Immunodetection and subcellular distribution of imidazoline receptor proteins with three antibodies in mouse and human brains: Effects of treatments with I 1 - and I 2 -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.