Mas receptor is translocated to the nucleus upon agonist stimulation in brainstem neurons from spontaneously hypertensive rats but not normotensive rats

Abstract

Aims Activation of the angiotensin (Ang)-(1-7)/Mas receptor (R) axis protects from sympathetic overactivity. Endocytic trafficking is an essential process that regulates receptor (R) function and its ultimate cellular responses. We investigated whether the blunted responses to Ang-(1-7) in hypertensive rats are associated to an alteration in MasR trafficking. Methods and results Brainstem neurons from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) were investigated for (i) Ang-(1-7) levels and binding and MasR expression, (ii) Ang-(1-7) responses (arachidonic acid and nitric oxide release and Akt and ERK1/2 phosphorylation), and (iii) MasR trafficking. Ang-(1-7) was determined by radioimmunoassay. MasR expression and functionality were evaluated by western blot and binding assays. MasR trafficking was evaluated by immunofluorescence. Ang-(1-7) treatment induced an increase in nitric oxide and arachidonic acid release and ERK1/2 and Akt phosphorylation in WKY neurons but did not have an effect in SHR neurons. Although SHR neurons showed greater MasR expression, Ang-(1-7)-elicited responses were substantially diminished presumably due to decreased Ang-(1-7) endogenous levels concomitant with impaired binding to its receptor. Through immunocolocalization studies, we evidenced that upon Ang-(1-7) stimulation MasRs were internalized through clathrin-coated pits and caveolae into early endosomes and slowly recycled back to the plasma membrane. However, the fraction of internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in WKY neurons. Surprisingly, in SHR neurons but not in WKY neurons, Ang-(1-7) induced MasR translocation to the nucleus. Nuclear MasR expression and Ang-(1-7) levels were significantly greater in the nuclei of Ang-(1-7)-stimulated SHR neurons, indicating that the MasR is translocated with its ligand bound to it. Conclusion MasRs display differential trafficking in brainstem neurons from SHRs, which may contribute to the impaired responses to Ang-(1-7).

Angiotensin Receptors Heterodimerization and Trafficking: How Much Do They Influence Their Biological Function?

“…Regarding MasR, upon Ang-(1-7) stimulation, MasR is endocyted by CCP and caveolae in a mechanism dependent on dynamin, and then the receptor is directed to the cell surface by slow recycling vesicles (Cerniello et al, 2017). This mechanism of internalization was observed in transfected HEK293T cells and in cultured brainstem neurons from Sprague-Dawley rats, Wistar-Kyoto rats, and spontaneously hypertensive rats (SHRs) (Cerniello et al, 2017;Cerniello et al, 2019). The interesting observation is that MasR undergoes a unique trafficking in brainstem neurons from SHR: The number of MasRs internalized through caveolae was greater compared to that internalized by CCP, and the number of receptors that were returned to the cell membrane was smaller, resulting in a lower amount of resensitized MasRs present at the plasma membrane.…”

“…The interesting observation is that MasR undergoes a unique trafficking in brainstem neurons from SHR: The number of MasRs internalized through caveolae was greater compared to that internalized by CCP, and the number of receptors that were returned to the cell membrane was smaller, resulting in a lower amount of resensitized MasRs present at the plasma membrane. Furthermore, a fraction of MasRs is translocated to the nucleus bound to its ligand only in brainstem neurons obtained from SHRs and not from normotensive rats (Cerniello et al, 2019). Figure 3 represents MasR trafficking in brainstem neurons from both strains.…”

“…It has also been postulated that nuclear GPCRs could be located in the nucleoplasm, particularly in the network of invaginations of the inner and outer nuclear membranes ( Jong et al, 2018 ; Bhosle et al, 2019 ; Ribeiro-Oliveira et al, 2019 ). AT1R and MasR have been described to colocalize with a nuclear pore complex marker in brain neurons, suggesting the presence of both receptors in the nuclear pore complex ( Lu et al, 1998 ; Cerniello et al, 2019 ). In cardiomyocytes, nuclei GPCRs have been described to be present in both inner and outer membranes ( O’Malley et al, 2003 ; Jong et al, 2005 ; Vaniotis et al, 2011 ; Bkaily et al, 2012 ; Tadevosyan et al, 2012 ).…”

“…MasR is another example of cell specific receptor nuclear translocation. Agonist stimulation induces MasR translocation to the nucleus in brainstem neurons from SHR but not from normotensive rats ( Cerniello et al, 2019 ). Physiological consequences of agonist-dependent MasR trafficking to the nucleus only in neurons from SHR need to be elucidated.…”

“…Ligands from the extracellular space might reach those receptors by cellular uptake using selective membrane transporters, membrane exchangers, or via receptor endocytosis ( Ribeiro-Oliveira et al, 2019 ). In fact, the components of the RAS are present in the nucleus of several cells types ( Tang et al, 1992 ; Jimenez et al, 1994 ; Camargo de Andrade et al, 2006 ; Cook and Re, 2012 ; Gwathmey et al, 2012 ; Kumar et al, 2012 ; Alzayadneh and Chappell, 2015 ; Costa-Besada et al, 2018 ; Cerniello et al, 2019 ). Thus, nuclear Ang receptors may be activated by endogenously synthetized Angs ( Tadevosyan et al, 2012 ).…”