Differential Regulation of Nicotinic Acetylcholine Receptors in PC12 Cells by Nicotine and Nerve Growth Factor

Abstract

ABSTRACT3 H]epibatidine were found: one that was increased about 4-fold in cells grown for 2 to 4 days in the presence of nicotine and one that was increased 5-fold in cells grown for 2 to 4 days in the presence of nerve growth factor (NGF). The actions of the two treatments were superadditive, resulting in approximately a 13-fold increase in binding sites in cells grown in the combination of the two treatments. The pharmacology of the binding sites in the nicotine-and NGF-treated cells was compared with the pharmacology of defined ␣3␤2 and ␣3␤4 nicotinic acetylcholine receptor (nAChR) subtypes heterologously expressed in human embryonic kidney 293 cells. Nicotine treatment predominantly increased a receptor with characteristics of an ␣3␤2 subtype, whereas the NGF treatment exclusively increased a receptor with characteristics of an ␣3␤4 subtype. Nicotinic receptormediated function measured with the 86 Rb ϩ efflux assay was evident only in the NGF-treated cells, and it had a pharmacological profile that was, again, nearly identical to that of the heterologously expressed ␣3␤4 receptor subtype. Receptor function measured with the [ 3 H]norepinephrine release assay was measurable in both nicotine-treated and NGF-treated cells; however, cytisine-stimulated [ 3 H]norepinephrine release indicated that nicotine treatment increased an nAChR containing ␤2 subunits, whereas NGF increased a receptor containing ␤4 subunits. NGF treatment increased mRNA only for ␤4 subunits in these cells, whereas nicotine treatment did not affect mRNA for any of the subunits measured. After withdrawal of the treatments, the receptors increased by nicotine were much less stable than those increased by NGF.Neuronal nicotinic acetylcholine receptors (nAChRs) are found throughout the central nervous system as well as in autonomic ganglia and the adrenal gland, where they mediate cholinergic neurotransmission critical to the functions of the autonomic nervous system. These receptors are composed of two types of subunits, ␣ and ␤. To date, nine neuronal ␣ subunit genes (␣2-␣10) and three ␤ subunit genes (␤2-␤4) have been found in vertebrate tissues. Different combinations of subunits compose subtypes of nicotinic receptors, all of which are ligand-gated cation channels that conduct Na ϩ , K ϩ , and Ca 2ϩ when activated by the endogenous neurotransmitter acetylcholine or by nicotine or other nicotinic agonists. However, the different receptor subtypes have distinguishing biophysical and/or pharmacological properties, including channel conductances, rates of desensitization and recovery, and sensitivity to drugs (for reviews, see Sargent, 1993;Colquhoun and Patrick, 1997;Lukas, 1998).Certain subtypes of nAChRs are strongly regulated by nicotine. Previous studies have shown that chronic administration of nicotine increases nicotinic receptor binding sites in rat and mouse brain (Schwartz and Kellar, 1983;Marks et al., 1983; for review, see Gentry and Lukas, 2002), and a similar increase is found in human brains from people who smoked tobacco (Benwe...

Targeting of neurotrophic factors, their receptors, and signaling pathways in the developmental neurotoxicity of organophosphates in vivo and in vitro

“…The selective suppression of only one out of the three ngf genes may have an important impact, since the γ subunit regulates the binding of NGFβ to its receptors [99]. Given the vital role of NGF in neuronal differentiation into the cholinergic phenotype [5,19,36], adverse effects on ngfg expression may contribute to the particular sensitivity of these neurons to organophosphates [4,42,72,73]. Similarly, BDNF plays critical roles in neuronal cell differentiation, brain development and synaptic plasticity [51], and alterations in this trophic factor have been implicated in psychiatric diseases, neurodegenerative disorders and epilepsy [34,35,44,67,68,83].…”

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.…”

Mouse β-TC6 Insulinoma Cells: High Expression of Functional α3β4 Nicotinic Receptors Mediating Membrane Potential, Intracellular Calcium, and Insulin Release

“…The neuromuscular subtype (␣ 1 ␤ 1 ␥␦) is expressed in human TE-671 rhabdomyosarcoma cells (Lukas, 1989). The ganglionic subtypes (␣3␤2* and ␣3␤4*) are expressed in rat PC-12 pheochromocytoma cells (Avila et al, 2003). Human IMR-32 (Nelson et al, 2001) and human SH-SY5Y neuroblastoma cells (Dajas-Bailador et al, 2002), express a ganglionic subtype, but the subunit compositions are not known with certainty, because several subunits are expressed in these cells.…”