Role of Nitric Oxide and Cyclic GMP in Cell Signaling and Drug Development
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Lindau Nobel Laureate Meetings282 / 340
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Short circuitLecture/Conference
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Musical developmentRRS DiscoveryFinishing (textiles)Lecture/Conference
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Musical developmentRRS DiscoveryTrainYearTissue paperMultiplizitätLecture/Conference
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Transcript: English(auto-generated)
00:15
It's always a treat to come to the Lindau meetings, to meet your Nobel laureate colleagues
00:22
and interact with a lot of talented young people. It's a lot of fun. I'm a clinical pharmacologist, and I'll tell you what that means shortly. But I was planning to go to medical school, and as I finished college, I learned about a new program
00:41
that was just beginning in Cleveland. The chairman of the pharmacology department, Earl Sutherland, who was an MD, decided that future scientists and professors should be trained both in medicine and basic science. And he was instrumental in creating the MD PhD program in Cleveland,
01:00
the first one to happen in the United States, and I decided to join that program. So I've trained in medicine, pharmacology, biochemistry. My research has been in endocrinology, cardiology. I've had some training in physiology. Probably trained too long. My family got a little concerned
01:20
about how many years I trained. But by becoming a clinical pharmacologist, it permitted me to chase problems into multiple tissues and organs, and I enjoyed that because it was very rewarding to learn these new disciplines and languages to communicate both in medicine and in basic science.
01:42
Sutherland and Rall in 1957 discovered the first intracellular second messenger, cyclic AMP. They were interested in the mechanism of epinephrine and glucagon-induced glycogen degradation to produce glucose. I joined them in 1958,
02:01
and for the next 12 years as a student and a fellow, worked with cyclic AMP to show that numerous hormones in many different tissues would enhance cyclic AMP production to produce a variety of physiological and biological effects.
02:20
In 1971, Earl Sutherland got the Nobel Prize for that discovery. And I'll show you shortly, there are other messengers, and most of them have received Nobel Prizes. But in the late 1960s, another cyclic nucleotide was discovered by some chemists, cyclic guanosine monophosphate.
02:41
And as a student, one of my projects was to search for compounds that might inhibit cyclic AMP production and activity. And when cyclic GMP came along, I thought perhaps it would antagonize the effects of cyclic AMP, so I switched my interest. As I was finishing my fellowship at NIH
03:01
and going to the faculty at the University of Virginia, more and more of my interest turned to cyclic GMP. And that led, a few years later, to the discovery of nitric oxide. And I'll review some of that history for you. I'm gonna try to avoid a lot of biochemistry, but I want to present the concepts of cell communication and how it often works.
03:23
And how this information can lead to the discovery and development of many drugs. And if we look back, perhaps 40 or 50% of all the pharmaceuticals on the market relate to cell signaling messengers,
03:41
either mimicking their effects or blocking their effects in these biochemical pathways. So it's been a gold mine for drug development and discovery. The first slide summarizes this concept of cell communication. These are three populations of cells
04:02
that will talk to each other. With evolution, we went from single cells to multicellular organisms. And cells had to learn how to talk to each other to survive. In this cartoon, there are three different populations of cells.
04:21
I'm gonna call cell type one a neuronal cell. It could be a central neuron in your brain or it could be a peripheral neuron in one of your nerves. I'm gonna call cell type two an endothelial cell, lining all of your blood vessels. It's only one cell thick. It was thought to be inert.
04:40
We know that's not true. There are many interesting messengers that come from these endothelial cells that regulate smooth muscle, regulate formed elements, platelets, white cells, et cetera. And I'm gonna call cell type three a smooth muscle cell in the wall of that blood vessel. And they're all gonna communicate.
05:03
If cell type one is a neuronal cell, the messenger that it produces and releases we call a neurotransmitter. If it's an endocrine cell, we call the messenger a hormone. If it's a macrophage or lymphocyte, we call them growth factors, cytokines or chemokines.
05:21
And these messenger molecules that come out of these cells into the synaptic space or bloodstream can be very simple, amino acids. They can be lipids. They can be peptides. And they can even be big hormones such as the gonadotropins from a pituitary. If they're small molecules, they're usually bound to a carrier
05:41
to protect them from degradation and prolonging their half-life. The larger molecules are usually protected quite well. And what they will do is home throughout the body to adjacent tissues or at a distance, either way, to go find their target. And they identify their target
06:02
by the presence of a protein, often, that's an integral membrane protein embedded in that membrane. And these first messengers bind to this protein. We call it receptor. And they bind by a three-dimensional conformational fit like a key in a lock.
06:21
So only the right messenger talks to the right receptor and only the right receptor identifies the right messenger. Now, sometimes, the receptors are inside of the cell as with thyroid hormone and some of the steroids. But most often, they're in the membrane. And this first messenger
06:40
doesn't necessarily have to go inside of the cell to do its business. By binding to its receptor, it induces a biochemical cascade that often enhances the formation and accumulation of an intracellular second messenger. The first such second messenger was cyclic AMP.
07:01
Then came calcium. Then came cyclic GMP. Then came the acoustinides, the prostaglandins. Then came diacylglycerol. Then came nitric oxide. So there are now a half a dozen or so intracellular messengers in hundreds of these first messengers.
07:21
And many of the messengers resulted in drugs and many of them resulted in Nobel Prizes. Nitric oxide is a very unique messenger because it's a free radical with an unshared electron in its outer shell. So it's very reactive. It's also a gas. It's also lipid soluble, so it can go wherever it wants.
07:43
It can go into the mitochondria, go into the nucleus, it can come out of the cell, go back in. It can come out and attach to another carrier and go to a distance, so it's also a hormone. It's the first messenger that functions as an intracellular messenger, an autochoid, a paracrine substance,
08:01
a local acting material, as well as a hormone. No other molecules can do that. The only thing that comes close are perhaps the prostaglandins. These messengers can function inside of the cell in which they're made, but with nitric oxide, it can come out and regulate the biochemistry and biology
08:21
of adjacent cells or distant cells. And when it goes over to cell type three, the smooth muscle cell in the wall of that blood vessel, it causes relaxation. And by dilating the blood vessels and relaxing the smooth muscle, you enhance blood flow, oxygen delivery, nutrient delivery, lower blood pressure.
08:46
I think nitric oxide is a very old molecule. I think as life began, the messengers had to be simple initially. They couldn't be complicated. And they happened to occur in organisms
09:00
that were probably in the deep grips of the ocean near all of this magma that was very toxic with transition metals and all sorts of gases. And to survive, they had to detoxify these materials. And once you create the biochemistry to detoxify, why not turn it around and start using it to send information to each other?
09:21
And I think that's how it happened. I can't prove that, but I think that makes sense to me biologically and biochemically. Perfect. We know today that nitric oxide is released into the atmosphere whenever you combust fossil fuels. Any substance with nitrogen when it's burned
09:40
makes a family of nitrogen oxides. NO, N2O, NO2, N2O3, et cetera. They're called NOx's, N-O-X-apostrophe-S. They're released into the atmosphere, and as you heard from Dr. Molina's discussion yesterday, they can interact with the ozone layer, which is the ultraviolet filter to prevent global warming.
10:03
So it depletes ozone and participates in global warming, and that's one of the effects of fossil fuels. We also know that it's how some very important cardiovascular drugs work from our research in the 70s. Nitroglycerin, nitroprusside are pro-drugs
10:22
that are converted to nitric oxide in the body, and I'll tell you more about that shortly. So nitric oxide was viewed as a toxic pollutant for many years, and when we came along and said
10:41
it's also an interesting messenger in the body, it does all these marvelous things, nobody believed us for 10 or 15 years. It was very difficult to convince the world that this was gonna be an important messenger. It took a while. That was disappointing, but it also permitted us to do all the important work during that period of time.
11:01
And we did an awful lot of work because we were very excited about our findings. But as you heard, I think, yesterday from Dr. Aron Hirschko, you gotta believe in your work and you gotta persist and do something that hasn't been done before because that makes it more fun, I think. On the right-hand side of this slide
11:21
are some of the biological effects of nitric oxide. It's by no means a complete list. We discovered the first biological effects in 1976, 77. Today, there are 150,000 research publications in nitric oxide. It's become one of the most popular areas
11:42
of medical research. There are about 80 or 90,000 publications on cyclic AMP, a similar number with cyclic GMP, so these areas have become quite popular, and they're popular because they've led to the discovery and development of a lot of important molecules for medical diseases.
12:01
And this is a partial list, and that time doesn't permit me to go through everything that it does, but I'll try to give you an overview and a flavor in the next 20 or 30 minutes. This is how cyclic nucleotides are produced. As a student, I worked with cyclic AMP. I found a lot of hormones
12:22
regulating cyclic AMP production. I switched to cyclic G. It turns out the pathways are very, very similar. In fact, the adenylate cyclases that make cyclic AMP and the guanylate cyclases that make cyclic GMP have a lot of homology. They probably originated from the same ancestral gene, I think.
12:40
There probably was a gene and protein at one point that made either cyclic nucleotide. If you mutate one amino acid in the catalictomide, the guanylate cyclase can make cyclic A and the adenylate cyclase can make cyclic G. And they convert nucleotides, either ATP to cyclic AMP or GTP to cyclic GMP in pyrophosphate.
13:03
The alpha phosphate cyclizes back on the two position of ribose, on the three position of ribose to form the cyclic nucleotide. It turns out that there are seven isoforms of guanylate cyclase, seven different gene products.
13:21
The soluble isoform is the heterodimer with an alpha beta subunit and it's the receptor for nitric oxide. Nitric oxide binds to a heme prosthetic group. Heme pops off of the pocket and activates the enzyme several hundred fold to make cyclic GMP.
13:41
Incredible amplification. And that's often the way signaling pathways work. A single molecule can affect an enzyme with a high turnover number to make lots of other messengers. So there's amplification all the way along the step. A kinase phosphorylates lots of proteins.
14:00
They regulate lots of other activities. There's also redundancy because all of these messengers talk to each other. That makes it a lot more complicated but also more interesting and a lot more fun, I think. The cyclic nucleotides are regulated, their levels and tissues, by the rate of formation
14:20
as well as the rate of degradation. And they're destroyed by a family of enzymes called cyclic nucleotide phosphodiesterases that cleave the phosphodiester bond to make the corresponding monophosphate. There are 11 cyclic nucleotide phosphodiesterases. Some hydrolyze cyclic AMP,
14:41
some hydrolyze cyclic GMP, some hydrolyze both. The one that's familiar to all of you in the audience is the type five phosphodiesterase, which selectively hydrolyzes cyclic GMP. And the reason you know about it is because that's the target for Viagra. That's how it works.
15:01
Viagra inhibits that enzyme, permitting the nerves in the blood vessels of the penis to release nitric oxide, make more cyclic GMP, more vasodilatation as you dilate the blood vessels, they fill with blood. That's the mechanism and physiology of the penile erection. It also does the same thing in other blood vessels.
15:21
And this area has become an interesting field for drug discovery, obviously. Now many of the messengers, cyclic AMP, cyclic GMP, calcium, disoglycerol, activate protein kinases. There are several hundred protein kinases.
15:42
Some of them transfer the gamma phosphate of ATP to serine or threonine residues of proteins. Some transfer the phosphate from ATP to tyrosine residues, they're called tyrosyl kinases. The serine, threonine kinases are regulated by intracellular messengers.
16:00
Cyclic AMP, cyclic GMP, calcium, disoglycerol. The tyrosine kinases are regulated by growth factors in cytokines. They don't have messenger molecules. These kinases phosphorylate a variety of proteins. The proteins can be enzymes. They can be structural proteins.
16:21
And when they're phosphorylated or dephosphorylated, their activities can change. They can be activated or inhibited and their structure and their function can change as well. So this basically is the mechanism for signal transmission and transduction communication. Simple. The problem is you have all these isoforms
16:43
in a variety of compartments and you don't know who's talking to who sometimes. That's what makes it difficult because what you'd like to have are drugs that are selective and specific to influence one pathway or one physiological effect or biological effect and not another to avoid side effects and toxicities.
17:06
I'm gonna skip through some biochemistry. Here we go. In the process of studying guanylate cyclase and discovering there were soluble
17:20
and particulate isoforms, we accidentally found azide, hydroxyl amine and sodium nitrite activating the soluble isoform of the enzyme. Big surprise. We often included azide in our buffers in the refrigerator cold room to inhibit bacterial growth.
17:41
That's how we stumbled onto it. My goal was to figure out how hormones regulated cyclic GMP formation. But when you homogenize tissues, the hormone effects were lost. You uncoupled the signaling pathway. But we had now three small molecules,
18:00
azide, hydroxyl amine and sodium nitrite, that would increase cyclic GMP in intact tissues and slices and cultures and activate the enzyme in cell-free extracts. And I thought if we understood how they activated the enzyme, perhaps we can reconstitute a hormone effect, which was my goal. I wanted to find hormone signaling pathways
18:23
so I can interrupt or accelerate the pathway as possible therapeutic agents for other diseases as we did with cyclic AMP. Well, it became quite a mystery story because we put these on all sorts of tissues and cells.
18:44
And one of the tissues I worked with at the time, because I knew cyclic AMP relaxed smooth muscle, I suspected the cyclic GMP would contract smooth muscle. And we developed a tracheal smooth muscle bottle and we put azide, sodium nitrite,
19:02
hydroxyl amine on these smooth muscle preparations. They increased cyclic GMP as they did in other tissues, but the tissue didn't contract, it relaxed. Big surprise again. I then remembered as a resident working in the intensive care units, taking care of patients with heart attacks.
19:22
And what we do with patients with heart attacks is give them vasodilators, nitroglycerin, nitroprusside to lower blood pressure. So the part doesn't have to work so hard and can heal faster. We call it decreased afterload, the workload on the heart. So we put nitroglycerin and nitroprusside
19:41
on these smooth muscles. They cause relaxation as they were thought to from work of others. The big surprise was they too activated quite a lot of cyclase and increased cyclic GMP. So now we had a family of compounds that were vasodilators, smooth muscle relaxants
20:00
that all activated guanylate cyclase, the soluble isoform. And we coined the term nitrovasodilators. And the question was, they're so different in structure, there has to be a common intermediate in what is it? And it took us about three or four years to figure that out, and it turned out to be nitric oxide.
20:21
And we first generated nitric oxide in early December 1976 in the fume hood. It activated all of our soluble guanylate cyclase preparation. So that was the eureka moment, and we realized, my goodness, a free radical, a gas, could activate an enzyme that had never been shown before. And I thought, what interesting chemistry this is going to be.
20:41
It's gonna be complicated because it's a free radical in the gas, but isn't it gonna be different and interesting? So we chased it. As I said, for 10 or 15 years, nobody would believe it. This is what a nitrovasodilator will do to a blood vessel. If you pre-contract the blood vessel with norepinephrine,
21:04
and then after the contraction is stable a couple minutes later, you add a nitrovasodilator, you increase cyclic GMP in 10 or 20 seconds, this is followed by relaxation. And after a couple minutes, cyclic GMP returns to basal levels, but relaxation persists.
21:22
Does that mean cyclic GMP is not related to relaxation? No, that's not true. Messengers have short half-lives. Cyclic AMP, GMP, nitric oxide don't last very long. But activation of the protein kinase in the phosphorylation of proteins lasts much longer, longer half-lives, and that's what's going on here.
21:44
So we realized that we had figured out how nitrovasodilators work. And that was one of the reasons I was invited to go to Stockholm. In 1847, Italian chemists made nitroglycerin.
22:02
What chemists often do is lick their fingers, stick it in the powder, and taste it. I'm not sure why they do that, but they do. And they got vascular headaches, like migraine. Some of the physicians in Europe realized that nitroglycerin, besides being an explosive, was also a vasodilator, and started using it for angina pectoris.
22:22
And it was used for 100 years to treat angina, not knowing how it worked. And in 1977, 78, we figured out there was a prodrug to make nitric oxide. Alfred Nobel made his fortune by making nitroglycerin, making it less explosive,
22:42
by combining it with a diatomaceous earth to make dynamite. And as you know, the history, and some of the young people should go to the internet and look up the Nobel Foundation internet, the Electronic Museum, and learn an awful lot about all the laureates, but also the history of Alfred Nobel. Interesting story.
23:01
Now, as a pharmacologist, you also learn that when you have an exogenous material that does something of interest, you should ask yourself, is it mimicking an endogenous pathway? And I proposed in 1978 that hormones must be acting, perhaps,
23:21
by converting some endogenous substance into nitric oxide, which also was a messenger. That was heresy. A gas, a free radical is a messenger, you're right, you're crazy. We couldn't prove it because the concentration required was very low, and the technology did not permit us to assay nitric oxide or its oxidation products
23:42
at dantamolar concentrations. So it took a few years to develop some new technology to do that, and we succeeded. And by the late 80s, people began to believe us because we had assays now to measure it, and because of another scientist, Robert Furchgott.
24:01
Robert Furchgott is a professor at SUNY Downstate in Brooklyn, a vascular biologist who I happen to know, who was interested in contraction in relaxation of blood vessels. We knew that a number of drugs would cause relaxation of blood vessels and lower blood pressure in animals,
24:21
acetylcholine, bradykinin, a number of others. Histamine. But when you put them on vessels in organ baths, they failed to cause relaxation. And he discovered the reason for that was because you were destroying the integrity of the endothelium. These endothelial-dependent vasodilators
24:42
required the integrity of the endothelium. And without the endothelium, they did nothing or they contract instead of relaxed. And he presented that work at a seminar in Virginia while I was still there, in 1980, and it turned out that much of his story was similar to our story with nitro vasodilators.
25:03
And after the seminar, I took them off to my office, and I said, Bob, there are three ways to relax smooth muscle. You increase cyclic AMP, or cyclic GMP, or decrease calcium, and that's the way the substance is working. Let's go figure this out. We agreed to collaborate. He called the material that came out of endothelial cells
25:23
EDRF, endothelial derival action factor. It had a short half-life. Turned out we did not collaborate. We moved to Stanford. He went back to New York. His wife developed breast cancer. He was distracted for a while. EDRF is nitric oxide, and it works through cyclic GMP,
25:42
to make a long story short. Now this is a blood vessel cartoon to show you how these pathways work. On the left is the endothelial lining of all of your blood vessels. On the right is the smooth muscle in the wall of that blood vessel. And in red are three categories of compounds
26:02
that work by increasing cyclic GMP production. In the top middle, the nitrovasodilators are prodrugs or precursors to make nitric oxide. Some require enzymes to do it. Some do it spontaneously in the blood based on pH and oxygen tension. The NO activates the soluble isoformic
26:22
to make cyclic GMP. That activates the cyclic GMP-dependent protein kinase. We found that that phosphorylates a variety of proteins in smooth muscle. We did that with P32 incorporation by pre-labeling the ATP pool. You can display those proteins on gels.
26:41
You then try to determine what they are, where the antibodies are taking the spots and sequencing with mass spec. It turns out that many proteins get phosphorylated. Some of them lose their phosphate, and two proteins lost their phosphate, and we identify them as myosin light chain. So what happens when you increase NO?
27:04
You make cyclic G. Cyclic G lowers cytosolic calcium. It influences phosphoenositide in intracellular calcium stores in their mobilization and the entry of calcium from the outside. And myosin light chain kinase
27:20
is a calcium-calmodulin-dependent enzyme, and with lower calcium, its activity drops, and myosin light chain loses its phosphate. These are active filaments, myosin filaments. When myosin's phosphorylated, they slide together a latch, you have contraction. You take off the phosphate, they slide apart, and you get relaxation. That's the mechanism.
27:40
The endothelial-dependent vasodilators only work if the endothelium is intact because they have the receptors, where acetylcholine, histamine, bradycardate, et cetera, smooth muscle doesn't have receptors for those ligands. And when they interact with the endothelial cell, calcium enters the cell,
28:01
activates an enzyme called nitric oxide synthase that converts the amino acid l-arginine to nitric oxide, which is EDRF. That goes over to the smooth muscle compartment. It works just like a nitro vasodilator. That tells us, when you have patients with heart attacks,
28:23
you don't wanna use endothelial-dependent vasodilators to lower their blood pressure and decrease afterload because they have endothelial dysfunction, and I'll tell you more about that shortly. You wanna use direct-acting nitro vasodilators to lower blood pressure. And that's what I learned as a house officer.
28:42
So it all began to come together again. Your medical training, your research training, often overlaps. The third category of these dilators are the atriopeptins. When Polanyi was studying the histology of the cell, he noticed that cardiac atria had granules.
29:01
And when you see granules in tissue, it's usually indicative of stored peptide hormones. The Bold in Canada isolated those granules, sequenced the protein as peptide, they called it atrionatriidic factor. It turns out there are three peptides in that family, AMP, BMP, CMP, and we discovered they all work
29:22
by activating the particulate isoforms of guanylate cyclase. So they too work through cyclic GMP, but through a different receptor. Now that gives us ways to elevate cyclic GMP in cells by going through the soluble pathway or through the particulate enzyme pathway or through the phosphodiesterases
29:41
or the protein kinases. I'll summarize that for you a little bit later. There are three isoforms of nitric oxide synthase, three different gene products. Most tissues have these enzymes. They convert arginine to nitric oxide and citrulline.
30:03
They're about 50 or 60% homologous. NOS2 is an interesting one because normally the transcript of protein is not present in tissues unless there's been an inflammatory insult. And pro-inflammatory cytokines, IL-1, interferon gamma, TNF alpha,
30:23
activate NF kappa B, which regulates transcription of NOS2. So if you see NOS2 transcriptor protein is indicative of an inflammatory insult. It becomes an inflammatory biomarker. Very important. They all catalyze this reaction.
30:43
They oxidize the guadadino nitrogen of arginine to hydroxyarginine, and that's further oxidized to form citrulline and nitric oxide. The enzymes have a very complicated array of cofactors. They are heme-containing proteins.
31:02
They utilize calcium calmodulin. They utilize FMN-FAD. The substrates besides L-arginine are oxygenated. Oxygen and NADPH. And they utilize tetrahydrobiopterin. If you oxidize those cofactors,
31:22
the enzyme no longer makes nitric oxide. That's very important. Instead, it makes superoxide anion, which will scavenge the nitric oxide, which is the last thing in the world you wanna do. Because when NO binds to a superoxide, it makes peroxynitrite, which is incredibly toxic.
31:41
And I'll say more about that shortly. So, okay, I'm a little bit, I'm almost done here. This is the pathway that we've worked out in many tissues and by understanding the elements and components in the pathway, the biological, biochemical sequence,
32:02
each step becomes a molecular target for you to go discover chemicals as potential drug leads to develop products. The pathways are always a little more complicated. Patients with hypertension, diabetes, atherosclerosis, tobacco use, and perhaps obesity have blood vessels
32:23
that do not make enough nitric oxide. And they don't make it for several reasons, and I won't go into the details, but by knowing the biochemistry and the problems in these patients, we can figure out better ways to treat their diseases. And we do it with nutritional supplements as well as drugs.
32:40
It's a very important disease that most of you in the audience have. And when I was at Stanford, I was able to work with coronaries from young motorcycle students who killed themselves on the motorcycle. And at 17, 18, 19, their coronaries were abnormal biochemically. They looked normal histologically, but they weren't making enough nitric oxide.
33:01
So it's there. These are some of the diseases and opportunities in clinical medicine where this story can be utilized to start treating patients. And it's a diverse list, neuroscience,
33:20
stem cells, cancer, gene transcription, hormone production. The list goes on and on. Now, I've not worked with all of these, but I worked with many of them. And it's been quite exciting. This is why there are 150,000 papers out there, and you can't keep up with the story anymore. It's too complicated. Thank you very much.