Journal of Cellular Biochemistry 89:1030–1043 (2003) Constitutive NO Synthase Regulates the Na þ /Ca 2þ Exchanger in human T cells: Role of [Ca 2þ ] i and Tyrosine Phosphorylation Juliann G. Kiang, 1,2,3 * David E. McClain, 4 Vishal G. Warke, 1 Sandeep Krishnan, 1 and George C. Tsokos 1,2 1 Department of Cellular Injury, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland 20910-7500 2 Department of Medicine, Uniformed Services University of The Health Sciences, Bethesda, Maryland 20814-4799 3 Department of Pharmacology, Uniformed Services University of The Health Sciences, Bethesda, Maryland 20814-4799 4 Applied Cellular Radiobiology Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20889-5603 Abstract For many types of cells, heat stress leads to an increase in intracellular free calcium concentration ([Ca 2þ ] i ) that has been shown to trigger a wide variety of cellular responses. In T lymphocytes, for example, heat stress stimulates pathways that make them more susceptible to Fas/CD95-mediated apoptosis. Because of our interest in understanding more about the response of lymphocytes to various stressors, we used human peripheral and Jurkat T lymphocytes to investigate the effect of heat stress on calcium homeostasis. We found that peripheral and Jurkat T cells both exhibit cNOs activity but not iNOs activity. Heat stress increased NO production, which was inhibited by LNNA (a cNOs inhibitor) but not L-NIL (an iNOs inhibitor). Heat stress increased [Ca 2þ ] i in Jurkat T cells by decreasing the K m of the cell surface membrane Na þ /Ca 2þ exchanger for extracellular Ca 2þ . Heating also increased cNOs phosphorylation at tyrosine residues. In cells incubated with LNNA, heat stress promoted an increase in [Ca 2þ ] i and a decrease in [Na þ ] i greater than in cells heated without LNNA, a larger decrease in K m of the Na þ /Ca 2þ exchanger for extracellular Ca 2þ , and decreased phosphorylation of cNOs. Our results suggest that cNOs plays an important regulatory role after heat stress. Heating appears to increase the phosphorylation of cNOs that is complexed with the Na þ /Ca 2þ exchanger to decrease its activity. This process is related to increased expression of Fas/CD95 on the cell surface, which might explain the apoptotic diathesis of lymphocytes after heat stress. J. Cell. Biochem. 89: 1030 – 1043, 2003. Published 2003 Wiley-Liss, Inc. { Key words: Na þ /Ca 2þ exchanger; Ca 2þ ; NO synthase; heat stress; lymphocytes; T cells; tyrosine phosphorylation; CD95; Fas Cells regulate intracellular Ca 2þ concen- tration ([Ca 2þ ] i ) in a variety of ways. Systems located at the cell membrane include ATPase- mediated Ca 2þ pumps, Na þ /Ca 2þ antiporter (exchanger), Ca 2þ -adenosine triphosphatase- regulated pumps, and Ca 2þ channels (including voltage-gated, second messenger-operated, and receptor-operated channels). Intracellular sys- tems include Ca 2þ mobilization from intracel- lular pools and cytosolic Ca 2þ -binding proteins such as calmodulin and calcineurin [Waldron et al., 1997; Kiang and Tsokos, 1998; Ma et al., 2002]. When resting [Ca 2þ ] i increases, it trig- gers the activity of Ca 2þ -dependent enzymes involved in many cellular biochemical reactions [Kiang and McClain, 1993; Kiang et al., 1994; Published 2003 Wiley-Liss, Inc. { This article is a US Govern- ment work and, as such, is in the public domain in the United States of America. Abbreviations used: [Ca 2þ ] i , intracellular Ca 2þ concentra- tion; cNOs, constitutive nitric oxide synthase; iNOs, inducible nitric oxide synthase; LNNA, No-nitro-L-argi- nine; L-NIL, L-N 6 -(1-iminoethyl)lysine; NO, nitric oxide; XIP, exchanger inhibitory peptide, PMSF, phenylmethyl- sulfonyl fluoride; DTT, dithiothreitol; HSP-70, heat shock protein 70 kDa. Grant sponsor: Department of the Army RAM II STO R and AFRRI Work Unit 00150. *Correspondence to: Dr. Juliann G. Kiang, Department of Cellular Injury, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Room 1N07, Silver Spring, MD 20910-7500. E-mail: Juliann.Kiang@na.amedd.army.mil Received 31 October 2002; Accepted 17 April 2003 DOI 10.1002/jcb.10564