Scavenging Effects of Dopamine Agonists on Nitric Oxide Radicals

Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1996 International Society for Neurochemistry Rapid Communication Scavenging Effects of Dopamine Agonists on Nitric Oxide Radicals *Sakiko Nishibayashi, *Masato Asanuma, *~MasahjroKohno, *Marvin Gómez-Vargas, and *Norio Ogawa * Department of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, Okayama; and tApplication and Research Center, JEOL Ltd., Tokyo, Japan Abstract: It has recently been considered that free radicals are closely involved in the pathogenesis of Parkinson’s disease (PD), and the level of nitric oxide radical (N0), one of the free radicals, is reported to increase in PD brain. In the present study, we established a direct detection system for N0 in an in vitro •NO-generating system using 3- (2-hydroxy-1 -methylethyl-2-nitrosohydrazino)-N-methyl-1-propanamine as an N0 donor and 2- (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1 -oxyl 3oxide (carboxy-PTIO) by electron spin resonance (ESR) spectrometry and examined the quenching effects of the dopamine agonists pergolide and bromocriptine on the amount of N0 generated. N0 appeared to be scavenged by pergolide and, to a lesser extent, by bromocriptine. In the competition assay, the 50% inhibitory concentration values for pergolide and bromocriptine were estimated to be —23 and 200 j.tM, respectively. It was previously reported that in vivo treatment of pergolide and bromocriptine completely protected against the decrease in levels of striatal dopamine and its metabolites in the 6-hydroxydopamine-injected mouse. Considering these findings, pergolide and probably bromocriptine may also protect against dysfunction of dopaminergic neurons because of its multiple effects; not only does it stimulate the presynaptic autoreceptors, but it also directly scavenges N0 radicals and hence protects against N0related cytotoxicity. This ESR spectrometry method using carboxy-PTIO may be useful for screening other drugs that can quench N0. Key Words: Nitric oxide—Free radical—Pergolide— Dopamine agonist—Scavenging effect—2- (4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide—Electron spin resonance. J. Neurochem. 67, 2208—2211 (1996). Parkinson’s disease (PD) is characterized by a slowly progressive degeneration of dopaminergic neurons in the nigrostriatal system. Levodopa is the commonly used drug of choice for the treatment of PD patients. The ergot derivatives, pergolide and bromocriptine, have pharmacological potency to act as dopamine (DA) receptor agonists, and they have been proposed as alternative drugs to overcome various problems attributable to long-term treatment of PD with 1evodopa and to prevent observable clinical progression of the disease (Lieberman et al., 1983; Jankovic and Orman, 1986; Goetz, 1992). 2208 Oxidative stress may play a principal role in the degeneration of DA neurons in the PD, and the following data have been reported: (a) Levodopa has the potential to become a levodopa radical (Ogawa et al., 1993). (b) There is an increase in iron deposition, which promotes free radical generation (Dexter et al., 1989b). (c) A high level of lipid peroxi- dation occurs (Dexter et al., 1989a). (d) Decreased activity of complex I in the mitochondrial respiratory chain is observed (Schapira et al., 1990). (e) Glutathione, glutathione peroxidase, and catalase levels are reduced in the brain of PD (Ambani et al., 1975; Kish et al., 1985). In the CNS, attention has recently been focusedon the nitric oxide radical (N0) as a neurotoxic mediator of neuronal cell death (Dawson et al., 1991; Zhang et al., 1994) and as a neuromodulator that regulates the release and uptake of neurotransmitters (PogUn and Kuhar, 1994; Ramassamy et al., 1994). More recently, an increased density ofNADPH-diaphorase-positive glial cells in the mesencephalon ofparkinsonian patients has been identified (Hunot et al., 1996), and the concentration of nitrite, a metabolite of nitric oxide, was also increased in the CSF of PD patients (Qureshi et al., 1995). Taken together, these results support the possibility that production of N0 is increased in PD. To our knowledge, the properties of currently used therapeutic agents on N0 have not been discussed thoroughly, basically because ofthe methodological problems arising in the N0 assays. To clarify the effects of DA agonists on N0, in the present study we report a simple method for the direct detection of N0 in an in vitro N0-generating system using 2-(4- Resubmitted manuscript received July 30, 1996; accepted August 5, 1996. Address correspondence and reprint requests to Dr. N. Ogawa at Department of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, 2-5-1 Shikatacho, Okayama 700, Japan. Abbreviations used: carboxy-PT1, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl; carboxy-PTIO, 2-(4-carboxyphenyl ) -4,4,5,5- tetramethylimidazoline-1-oxyl 3-oxide; DA, dopamine; ESR, electron spin resonance; N0, nitric oxide radical; NOC7, 3-(2-hydroxyl-methylethyl-2-nitrosohydrazino) -N-methyl-i -propanamine; 02, superoxide anion; 0H, hydroxyi radical; 6-OHDA, 6-hydroxydopamine; PD, Parkinson’s disease. DA AGONISTS QUENCH NITRIC OXIDE RADICAL 2209 carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3oxide (carboxy-PTIO) as the N0 detecting reagent and 3(2-hydroxy- 1 -methylethyl-2-nitrosohydrazino)-N-methylI-propanamine (NOC7) as the N0 donor by electron spin resonance (ESR) spectrometry and examine the quenching activity ofpergolide and bromocriptine on the generated N0. MATERIALS AND METHODS Reagents The N0-detecting agent carboxy-PTIO and NOC7 were purchased from LABOTEC (Tokyo, Japan) and from Dojin Laboratories (Kumamoto, Japan), respectively. The DA agonists pergolide mesylate and bromocriptine mesylate were supplied by Eli Lilly Co. (Indianapolis, IN, U.S.A.) and by Sandoz Pharmaceutical Co. Ltd. (Basel, Switzerland), respectively. The metabolites of pergolide, pergolide sufoxide and pergolide sulfone, were also supplied by Eli Lilly Co. Methyicellulose, used as the vehicle for pergolide and bromocriptine, was purchased from Wako Pure Chemical Industries (Tokyo). 4-Hydroxy-TEMPO was purchased from Sigma (St. Louis, MO, U.S.A.). ESR spectrometry in N0-generating system Carboxy-PTIOwas dissolved in 250mM phosphate buffer solution (pH 7.6), and NOC7 was dissolved in 1 mM NaOH. In a test tube, 0.5% methylcellulose and 5 ~iM NOC7 were mixed for 30 mm at room temperature, and then 5 1.sM carboxy-PTIO was added to the mixture. The spectra were recorded every 20 mm, up to 190 mm after addition of carboxy-PTIO, with an ESR spectrometer (model JES-FR3O; JEOL Ltd., Tokyo) using a flat quartz cuvette. The signal intensities were evaluated by the relative peak height of the 2~ first signal of the 2-phenyl-4,4,5,5-tetramethylimidazoline1-oxyl (carboxy-PTI) to thestandard intensitytoofcorrect Mn signal, which was usedspin as adduct the internal for measurement error. Regarding the conditions of the ESR spectrometer to estimate the N0 content, the magnetic field, power, modulation frequency, modulation amplitude, response time, temperature, amplitude, and the sweep time were 335.6 ±5 mT, 4 mW, 9.41 GHz, 1 X 0.1 mT, 0.1 s, 25°C, 1 x 250, and 1 mm, respectively. Pergolide and bromocriptine in NO-generating system Pergolide and bromocriptine were suspended in 0.5% methylcellulose. Pergolide or bromocriptine (final concentrations ranging from 10 nM to 1 mM in triplicate) and 5 1.tM NOC7 were mixed in a test tube and then added with 5 ~iM carboxy-PTIO. The signal intensities of the carboxy-PTI were measured 90 mm after addition of carboxy-PTIO under the same ESR conditions as described above. RESULTS FIG. 1. ESR signals of carboxy-PTIO and carboxy-PTI in the N0-generating system and the effects of pergolide: (A) carboxy-PTIO without NOC7, (B) control (0.5% methylcellulose), (C) 10 1iM pergolide, (D) 33 ~sMpergolide, and (E) 1 mM pergolide. B—E: At 90 mm after addition of carboxy-PTIO to the mixture of NOC7 and 0.5% methylcellulose or 10 jsM, 33 1iM, or 1 mM pergolide, carboxy-PTI signals were recorded by ESR spectrometry. The dot indicates a monitored carboxy-PTI signal. mT). In the process of their transformation, carboxy-PTIO and carboxy-PTI present nine mixed signals, including two components of each. The carboxy-PTI signal indicates the N0 generation. It is easy to distinguish between the signals of carboxy-PTIO (Fig. IA) and carboxy-PTI (Fig. lB. dot) using the ESR spectrometer, because these radicals have different g values. In this way, the concentration of N0 can be calculated, and it was confirmed by comparison with a suitable standard (4-hydroxy-TEMPO). After addition of carboxy-PTIO to the mixture of 0.5% methylcellulose and NOC7, a time-dependent increase in the carboxy-PTI signal, which reflects N0 production from NOC7, was observed by ESR. The NO generated was first detected 10 mm after addition of carboxy-PTIO and steadily increased until it reached a plateau (Fig. 2). According to this result, the following experiments were performed at 90 mm after addition of carboxy-PTIO. ESR quantification of N0 produced Pergolide and bromocriptine in N0-generating system NOC7, developed as an NO donor, is stable in an alkaline solution, and it starts to produce N0 under neutral pH (Harabie and Klose, 1993). The ESR signal of carboxy-PTIO is characterized by five lines (1:2:3:2:1, a~= 0.82 mT; Fig. 1A). The generated N0 from NOC7 was oxidized by carboxy-PTIO, carboxy-PTIO was then transformed to carboxyPTI, and N02 was produced from N0. The carboxy-PTI shows seven-line signals when carboxy-PTIOhas been completely converted to carboxy-PTI (aNt = 0.92 mT, aN2 = 0.54 Pergolide at concentrations of 10 nM up to 1 ~aMhad no scavenging activity in the N0-producing system. After addition of 3 ,.sM pergolide, N0 quenching began dosedependently, and a pergolide concentration of 333 ,aM completely scavenged the generated N0 (Figs. lB — I E and 3). On the other hand, bromocriptine showed no apparent quenching activity at 10 nM— 100 tiM. However, at the relatively high doses of 333 ,uM and 1 mM, bromocriptine showed a quenching effect on NO (Fig. 3). The IC50 for J. Neurochem., Vol. 67, No. 5, 1996 S. NISHIBAYASHI ET AL. 2210 FIG. 2. Time course of N0 generation following addition of carboxy-PTIO to NOC7 and 0.5% methylcellulose. Carboxy-PTI signals were immediately recorded every 20 mm up to 190 mm after carboxy-PTIO addition to the mixture of NOC7 and 0.5% methylcellulose. Data are mean ± SEM (bars) values of triplicate assays. pergolide and bromocriptine was estimated to be —-~23and 200 ~tM, respectively (see Fig. 3, inset). To evaluate the kinetics of the reaction between NO and DA agonists, we modified the kinetic competition model for our experiment based on the obtained results (Mituta et al., 1990). We assumed that the reduction of carboxy-PTIO to carboxy-PTI was linked and competed with the reaction of DA agonists with generated N0, as shown in Fig. 4. The reactions can be described as follows: carboxy-PTIO + NO —L.. carboxy- PTI + N0 2 and drug + N0 —~ drug- N0, where k1 and k2 are the second-order rate constant for each reaction, respectively. The reaction rate constant (k2) for the drug can be expressed by the equation k2 = k1 [carboxy-PTIO]/IC50 (Mituta et al., 1990), using the IC50 for each drug and k1 = 1.01 x l0~M’ s’ from Hogg et al. (1995). Therefore, we can estimate the value of k2 for pergolide and bromocriptine to be -‘-2.2 X l0~and 2.5 X 102 M~s~, respectively (see Fig. 3, inset). In this system, these two drugs do not interfere with any reagent. There are no chemical reactions between carboxy-PTIO and pergolide at any dose or bromocriptine at the 10 nM— 100 ~.tMconcentration range; however, 333 ~M and 1 mM bromocriptine reduced the signal of carboxy-PTIO itself to 75 and 65%, respectively (data not shown). Therefore, we adjusted the resulting signal intensity of carboxy-PTI at these two points (Fig. 3). FIG. 3. Quenching effect of pergolide (0) and bromocriptine (.) on the formation of carboxy-PTI. ESR signals of carboxy-PTI were recorded 90 mm after addition of carboxy-PTIO to the mixture of NOC7 and pergolide or bromocriptine (final concentration ranging from 10 nM to 1 mM). Data are means of the percent signal intensity of carboxy-PTI in three independent experiments. The shaded circles represent the adjusted percentages of two doses of bromocriptine (333 1sM and 1 mM), because they reduced the signal of carboxy-PTIO itself to 75 and 65%, respectively. Inset: Calculated values of IC50 and k2 for pergolide and bromocriptine. J. Neurochem., Vol. 67. No. 5, 1996 FIG. 4. Schematic model of the reaction of DA agonists with generated N0 linked with the reduction of carboxy-PTIO. DISCUSSION The current view is that free radicals are involved in the pathogenesis of Parkinson’s disease. An increased N0 level has been reported in the brain of patients with PD (Qureshi et al., 1995; Hunot et al., 1996). In the present study, we established a method for directly detecting NO in an in vitro ~NO-generating system with the NO donor NOC7 by ESR spectrometry using carboxy-PTIO (Fig. 2) and examined the quenching effects of pergolide and bromocriptine on the generated N0. According to the results, both pergolide and bromocriptine quenched N0, but pergolide did so to a much greater extent (Figs. 1 and 3). We also examined the effects of the sulfoxide and the sulfone metabolites of pergolide in this NO-generating system. There are no significant differences in the scavenging ability against N0 among the three types of pergolide compounds (data not shown). Bromocriptine specifically acts on D2 receptors, whereas pergolide has high affinity for D2, D3, and Dl receptors (Sokoloff et at., 1990; Fuller and Clemens, 1991; Seeman and Van Tol, 1994). It is known that some neurotoxins like 6-hydroxydopammne (6-OHDA) and MPTP, which exert effects on DAergic neurons by autoxidation, can be converted into or produce superoxide anion (02) and hydroxyl radical (OH) (Heikkila and Cohen, 1972; Sinha et al., 1986). We previouslyreported that in vivo treatment with bromocriptine completely protected against the 6-OHDA-induced decrease in DA levels in the mouse striatum (Ogawa et al., 1994). Although bromocriptine showed dose-dependent scavenging activity in an in vitro OH-generating system (Ogawa et al., 1994), bromocriptine had a lesser quenching effect on the N0-generating system than pergolide in the present study (Fig. 3). Besides its stimulation on the pre- and postsynaptic D2 receptors, bromocriptine may also protect DAergic neurons by its direct scavenging activities on the cytotoxic 0H radicals, On the other hand, in vivo treatment with pergolide also completely protected against the decrease in content of striatal DA and its metabolites in the 6-OHDA-injected mouse (Asanuma et al., 1995). Similarly, chronic administration with pergolide protected against age-related decreases in levels of nigrostriatal DAergic neurons in the rat brain (Felten et al., 1992). Therefore, pergolide protection against 6-OHDAinduced DAergic dysfunction to normalize the decreased DA synthesis or DA storage may be due in part to inhibition of the formation of toxic 6-OHDA metabolites or of 6-OHDA uptake by its stimuiatory effect on presynaptic autoreceptors. Furthermore, in the present study, we found that pergolide scavenges N0 radicals. In recent reports, it has been proposed that N0 radicals rapidly react with 02 to produce a powerful oxidant, peroxynitrite, and consequently leads to initiation of the lipid peroxi- DA AGONISTS QUENCH NITRIC OXIDE RADICAL dation cascade and subsequent cell death (Lipton et al., 1993; Beckman, 1994). Treatment with pergolide in vivo reportedly increased Cu/Zn superoxide dismutase activity in the rat striaturn (Clow et al., 1992). Considering these findings, pergolide may well protect against and ameriolate existing dysfunction in DAergic neurons because of its multiple effects, not only by stimulating the presynaptic autoreceptors and inhibiting peroxynitrite production via induction of superoxide dismutase activity, but also by its direct scavenging activities on NO radicals to protect NO-related cytotoxicity. The quenching activities of pergolide on the in vitro NOgenerating system was ‘-‘~l0-foldhigher than that of bromocriptine in the present study (Fig. 3). 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