 So, I'm going to talk to you about the history and discovery of this unique molecule, which by the way is a gaseous molecule, like oxygen, carbon dioxide, nitrogen, N-O is a gas. Now, it doesn't work as a gas in the body. Oxygen doesn't work as a gas in the body. These gases are soluble in liquids, in water, in the blood, and so on. So they work in solution. They don't float around as bubbles producing effects in the body. So this molecule is nitric oxide, also abbreviated N-O. And this is, I don't think you have to be a chemist to understand this. This is a very simple molecule. It consists of one atom of nitrogen and one atom of oxygen, very simply. But it's more complex than meets the eye, especially if you're not a scientist. This is what we call a radical, a free radical, and the reason is that it has this electron. Atoms and molecules have electrons that rotate around them. And in most cases, when the molecules are stable, they need to have two electrons paired together like this. However, when you have one single electron by itself, these molecules are very unstable. They don't like to exist in that form. They're always running around looking for other molecules that also have a single electron so they can react in order to form an electron pair, and that makes them stable. And so for that reason, nitric oxide is fairly reactive, which accounts for all the different things that it does in the body, as I will explain to you. And I think that nitric oxide is the most widespread signaling molecule in the body. And I hope to convince you of that. It's also, I think, the most widespread anti-aging molecule in the body. Having said those two, what I come to realize is that it's really nice when you're a Nobel laureate because you can say whatever you want and very few people question you. So how does nitric oxide elicit its effects in the body? Well, there are probably two major mechanisms. One is because of the chemistry of nitric oxide, as I explained earlier. It's reactive. It can undergo direct chemical interactions with a variety of other molecules and bind to those other molecules and alter the function of those other molecules. But another mechanism, the first one described, and one that I was involved with very early on, is that it can stimulate the production in cells of another signaling molecule, which has the name of cyclic GMP. And this is a signaling molecule inside cells. And so nitric oxide can activate an enzyme. The name of the enzyme is guanylate cyclase. You don't have to ever remember that. It's kind of a tongue twister. But this enzyme catalyzes the formation of cyclic GMP in cells. And it kind of looks like this, if I may show this cartoon or schematic. But this enzyme actually can catalyze the conversion of something called GTP to the important molecule here, cyclic GMP. Enzymes are like catalysts. They speed up a reaction. Nitric oxide can activate this enzyme guanylate cyclase so that the enzyme actually makes three or four hundred fold more cyclic GMP than in the absence of the nitric oxide. So that's really one of the main mechanisms of nitric oxide in the body. So whatever effects that nitric oxide produces, many of those effects are actually produced by cyclic GMP. In other words, NO activates the enzyme, it elevates cyclic GMP, and it's the cyclic GMP that produces some of the effects. This is very popular in biochemistry and physiology. A cascade of reactions and then the final step actually is the response, so to speak. So we were doing that work in about 1979, 1980, and that's when we also started to look at nitroglycerin. So let me explain nitroglycerin. You all know this. Nitroglycerin is both a drug, right, and an explosive. Nitroglycerin is the active explosive in dynamite that Alfred Nobel invented back in the 1800s. But nitroglycerin is also a drug, right? It comes as tablets, and if you place those tablets under your tongue, if you're suffering from angina pectoris or chest pains due to an impending heart attack, nitroglycerin dilates certain blood vessels and just kind of relieves the stress on the heart and that pain disappears. So when you widen the arteries or cause the drop in blood pressure, this is what we call a vasodilator effect, and this vasodilator effect of nitroglycerin was actually discovered in Alfred Nobel's dynamite factories. So when the workers came into the factories, they noted that on Mondays when they began their job, they would develop tremendous migraine-like headaches, which lasted all week until they went home on the weekend. In addition, people with chest pains noticed that the pains would disappear while they were working in the dynamite factories, but on the weekends those pains would come back. And that's due to the fact that nitroglycerin is a volatile liquid. In other words, fumes can come off the nitroglycerin, get into the air, people can breathe those fumes. And so because of the vasodilator effect on the cerebral arteries, you can get a headache. And because the nitroglycerin is a vasodilator and the peripheral vessels near the heart, that can relieve the anginal pain, if you will. But the mechanism of action of nitroglycerin as a vasodilator remained unknown for well over a hundred years until we worked it out in our lab. And we often asked the question, could nitroglycerin work by a nitric oxide cyclic GMP mechanism? And the reason we brought that up, I'll show you. In other words, we asked the question, could nitroglycerin work by being converted or metabolized to nitric oxide, let's say, in blood vessels? And the reason I thought that is just by looking at the chemical structure. So here's where there's a little bit of science, and I'm going to focus a little bit on the scientists in the audience. So this is nitroglycerin. We have a three-carbon backbone, and we have three nitrate ester groups. And of course, as you can see, these are ester groups, nitrate esters. It's not a nitrate. The oxygen actually acts as a bridge between the carbon and the nitrogen. The nitrogen is not bound directly to the carbon. So it's an estrogen. But before I describe the experiment we did, let me just show you how nitroglycerin has been synthesized as an explosive ever since the days of an Italian. His name was Ascanio Sobrero, who actually first synthesized nitroglycerin, but he had so many accidents and explosions, he couldn't contain it. It was Alfred Nobel, who then worked with nitroglycerin and figured out how to make it safe by developing or inventing dynamite. But anyway, the way you make nitroglycerin simply is you take the starting material glycerol. This is a very safe substance. This isn't like an alcohol or a sugar. It's actually very sweet. It's a thick liquid. You could put it on your tongue. It's very sweet. It's used in many pharmaceutical preparations to sweeten the flavor of bad-tasting cough syrups and so on and so forth. So it's very safe. The nitroglycerin is made by adding glycerol to a mixture of two substances that are not so safe, and these are concentrated nitric acid and concentrated sulfuric acid. When these three are mixed together, the result is this trinitroglycerol or nitroglycerin. It's a very simple reaction. The only requirement for this reaction is that it be conducted carefully. So if we get back to nitroglycerin, we suspected that maybe NO could be formed from nitroglycerin. If you look at one of these groups here, you could see that the NO is sort of built in to the molecule. And so we had an idea that one of these or all of these might actually somehow be converted to NO. And without explaining any details, that's what we found. We found that when nitroglycerin comes in contact, for example, with vascular arterial tissues, smooth muscle, the result is a formation of NO, and this is the second product of the reaction. It's the dinitro alcohol, if you will. And this product here is not active as a vasodilator. It's nitragoxide that's quite active as a vasodilator. And about the same time, this was 1980, we made another discovery that nitragoxide can inhibit the clotting of human blood. And it can do that by inhibiting the function of platelets. It prevents platelet aggregation. When your blood clots, usually at a site of injury somewhere, the platelets stick to the inside of the blood vessel and they begin to clump together and form a plug which gets larger and larger and then actually plugs the injury so that you don't have a massive loss of blood. Well, nitric oxide can inhibit that entire process. And its mechanism is through the cycle GMP that I was talking about. So by 1980, this was the pharmacology of NO, which I thought was very interesting. It's a vascular smooth muscle relaxant. That means vasodilator. But it's also non-vascular smooth muscle relaxant. It could relax the tracheal smooth muscle or the bronchi smooth muscle. In other words, it can relax airway smooth muscle, even gastrointestinal smooth muscle. Any smooth muscle can be relaxed by nitric oxide. And when you test it in the animal, it's a vasodilator. It lowers the blood pressure, especially when the blood pressure is elevated like in hypertension. And because it's a vasodilator, it improves local blood flow to different organs in the body. And I already told you it inhibits blood clotting. And all of these effects are mediated by cycle GMP. So this was beginning to become a really fascinating story that we have when you add nitric oxide to human tissues, you can elevate cycle GMP and produce all of these effects. But the one thing that bothered me was the physiological relevance of nitric oxide. So let me explain to those of you who don't work in the laboratory or clinically. Pharmacology means the effect of the chemical or drug on the cells, on the tissues. That's an outside chemical. You add it to tissues, you get an effect. That's a pharmacological effect. Pharmacological effect means something exists in the body and then interacts with other cells in the body and produces an effect, a response. That's physiological because it's inside the body. So what about nitric oxide? At this point in time, NO is not known to exist in the mammalian species. It was well known to be a gas in the Earth's atmosphere. It occupies quite a high percentage of gas in the atmosphere and in the atmosphere it reacts, nitric oxide reacts with oxygen to form NO2 or nitrogen dioxide also known as acid rain. Not a very safe gas to be around. And because of that well known reaction, no one even suspected that NO would be present endogenously, that is inside mammals. No one was thinking about nitric oxide as a naturally occurring signaling molecule. And so nobody was thinking about the physiological relevance of nitric oxide. But in our laboratory discussions, we always said, wait, not so fast. Why would a pure outside exogenous molecule be such a potent vasodilator? And it works at very, very, very low concentrations to produce this effect. Also, why would well-defined receptors exist for nitric oxide inside cells? And those receptors are on guanylate cyclase. Remember I said NO activates, it binds to and activates guanylate cyclase to elevate cycle GMP. Why would we have receptors on guanylate cyclase to react to a chemical that's only present in the atmosphere? That didn't make sense. And so we thought, well, maybe NO does exist in cells, but we just did not know that at the time. Certainly if NO existed in cells, that would explain the physiological relevance of the NO cycle GMP system. But our conclusions nevertheless were that the chemistry of NO revealed that it was highly unlikely that NO could exist much less function in mammalian cells. Because it's too unstable and highly reactive with other molecules. But then something happened also in 1980. Someone else made another discovery. His name was Robert Furchkott, or Bob Furchkott. So what the discovery he made appeared to have nothing to do with nitric oxide at the time. And what he did is he showed the mechanism of vasodilation by another molecule called acetyl choline. Acetyl choline is a chemical in the body that's released from many different nerves that causes vasodilation inside the animal. But whenever people would try to see if acetyl choline could cause relaxation of the artery in vitro outside the animal, it would never work. And Bob Furchkott figured out how he can get it to work. That took about 100 years. Not of his own time, of course, but since acetyl choline was first tested. And so what I want to do is I want to take you through a series of cartoons or schematics that will eventually illustrate our progression of thinking or thoughts that culminated in our discovery that nitric oxide is indeed an endogenous signaling molecule. So allow me, please, to take you through this chronologically. This is the first setup, the first experiment that Bob Furchkott did. This is a, he would take a small segment of artery and then using a very sharp razor make rings, cross-section rings, and then put each in a tissue bath and hook it up so that you could record contraction or relaxation. Then he would add various drugs and chemicals to that to see whether the rings would relax or contract. And it's a pretty good way to study vascular function. So this schematic shows a cross-section of that. This would be where the blood flows. This is the lumen of the blood vessel where the blood flows. This would be one of many layers of smooth muscle cells. When those cells are relaxed, the artery dilates or widens, vasodilation. When those muscle cells contract, you get vasoconstriction or tightening of the blood vessel. And there are many layers of smooth muscle cells, but the endothelial cells or endothelium consists only of a single layer of cells that separate the blood from the underlying smooth muscle. So what Bob Furchkott found was that when he added acetylcholine, that's abbreviated ACH, he got a relaxation as long as the endothelial cells were present. And he showed by experimentation that acetylcholine elevated something in the endothelial cells. He didn't know what it was, so he called it endothelium-derived relaxing factor because it was a relaxing factor that came from the endothelial cells. And somehow this EDRF moved into the smooth muscle to cause relaxation. Now he did some really interesting experiments. If he took, for example, a cotton swab and he gently removed the endothelial cells without hurting the smooth muscle cells, if he did that and then added acetylcholine, there was no relaxation. So the relaxation was endothelium-dependent and some relaxing factor was being made in the endothelial cells. That's why he called it EDRF or endothelium-derived relaxing factor. He had no idea what it was. And he tried to determine what it was and he couldn't because when the EDRF is made, it lasts for one or two seconds and then vanishes. It's destroyed. And so you need more than a couple of seconds to be able to isolate something and identify it. That's why he couldn't do it. A lot of other laboratories tried to repeat his experiments, which we all did, and then we went after EDRF to see what it might be. So one of the first experiments we did, we knew that cyclic GMP was involved in vasodilation in relaxation. We had just shown that for nitric oxide, elevate cyclic GMP causes relaxation. So we measured cyclic GMP levels and we found that they went up. Great. But that does not tell you that cyclic GMP was responsible for the relaxation. It just tells you that acetylcholine in the presence of the endothelium can elevate cyclic GMP. But we had to determine whether the cyclic GMP was actually causing the relaxation or was going up for some other reason. And so we figured if cyclic GMP was going up, that enzyme guanylate cyclase that I talked to you about must be there in order to raise cyclic GMP. So we tested an inhibitor of this enzyme guanylate cyclase, which happens to be called methylene blue. So when we added methylene blue to this system, what happens is we were able to block, these hash marks means blockade. We blocked the ability of acetylcholine to elevate cyclic GMP. And by blocking cyclic GMP, we also blocked relaxation. So that told you that cyclic GMP is required for the relaxation. And that guanylate cyclase, which I abbreviate GC, must be here. So when you look at this, you're thinking, well, okay, whatever this EDRF is, it must be activating this guanylate cyclase to increase cyclic GMP and cause relaxation. And this whole process, once again, was endothelium dependent. Well, at the time, the only other substance I knew of that could activate guanylate cyclase, elevate cyclic GMP, and cause relaxation was what? Nitric oxide. I told you about that. So in this experiment, if we add nitric oxide, nitric oxide is a very small, lipid-soluble molecule. That means it can diffuse through all the membranes. It just goes sailing through membranes. So nitric oxide can get into the smooth muscle to elevate cyclic GMP and cause relaxation. Nitric oxide does not need the endothelium to do this. It's endothelium independent. It just gets right in and produces cyclic GMP and relaxation. So take a look at these data. I took a good look at this data, trying to conclude what EDRF might be. So having this all in front of you, if you would look at this data, what do you think the chemical nature of EDRF is? What does it resemble? Clearly, it resembles nitric oxide. Clearly, it resembles nitric oxide. So we were lucky that we did these experiments. But before we could publish such a provocative conclusion, you can't publish provocative conclusions unless you have the experimental evidence to back it up. So we had to quickly design some chemical experiments and other biochemical pharmacological experiments to prove, to show the identity of the EDRF. And when we did that, we could see that EDRF was clearly nitric oxide, which I have to admit came as somewhat of a surprise. But then once we looked at all the data, it was not that surprising. Of course, you're always surprised when you see something for the first time. But now, what we can do is you see this EDRF? We don't have to use that anymore. We can now change it to NO, because EDRF is NO. And this was the first demonstration that mammalian cells could actually produce nitric oxide. And remember earlier I said, because of the chemistry of NO, NO could never exist inside cells, much less work as a signaling molecule. And the one thing I learned from this experiment, as well as many others in my lifetime, is never say never. Just do the experiments. And we got into looking at, so as I said, this was the first demonstration that NO does exist in cells. We looked at antioxidants, and the reason we did that was because in order for us to identify EDRF, we had to stabilize it somehow, make it stick around for maybe 20, 30 seconds instead of 2 or 3 seconds. That would give us enough time to do our reactions to try to identify the EDRF. And so we had to add antioxidants or reducing agents, if you will, to enable this identification. We tested a whole bunch of different ones. And I'm not going to rattle them off here because we have a lot of people who won't understand that. But suffice it to say that the most effective antioxidant that we used in our hands was good old as ascorbic acid or vitamin C. And for those of you who are interested, we had 5 millimolar ascorbic acid present. We tried a variety of other ones. They didn't work. Oh, they worked a little bit. But vitamin C was great. And today, back then, we didn't have all these polyphenolic antioxidants like elagic acid and pomegranates or all the polyphenols or the other antioxidants that we heard about from blueberries and so on. I'm sure they would all work and probably better, perhaps, than vitamin C. I'm not sure. Nobody's ever done these experiments. And so once we made this discovery, of course, many, many people jumped into the field. I mean, we had a new player on the block, nitric oxide produced in mammalian cells. My goodness, what else can it do? And on and on. How is it made? We didn't show how it was made. We just showed that it was made. Then many incredible laboratories, not mine, jumped into the field and described the enzymes that exist to make nitric oxide. And this is called anosynthase is the name of the enzyme. And I'm showing this reaction. I don't want anybody who's not a scientist to leave the room. But some of you others may be interested. But nitric oxide comes from a very simple amino acid that we consume every time we have protein. Arginine is one of 20 or so amino acids present in proteins that we have in the body, proteins that we consume. And one of these amino groups here actually is converted to nitric oxide, leaving you with the second product of the reaction, which is another amino acid called citrulline. And so the sequence of steps, which I will not explain. I'll explain it to you later, if you'd like. But the first step is a conversion of the NH to the NOH. And then the next step is putting the oxygen on this molecule to form citrulline. And then the N is cleaved, removed from here, to produce nitric oxide. And really, that's the major reaction. And I'm not going to say more about that. Now here's a slide. Maybe I should have shown you at the beginning. So I apologize. But this is another cartoon of a cross-section of the artery. So we have the blood that's in the lumen that circulates. Hemoglobin, that's what makes it red. And then we have the smooth muscle cells here. When they relax, the vessel widens or dilates, vasodilation. When these muscles contract, you get vasoconstriction. It tightens. Vasoconstriction increases blood pressure. Vasodilation lowers blood pressure. The endothelial cells I was talking about form a single layer right here as a barrier between the blood and the smooth muscle cells. Every single endothelial cell makes NO. The average adult has 60 trillion endothelial cells. Now don't ask me how they counted them. I have no idea. But you can estimate that. And what the nitric oxide does in this diagram is a little bit incorrect. I plagiarize this from another book. But the endothelial cells make NO. And that NO diffuses backwards into the muscle cells to relax them. The NO also diffuses in the front of the endothelial cells right at the barrier between blood and endothelial cell. And very importantly, that nitric oxide prevents platelets from sticking to the surface. You don't want your blood to clot for no apparent reason. I mean, that would cause a heart attack, a stroke, and other things. Nitric oxide protects against that. Also, the nitric oxide released by the endothelial cells prevents other cell types from getting through into the smooth muscle that could cause cholesterol plaque deposition, atherosclerosis, coronary artery disease. NO is very protective against that. Sometimes I think the most important effective NO is as an anti-inflammatory to prevent inflammation of the arterial wall, even more importantly, than as a regulator of blood pressure. Again, that's my opinion. And I can say whatever I want, as I told you earlier. But this arrow shows NO going into the blood. It really doesn't do that. When NO goes into the blood, it's immediately destroyed within nanoseconds because NO reacts with hemoglobin, oxihemoglobin. Hemoglobin binds oxygen. If NO hits that hemoglobin, it's immediately destroyed. So NO can't travel down the bloodstream to produce an effect anywhere. The hemoglobin is what keeps the action of nitric oxide local right at the spot that it is produced. It's sort of like a locally acting hormone or substance.