Evidence for mediation of L-2-chloropropionic acid-induced delayed neuronal cell death by activation of a constitutive nitric oxide synthase

1 Delayed neuronal cell death elicited by excess excitatory amino acid concentrations has been strongly implicated in many neurological disorders including head trauma, stroke, motor neurone disease and Huntington's disease. We have used the neurotoxin, L-2-chloropropionic acid (L-CPA) to model cellular events in vivo leading to delayed neuronal cell loss which is confined to the cerebellar cortex and can be prevented by inhibitors of nitric oxide synthase such as N-G-nitro-L-arginine methyl ester. 2 Experiments were performed to determine whether the constitutive nitric oxide synthase (NOS) or inducible form of NOS (iNOS) was responsible for the neuronal cell death. Activation of NOS was confirmed by a 39% increase in cerebellar total nitrate and nitrite concentrations in L-CPA-treated brains, as compared to controls (controls = 2.53 +/- 0.10; L-CPA treated = 3.51 +/- 0.31 nmol mg(-1) protein, P<0.01 Student's t tests, n = 6, mean) +/- s.e.mean). Biochemical measurements of total NOS activity were made in homogenates of cerebellum 6 h and 48 h following L-CPA administration, times at which L-CPA concentrations are maximal in brain and a time when there is a high proportion of cerebellar granule cell death, respectively. NOS activity as measured by the amount of [H-3]-arginine converted to [H-3]-citrulline, did not reveal any difference between controls (rats dosed with water) and animals dosed with L-CPA at either 6 or 48 h following dosing. Furthermore the ability of three NOS inhibitors, N-G-nitro-L-arginine, 7-bromo-3-nitroindazole and S-methylisothiourea to block the conversion of [H-3]-citrulline to [H-3]-arginine was identical at 6 and 48 h time points in control and L-CPA treated rats. 3 Quantitative autoradiography using [H-3]-N-G-nitro-L-arginine was used to measure the relative anatomical distribution and amount of NOS enzyme in the cerebellum of controls and L-CPA-treated rats 48 h following dosing. There was no significant alteration in the binding of [H-3]-N-G-nitro-L-arginine to granular and molecular layers of the cerebellum of control and L-CPA-treated rat brains. 4 Western blotting using antibodies against the inducible NOS enzyme failed to detect the protein in cerebellums of L-CPA-treated rats when measured 48 h after L-CPA dosing. 5 In conclusion, the increase in cerebellar nitrate/nitrite concentrations in L-CPA-treated rats provides further evidence for activation of NOS in the cerebellum following administration of L-CPA. The failure to demonstrate an increase in NOS activity at 6 or 48 h in L-CPA-treated rats as compared to controls suggests that the source of nitric oxide responsible for the granule cell death must originate from the constitutive NOS enzyme, probably the neuronal form which is highly enriched in the cerebellum. This hypothesis was further substantiated by Western blotting and quantitative autoradiography.