Expression of AT1, AT2 receptors and a non-AT1, non-AT2 angiotensin II binding site in rat brain after endothelin-1 induced ischemic stroke

New findings suggest that the activation AT1 receptor decreases cerebral perfusion after an ischemic stroke in the brain while the activation of AT2 receptor opposes those actions providing neuroprotective effects. Recent discoveries reveal the existence of a novel non-AT1, non-AT2 binding site for angiotensin II (Ang II) in the brain which may indicate additional effects of the brain angiotensin system after ischemic damage. To assess this, 5 rats were microinjected with 3 μl of 80 μM endothelin-1 (ET-1) to stimulate middle cerebral artery occlusion in the right hemisphere of the forebrain. 24 hours later, the rats were sacrificed and the brains were removed and frozen. The brains were analyzed by observing the binding of 125I-Sar1 Ile8 Ang II to the tissue by receptor autoradiography. Using quantitative densitometric analysis of the 125I-Sar1 Ile8 Ang II binding (MCID) to the forebrain caudal to the ischemic region of the brain, no differences in AT1, AT2 or non-AT1, non-AT2 binding was observed between the hemispheres in either the density of the receptor binding or the area encompassed by each hemisphere. The results suggest that the ischemia does not alter the expression of angiotensin binding proteins in the region posterior to the stroke zone at 24 hours post-ischemia

Identification of Membrane-bound Variant of Endopeptidase 24.16 as the non-AT1, non-AT2Angiotensin II Binding Site

Objective. To determine the identity of the non-AT1, non-AT2 angiotensin II (AngII) binding site in the mammalian brain. Background. New discoveries about the renin-angiotensin system continue to abound in the 21st century, e.g., discovery of a renin receptor, identification of the mas oncogene protein as the angiotensin 1-7 receptor, discovery of an enzyme (angiotensin-converting enzyme-2, ACE-2) that converts AngII to angiotensin 1-7, use of AT2 receptor agonists as therapeutic agents, and discovery of a novel non-AT1, non-AT2 angiotensin binding site in the brain. Methods. An angiotensin analog, photoaffinity probe 125I-sarcosine1,benzoylphenylalanine8-AngII was used to specifically label the non-AT1, non-AT2 AngII binding site in membranes prepared from mouse forebrain and HEK293 cell overexpressing mouse neurolysin in the presence of AT1 receptor-saturating concentrations of losartan, AT2 receptor-saturating concentrations of PD123319, and 150 μM parachloromercuribenzoate. The 125Isarcosine1, benzoylphenylalanine8-AngII radiolabeled binding site was purified by 2-Dimensional electrophoresis and analyzed by mass spectrometry, or solubilized and immunoprecipitated. Saturation binding assays or in vitro autoradiography with 125I-sarcosine1,isoleucine8-AngII measured expression of the binding site in brain and HEK293 cell membranes. Results. The binding site is a ~75kDa membrane protein that fits a mass spectrometric profile of neurolysin. An antibody to neurolysin precipitated the 125Isarcosine1, benzoylphenylalanine8-AngII labeled protein. Overexpression of neurolysin in HEK293 cells increased non-AT1, non-AT2 125I-sarcosine1,isoleucine8-AngII binding. Binding of 125I-sarcosine1, isoleucine8-AngII to the non-AT1, non-AT2 binding site in neurolysin knock-out mouse brains was dramatically decreased compared to wild-type brains. Conclusion. A membrane-bound variant of the metalloendopeptidase neurolysin (E.C.3.4.24.16) is the novel AngII binding protein. Grants. Supported by NHLBI HL-096357

A Web-Based Pharmacogenomics Search Tool for Precision Medicine in Perioperative Care

Background: Precision medicine represents an evolving approach to improve treatment efficacy by modifying it to individual patient’s gene variation. Pharmacogenetics, an applicable branch of precision medicine, identifies patient’s predisposing genotypes that alter the clinical outcome of the drug, hence preventing serious adverse drug reactions. Pharmacogenetics has been extensively applied to various fields of medicine, but in the field of anesthesiology and preoperative medicine, it has been unexploited. Although the US Food and Drug Administration (FDA) has a table of pharmacogenomics biomarkers and pharmacogenetics, this table only includes general side effects of the included drugs. Thus, the existing FDA table offers limited information on genetic variations that may increase drug side effects. Aims: The purpose of this paper is to provide a web-based pharmacogenomics search tool composed of a comprehensive list of medications that have pharmacogenetic relevance to perioperative medicine that might also have application in other fields of medicine. Method: For this investigation, the FDA table of pharmacogenomics biomarkers in drug labeling was utilized as an in-depth of drugs to construct our pharmacogenetics drug table. We performed a literature search for drug–gene interactions using the unique list of drugs in the FDA table. Publications containing the drug–gene interactions were identified and reviewed. Additional drugs and extracted gene-interactions in the identified publications were added to the constructed drug table. Result: Our tool provides a comprehensive pharmacogenetic drug table including 258 drugs with a total of 461 drug–gene interactions and their corresponding gene variations that might cause modifications in drug efficacy, pharmacokinetics, pharmacodynamics and adverse reactions. This tool is freely accessible online and can be applied as a web-based search instrument for drug–gene interactions in different fields of medicine, including perioperative medicine. Conclusion: In this research, we collected drug–gene interactions in a web-based searchable tool that could be used by physicians to expand their field knowledge in pharmacogenetics and facilitate their clinical decision making. This precision medicine tool could further serve in establishing a comprehensive perioperative pharmacogenomics database that also includes different fields of medicine that could influence the outcome of perioperative medicine

Histology WT1 WT2 KO1 KO2

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

Autoradiographic film for WT1 KO1

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

AT₁ and AT₂ receptor binding comparison.

<p>Comparison of ¹²⁵I-SI Ang II binding to the AT₁ and AT₂ receptors of neurolysin KO and WT mouse strain brains in the presence of PD123319 or losartan, respectively. Bregma +0.14 (histology and AT₁) and +0.02 mm (AT₂ and non-specific) for KO, and +0.14 (histology, AT₁, and non-specific) and +0.26 mm (AT₂) for WT.</p

Autoradiographic film with KO1 and WT2

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

Histology WT3 KO3

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

Autoradiographic film for WT3 KO3

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

Regional distribution: non-AT₁, non-AT₂ binding.

<p>Regional distribution of non-AT₁, non-AT₂ Ang II binding in neurolysin KO and WT mouse brains. Brain regions were divided into cerebellum, brainstem and midbrain (Panel A), hypothalamic nuclei (Panel B), thalamoseptalstriatal regions (Panel C), and telencephalic regions (Panel D). In all but two regions, a priori t-tests showed significant reduction in ¹²⁵I-SI Ang II binding in the brains of the neurolysin KO mice. * p<0.05. AH, Anterior Hypothalamus; AMYG, Amygdala; ARC, Arcuate Nucleus; CCTX, Cingulate Cortex; CP, Choroid Plexus; CPu, Caudate Putamen; CRBLM, Cerebellum; DMH, Dorsomedial Hypothalamus; DTLCMe5, Dorsal Tegmentum, Locus Coeruleus and Mesencephalic Nucleus of the Trigeminal Nerve; ETC, Entorhinal Cortex; HPC, Hippocampus; IPN, Interpeduncular Nucleus; LMC, Limbic Cortex; LS, Lateral Septum; ML BRST, Mediolateral Brain Stem; MnPO, Median Preoptic Nucleus; MPOH, Medial Preoptic Nucleus; NACC, Nucleus Accumbens; NTS, Nucleus Tractus Solitarius; PAG, Periaqueductal Gray; PH, Posterior Hypothalamic Area; PMN, Premamillary Nucleus; PVA-THAL, Paraventricular Thalamic Nucleus, Anterior; PVH, Paraventricular Hypothalamic Nucleus; PVTHAL, Paraventricular Thalamic Nucleus; Red N, Red Nucleus; RSPC, Retrosplenial Cortex; SC, Superior Colliculus; SCN, Suprachiasmatic Nucleus; SN, Substantia Nigra; TSN, Triangular Septal Nucleus; VMH, Ventromedial Hypothalamic Nucleus.</p