Immunocytochemical study of the glucocorticoid receptor in rat liver nuclei after hyperthermic stress Aleksandra C ˇ voro 1 , Aleksandra Korac ´ 2 , Gordana Matic ´ 1 * 1 Department of Biochemistry, Institute for Biological Research, 29 Novembra 142 Belgrade, Belgrade 11060, Yugoslavia 2 Institute of Zoology, Faculty of Biology, University of Belgrade, Belgrade, Yugoslavia Received 13 June 2002; revised 6 November 2002; accepted 14 January 2003 Abstract The presence of glucocorticoid receptors (GR) in rat liver nuclei over a 24 h time period following hyperthermic stress at 41 (C was immunocytologically studied using unfixed nuclear smears. Liver nuclei in unstressed animals were found to be immunonegative for GR. However, intense GR immunopositivity followed by a subsequent gradual decrease in receptor levels was observed in the nuclei of test animals during the first 2 h after stress. This stress-related increase in the receptor nuclear level was greater than the increase seen after dexamethasone administration. These results suggest that hyperthermic stress could potentiate the hormonal stimulation of receptor nuclear translocation.  2003 Elsevier Science Ltd. All rights reserved. Keywords: Glucocorticoid receptor; Hyperthermic stress; Immunocytochemistry 1. Introduction Glucocorticoid hormones are known to be vital for the maintenance of life, and are especially crucial during various stressful conditions. The cellular effects of gluco- corticoids are mediated through the glucocorticoid receptor (GR) and involve the regulation of gene tran- scription, and thereby protein synthesis (Yamamoto, 1985). In the absence of the hormone, the GR is associated with specific molecular chaperones and other proteins that are required to maintain the GR protein in a conformation capable of hormone binding, and are essential for the proper functioning of the signaling pathway (Pratt and Toft, 1997). The subcellular distri- bution of the GR has been studied for many years, but has remained controversial. General opinion is that unliganded GR, in contrast to estrogen and progester- one receptors, is mainly localized within the cytoplasm of target cells. Hormone binding induces transformation of the GR, leading to its translocation from the cytoplasm to the nucleus. It has recently been demonstrated in cultured cells (Hu et al., 1996; Sanchez, 1992) that heat stress could potentiate the effect of dexamethasone on GR nuclear translocation, thus leading to an increase in the level of GR-mediated gene expression. Such a response has been referred to as the ‘heat shock potentiation effect’ (Hu et al., 1996). Our previous immunochemical analyses of GR in the liver of rats exposed to 41 (C hyperthermic stress revealed a significant decrease in its cytoplasmic levels in response to the stress (C ˇ voro et al., 1998). However, Western blot analysis of the receptor in liver nuclei failed to provide unambiguous data, probably because of the low efficiency of receptor immuno- purification from the nuclei. The conflicting evidence regarding immunolocalization of GR may also result from the varying specificity of different techniques applied. Immunocytochemical techniques have several advan- tages over immunochemical ones, including the use of highly specific monoclonal antibodies, good reproduc- ibility and the ability to identify total immunologically reactive GR. The major disadvantage of these tech- niques, however, is that the cells have to be fixed and permeabilized in order to allow the antibodies access into the cell. The processing technique employed may * Corresponding author. Tel.: +381-11-764-422x103; fax: +381-11-761-433 E-mail address: gormatic@ibiss.bg.ac.yu (G. Matic ´). Cell Biology International 27 (2003) 403–407 Cell Biology I nternational www.elsevier.com/locate/cellbi 1065-6995/03/$ - see front matter  2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00019-2