In vitro and in vivo immunocytochemistry for the distribution of mineralocorticoid receptor with the use of specific antibody.
To examine the distribution of mineralocorticoid receptor (MR) and the interactions with glucocorticoid receptor (GR) in the brain, we raised a polyclonal antibody against the transcriptional modulation domain of rat MR using the GST-fusion system. Immunoblotting analysis revealed that this antibody recognized a band with the molecular mass of MR in MR-transfected COS-1 cells and in a homogenate of rat hippocampus, and showed no cross-reactivity with GR. In vitro immunocytochemistry of both primary cultured hippocampal neurons and MR-transfected cells revealed immunoreactivity detected by this antibody in both the cytoplasm and nucleus in the absence of aldosterone (ALD), a specific agonist of MR. After 1 h of treatment with 10(-7) M ALD, the MR-immunoreactivity was accumulated in the nuclear region. In the case of GR-transfected cells, our anti-MR antibody either detected no immunopositive cells in the presence or absence of GR agonist. In our in vivo study, MR-immunoreactivity was observed in the rat hippocampus, where cell nuclei showed immunopositive reactions. These results suggest that our antibody against rat MR shows high specificity for the receptor both in liganded and unliganded forms, with no cross-reactivity to GR, and will be useful for cell biological and neuroanatomical investigations of MR.
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Japan.
T
T
Ito
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N
Morita
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M
Nishi
M
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Kawata
D000450:Aldosterone / Q000494:pharmacology; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D000906:Antibodies / Q000276:immunology; D019556:COS Cells; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D018161:Receptors, Mineralocorticoid / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
D000906:Antibodies; D018076:DNA, Complementary; D018161:Receptors, Mineralocorticoid; D011993:Recombinant Fusion Proteins; D000450:Aldosterone