Nitric Oxide and Cyclic GMP in Cell Signaling and Drug Development
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00:00
Lecture/ConferenceMeeting/Interview
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Lecture/ConferenceMeeting/Interview
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Computer animation
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Computer animation
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Computer animation
Transcript: English(auto-generated)
00:14
Hello to our GORA talk from 11 to 11.40.
00:20
My name is Tobias Meyer. I'm the scientific head of the National Institute for Science Communication and I'm hosting this talk today. I'm hosting Ferit Murat. He won the Nobel Prize in 1998 together with Robert Furchgott and Louis Ignaro.
00:42
On, actually, also communication. It's not science communication, it's cell-to-cell communication, particularly second messengers, nitric oxide as a core molecule there. He will tell you all about it now. We have planned around a 20-minute talk. We'll see how long it goes
01:00
and then we go to some questions and answers after that. Ferit, the stage is yours. Thank you very much. When I prepared the summary abstract for this meeting a couple of months ago, there were only 150,000 publications on nitric oxide.
01:20
Today I've learned there are 160,000, so I've missed the last 10,000. It's a remarkable field. I began working in messenger systems 60 years ago and while I'm not the oldest scientist here, I've probably been working with cell signaling longer than anybody at this meeting.
01:43
I began as an MBPhD student working with Earl Sutherland and Ted Rolle as a combined degree student and I joined their laboratory the year after they discovered cyclic AMP. And over a seven-year period, which is how long the program was,
02:00
we discovered numerous hormones that regulated adenylate cyclase and cyclic AMP, many, many hormones. And Lefkowitz discussed some of that the other day. My assignment was to show that the catacalls work through the beta receptor and then I went on to show that acetylcholine-inhibited,
02:21
ACTH-activated, parathyroid hormone, thyroid-stimulated hormone, the gadiotropins, a long, long list and it was a very exciting time over the next 10-year period. To get involved in the early phases of the cell communication system. And in the late 60s,
02:43
I was planning to leave my training to go to the University of Virginia and cyclic AMP had just been discovered and cyclic AMP was becoming rather crowded. A lot of people, very popular area. So I decided to switch to cyclic AMP and I'll tell you some of that story.
03:02
And that led us then to the discovery of nitric oxide a few years later. Now, in the process of evolution, as cells evolved in the deep grips of the ocean, I suspect, near the magma and all the transition metals and gases and poisons coming out, they really had to evolve their biochemistry
03:22
to handle this toxic environment. And then they coalesced to form multicellular organisms and they used many of these molecules to communicate with each other and I think the very first messengers were very simple molecules, transition metals, gases and other noxious substances.
03:43
The concept is summarized for you on the first slide. Cells, multicellular organisms have cells that talk to each other. And I'm gonna present three different populations of cells that are gonna communicate.
04:00
I will call cell type one a neuronal cell. It could be in your brain or peripheral neuron. It doesn't matter. I'll call cell type two an endothelial cell lining all of your blood vessels. And if you add up all the endothelial cells, it's probably one of the largest organs in the body. It's bigger, bigger than the liver, heart or brain.
04:20
And we used to think that it was inert. It didn't do much. But now we know it produces a lot of important molecules and messengers. And cell type three, I will call the smooth muscle cell on the wall of that blood vessel. And they're gonna communicate. Cell type one is gonna produce a substance, a messenger,
04:40
that is released into the synapse or into the bloodstream. If it's a small molecule, it often has a protein carrier, a transporter, to protect it from degradation and prolong its half-life. If it's a nerve cell, a neuron, we call that messenger a neurotransmitter. If it's an endocrine cell, we call that messenger a hormone.
05:04
If it's a macrophage or a lymphocyte or an inflammatory cell, we call them growth factors, cytokines, chemokines. So they come in various sizes and shapes. Some are simple, some are complicated. Some are large proteins, like the catastrophes. But the concept is the same. They're all messenger molecules.
05:22
And they want to go find their target. So they home throughout the body and identify their target by the presence of a protein, often in the cell membrane, of the target cell. Sometimes those proteins are inside of the cell, as with steroids or thyroid hormone,
05:40
but most often they're integral membrane proteins. And these messengers will bind with that protein in a three-dimensional conformational fit, like a key in a lock. And that's where the specificity of the interaction takes place. Only the right messenger or ligand combines with the right receptor, and only the right receptor identifies the right ligand.
06:04
That ligand doesn't necessarily have to go inside of the cell to do its business. By combining with its receptor, it induces a biochemical cascade, resulting often in the production and accumulation of another intracellular second messenger. The first second messenger discovered,
06:22
cyclic EMP, was discovered in 1957. Today we know there are a handful of other intracellular second messengers. Calcium, isoglycerol, cyclic GMP, nitric oxide, some of the acoustinoids. And while there are hundreds of the first messengers,
06:41
there are a small number of intracellular second messengers. And I can tell you that most of these messengers have resulted in Nobel Prizes. Probably half of the Nobel Prizes have come from messenger systems. You heard a little bit about it earlier today with regard to protein synthesis.
07:01
But think of all the hormones, all the agonists, all the antagonists that are used in endocrinology and cardiology and immunology that regulate the transmission of this information, the cell signaling phenomenon. Nitric oxide is a unique intracellular messenger. It's a free radical with an unshared electron,
07:21
very reactive. It's also a gas. It's the only gas messenger that we've identified so far that's present in cells. It can function in the cell in which it's made, as other second messengers usually do. But it could come out of cells, unlike other second messengers,
07:40
and regulate the biology of adjacent cells or cells at a distance. So not only is it an intracellular messenger, it's an extracellular messenger. It's a paracrine substance. It's an autochoid. It's probably a hormone. It can bind to other carriers and be delivered at a distance, as other hormones can be.
08:03
So it's a very unique molecule. When it comes out of the endothelial cell, it goes over to the smooth muscle cell. Nearby regulates the production of another messenger, cyclic quantosine monophosphate, cyclic GMP. And I'm going to put that story together for you.
08:21
And most of this work began around 1970, as I left NIH to switch my interest from cyclic AMP to cyclic GMP. I'm going to tell you that nitric oxide is a natural endogenous substance in many, many cells.
08:41
It's a messenger. But it's also how some very important drugs work. Nitroglycerin, which made Alfred Dobell an awful lot of money making nitroglycerin into dynamite. He had chest pain. He was advised to take nitroglycerin if he refused because of the vascular headaches. It was a vasodilator.
09:01
But it's also a pollutant. Whenever you combust fossil fuels that possess nitrogen in their structure, you produce a family of nitrogen oxides that go off in the atmosphere, interact with ozone and deplete ozone, and ozone is the ultraviolet filter to prevent global warming. So nitric oxide and its oxidation products
09:21
really had a bad reputation for many years. That began in the 1940s and 50s as the chemistry was being understood. And when we came along and suggested it's how some important drugs work or it's a natural substance in the body, nobody would believe us. And it took 10 or 15 years
09:40
to convince the world it was important. And that was rather frustrating for me because I was very excited about it. I'm going to tell you some of the story. And others didn't believe it, didn't think it made sense. How can a free radical in the gas be a messenger? That's silly, Murad. Something's wrong here.
10:01
But what happened because of that, because others were not interested, it allowed us to dominate the field for 10 or 15 years. So we basically did all the early important stuff. It finally turned out pretty positive for us. But it took a while to get the world to believe what we were doing.
10:24
I was interested in how hormones might regulate cyclic GMP production similar to what I had done as a student with hormones regulating cyclic AMP production to mediate various biological effects. I thought cyclic GMP would be
10:40
another intracellular messenger. Well, we know that there are enzymes that make it from GTP. We call those enzymes guanylate cyclases. They have a lot of homology with the adenylate cyclase. In fact, their catalytic domains are quite similar. But unlike adenylate cyclase, which comes in a single isoform, a single gene product,
11:01
there are multiple isoforms of guanylate cyclase. There's soluble isoforms and particulate isoforms. And there are about seven different gene products. And that was something that we found quite early. And they all convert GTP to cyclic GMP. But they're also very permissive.
11:21
When they're activated, they can convert ATP to cyclic AMP. So it's an alternate pathway for cyclic AMP synthesis as well. But it prefers to make cyclic GMP. The soluble guanylate cyclase is the heterodimer with two subunits, alpha and beta. And on the beta subunit is a heme prosthetic group on the histidine 105.
11:43
And that heme prosthetic group has a ferrous reduced iron, which is the target for nitric oxide. So soluble guanylate cyclase is the receptor for nitric oxide. The particulate isoforms we learned were receptors for other protein peptide hormones.
12:03
The atropectins, E. coli, heat-stable neurotoxin, which causes diarrhea. Guanylate, uroguanylate, new hormones that were discovered after our findings with E. coli heterotoxin. So most of the G. cyclases, unlike the adenylate cyclases, are receptor proteins.
12:21
You heard with Lefkowitz's talk that hormones interact with adenylate cyclase through the G protein pathway. We suspected that there were such mediators with all the hormone effects I found as a student, which encouraged Gilman and Rodbell to go looking for these coupling proteins, the G proteins that came after our earlier work.
12:43
The cyclic nucleotides are inactivated by a family of cyclic nucleotide phosphodiesterases. There are 10 or 11 different isoforms of phosphodiesterase. They cleave the phosphodiester bond to convert it to the corresponding monophosphate, AMP for cyclic AMP,
13:02
GMP for cyclic GMP. Of all the isoforms, some selectively hydrolyzed cyclic AMP, some selectively hydrolyzed cyclic GMP, some hydrolyzed both. And the form that's become most popular is the type 5 phosphodiesterase, which Lou Agdero talked about the other day,
13:22
which is the target for a sildenafil. It inhibits that enzyme. And by inhibiting the type 5 phosphodiesterase found in blood vessels, it permits more cyclic GMP to accumulate in the blood vessel under the influence of nitric oxide or other vasodilators to create more vasodilation
13:41
to increase blood flow, lower blood pressure. That's how it works. We predicted there would be such a drug as Viagra 15 years before it was marketed, knowing that nitrovasodilators, which I'll tell you about, and phosphodiesterase inhibitors permitted the accumulation of both cyclic nucleotides, cyclic AMP and cyclic GMP.
14:01
Most of the intracellular messengers regulate a family of protein kinases. We call them serine threonine protein kinases when they phosphorylate, transfer the terminal gamma-phosphate ATP to a protein substrate on the serine or threonine residue. Cyclic AMP does that. Cyclic GMP does that.
14:21
Cyclic AMP, calcium calmodulin does that. Diacylglycerol does that. When these proteins are phosphorylated, and there are hundreds that can be phosphorylated, if they're enzymes, their activity can go up or go down, depending on the protein. If it's a structural protein, its function could change,
14:41
motility of the cell, transfer of perennials with the inside of the cell, the cytoskeleton, et cetera. There are other kinases that phosphorylate on the tyrosine residues adjacent to the hydroxyl group on tyrosine. We call those tyrosine kinases. Those are not regulated by messengers. They're regulated by growth factors and cytokines
15:01
because they are already tyrosine protein kinases. So this is the concept of cell signaling. It's pretty straightforward. Easy. Ligands alter the production of a messenger. The messenger regulates the kinase. The messenger can be inactivated by phosphodiesterases. The problem to put this all together
15:21
is because you're dealing with all of these isoforms. Multiple isoform, toluene cyclase, phosphodiesterase, protein kinase, and a variety of protein substrates. But once you understand all the detailed biochemistry, drug discovery is pretty simple. And I want to convince you of that in just a few minutes.
15:43
So we set out to see if hormones could regulate cyclic GMP formation similar to what I had done as a student with cyclic AMP formation without dealing with cyclase. And I wanted to know if cyclic GMP was a messenger, and if so, what biology would it regulate? That was our goal.
16:03
It turned out in our early experiments there were multiple isoforms of guanylate cyclase, soluble and particulate. We found that the activities could be regulated independently of each other as we looked at regenerating the liver, fetal liver, hepatic tumors, kidney tumors.
16:20
The soluble activity was lowered, the particulate activity was normal. They were regulated differently from each other. And accidentally in the process we discovered that some small molecules activated the soluble guanylate cyclase. That was quite an accident. We found that azide hydroxyl amine and sodium nitrite would activate soluble enzymes,
16:41
not the particulate enzyme. The kinetic properties of the soluble and particulate isoforms were quite different. So we were convinced that there were multiple genes regulating the production of guanylate cyclases. We were hoping to find hormone activation in the cyclase, but we failed.
17:01
Hormones, some hormones, would increase cyclic GMP accumulation in intact tissue, slices, cell cultures, but they failed to work when we homogenized the cells in cell-free extracts. We disrupted the pathosycline pathway. However, accidentally we found that azide hydroxyl amine
17:21
and sodium nitrite would activate the soluble isoform. Quite an accident again. We used to put azide in our buffers that we stored in the refrigerator or cold room and found that it would activate guanylate cyclase, but it only activated the soluble isoform. And we did this simple experiment. This is to show
17:44
some of the kinetic differences in these isoforms. We did this experiment. Azide would activate soluble guanylate cyclase in liver extracts, but it failed to activate heart extracts or cerebral cortex extracts. Because we were working with soluble fractions, we
18:02
did reconstitution. We mixed them. And when we mixed liver with heart, the azide effect disappeared. Heart possessed an inhibitor of the azide activation. It possessed two inhibitors. We purified them. They were hemoglobin and myoglobin. When we mixed liver with cerebral cortex,
18:23
the azide effect was enhanced because azide was converted to some activator by liver that activated guanylate cyclase, contributed by the liver, and also contributed by the cerebral cortex. We purified the factor from liver. It turned out to be catalase. Some of the cytochromes would do it too.
18:42
So we now knew that these small molecule activators were being converted to something else. And we thought if we understood what that pathway was and what they were, perhaps we could be clever enough to reconstitute a hormone effect, which was our goal.
19:03
We put azide, hydroxylamine, sodium nitrite on a variety of tissue slices, cell cultures, and one of the preparations we worked with was tracheal smooth muscle. I knew as a student cyclic AMP, epinephrine, other agents, would relax smooth muscle, airway smooth muscle,
19:20
GI smooth muscle, vascular smooth muscle. And I thought that cyclic AMP would antagonize the effects of cyclic AMP. When we put azide hydroxylamine, sodium nitrite, on tracheal smooth muscle, which was rather homogeneous, and you wanted homogeneous cell types when you do biochemistry.
19:40
So you know what cells are doing the biochemistry. It's difficult if you're working with heterogeneous systems to know who's doing what. They all caused elevations of cyclic AMP, but they also caused relaxation. That was a surprise. I expected the elevation of cyclic AMP to antagonize the effects of cyclic AMP and cause contraction.
20:03
They didn't. And then I remembered as a resident taking care of patients with heart attacks in the intensive care unit. And what we would do is administer intravenous nitroglycerin or nitroprusside to lower blood pressure, we called a decrease afterload, so the heart wouldn't have to work so hard
20:22
and could recover after a monocardial infarction. And they were vasodilators. So we then put nitroglycerin and nitroprusside on our muscles. They caused relaxation as expected. They relax all smooth muscles, whether it's tracheal smooth muscle,
20:41
biliary tract, intestinal smooth muscle, vascular smooth muscle. They all respond the same way in terms of the biochemistry. The big surprise was when we elevated cyclic AMP with these agents, there was relaxation, and we convinced ourselves that cyclic AMP
21:00
was a smooth muscle relaxant. They cause relaxation as opposed to contraction. Surprise. So then we tested a variety of nitro and nitroso compounds, nitroglycerin, nitroprusside, azide, et cetera, nitrosamines, nitrosouris, and they all activated soluble
21:22
glomerulone cyclase, and they all caused relaxation of smooth muscle. But we knew that hemoglobin and myoglobin were inhibitors. We knew that some of them required enzymes for their conversion to some activator. So now we had some activating intermediate being produced in our incubations, and that was an incredible
21:41
mystery story. And for a couple of years, we tried to figure out what that substance was. And the big clue was that the inhibitors, the nitrobasodilators, as we call them, coined a new term, were all blocked by hemoglobin and myoglobin, which we knew had a high affinity for nitric oxide.
22:03
So we generated nitric oxide in the fume hood, a simple chemical reaction. We mixed ferrous sulfates, sodium nitrite, and sulfuric acid, stirred it, ventilated it with a catheter into our incubations. We activated all of our soluble guanylate cyclase preparations.
22:21
Eureka, we discovered that nitric oxide was an activator of soluble guanylate cyclase, a free radical, a gas, activated an enzyme for the first time, and that was exciting biochemistry. We then went ahead and applied it to smooth muscles and other tissues, and we could cause relaxation and increase
22:40
cyclic GMP. And that was the eureka moment in December of 1976. And we took off. The world wouldn't believe us for a long time, for the next 10 or 15 years. But it was obvious that we now had a new pathway of cell signaling that entailed gases and free radicals.
23:02
This is what nitroprusside does to a ratty or a segment. Within 10 or 15 seconds, cyclic GMP levels are elevated. After it reaches a peak in one or two minutes, they return to basal levels. The elevation of cyclic GMP is followed by relaxation. Cyclic GMP precedes the biology,
23:22
the relaxant event. Relaxation persists for some period of time, 10 or 20 minutes. You might argue that cyclic GMP is not related to relaxation. Well, I'm going to tell you it is, but what's different is that the messenger has a short half-life, and most of them do. They don't last but 10 or 20 or 30 seconds
23:42
or so, whereas the downstream effectors, the protein kinases and phosphoryl proteins, have a longer half-life and relaxation will persist for 10 or 20 minutes. So there is an association. And a lot of people working with cyclic GMP systems previously
24:01
forgot to do it properly with time courses and convinced themselves that cyclic GMP didn't mediate the effects of their hormones when in fact it did. Because they were working with heterogeneous cell types or they were looking at the wrong time courses. So we realized in 1978 that we
24:21
had figured out how the nitrobasidiolators, nitroglycerin, nitroprusside, and others, the inorganic, organic nitrites and nitrates, worked. They were all pro-drugs or precursors converted to nitric oxide. Now as a pharmacologist, when you have a drug that does something interesting,
24:42
the opiates for example, you wonder if there is an endogenous substance that does the same thing. The discovery of enkephalins came from the previous work with opiates. People looked for such a substance. That's happened in other areas of pharmacology. And we wondered by forming nitric oxide
25:02
from nitrobasidiolators, is it possible that nitric oxide was being made in tissues endogenously and acted as a messenger. We suggested in 1978 it was going to be a second messenger. That was heresy. Free radicals can't be second messengers. Gases can't be second messengers.
25:21
Be right, you're crazy. The problem in proving it is the concentration of nitric oxide required to activate guanylate cyclase was very low. Nanomolar concentrations. There were no assays to measure nitric oxide and its oxidation products nitride and nitrate at such low concentrations.
25:42
So we had to spend a couple of years creating new technology to measure it. And we found that rat lung fibroblasts had no enzyme to make NO but most other tissues could make it. But because they didn't make it, they were very sensitive to nitric oxide production to elevate cyclic GMP
26:02
levels in those cells. And we use those as a bioassay, a biomarker of nitric oxide formation and they were quite sensitive. And we then screened lots of cell extracts and tissues, cell cultures and found that almost everything we looked at made nitric oxide. It was very ubiquitous.
26:21
It was present everywhere we looked. And that was quite exciting. But it was also obvious that it was going to create a very complicated story as to how to put the biology and biochemistry together to figure out what it does. And that took a few more years. About this time, 1980, Robert Furchcott
26:42
gave a lecture at the University of Virginia and I attended that seminar. And he talked about endothelial dependent vasodilators acetylcholine, histamine, bradykinin were known to cause hypotension, blood vessel relaxation in animals. When you tested those agents in the laboratory on blood
27:02
vessels, they failed to do anything. And that's because you destroyed the integrity of the endothelium. If you were careful to preserve the integrity of the endothelium, they now caused relaxation and he called them endothelial dependent vasodilators. And they produced a substance endothelial EDRF, endothelial dependent
27:22
relaxing factor, which Ignaro talked about the other day. And that caused relaxation. And I heard that seminar and learned from Furchcott that he could block the effects of this pathway with inhibitors that we were using to inhibit guanylyl cyclase activation. Hemoglobin, myoglobin, methylene blue, other agents that we were using.
27:42
Also, EDRF had a very short half-life. It was very reactive and I suspected it was a free radical. And I suggested to Furchcott it might be nitric oxide or a complex of nitric oxide. And we were going to work together to figure that out. We, a few months later, moved to Stanford. He went back to New York. His wife developed breast cancer. We never collaborated. So we went
28:02
ahead finally to do the work ourselves and sure enough, we were right. EDRF does increase cyclic GMP production. EDRF turns out to be nitric oxide or a complex of nitric oxide. And Ignaro sort of summarized that for you the other day. So this is a blood vessel now to put the story
28:22
together with an endothelium on the left, a smooth muscle compartment on the right. And in red are three categories of vasodilators that work by enhancing cyclic GMP formation. In the middle and the top are the nitro vasodilators. Nitroprusside creates
28:40
nitric oxide spontaneously in the bloodstream, depending on pH and oxygen tension. Whereas nitroglycerin or some of the other nitroglycerin dilators require an enzyme to do it. There's a denitrase. People have worked on that and it produces nitroglycerin tolerance, which is another story. But they all convert to nitric oxide, which activates
29:02
soluble gloyally cyclase to make cyclic GMP. It activates the protein kinase. The protein kinase phosphorylates a variety of smooth muscle proteins that we've identified with phosphate incorporation and 2D gels. And it turns out that decreases the phosphorylation
29:20
of two proteins, myosinol and light chain, on our gels. So this is the pathway. Cyclic-G regulates intracellular calcium, which regulates the activity of myosin light chain kinase, which is a calcium calmodulin dependent enzyme. So when you have in myosin filaments, if you phosphorylate myosin, they slide
29:42
together, you get contraction. If you dephosphorylate, they slide apart and you get relaxation. That's how cyclic AMP and cyclic GMP regulate smooth muscle motility. On the left hand part of this slide are the endothelial dependent phase dialers, histamine, bradykinin,
30:01
acetylcholine. They work on the endothelial cells because that's where the receptors are. Their receptors are not on the smooth muscle cell. And when they interact with their receptors, they permit calcium to enter the endothelial cell to activate an enzyme to make EDRF. That enzyme is nitric oxide synthase, which converts
30:21
l-arginine to nitric oxide, which goes over to the smooth muscle compartment and works just like a nitrobasidiolator does. So the pharmacology biochemistry of the two pathways are quite similar. And that's why we thought that EDRF could be a nitric oxide or complex. A third category of vasodilators
30:42
are the atropeptins. When Pilati was looking at the histology of cells, he found that cardiac atria contain granules, and when cells have granules it's usually indicative of stored peptide hormones like chromatin tissue. DeBolt in Canada isolated those granules, purified the peptides, they turned out to be the atropeptins.
31:03
A and F, B and F, C and F. They are short peptides that cause vasodilation and they also have natriuretic effects on the kidney to enhance sodium chloride and water excretion. So they had some of the properties of the nitropated
31:20
dilators and some of the properties of E. coli heat stable neurotoxin, which we found to activate guanylase cyclase in the intestinal mucosa to cause diarrhea. So we tested the atropeptins and sure enough they activate the particulate guanylase cyclase and one of the particulate isoforms is the receptor for the atropeptins and another
31:41
particulate G. cyclase is a receptor for E. coli heat stable neurotoxin and we purified all these isoforms along the way. So we purified lots of enzymes. And some work directly through nitric oxide and some work directly on the proteins. It's because the protein cyclases are receptors themselves
32:00
to make cyclic GMP. A couple of other laboratories did some very important experiments and I don't have time to review that for you. But let me jump in. There are three different isoforms of enzyme that make nitric oxide. We call it NAS-1, 2, and 3. We purified all of them. We were the first to
32:21
purified NAS-1 and NAS-3. The Cornell group purified NAS-2. NAS-2 is interesting because normally tissues don't have protein or transcript for NAS-2. The NAS-2 transcription is regulated by pro-inflammatory cytokines that activate NF-kappa-B to
32:41
regulate the transcription of NAS-2. So when you see NAS-2 protein or transcript, it's indicative of an inflammatory insult in that tissue. It's a biomarker. Most tissues have one or another or sometimes all three of the isoforms of nitric oxide. There's about 50-60% homology between the
33:01
isoforms. The homology is predominantly in the catalytic domain where arginine binds a substrate to be converted to citrulline and nitric oxide. I'll show you that. There's a stick figure showing arginine on the left with two guanadino nitrogens. One of them gets oxidized to hydroxyarginine. That gets
33:20
further oxidized to form nitric oxide and citrulline. The enzymes have a very complicated array of cofactors that we learned about as we purified them. They require oxygen. They require NADPH. They're calcium calmodulin dependent. They have a heme prosthetic group. They require tetrahydrobiopterin.
33:42
They're active as homodimers typically in tissues to make nitric oxide. That's very important. By understanding now the biochemistry and the cofactors it allows you to manipulate tissues to produce nitric oxide. That can be important therapeutically.
34:01
I'll put this story together for you now. Quickly. Hormones, ligands interact with their receptors. Those receptors control the intracellular environment, the cofactors for nitric oxide synthase, which converts arginine to nitric oxide,
34:21
which is EDRF. That activates soluble guanoacyclase to make cyclic GMP to activate a protein kinase, cyclic G-dependent protein kinase that phosphorylates a variety of proteins that produce a biological effect. Each step in the pathway becomes a molecular target.
34:40
You can now go to chemical libraries and screen compounds that alter that biochemical step, either activate or inhibit. That's how you discover drug leads. You then take those drug leads and modify structures to optimize the pharmacokinetics, the half-lives,
35:01
the potency, etc. Nitric oxide prefers to make cyclic GMP, but it can be pulled off in other directions. It can be oxidized. It can form complexes with other transition metals besides iron. It can form complexes with thiol groups and proteins. There are hundreds of proteins that get nitrosylated.
35:22
It can also interact with other free radicals, superoxide anion, and it forms a very toxic species, peroxynitride. When you have inflammation, you induce, as I told you, NF-kappaB, which induces the expression of most of the inflammatory genes, including NOS2.
35:41
You make a lot of NO. You make a lot of hydrogen peroxide and a lot of superoxide anion. You make a lot of peroxynitride, which is really a poison. It phosphorylates nitrates protein residues adjacent to tyrosine hydroxyl, blocking tyrosine kinase pathways.
36:00
So the signaling pathways with tyrosine kinases and nitric oxide and cyclic G are all interactive. They're interactive with calcium. They're interactive with cyclic EMP through the phosphine anstrases. They're all talking to each other, which makes life complicated, but also interesting. The more complexity you have, the more opportunities
36:20
you have to manipulate the pathways for therapy. And that's obviously exciting, but it takes a lot more effort to figure it all out. There's a disease called endothelial dysfunction. Patients with hypertension, atherosclerosis, diabetes,
36:41
tobacco use, and probably obesity have blood vessels that don't make enough nitric oxide. And they don't make enough for a variety of reasons. We now know the biochemistry of that. They make inhibitors of nitric oxide synthase, a methylated arginine analog. They make free radicals that scavenge nitric oxide.
37:00
You oxidize the tetrahydrobioptrin to a dihydrobioptrin. It's no longer effective as a co-factor for NAS. And instead of making NO, it now makes superoxide, which scavenges and traps nitric oxide. So by understanding the biochemistry of endothelial dysfunction, we now can figure out better ways to treat these diseases.
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And people and companies are, with arginine supplementation and various antioxidants, many of the traditional Chinese medicines for antioxidants, many of them work through nitric oxide and cyclic GMP. As a matter of fact, I have a research center in Shanghai that we've worked on that.
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And let me show you two slides of some of the biological effects and therapeutic uses of this pathway. It's really quite vast with 150 or 60,000 papers. I can't review all of it for you. But it's been very exciting for me to see our fundamental
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research have such broad application in clinical medicine. It regulates smooth muscle. It regulates platelet aggregation. It regulates angiogenesis. It regulates gene expression. It regulates stem cell differentiation and proliferation. It regulates cancer proliferation.
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The list goes on and on and on. And you can't do everything. We work on many of these things, but not all of it. You've got to focus. But it's been quite exciting to see this all take off now. It already has provided a lot of important medicines and it's going to provide a lot more.
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There are probably 70 to 100 biotech companies around the world working on some aspect of nitric oxide and cyclic GMP research. Thank you for your attention. We can have some questions if you'd like. So, thanks Ferit. Fantastic talk. I like the whole
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loop basically that you draw from starting out in the lab, how you discovered all the details, to then integrating it with the whole signal transduction process and up to diseases. We are fairly short on time. There are still there is still time for say a couple of questions.
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There's a microphone standing over here. So if anyone of you has a question can go over to the microphone. Alternatively this young gentleman here has a microphone in his hand who would be happy to come over to your place. There's one question here at the back.
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Hello, sir. Very nice talk. I was just curious because I'm working with stem cells and it's how does it regulate towards adult neurogenesis, particularly neural stem cells. And as you mentioned there are so many papers with nitric oxide role and its effect but there are also very
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lot of papers which has a conflicting data where anode is responsible to maintain the stem cellness of the cell but some paper also mentioned that it's promoting differentiation into neurons. So what's your take on it? Thank you. Most of the beneficial effects of
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nitric oxide occur at very low concentrations. Probably nanomolar concentrations to activate guanylate cyclase to make cyclic GMP. And when you do that you enhance proliferation you regulate lots of genes. We don't know precisely how it regulates genes but with microarrays we know that many genes are activated and modified.
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At high concentrations it's very toxic because it does other things. It can interact with peroxy nitride, it can nitrate proteins that can form nitrosylation products and thiol groups and cysteines and proteins. So there are many effects and it depends on whether you're working with low concentrations or high concentrations. And that's true with most messengers.
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You know most diseases with messengers are because you have too little or too much. Think of all the endocrinopathies. Too little or too much causes a problem. The same is true with messengers and certainly with nitric oxide. So I think that's one of the problems. With our experience we have found that nitric oxide enhances the
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proliferation of embryonic stem cells, both human and mouse embryonic cells. And it also pushes the cells into myocardial cells or neuronal cells. Now it turns out that these cells have an abnormal guanylate cyclase. They lack the alpha subunit so they're not activated by you know but they're making a lot of nitric oxide.
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They have a lot of NOS activity. So it must be doing other things in these cells to influence differentiation rather than working through cyclic GMP. Maybe it's working directly on transcription. I don't know. We're trying to sort that out. So it's a tumor cyclic GMP is a tumor suppressor. Tumors make a lot of
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NO but they too have an abnormal guanylate cyclase. They lack alpha. And without alpha you can't make cyclic G. You need the heterodimer alpha plus beta. So maybe it's doing other things in tumors to regulate transcription as well. There's one question over there. Hi.
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Thank you for the nice talk. You mentioned a couple of times that it took 10 years for the science community to believe. Was there something that happened specifically or it was just a matter of time and more data for them to believe you? What really happened was that Furchcott's observations with endothelial
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cell dependent relaxation to make EDRF was a stimulus in vascular biology because now we could explain how a number of agents caused relaxation and why they failed previously in vascular preparations without endothelial. That was an important observation. It also told us
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that muscle cells and blood vessels don't have the receptors for the endothelial dependent vasodilators. If you have a patient with a heart attack and you want to lower blood pressure afterload, you can't use acetylcholine, britakine and histamine because the smooth muscle, the endothelium is defective, maybe absent, destroyed
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and they don't have receptors on the smooth muscle so you have to use nitro vasodilators. So it taught us a lot about therapy and the intensive care unit, how to manage blood pressure, blood flow, etc. And because of that transition between about 1980 and 1988-89 people then became
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interested in the whole field, in oocyclology and it sort of took off in the late 1980s. Okay, I have one last question and I think then we should close it. And that is there is a lot of young researchers here who are at the start of their academic careers. Now you
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had an illustrious academic career, you shifted to industry and back, you're still doing research. Maybe you can give one word of advice to the young researchers. What is the most important factor for a successful academic career? Well, I've trained in multiple areas, not only pharmacology, biochemistry, but also
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medicine. And I go back and forth, basic research, clinical research and I love research. And although I made lots of moves from various universities and to industry and back again, I've always taken my research with me. When I was at Abbott running their pharmaceutical research,
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I had a laboratory of 20 people working. That's where we purified the NOS isoforms and worked out a lot of the biochemistry. So to have the research program move with me, it was possible to go back and forth. The people who have given up their research when they go to industry have more difficulty
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coming back to academics because it takes time to get refunded and build your program again. But it's doable. It's possible. And a lot of people ask me, you know, should I go to industry? Should I stay in academics? And they're different. In academics, you have much
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more independence to select what you want to work on. But you have to find funding to do it. In industry, there's more security. You don't have to go get all the grants. You don't have to publish so much to protect your position, but you don't have the freedom to pick your problem. You may be reassigned to another project if
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that project's not working well. So they are different, but they offer advantages, and it depends on what you're looking for. It's doable to do both of them and back and forth, but you've got to preserve your research interests. All right. Thanks again, Ferit. Thank you for coming to the Agora session. Enjoy the rest of your day.