Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Abstract

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca2+ influx. However, the study of recombinant alpha7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha3, alpha5, alpha7, beta2 and beta4 subunits was present during the course of differentiation, while mRNAs coding for alpha2, alpha4 and beta3 subunits were not expressed in PC12 cells. alpha7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha7 agonist choline. Increased alpha7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

Differences in the Cellular Uptake and Intracellular Itineraries of Amyloid Beta Proteins 40 and 42: Ramifications for the Alzheimer’s Drug Discovery

“…29,30 It was also shown that the differentiation of PC12 cells with the nerve growth factor increases the expression of various receptors implicated in A β 42 endocytosis to the levels observed in central nervous system derived neuronal cell lines. 31,32 Our studies have clearly demonstrated that A β 40 uptake mechanisms observed in PC12 cells are verifiable in primary rat embryonic cortical neurons. 9 …”

Molecular mechanism of interactions of the physiological anti-hypertensive peptide catestatin with the neuronal nicotinic acetylcholine receptor

“…S4). In these experiments, we used neuronally differentiated PC12 cells that predominantly express a3b4 nAChR subtype (Avila et al, 2003;Henderson et al, 1994) and gave larger and more reproducible amperometric responses to ACh as found by us and other investigators (Nery et al, 2010) compared with un-differentiated PC12 cells.…”

Rescue of Amyloid-Beta-Induced Inhibition of Nicotinic Acetylcholine Receptors by a Peptide Homologous to the Nicotine Binding Domain of the Alpha 7 Subtype

“…RT-PCR analysis revealed that such cells express α3, α5, α7, β2 and β4 nAChR subunits, and measurements in the presence of methyllycaconitine (MLA) indicated that, on day 2 following induction to neuronal differentiation, α7 receptors contributed about 50% of cholinergic receptor-mediated whole cell currents [36].…”

“…PC12 cells (ATCC, catalogue # CRL-1721) were cultured and induced to neuronal differentiation as described [24], [36]. Briefly, PC12 cells were cultured in DMEM (Invitrogen, Life Technologies, Carlsbad, CA, USA) in the presence of 10% FBS (Cultilab, Campinas, São Paulo, Brazil), 5% horse serum (Invitrogen, Life Technologies, Carlsbad, CA, USA), streptomycin (100 µg/ml), penicillin (100 U/ml – Sigma-Aldrich, St. Louis, MO, USA) and 1 mM sodium pyruvate (Invitrogen, Life Technologies, Carlsbad, CA, USA).…”

“…For differentiation, 2.5×10 5 cells/ml, as determined by Neubauer chamber counting, were induced to neuronal differentiation for up to 6 days in DMEM containing 30 ng/ml FGF-2 (Sigma-Aldrich, St. Louis, MO, USA) and 250 µM dibutyril cAMP (Sigma-Aldrich, St. Louis, MO, USA). Under these conditions, differentiated PC12 cells express α3, α5, α7, β2 and β4 nicotinic receptor subunits [36]. For evaluation of cell viability, PC12 cells on day 3 of neuronal differentiation were exposed for 48 h to different peptides at 1 and 100 µM concentrations, then washed with PBS and stained with trypan blue.…”