Discussion and perspectives

The three different methodologies used during my thesis, Ca²⁺-imaging, single channel and whole cell, have allowed a better understanding of the Ca²⁺ entry pathways in EC. Thanks to the Ca²⁺-imaging, we obtained good evidence that RACE and SOCE are different routes for Ca²⁺ entry, but due to lack of specific inhibitor, they were difficult to differentiate using this approach. The patch clamp in single channel configuration has allowed us to record and characterize a non-specific cationic channel, activated by Ca²⁺ and stimulated directly after the addition of histamine but not by passive depletion of the stores. A second and different channel was activated by histamine, but with a delay of approximately three minutes and a full characterization could not be done. Indeed, the expression level of channels was weak and variable during time, making this approach too restrictive. The whole cell recording was the technique which has enabled us to differentiate the RACE from the SOCE currents. SOCE current in EA.hy926 cells has almost all the electrophysiological characteristics of the ICRAC, except the Ca²⁺ selectivity. So SOCE current on these cells is an ICRAC-like. The IRACE recorded after stimulation with histamine corresponds to the INCX, showing that the exchanger has a role in the RACE pathway.

It was difficult to find the good technique and the good conditions to differentiate the two pathways. Indeed the pharmacological study with the Ca²⁺ imaging approach has confirmed the lack of specific inhibitors. Initially, the La³⁺ appeared to be a useful tool to discriminate between RACE and SOCE, because it fully blocked the SOCE without affecting the RACE. But it turned out that the lack of inhibitory effect on RACE was an artefact, due to the use of histamine containing phosphate. The phosphate formed a complex with La³⁺ that

0 250 500 750 1000 1250 1500 10µM of La³⁺ inhibits totally this Ca²⁺ entry. The stimulation of

precluded its inhibitory effect on RACE. The effect of phosphate on La³⁺ was clearly identified. Indeed, after the inhibition with La³⁺ of the Ca²⁺-entry activated by histamine-chloride, the addition of phosphate is sufficient to remove the inhibition (fig 1). After the discovery of this unfortunate artefact, the histamine used was the classical chloride salt.

It remained however one point that could not be solved so far. In our experiment in presence of high K⁺ (fig 7, Jousset et al. 2008) the addition of La³⁺ inhibited the Ca²⁺ entry even though histamine-phosphate was present. That was the only experimental conditions where La³⁺ had an effect in presence of phosphate. We do not have explanation but it might be that the high K⁺ has an effect on the chelator power of the phosphate, but this point remains to be clarified.

The patch clamp technique in single channel configuration was performed in parallel to the study in Ca²⁺ imaging. The main problem with this approach was the difference of channel expression over time. The channel A was quickly identified as a channel activated by histamine and Ca²⁺ but not by TG. But this channel has not been fully characterized. Indeed, while it has been recorded about twenty times in a very short period (~ 3 months), it was almost absent from the recording in the following months. It is known that the channels expression may change in culture conditions and during serial passage of cultured cells.

Moreover, expression pattern from cultured cell lines does not always reflect the in vivo expression of channels in native tissue (Yao and Garland 2005); (Garcia and Schilling 1997).

So, it would be wise to repeat this approach on freshly harvested HUVEC.

The patch clamp in whole cell configuration was the best approach to differentiate the RACE from the SOCE. But it took us a rather long time until we found the good experimental conditions. The difficulty was to avoid a depletion of the ER. Indeed, the Ca²⁺ imaging approach showed us in the first publication that the addition of histamine in presence of 2mM Ca²⁺ in the bath leads to a very low depletion of the stores (fig. 6, Jousset et al. 2008). We thought that in our electrophysiological experiments as in Ca²⁺ imaging, the presence of 2mM Ca²⁺ in the bath (with the 10mM Ba²⁺) would be sufficient to avoid the depletion of the ER upon histamine stimulation. But the clamp of the voltage at 0mV between two voltage ramps together with the inhibitory effect of Ba²⁺ on Ca²⁺ entry pathway activated by histamine leads to a greater depletion of the ER than in the conditions used in Ca²⁺ imaging (fig 3, manuscript). This shows that it is crucial to pay attention to the experimental conditions used.

The involvement of NCX in the RACE has been shown electrophysiologically. We wanted to confirm this implication by Ca²⁺ imaging, first by using the known inhibitors KB-R7943 and Ni²⁺ (fig. 4, manuscript). In addition, we also used siRNA against NCX to

demonstrate its implication. The first step was to determine the NCX isoforms expressed in EA.hy926. A RT-PCR was performed and has revealed the presence of NCX1 and NCX3 but not NCX2. While the expression of NCX1 is strong, NCX3 is weakly expressed. The use of siRNA against the NCX1 or NCX3 has no visible impact on the RACE or the SOCE pathways. But various problems arise. To quantify the downregulation of the exchanger we performed a real time PCR. Whereas the downregulation of around 80% of the NCX1 mRNA was proved with this method, the presence of the NCX3 mRNA in control or in downregulated conditions turned out to be difficult. We could not verify the silencing of NCX3 with the quantitative PCR, maybe due to its low basal expression level. Thus, while the silencing of NCX1 has no visible effect on the Ca²⁺ entry after agonist activation or the depletion of the stores, an interpretation of the Ca²⁺ imaging experiments after downregulation of NCX3 was impossible. It would be worthwhile to solve this problem in order to assess the real impact of the NCX3 downregulation on the Ca²⁺ entry after histamine activation. The western blot technique with specific antibody is probably the best approach to estimate the real expression level of the protein, but it might be also difficult with low protein level.

A possible compensatory system used by the cell may be another explanation for the lack of visible inhibition when NCX is downregulated. The downregulation of NCX could lead to the upregulation of other proteins such as TRP channels, STIM or ORAI. So it will be essential to check the expression level of the other potential channels involved in RACE pathway when NCX is silenced. Furthermore, Ca²⁺ entry after histamine activation is probably due to the simultaneous involvement of different processes. The block of one player is compensated possibly by the activation of other channels involved in RACE such as TRPC members or ORAI1. We have also seen with the patch clamp technique that the inhibition with Ba²⁺ of the NCX current and the clamp of cells at 0mV lead to the depletion of the stores and to the SOCE current activation (fig. 1, manuscript). So the downregulation of NCX possibly depletes the ER sufficiently to activate the SOCE channels. Thus, it is possible that the downregulation of NCX has an effect but that this one is simply not visible in Ca²⁺

imaging.

Our experiments point to an involvement of NCX in RACE, but its mode of activation remains unknown. We have two different hypothesis. The first hypothesis, as it has been shown in platelets, HEK293 cells and smooth muscle cells, implies a local Na⁺ loading through a NSCC. This Na⁺ increase near the NCX reverses its mode, leading to the entry of Ca²⁺ (Harper and Sage 2007); (Rosker et al. 2004); (Poburko et al. 2007); (Lemos et al.

2007). This mode of activation requires a restricted environment close to the NSCC and the NCX in order to have a sufficient increase in the concentration of Na⁺ near the NCX to reverse its mode. We have not recorded any other current activated by histamine in our patch clamp experiments than the INCX. The explanation is maybe that this current is too small to be recorded, or that the outwardly rectifying current activated by histamine is a mix of currents due to the NCX and due to the NSCC responsible for the Na⁺ loading. This NSCC could be a member of the TRP family as shown in platelets, HEK293 cells or smooth muscle cells (Harper and Sage 2007); (Rosker et al. 2004); (Poburko et al. 2007); (Lemos et al. 2007). It will be important to reveal this Na⁺ entry, in order to confirm this hypothesis. Imaging with a fluorescent Na⁺ probe such as SBFI or CoroNa green would be a good method to assess this point (Poburko et al. 2007); (Harper and Sage 2007). The patch clamp method in whole cell configuration will be another possibility to test this hypothesis. Indeed with this approach we could test different concentrations of intracellular Na⁺ by increasing the Na⁺ concentration in the pipette. By simulating a Na⁺ loading, the reverse mode of the NCX should be activated.

The second hypothesis is the direct activation of the NCX in the reverse mode by a second messenger produced after histamine stimulation. This hypothesis would explain the absence of another visible current after histamine activation in our patch clamp experiments.

A number of factors are known to directly modulate the NCX, such as ATP (Baker and Glitsch 1973); (Dipolo 1974), phosphatidylserine, and some inositol phosphates including PIP2 (Collins and Hilgemann 1993); (Luciani et al. 1991) or cGMP (Furukawa et al. 1991).

PKC and PKA contribute also to the modulation of the NCX in a variety of tissues by phosphorylating and dephosphorylating the exchanger (Iwamoto et al. 1996) (for review see (Blaustein and Lederer 1999); (Lytton 2007)). So it is possible that a factor produced after histamine activation directly modulates the exchanger. To test a direct activation, the patch clamp in real whole cell configuration would allow a direct application in the cytosol of potential second messengers known to activate the NCX.

In parallel to the electrophysiological characterization of RACE and SOCE current, we also performed Ca²⁺ imaging experiments. We also initiated study regarding the molecular identity of RACE and SOCE. First we tested the involvement of STIM1 and ORAI1 in RACE and SOCE. The downregulation of one or other of these proteins has an impact on Ca²⁺ entry activated by agonist and by depletion. The other family of proteins potentially involved in Ca²⁺ entry is the TRP channels, especially the TRPC subfamily. Thus we also tested the involvement of the TRPC members expressed in EA.hy926 cells, which are TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6. The silencing of these TRPC has no effect on the histamine or

TG stimulation. The concentration of histamine used in these experiments was 100µM.

Fabrice Antigny, a post doc in the lab, continues this study but using a more physiological concentration of agonist, 1µM. His results show that the Ca²⁺ entry in EA.hy926 cells is difficult to resolve, because many proteins appear to be implicated in the different pathways.

Indeed, whereas the downregulation of TRPC6 has no effect on the histamine-activated or TG-activated Ca²⁺ entry, the TRPC3 silencing has an impact on both pathways. The downregulation of TRPC1, TRPC4, TRPC5 and STIM2 inhibits partially the RACE but has no effect on the SOCE. At this stage of the study, among the members of the TRP channels, only the TRPC expressed in EA.hy926 cells have been tested. We can already see that the Ca²⁺ entry in these cells is supported by a great diversity of proteins. But other TRP family members are good candidates to support RACE, as TRPM2, TRPM4 and TRPM5 that have been shown to be activated by intracellular Ca²⁺ (Kraft and Harteneck 2005). So there are possibly more channels with a role in RACE or SOCE pathway. The involvement of these different players, especially in RACE, shows that possibly agonist-induced Ca²⁺ influx is the result of several molecules simultaneously engaged. Moreover the cells have a compensatory system, when one of the players is silenced. For example, the downregulation of TRPC3 leads to a change in expression level of the other TRPC members. Hence, in addition to the involvement of different players, the modifications of TRP expression pattern could change the channel composition. The amount of STIM and ORAI mRNA when the TRPCs are silenced was not tested. But compensation is also possible by increasing expression of these two proteins. Thus even if the Ca²⁺ imaging is an excellent tool to study the Ca²⁺ entry, the complexity of the machinery engaged after an agonist activation and the lack of specific blockers make the understanding of Ca²⁺ entry very challenging.

This study has revealed that the channels involved in the RACE and the SOCE pathways are different, but both leads to an increase of cytosolic Ca²⁺. So, why the cells need different routes for Ca²⁺ entry?

The activation of cells with an agonist is followed simultaneously by the production of second messengers and the release of Ca²⁺ from ER via the opening of IP3 receptor. So the RACE and SOCE pathways are potentially activated together during a physiological response.

In these conditions it is difficult to evaluate the specific function of one or the other pathway.

The best known function of the SOCE is the refilling of the stores when they are depleted (Putney 1986); (Parekh and Putney 2005). A physiological activation does not cause a significant depletion of the ER, at least in our cellular system. In our experiments we used

100µM histamine and even at this high concentration of agonist the depletion was not important (Jousset et al. 2008). But it has been shown that the activation of SOC channels requires a significant depletion of the ER (Parekh et al. 1997); (Fierro and Parekh 2000);

(Lewis 2007); (Park et al. 2009). So in our cells the SOCE is possibly not activated during histamine stimulation, but would be activated only in emergencies to compensate a lower Ca²⁺ entry due to the inhibition of channels involved in RACE pathway. So we can speculate that in vivo, only pathologic conditions resulting in significant depletion of the ER would activate SOCE channels.

The refilling of the stores is not the only known function of SOCE pathway. In T lymphocytes, Ca²⁺ entry through CRAC channels plays also a significant role in gene regulation. Indeed, the elevated cytoplasmic Ca²⁺ due to the opening of CRAC channels binds to calmodulin and the complex formed activates the phosphatase calcineurin leading to the dephosphorylation of nuclear transcription factors such as NFțB and NF-AT (Dolmetsch et al. 1998). It has also been shown that SOCE could have a role in apoptosis. For example, in thymocytes the Ca²⁺ entry activated by TG is a signal for the activation of apoptosis (Jiang et al. 1994) (for review see (Parekh and Putney 2005)). But in normal physiologically relevant responses, evidences of a functional role of the SOCE is largely limited to hematopoetic cells (Zweifach and Lewis 1993); (Lewis and Cahalan 1989); (Hoth and Penner 1993).

The increase of cytosolic Ca²⁺ due to the RACE can be used to refill stores. We have seen that various channels or exchangers are involved in the histamine as well as in the TG-activated Ca²⁺ entry in Ea.hy926 and among them members of the TRPC family. An excellent review from Yao and Garland points on the involvement of TRP family members in various functions of the EC. The different TRP isoforms are implicated in different functional responses, due to the heterogeneity of the channel properties, such as the ion permeability and the Ca²⁺ sensitivity or the differential expression within different subcellular compartments (Yao and Garland 2005) or the different mode of activation. The channels or exchangers involved in SOCE and RACE pathways could generate a different Ca²⁺ signal in the cells, so that the activation of one or the other pathway could lead to different functional responses.

This aspect potentially could lead to new exciting findings in the field of EC physiology.

Ca²⁺ signal is crucial for cellular function in all types of cell, but maybe even more in EC that fulfills so many tasks. It is therefore possible that the cell has several pathways leading to Ca²⁺ entry so that in case one of them would be impaired, the other could take over.

Moreover various proteins are involved in each pathway, which is another ““safety”” mecanism.

Indeed, if an expression problem of one protein occurred, the others may have a compensatory role.

Our initial hypothesis was that at least two different pathways co-existed in EC: the RACE and the SOCE pathways, and that the channels supporting these influxes are molecularly distinct. Until recently, the assumption was that the SOCE implied STIM1 and ORAI1, while the RACE implies other channels and/or exchanger. Indeed, our experiments point to the existence of different molecule implicated in RACE and SOCE as shown by the different electrophysiological profiles of histamine versus TG-induced currents. In the recent literature, it was shown that the molecules involved in Ca²⁺ entry actually can interact with each other, such as STIM and ORAI forming trimers with some TRPC channels (Liao et al.

2009); (Wang et al. 2008); (Liao et al. 2008). Thus, the RACE and the SOCE might be more interconnected than are though, both at the level of the activation mechanism and on the molecules involved. That would certainly explained, at least in part, the difficulties we had to separate both types of Ca²⁺ influx in EC.

X. Remerciements

J’’exprime ma profonde reconnaissance au Dr. Maud Frieden. Tout d’’abord pour l’’excellente formation qu’’elle m’’a apportée, tout le savoir transmis avec pédagogie et patience ainsi que le suivi bienveillant durant ma thèse. Mais je tiens aussi à exprimer ma sincère gratitude pour sa compréhension et son soutien dans les périodes difficiles et les moments de doutes, que ce soit professionnels ou plus personnels. Je salue donc ses capacités scientifiques aussi bien qu’’humaines.

Le Professeur Demaurex a toute ma reconnaissance pour son soutien scientifique et ses conseils avisés sur mon projet. J’’ai aussi beaucoup apprécié sa disponibilité et son écoute.

Je tiens à remercier mon répondant en faculté des sciences, le professeur Jean-Louis Bény ainsi que le professeur Jean-Yves Chatton, pour avoir accepté de faire partie de mon jury de thèse.

Je remercie chaleureusement Cyril Castelbou pour son excellent soutien technique, mais surtout pour son amitié, son écoute et sa disponibilité.

Un grand merci à Fabrice Antigny, avec qui une entente scientifique et amicale s’’est installée dès son arrivée grâce à sa bonne humeur.

Merci à Hélène Jousset pour ses conseils scientifiques, son soutien indispensable, toutes les discussions chaleureuses et pour tous ces moments d’’amitié que nous avons partagés.

Mes collègues passés et présents ont été aussi d’’un grand soutien, sur le plan scientifique mais ils ont surtout contribué à la bonne ambiance générale. Je remercie donc Gabor, Serge, Damon, Antoine, Wei Wei, Samira et Paula. Un merci particulier à Yves, Umberto et Jaime qui partagent le bureau avec moi, pour tous les partages professionnels et amicaux.

Un grand merci aussi à Stéphane König, pour son aide scientifique précieuse mais aussi pour tous les moments de détentes et de « franches rigolades ».

Enfin, je souhaite remercier toutes ces belles rencontres faites durant ces quelques années, qui

Enfin, je souhaite remercier toutes ces belles rencontres faites durant ces quelques années, qui