DIFFERENTIAL ROLES OF NEURONAL AND ENDOTHELIAL NITRIC OXIDE SYNTHASES DURING CARRAGEENAN-INDUCED INFLAMMATORY HYPERALGESIA F. TAO, Y.-X. TAO, C. ZHAO, S. DORÉ, W.-J. LIAW, S. N. RAJA AND R. A. JOHNS* Department of Anesthesiology and Critical Care Medicine, Johns Hop- kins University School of Medicine, 600 North Wolfe Street/Blalock 1415, Baltimore, MD 21287, USA Abstract—The present study investigated the role of neuronal nitric oxide synthase (nNOS) in carrageenan-induced inflamma- tory pain by combining genomic and pharmacological strategies. Intrathecal injection of the nNOS inhibitor 7- nitroindazole dose-dependently inhibited carrageenan-induced thermal hyperalgesia in both early and late phases in wild-type mice. However in nNOS knockout mice, carrageenan-induced thermal hyperalgesia remained intact in the early phase but was reduced in the late phase. Spinal Ca 2 -dependent nitric oxide synthase (NOS) activity in nNOS knockout mice was signifi- cantly lower than that in wild-type mice. Following carrageenan injection, although the spinal Ca 2 -dependent NOS activity in both wild-type and knockout mice increased, the enzyme activ- ity in nNOS knockout mice reached a level similar to that in wild-type mice. On the other hand, no significant difference in spinal Ca 2 -independent NOS activity was noted between wild- type and nNOS knockout mice before and after carrageenan injection. Furthermore, intrathecal administration of the endo- thelial NOS (eNOS) inhibitor L-N 5 -(1-iminoethyl)-ornithine in nNOS knockout mice inhibited the thermal hyperalgesia in both early and late phases, though this inhibitor had no effect in wild-type mice. Meanwhile, Western blot showed that eNOS expression in the spinal cord of nNOS knockout mice was up-regulated compared with wild-type mice; immunohisto- chemical staining showed that the spinal eNOS was mainly distributed in superficial laminae of the dorsal horn. Finally, double staining with confocal analysis showed that the en- hanced spinal eNOS was expressed in astrocytes, but not in neurons. Our current results indicate that nNOS plays different roles in the two phases of carrageenan-induced inflammatory pain. In this model, enhanced spinal eNOS appears to compensate for the role of nNOS in nNOS knockout mice. © 2004 IBRO. Pub- lished by Elsevier Ltd. All rights reserved. Key words: nitric oxide synthase, carrageenan, thermal hy- peralgesia, intrathecal injection, peripheral inflammation, central sensitization. Several lines of evidence have accumulated suggesting a role for nitric oxide (NO) as a mediator of inflammation (Nussler and Billiar, 1993; Lyons, 1995; Miller and Grisham, 1995). NO also plays a significant and pivotal role in the development and maintenance of hyperalgesia (Kitto et al., 1992; Malmberg and Yaksh, 1993; Meller and Gebhart, 1993). Unlike most other endogenous chemical mediators, which are stored in vesicles and secreted in a controlled fashion, NO is a diffusible gas that readily per- meates cell membranes. Because NO cannot be stored, its signaling specificity must be controlled at the level of syn- thesis. Indeed, the members of the NO synthase (NOS) family are among the most highly regulated of known enzymes (Bredt, 2003). To date, three isoforms of NOS can be distinguished, namely the constitutive neuronal NOS (nNOS), endothelial NOS (eNOS), and the inducible NOS (iNOS; Forstermann et al., 1995; Mayer, 1995; Paakkari and Lindsberg, 1995). nNOS and eNOS are Ca 2+ /calmodulin-dependent and present in both spinal cord and brain (Wei et al., 1999; Tao et al., 2000), whereas iNOS is functionally Ca 2+ -independent and normally present in macrophages and inflammatory cells. Under certain conditions, iNOS expression is induced in the spi- nal cord and brain (Barker et al., 1998). Considerable evidence has demonstrated that NO and its synthases are involved in the central mechanisms of inflammatory hyper- algesia at the spinal cord level (Moore et al., 1991; Malm- berg and Yaksh, 1993; Meller et al., 1994a; Roche et al., 1996; Handy and Moore, 1998a,b; Osborne and Coderre, 1999; Guhring et al., 2000, 2001; Lin et al., 1997; Tao et al., 2000; Tao and Johns, 2000, 2002). Among the three isoforms of NOS, nNOS is the most abundant in the CNS and is localized mainly in neurons (Dawson and Snyder, 1994). It has been reported that spinal nNOS plays a critical role in the development and maintenance of inflammatory hyperalgesia (Handy and Moore, 1998a,b; Osborne and Coderre, 1999). Peripheral inflammation can up-regulate the expression of nNOS in the spinal cord (Wu et al., 1998, 2001). However, trans- genic mice deficient in nNOS exhibit intact formalin-in- duced pain behaviors (Crosby et al., 1995). Therefore, it seems that nNOS is not essential for the mechanisms of inflammatory hyperalgesia. The exact role of nNOS in the central sensitization of inflammatory hyperalgesia still re- *Correspondence to: R. A. Johns, Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, 720 Rutland Avenue/Ross 361, Baltimore, MD 21205, USA. Tel: +1- 410-614-1848; fax: +1-410-614-7711. E-mail address: rajohns@jhmi.edu (R. A. Johns). Abbreviations: ANOVA, analysis of variance; eNOS, endothelial nitric oxide synthase; GFAP, glial fibrillary acidic protein; iNOS, inducible nitric oxide synthase; L-NAME, N-nitro-L-arginine methyl ester; L-NIO, L-N 5 -(1-iminoethyl)-ornithine; LTP, long-term potentiation; nNOS, neu- ronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide syn- thase; NSE, neuronal specific enolase; PBS, phosphate-buffered sa- line; PWL, paw withdrawal latency; PWLs, paw withdrawal latencies; 7-NI, 7-nitroindazole. Neuroscience 128 (2004) 421– 430 0306-4522/04$30.00+0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.06.038 421