Immunodetection and subcellular distribution of imidazoline receptor proteins with three antibodies in mouse and human brains: Effects of treatments with I₁- and I₂-imidazoline drugs

Abstract

Various imidazoline receptor (IR) proteins have been proposed to mediate the effects of selective I1- and I2-IR drugs. However, the association of these IR-binding proteins with classic I1- and I2-radioligand binding sites remains somewhat controversial. In this study, three IR antibodies (anti-NISCH and anti-nischarin for I1-IRs; and anti-IRBP for I1/I2-IRs) were used to immunodetect, characterize and compare IR protein patterns in brain (mouse and human; total homogenate, subcellular fractionation, grey and white matter) and some cell systems (neurones, astrocytes, human platelets). Various immunoreactive IRs (specific molecular weight bands coincidently detected with the different antibodies) were related to I1-IR (167 kDa, 105/115 kDa and 85 kDa proteins) or I2-IR (66 kDa, 45 kDa and 30 kDa proteins) types. The biochemical characterization of cortical 167 kDa protein, localized in the membrane/cytosol but not in the nucleus, indicated that this I1-IR also forms part of higher order nischarin-related complexes. The contents of I1-IR (167 kDa, 105/115 kDa, and 85 kDa) proteins in mouse brain cortex were upregulated by treatment with I1-drugs (moxonidine, efaroxan) but not with I2-drugs (BU-224, LSL 61122). Conversely, the contents of I2-IR (66 kDa, 45 kDa and 30 kDa) proteins in mouse brain cortex were modulated by treatment with I2-drugs (decreases after BU-224 and LSL 61122, and increases after idazoxan) but not with I1-drugs (with the exception of moxonidine). These findings further indicate that brain immunoreactive IR proteins exist in multiple forms that can be grouped in the already known I1- and I2-IR types, which are expressed both in neurones and astrocytes.

Effect of imidazoline‐1 receptor agonists on renal dysfunction in rats associated with chronic, sequential fructose and ethanol administration

“…In accordance with our findings, a 2015 study by Keller and Garcia‐Sevilla showed I1R upregulation in the mouse brain cortex after treatment with moxonidine (I1R agonist) . Additionally, El‐Mas et al showed that I1R pathway activation mediates renal protection in rats …”

“…Garcia-Sevilla showed I1R upregulation in the mouse brain cortex after treatment with moxonidine (I1R agonist). 50 Additionally, El-Mas et al showed that I1R pathway activation mediates renal protection in rats. 51 In agreement with the improvements in kidney function, rilmen- Our results are in accordance with the findings of Aceros et al, who reported that moxonidine (I1R agonist) reduces oxidative stress by increasing nischarin expression.…”

Imidazoline I 2 receptors: An update

“…A biochemical investigation using a polyclonal antiserum againist imidazoline receptor proteins revealed four different protein bands and the bands were not identical between rat and rabbit brain tissues (rat brains: ~ 30, ~ 45, ~ 66, ~ 85 kD; rabbit brains: ~30, ~ 57, ~ 66, ~ 85 kD) (Olmos, Alemany, Boronat, & Garcia-Sevilla, 1999). In addition, chronic treatment with selective I 2 receptor ligands such as BU224 and tracizoline (also known as LSL61122, valldemossine) significantly reduced while treatment with idazoxan increased the 30 kD, 45 kD and 66 kD protein levels in the mouse brain (Keller & Garcia-Sevilla, 2015), indicating the biochemcial relevance of I 2 receptor ligands and these proteins. Using a selective and high affinity I 2 receptor ligand 2-BFI to generate affinity column, a protein was successfully isolated and sequenced which was identified as the brain creatine kinase (Kimura et al, 2009).…”

Molecular aspects of monoamine oxidase B

“…New ligands enable detection and imaging studies for distribution and occupancy of I 2 sites (Tyacke et al, 2012, Kealey et al, 2013, Keller and Garcia-Sevilla, 2015. Further development will be aided by computational methods, both for structure-function relationships and for drug-like properties to ensure brain permeation (Moraes and de Azevedo, 2012, Nikolic and Agbaba, 2012, Vucicevic et al, 2015.…”