High-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in neuronal cultures for mechanistic and drug discovery studies

Balmiki Ray, Jason A. Bailey, Jay R. Simon, Debomoy Lahiri

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzymeAby the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.

Original languageEnglish
Article number7.23
JournalCurrent Protocols in Neuroscience
Volume1
Issue numberSUPPL.60
DOIs
StatePublished - Jul 2012

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Choline O-Acetyltransferase
Drug Discovery
Choline
Acetylcholine
Cultured Cells
Hemicholinium 3
Cholinergic Neurons
Synaptosomes
Neuromuscular Junction
Myasthenia Gravis
Extracellular Space
Neuroblastoma
Peripheral Nerves
Radioisotopes
Cholinergic Agents
Neurotransmitter Agents
Alzheimer Disease
Cardiovascular Diseases
Brain

Keywords

  • Brain enzyme
  • Cell culture
  • Cholinesterase
  • CNS
  • Enzymatic activity
  • Neuronal differentiation
  • Synapse

ASJC Scopus subject areas

  • Neuroscience(all)
  • Medicine(all)

Cite this

High-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in neuronal cultures for mechanistic and drug discovery studies. / Ray, Balmiki; Bailey, Jason A.; Simon, Jay R.; Lahiri, Debomoy.

In: Current Protocols in Neuroscience, Vol. 1, No. SUPPL.60, 7.23, 07.2012.

Research output: Contribution to journalArticle

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