Discovery and pharmacological characterisation of angiotensin-(1-7) receptors and identification of their importance in diabetes mellitus

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Tetzner, Anja
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University College Cork
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The renin-angiotensin system (RAS) is known to be the main regulator of blood pressure and fluid balance. Within the RAS, angiotensin (Ang)-(1-7) is known to have cardiovascular protective effects. It represents the opponent of the often detrimental Ang II, which is known to stimulate the angiotensin receptor type 1 (AT1), causing negative effects such as a pathological rise in blood pressure. Beside the well accepted fact that the G-protein coupled receptor Mas is a receptor for the heptapeptide, it was not possible to characterise the ligand/receptor pharmacology or to identify further receptors, since there was a lack in the understanding of the initial intracellular signalling pathways stimulated by Ang-(1-7). In this study, cyclic adenosine monophosphate (cAMP) was identified as a second messenger stimulated by Ang-(1-7). The heptapeptide elevates cAMP concentration in various cell lines, as well as in Mas transfected HEK293 cells, confirming that Mas is a functional receptor for Ang-(1-7). Even more important, MrgD was identified as a second receptor for the peptide, while AT2 could be excluded to be targeted by the heptapeptide. It was also examined, if there are any changes in the intracellular signalling if the first amino acid of the peptide is decarboxylated. The receptor fingerprint for Ala1 -Ang-(1-7) was discovered, and the consequences for pharmacodynamics characterised. The dose-response curves were clearly different from the curves generated with Ang-(1-7). They showed a much lower EC50 and a bell-shaped curve for Ala1 -Ang-(1-7). Furthermore, pharmacological proof was provided that both, Mas and MrgD, are functional receptors for Ala1 -Ang-(1-7). Interestingly, it was also discovered that the AT2 receptor blocker PD123319 is not AT2 specific, but can also block the effects of Ang-(1-7) and Ala1 -Ang(-1-7) in Mas and MrgDtransfected and in primary cells. This raised the question whether the selective nonpeptidic AT2 receptor agonist, Compound 21 (C21), is also unspecific and stimulates Mas and MrgD too. This hypothesis was supported by the fact that the chemical structure of C21 is similar to the Mas receptor specific, non-peptidic agonist AVE0991. Using cAMP and downstream molecules as readouts, pharmacological proof that Mas and MrgD are functional receptors for C21 was generated. The last part of the study examined the role of Ang-(1-7) and its receptors in diabetes mellitus (DM). Previous studies demonstrated that the ACE2/ Ang-(1–7)/ Mas axis has beneficial effects on glucose homeostasis, but the underlying mechanisms remained unknown. The effects of Ang-(1–7) and its receptor Mas on the function of β-cells were investigated. Islets isolated from Mas-deficient and wild-type mice were stimulated with Ang-(1–7) or its antagonists and effects on insulin secretion were determined. It was found that Ang-(1–7) was able to increase the insulin secretion from wild-type islets, but not from islets derived from Mas deficient animals. Interestingly, Ang-(1-7) antagonist DPro, but not A779 could block the Ang-(1-7) mediated effects indicating the involvement of another Ang-(1-7) receptor. However, the heptapeptide did not affect the insulin gene expression or the excitation-secretion coupling, but increased intracellular cAMP involving exchange protein activated directly by cAMP (EPAC), leading to a higher insulin secretion by the β-cells. Ang-(1–7) was also applied to normo-glycaemic mice for 14 days using osmotic pumps. The effects of the heptapeptide in vivo had only marginal effects on glucose tolerance in wild-type mice. However, Ang-(1-7) had improved the insulin secretion in islets isolated from these mice. Interestingly, although less pronounced than in wild-types, Ang-(1–7) still affected insulin secretion in islets derived from Mas deficient mice. The effect of Ang- (1-7) in mice with STZ-induced diabetes was marginal, as in normo-glycaemic mice. Taken together, these results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for Ang-(1-7), and by excluding AT2 as a receptor for the heptapeptide. Furthermore, the identification of Ala1 -Ang-(1-7) as a peptide with specific pharmacodynamic properties can be used as a basis for the design of more potent and efficient Ang-(1-7) analogues, which can be useful in therapeutic interventions in a rapidly growing number of diseases. The proof that C21 and PD123319 are not AT2 receptor specific as generally assumed, but also interact with the two Ang-(1- 7) receptors, Mas and MrgD, might be an explanation for the partial overlap in beneficial effects of both compounds. Thus, the better understanding of the interaction of small molecules like C21 with their receptors, lays the foundation for the development of small molecules which stimulate all or just one of the Ang-(1-7) receptors, which may be beneficial in diseases like diabetes mellitus. Since it could be shown that Ang-(1–7) plays a significant role in the regulation of insulin secretion from mouse islets in vitro and in vivo, mainly, but not exclusively, by Mas-dependent signalling, modulating the accessory pathway of insulin secretion via increase in cAMP, makes clear that Ang-(1-7) and its receptors are very promising therapeutic targets.
Renin-angiotensin System , Angiotensin-(1-7) , Mas receptor , MrgD receptor , AT2 receptor , G-proteins , Ala1-Angiotensin-(1-7) , C21 , Ang-(1-7)/Mas axis , Dose-response curve , Diabetes mellitus
Tetzner, A. 2018. Discovery and pharmacological characterisation of angiotensin-(1-7) receptors and identification of their importance in diabetes mellitus. PhD Thesis, University College Cork.