Increased Striatal Dopamine Efflux in Vivo Following Inhibition of Cerebral Nitric Oxide Synthase by the Novel Monosodium Salt of 7-Nitro Indazole M.T

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Brftish Joumal of Pharmacology (1995) 114, 257-258 r 1995 Stockton Press All rights reserved 0007-1188/95 $9.00 x SPECIAL REPORT Increased striatal dopamine efflux in vivo following inhibition of cerebral nitric oxide synthase by the novel monosodium salt of 7-nitro indazole M.T. Silva, S. Rose, J.G. Hindmarsh, G. Aislaitner, J.W. Gorrod, P.K. Moore, 'P. Jenner & C.D. Marsden

Neurodegenerative Diseases Research Centre, Pharmacology Group, Biomedical Sciences Division, King's College London, Manresa Road, London The role of nitric oxide (NO) in striatal dopamine release has been controversial. Most NO synthase inhibitors affect more than one isoform of the enzyme and exert vasoconstrictor effects which may also affect striatal dopamine function. We now report on the effect of a soluble monosodium salt of the selective brain NO synthase inhibitor 7-nitro indazole (7-NINA). Using 7-NINA the first study of selective inhibition of the brain isoform of NO synthase on dopamine efflux in rat striatum was undertaken by use of in vivo microdialysis. Perfusion with 7-NINA (1 mM) increased striatal dopamine efflux. The effect of 7-NINA was partially antagonized (67%) by co-perfusion with L-arginine (1 mM), the precursor of NO formation in vivo. This suggests that 7-NINA induces a competitive inhibition of NO synthase activity. These data show that endogenous NO has an inhibitory effect on striatal dopamine efflux in vivo. Keywords: Nitric oxide synthase; dopamine; microdialysis; 7-nitro indazole; striatum; L-arginine

Introduction Nitric oxide (NO) acts as a neuromodulator in evaporated to dryness in a rotary film evaporator at 35°C the brain and functions as a retrograde neuronal messenger under reduced pressure. The red solid formed was dissolved following N-methyl-D-aspartate (NMDA) receptor stimula- in the minimum amount of methanol, filtered and again tion (Hanbauer et al., 1992; Lei et al., 1992). In this capacity, evaporated to remove water. This procedure was repeated NO may mediate cortico-striatal glutamatergic modulation of once more with methanol and then twice with diethyl ether. striatal dopamine release (Rose et al., 1994a). However, there Structure and purity was determined by thin layer is confusion over the manner in which NO modulates chromatography, nuclear magnetic resonance and mass spec- dopamine release. In vitro studies using tissue slices have troscopy. The resulting salt was shown to be the shown NO to increase dopamine efflux in the striatum monosodium salt of 7-NI and the solid was determined to be (Lonart et al., 1993), whereas in vivo microdialysis studies 96% pure. 7-NINA was prepared freshly every two weeks using NO synthase inhibitors or exogenous NO suggest the and stored in a dessicator at 4°C. opposite to be true (Guevara-Guzman et al., 1994; Rose et al., 1994b). Microdialysis procedure Male Wistar rats (280-320 g, Ban- The use of NO donors such as S-nitroso-N-acetylpeni- tin and Kingman, Hull, UK) were anaesthetized with chloral cillamine, S-nitrosoglutathione and sodium nitroprusside in hydrate (500 mg kg-', i.p.) and anaesthesia was maintained brain is limited as the carrier molecules (penicillamine, throughout the experiment. Microdialysis probes (3 mm glutathione and ferricyanide respectively) alter striatal membrane; Hospal AN 69 HF) were implanted into the neurotransmitter release in vivo (Guevara-Guzman et al., striatum by standard stereotaxic techniques (coordinates 1994). Similarly, the use of NO synthase inhibitors in brain is A + 2 mm, L + 3 mm from bregma and V-6.5 mm from complicated by their effect on peripheral and cerebral blood dura; Pelligrino et al., 1979). Body temperature was main- flow. tained at 37°C with a homeothermic blanket (Harvard Recently, Moore and colleagues (1993) reported inhibition Apparatus Ltd, Watford, UK). The microdialysis probes of neuronal NO synthase activity by 7-nitro indazole (7-NI) were continuously perfused with artificial CSF (composition, with no effect on mean arterial blood pressure. This suggests mM: NaCl 138, KCI 5, CaCl2 1.2, NaH2PO4 1, NaHCO3 10; that 7-NI is a selective inhibitor of neuronal NO synthase so pH 7.5) at a rate of 2 ,sl min-'. At least 1 h following implan- providing a tool with which to study the effects of NO in tation, 10 min fractions of dialysate (20 ld) were collected brain. However, until now the insolubility of 7-NI at concen- into 0.25 M acetic acid (25 jil). Following a 1 h period to trations above 100l M in physiological media has precluded determine basal dopamine efflux, 7-NINA (1 mM), L-arginine its use in in vivo studies of neurotransmitter release. For this (1 mM), or a combination of 7-NINA (1 mM) and L-arginine reason, we report on the preparation of a more soluble (1 mM) were included into the artificial CSF for a period of monosodium salt of 7-NI, termed 7-NINA, and on the effect 90 min. Control animals were perfused continuously with of selective inhibition of brain NO synthase on striatal artificial CSF. Dialysate samples were analysed for levels of dopamine release. dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) by high performance liquid Methods Preparation of monosodium salt of 7-nitroindazole chromatography (h.p.l.c.) with electrochemical detection. (7-NINA) 7-NI (1.625 mmol; Lancaster Synthesis Ltd, Morecambe, UK) was dissolved in 20 ml of methanol:chloro- Results Perfusion of rat striatum with 7-NINA immediately form (3:1 v:v). To this was added 3.385 ml of 0.48 M sodium increased dopamine efflux to between 203 and 229% of basal hydroxide (1.625 mmol). The colour of the solution changed and control values (Figure la). In the presence of L-arginine, immediately from yellow to red. The red solution was 7-NINA increased striatal dopamine efflux to between 132 and 191% of basal values (Figure la), such that total dopamine efflux over the 90 min period of infusion was ' Author for correspondence. significantly less (67%) than that produced by 7-NINA alone 258 M.T. Silva et al Special report

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0 *~~~~~~~~~~~~~~. 0 ~~~~~~~~~~~0 X E~~~~~~~~~~~~~~~~( 200~~~~~~~~~~~ * * ~ ~ ~ .E150 rE C.L (U 0.. 0 C0 100- *.* - 2 5 0

50 1-. 0 3 6 9 1'2 15 Cohtrol L-ARG 7-NINA 7-NINA Fraction number + L-ARG

Figure 1 (a) The effect of the monosodium salt of 7-nitro indazole (7-NINA, U, n = 8), 7-NINA plus L-arginine (, n = 10), L-arginine (A, n = 9) and artifical CSF (0, n = 9) on dopamine efflux from rat striatum in vivo. Results are expressed as % basal efflux ± s.e.mean. *P<0.05 compared to basal dopamine efflux (Dunn's test); NP < 0.05 compared to control (Dunn's test). (b) The effect of 7-NINA (n = 8), 7-NINA plus L-arginine (L-Arg) (n = 10), L-arginine (n = 9) and artificial CSF (control, n = 9) on total dopamine efflux above basal over the 90 min perfusion period. Results are expressed as mean ± s.e.mean. *P < 0.05 compared to artificial CSF (Dunn's test); tP <0.05 compared to L-arginine, $P< 0.05 compared to 7-NINA (Dunn's test).

(Figure Ib). Perfusion with artificial CSF and L-arginine ever, they are in agreement with Guevara-Guzman et al. alone did not alter striatal dopamine efflux compared to (1994) who reported decreased dopamine release in vivo fol- basal values (Figure 1 a and b). DOPAC and HVA levels lowing local administration of NO donors using mic- were unaltered (data not shown). rodialysis. The reason for the difference between the in vitro and in vivo studies is not known. It may be due to an Discussion The novel monosodium salt of 7-NI, 7-NINA, inhibition of NO release following cortical glutamatergic inhibits cerebellar NO synthase activity in vitro with a similar stimulation of striatal NMDA receptors which can occur in IC50 to that reported for 7-NI (Rose et al., 1994b). Inhibition the in vivo but not the in vitro situation (Lei et al., 1992). of striatal NO synthase by 7-NINA resulted in increased In conclusion, for the first time we have shown that selec- striatal dopamine efflux which was partially inhibited by tive inhibition of brain NO synthase activity by 7-NINA L-arginine, the substrate for NO synthase. This inhibition of increases striatal dopamine efflux in vivo, suggestig an 7-NINA-induced increased striatal dopamine efflux is consis- inhibitory influence of endogenous NO on dopamine release. tent with an action as a competitive antagonist of brain NO This may be a direct effect of NMDA-evoked NO on striatal synthase activity and confirms that 7-NINA exerts its action dopaminergic neurones, or mediated by striatal cholinergic or on dopamine release via the inhibition of NO formation. GABAergic interneurones. These data suggest that endogenous NO has an inhibitory influence on striatal dopamine release in vivo. This is contrary to the findings of Lonart et al. (1993) who reported increased This study was supported by the Medical Research Council. M.T.S. dopamine efflux from striatal slices by donors of NO. How- holds a M.R.C. Training Fellowship.

References

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