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 diso rders including head trauma, stroke, motor neurone disease and Hunting ton's disease. We have used the neurotoxin, L-2-chloropropionic acid ( L-CPA) to model cellular events in vivo leading to delayed neuronal ce ll loss which is confined to the cerebellar cortex and can be prevente d by inhibitors of nitric oxide synthase such as N-G-nitro-L-arginine methyl ester. 2 Experiments were performed to determine whether the co nstitutive nitric oxide synthase (NOS) or inducible form of NOS (iNOS) was responsible for the neuronal cell death. Activation of NOS was co nfirmed by a 39% increase in cerebellar total nitrate and nitrite conc entrations 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 measur ements of total NOS activity were made in homogenates of cerebellum 6 h and 48 h following L-CPA administration, times at which L-CPA concen trations are maximal in brain and a time when there is a high proporti on of cerebellar granule cell death, respectively. NOS activity as mea sured by the amount of [H-3]-arginine converted to [H-3]-citrulline, d id not reveal any difference between controls (rats dosed with water) and animals dosed with L-CPA at either 6 or 48 h following dosing. Fur thermore 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 autoradiogra phy 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 si gnificant alteration in the binding of [H-3]-N-G-nitro-L-arginine to g ranular and molecular layers of the cerebellum of control and L-CPA-tr eated rat brains. 4 Western blotting using antibodies against the indu cible 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, t he increase in cerebellar nitrate/nitrite concentrations in L-CPA-trea ted rats provides further evidence for activation of NOS in the cerebe llum 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 compare d to controls suggests that the source of nitric oxide responsible for the granule cell death must originate from the constitutive NOS enzym e, probably the neuronal form which is highly enriched in the cerebell um. This hypothesis was further substantiated by Western blotting and quantitative autoradiography.