283 Original Paper Cell Physiol Biochem 2007;20:283-292 Accepted: April 13, 2007 Cellular Physiology Cellular Physiology Cellular Physiology Cellular Physiology Cellular Physiology and Biochemistr and Biochemistr and Biochemistr and Biochemistr and Biochemistry Copyright © 2007 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com © 2007 S. Karger AG, Basel 1015-8987/07/0205-0283$23.50/0 Accessible online at: www.karger.com/cpb Cross-Regulation of iNOS and COX-2 by its Products in Murine Macrophages Under Stress Conditions Luiz Euribel Prestes-Carneiro a,* , Marina Tiemi Shio b,* , Patrícia Dias Fernandes c and Sonia Jancar b a Department of Immunology, University of Oeste Paulista, Presidente Prudente, São Paulo , b Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, c Department of Pharmacology, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, *LEPC and MTS contributed equally to this work Dr. Sonia Jancar Departamento de Imunologia, ICB, Universidade de São Paulo Av. Prof. Lineu Prestes 1730, São Paulo, SP, CEP: 05508-900, (Brazil) Tel./Fax. +55-11-3091 7744 E-Mail sojancar@icb.usp.br Key Words Heat shock  Nitric oxide  Prostaglandins  Macrophages  LPS  COX-2  iNOS Abstract Exposure of macrophages to heat shock induces rapid synthesis of heat shock proteins (HSPs) which are important for cell homeostasis. Prostaglandins (PGs) and nitric oxide (NO) are important cell regulatory molecules. We have therefore investigated the interactions between these molecules in the LPS- induced expression of iNOS and COX-2 and in the mitochondrial activity of macrophages. Cultures of the murine macrophage cell line, J774, were exposed to heat shock (43°C, 30min) and stimulated with LPS (1 µg/ml), concomitantly or after 8h of cell recovery. NO production was measured by Griess reaction; PGE 2 by ELISA; HSP70, iNOS and COX-2 by immunobloting; mitochondrial activity by MTT assay. Heat shock induced HSP70, but not iNOS or COX-2 whereas LPS induced iNOS and COX-2 but not HSP70. When heat shock and LPS were given concomitantly, iNOS but not COX-2 expression was reduced. When a period of 8h was given between heat shock and LPS stimulation, iNOS, COX-2, PGE 2 and NO levels were significantly increased. Under these conditions, the expression of COX-2 was reduced by L-NAME (NO- synthesis inhibitor) and of iNOS by nimesulide (PGs- synthesis inhibitor). Such cross-regulation was not observed in cells at 37°C. These treatments significantly reduced MTT levels in cells at 37°C but not in cells submitted to heat shock. These results suggest that HSPs and cross-regulation of iNOS and COX-2 by their products might be of relevance in the control of cell homeostasis during stress conditions. Introduction The observation that an increase in temperature of a few degrees above the physiological level induces the synthesis of a small number of proteins in Drosophila salivary glands, lead to the discovery of a universal protective mechanism which prokaryotic and eukaryotic cells utilize to preserve cellular functions and homeostasis [1]. These proteins were called heat shock proteins (HSP) and were shown to prevent protein aggregation during stress conditions and contribute to refolding of damaged proteins [2]. In mammalian cells, several HSP genes are