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Thomas Unger, M.D., Ph.D. Professor Unger attended Medical Schools at the Universities of Munich, Leeds, and Heidelberg, and graduated from the Medical Faculty of the University of Heidelberg in 1975. In 1976-78 he worked as a Research Fellow at the Clinical Institute of Montreal at McGill University. He then came to the University of Heidelberg, where he became a Professor of Pharmacology in 1986, and Professor of Experimental Hypertension Research in 1990. Since 1994 he is the Director of the Institute of Pharmacology at the University of Kiel. He is a member of various Societies such as the German Institute for High Blood Pressure Research (Chairman since 1994), the German Hypertension League, the International Society of Hypertension, and the European Society of Hypertension. |
Introduction of Dr. Thomas Unger
Dr. Weber: We are now going to start talking about the angiotensin II receptors, their signaling pathways and getting into the issues of cell growth and cell differentiation. It's a pleasure to invite Thomas Unger, who is the Chairman of the German Institute for Hypertension Research and is also Director of the Institute for Pharmacology at the Christian Albrecht University in Kiel. Dr. Unger has been a major leader in this field, and it's a real pleasure to have him join us this morning. Welcome.
AII Receptors: Signalling Pathways, Cell Growth and Differentiation
Dr. Thomas Unger
28.8 modem
14.4 modemReal Audio recording of Dr. Unger's Presentation
Introductory remarks
Dear Michael, ladies and gentlemen, it is a pleasure to be here and to be invited to New York, of course. The first time I have my two daughters with me--the first time here in the United States. And we can spend the rest of the week, I hope, to see what can be bought and not in terms of Levi jeans and so on.
Unfortunately, I can not invite you to Keil or any other place in Germany for the next ISH meeting. Some of you may remember, ten years ago, exactly ten years ago we had the same meeting in Heidelberg. It was in '86. We were 3,000 people there, which was a little bit too large for a little university town like Heidelberg, but we managed somehow. So I'm convinced that Glasgow, being a bigger city, is able to provide a very, very nice European-style meeting; and, of course, you are all welcome to Europe.
Short and long-term effects of angiotensin
My task today is to get you more involved into the basics of angiotensin receptors. So let's start with a slide which is just a variation on the theme that has already been played on by John Reid in his introductory lecture. It is that angiotensin, of course, has a lot of actions. These actions can be divided into short-term and long-term actions. The hypertrophy which occurs in vessels may also occur in the heart, leading to left ventricular hypertrophy. Angiotensin may be a player in these pathophysiological games. Of course, if we treat patients with any blocker of the renin angiotensin system, we always hope that we not only lower blood pressure but that we specifically interfere with these pathological events which are risk factors by themselves.
Angiotensin receptor subtypes
Until 1989, actually, those of us working in the field--we all thought that all these divergent actions of angiotensin would be brought about by one simple receptor, which was unusual if you think of so many different receptor- mediated effects. Think of serotonin or muscarinic receptors or alpha beta receptors. They all have subtypes. But the literature was unifying this concept to one receptor model until '89. Then those compounds came. You have already heard about losartan and this compound--PD123177. They were the first ones, non-peptidergic compounds to interfere specifically with what were then called subtypes of the angiotensin receptor. With these compounds, with these tools, it was able to clearly distinguish between at least two receptor subtypes, the angiotensin AT1 and the AT2 receptor. And you can see how selective these compounds are. In the middle you have angiotensin peptides, ang I, ang II, and ang III. They are not selective. They bind with the same affinity to the AT1 and the AT2 receptor. And so does saralasin, a peptide analogous to angiotensin which was developed in the '70s and which is certainly a partial antagonist under certain circumstances. But disappointingly, this compound was not able to do the job of angiotensin blockade under all circumstances. Now, losartan and its active metabolite, E3174, are actually able to selectively block the AT1 receptor, as you can see here. Two compounds, CGP42112A and PD123177, on the other hand selectively block the AT2 receptor. The difference between the two groups of compounds is large enough to give you a highly specific blockade of one or the other of these two subtypes.
At that point we came to the concept of having two subtypes: the AT1 and the AT2. In addition to blockade by the compounds which we have mentioned already, others could show that they were DDT sensitive concerning the AT1 and DDT resistant concerning the AT2 receptors. So there were means to distinguish now between these two receptor subtypes. Of course, with subtypes the situation becomes more complicated. Today we speak not only about the AT1 and the AT2, but we speak already about unknown subtypes and the AT4 subtype and so on. Probably we will find a few more of these subtypes as we did with, for instance, the serotonin receptors and so on. But we can concentrate for the purpose of today on the AT2 and on the AT1 receptor.
AT1 receptor and its actions
The AT1 receptor, as you can see here, is the one which mediates all the classical effects of angiotensin II that have already been described by John Reid in his lecture: vasoconstriction, aldosterone release, and other known effects of angiotensin, for instance the release of vasopressin and also the hypertrophic actions in the vasculature in the heart and in the kidney. The AT2 receptor, until now, is much less known with respect to its function.
What type of receptors do we encounter when we talk about angiotensin AT1 and AT2? This picture just gives you an idea of different families of receptors which we know today. On the left-hand side, you see this snake-like receptor, which goes seven times through the membrane and is called a 7-transmembrane-domain-receptor type. Usually it's G protein coupled, and the G protein then mediates further signaling in the cell. But you see there are also other cell receptor types, for instance the tyrosine kinase receptor, which is associated with growth factors. Then there is a very interesting receptor which is coupled to a guanylate cyclase and increases cyclic GMP production, and other receptors, such as the LDL receptor, merely transport receptors--they take the agonists, transport it into the cell, and then the agonist together with the receptor can do its job, whatever it is.
Now, the angiotensin receptors AT1 and AT2 belong to this group here, to the G protein coupled 7-transmembrane- domain receptors; although with respect to the AT2, the coupling to the G protein is still a bit controversial. And this is what the AT1 receptor looks like: It goes 7 times through the membrane, has a carboxyl terminal inside and an N-terminal outside, as you would expect from a protein receptor. Angiotensin actually binds to the outer domains 1, 3, and 7 of this receptor and possibly also to domain 5. So it has specific binding sites. Obviously, between the AT1 and the AT2 receptor, although they don't share much homology, the sites where angiotensin binds with the same affinity nned to have similar features. But the other sites inside the membrane, where the antagonists and the specific binding compounds bind, are quite different between AT1 and AT2.
Intracellular actions of the AT1 receptor
Now if you stimulate an AT1 receptor, a very complicated cascade of events occurs. This is just a very simplified picture, although you may think it is already complicated enough, and it is indeed. But what it means is that basically you get an increase of intracellular calcium. The calcium may come from intracellular stores or it may also come from the extracellular space and be directed into the cell through gates, through voltage regulated calcium channels, for instance, here, and other channels into the cell. This pathway involves G proteins, stimulatory G proteins in this case, and it involves PLC (phospholipase C) and also phosphokinase C -- those enzymes stimulate IP3 metabolism or changes in transmembrane electrolyte transport. And finally, you will end up with an increase of calcium in the cell. That is the common pathway.
There are other pathways that are engaged by angiotensin and that have been disclosed in recent years, for instance the Jak STAT 2 pathway, but I'm not going into details here.
Calcium, phosphorylation, vasoconstriction, and growth
Now if calcium then is stimulated in the cell, it can do a lot of things as you are probably aware of. It can, for instance, lead to vasoconstriction if the cell, like a smooth muscle cell of the vessel, has a contractile apparatus and can respond to calcium increase with vasoconstriction. If the cell does not have this apparatus, it may respond with something else, for instance with the stimulation of certain enzymes. Many cells including smooth muscle cells also have the ability to respond to calcium increase with cell growth and with the stimulation of growth regulatory genes. So you can have both. You can have the contraction, the short-term effect of angiotensin on one hand; and you may have the stimulation or modulation of growth, the long-term effect on the other hand. And through calcium in some other parts of the pathway, they may be connected with each other.
Is this really the case? Just an example how we can envision this event. This part you have already seen and heard about. now we have calcium stimulation, we have a phosphorylation of proteins through several kinases. And these can feedback to the nucleus of the cell. In this nucleus, they may act on the DNA to increase the expression of growth factors. These intermediate growth factors could be, for instance, the classical proto oncogenes or immediate early genes . C fos, c myc, and c jun are just examples of this growing family. Those proto oncogenes may then be transformed to proteins. They may form dimers. One example is given here. But that is only one example. They may feedback to the DNA again in the nucleus and find a sensitive element in the promotor area of the DNA of growth factors. For instance, here the AP1 area. And if they interact, they can stimulate the expression of growth factor proteins. This eventually may lead to growth of the cells. So we have a pathway which involves the receptor, which involves the stimulation of calcium and the phosphorylation of proteins, and finally the generation of growth factors and growth of the cells.
Angiotensin and growth of vascular smooth muscle cells
Now can this actually happen? Yes, this is just one example from our lab, but this type of experiment has been done early, for instance Allan Naftilan, while in Victor Dzau's lab. What this tells you is that angiotensin can indeed induce c fos in vascular smooth muscle cells, and this is a time-dependent phenomenon. Does this actually lead to growth? Yes. Again, an experiment from our lab which just confirms what had been done before by others shows you that angiotensin can increase the thymidine incorporation in quiescent vascular smooth muscle cells. And just to demonstrate that this is an AT1 effect, you can block this effect with losartan, as you can see here. It goes back to quiescent levels. Interestingly, this effect of angiotensin, which is mediated by the AT1 receptor is not always present. Bovine aortic endothelial cells, for instance, do not respond to angiotensin with this growth response. And, of course, then this can also not be blocked by the AT1 blocker losartan. So it really depends upon what kind of receptor is present on these cells.
AT2 receptor and its actions
Now coming to the AT2 receptor. On first glance this AT2 receptor looks very similar to the AT1. And indeed, as I said in the beginning, it is also a 7-transmembrane domain receptor although it has only 33 to 34 percent homology to the AT1. But obviously those domains where angiotensin binds extracellularly they have to be similar so that angiotensin can bind with the same affinity, but inside there is quite a difference between AT1 and AT2. This AT2 receptor is still an enigma for all of us working with it. We know that this receptor is expressed in embryonic and fetal tissue. It is markedly expressed there, over expressed if you want. In some instances much more than the AT1 receptor. But after birth, we all lose this receptor in some tissues, and in other tissues we have very little left. The AT1 receptor is the one which dominates then. The AT2 receptor may come up again in wound healing and tissue repair, for instance after myocardial infarction or after intima lesions of vessels. It may have a role, at least some people think it has, in angiogenesis, and it may also play a role in differentiation of cells, We have evidence in favor and some evidence against G coupling of this receptor subtype.
AT2 receptor signalling mechanisms
Its mechanism of signaling--there is a lot that has been published in the literature, but still we don't really know what the final mechanism is. Maybe this receptor engages several mechanisms and not only one. And, of course, you can put that together. This is from a recent review paper from Clara Nahmias, Paris, published in TIPS last year. It just shows you that signaling mechanisms reaching from T-type calcium channels down to phosphotyrosine phosphatase stimulation and inhibition have been implicated in the signaling pathways of the AT2 receptor. And, of course, this depends very much on the system in which those investigators have worked and on the cells they have used for their experiments. This is also reflected in a long list of possible effects that may be mediated by the AT2 receptor, starting from depressor responses to angiotensin II that some people have claimed, which I think is rather unlikely, and going to interference with cognitive function or central pressor actions and so on. Many, many effects have been attributed to the AT2 receptor by using specific ligands or antagonists. I have to say there is also still a lot of confusion about the action of these ligands or antagonists though they are very specific for the AT2 receptor. On this coming Sunday we will have here in New York a workshop meeting organized by Victor Dzau and myself, on the AT2 receptor. This workshop, I have to say, was generously helped and funded by Merck. In this workshop we want to clarify some of the effects of the AT2 receptor and come to a more comprehensive picture.
Anti-proliferative effects of AT2 receptors
Well, we have worked with the AT2 receptor, too, and shown that something else may even happen with this receptor, and that is that it may mediate anti- proliferation as opposed to the AT1 receptor, which of course you have seen causes proliferation, at least in some cell types. And this experiment shows you two things. In endothelial cells from the rat (microvascular endothelial cells) angiotensin reduces growth induced by the growth factors such as bFGF. We wanted to find out also which receptor subtype was involved. We blocked this effect with PD123, the AT2 antagonist; but we were unable to block the effect with losartan. This demonstrated to us that obviously angiotensin through the AT2 was able to block this proliferation in endothelial cells. We did not see this effect in vascular smooth muscle cells. But no wonder, because at least in culture, these VSMC do not harbor the AT2 receptor, only the AT1. But endothelial cells have both receptors on board. So there is an effect which is completely different from what we have learned about the AT1 receptor. And also, one other effect which is very interesting which we observed.
Remember that on one slide we had the word "differentiation". These are PC-12 cells, a cell line which is derived from rat pheochromocytoma cells. This is a cell line and it has all the benefits and all the disadvantages of working with cell lines, but it has only the AT2 receptor and no AT1 receptors on board. That's why we used it. We looked through the phase contrast microscope at these cells and we could see that under the influence of angiotensin, they started to differentiate, as you can see by this growing of neurites of these cells. Just to show you that this differentiation of these PC12 cells is an AT2 receptor mediated effect--if you pretreat the cells with an AT2 blocker, PD123177, you don't have this differentiating effect any more in response to angiotensin II... that is the AT2 receptor.
Angiotensin and AT1-AT2 receptor actions
What is actually the mechanism by which angiotensin acts through AT1 and AT2? And how can they actually interact? Well, that's a very complicated story. Just one possibility, one attempt to make that clear. If we have the AT1 receptor and the AT2 receptor on cells, the AT1 is of course the one which you have seen which produces growth or hypertrophy through several mechanisms, for instance the protein kinase C mediated mechanism, the jak STAT mechanism, and other mechanisms going through MEK kinases. So finally they end up with immediate early genes that you have seen on one of the slides and with growth factors which are stimulated to induce growth. That is what we know and what several labs in the world have shown. Now if for instance the AT2 receptor would indeed stimulate a phosphotyrosine phosphatase, then this phosphotyrosine phosphatase could dephosphorylate at various sites where growth factors may phosphorylate. And it could actually stop at those levels but could also stop through an unknown mechanism at this level and even down there at the molecular level, here, and have nuclear targets to interfere with. For instance, we have recently shown that the AT2 receptor can indeed stimulate the DNA for factors which initiate differentiation of cells and which also may inhibit the growth of cells. So the message of this slide is that the interactions can occur at various levels down to the nucleus and can even have some bearing on apoptosis, or programmed cell death. I hope Victor Dzau will give us some further clues about that work this issue.
Summary
Taking these things together, we have at the present time two major receptor subtypes for angiotensin: AT1 and AT2. They are distributed in a differential fashion in the organism. The AT1 receptor is distributed in the adult vasculature, in the kidney, in the adrenal gland, in the heart, in the liver, and in the brain. The AT2 receptor is distributed rather, but not exclusively, in fetal tissues. But it can also be seen in adult brain, in adrenal gland, in the ovary, in the uterus, and in endothelial cells, I should add. Actually this slide can be written in a different fashion every month. It can also be shown, for instance, in the heart. A recent paper shows that angiotensin AT2 receptors dominate the AT1 receptor in the failing human heart. So under pathophysiological conditions, this receptor may come and go in different organs.
The function of the AT1 receptor is, among others, immediate vasoconstriction. At1 has to do also with cardiac contractility with aldosterone release, glomerulofiltration, renal blood flow and vascular and cardiac hypertrophy. The AT2 receptor may inhibit cell proliferation and may have a role in growth and development, as you've seen, and differentiation. The structure of the AT1--it's a 7-transmembrane receptor, and it's certainly G protein coupled. The AT2 receptor is also 7-transmembrane, but may or may not be G protein coupled. The ligand specificity of compounds binding to the AT1 and to the AT2 receptors is very high. We have isoforms of the AT1 receptor in rats and mice but not in man. For the AT2 receptor, we don't have isoforms so far.
That brings me to the end. I hope I could give you some insight into the story of the AT1 versus AT2 and show you that we still have a lot to learn with respect to both receptors. Thank you very much.QUESTIONS AND DISCUSSION Dr. Weber:
Thank you, Thomas. That was just outstanding. We have a few seconds for one or two quick questions. Thomas, if I were a pharmaceutical company, should I be investing in developing an AT2 agonist?
Dr. Unger:
Well, this is not the first time that I've been asked this question. I am glad you are not the company and you don't have to make this decision. I think it's not necessary because if you treat with losartan, you block, of course, directly the AT1 receptors. We all know that. On the other hand and for reasons of time I didn't show that slide, you may have, indirectly, a stimulation of the AT2 receptor due to the increased angiotensin levels under blockade of AT1, because you lose this negative feedback in the kidney on renin release which is AT1 mediated. So you have increased levels of angiotensin and you have the AT2 receptor unopposed. So if the At2 receptor happens to be present, then you may have in one drug a combined direct AT1 blocker plus an indirect AT2 agonist.
Dr. Weber:
We'll hear a little later from Dick de Zeeuw about some of the experience looking at the effects of blocking the renin angiotensin system within the kidney. Do you think there is some special role of the AT2 receptor in renal function which may differentiate drugs such as losartan from the ACE inhibitors?
Dr. Unger:
Well, this is something which I hope we can clarify in the next months or so, at least a little bit on Sunday. There are specific renal actions that have been described with respect to the AT2, for instance, by the group of Jean Sassard in France, but I think it is too early to ascribe specific functions of angiotensin to the AT2 receptor in the kidney.
Dr. Weber:
Thank you, Thomas. I really appreciate your excellent presentation.
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