A comparison of the effects of L‐NAME, 7‐NI and L‐NIL on carrageenan‐induced hindpaw oedema and NOS activity

Abstract

Intraplantar injection of carrageenan (150 μl, 1–3% w/v) in the rat resulted in a dose‐related increase in hindpaw weight (oedema) characterized by a rapid ‘early’ phase (up to 2.5 h) response followed by a more sustained ‘late’ phase (2–6 h) response. No change in weight of either the contralateral (i.e. noninjected) hindpaw or hindpaws injected with saline was observed. Six hours after intraplantar injection of carrageenan (1–3% w/v) hindpaw constitutive (i.e. calcium‐dependent) nitric oxide synthase (cNOS) activity (determined ex vivo as the conversion of radiolabelled L‐arginine to radiolabelled citrulline) was increased (e.g. 2% w/v; 0.64±0.08 pmol citrulline mg−1 protein 15 min−1 c.f. 0.08±0.04 pmol citrulline mg−1 protein 15 min−1 in saline‐injected, control animals, n=4, P<0.05). Carrageenan injection also resulted in the appearance in hindpaw homogenates of inducible (i.e. calcium‐independent) nitric oxide synthase (iNOS, e.g. 2% w/v; 0.67±0.14 pmol citrulline mg−1 protein 15 min−1, n=4). Hindpaw cyclic GMP concentration was also significantly increased 6 h after intraplantar injection of carrageenan (e.g. 2% w/v; 379.6±26.8 fmol mg−1 protein c.f. 261.8±42.2 fmol mg−1 protein, in saline‐injected, control animals, n=4, P<0.05). Pretreatment (5–25 mg kg−1, i.p., 30 min before carrageenan, 2% w/v) of animals with L‐N G nitro arginine methyl ester (L‐NAME; isoform nonselective inhibitor of NOS) or 7‐nitro indazole (7‐NI; inhibitor of neuronal NOS, nNOS) caused dose‐related inhibition of both the early (2 h) and late (6 h) phase hindpaw oedema, associated with reduced hindpaw iNOS and cNOS activity and cyclic GMP concentration in animals killed at 6 h. Administration of 7‐NI (5–25 mg kg−1, i.p.) to animals 2.5 h after intraplantar carrageenan (2% w/v) injection (i.e. at the end of the early phase oedema response) produced dose‐related inhibition of the late phase response. Pretreatment (5–25 mg kg−1, i.p., 30 min before carrageenan, 2% w/v) of animals with L‐N6‐iminoethyllysine (L‐NIL, selective inhibitor of iNOS) (5–25 mg kg−1) failed to affect the early phase hindpaw oedema response but did produce a dose‐related inhibition of the late phase oedema. L‐NIL pretreatment also inhibited the carrageenan‐induced increase in both hindpaw iNOS and cNOS activity as well as the rise in hindpaw cyclic GMP concentration. The present experiments demonstrate an anti‐inflammatory effect of 7‐NI as evidenced by inhibition of carrageenan‐induced hindpaw oedema in the rat. Inhibition of nNOS (early phase) and iNOS (late phase) at the site of inflammation most probably accounts for the anti‐inflammatory activity observed. These data suggest a role for nitric oxide synthesized by the nNOS isoform (most probably within sensory nerves) in this model of inflammation. British Journal of Pharmacology (1998) 123, 1119–1126; doi:10.1038/sj.bjp.0701735