Inhibition and inactivation of two presynaptic
cholinergic "markers",
choline acetyltransferase and
high affinity choline transporter, has been investigated using inhibitors designed with a redox-reactive
catechol tethered to a quaternary
ammonium group. Two quaternary
ammonium alkyl-substituted
catechols, 3[(trimethylammonio)
methyl]catechol (TMC, 1) and
N,N-dimethylepinephrine (
catecholine, 2) were shown to bind weakly and noncompetitively to bovine
choline acetyltransferase yet inactivated the
enzyme in a time course consistent with the involvement of early intermediates in the spontaneous oxidation of these
catechols. Both agents also inhibited high-affinity
choline uptake. The time course of TMC and
catecholine spontaneous oxidation-dependent inactivation of high affinity
choline uptake sites was slower than, if it occurred at all, the spontaneous degradation of measurable
choline transport in synaptosomes. When compared with inhibition of uptake of other
neurotransmitters, it was shown that
catecholine demonstrated more selectivity than TMC toward inhibition of
choline transport. Km (microM) and Vmax (pmol/min per mg of
protein) were measured for high affinity transport of
choline,
dopamine, and
serotonin and were observed to be Km = 2.04 +/- 0.31, Vmax = 22 +/- 1; Km = 1.4, Vmax = 53; and Km = 0.15, Vmax = 23, respectively, in good agreement with published literature values. Ki's (mM) for
catecholine and TMC, calculated from experimentally determined IC50's, were for
catecholine 0.13 +/- 0.06, 0.53 +/- 0.09, and 0.39 +/- 0.10, and for TMC 0.06 +/- 0.03, 0.09 +/- 0.03, and 0.09 +/- 0.08, for
choline,
dopamine, and
serotonin transport, respectively. In vivo studies using
catecholine suggest that this compound impairs learning ability associated with long-term memory. Thus,
catecholine represents a lead compound in a potential series of redox-reactive
choline analogs, which may become useful irreversible antagonists of the critical
cholinergic macromolecular targets underlying
cholinergic hypofunction in disorders such as
Alzheimer's disease.