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Published online before print December 30, 2005

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(Journal of Leukocyte Biology. 2006;79:519-528.)
© 2006 by Society for Leukocyte Biology

Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies

R. Saini^*, S. Patel^*, R. Saluja^*, A. A. Sahasrabuddhe, M. P. Singh, S. Habib, V. K. Bajpai and M. Dikshit^*,1

^* Cardiovascular Pharmacology Unit,
Electron Microscopy Unit, and
Division of Structural and Molecular Biology, Central Drug Research Institute, Lucknow, India; and
Industrial Toxicology Research Centre, Lucknow, India

¹ Correspondence: Cardiovascular Pharmacology Unit, Central Drug Research Institute, Lucknow-226001, India. E-mail: madhudikshit{at}yahoo.com

Nitric oxide (NO) modulates diverse functions of polymorphonuclear neutrophils (PMNs), but localization of NO synthase (NOS) and identification of its interacting proteins remain the least defined. The present study discerns subcellular distribution of NOS and caveolin-1, a prominent NOS-interacting protein in rat PMNs. Localization of NOS was explored by confocal and immunogold electron microscopy, and its activity was assessed by L-[³H] arginine and 4,5-diaminofluorescein diacetate (DAF-2DA). Reverse transcriptase-polymerase chain reaction using NOS primers and Western blotting demonstrated the presence of neuronal NOS (nNOS) and inducible NOS (iNOS) in PMNs. Immunocytochemical studies exhibited distribution of nNOS and iNOS in cytoplasm and nucleus, and L-[³H] citrulline formation and DAF fluorescence confirmed NOS activity in both fractions. NOS activity correlated positively with calmodulin concentration in both of the fractions. nNOS and iNOS colocalized with caveolin-1, as evidenced by immunocytochemical and immunoprecipitation studies. The results thus provide first evidence of nNOS and iNOS in the nuclear compartment and suggest NOS interaction with caveolin-1 in rat PMNs.

Key Words: polymorphonuclear leukocytes • nitric oxide synthase • diaminofluorescein diacetate • caveolin-1 • nucleus • subcellular localization

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