Journal of Neitrochemistry Raven Press, Ltd., New York 0 I992 International Society for Neurochemistry

Effect of Nitroprusside (Nitric Oxide) on Endogenous Dopamine Release from Rat Striatal Slices Xing-Zu Zhu and Lu-Guang Luo Department of Pharmacology, Shanghai Institute of Materia Mrdica, Chinese Academy of Sciences, Shanghai, China

Abstract: It is becoming apparent that the synthesis of nitric oxide (NO) from L-arginine not only explains endotheliumdependent vascular relaxation, but is a widespread mechanism for the regulation of cell function and communication. We examined the role of NO on the endogenous dopamine (DA) release from rat striatum. Nitroprusside, in the concentration range of 3-100 pM, induced a dose-dependent increase in the endogenous DA release from rat striatal slices. The maximal response was 330% over the baseline release. A higher concentration of nitroprusside (300 p M ) produced an inhibitory effect on the spontaneous release of

DA. L-Arginine (10 and 100 p M ) , a substrate in the NOforming enzyme system, also produced an elevation of DA release. L-Arginine-induced DA release was attenuated by p-monomethyl-L-arginine. an inhibitor of NO synthase. NADPH ( 1 p M ) , a cofactor of NO synthase, enhanced L-arginine-induced DA release. These results suggest a possible involvement of N O in the DA release process in rat striatum. Key Words: Nitric oxide-Dopamine release-Striatum. Zhu X.-Z. and Luo L.-G. Effect of nitroprusside (nitric oxide) on endogenous dopamine release from rat striatal slices. J. Neiirochern. 59, 932-935 ( 1992).

Initially identified as a mediator for macrophages and endothelial cells, nitric oxide (NO) has been recognized recently as a prominent neuronal messenger (Garthwaite, 1991; Snyder and Bredt, 1991). NOforming enzymatic activity has been demonstrated in brain extracts (Knowles et al., 1989). Immunohistochemical studies of NO synthase in rat brain have shown that the enzyme is localized only in neurons (Bredt et al., 1990). It is highly concentrated in the molecular and granule cell layers of the cerebellum, superior and inferior colliculi, and the granule cell layer of the olfactory bulb. It also occurs in scattered neurons, appearing in apparent isolation throughout the cerebral cortex and corpus striatum (Snyder and Bredt, 1991). A function for NO in the CNS has been suggested by the observation that cell suspensions from the cerebellum of 8-9-day-old rats release a factor that acts upon blood vessels in a fashion reminiscent of NO (Garthwaite et al., 1988). Evidence has also been obtained that NO mediates glutamate stimulation of cyclic GMP formation in the cerebellum via N-methyl-D-aspartate (NMDA) receptors (Bredt and Snyder, 1989; Garthwaite et al., 1989). In this study,

we have examined the effect of nitroprusside on the endogenous dopamine (DA) release from rat striatum. The results provide the first evidence that NO may play a role in the control of DA release in rat striatum. MATERIALS AND METHODS Chemicals DA, dihydroxybenzylamine (DHBA), L-arginine, and p-monomethyl-L-arginine (L-NMMA) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). NADPH was purchased from Shanghai Dong Feng Biochemical Technology Company, Shanghai Institute of Biochemistry (Shanghai, China). Sodium nitroprusside was obtained from Wuhan Second Pharmaceuticals (Wuhan, China).

Superfusion medium Krebs-Henseleit medium (pH 7.4) was used. Unless otherwise noted, this medium contained 113 mM NaCI, 4.7 mM KCI, 1.2 m M MgSO,, 1.2 m M KH,PO,, 25 mM NaHCO,, 2.5 mM CaCI,, 0.029 mM Na,EDTA, 0.29 mM ascorbic acid, and 1 1.1 mMglucose, and was gassed continuously with a 95% o,/% C 0 2 mixture.

Received October 24, I99 1 ;revised manuscript received January 20, 1992; accepted February 27, 1992. Address correspondence and reprint requests to Dr. X.-Z. Zhu at Department of Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 319 Yue-Yang Rd., Shanghai 20003 I. China.

Abbreviations used; DA. dopamine; DHBA. dihydroxybenzylamine: NMDA. N-methy1-D-aspartate; L-NMMA. fl-monomethyl-L-arginine.

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Superfusion system

Data analysis

The chamber with an internal volume of 150 p1 was suspended over a heated water bath that maintained a buffer temperature of 37°C within the chamber. Segments of Teflon tubing were used to connect the buffer reservoir to the manifold tubing of a peristaltic pump (LKB), and Tygon tubing connected the pump to the bottom of the chamber. Solution flowing out from the top of the chamber was collected as described previously (Zhu et al.. 1985). Buffer was passed through the system at a rate of 120 pl/min and maintained at this rate for the entire experiment.

DA release was calculated as nanograms of DA per 100 mg of tissue per 5 min, and the results were expressed as "overflow." Overflow represents the amount of DA released in excess of baseline levels after stimulation. This value was standardized in each experiment by expressing the overflow as a percentage increase over the baseline. All values are expressed as means f SE. Data were subjected to Student's f test, and the acceptable level of significance was 0.05 or less.

Preparation of slices for superfusion

Spontaneous release and nitroprusside-induced overflow of DA DA was released spontaneously from superfused striatal slices at a rate of 0.19 f0.02 ng of DA/l00 mg oftissue/5 rnin (n = 45). Nitroprusside, in the concentration range of 3- 100 pM, produced a dose-dependent increase in the DA release, the maximal response being 330% over the baseline release (Fig. 1). The concentration of DA in the superfusate began to rise during the first 5 rnin of the treatment period and peaked in the next 5 min. When the treatment was terminated after 10 min, overflow began to decline toward baseline within 20 min. However, the spontaneous release of DA was reduced to 50% by 300 pM nitroprusside (data not shown).

Male Sprague-Dawley rats (weighing 200-300 g), bred by Shanghai Experimental Animal Center (Shanghai, China), were killed by decapitation. The corpus striatum was dissected on ice and coronally sliced using a McIlwain-Brinkmann tissue chopper (Mickle Laboratory Engineering Co.. Gomshall, Surrey, U.K.). Slices were then transferred to the chamber and superfused at 37°C with Krebs-Henseleit medium. The medium was bubbled with 95% 0 2 / 5 % C 0 2for at least 30 rnin immediately before use.

Sample collection The slices were first superfused for 60 rnin for equilibration. and then 5-min fractions (600 pI each) of superfusate were collected into tubes which were kept in ice. After two tubes of eluates were collected as pretreatment controls, the slices were treated for 10 min with a Krebs solution containing drugs. From each fraction of superfusate, 500 pl of solution were transferred into a tube containing 50 pl of 1. I M perchloric acid (pH 1.5) with 2.2 m M Na,S,O,. These samples then were frozen at -30°C for later analysis of endogenous DA.

Measurement of DA The DA concentration of superfusate was determined by a modification of the methods of Keller et al. (1976) and Mefford et al. ( 1 980). Superfusate and tissue samples were partially purified by alumina extraction. To each sample were added 20 pl of an internal standard, DHBA, 10 mg of activated alumina, and 150 pl of 2 M Tris buffer (pH 8.6) containing 0.2 m M EDTA. Tubes were capped and shaken for 20 min, centrifuged, and the supernatant aspirated. Dilute Tris buffer (200 p1 of 1 mMTris, pH 8.6) was added to the alumina. After being shaken for 1 rnin and centrifuged for 30 s, the supernatant again was aspirated and the wash procedure repeated. DA and DHBA were then eluted from the alumina with 60 pl of 0.1 A4 perchloric acid. A portion of the eluate (20 pl) was injected onto an HPLC system (Waters Associates). DA and DHBA were separated on a RESOLVE C18 Radial-PAK cartridge, 8 mm X 10 cm (Waters Associates), using a mobile phase (pH 3.0) containing monochloroacetic acid, (+)- 10-camphorsulfonic acid, Na, EDTA, NaOH. and 8% methanol. The flow rate was 1.1 ml/min. Electrochemical detection was accomplished with a BAS LC4B. The potential of the electrode was set at +0.7 V. DA and DHBA were quantified in individual samples by comparing DA and DHBA peak areas with those of standard solutions containing DA and DHBA using a Waters Baseline 8 10 Chromatography Workstation (Waters Associates). The DA value was adjusted for recovery from alumina. which was 60-80% for DA. This system possessed sufficient sensitivity to detect 10-pg quantities of these compounds.

RESULTS

L-Arginine-induced overflow of DA L-Arginine also induced a dose-dependent increase in the DA release (Fig. 2). However, a delay in the increase of DA release was observed. The concentration of DA in the superfusate did not change significantly during the first 5 rnin of the treatment period. During the second 5 rnin of the treatment period, it began to rise and peaked in the next 5 min, after the

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FIG. 1. Efflux of DA from superfused striatal slices that were treated with 3 (0),10 (O),30 (A), and 100 (A)pM nitroprusside for 10 min. The ordinate represents induced DA overflow expressed as a percentage of the basal release, and the abscissa represents successive 5-min collections. Slices were exposed to nitroprusside for 10 min at the beginning of the third collection. Each point is the mean SE of eight separate experiments. *p < 0.05; **p < 0.01, significant difference from the corresponding fraction in the control group.

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X.-Z. ZHU AND L.-G. LUO

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treatment was terminated. The DA overflow induced by 10 pLM L-arginine was inhibited by 50 pM LNMMA, an inhibitor of NO synthase, by 90%(Fig. 2), but was not affected by 1 pM hemoglobin (data not shown). The overflow of DA induced by 10 pM L-arginine was enhanced in the presence of exogenous 1 y M NADPH, a cofactor of NO synthase (Fig. 3).

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DISCUSSION

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The major findings of the present study are as follows: (a) nitroprusside, which generates NO, induces a dose-dependent increase in the endogenous DA release from the striatal slices; (b) L-arginine also produces an elevation of DA release; (c) L-arginine-induced elevation of DA release is attenuated by LNMMA, an inhibitor of NO synthase; and (d) NADPH, a cofactor of NO synthase, enhances L-arginine-induced DA release. Several convergent lines of research in apparently disparate fields have led to a realization that NO has physiological roles. The present result that nitroprusside produces DA release suggests a possible involvement of NO in the release process. Evidence has shown the following: (a) NO is formed from L-arginine in the brain through an enzymic reaction similar to that in vascular endothelial cells, neutrophils, and macrophages; and (b) the enzyme is soluble and NADPH-dependent, requires a divalent cation, and is inhibited by L-NMMA, an inhibitor of NO synthase (Marletta et al., 1988; Knowles et al., 1989; Palmer and Moncada, 1989). Although the precise details of the mechanisms involved in the NO-induced DA release in the striatum remain to be elucidated, evidence warrants the existence in the striatum of an

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FIG. 3. Efflux of DA from superfused striatal slices that were treated with 10 pM L-arginine (0)and 10 pM L-arginine plus 1 pM NADPH (0)for 10 min. The ordinate represents induced DA overflow expressed as a percentage of the basal release, and the abscissa represents successive 5-min collections. Slices were exposed to treatment for 10 min at the beginning of the third collection. Each point is the mean & SE of eight separate experiments. *p i 0.05, significant difference between L-arginine alone and L-arginine plus NADPH.

enzymic system capable of converting L-arginine into NO (Snyder and Bredt, 1991). Recently, NMDA, an excitatory amino acid known to elevate cyclic GMP levels in the brain, has been shown to induce the release of NO from rat cerebellar cells. The release of NO accounts for the elevation in cyclic GMP levels that follows NMDA-receptor activation (Garthwaite et al., 1989). It is possible that in the striatum receptor-mediated activation of the formation of NO from L-arginine leads to stimulation of the soluble guanylate cyclase, resulting in a DA release. To analyze this possibility, further studies must be conducted to determine which type of neurotransmitter receptor is linked to the activation of the formation of NO from L-arginine and to measure the changes of the cyclic GMP level in the striatum following these activations. It must also be determined whether cyclic GMP induces DA release in the striatum, although cyclic GMP has been shown to activate tyrosine hydroxylase in the striatal synaptosomes (Roskoski and Roskoski, 1987).

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Acknowledgment: This work was supported by a grant from the Chinese Academy of Sciences.

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REFERENCES 1

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FIG. 2. Efflux of DA from superfused striatal slices that were treated with 10 (0)and 100 ( 0 )pM L-arginine or I0 pM L-arginine plus 50 pM L-NMMA (A) for 10 min. The ordinate represents induced DA overflow expressed as a percentage of the basal release, and the abscissa represents successive 5-min collections. Slices were exposed to L-arginine or L-arginine plus L-NMMA for 10 min at the beginning of the third collection. Each point is the mean f SE of eight separate experiments. **p 0.01, significant difference from the corresponding fraction in the control group.

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