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 cell 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 was 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 Ca2+ 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.

What is hydroxynorketamine and what can it bring to neurotherapeutics?

“…We have recently demonstrated that intracellular D-Ser concentrations in PC-12 cells can be attenuated by the inhibition of cellular receptors and transporters [23–25]. Incubation with ( 2S,6S )-HNK decreased the intracellular concentration of D-Ser [23,25], which was attributed to ( 2S,6S )-HNK-mediated inhibition of the basal activity of α7-nAChR.…”

“…Incubation with ( 2S,6S )-HNK decreased the intracellular concentration of D-Ser [23,25], which was attributed to ( 2S,6S )-HNK-mediated inhibition of the basal activity of α7-nAChR. This inhibition lowers intracellular Ca 2+ , which, in turn, reduces the magnitude of Ca 2+ -activated SR and consequently the intracellular D-Ser concentrations.…”

“…The incubation of PC-12 cells with (2 S ,6 S )-HNK also led to de novo protein synthesis of the monomeric form of SR [23,25]. The effect was associated with the mammalian target of rapamycin (mTOR) signaling pathway and involved phosphorylation-dependent modulation of multiple intracellular proteins, including extracellular signal-regulated kinases (ERK1/2), protein kinase B (Akt), eukaryotic initiation factor 4E binding protein (4E-BP1) and p70S6 kinase (p70S6K).…”

Interactions of (2S,6S;2R,6R)‐Hydroxynorketamine, a Secondary Metabolite of (R,S)‐Ketamine, with Morphine

“…Negative allosteric modulation of a7-nACh receptors proposedly leads to multiple effects that may have opposite actions regarding nociception and opioid tolerance. decreased production of D-serine [31][32][33], an important NMDA receptor co-agonist. Decreased D-serine levels have previously been associated with attenuation of morphine tolerance [34,35].…”

Are basal D-serine plasma levels a predictive biomarker for the rapid antidepressant effects of ketamineand ketamine metabolites?

“…The latter effect mirrored the affinities of (S)-ketamine and (R)-ketamine at the NMDA receptor, 0.7 and 2.6 μM, respectively . We have also shown that in PC-12 and 1321N1 human astrocytoma cell lines, the inhibition of basal α7-nAChR activity by (R,S)-dehydronorketamine and (2S,6S)-hydroxynorketamine leads to decreased intracellular D-serine concentrations and de novo protein synthesis associated with the activation of the mammalian target of rapamycin (mTOR) pathway (Singh et al 2013;Paul et al 2014). A recent study suggested that mTOR activation in the frontal cortex of the Wistar rat was responsible for the rapid antidepressant effect of (R,S)-ketamine (Dwyer and Duman 2013).…”