CME Slides Forum - S. Lamas

A joint Congress by ERA-EDTA and ISN

CLASSICAL AND NON CLASSICAL NITRIC OXIDE SIGNALING IN VASCULAR DISEASE

Santiago Lamas, Madrid, Spain

Chair: Francesca Mallamaci, Reggio Calabria, Italy

Toshio Miyata, Sendai, Japan

lamas

Dr S. Lamas
Centro Nacional de Investigaciones Cardiovasculares
Consejo Superior de Investigaciones Científicas
Madrid, Spain

Doctor Miata, Doctor Mallamaci, Ladies and Gentlemen, Dear friends, good afternoon to everybody. I think we were told that we only have about 15 minutes, so I think I’m going to need to rush a part of my presentation but I’ll be pleased to answer any questions if it isn’t clear. Basically I’m going to talk about classical and non-classical nitric oxide signalling in endothelial cells as a sort of introduction to the session which is endothelial dysfunction which will be delivered in other talks.

The field of nitric oxide in the vascular wall all started with this classic experiment by Bob Furchgott who showed that in an intact ring there is relaxation with acetylcholine, whereas in a ring where there is no endothelium we do not find relaxation. Therefore, he posed the theory of endothelium derived relaxing factor which was shown to be nitric oxide.

We now know that nitric oxide is involved in neurotransmission, is an immune effector also and is a vasorelaxing substance involved not only in regulating vascular tone but also angiogenesis and platelet aggregation. Basically this is the scheme by which nitric oxide works.

We have the nitric oxide synthase, the endothelial nitric oxide synthase. There are also claimed nitric oxide synthase in the mitochondria. NO is generated and stimulates sGC in the smooth muscle cell which generates cGMP.

The classical NO activation in smooth muscle is more complicated because once cGMP is generated, it’s able to stimulate here the cGMP-dependent kinases which exert different effects. Basically the most important effects are related to the regulation of the L-type calcium channels through the hyperpolarisation of the potassium channels. This involves that there’s a final decrement of intracellular calcium and this contributes to the uncoupling of actin/myosin proteins and therefore, to the relaxation of the vascular smooth muscle.

But we may find other non-classical signalling which is related basically to the interaction of NO with other free radicals or to the action of NO itself. NO may interact with superoxide and generate peroxynitrite which may lead to post-translational modifications, the best known of which is tyrosine nitration. NO may lead to the formation of nitrosothiols leading to S-nitrosylation and of course, the reactive oxygen intermediates and also peroxynitrite may contribute to oxidation. I’m also going to mention later another post-translational modification.

The whole concept then is that of nitroxidative stress and the current definition, which is accepted, is the altered production of reactive oxygen and nitrogen species and impairment of cellular antioxidant defences leading to a destruction of redox signalling and molecular damage.

Basically in endothelial cells we may find several sources for the production of superoxide anion, including stimuli such as the shear stress, hypercholesterolemia, several growth factors and the oxidised lipoproteins. The two major sources for the production of a superoxide anion are NADPH oxidase, then eNOS which are expressed in the endothelial cells and mitochondria but there are also other enzymatic systems such as xanthine oxidase or the NO synthase itself which needs an uncoupled state which may lead to the generation of superoxide which when combined again with NO may lead to the production of peroxynitrite.

So here’s the basic picture, the most important message I would like to convey to the audience. On the upper part we have the classical NO signalling which is basically sGC-dependent. Some investigators claim that also because of the capacity of NO to interact with cytochrome C oxidase, CcOx inside the mitochondria this contributes to regulate respiration at the mitochondrial level. However, we have in the lower part of the slide, like I said the capacity of NO to interact with other free radicals and generate post-translational modifications. The 3 most important of which are tyrosine nitration, S-nitrosylation and S-glutathionylation or S-thiolation. I’ll give you some examples of work done in our laboratory regarding tyrosine nitration and S-glutathionylation.

Basically the story of tyrosine nitration began with cyclosporine A many, many years ago which as you very well know is basically a calcineurin inhibitor which after binding to cyclophilin promotes inhibition of the NF-AT family of transcription factors. Then they don’t translocate to the nucleus and they interfere with the immune response.

But there are vascular side effects relating to cyclosporine A the most important of which in the vascular field, are increased vascular resistance and this is fundamentally related to the presence of endothelial dysfunction. Some years ago it was claimed that maybe NO absolutely was decreased in the context of cyclosporine.

We and other laboratories showed that this was not the case. This is actually showing a Northern Blot in which the expression of endothelial NO synthase comes up when cells are treated with cyclosporine and this is due to transcription and regulation as shown here in this experiment with nuclear extracts of endothelial cells.

We were able to show that when you expose endothelial cells to cyclosporine A it induces tyrosine nitration as shown here with an antibody and this is due to the fact that in the short-term both NO and superoxide anion are formed inside endothelial cells and this leads to the production of peroxynitrite and to tyrosine nitration.

Actually when you give N-acetylcysteine you are able to prevent endothelial toxicity, as shown here and measured by 2 different techniques --- and cellular survival of endothelial cells which when we give this anti-oxidant, then the decrease in survival of the cells is prevented.

Interestingly also when we used cyclosporine A in an animal model in which we administered cyclosporine A to mice we showed increased tyrosine nitration in the vascular wall of the mice which was also prevented by the concomitant use of N-acetylcysteine.

Lately we have devoted some efforts to clarifying which were the sources of production of superoxide inside endothelial cells regarding the use of cyclosporine A. Basically there are two main sources for the production of superoxide which are complex I and complex III inside the mitochondria and we did some experiments, some of them I’m not going to show.

The most important is that we did direct and specific determination of superoxide inside endothelial cells using this technique of spin trapping which is exquisitely specific for superoxide anion. Therefore, we have a free radical which in the vicinity of a spin trap forms a stable free radical or a spin adduct which we can measure using electrospin resonance.

In the presence of cyclosporine A this is a positive control with DMNQ we observed the signalling endothelial cells which is clearly specific for hydroxyl anion or for superoxide anion. We discarded the possibility that it was hydroxyl anion and we used tiron which suppresses this specific signal for superoxide anion.

This is basically the detection of specific mitosox fluorescent probe inside endothelial cells and we observed an increased presence of superoxide anion when we used these mitosox fluorescent probes.

When we suppress functional mitochondria in endothelial cells, basically what we see is that there is a difference between these black bars in which endothelial cells have no functional mitochondria compared to these wild type cells in which they still can respond to cyclosporine A.

So this means that the major source of superoxide anion inside endothelial cells is probably mitochondria in relationship to the use of cyclosporine A.

So what we tried to do is to establish like a target for this tyrosine nitration in endothelial cells and we looked for this tyrosine nitration. It has been known for many years that mitochondrial MnSOD, superoxide dismutase is an important biochemical target for tyrosine nitration because nitration inactivates MnSOD very exquisitely due to its interaction with a tyrosine inside its catalytic centre.

Basically what we did is some studies using cross-immunoprecipitation in the presence of cyclosporine A with an antibody directed for MnSOD or for an anti-nitrotyrosine and we observed that in the presence of cyclosporine A there was increased detection of MnSOD nitration both using MnSOD or the anti-nitrotyrosine nitration here.

So we looked for functional consequences of this situation and we observed that both aconitase and manganese superoxide dismutase were clearly diminished concerning its activity in the presence of cyclosporine A.

We finally moved to study which would be the residue inside MnSOD which would be the specific target for cyclosporine A. Tyrosine 34 had been shown to be specifically targeted in the context of nitration. So we did some experiments specifically looking for the nitration of tyrosine 34 in MnSOD and we used mass spectrometry analysis.

We observed, this is our positive control, this is Sin-1, this is vehicle, this is cyclosporine A that in the presence of both our positive controls and with cyclosporine A we got two nice peaks which identified tyrosine nitration in the recombinant protein MnSOD.

We also overexpressed the protein inside BAEC and this is the overexpression using an adenoviral constructs

and again, we were able to see that the nitrated peptide for the MnSOD was present in the cyclosporine A treated samples.

So in summary for this part of the talk and I think the second part of the talk I’m going to really briefly go through it because of time constraints, we showed that in cyclosporine A treated endothelial cells an increase in the intracellular formation of superoxide was detected. That this produces an antioxidant sensitive nitration of tyrosine which is a marker for endothelial damage by nitrosative stress. Superoxide is produced at the levels of the mitochondria complexes I and II. Treatment with cyclosporine A may lead to the nitration of specific proteins, such as MnSOD. Peroxynitrite and tyrosine nitration may represent mechanisms of damage in pathophysiological situations where superoxide anion generation is increased.

I’m just going to briefly comment on this other modification which is S-glutathionylation, we have done work with it recently regarding specifically endothelin-1.

What is S- glutathionylation? It’s basically the formation of a mixed disulfide oxidised with a glutathione that reacts with a --- protein and it forms this mixed disulfide and this is able to regulate many enzymatic systems.

In the vascular wall we’ve been interested for some years in the physiology of enodhtelin-1 which is not only a vasoconstrictor but it’s also a regulator of fibrosis.
Now it’s also important when we mention endothelin-1 not to think only about it in terms of its vasoconstrictor capacity but also in terms of its profibrotic ability.

and we were able to characterise the specific three prime UTR elements inside the endothelin-1 gene which were important for this stabilisation.

Recently we have looked at the proteins which bind to RNA to the 3 prime UTR regions of endothelin-1 and we showed that GAPDH was one of the proteins.

Initially we thought this was an artefact but now we are fully convinced due to some kinetic studies that we have done that GAPDH binds to this RNA.

Interestingly enough when in endothelial cells we silence GAPDH, we observe that there is increased endothelin-1 gene and that there is increased endothelin-1 mRNA and that there is increased endothelin-1 peptide.

The part that I would like to tell you about concerns the fact that GAPDH can also be regulated by the redox signalling.

It has been shown to become both denytrosylated and S-thiolated. In fact it has a sustained residue which is important for its redox binding.

We did some studies specifically concerning this sustained within GAPDH and we showed that the oxidant agents, the oxidised glutathione and S-nitrosoglutathione were able to inhibit GAPDH binding to mRNA not having effects in the mutant when the cysteine had been mutated for this serine.

Interestingly these are basically all studies that I’m not going to go into detail because of shortage of time but let me show you here how endothelin-1 mRNA is increased in the presence of hydrogen peroxide when this only occurs when we have an intact 3 prime UTR region.

We showed that the half-lives of values of mRNA were increased in the presence of pro-oxidant agents only with an intact 3 prime UTR region.

We came up with this model and this is a basically the last part of my talk in which we showed that native GAPDH is able probably to interact with the 3 prime UTR region of the endothelin-1 mRNA and this leads to accelerated degradation. However, when GAPDH becomes thiolated, modified in this cysteine its capacity to bind GAPDH is limited and then endothelin-1 mRNA is stabilised.

So in summary I would like also to convey the message that S-glutathionylation is a relevant post-translational modification with potential functional consequences. Exogenous or endogenous NO may promote S-glutathionylation of proteins. This can be due to either a direct reaction with S-nitrosoglutathione or denitrosylation by reduced GSH.
S-glutathionylation of GPADH may represent a novel mechanism regulating mRNA stability and degradation.

This is my current group. I also want to mention my special thanks for all the mass spectrometry studies to Jesus Vazquez at Fisiopatologia molecular and thank you very much for your attention.

Question: Massy from France. We have learnt from some old data that peroxynitrite can be metabolised to be nitrosothiol is this is one way cells can detoxify this peroxynitrite? Sometimes if you look at the level of peroxinitrate going tyrosine you find there’s no elevation but in another way you have an increase of nitrosothiol. In your model did you look at this change in peroxynitrite and nitrosothiol to see if they are balanced between the two of them?

Dr. Lamas: Well, what you say is possible but you have to take into account these are all studies in endothelial cells and I think that there is a balance in the fate of NO inside the cell in the sense that it very much depends if it’s going to encounter another free radical or not and also about the microenvironment. The formation of nitrosothiols may take place within limited and reduced environment because NO is not that able to react with cysteines they need a specific -- and it also has a limited diffusion. The other point is that when it encounters superoxide for example, peroxynitrite may be formed and then it can lead to tyrosine nitration. The idea of the balance you proposed may be true but with endothelial cells I think we still to do much more work.

Question: Do angiotensin receptor blockers, known to interfere with oxidative stress, have some impact on the modification of the tyrosine nitration in vivo?

Dr. Lamas: Yes in people. I think it has been shown in some models by the group of Richard Coen in Boston that actually in some angiotensin II related vascular toxicity there’s tyrosine nitration but I’m not sure if blocking the angiotensin II has shown to decrease that tyrosine nitration.

Question: Among the current available medical agents what are the most effective drugs?