title: Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells.
abstract: Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cells primarily express the α7-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular d-Ser levels and SR protein expression. Incubation with (S)-nicotine increased d-Ser levels, which were attenuated by the α7-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of d-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α7-nAChR. The nAChR subtype selectivity was probed using α7-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α3β4-nAChR specific inhibitor AT-1001. The compounds reduced d-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca(2+) flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the α7-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels.
authors.affiliation: Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: Nagendra S
authors.initials: NS
authors.lastname: Singh
authors.forename: Rajib K
authors.initials: RK
authors.lastname: Paul
authors.forename: Anuradha
authors.initials: A
authors.lastname: Ramamoorthy
authors.forename: Marc C
authors.initials: MC
authors.lastname: Torjman
authors.forename: Ruin
authors.initials: R
authors.lastname: Moaddel
authors.forename: Michel
authors.initials: M
authors.lastname: Bernier
authors.forename: Irving W
authors.initials: IW
authors.lastname: Wainer
mesh_terms: D000157:Aconitine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008464:Mecamylamine / Q000494:pharmacology*; D009538:Nicotine / Q000378:metabolism; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D016716:PC12 Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D019998:Racemases and Epimerases / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D000099300:Serine Racemase
chemical_list: D000157:Aconitine; D008464:Mecamylamine; D009538:Nicotine; D018733:Nicotinic Antagonists; D019998:Racemases and Epimerases; D011978:Receptors, Nicotinic; D058570:TOR Serine-Threonine Kinases; D000099300:Serine Racemase; C054634:methyllycaconitine
keywords: DHNK; MEC; MLA; Nicotinic acetylcholine receptors; Serine racemase; d-SR; d-Ser; d-Serine; d-serine; dehydronorketamine; dimeric serine racemase; m-SR; mTOR; mecamylamine; methyllycaconitine; monomeric serine racemase; nAChR; nicotinic acetylcholine receptor