I am absolutely delighted to be able to return to Virginia to meet a lot of friends and colleagues. See a lot of old faces and some new faces and share with you this area that I have been involved with. A lot of it has been, as it would be, luck and serendipity. Sort of work on the right thing at the right time that turned out to be important. And it has been absolutely marvelous and exciting to see all of this move forward. I tell people that most scientists do not have an opportunity to see their fundamental basic research have such an impact in clinical medicine and it’s very exciting to see that happen. And I am going to try to convince all of you that this field has really got so much more to contribute to patient care in the future.

Let me tell you a little bit about my research and why I got the prize, having worked on a problem that turned out to be very important. I knew it was interesting, I thought it would be important. I hoped it would be important, but I didn’t realize how important at the time. I grew up as an M.D., Ph. D. student in the Sutherlands Laboratory in Cleveland. I became very interested in cell communication - how cells talked with hormones and second messenger systems. And when I finished my training, I came here to Virginia for my first faculty position. And as a young faculty member, I was concerned about being productive, competitive, successful. And I saw the cyclic AMP field growing furiously with lots of people in big laboratories. I did not know how I was going to compete with these big operations. And about this time, cyclic GMP emerged as another potential intercellular second messenger. So I switched gears from one second messenger to another. And that turned out to be, obviously very important. We tried to decide how hormones and drugs regulated cyclic GMP production and what functions it had in cells. Is it too a messenger or not?

Our first publication with nitric oxide biology to activate and to cause smooth muscle relaxation was in 1977. Today, twenty-eight years later, there are seventy thousand to eighty thousand publications in the field of nitric oxide research. That is incredible. I would have never dreamed that. And there are very few areas of biology that it does not impact. Almost any system you think of, I would bet that it is going to influence it in one way or another. And makes it exciting. You say, well how can it be specific now for drug discovery and development? Well that is just clever chemistry and drug delivery systems from pharmacokinetics. There are tricks for all of that. The important thing is to understand all of the detailed pathways and many have identified molecular targets in which to design drugs and manipulate those pathways. These cells are going to talk to each other. They are going to communicate with each other. And they do it by producing molecules. They call those molecules first messengers or hormones or growth factors - they give them all sorts of names, but basically what they do is permit communication between cells where these molecules, we call them liggins are recognized in a target cell by the presence of an appropriate receptor in the surface of that target cell. It’s that key and lock fit or the hand in the glove that produces the specificity of that communication. And when those molecules plug into the right receptors in these cells, a biochemical cascade ensues permitting various intracellular messengers to accumulate and then do what the hormone instructs the cell to do. To regulate some physiologic or biochemical process through these second messengers. The first such second messenger was cyclic AMP, discovered by Roland Sutherland. Sutherland got the Nobel Prize in 1971 for that discovery. We know today that there are other second messengers. Cyclic GMP, calcium, acylglycerol, some peptide, and nitrite oxide. What is unique about nitrite oxide as a messenger is that it is a gas. It is a free radical with an unshared electron. It is very permeable in a liquid bi-layer so it can go wherever it wants. It does have a short half-life because it’s reactive as a free radical. But not only does it function intracellularly as a messenger, it can leave the cell and govern the activity of adjacent cells or even distant cells. It’s a unique molecule. It is the only messenger that is un-charged, therefore, it does not need transporters and carriers. It can go where it wants, which makes it unique. When we discovered that a free radical could activate gly-cyclades, a lot of people thought that was heresy. Free radicals are not supposed to activate insides. That is just unheard of. And then I proposed that not only does it activate glynlysiclids, it’s probably a second messenger in a lot of cells to mediate a lot of hormone effects. Not that was real heresy. And it was not a popular notion, but fortunately, we were right. It took us a long time to prove all of that, but we were right. It’s a free radical with an unshared electron. It became popular about fifty years ago when it became recognized that fossil fuels or anything combustible that possesses nitrogen, when it is combusted with oxygen will generate a family of nitrogen oxides. So they come out of automobile exhaust, cigarette smoke, smoke stacks. These nitric oxides and nitrogen oxides dry a valency so there are families of these molecules, are important because they interact with the ozone and deplete the ozone layer, therefore removing the ultraviolet filter from its global warming. That’s why it was interesting. We come along all of a sudden years later and say, “My God. This pollutant and gas is not only toxic, but it’s an activator and a messenger in the body and it does lots of other things.” And it took awhile to convince people of that, but finally they came around. We recognized that it was the way some important drugs worked - Nitroglycerin, which helped make Alfred Nobel famous and wealthy. He figured out how to formulate nitroglycerin to make it less explosive. Nevertheless, he had a number of explosions in his factories. One of which killed his brother, but he made dynamite. Became a rich man blowing up mountains and tunnels and railroad passes and whatever. And he also invented the fuse and detonator for dynamite. He had numerous patents as a chemical engineer, a very clever fellow. These are some of the processes that it regulates. I am going to skip through that. I am going to show you some more in a moment. We know in these second messenger systems, what happens invariably is a liggin, a hormone, a first messenger, whatever you want to call it enhances the production of an intercellular second messenger, whether it is cyclic AMG, cyclic GMP, diglycerol, some lipid, all sorts of intercellular messengers. These accumulate and they are going to do the business for the messenger. They are going to transmit inside of the cell what it needs to do without the hormone having to get inside the cell necessarily. And very often they activate various proteinases that phosphorically protein substrates. And when these protein substrates are phosphorylated, whether it is structural proteins or enzymes, their shape, their confirmation, their mortality, their activity changes. Can be activated or inhibited. That’s fundamentally it. There are also families of enzymes that hydrolyze and inactivate these messengers. Today we know, there are probably about seven or eight different isoforms for glycerol, different gene products. We’ve purified them. We’ve cloned them. We manipulate them. We know their promoters. We know their genomic structures. We make mutants. We make knock-out mice and all that sort of stuff. There are about ten or eleven phosphodiesterases that either hydrolyze cyclic A or cyclic GMP. The one you all are familiar with is the Type 5 phosphodiesterase. Why is that one important? That one is found predominantly in blood vessels including the blood vessels of the penis. That’s the target for Viagra. You inhibit that enzyme, you potentiate the endogenous nitrite oxide released from the nerve terminals. You make a lot more cyclic G and it accumulates and it relaxes the blood vessels so they are gorged with blood and that is the mechanism for erection. We used to have wisecracks at the laboratory like filling condoms with phosphodiesterase and goodies. It was a wisecrack. A joke. I wish I had patented it. I didn’t bother at the time. But anyway, it is a fun system because there are complicated systems. Lots of players, lots of isoforms, different compartments. So while it appears simple, there’s much more to it in this complicated matrix. And there are one hundred fifty different protein cyesis. The phosphorate of a variety of different proteins. As we were looking at GMP in its regulation, trying to figure out how hormones regulated it, we accidentally found some simple molecules activating it. I am not going to tell you why we did those experiments. But we found that azide hydroxide sodium nitride would activate the enzyme. We began to use those activators as surrogate replacements for hormones and tissues. They would activate when hormones wouldn’t in self-resistance. They would also elevate cyclic GMP levels and attack cells. We learned that a variety of compounds would do the same thing. Not only azide hydroxide sodium nitride, but nitroglycerin would do it because it too relaxes the muscle. Nitroprusside, another very important cardiovascular drug we use in intensive care units to lower blood pressure and relieve the workload on the left heart after cardio malfunction. These are all pro-drugs using nitric oxide. Why did we suspect nitric oxide is the intermediate? Because we knew that some tissues possessed inhibitors of this pathway. We purified those inhibitors. And we knew from the literature that nitric oxide had a very high affinity for the hemibody of human blood. We said the intermediate has got to be nitric oxide. We generate nitric oxide gas in a fuel hood over in the medical school building and I’ll be darn if we didn’t activate the enzyme. That was an exciting moment. That experiment was done late at night by a Japanese fellow who got on an airplane the next day to go home. Incredible. He could have just as well gone home and packed his bags, but he wanted to get that experiment done before he returned to Japan. And it was a very exciting time. He was so important to this field that I invited him as well as another four or five trainees to go with me to Stockholm. Carol and I took fifty-one people to Stockholm. I spent an awful lot money of the prize to get our family, our five kids, their spouses, grandkids, and some others over there. It was exciting. Wonderful party. It goes on for nine or ten days. I didn’t get more than three or four hours of sleep for nine or ten days, but dances and parties by the medical school. Beautiful young students. It was fun. Dinner with the king and queen. So this is how we put the story together some years ago. We now know that nitric oxide is a very important molecule in vascular biology. It is produced by the endothelial lining under the influence of various hormones. It’s produced in lots of other tissues. We have worked out the detailed biochemistry of all of this. That permits us now to identify molecular targets to develop drugs, to figure out how they are working. To elevate blood pressure or reduce blood pressure. influence cardiac output. All of these things. It’s obvious once you know the pieces. The enzymes in the body that make nitric oxide are called nitric oxide synthases. We have purified all of these, as have some other laboratories. They are very complicated enzymes. They’ve a very complicated co-factor requirement. What they do is they oxide the terminal nitrogen to a hydroxy argeny that is further oxidized to nitric oxide. Turns out there are some very important diseases in which you don’t make sufficient quantities of nitric oxide. We call those diseases endothelial dysfunction. Hypertension. Diabetes. Obesity. Tobacco use. Athosclerosis. You don’t make enough for various reasons. I will summarize some of that for you in a moment. This is a scheme that sort of helps us design experiments. We now know that a variety of hormones and liggins interact with their appropriate receptors to influence the activity of nitric oxide synthases to make N-O from LRG. And it does it by manipulating the concentrations of various cofactors. The oxidized or reduced state or availability of calcium. Etcetera, Etcetera. The nitric oxide activates to give you a function. That function can be a lot of different things. We can manipulate this pathway now with receptor agonist, antagonist by changing the availability of cofactors. By slipping in genes to modify these players. By throwing in compounds that are inhibitors of nitric oxide. By throwing in molecules to suck up and scavage nitric oxide. By throwing in activators and inhibitors. All of those are potential targets now to discover drugs. It’s pretty easy to discover drugs once you know all the pathways. It’s not so complicated. Our success rate when I was at Abbott was remarkable. We only had a couple failures. It doesn’t have to be eighty and ten percent successes. It can be eighty and ninety percent if you really think through the project and problem carefully. There are other participants in nitric oxide biology. It can do other things. It can be oxidized to nitride and nitrate, which are inactive, but they are markers of N-O production. So by measuring it in error, you can tell basically how much nitric oxide you are making in various diseases. The nitric oxide likes to interact with other transition metals besides iron. It also likes to react with proteins. One hundred fifty different proteins so far. They get nitrosated. Summer transcription factors. Summer receptors. A lot of interesting possibilities. And those are reversible reactions. And another very important reaction is the interaction of the nitric oxide free radical with the super-oxide anion free radical. In any situation of inflammation, whether it is Alzheimer's or arthritis - I don’t care what your model of inflammation is, there’s evidence that you are making a lot of N-O and a lot of super oxide, which results in the production of peroxy nitride. It’s almost a diffusion limited reaction. That sucks up the N-O and prevents its activation of cyclydes, So the beneficial effects of N-O are to make cyclic G. The adverse effects of N-O are to combine with super-oxide and make peroxy-nitride and screw up proteins. So we do lots of to identify those proteins and see how they are modified. I think if we understand this pathway, we will come up with whole new approaches to anti-inflammatory therapy. If these are really causal inflammation, and we can figure out how to stop it or reverse it, we’ll have new approaches. We won’t have to worry about COX-2 inhibitors and all their problems. This is Endothelial Dysfunction that I mentioned. The bottom line is that under all of these conditions, there is oxidative stress, enhanced production of reactive oxygen species, therefore oxidation of the cofactors required both for the metabolism of a-symmetric dymethal argeny, which is a competitive inhibitor of this enzyme as well as depletion of the cofactors required for nitric oxide. You don’t make enough N-O. So how do you treat these diseases? Well you treat the underlying disease whether it is diabetes or cigarette smoking or whatever. But there are other ways to approach it as well with regard to nutritional supplementation perhaps. Anti-oxidants in the way of vitamins. Turns out, most people are probably depleted. Probably don’t have enough Vitamin E and all these other goodies. So there are a number of ways to supplement therapy and enhance improvement. And it has been shown in certainly animals and humans that these things improve vascular function and integrity. There are lots of potential applications now for this whole field. You can imagine with eighty thousand publications, you can almost go in any direction you want for clinic utility of this information. We know that nitric oxide is a neurotransmitter. There are nerves in the brain, the GI track, the penis that liberate nitric oxide as the neurotransmitter. We know also that nitric oxide participates in the size of an infarct in the brain. If you knock out NOS1in a mouse, they have a smaller area of infarct. If you knock out NOS3, they have a bigger area of infarct. This sort of fits what you might expect. It plays a role in affluent accumulation of production and reabsorption in the eye in glaucoma. It is responsible for retina degeneration in elevated eye pressures. We talked about the notion of blood flow, blood pressure. A fun one has been pulmonary hypertension. Children, babies, when they are premature, retain their fetal circulation. The fetal circulation is that they don’t need the oxygen at their lungs because the placenta in the mother’s blood supply provides the oxygen supply. So when they are born prematurely, their lungs are constricted. They are not making enough nitric-oxide. They don’t want to dilate those blood vessels so the blood supply becomes unsaturated, unoxidated. They are blue babies. If you give them inhaled nitric oxide by nasal catheter in very low concentrations, you dilate the pulmonary vessels. They no longer have pulmonary hypertension. They now perfuse their lungs. They oxidate their blood and their hypoxia improves. And you don’t have to put them on these horrible devices. It’s pretty devastating. Most of those babies end up getting strokes and so forth. So it has been fun to watch that. That’s been exciting. A lot more interesting I think than Viagra, which I think is trivial science. It plays a role in wound healing and angiogenesis. The list goes on and on and on. Here are some other things to look at. The production of nitric oxide in its induction of NOS2 by endotoxins and pro-inflammatory sidacons is a defense mechanism for infections. If an insect bites a plant, they induce nitric oxide production to heal that wound and kill the bugs invading through that stock. People do the same thing. The endotoxin and pro-inflammatory sidacons induce NOS2. You make a lot of N-O. It kills the invading organism as a self-defense mechanism to infection. The problem is that you make so much N-O that most of your blood vessels over-dilate. They pull your blood. Your blood pressure drops and we call it septic-shock. The mortality rate for septic-shock in intensive care units is about seventy percent. Pretty serious stuff. If you inhibit the NOS2 in those patients, you can enhance their blood pressure and hopefully improve their recovery. The same is true also in cardiogenic shock. So there are lots of applications. We are not over the hurdle because we need very selective compounds to inhibit the right nitric oxide cynthades in the right place. So we are not through with all the additional chemistry and everything else that needs to be done in these areas. Gene regulation: we know that there are lots and lots of genes regulated by nitric-oxide. Up and down and sideways. It’s been fun to do some of that. We are about to put our first paper together. Another area that’s been very exciting is this potential application in stem cell biology. A few years ago, we decided to take mouse and human embryonic stem cells in the laboratory and culture and see if we can influence their growth rates and differentiation into various cell types by manipulating nitric-oxide and cyclic G and it looks very encouraging and very promising. I hope some day that we’re going to be able to create a cocktail to influence stem cell proliferation, differentiation, and that we won’t need stem cells for treatment. All we’ll have to do is give the right cocktail to patients and recruit their own stem cells to make the right cell type. Now that’s pretty wishful thinking. That’s thirty years off in the horizon, but young people look back and remember I told you that was going to happen. But that’s what makes science fun is to think about crazy things and to go chase them and get those answers before anybody else. It is really exciting.