Brain Research 782Ž. 1998 337±342 Short communication Phenidone blocks the increases of proenkephalin and prodynorphin gene expression induced by kainic acid in rat hippocampus: involvement of Fos-related antigene protein Hyoung-Chun Kim a,), Jeong-Hye Suh a, Je-Seong Won b, Wang-Kee Jhoo a, Dong-Keun Song b, Yung-Hi Kim b, Myung-Bok Wie b, Hong-Won Suh b a College of Pharmacy, Kangwon National UniÕersity, Chunchon 200-701, Kangwon-Do, South Korea b Department of Pharmacology, Institute of Natural Medicine, College of Medicine, Hallym UniÕersity, Chunchon 200-701, Kangwon-Do, South Korea Accepted 11 November 1997 Abstract To determine the possible role of cyclooxygenaserlipoxygenase pathway in the regulation of proenkephalinŽ. proENK and prodynorphinŽ. proDYN gene expression induced by kainic acid Ž. KA in rat hippocampus, the effects of esculetin, aspirin, or phenidone on the seizure activity, proENK and proDYN mRNA levels, and the level of fos-related antigeneŽ. Fra protein induced by KA in rat hippocampus were studied. EsculetinŽ.Ž 5 mgrkg , aspirin 15 mgrkg . , or phenidone Ž 50 mgrkg . was administered orally five times every 12 h before the injection of KAŽ. 10 mgrkg, i.p. Seizure activity induced by KA was significantly attenuated by phenidone. However, neither esculetin nor aspirin affected KA-induced seizure activity. The proENK and proDYN mRNA levels were markedly increased 4 and 24 h after KA administration. The elevations of both proENK and proDYN mRNA levels induced by KA were inhibited by pre-administration with phenidone, but not with esculetin and aspirin. ProENK-like protein level increased by KA administration was also inhibited by pre-administration with phenidone, but not with esculetin and aspirin. The increases of proENK and proDYN mRNA levels induced by KA were well correlated with the increases of Fra protein level. Additionally, the induction of Fra protein was inhibited by pre-administration with phenidone, but not with esculetin and aspirin. The results suggest that blockade of both cyclooxygenase and lipoxygenase pathways appears to be responsible for increases of proENK and proDYN mRNA levels induced by KA via inhibiting the induction of Fra protein in rat hippocampus. q 1998 Elsevier Science B.V. Keywords: Kainic acid; Cyclooxygenase; Lipoxygenase; Proenkephalin; Prodynorphin; Fra protein; Hippocampus Kainic acidŽ. KA , a unique neuroexcitatoryrneurotoxic cortexwx 13,19 . In addition, the increases of c-Fos, Fra, and substance, produces seizure and neural degeneration in the Jun proteins induced by convulsant dose of KA correlate mammalian CNS, and appears to provide a good model for with AP-1 DNA binding activitywx 18,19 . The neurons of some aspects of human temporal lobe epilepsywx 3,17 . In the hippocampus express both proENK and proDYN the rat hippocampus, KA receptors are concentrated in the mRNA and their protein productswx 11,15 . These proENK CA3rCA4 regions, with moderate levels in the other areas and proDYN mRNA levels are also elevated by KAwx 9 . of the hippocampuswx 8,12 . Both proENK and proDYN genes contain an AP-1-like Several types of proto-oncogene product, such as Fos domain in their promoter regionswx 7,10 . and Jun family proteins, serve as the third messengers in Cis-unsaturated fatty acids can be liberated by phospho- wx the regulation of various types of genes 4,14 . These lipase A2 or diacylglycerol lipase and serve as a precursor, proteins interact with AP-1 domain in the promoter of and subsequently metabolized further via the action of various types of genes, in turn, modulate the transcrip- cyclooxygenase and lipoxygenasewx 5 . Recently, Suh et al. tional activitywx 16 . Convulsant doses of KA induce c-Fos, wx21,22 have reported that arachidonic acid or prosta- Fra, and Jun proteins in rat hippocampus and entorhinal glandin E2 increases proENK mRNA levels in bovine chromaffin cells. However, the involvement of cyclooxy- genase and lipoxygenase pathways in KA-induced increase ) Corresponding author. Fax: q82-361-55-7865. of opioid peptides mRNA in the hippocampus has not been 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S0006-8993Ž. 97 01401-7 338 H.-C. Kim et al.rBrain Research 782() 1998 337±342 characterized. In an attempt to examine the role of cyclo- sarcosine, 2% blocking reagent. , and hybridized overnight oxygenaserlipoxygenase in the regulation of proENK and in Dig-labeled proenkephalin and prodynorphin probes proDYN mRNA expression, the effects of phenidone, es- containing prehybridization buffer at 688C. Then mem- culetin, and aspirin on KA-induced proENK and proDYN branes were washed twice in 2= wash solutionŽ 2= SSC, mRNA levels, or proENK and Fra proteins were studied in 0.1% SDS. at room temperature, and further washed in the present study. 0.1= wash solutionŽ. 0.1= SSC, 0.1% SDS at 688C for 1 Male Sprague±Dawley rats weighing 200±280 g were h. After equilibrating in a buffer IŽ 100 mM maleic acid, used. Animals were housed two per group in a room 150 mM NaCl, pH 7.5. for 1 min, the membranes were maintained at 22"58C with an alternating 12 h light±dark gently agitated in buffer IIŽ 1% blocking reagent in buffer cycle. Animals were used only once. EsculetinŽ. 5 mgrkg , I. for 30±60 min and immuno reacted with the diluted aspirinŽ. 15 mgrkg , or phenidone Ž. 50 mgrkg was admin- anti-DIG-alkaline phosphatasew 1: 10,000Ž. 75 mUrmlx in istered orally five times every 12 h. Thirty minutes after a buffer II for 30 min. After washing twice for 15 min per the last administration of each drug, KAŽ. 10 mgrkg was wash in 0.3% Tween 20Ž. in buffer I , the membranes were administered intraperitoneally. Then, animals were sacri- equilibrated in a buffer IIIŽ 100 mM Tris±HCl, pH 9.5, ficed 4 and 24 h after KA administration. 100 mM NaCl, 50 mM MgCl2 . for 2 min and added 1:100 Seizure activity was rated during a 4-h period after the diluted CSPDŽ. Boehringer Mannheim, Germany solution, KA challenge according to the scale devised by Baran et a chemi-luminescent substrate solution, in buffer III. After al.wx 1 . The seizure rating scale were as follows: 0; normal, incubation at 378C for 15±20 min, the membranes were rare wet-dog shakesŽ. WDS , no convulsions, 1; intermedi- exposed to Hyperfilm-MPŽ Amersham, Buckinghamshire, ate numbers of WDS, rare convulsion type A, 2; frequent England. For re-hybridization, membranes were washed WDS, frequent type A convulsionsŽ no rearing or salva- for 20 min at room temperature in sterile millepore water, tion. , 3; frequent WDS, frequent type A convulsion, ap- then further washed for overnight at 658Cin50mM pearance of type B convulsions with rearingŽ but without Tris±HCl, pH 8.0, 50% dimethylformamide and 1% SDS, falling. , salivation, 4; WDS, type A convulsions, frequent and re-hybridized to Dig-labeled rat cyclophilin cRNA type B convulsions with falling, salivation, 5; continuous probe as a internal loading control. generalized limbic seizures, death within 3 h. Type A All data shown in the present study were repeated at convulsions: focal convulsions affecting head and extremi- least three-times and showed the same tendency of results. ties, starting 30 min after KA injection. Type B convul- No animal received phenidone, esculetin or aspirin alone sions: frequent generalized convulsions usually starting exhibited seizure behaviorŽ. data not shown . The rats 60±90 min after KA injection. Surviving rats expressed receiving KAŽ. 10 mgrkg manifested robust behavioral recurrent seizure behavior. seizures lasting 4 to 6 hŽ. Fig. 1A . Increased seizure scores Animals from each group were anesthetized with 50 induced by KA were significantly attenuated by the pre- mgrkg pentobarbital and then perfused transcardially with treatment with phenidoneŽ. Fig. 1A . However, neither a 140 ml syringe containing salineŽ 40 mlr100 g body esculetin nor aspirin affected KA-induced responseŽ Fig. weight.Ž followed by 4% paraformaldehyde 70 mlr100 g 1A. body weight. The brains were then removed, stored in 4% Both hippocampal proENK and proDYN mRNA levels paraformaldehyde overnight and then cut at 40 mm in the were not altered in the animals treated with saline, horizontal plane with a vibratome. Prior to overnight incu- phenidone, esculetin or aspirin aloneŽ. Fig. 1B . Both bation in primary antibody, sections were prewashed in proENK and proDYN mRNA levels were remarkably in- 0.2% Triton X-100 for 15 min, followed by 4% normal creased 4 and 24 h after KA administration. Six hours after goat serum for 20 min. After a 24-h incubation with the KA injection, in all nonseizing rats given KA plus primary Fra antiseraŽ. 1:1000 dilution or proENK antisera phenidone, esculetin or aspirin, the levels of proENK and Ž.1:1000 dilution , sections were then incubated with a proDYN mRNA was sightly increased. However, these secondary biotinylated antiserumŽ. 1:500 dilution for 1 h. levels were returned to the basal level after 24 h after KA Sections were always washed three times with PBSŽ pH administrationŽ. Fig. 1B . Seizing rats pretreated with 7.4. between each incubation step. As the chromogen, phenidone, but not esculetin and aspirin, exhibited de- X 3.3 -diaminobenzidine was used. creases in both proENK and proDYN mRNA levels at all Total cellular RNA and proteins were extracted from time pointsŽ. Fig. 1B . pooled rat hippocampus Ž.ns3rgroup using a rapid Hippocampal proENK-like immunoreactivity or Fra-im- guanidine thiocyanate±water saturated phenolrchloroform munoreactivity was not affected by the treatment with extraction and subsequent precipitation with acidic sodium phenidone, esculetin or aspirin aloneŽ. data not shown . As acetatewx 6 . A total of 10 mg of total RNA were elec- shown in Figs. 2 and 3, KA significantly increased proENK trophoresed and transferred on nylon membrane.