Nerve growth factor enhances [3h]nicotine binding to a nicotinic cholinergic receptor on pc 12 cells

Thelma C. Madhok, Burt Sharp

Research output: Contribution to journalArticle

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Abstract

Although nicotinic cholinergic agonists have functional effects on PC 12 cells, radioligand-binding sites have not been detected. We, therefore, studied PC 12 cells incubated in the presence of nerve growth factor (NGF) and determined that specific [3H] nicotine-binding sites were induced approximately 2.5-fold in the presence of NGF (50 or 100 ng/ml). Specific binding was maximal between the first (100 ng/ml NGF) and seventh (50 ng/ml NGF) days of treatment and was stable for 2 weeks with addition of NGF every 3 days. Using intact cells, average association and dissociation rates for [3H]nicotine were 0.00021 min-1nM-1(n = 2) and 0.048 min-1(n = 2), respectively, at 4 C, yielding an average apparent Kdof 229 nM. At 22 C, stable equilibrium was not attained during association studies. A similar Kdvalue for broken cell preparations was obtained by kinetic analysis (i.e. an average association rate of 0.00042 min-1nM-1and dissociation rate of 0.087 min-1), yielding an average Kdvalue of 207 nM (n = 2) at 4 C. By saturation binding analysis of intact cells, an average Kdof 292 nM (n = 2) and a binding capacity (Bmax) of 15, 118 molecules/cell were obtained. [3H]Nicotine binding was inhibited on an equimolar basis by L-(-)nicotine and N-methylcarbamylcholine. D-(+) Nicotine was 7-fold less potent, whereas α-bungarotoxin, meca-mylamine, and atropine showed no significant inhibition. [3H]Nicotine binding was also inhibited quantitatively by mono-specific polyclonal antibodies raised against the predicted α3-subunit sequence (amino acids 130-139) of the rat neuronal nicotinic cholinergic receptor. This study represents the first biochemical characterization of NGF-stimulated nicotine-binding sites on PC 12 cells and confirms previous evidence of the presence of functional nicotinic cholinergic receptors on these cells.

Original languageEnglish (US)
Pages (from-to)825-830
Number of pages6
JournalEndocrinology
Volume130
Issue number2
DOIs
StatePublished - Jan 1 1992

Fingerprint

Nicotinic Receptors
Cholinergic Receptors
Nerve Growth Factor
Nicotine
Binding Sites
Nicotinic Agonists
Bungarotoxins
Atropine
Amino Acid Sequence
Antibodies

All Science Journal Classification (ASJC) codes

  • Endocrinology

Cite this

Nerve growth factor enhances [3h]nicotine binding to a nicotinic cholinergic receptor on pc 12 cells. / Madhok, Thelma C.; Sharp, Burt.

In: Endocrinology, Vol. 130, No. 2, 01.01.1992, p. 825-830.

Research output: Contribution to journalArticle

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