of cGMP. They confirmed the finding that acetylcholine causes vascular relaxation indirectly by first causing the release of a relaxing factor from the endothelium. They then showed that this relaxation is associated with an increase in cGMP, and that the addition of methylene blue blocks cGMP accumulation by acetylcholine and blocks relaxation. They had already shown that methylene blue could block the cGMP accumulation and relaxation by NO.

A schematic published by Ignarro in 1984 compared NO and the relaxing factor. Both could elevate cGMP to cause relaxation, and methylene blue could inhibit the cGMP accumulation and therefore the relaxation response to both NO and the relaxing factor. Ignarro reviewed a few of the experiments that established in 1986 that EDRF was indeed NO.

EDRF and NO

Work published in 1986 showed that EDRF could directly activate GC and thus cause an increase in cGMP and the resultant relaxation in a fashion very similar to NO.

Bioassay cascade experiments were conducted with small segments of animal pulmonary artery or vein. After these segments were perfused, bradykinin and acetylcholine were added and allowed to drip over three strips of artery or vein that were endothelium-denuded. The denuded strip permitted only the relaxation by NO or EDRF to be seen. The addition of authentic NO caused nice relaxation of strip 1, less relaxation of strip 2, and even less of strip 3, due to the short 5-second half-life of NO.

The addition of acetylcholine to the perfused artery or the calcium ionophore A23 to the perfused vein showed similar decay in the relaxation and a 5-second half-life for EDRF. The addition of pyrogallol, which generates superoxide anion from solution that can destroy NO, to the perfusion fluid nearly abolished NO and EDRF relaxation. The addition of superoxide dysmutase to remove the superoxide to increase the stability of NO yielded a marked increase in the effects of NO. They also found that both the calcium ionophore, which releases EDRF, and the NO increases cGMP levels. Measuring the perfusion fluid containing calcium ionophore detected equivalent amounts of NO as with authentic NO.

The experiment that established EDRF is NO was based on a principle discovered in the 1930s. The addition of NO to deoxyhemoglobin results in a distinct shift in the absorbance maximum, from about 430 nanometers to about 409 nanometers. The addition of deoxyhemoglobin to freshly-isolated endothelial cells from bovine aorta had little effect. But, the addition of acetylcholine, bradykinin or calcium ionophore to release EDRF caused a characteristic shift identical to that caused by NO in the absorbance maximum. Only NO can cause this exact shift in absorbance maximum. Therefore, they had obtained direct proof that EDRF and NO were essentially the same substance.

The discovery by Ignarro that EDRF was indeed NO led to much work in this area, showing that NO played very important biologic or physiologic roles in many different areas, including the GI tract, central nervous system, pulmonary function, host defense, and so forth. Perhaps the most important role is the cardiovascular function of NO.

NO is synthesized in the body by NO synthase, of which there are three different isoforms. Neuronal NO synthase makes NO as a neurotransmitter. Endothelial NO synthase is present in the endothelial cells. Important in the cardiovascular field, and others, is inducible NOS (iNOS).

The biochemical mechanism to synthesize NO is rather complex. NO is derived from one of the two amino nitrogen atoms in the guanidino function of arginine. The enzyme NOS catalyzes the oxidation of one of these to the N-hydroxy intermediate, which is then converted to NO, which is cleaved. The other amino acid formed is citrulline. Oxygen is incorporated into both products of this reaction. This reaction involves NADPH as an electron donor, and many other co-factors such as tetrahydrobiopter, calmodulin, and calcium.

Effects of endothelial NOS

The isoforms of NOS look essentially the same, based on x-ray crystallographic studies conducted by Masters. The endothelial NOS has a homodimer and two identical monomeres. Each monomere contains one mole of heme and one molecule of tetrahydrobiopterin in the interface. Zinc is also present in the interface, which may play a role in holding the dimer together.

The vasorelaxation and cell adhesion functions of NO are very important.

The presence of endothelium-dependent vasodilators, acetylcholine, bradykinin, histamine, or the shear stress of blood flow, can eventually cause calcium to enter the endothelial cell. Calcium entry triggers binding of calmodulin onto the membrane-bound endothelial NOS, activating the e-NOS to make NO, which can diffuse into the nearby smooth muscle cells to cause cGMP-dependent relaxation. The NO can also diffuse luminally into the platelets and monocytes that will eventually enter the cell and be transformed into macrophages and atherosclerosis. NO can enter leukocytes near the intimal surface, and functions to prevent leukocyte adhesion to the intimal lining and to prevent aggregation.

NO in blood pressure regulation

NO was shown to contribute to blood pressure control by the continuous release of NO from the resistance arterials in animals by Ignarro's laboratory in the late 1980s.

Conscious rats were instrumented for the recording of systemic blood pressure and the intravenous injection of drugs. A marked increase in blood pressure was found with the injection of N-nitro-arginine, a NOS inhibitor. But, the addition of excess L-arginine, a substrate for NOS, but not D-arginine, could reverse this hypertension. L-arginine competes with the N-nitro-arginine and overcomes it, and thus the hypertension effect of N-nitro-arginine can be reversed.

Interfering with the continuous release of NO, which is trying to cause vasodilation, causes an increase in blood pressure. This occurs simultaneously with the continuous release of norepinephrine in the sympathetic nervous system, where it functions to cause vasoconstriction and increased vascular tone. NO and norepinephrine work to control blood pressure in this way. Therefore, it seems reasonable that in essential hypertension there is a defect in the NO pathway. However, the answer to this question is unknown. NO has been shown to be effective therapy to lower persistent pulmonary hypertension in newborn babies.

Inducible NOS can have profound effects on the body. The iNOS protein is induced when confronted by lipopolysaccharide and certain cytokines like interferon gamma or TNF-alpha or IL-1-beta, among other substances. After several hours, iNOS produces large amounts of NO. iNOS can affect target cells, like tumor cells, or even nearby vascular smooth muscle cells, which can undergo a great deal of relaxation in response to lipopolysaccharide. Excess NO can produce detrimental effects on certain target cells.

It is unknown whether NO itself causes cytotoxicity, or whether it is another product. NO can react with superoxide anion to form peroxynitrite, which is very cytotoxic. Although it must be proven, Ignarro thinks that NO is protective in the cardiovascular system in most cases. It is the reaction between NO and superoxide to form peroxynitrite, or perhaps other reactions, that is cytotoxic.

NO is an important neurotransmitter in many functions. In addition to being a mediator of erectile function, NO plays a role in the GI tract, genitourinary tract, airway function, peripheral sensory transmission, among others. NO may also be important in learning. More work is needed to understand the precise role of NO in these latter areas.

The role of NO as a neurotransmitter in some of the peripheral nerves is understood. Since 1990 Ignarro's laboratory has studied whether NO is responsible for the neuronally-induced relaxation of corpus cavernosum smooth muscle (erectile tissue) and therefore penile erection.

In the nerves that innervate the corpus cavernosum, they showed that calcium influx occurs in respone to nerve stimulation (sodium current) allowing for stimulation of neuronal NOS with the liberation of NO. The neurotransmitter NO diffuses into the smooth muscle, both vascular and non-vascular, and causes relaxation of this smooth muscle, allowing rapid filling of these blood vessels with blood--the physiological erectile response.

In the signal transduction system, NO, the neurotransmitter from the nerve, enters the smooth muscle to cause activation of GC and elevation of cGMP levels. The cGMP causes relaxation. Impotent patients make insufficient quantities of NO, therefore have insufficient quantities of cGMP. The cGMP signal is rapidly degraded in all tissues by phosphodiesterase (PDE). The small amounts of cGMP that are formed are quickly degraded by PDE. In patients who are impotent there is insufficient cGMP formed to cause an erectile response. Sildenafil inhibits PDE, allowing the cGMP to accumulate until a physiologic response is produced.