This past February the 14th Nitric Oxide Gordon conference was held in Ventura California. The conference was lucky enough to have Nobel Laureate Lou Ignarro attend, where he led much of the discussion and delivered great advice to young and old investigators alike. Recently, Dr. Ignarro has published a book about his career in science and the discovery of NO. At the conference, I was fortunate enough to interview him about his career and his views on the future of in vitro experimentation.
Dr. Andrew J. Gow: Welcome, to start can you give us some insight into your discovery of nitric oxide (NO) and what inspired you to look at NO in the first place?
Dr. Louis J. Ignarro: Yeah. Well, it is very easy. And it is fun to try to recall these things as well. The person who really started off this whole field was Fred Murad, who shared the Nobel Prize, because he showed, accidentally actually, that NO could activate guanylate cyclase and elevate cyclic guanosine monophosphate (GMP) levels.
At the time I was interested in cyclic GMP and for several years I had been looking at cyclic GMP levels and trying to understand what the physiological significance of this cyclic GMP was. But there was no way at the time to elevate and stimulate the production of cyclic GMP in tissues or cells or animals and then try to observe any overt effects. You could do that with cyclic adenosine monophosphate but not cyclic GMP. Then Murad discovered that NO stimulates cyclic GMP formation. So, that was a great tool that one could use to stimulate cyclic GMP formation and see what it does.
And so we started working on this in 1979. And that is when we found that NO was actually a vascular smooth muscle relaxant, in vitro. And then we were able to show that in vivo as well. And all of this was through a cyclic GMP mechanism. One of my postdocs decided to study the effect of NO on platelets because he read somewhere that sodium nitroprusside, a nitro vasodilator… That is a phrase coined by Murad… prolonged prothrombin bleeding time. And so we figured it was a NO mechanism, so we studied NO and found that NO elevated platelet cyclic GMP and inhibited aggregation and inhibited adhesion to the internal lining of blood vessels. At the time, I did not realize how important that was going to turn out to be.
And so, just making a long story short, being a pharmacologist, I decided to study more of the pharmacology of NO. We knew it was a vascular smooth muscle relaxant, an inhibitor of platelet aggregation function. So, what else did NO do? So, we started exploring different things and so on, found out it had anti-inflammatory properties, and so on. And so I kept asking myself and bringing this to the attention of the group that here we have an exogenous component of air pollution that produces all these incredible effects, which are protective effects in the cardiovascular system.
Dr. Gow: You just pointed out that NO was an exogenous part of what we consider to be air pollution. When in the process did it become clear that it was actually naturally produced, which we made our own.
Dr. Ignarro: I kept looking at these effects and I said to myself, “What is the physiological significance of cyclic GMP?” Why do we have guanylate cyclase? The only known substance that could activate it at the time was NO. Where is the NO? Do we have guanylate cyclase because somehow we were going to get exogenous NO to elevate our cyclic GMP levels to do something? And that is when I began to think that we must have endogenous NO and that is why we have guanylate cyclase. Then we could say, “Okay, we have this NO cyclic GMP pathway, and it's really a signaling pathway.”
And we started looking for NO. The methods were not so sophisticated back then. This was the early 1980s. We really could not find anything. But I just believed it was there. Then we were lucky because Bob Furchgott, who is the third person who shared the prize, discovered this endothelium-dependent relaxation by acetylcholine and bradykinin and other naturally occurring substances. And he showed that somehow these agents interact with the endothelium to generate a factor, a relaxing factor, which he called endothelium-derived relaxing factor or EDRF. And he published that in 1980. But he did not know what it was, but he knew it was very unstable.
So, we jumped in on that, because I am a vascular pharmacologist and I figured, “Well, let's look at that and just try to determine what it is.” I had no idea that it could be NO, but we started to look at EDRF and study its release, with a variety of experiments. One thing we found was that EDRF had a half-life of 2 to 5 seconds that was a very short half-life. But we did not know what it was. And so, after a while, because we were experts on NO working with NO in the laboratory, we became experts in working with an EDRF.
And it was very obvious that the properties, the physical, the pharmacological, and the chemical properties of EDRF and NO, were virtually identical. And that is when I pulled my group together and I said… This was in 1985… That EDRF must be NO. And there was a lot of commotion in the group, just like I had from the outside. “No, it can't be.” However, we decided after comparing EDRF and NO that there is a strong likelihood that EDRF was NO. So, I designed some chemical experiments to isolate and identify EDRF.
Dr. Gow: So that was when you used the reaction with hemoglobin as the identification of NO?
Dr. Ignarro: Yes, exactly. And EDRF turned out to be NO. And we were going to use chemiluminescence. This is what another group used. But it was just a big machine, and we did not think it would be sensitive enough. So, we had experience doing spectrophotometry on hemoglobin and nitrosyl hemoglobin. When NO reacts with hemoglobin, it causes a shift in the peak, if I remember correctly, from 430 nm to, I think, 398 nm or something. A distinct shift.
And so we thought “Ah, let's perfuse an artery…,” collect the perfusate and try to measure EDRF. We took that perfusate and immediately added it to our cuvettes with the hemoglobin and measured them spectrophotometrically. And sure enough, I will never forget those experiments, we could see that shift in the spectrum. And only NO could produce that shift.
Dr. Gow: Its fascinating that you have that very clear memory of that moment.
Dr. Ignarro: Yes. Actually, the Nobel Committee singled out that experiment, I remember, and identified it at our meeting. We were each told separately why we received this accolade, and they pointed out that experiment to me. So, that made me feel pretty good.
Dr. Gow: I have heard you say before that to get the Noble Prize in physiology and medicine, you have to have a single seminal experiment that has improved human existence, is that right?
Dr. Ignarro: Exactly, there are two criteria. One is you have to have made an original discovery. It is not important how much you have advanced the field. There are other prizes that would be awarded for that, not the Nobel. It can be a single experiment or a few experiments, but it is not only the discovery that is important, it is the benefit that the discovery has to humankind. Alfred Nobel called it mankind but today, of course, we call it humankind.
Dr. Gow: So, when you were doing those experiments and seeing that spectral change, were you thinking, “Gosh, this could be Nobel worthy.”
Dr. Ignarro: Throughout my research career, the only thing I worried about was losing my tenure at the university. I did not think about a Nobel Prize. And I did some interesting experiments before identifying EDRF as NO that maybe were Nobel worthy. And other people said that, but I never paid attention to that. But I have to admit, when we realized that EDRF must be NO, even before doing the key experiments, that Stockholm ran through my mind. And I did not talk to anybody about it, but when that first experiment came off the recording spectrophotometer and we could see that shift by the EDRF, I went back to my office and I had tears in my eyes, and I said, “You know, this could be Nobel Prize material.” That was so incredible. I mean, I get goosebumps just thinking about it right now.
Dr. Gow: So, I know you have written a book that is very much a memoir, addressing your whole career and your experience as a scientist. Can you tell us a little bit about why you wrote the book and who you would like to read it?
Dr. Ignarro: Many of my friends, both scientific colleagues and just ordinary friends, urged me to write the book because they knew about my life; and I think, there were two reasons I wanted to write the book.
One was for the general public to understand that one does not have to be the child of a professional person to climb to the top of his or her profession and get awarded something like the Nobel Prize. And I point out in the book that my parents were immigrants from Italy. They met each other in Brooklyn, New York. They got married there and I came along in 1941. They could not speak English and so my English was terrible when I went to school. Upon entering elementary school, the teachers told my parents that unless my English picks up, I am going to have a lot of trouble. And so, my mom quit speaking Italian at home and spoke only English.
This helped me pick up my English. My parents motivated me to study because they did not want me to be a carpenter like my father, who never went to school and worked his hands to the bone every day. And he would always tell me in Italian, “I want you to have a profession, to go to college, and all that.” That really motivated me, and I was able to do well at school and go on to have a profession.
Point number 2 is I always had a passion for science. I mean, some kids do, most kids do not. But I had a passion for all kinds of science, whether it was chemistry, biology, or physics. Even before taking any of those courses, I wanted to get my fill of science. So, that certainly motivated me in my profession. And I think that is a good example to point out to everyone. I often get invited to elementary schools and high schools globally, not just in the United States, to talk about my experience, to talk about how I was able to go on this rollercoaster ride and overcome seemingly insurmountable obstacles to climb to the top of my profession. And I found that to be a very valuable talk to give. And so I wanted to express that in the first part of my book.
Dr. Gow: Do you think the book also is there to help the younger scientists? People who are trying to think about what their career might be and what they can achieve.
Dr. Ignarro: Exactly. I think so. Because in the book, I point out in detail my professional career. In other words, what is it exactly that I did to deserve the Nobel Prize? So, what I have tried to do, and it was difficult taking a lot of time, is to describe the experiments on NO in such a manner so that anyone can understand it. You do not have to be a scientist experienced in the field. You did not even have to be a professional person.
And as it turns out, the feedback I get back from young scientists is that it is very rewarding to read that if you have a clear mind and you think logically you do not have to do sophisticated experiments to make discoveries. It is the logic that drives. And taking chances a little bit, thinking outside the box. But most importantly, never giving up. Never giving up. So, I think I get those points across pretty clearly in the book.
Dr. Gow: You are a classically trained pharmacologist. And as you say in your book, you like pharmacology because it gave you the chance to do both chemistry and biology. Can I ask you to describe what you consider to be the biggest technical change in the field of pharmacology, since you started in the 60s?
Dr. Ignarro: Molecular biology and genetics. Nobody even knew what a Western blot was when I started doing my work. I mean, that became popular later. And I have to tell you, in all the experiments that my laboratory had done that resulted in this 1998 Nobel Prize, we never did a molecular biology experiment. Number 1, because we did not have to. And number 2, because it was just starting up and I did not know what to do. It was later that we started doing that kind of work. If I had access to knockouts and overexpressed enzymes back then, that would have been great. I do not think it would have sped up identifying EDRF as NO, because I was lucky, in the field of NO, vascular biology, those discoveries really had to come from classical pharmacology.
Dr. Gow: But do you think that maybe we are relying too much on our genetic models now?
Dr. Ignarro: I am not sure. When I go to a talk, I hear about many experiments based on genetic models. But it is hard to answer that question. I think a scientist must never forget the classical pharmacology and biochemistry. Because you can answer many questions that way. I mean, one of the things we did was to identify NO as a neurotransmitter that causes penile erection. And that work led to Viagra. Again, no molecular biology involved. There was just classical experiments, chunks of erectile tissue, corpus cavernosum subjected to electric field stimulation, because the nerves have to stimulate. And we discovered that during that stimulation the nerves released NO. We identified it. And the rest is history.
So, again, I think you have to do that experiment. How could you do that experiment to show that the neurotransmitter was NO with a molecular biology or a knockout experiment? You still have to do it and identify the neurotransmitter chemically. So, there is always room for classical pharmacology. You could make the important discovery using classical pharmacology and then you can go to town and put the icing on the cake with all of the more modern advances in science that we know.
Dr. Gow: So, I know from reading your work and also reading your book that a lot of your early work on NO was done with tissue baths, is that right?
Dr. Ignarro: Right. Within the entire pharmacology department, I was known as the twitch pharmacologist.
Dr. Gow: Today, we are concerned with novel alternative methods, experimentation that does not directly involve animals. Would you say that the tissue bath was one of the earliest forms of in vitro experimentation?
Dr. Ignarro: Yeah, I never used live animals. I have always had pets. And I have to tell you, I always had a problem with some of the animal usage. I would collaborate with the physiologists who would do the live animal experiments, but in my laboratory the furthest we went was to take tissues from small laboratory animals and use that.
Dr. Gow: Is there any one of the alternative type methods that you think is useful for modern pharmacology?
Dr. Ignarro: I think the advances made in mass spectrometry, for example, have been incredible, allowing for the identification of chemical components. I mean, those procedures have been refined tremendously, at least in the chemical pharmacology field and so on. But I am very impressed with the extensive knowledge that has been learned from using genetic knockouts.
Dr. Gow: Say we did not know about NO and you were starting now, would you still use the tissue bath route? Or would you think about other techniques that you might use when you are trying to figure out what this elusive EDRF was?
Dr. Ignarro: What the elusive EDRF was? Well, I mean, science progresses by certain experiments establishing a baseline or a foundation from which you move further in a particular direction. If you want to go from A to C, you really have to go through B first. It is extremely rare that you can go from A to C directly. So, I think that you need to perform the obvious experiments to answer questions. So, yes, if today right now somebody identified this magical substance that was released from endothelial cells and we wanted to go after it to figure out what it was, sure, I would still start off with isolated arterial or venous preparations in vitro.
I might also set up classical experiments such as those developed by John Vane, for which he was awarded the Nobel Prize, when he was studying prostaglandins. And that is to profuse a small segment of artery and have the perfusate drip on isolated blood vessels. In that way you can study the half-life of your agent. I do not know any other way to approach the problem when you do not know what the substance is. Now, if you had a hunch, it might be NO, sure, you could go directly to a chemical assay, but you still have to set up the apparatus to form the NO so you could collect it and measure it.
Dr. Gow: What about approaches like organ on a chip, where you could recreate a blood vessel, with smooth muscle cells and endothelial cells in three dimensions?
Dr. Ignarro: Well, I certainly would not know how to do that, but that would be incredible. Maybe with artificial intelligence (AI), eventually we will be able to establish something like that. That would be fantastic. And then we can avoid altogether using laboratory animals to do work. That is probably where the field is progressing, but being 82 years of age I do not think I am going to be able to see that. But maybe.
Dr. Gow: You also made a comment early on which I think was very insightful about the amount of information that we have learned from all of these different models, and the massive, effectively, in silico information that we have. Do you think there is a way in this informatic era, that we can bring these data together, as you say, with AI and modeling approaches?
Dr. Ignarro: Yeah, I do not know enough about that to be able to answer that question specifically because I have never used those approaches. But I read as much as I can, talk to people who are using AI and other procedures. It seems to me that eventually we should be able to use that AI to answer many questions. But what you are going to get out of AI is what you put into it.
So, all the data have to be put into it, and the system is going to have to be developed, such that you give it certain questions and it answers the questions correctly. And then you could move on perhaps to ask AI to predict, what is logical to come next. That I would love to see. I think that, and genetic engineering, will rid us from all hereditary diseases. And I would certainly love to see at least a little bit of that before I leave the world. I think those would be incredible advances.
Dr. Gow: Do you have any other advice for young scientists about their career or the field in general?
Dr. Ignarro: There is one piece of advice that I like to give to young and established investigators. And that is to never ever give up. I always tell people, and I have told several people at this meeting, that if you feel that your idea is valid, if you really think it is a good idea and you have done certain experiments to convince yourself that this is valid, do not let anybody change your mind. Listen to what they have to say, but do not stop doing your experiments just because someone else said that is not possible. And I always give them the example of how I could not get my NO work published. Nobody believed it. I could not get a grant as nobody believed it. But as I mentioned the other day I never gave up.
Also, its important to be imaginative when you do experiments, it is okay to think outside the box. You can test these experiments and test your hypothesis to see where it takes you and not to be shy about doing such experiments. And I think that is what all investigators need to do. You have an idea, you need to pursue it no matter what, until you have disproven your hypothesis. If you are convinced that your hypothesis is wrong, then go somewhere else. Finally, if you have a hypothesis and you think it is an incredible hypothesis and you do four experiments, two work, and two do not work.
Do not dismiss the two experiments that do not work to look for another two experiments that work, so you can say you have four experiments that support your hypothesis because there are two that did not, which means your hypothesis is null void. There is a lot of pressure in research today. The publish or die concept was around when I first started my career, and it is around now more than ever. And so, you know, you have to be very critical of your hypothesis and test it carefully so that you do not give in to that pressure.
Dr. Gow: Thank you, this has been very informative and thank you for all you do for the field.
Dr. Ignarro: You are welcome.
