The Road to Stockholm - A Nobel Mission
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Transcript: English(auto-generated)
00:14
Thanks very much, Wolfgang, and thanks to all of you for being here instead of being outside in that wonderful, wonderful weather.
00:24
You know, unlike some of the other speakers, I'm retired, retired about four years ago, and no longer active in the laboratory setting. But I assure you that not too long ago, I was pretty active. And so my plan, actually, today, at this very special event
00:42
in Lindau with so many young investigators is to tell you about some of my own early thoughts and how I addressed various questions as they popped up in order to answer key critical questions to better understand a most unique signaling molecule,
01:04
namely nitric oxide. And then later at this conference, you can tell me whether or not my thinking was logical or just plain lucky or both. So please pay attention to my chronological train of thought. And I'm going to present you mostly with concepts and schematics
01:23
rather than hard data and facts. I decided to do that for this particular audience. But before I begin, I just want to say how wonderful it is to be here in this incredible village of Lindau with all of you people. And I say this because it's so much nicer to be here this week
01:44
than back at home, where things are very stormy indeed, mainly because we have a president who thinks he's the king of the United States of America. But let's get on to more pleasant things, and that is to talk about this wonderful molecule, nitric oxide,
02:04
which is a very simple molecule, as you can see, one atom of nitrogen, one atom of oxygen, very simplistic. It is a free radical, it has an unpaired electron, which accounts for its pharmacological activity, as I'll begin to discuss. So let's start from the beginning of time.
02:22
At about 40 years ago or so, the field of nitric oxide was created by Fred Murad, who is with us at this meeting. He demonstrated specifically that nitric oxide gas can activate an enzyme, guanylate cyclase, actually the soluble isoform of guanylate cyclase,
02:43
which catalyzes the conversion of GTP to cyclic GMP, which is demonstrated here. Now, cyclic GMP had been suspected to be a signaling molecule much the same as cyclic AMP, but perhaps with a different function.
03:02
The discovery of cyclic AMP, which is formed from ATP in an analogous enzymatic reaction, was awarded the Nobel Prize, as you heard earlier, in Physiology or Medicine in 1971, the prize going to Earl Sutherland. Now, cyclic GMP levels in tissues could be elevated
03:23
not only by nitric oxide gas itself, but also by certain drugs or chemicals that contained or generated nitric oxide. And again, Fred Murad first showed this. One good example is nitroglycerin. So let's talk about nitroglycerin for a moment.
03:41
Nitroglycerin, of course, is the explosive used by Alfred Nobel to make dynamite back in the 1860s and 70s. And it was a known vasodilator since the 1870s, because that was discovered in Alfred Nobel's dynamite factories in Stockholm. So when people went to work, and they would breathe in the fumes of the nitroglycerin,
04:03
which is a volatile, oily substance, they would get tremendous headaches because of the vasodilation of the cerebral arteries. But some of those workers had heart problems, they had angina, they suffered chest and arm pains. And when they would be working in the dynamite factories, those pains dramatically went away until the weekend,
04:22
when they were away from the dynamite factories and those pains came back again. So within a few years, the physicians and the community recognized what that was all about, and very carefully, they made tiny, tiny pills of nitroglycerin mixed with sugars and other materials that you would place under the tongue,
04:40
and within 20 seconds, you'd have a clinical effect. And of course, we still use nitroglycerin today for that purpose. And one of the names of the drugs is nitrostat, which some of you may be taking. Not me, not yet, anyway. And so, you know, the mechanism of action was unknown.
05:03
In fact, it remained unknown for over 100 years. And so we thought, as Fred Meerad suggested, could it work by a nitric oxide cyclic GMP mechanism? And at about this time, this was timely because I was teaching medical pharmacology at UCLA
05:20
at Tulane University in New Orleans, and a medical student raised her hand and asked me what the mechanism of action was of nitroglycerin as a vasodilator. And I responded, you know, that's a great question, but it's not known. We don't know how it works. And she was puzzled by that answer and said, well, why don't you know how it works? How come the mechanism of action is not known?
05:42
Well, I thought about that, and I decided to do some experiments to see if nitroglycerin could actually be metabolized or converted to nitric oxide in the presence of arterial smooth muscle, venous smooth muscle. Nitroglycerin is very stable in aqueous solution,
06:00
but what about when it's incubated with the arterial preparations? And what we showed, without getting into any details, is that nitroglycerin is actually converted to NO. Now, look at this structure. I like to use a lot of chemistry in my input to pharmacology, chemical pharmacology. So nitroglycerin is really glycerin, or glycerol,
06:23
which is a nitrate, it's a nitrated ester, has three ONO2 groups. And so what we found, without going into any detail, is that one of these nitrate ester groups is actually cleaved and converted to NO, nitric oxide, and the second product of the reaction is the dinitroalcohol.
06:43
And when we tested the dinitroalcohol, it was inactive as a vascular smooth muscle relaxant, and so the obvious question was, well, is NO the active vasodilator species of nitroglycerin? And of course, the obvious answer was going to be yes. We knew that from the studies of Murad studying nitric oxide
07:04
as a relaxant of nonvascular smooth muscle, but we bought some authentic nitric oxide gas, tested it on vascular smooth muscle, and found it also to be a very potent relaxant on those tissues as well. Now, during this time,
07:24
we decided to better understand how a specific vasodilator drug called nitroprusside also inhibits blood clotting. There was a clinical report or two showing that nitroprusside, in addition to being a vasodilator to control the blood pressure,
07:40
actually inhibited blood clotting. And so we wanted to determine whether or not somehow the nitric oxide, that nitroprusside releases the cause of vasodilation, was also responsible for the inhibition of platelet aggregation. And we found that that was the case. Nitric oxide inhibits human platelet aggregation,
08:02
and it does so via a cyclic GMP mechanism. And we didn't think much of that when we published it in 1980. We didn't realize how incredibly important, clinically, that was going to turn out to be much later. And so we're at about 1980,
08:22
which is about four years after the discovery of nitric oxide, and this is what we appreciated about its pharmacology. It's a vascular and nonvascular smooth muscle relaxant in vivo. It's a vasodilator, lowers the blood pressure, improves blood flow. It inhibits blood clotting by interfering with platelet aggregation,
08:44
and all of these effects are mediated by cyclic GMP. But there was a problem, and the problem is, what about the physiological relevance of nitric oxide? I mean, the pharmacological relevance was clear, but what about the physiology?
09:03
Nitric oxide was well known to be a gas present in the Earth's atmosphere, and in the air, it undergoes oxidation by oxygen to form the very noxious nitrogen dioxide, or NO2, also known as acid rain. And because of this reaction between NO and oxygen,
09:24
NO was not known or even suspected to be present endogenously in mammals, much less act as a signaling molecule. In other words, no one in his right mind was thinking that nitric oxide could be working as a signaling molecule
09:43
in vivo, in cells. But not so fast, because when we had our laboratory discussions every week or every two weeks, we would often ask questions like, why would a purely exogenous molecule be such a potent vasodilator?
10:02
And even more importantly, why would well-defined receptors exist in mammalian cells for an outside gas, nitric oxide? And the receptors for nitric oxide are the guanylate cyclase, the active site of the guanylate cyclase, which NO activates.
10:23
And so we thought, all right, maybe nitric oxide does exist in cells, it's just that no one's demonstrated this yet. Perhaps it does exist, and certainly if it did, that would explain the physiological relevance of the nitric oxide cyclic GMP system.
10:40
And so we decided that we were going to set up some assays and look for nitric oxide in cells and tissues. But before we embarked on those studies, we once again reviewed the chemical properties of nitric oxide. And I want to go over that with you,
11:01
because it's because of these chemical properties that we decided perhaps we should not do these experiments and look for nitric oxide within cells. For example, nitric oxide is a very small lipid-soluble molecule that readily diffuses through cell membranes and therefore cannot be stored in any organelles.
11:22
And so at that time, when you think about this, you think, well, how could a signaling molecule function in this way, not stored in organelles, how would it be released, how could it simply just diffuse through all the cell membranes and organelle membranes? Also, nitric oxide is a reactive free radical,
11:42
interacts with other radicals that have unshared electrons so that a stable electron pair can form covalently. And a good example is oxygen radicals, like superoxide anion, O2 dot minus. In fact, the reaction between NO and superoxide is one of the fastest reactions known.
12:02
In addition, nitric oxide reacts with iron, it reacts with sulfur and other substances, other elements. So how in the world could such a molecule like nitric oxide even exist in cells, much less act as a signaling molecule?
12:21
But then we got some help. We got a helping hand from Bob Furchcott, who shared in the prize with Fred Murad and I. He made a discovery in 1980, he discovered EDRF, which is an abbreviation for endothelium-derived relaxing factor.
12:40
And this explained a mystery that lasted for nearly 100 years. Acetylcholine, which is a neurotransmitter in the body, causes well-known to cause vasodilation in vivo. But in vitro, you couldn't get it to relax vascular preparations. It either would do nothing or cause a contraction,
13:02
until Bob discovered the mechanism of acetylcholine, which I can demonstrate or show you in this cartoon, this schematic. So here we have a schematic of a cross-section of an artery, small artery. On the right side is the vascular smooth muscle cell. There are many cells, there are multiple layers of vascular smooth muscle.
13:23
On the left side, we have the lumen, where the blood flows. OK, and in the middle, we have the single layer of endothelial cells. There's only a single layer of endothelial cells lining the inside of the blood vessels. And what Furchcott found was that when acetylcholine was added to this in vitro preparation
13:43
of helical strips or rings of artery, the acetylcholine produced relaxation of the vascular smooth muscle only when the endothelial cell layer was present. So if you remove those endothelial cells by just scraping the internal surface with some cotton or a cotton swab
14:04
to remove the endothelial cells, that acetylcholine did not produce the relaxation. And so he showed through a series of very nice experiments that acetylcholine relaxed the smooth muscle by first generating a relaxing factor in the endothelial cells. And he didn't know what this was, and he called it EDRF,
14:21
or endothelium-derived relaxing factor. Now, the obvious question became, of course, what is the chemical nature of EDRF? And we jumped into that, as did many other people. And the following is the way we approached the problem, and I think somewhat logically,
14:42
but we'll see what you have to say. Since both cyclic AMP and cyclic GMP were known to be prominent vascular smooth muscle relaxants, and because we had good radioimmunoassays at the time for cyclic AMP and cyclic GMP, we measured both cyclic nucleotides in this model system.
15:03
And we found that when acetylcholine was added here, we got an increase in cyclic GMP, no increase in cyclic AMP. So that was good. And then we said, well, does this occur when the endothelium is removed? No. In other words, the increase in cyclic GMP by acetylcholine
15:24
was endothelium-dependent. And so the next question was, was cyclic GMP causative? That is, was cyclic GMP required for this relaxation effect, or did it just go up for some other reason? And so to test this hypothesis,
15:40
we used the substance methylene blue, which was shown earlier to be an inhibitor of guanylate cyclase, blocked the action of cyclic GMP. So when adding methylene blue, it caused a blockade of the increase of cyclic GMP and also blocked relaxation. So this indicated clearly that cyclic GMP was causing the relaxation.
16:04
Now, this implied, of course, that guanylate cyclase had to be present in the vascular smooth muscle cells for methylene blue to inhibit. Now, this, of course, had been shown earlier. This was known, but what we wanted to do next
16:21
was to determine whether the EDRF itself could activate the guanylate cyclase. This was a tedious series of experiments, but we were able to show that the EDRF formed, actually activated the guanylate cyclase to increase cyclic GMP. So we could add the guanylate cyclase there,
16:41
and so, so far, what you see is we have acetylcholine generating EDRF. The EDRF activates the guanylate cyclase to elevate cyclic GMP to cause relaxation. Now, when we tested nitric oxide by itself,
17:00
as shown here, the nitric oxide, you can see that right here, nitric oxide is a lipid-soluble molecule diffuses through cells. It gets right into the vascular smooth muscle cell, gets to the guanylate cyclase, activates it, produces relaxation. NO does not require the endothelial cells. It's endothelium-independent.
17:22
It just goes directly into the smooth muscle cell to cause that relaxation. And so we looked at our data carefully, as you are looking now, and so what did we conclude? I ask you, what would you conclude
17:41
by looking at that slide? What is EDRF? I think that it's very clear that EDRF must be NO. And so in a series of some of the most difficult experiments I've ever conducted in my career,
18:01
using biochemical, pharmacological and especially chemical experiments, we demonstrated unequivocally that EDRF is NO, so now we can modify the slide and change EDRF to nitric oxide. And this was the first demonstration
18:22
that mammalian cells could produce nitric oxide. And Randy, we published this in PNAS. So... So much for the supposition that nitric oxide could never function as an endogenous signaling molecule.
18:42
Never say never. And ironically, the very chemical reasons we believe that NO could not be an endogenous signaling molecule turned out to become the most salient chemical reasons why NO is the perfect signaling molecule.
19:01
So let's just revisit this slide again. NO is lipid-soluble, it diffuses through membranes, it doesn't have to be stored in organelles, it's a reactive free radical for numerous reasons, it is inactivated quickly, that's what you want. You don't want a signaling molecule to last and be around and around.
19:24
Once that signaling molecule is generated, it'll produce its ultimate effect immediately, and then you want it removed, so it's readily inactivated. OK, nitric oxide reacts with iron. Of course it does, because the receptor for nitric oxide
19:41
is the heme iron in the catalytic site of guanylate cyclase. Nitric oxide reacts with sulfur. Of course it does. NO reacts with SH groups covalently and can modify sulfhydryl groups in numerous proteins, changing their structure, changing their function, and if it's an enzymatic protein with a sulfhydryl group at the catalytic site,
20:03
NO will inhibit that enzyme. And so the moral of the story is, if the chemical properties of a molecule turn you off, just step outside the box and think again.
20:20
Now, subsequent studies after this, some really beautiful studies not done by our laboratory at all, showed that endothelial endosynthase was present in the cells, in the endothelial cells, that catalyzes the conversion of arginine to nitric oxide. OK, so moving on then,
20:41
and those studies were made and published in 1986. So moving on to 1990 or 1992, we made another interesting discovery. We found that nitric oxide is the long-sought-after peripheral neurotransmitter that promotes erectile function.
21:03
Now, what possessed us to even address this project? I am not a neuroscientist, and I'm certainly not a urologist, but I was aware that the neurotransmitter released from the nerves that innervate the corpus cavernosum,
21:21
or erectile tissue in males, was completely unknown at the time. This is 1990. And because of that, there wasn't a single drug one can take orally to treat erectile dysfunction, which afflicts more than 10 percent of the male population worldwide.
21:41
So what caught my attention was that NO had just previously, a few months before that, been shown to be a possible neurotransmitter in the brain, having something to do, perhaps, with promoting memory and learning. And so I thought, well, if NO could be a neurotransmitter in the brain,
22:00
why couldn't it also be a neurotransmitter peripherally? And so we performed the appropriate experiments using erectile tissue from rabbits and from humans, and the rest is history. And so our paper in human tissue was published in the New England Journal of Medicine in 1992.
22:21
This is the first and last paper that I have in the New England Journal of Medicine. And the New England Journal of Medicine, as many of you probably are aware, is intimately connected to the news media. And in the morning of the appearance of our paper, I received numerous phone calls to be interviewed.
22:42
The first one was from the editor of Hustler magazine. And that was a difficult one to deal with. I had to decide whether or not I wanted to go through this interview. And then I remembered that my mom at the time was still alive and doing well, so I decided to decline that interview.
23:03
Being Italian, my Italian mother would not favor such an interview. And so I also got interviewed by the editors from the New York Times, which I accepted, and they published our interview in the left-hand column of the front page of the New York Times.
23:20
And, you know, that's not bad for a pharmacologist receiving that kind of attention. Now, if I would have shown that NO was something else, it probably wouldn't have made the newspapers. But of course, when you talk about molecules and erectile function, that always makes front pages of all the newspapers. And so a similar article was published in an Italian newspaper.
23:44
And so being Italian, I can tell you that many times, Italians can express themselves better in pictures than in words. And so here's a cartoon in the Italian newspaper, which shows a gentleman in bed with his lady, and he's wearing a nitric oxide gas tank.
24:03
I couldn't tell you how upset my mom was when she saw that. And so a few months after the New England Journal of Medicine paper, the Journal of Science saw fit to declare nitric oxide
24:21
as Molecule of the Year. And of course, what was present in the nerves was a neuronal isoform of endosynthase, which catalyzes the conversion of arginine to nitric oxide. So what happened six years later? The New England Journal paper was 92.
24:41
Six years later, it was 98. Sildenafil gets marketed as Viagra. FDA puts that on fast track, and it gets developed in a very short period of time. And of course, sildenafil, as you probably all know, works by increasing the actions of cyclic GMP and therefore NO.
25:00
Sildenafil is a phosphorasterase inhibitor, more specifically a PDE5 inhibitor, which is concentrated in the corpus cavernosum. And so if you block the degradation of cyclic GMP, it goes up. And while you have the inhibitor present, if nitric oxide comes along, you get a marked increase in cyclic GMP.
25:23
Now, let's look at the dates more carefully. March in 98, that was the marketing of Viagra. A few months later, in October of 98, the Nobel Prize for nitric oxide gets announced.
25:44
Hmm, I thought, was that a coincidence, or what? So I decided to look up the members of the Nobel Committee for Physiology or Medicine, and I found that the majority of them were men over the age of 60.
26:05
It's really amazing, but we'll take the Nobel. And because of all that work, I have to tell you that the newspapers gave me the acronym of the Father of Viagra.
26:20
I had nothing to do with the development of Viagra. Believe me, another company made the billions, but I got the Father of Viagra. So I'm almost finished. This is the next to last slide. This is a Nobel poster. Now, every year, the Nobel Foundation makes a poster of every single Nobel Prize.
26:42
This particular one is for nitric oxide, and it shows the three of us up there sharing the prize. And what we have is we have a heart in the middle, and then on the left and right, we have coronary arteries. The coronary artery to the left, as you can see, is quite healthy. You don't see any atherosclerosis. There's no inflammation.
27:01
There's a large lumen for blood to flow through. This artery probably came from, it's a cartoon, but in the real world, this artery probably came from someone who was on a healthy diet, lots of physical activity, very healthy, and so on. But look at the artery on this side, a very sick artery. The artery on the left is making lots of nitric oxide.
27:21
The artery on the right is not. There's a deficiency in nitric oxide. When you have a deficiency of nitric oxide, many things happen. Pathology changes, different kinds of cells infiltrate the arteries. You get deposition of cholesterol plaques or atherosclerosis. And remember, nitric oxide is a pretty potent inhibitor of vascular smooth muscle cell proliferation.
27:43
So in the absence of NO, or when NO goes down, you get overgrowth of the vascular smooth muscle, thereby further occluding the lumen. Big problems when you have a deficiency in NO. And that artery, in the real world, probably came from someone who was not eating a healthy diet
28:02
and who led mainly a sedentary lifestyle. And I think this is very important to remember, but it's a subject for another meeting. And so I, you know, based on my many years of experience in conducting basic research on nitric oxide, and being a Nobel laureate, I guess I can say whatever I want,
28:23
I truly believe that NO is not only the most widespread signaling molecule in the body, but also the most widespread anti-aging molecule in the body. So please keep that in mind. And I'm finished, and thank you so much for your attention.