Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells

作者：Singh Nagendra S; Paul Rajib K; Ramamoorthy Anuradha; Torjman Marc C; Moaddel Ruin; Bernier Michel; Wainer Irving W^*

来源：Cellular Signalling, 2013, 25(12): 2634-2645.

DOI：10.1016/j.cellsig.2013.08.025

• Serine racemase
• D-Serine
• Nicotinic acetylcholine receptors
• mTOR

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 alpha(7)-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 alpha(7)-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of D-Set reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of alpha(7)-nAChR The nAChR subtype selectivity was probed using alpha(7)-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and alpha(3)beta(4)-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 Ca2+ flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the alpha(7)-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels. Published by Elsevier Inc.

• 出版日期2013-12