The role of angiotensin IV and identification of its targets

Evidence is accumulating that Ang IV, a major metabolite of Ang II, exerts physiological effects in its own right. Its central actions are already well-known to include the increase of memory recall and learning in passive and conditional avoidance behavioural studies. Additionally, we also found that i.c.v. administered Ang IV protected rats against pilocarpine-induced seizures. In the periphery, in vivo as well as in vitro studies with vascular smooth muscle cells have also pointed at the propensity of Ang IV to affect the cardiovascular system.
The beneficial effects of Ang IV on memory and learning may herald new therapeutic strategies to help patients with cognitive deficits. In the same vein, the anticonvulsant properties of Ang IV could also lead to novel therapeutic applications. Central to these issues is the unequivocal identification of the cellular targets that are implicated in these beneficial effects of Ang IV as well as in its potential side-effects. During these last four years, we have made substantial progress in this respect (see part 2.) and we are now in a comfortable position to re-actualise the initial working hypotheses.
The initial proposal for the existence of "AT4 receptors" as cellular targets for Ang IV (DeGasparo et al., 2000) was based on radioligand binding studies, namely on the discovery of high affinity binding sites for radioiodinated Ang IV (and analogues) in the CNS as well as in peripheral tissues and cells thereof. Subsequently, Albiston et al. (2001) reported that these binding sites correspond to the insulin-regulated aminopeptidase (IRAP) enzyme. Our studies on native and recombinant cell lines confirmed the widespread occurrence of IRAP and also that it constitutes the major (if not the sole) high affinity binding site of Ang IV. While these binding studies represent a most helpful tool for the purpose of IRAP detection and quantification, we also clearly established that this high affinity binding only takes place to the apo-form (i.e. inactive, catalytic zinc-depleted form) of IRAP. We therefore believe that, unless apo- IRAP acts as a cellular receptor, Ang IV-based binding studies are inadequate screening tools for the detection of Ang IV-derivatives with potential therapeutic interest.
Nonetheless, we and others (Lew et al., 2003) have clearly established that Ang IV can also be recognised by the physiologically relevant (i.e. catalytically active) form of IRAP, albeit with ± 20-fold lower potency than the apo-enzyme. Yet, we have also become increasingly aware that Ang IV is rapidly degraded in vivo as well as in vitro and that some of its (patho)physiological effects could also involve its interaction with other cellular targets. While Ang IV produces a full, dose-dependent decrease of IRAP's catalytic activity, similar effects of Ang IV have now also been observed while studying the related aminopeptidases AP-N (Garreau et al., 1998) and AP-A (Goto et al., 2006). Moreover, as we found that some of the in vivo effects of Ang IV (e.g. on the hippocampal acetylcholine concentration and on renal blood flow) can be blocked by the AT1 receptor-selective antagonist candesartan, due attention should also be paid to the potential contribution of the by now "classical" AT1 and AT2- receptors.
The present project is based on two highly complementary approaches/strategies.
(i) The first is based on the now well documented role of IRAP as an Ang IV target and will further explore by which mechanisms the Ang IV-IRAP interaction is able to trigger physiologically relevant intra- as well as extracellular processes (outlined in parts 4.3 to 4.5). This approach will be intially focussed on intact-cell experiments and relevant outcomes will serve as a rationale (or tools) for dedicated in vivo tests.
(ii) Secondly and in parallel the investigations aim to acquire a better understanding of the physiological role of Ang IV in the CNS (outlined in part 4.6). This will be carried out in the spirit that IRAP may not be the (sole) physiological target for Ang IV. Here, in vivo research will first focus on comparative studies with wild-type vs. KO animals and with Ang IV versus its most interesting synthetic analogs.
Mechanism of action underlying the physiological effects of Ang IV via IRAP
So far, three potential mechanisms have been put forward to explain the role of IRAP in the increased cognitive performance after Ang IV administration (summarised in Chai et al., 2004). We aim to elucidate which of these mechanisms is involved and what is their relative contribution in the in vivo effects of Ang IV, with particular emphasis on those effects that were already disclosed in our previous work. This approach thus allows to to perform a critical evaluation of the (patho)physiological relevance and molecular peculiarities of each of these working hypotheses. As outlined in the individual descriptions hereunder, the outcome of these studies will have a profound impact on the screening strategies to be adopted in our quest for useful therapeutic drugs.
(i) The ability of Ang IV to inhibit the catalytic activity of IRAP has already been well documented in in vitro conditions and some of the IRAP substrates in those studies (somatostatin, vasopressin and oxytocin) are known for their memory-facilitating or anticonvulsant properties. According to this hypothesis, any compound that selectively inhibits the catalytic activity of IRAP could increase cognitive performance. In addition to the already obtained indirect evidence that Ang IV also prevents neuropeptide degradation in vivo, we wish to obtain direct/conclusive evidence for such mechanism. Therefore, we will monitor changes in various neuropeptide concentrations in hippocampal and striatal dialysates (microdialysis experiments in combination with LC-MS/MS detection) following the in vivo administration of Ang IV and synthetic analogues of interest to control and knock-out animals. Similar experments will also be done in vitro with cultured cells either under native or IRAP-overexpressing conditions.
(ii) As second hypothesis, we have been the first to propose that IRAP should be capable of triggering intracellular events on top of its enzymatic activity (Vauquelin et al., 2002). This hypothesis was based on the structural properties of IRAP (i.e. its occurrence as dimers at the cell surface and, hence, the presence of 2 membrane-spanning _-helices) and on previously reported receptor-like properties of other aminopeptidases (AP-N and dipeptidase IV). Meanwhile, we have gathered robust evidence in favor of this receptor-hypothesis by showing that Ang IV elicits non-AT1 receptor mediated signalling events in CHO cells (increased thymidine incorporation) and vascular smooth muscle cells (collaboration with Prof. Ruiz-Ortega - Spain). This could imply that the in vivo effects of Ang IV analogues should not be simply dependent on their ability to bind to IRAP but also on propensy to stimulate cell signalling. To this end, in vitro experimental systems will be further developed to test Ang IV analogues for their intrinsic activity (i.e. agonistic vs. antagonistic properties), to elucidate the chain of IRAP-mediated cell signalling events and to compare these events in different cell types.
(iii) Finally, the possibility arises that the beneficial effects of Ang IV on memory and learning involve an increased glucose uptake in hippocampal neurones. The potential contribution of IRAP in such mechanism is based on the demonstration that IRAP and the GLUT4 glucose transporter co-exist in hippocampal neurones (Fernando et al., 2005) and on the rationale that both proteins are co-transported between intracellular stores and the cell surface. This opens the possibility for Ang IV to interfere with IRAP recycling and by this way to enhance/prolong the exposure of GLUT4 at the cell surface. To explore this intriguing possibility, in vitro experiments will first explore the ability of Ang IV to affect IRAP transport on intact cells. To this end, we will develop an immunochemical approach (collaboration with Prof. Bottari - France) and already established a radioligand binding approach for the dectection of IRAP at the cell surface. We will also explore for the aptitude of Ang IV and related ligands to trigger glucose uptake into GLUT4 containing cells and into the hippocampus of rodents (collaboration with Prof. Albiston and Chai - Australia).
The (patho)physiological role of Ang IV in the CNS
The further in vivo investigations are, in the first place, aimed to acquire a better understanding of the (patho)physiological role of Ang IV in the CNS with special reference to memory, epilepsy, neuroprotection and central modulation of blood pressure. Since the initial demonstration by Braszko et al. (1988) that i.c.v. injection of Ang IV enhances memory retention in a passive avoidance task, numerous other studies further stressed the positive role of this peptide in memory and cognition. We wish to inquire whether IRAP is truly a central player in these processes and, if so, by which molecular mechanism(s). Our interest in the link between Ang IV and epilepsy sprouted from our observation that i.c.v. Ang IV infusion attenuates the severity of limbic seizures in a pilocarpine-induced rat model for limbic epilepsy (Stragier et al., 2006). In this respect, we would especially like to test our hypothesis that this effect of Ang IV are mediated by augmentation of DA and 5-HT levels in the hippocampus. The possible role of Ang IV in neuronal regeneration/protection is based on the finding that Ang IV promotes cell survival in the hippocampus (Kakinuma et al. (1997; 1998) and, more recently, that it confers neuroprotection in a model for experimental ischemic stroke (Faure et al., 2006). To get more insight in this potential therapeutically important issue, we plan to test neuroprotective effects of Ang IV in established models of cerebral ischemia and Parkinson's disease along with the in vivo role of IRAP in the regulation of cell survival and proliferation. Related to our preceding work in renal blood flow, we would like to address the potential implication of central Ang IV in the regulation of blood pressure. Finally we will further elucidate the mechanism(s) by which Ang IV affects renal function. Obviously we will also remain very attentive to applications in alternative therapeutic area`s (such as diabetes) that could emerge in the literature or during the elaboration of our research program. Also, some of the newly developed research tools could find a potential use as diagnostic tools.
Because of the complexity inherent to in vivo models, doubt may be raised whether IRAP represents the only cellular recognition site for the above-mentioned actions/implications of Ang IV. In preceding work, we have tried to tackle this problem by using selective antagonists/inhibitors for known non-IRAP targets such as AP-N and AT1 and AT2- receptors. Being aware of the sometimes limited efficacy of this approach, we have now initiated collaborative efforts with other research teams to acquire/develop additional tools (outlined in part 4.2) to discriminate IRAP from the other targets and to increase the in vivo stability of Ang IV. In the first place, we will conduct parallel studies on KO mice either lacking IRAP (collaboration with Prof. Albiston and Chai - Australia) or AT1 /AT2- receptors (collaboration with Prof. T. Walther - Univerisity of Berlin, Germany). These studies will obviously also include a behavioural phenotyping of the knock-out mice. The use of organ-specific knock-out mice could represent a most valuable extension to these studies. We can now also count on the collaboration of three chemistry departments (Prof. Tourwé - VUB, Prof. Hallberg - Sweden, Prof. Yiothakis - Greece) to provide us with novel synthetic Ang IV analogues. After an initial in vitro screening for stability, target-selectivity and potency, the most interesting ones will be studied in vivo for their ability/inability to mimick Ang IV-mediated responses. Obviously, these collaborations are of mutual benefit as our feedback to the chemists may lead to the synthesis of potential lead compounds for the development of therapeutic drugs. Efforts have also been undertaken to decrease the metabolic breakdown of Ang IV and, in this respect we have been the first to show that selective AP-N blockade (by compound 7B, provided by Prof. Yiothakis - Greece) increases the stability of Ang IV in vitro and amplifies the Ang IV elicited increase in striatal extracellular dopamine concentration. This strategy will certainly be extended to all in vivo studies to come.