Systematic Administration of Iptakalim, anATP-Sensitive Potassium Channel Opener,Prevents Rotenone-Induced Motor andNeurochemical Alterations in Rats

Yong Yang,1,2 Xing Liu,2 Yan Long,2 Fang Wang,2 Jian-Hua Ding,2 Su-Yi Liu,2

Ye-Hong Sun,2 Hong-Hong Yao,2 Hai Wang,3 Jie Wu,2,4 and Gang Hu1,2*1Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, PR China2Department of Pharmacology and Neurobiology, Nanjing Medical University, Nanjing,Jiangsu, PR China3Beijing Institute of Pharmacology and Toxicology, Beijing, PR China4Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, Arizona

Our previous studies revealed that iptakalim, a novelATP-sensitive potassium channel opener, has a signifi-cant neuroprotective function against ischemia in vivoor rotenone-induced neurotoxicity in vitro. To investi-gate the potential pharmaceutical benefit of ATP-sen-sitive potassium channel openers on neurodegenera-tive diseases, we studied the effects of iptakalimand diazoxide, a selective mitochondrial ATP-sensitivepotassium channel opener, on the rotenone-inducednigrostriatal degeneration in rats. Iptakalim (1.5 mg/kg/day, orally) or diazoxide (1.5 mg/kg/day, orally) alonewas administered to rats for 3 days, and then for4 weeks was used daily with an injection of rotenone(2.5 mg/kg/day, subcutaneously) 1 hr later each time.The results showed that rotenone-infused rats exhibitedparkinsonian symptoms and had dopamine depletion inthe striatum and substantia nigra. Pretreatment withiptakalim or diazoxide prevented rotenone-induced cat-alepsy and the reduction of striatum dopamine con-tents. Moreover, iptakalim and diazoxide reduced theenzymatic activities and mRNA levels of inducible nitricoxide synthase elicited by chronic administration ofrotenone. These neuroprotective effects of iptakalimand diazoxide were abolished by 5-hydroxydecanoate,a selective mitochondrial ATP-sensitive potassium chan-nel blocker. In conclusion, our data suggested that mito-chondrial ATP-sensitive potassium channels might play akey role in preventing both parkinsonian symptoms andneurochemistry alterations induced by rotenone in rats.The selective activation of mitochondrial ATP-sensitivepotassium channels may provide a new therapeutic strat-egy for prevention and treatment of neurodegenerativedisorders such as Parkinson’s disease.VVC 2005 Wiley-Liss, Inc.

Key words: diazoxide; iptakalim; inducible nitric oxidesynthase; mitochondrial ATP-sensitive potassium chan-nels; Parkinson’s disease

Iptakalim is a novel ATP-sensitive potassium channel(KATP) channel opener used as an antihypertensive drug(Wang, 1998, 2003). Our recent research has revealed thatiptakalim exhibits significant neuroprotection, not only inpromoting behavioral recovery but also in protecting neu-rons against necrosis and apoptosis in different animal mod-els of stroke, as well as in cultured cells (Wang et al., 2004).We also found a neuroprotective role of iptakalim againstrotenone-induced PC12 cells death by activating mito-KATP channels, which may also be employed to potentiallytreat neurodegenerative disease, such as Parkinson’s disease(PD) (Yang et al., 2004).

PD is a common neurodegenerative disorder ofunknown etiology. Genetic mutations and environmentalfactors have been considered thus far as contributors tocertain forms of this disorder (Steece-Collier et al., 2002).

Contract grant sponsor: National Natural Science Foundation of China;

Contract grant number: 39970846; Contract grant sponsor: National

Ministry of Science and Technology of China; Contract grant number:

969010101.

*Correspondence to: Gang Hu, MD, PhD, Department of Pharmacology

and Neurobiology, Nanjing Medical University, 140 Hanzhong Road,

Nanjing 210029, Jiangsu, PR China. E-mail: ghu@njmu.edu.cn

Received 8 December 2004; Revised 26 January 2005; Accepted 1

February 2005

Published online 28 March 2005 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.20467

Abbreviations: 5-HT, serotonin; 5-HIAA, 5-hydroxyindoleaceticacid; 5-

HD, 5-hydroxydecanoate; ANOVA, analysis of variance; DA, dopamine;

DMSO, dimethylsulfoxide; DOPAC, dihydroxyphenylacetic acid; eNOS,

endothelial nitric oxide synthase; HVA, homovanillic acid; Ipt, iptakalim;

iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; mito-

KATP channels, mitochondrial ATP-sensitive potassium channels; NE,

noradrenalin; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide

synthase; KATP channels, ATP-sensitive potassium channels; Rot, rote-

none; PD, Parkinson’s disease; PEG, polyethylene glycol; Sal, saline;

TNF-a, tumor necrosis factor-a; Veh, vehicle.

Journal of Neuroscience Research 80:442–449 (2005)

' 2005 Wiley-Liss, Inc.

Its pathologic hallmarks are loss of dopaminergic neuronsin the substantia nigra pars compacta and the presence ofneuronal proteinaceous cytoplasmic inclusions, commonlyknown as Lewy bodies (Fahn and Przedborski, 2000).Increasing evidence demonstrates that the ongoing inflam-matory process in the brain is critically involved in thepathogenesis of PD (McGeer et al., 1988, 2001; Vila et al.,2001). Microglia, the major resident immunoreactive pop-ulation in the brain, are activated promptly in some patho-logic conditions and produce an excess of proinflamma-tory factors, including nitric oxide (NO), tumor necrosisfactor-a (TNF-a), interleukin 1b (IL-1b), and reactiveoxygen species, which may trigger or exacerbate neuronaldeath (Hunot et al., 1996; Le et al., 2001).

Rotenone, an inhibitor of mitochondrial NADHdehydrogenase, has been used widely as a herbicide andfish killer in reservoirs (Horgan et al., 1968; Gutman etal., 1970). Chronic administration of rotenone reprodu-ces some features of PD: loss of dopaminergic neuronsin the substantia nigra (SN) as a result of microgliaactivation in this region and the formation of Lewybody-like inclusions in the substantia nigra. Behavio-rally, rotenone-infused rats exhibit reduced mobility,flexed posture, and in some cases, rigidity and catalepsy(Betarbet et al., 2000; Alam and Schmidt, 2002; Shereret al., 2003; Fleming et al., 2004; Zhu et al., 2004).Rotenone is therefore a suitable neurotoxin to investi-gate new possible therapeutic compounds for PD.

Our present study showed that iptakalim could protectrotenone-induced motor and neurochemical alterations inrats by activation of mitochondrial KATP channels, whichmay inhibit the glial inducible nitric oxide synthase (iNOS)expression and alleviate the product of NO, thereby protect-ing dopaminergic neuron against rotenone neurotoxicity.

MATERIALS AND METHODS

Materials

Rotenone, diazoxide, l-dopa and all the solvents forHPLC coupled to an electrochemical detector (HPLC-ECD)were obtained from Sigma (St. Louis, MO). Sodium 5-hydroxydecanoate (5-HD) was purchased from Valeant Phar-maceuticals International (Costa Mesa, CA). All PCR primerswere purchased from Sangon (Shanghai, China). All reagentsfor reverse transcription (RT)-PCR were purchased fromPromega (Madison, WI).

Animals and Treatment

Male Sprague-Dawley rats aged 7 weeks were chosenfor experiments. At the beginning of the experiments, theyweighed 220–240 g. Rats were kept six to a cage understandard laboratory conditions with free access to standard lab-oratory food and tap water, constant room temperature of228C, 50–60% humidity, a natural day–night cycle, etc. Allexperiments were carried out according to the National Insti-tutes of Health Guide for the Care and Use of LaboratoryAnimals (publication no. 85-23, revised 1985) and the Guide-lines for the Care and Use of Animals in NeuroscienceResearch by the Society for Neuroscience and approved by

IACUC (Institutional Animal Care and Use Committee ofNanjing Medical University).

Dimethylsulfoxide (DMSO)/polyethylene glycol (PEG;1/1) was used as vehicle for rotenone. Diazoxide was dissolvedin 100% DMSO as vehicle and diluted with sterile saline to aconcentration of 1 mg/ml (0.2% DMSO) for administration torats. Iptakalim and 5-HD, a selective mito-KATP channelblocker, was dissolved in saline.

Rats were pretreated with iptakalim (1.5 mg/kg/day,orally) or diazoxide (1.5 mg/kg/day, orally) with or without 5-HD (3.0 mg/kg/day, orally) for 3 days. After the 3 days, theywere administered daily for 4 weeks with an injection of rotenone(2.5 mg/kg/day, subcutaneously) 1 hr later each time. Controlrats were treated with an appropriate solvent of rotenone.

Behavioral Tests

The catalepsy test was chosen for the assessment of theeffects of drugs on rotenone-induced parkinsonian symptoms.The rats were placed with both forepaws on bars 9 cm aboveand parallel from the base and were in a half-rearing position.Latency time of the removal of the paw was recorded.

Neurochemistry Procedure

Animals were sacrificed after the behavioral test and theirbrains were removed rapidly (within 25–40 sec) and placed inice-chilled 0.9% NaCl solution for 1 min. Tissue samples of thestriatum and substantia nigra were taken bilaterally, weighedimmediately, and stored in liquid nitrogen until assay.

Frozen tissue was sonicated in 0.1 M HClO4 containing0.5% Na2EDTA and 0.1% Na2S3O5 (30 ml for every 10 mgtissue) and centrifuged at 12,000 � g for 20 min at 48C.Supernatant was centrifuged again for another 10 min underthe same conditions. Sample supernatant was analyzed directlyfor monoamine neurotransmitter contents, including dopa-mine (DA), dihydroxyphenylacetic acid (DOPAC), homova-nillic acid (HVA), noradrenalin (NE), 5-HT (serotonin), and5-hydroxyindoleaceticacid (5-HIAA, metabolite of 5-HT) byreverse-phase HPLC-ECD.

HPLC-electrochemical detector (ECD) (BAS Corporation;USA) for monoamine neurotransmitters consisted of BASLC-4C, a reverse-phase C18 column (Ultrasphere ODS, 4.6 mm� 250 mm, 5 mm), chromograph interface DA-5, and solventdelivery system. The mobile phase consisted of 0.1 M citrate, 75mM Na2HPO4, 0.1 mM EDTA, 1.0 mM 1-heptanesulfonicacid, and 10% methanol, pH 3.9. Chromatograms were analyzedwith the aid of a chromatographic data system (BAS) (Yanget al., 2004). Sample results were expressed as picogram permilligram (pg/mg) of tissue.

NOS Activity Assay

NO is synthesized from l-arginine by the enzyme NOS(Knowles and Moncada, 1994). Three main types of NOSenzyme are associated with differing roles: endothelial NOS(eNOS), which is expressed in endothelial cells; iNOS, whichis not expressed in the central nervous system (CNS) exceptin the inflammatory state after lipopolysaccharide or cytokineinduction in glial cells; and neuronal NOS (nNOS), which islocalized in neurons (Vincent and Kimura, 1992). In this

Neuroprotective Effects of Iptakalim 443

study, NOS activities in the striatum and substantia nigrawere determined as described previously (Zhu et al., 2002).Briefly, brain samples were homogenized in a buffer contain-ing 50 mM HEPES, pH 7.4, 1 mM dl-dithiothreitol, l mMEDTA, 0.32 M glucose, 10 mg/ml leupeptin, 10 mg/ml soy-bean trypsin inhibitor, 2 mg/ml aprotinin, and 1 mM phe-nylmethylsulfonyl fluoride. The homogenates were centri-fuged at 1,200 � g for 20 min at 48C. NOS activities insupernatants were measured spectrophotometrically using acommercially available kit (Juli Bioengineering Co., Nanjing,China) that is based on the oxidation of oxyhemoglobin tomethemoglobin by NO as described previously (Knowleset al., 1990). Ca2þ-independent NOS activity was measuredby adding EGTA (3 mM) to chelate-free Ca2þ from thereaction mixture. Ca2þ-dependent NOS activity was com-puted by subtracting Ca2þ-independent NOS activity fromtotal NOS activity. NOS activity is expressed as picomolesof NO formed in 1 min by 1 mg of protein (pmol/min/mg). Calcium-dependent activity in cytosolic samples is con-sidered to represent nNOS because eNOS is associated pri-marily with membranes (Johansson et al., 2002). Proteinconcentration in the samples was determined by the Bradfordmethod (Bradford, 1976).

RNA Extraction and RT-PCR

A 30-mg tissue sample was homogenized in 1 ml of thedenaturing solution (TRIZOL; Invitrogen Life Technologies,Carlsbad, CA). The RNA pellet was dissolved in 10 ml ofnuclease-free water. The purity of isolated RNA was esti-mated spectrophotometrically by measuring absorbency at260 nm and the ratio of 260 nm/280 nm. The PCR primerswere designed to amplify the iNOS product (230 base pairs[bp]). The composition of iNOS was selected from thesequence (GenBank accession number NM_012611). Thesense primer for iNOS was 50-CTGCATGGAACAGTA-TAAGGCAAAC-30, and the antisense primer was 50-CAGA-CAGTTTCTGGTCGATGTCATGA-30.

For assessment of cDNA quality, a pair of glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) primers wasdesigned (GenBank accession number BC059110). The for-ward primer was 50-TGGTGCCAAAAGGGTCATCTCC-30and the reverse primer was 50-GCCAGCCCCAGCAT-CAAAGGTG -30 to amplify a 559-bp product. Aliquots(4 ml) of transcription product was added to 1 ml of 2.5 mMoligo d(T)15 prime, incubated for 5 min at 708C to removecontaminating DNA from the isolated RNA, and then cooledimmediately on ice for 5 min. The cDNA synthesis was car-ried out in a volume of 20 ml containing 5� Moloney murineleukemia virus (M-MLV) buffer (4 ml), 10 mM each of deoxy-nucleotide triphosphate (0.25 ml each), 40 U of RNase inhibi-tor (0.5 ml), and 200 U reverse transcriptase (M-MLV; 1 ml),at 428C for 60 min, 958C for 10 min and at 48C untilremoved. The cDNA was normalized by PCR for constantGAPDH and stored at �708C for PCR. A 20-ml mixture sys-tem of PCR reaction was prepared for all PCR analysesincluding 100 U Taq DNA polymerase (0.3 ml), 10� reactionbuffer (2 ml), 25 mM MgCl2 (1.2 ml), 10 mM dNTP (0.4 ml),0.5 ml of each primer, and 1 ml of the synthesized cDNA; the

remainder was RNase-free water. PCR conditions for iNOSwere 35 cycles of denaturation at 958C for 45 sec, annealingat 638C for 45 sec, and extension at 728C for 45 sec. PCRconditions for GAPDH were 25 cycles of denaturation at958C for 45 sec, annealing at 608C for 45 sec, and extensionat 728C for 45 sec. After the last cycle, the final extension wascarried out at 728C for 10 min for all PCR analyses. Theamplified products were separated by electrophoresis in 1�Tris-acetate EDTA buffer with a 2% agarose gel containing0.1 mg/ml of ethidium bromide (4 ml). DNA bands werevisualized and analyzed by JD-801 Gel Electrophoresis Imageanalytic system (Jiangsu, China). The ratio of NOS toGAPDH mRNA was calculated.

Fig. 1. Effects of iptakalim and diazoxide on catalepsy (latency timein sec) of rotenone-treated rats. Data are expressed as latency time(sec) of the removal of the paw and given as mean 6 SEM; n ¼ 8;*P < 0.001 vs. lane 1; #P < 0.001 vs. lane 2; þP < 0.001 vs. lane 3;yP < 0.001 vs. lane 4 (one-way ANOVA followed by the Newman-Keuls test). Lane 1, vehicle þ 0.2% dimethyl sulfoxide in saline; lane2, rotenone 2.5 mg/kg/day þ 0.2% dimethyl sulfoxide in saline; lane3, rotenone 2.5 mg/kg/day þ iptakalim 1.5 mg/kg/day; lane 4: rote-none 2.5 mg/kg/day þ diazoxide 1.5 mg/kg/day; lane 5: rotenone2.5 mg/kg/day þ 5-HD 3.0 mg/kg/day þ iptakalim 1.5 mg/kg/day; lane 6: rotenone 2.5 mg/kg/day þ 5-HD 3.0 mg/kg/day þdiazoxide 1.5 mg/kg/day.

TABLE I. Effects of Iptakalim and Diazoxide on the Weight and

Mortality of Rotenone-Treated Rats*

Treatment Weight variation (g) Mortality (%)

Vehicle þ saline þ120 6 32 0

Rotenone þ saline �22 6 8 51

Rotenone þ iptakalim þ68 6 28 13

Rotenone þ diazoxide þ52 6 34 15

Rotenone þ 5-HD þ iptakalim �12 6 9 48

Rotenone þ 5-HD þ diazoxide �25 6 7 43

*Rats were chronically, continuously and systemically exposed to dimeth-

ylsulfoxide (DMSO)/polyethylene glycol (PEG) (1/1) or rotenone

(2.5 mg/kg/day, subcutaneously) for 4 weeks with 0.2% DMSO in saline,

iptakalim (1.5 mg/kg/day, orally), or diazoxide (1.5 mg/kg/day, orally)

with or without 5-hydroxydecanoate (5-HD; 3.0 mg/kg/day, orally).

Observations were made on the last day of this experiment.

444 Yang et al.

Statistical Analysis

Data are expressed as mean 6 standard error of themean (SEM) and were analyzed between groups byOrigin v7.5 (Originlab Corporation, Northampton, MA),with one-way analysis of variance (ANOVA) followed bythe Newman-Keuls test. P < 0.05 was considered statisti-cally significant.

RESULTS

Behavioral Tests

Using the bar test, rotenone-treated rats showedprolonged latency time compared to that in the vehicle-treated control group. The latency time of rats pretreatedwith iptakalim (1.5 mg/kg/day, orally) or diazoxide(1.5 mg/kg/day, orally) decreased 61.0% and 55.7%,respectively, compared to that of the rotenone-treatedrats (Fig. 1). The latency time of the rats preadminis-trated with 5-HD (3.0 mg/kg/day, orally) showed nosignificant difference compared to that for the rotenone-treated rats (P > 0.05).

Rotenone-treated rats underwent significant weightloss and significant mortality (51%) during the treatment.Rats with iptakalim (1.5 mg/kg/day, orally) and diaz-

oxide (1.5 mg/kg/day, orally) pretreatment significantlyincreased their weight and had lower mortality (13% and15%, respectively) compared to that in the rotenone-treated rats. 5-HD (3.0 mg/kg/day, orally) pretreatedrats had weight and mortality similar to that of the rote-none-treated rats (Table I).

Neurochemical Changes

In the second experiment, levels of DA and itsmetabolites (DOPAC and HVA), NE, and 5-HT and itsmetabolite (5-HIAA) were measured in the striatum(Table II) and substantia nigra (Table III) for each groupof animals. Rats chronically treated with rotenonereduced the striatum DA content by 62.5%, NE contentby 48.9%, 5-HT content by 40.1% and its metabolite(5-HIAA) by almost 30%, similar to the reductionobserved in DA metabolites (such as DOPAC and thefinal metabolite, HVA). DA content in the substantianigra was reduced by 47.2%, DOPAC contentby 25.2%, HVA content by 19.8%, 5-HIAA contentby 27.9%, NE content by 49.9%, and 5-HT content by40.2%. Pretreatment with iptakalim (1.5 mg/kg/day,orally) or diazoxide (1.5 mg/kg/day, orally) significantlyelevated the contents of DA, NE, and 5-HT and their

TABLE II. Effects of Iptakalim and Diazoxide on Monoamine Levels in the Striatum of Rotenone-Treated Rats*

Treatment DA DOPAC HVA NE 5-HT 5-HIAA

1 Vehicle þ saline 7,987 6 548 1,869 6 359 496 6 101 3,697 6 269 498 6 68 398 6 95

2 Rotenone þ saline 2,995 6 287c 1,398 6 202a 398 6 62b 1,889 6 359c 298 6 85c 287 6 54c

3 Rotenone þ iptakalim 6,045 6 654f 1,685 6 198d 441 6 79d 3,245 6 264f 400 6 98f 345 6 61f

4 Rotenone þ diazoxide 5,915 6 495bf 1,784 6 210d 452 6 54e 3,104 6 268f 440 6 38f 320 6 79f

5 Rotenone þ 5-HD þ iptakalim 3,319 6 359ci 1,421 6 187bg 400 6 57ag 2,104 6 197ch 321 6 50cg 324 6 45bg

6 Rotenone þ 5-HD þ diazoxide 3,224 6 398cl 1,430 6 187bj 422 6 59aj 2,287 6 287cl 350 6 49ck 304 6 69cj

*Rats were chronically, continuously, and systemically exposed to dimethylsulfoxide (DMSO)/polyethylene glycol (PEG) (1/1) or rotenone (2.5 mg/

kg/day, subcutaneously) for 4 weeks with 0.2% DMSO in saline, iptakalim (1.5 mg/kg/day, orally), or diazoxide (1.5 mg/kg/day, orally) with or

without 5-hydroxydecanoate (5-HD; 3.0 mg/kg/day, orally). The monoamine transmitters and their metabolites in the striatum were measured with

HPLC-ECD. Data are expressed as picogram per gram (pg/mg) of fresh brain weight and given as mean 6 standard error of the mean; n ¼ 8. DA,

dopamine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; NE, noradrenalin; 5-HT, serotonin; 5-HIAA, 5-hydroxyindoleacetic acid.aP < 0.05, bP < 0.01, cP < 0.001 vs. group 1; dP < 0.05, eP < 0.01, fP < 0.001 vs. group 2; gP < 0.05, hP < 0.01, iP < 0.001 vs. group 3; jP <0.05, kP < 0.01, lP < 0.001 vs. group 4 (one-way ANOVA followed by Newman-Keuls test).

TABLE III. Effect of Iptakalim and Diazoxide on Monoamine Levels in the Substantia Nigra of Rotenone-Treated Rats*

Treatment DA DOPAC HVA NE 5-HT 5-HIAA

1 Vehicle þ saline 398 6 54 168 6 19 100 6 17 179 6 24 463 6 27 424 6 59

2 Rotenone þ saline 210 6 48c 101 6 12c 60 6 7c 90 6 14c 341 6 56c 320 6 36b

3 Rotenone þ iptakalim 342 6 38e 150 6 23d 80 6 9d 154 6 21e 440 6 35d 396 6 46d

4 Rotenone þ diazoxide 306 6 57c 140 6 27d 81 6 10d 136 6 18d 427 6 47d 410 6 36d

5 Rotenone þ 5-HD þ iptakalim 249 6 39cg 110 6 12bg 68 6 5bf 100 6 10cg 371 6 30bg 318 6 40bg

6 Rotenone þ 5-HD þ diazoxide 266 6 41ch 129 6 19ai 55 6 8cj 97 6 9ci 350 6 42ci 334 6 38bi

*Rats were chronically, continuously, and systemically exposed to dimethylsulfoxide (DMSO)/polyethylene glycol (PEG) (1/1) or rotenone (2.5 mg/

kg/day, subcutaneously) for 4 weeks with 0.2% DMSO in saline, iptakalim (1.5 mg/kg/day, orally), or diazoxide (1.5 mg/kg/day, orally) with or

without 5-hydroxydecanoate (5-HD; 3.0 mg/kg/day, orally). The monoamine transmitters and their metabolites in the substantia nigra were measured

with HPLC-ECD. Data are expressed as picogram per gram (pg/mg) of fresh brain weight and given as mean 6 standard error of the mean; n ¼ 8.

DA, dopamine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; NE, noradrenalin; 5-HT, serotonin; 5-HIAA, 5-hydroxyindoleacetic

acid.aP < 0.05, bP < 0.01, cP < 0.001 vs. group 1; dP < 0.01, eP < 0.001 vs. group 2; fP < 0.05, gP < 0.01, vs. group 3; h P < 0.05, iP < 0.01, jP <0.001 vs. group 4 (one-way ANOVA followed by Newman-Keuls test).

Neuroprotective Effects of Iptakalim 445

metabolites in the striatum and substantia nigra com-pared to that in the rotenone-treated rats. Pretreatmentwith 5-HD (3.0 mg/kg/day, orally) abolished the effectsof iptakalim and diazoxide.

Identification of NOS Activity and mRNA Levelsin the Striatum and Substantia Nigra

NOS activity and mRNA levels in the striatumand substantia nigra were measured. The iNOS activitiesand mRNA levels in rotenone-treated rats were severaltimes higher than those in vehicle control rats (Fig. 2and 3). There was no significant difference betweenthe vehicle control group and iptakalim- or diazoxide-treated groups. 5-HD abolished the effects of iptakalimand diazoxide.

DISCUSSION

Our results indicated that chronic administration ofrotenone dissimilarly decreased DA, NE, and 5-HT con-tents, but did not deplete glutamate and �-aminobutyric

acid (GABA) contents (data not shown) in the nigros-triatal system of rats. These changes of transmitters weresimilar to that found in parkinsonian symptoms.Although dopaminergic neuron loss is characteristic forPD, the neurodegeneration extends well beyond dopa-minergic neurons. Neurodegeneration and Lewy bodyformation are found in noradrenergic (locus coeruleus),serotonergic (raphe), and cholinergic (nucleus basalis ofMeynert and dorsal motor nucleus of vagus) systems,and in the cerebral cortex (especially the cingulate andentorhinal cortices), olfactory bulb, and autonomic ner-vous system. The lesions in cholinergic, serotonergic,and noradrenergic pathways are not characterized asclearly as those in the dopaminergic systems (Schulz andFalkenburger, 2004). Our results provided new evidencefor rotenone as a valid toxin to produce a rat model ofPD although the exact mechanism underlying how rote-none selectively damages monoamine systems remainsunknown.

Pretreatment with diazoxide and iptakalim pre-vented rotenone-induced behavioral alterations and DA,NE, and 5-HT deletion in the nigrostriatal system ofrats. Those effects could be abolished by the pretreat-ment of 5-HD, a selective mito-KATP channel blocker.5-HD alone had no effect on the normal rats or on rote-none-induced motor deficit or neurochemical changes

Fig. 2. iNOS activity in the striatum (A) and substantia nigra (B). Dataare presented as mean 6 SEM; n ¼ 5; *P < 0.001 vs. lane 1; #P <0.001 vs. lane 2; þP < 0.001 vs. lane 3;

yP < 0.001 vs. lane 4 (one-way

ANOVA followed by the Newman-Keuls test). Lane 1, vehicle þ0.2% dimethyl sulfoxide in saline; lane 2, rotenone 2.5 mg/kg/day þ0.2% dimethyl sulfoxide in saline; lane 3, rotenone 2.5 mg/kg/dayþ iptakalim 1.5 mg/kg/day; lane 4, rotenone 2.5 mg/kg/day þ diazo-xide 1.5 mg/kg/day; lane 5, rotenone 2.5 mg/kg/day þ 5-HD3.0 mg/kg/day þ iptakalim 1.5 mg/kg/day; lane 6: rotenone 2.5 mg/kg/day þ 5-HD 3.0 mg/kg/day þ diazoxide 1.5 mg/kg/day.

Fig. 3. iNOS mRNA levels in the striatum (A) and substantia nigra(B). Data are presented as mean 6 SEM; n ¼ 5; *P < 0.001 vs. lane1; #P < 0.001 vs. lane 2; þP < 0.001 vs. lane 3;

yP < 0.001 vs. lane

4 (one-way ANOVA followed by the Newman-Keuls test). Lane 1,vehicle þ 0.2% dimethyl sulfoxide in saline; lane 2, rotenone2.5 mg/kg/day þ 0.2% dimethyl sulfoxide in saline; lane 3, rote-none 2.5 mg/kg/day þ iptakalim 1.5 mg/kg/day; lane 4, rotenone2.5 mg/kg/day þ diazoxide 1.5 mg/kg/day; lane 5, rotenone2.5 mg/kg/day þ 5-HD 3.0 mg/kg/day þ iptakalim 1.5 mg/kg/day; lane 6, rotenone 2.5 mg/kg/day þ 5-HD 3.0 mg/kg/day þdiazoxide 1.5 mg/kg/day.

446 Yang et al.

in this study. It suggested that the activation of mito-KATP channels might be involved in the neuroprotectiveeffects of diazoxide and iptakalim.

ATP-sensitive potassium channels are located invarious parts of the cells, including the surface of theplasmalemmal membrane and the inner mitochondrialmembrane (Busija et al., 2004). Although the preciseprotein composition of mito-KATP channels has notbeen determined, a distinct pharmacologic function ofmito-KATP channels has been found. They are activatedselectively by low concentration of diazoxide andblocked by 5-HD (Ghosh et al., 2000; Sato et al., 2000).Recently, some candidates for mito-KATP channel pro-teins have been identified. Immunopositive colloidalgold particles were scattered over the mitochondria, sug-gesting that Kir6.1 was located on the inner membrane(Suzuki et al., 1997). Bajgar et al. (2001) isolated andpurified a novel mito-KATP channel protein from ratbrain mitochondria that exhibited ligand-binding proper-ties similar to those of heart mito-KATP channels. Accu-mulating data have indicated that the amount of mito-KATP channels located in brain cells is at least sixfoldhigher than that in heart cells. Recent studies indicatedthat mito-KATP channels play an important role in main-taining mitochondrial function by regulating intracellularsignal transduction, mitochondrial volume, and calciumhomeostasis, which may be involved in cell survival anddeath, and the regulation of neuronal differentiationduring the development and synaptic plasticity in thephysiological conditions of the adult (Liu et al., 2002;Rajapakse et al., 2002; Horiguchi et al., 2003; Kis et al.,2003; Mattson and Liu, 2003; Teshima et al., 2003;Busija et al., 2004). The exact neuroprotective mecha-nism of mito-KATP channel activation against rotenonetoxicity, however, remains unknown.

Previous studies revealed that rotenone could acti-vate microglia (Fleming et al., 2004; Zhu et al., 2004).Reactive microglia can produce cytokines IL-1b andTNF-a, which further activate microglia and astrocytesto induce iNOS and produce NO, which play a pivotalrole in rotenone-induced selective nigrostriatal neurode-generation (Gao et al., 2002; He et al., 2003; Bashkatovaet al., 2004). NO can inhibit ribonucleotide reductase,superoxide dismutase, mitochondrial complexes I, II,and IV in the respiratory chain, GAPDH, activate or ini-tiate DNA strand breakage, lipid peroxidation, and pro-tein oxidation, and increase the generation of toxic radi-cals such as hydroxyl radicals and peroxynitrite (Brownand Borutaite, 2002; Ebadi and Sharma, 2003). All theseevents have been postulated to be involved in dopami-nergic cell loss in PD. Moreover, peroxynitrite formedas a result of the nearly diffusion-limited reaction of NOwith superoxide could deactivate the activity of tyrosinehydroxylase, the initial and rate-limiting enzyme in thebiosynthesis of DA (Beckman et al., 1993; Ara et al.,1998; Blanchard-Fillion et al., 2001). Dopaminergicneuron loss and inactivation of tyrosine hydroxylase maybe responsible for the decline in DA formation induced

by rotenone (Ischiropoulos et al., 1995; Abreu et al.,2000). We therefore hypothesize that activation of mito-KATP channels exerts neuroprotective effects on dopami-nergic neurons by inhibiting glial activation and reduc-ing the products of NO induced by rotenone.

We measured the iNOS activities and mRNAlevels in the striatum and substantia nigra. There was arobust increase in iNOS activities and mRNA levels,which were associated with behavioral abnormalities anddecrease of DA content in those regions of rats chal-lenged with rotenone. The increase of iNOS activitymight be due to the elevated mRNA levels. Theobserved increase in iNOS activity in the rat brainmight be the result of increased levels of NO and lipidperoxidation, which were produced after chronicadministration of rotenone. Interestingly, iptakalim anddiazoxide reduced the activity of iNOS, but had noeffect on the elevated activity of nNOS (data notshown) after chronic rotenone infusion, suggesting thatNO from iNOS expressed in glia is a key mediator ofrotenone-induced neuronal death. Similar results werealso found in previous studies (Sparrow, 1994; Brownand Bal-Price, 2003). These effects could be abolishedby 5-HD, suggesting that mito-KATP channels of gliamight contribute to the protective effect of iptakalimand diazoxide against rotenone toxicity. Our recentunpublished data also indicate that activation of mito-KATP channels could also inhibit the products of NOX

and TNF-a of cultured reactive microglia induced byMPPþ and lipopolysaccharide (LPS). The endogenousmechanism of how mito-KATP channels regulate gliafunction, however, is unknown. Further work will bedone to decide whether selective iNOS inhibitors canexert protective effects against rotenone-induced neu-ronal injury in rats and address the exact function of glialmito-KATP channels.

We demonstrate for the first time that rotenone-induced parkinsonian symptoms (e.g., catalepsy) in ratscould be prevented by iptakalim and diazoxide, support-ing the view that the protective effects triggered byactivation of mito-KATP channels may potentially andfeasibly serve therapeutic roles in PD. The regulation ofiNOS might be a neuroprotective target of mito-KATP

channel openers against rotenone toxicity. These find-ings also suggested that inhibiting reactive glia is a pro-spective target for dampening neurodegenerative disor-ders, especially PD.

ACKNOWLEDGMENTS

These studies were supported in part by grantsfrom the National Natural Science Foundation of China(No.39970846) and the state key project of new drugresearch and development (No.969010101) of theNational Ministry of Science and Technology of China.

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