First the complete canonical brain original RAS Angiotensino

First, the complete canonical brain original RAS (Angiotensinogen, Renin, Angiotensin I, Angiotensin Converting Enzyme (ACE), Angiotensin II) has not been conclusively identified in any specific cell type within the brain [1]. Validated evidence for the presence of brain Angiotensinogen and ACE was provided by in situ hybridization and quantitative autoradiography [2], [3], [4], [5]. Surprisingly, brain ACE and Angiotensinogen were found to be localized in separate cells; Angiotensinogen was predominantly or exclusively expressed in glial cells located in many brain structures, while ACE was predominantly expressed in the choroid plexus and the caudate nucleus and has, additionally, a predominant endothelial localization [1], [2], [3], [4], [5], [6], [7]. In the subfornical organ, in situ hybridization of Angiotensin II AT1 receptors was restricted to neurons, while Angiotensinogen was localized in glial cells [3]. Furthermore, ACE is not only responsible for Angiotensin II production, but it is also involved in the metabolism of several other peptides [8]. For these reasons, it is highly unlikely that brain Angiotensinogen, ACE and Angiotensin II are physiologically related as proposed in the canonical RAS [1], [2], [8]. Only immunocytochemistry using poorly characterized MLN 8237 has been used to determine the localization of Angiotensin II in specific brain cells, and these studies revealed a lack of association between Angiotensin II and ACE [9]. Quantitative Angiotensin II-like determinations in the brain reveal extremely low concentrations, at the low picogram level, and several times lower than those found in the circulation [10], [11], [12], [13]. However, many publications report acute intracerebral administration or effects in neuronal cultures using Angiotensin II at levels 1000-fold higher than endogenous levels [14], [15]. All results obtained with commercially available but not fully characterized antibodies for all RAS components need reexamination. Commercially available antibodies are sold with specific reassurance of specificity as stated in their instruction sheets. Although recognizing the peptide antigen, these antibodies are not selective and react with several other unidentified proteins. All tested commercially available antibodies for AT1 and AT2 receptors lack proper characterization, and unfortunately their use may generate erroneous results. For example, commercially available AT1 and AT2 receptor antibodies detect these receptors in cells devoid of receptor gene expression and in mice genetically engineered not to express AT1 or AT2 binding domains (AT1 and AT2 knockout mice) [16], [17]. Non-selectivity of commercially available AT1 receptor antibodies has been independently confirmed [18]. This problem, unfortunately, includes not only Angiotensin II receptors but also all GPCR receptors [19], [20], and may extend to other substances beyond the receptor field. Specific instructions to fully determine antibody specificity have been published before [19], [20]. Validated antibody characterization is now required to be included in research proposals to the US National Institutes of Health. The specific requirements are not different from those of the literature: “Key antibodies might be validated by Western blot, ELISA, immunoprecipitation, immunofluorescence, or flow cytometry using knockdown cells and positive and negative controls, depending on the assay proposed”. (https://nexus.od.nih.gov/all/2016/01/29/authentication-of-key-biological-andor-chemical- resources-in-nih-grant-applications/). Several more recent studies reported on RAS-related enzymes and peptides that may play important roles in the brain and interact with or be part of the canonical RAS components. Reports on this additional and increasing complexity of the canonical RAS are of major interest. Their physiological and pathological roles are hotly disputed, and more studies are needed to identify the roles and relevance of each novel enzyme or peptide reported. Readers are directed to authoritative reviews on this most interesting subject [21], [22], [23], [24], [25].