In Vivo and in Vitro Effects of Lysine Clonixinate on Nitric Oxide Synthase in LPS-Treated and Untreated Rat Lung Preparations A. M. Franchi,* ,1 G. Di Girolamo,† M. Farina,* A. R. de los Santos,‡ M. L. Martı ´,‡ and M. A. F. Gimeno* *CEFYBO (Pharmacological and Botanical Research Center) and CONICET (Scientific and Technical Research National Council), Buenos Aires, Argentina; †Pharmacology Department, School of Medicine, Universidad de Buenos Aires, Buenos Aires, Argentina; and ‡School of Medicine, Universidad de Buenos Aires, Hospital de Clı ´nicas Jose ´ de San Martı´n, Buenos Aires, Argentina Received in revised form June 7, 2000; published online February 28, 2001 Recent studies have shown that some nonsteroi- dal antiinflammatory drugs (NSAIDS) inhibited the inducible NO synthase (iNOS) without direct effect on the catalytic activity of this enzyme. This study was conducted to investigate the in vitro and in vivo effects of lysine clonixinate (LC) and indomethacin (INDO) on NOS activity in rat lung preparation. LC is a drug with antiinflammatory, antipyretic, and analgesic action. In the in vitro experiments, rats were injected with saline or lipopolysaccharide (LPS) and killed 6 h after treatment. Lung prepara- tions were incubated with LC at 2.3  10 5 M or 3.8  10 5 M. The minimum concentration did not modify NOS activity in control or LPS-treated rats but the maximum dose inhibited increased NO production induced by LPS. Furthermore, INDO at 10 6 M had no effect on enzymatic activity in control or LPS- treated rats. In the in vivo experiments, 40 mg/kg of LC were injected ip. Such a dose did not affect basal production of NO. When LC and LPS were injected simultaneously 6 h before sacrifice, a significant decrease in LPS-induced NOS activity was ob- served. INDO 10 mg/kg injected in control animals had no effect on NOS activity and did not block LPS induced stimulation of NO production when in- jected simultaneously. Finally, when LC (40 mg/kg) was injected 3 h after LPS, the enzymatic activity remained unchanged. Expression of iNOS was de- tected by Western blotting in rats treated with LPS plus 4, 10, 20, and 40 mg/kg of LC. The lowest dose was the only one showing no effect on LPS-induced increase of iNOS. In short, LC is a NSAID with in- hibitory action on the expression of LPS-induced NOS, effect that was not seen with INDO in our experimental conditions. © 2001 Academic Press Key Words: lysine clonixinate; iNOS; indometha- cin; rat lung. The therapeutic effect of the NSAIDs is thought to be due mainly to inhibition of cyclooxygenase en- zymes and subsequent reduction of prostaglandin synthesis (1, 2). Nitric oxide (NO), first identified as an endothelium-derived relaxing factor (3), has been proven to be an intra- and extracellular mediator of cell functions (4). NO produced by the constitutive isoforms of nitric oxide synthase (cNOS) is a key regulator of homeostasis, whereas production of NO by the inducible nitric oxide synthase (iNOS) plays an important role in inflammation, host defense re- sponse, and tissue repair. Following induction, iNOS is active for a period of 4 to 24 h and nanomo- lar concentrations of synthesized NO are more than 100 times those produced by cNOS (5). Recently, 1 To whom correspondence and reprint requests should be ad- dressed. Fax: 54-11-4856-2751. E-mail: cefybo@websa.com.ar. NITRIC OXIDE: Biology and Chemistry Vol. 5, No. 2, pp. 150 –157 (2001) doi:10.1006/niox.2000.0325, available online at http://www.idealibrary.com on 150 1089-8603/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.