Pharmacological evidence of specific acetylcholine transport in rat cerebral cortex and other brain regions

Abstract

Functional acetylcholine receptors (AChRs) were recently demonstrated to exist not only in the plasma membrane but also intracellularly in brain tissues. In order to activate intracellular AChRs, endogenous hydrophilic ACh must cross the plasma membrane. Here, we examined the pharmacological characteristics of this process, including whether it is mediated by active ACh uptake. When ACh esterase (AChE) was suppressed by diisopropylfluorophosphate, [ H]ACh was effectively taken up into segments of rat cerebral cortex and other brain regions, in contrast to peripheral tissues such as liver and kidney. The uptake of [ H]ACh in rat cerebral cortex was temperature-dependent, and the uptake capacity was comparable to that of [ H]choline. However, [ H]ACh uptake was inhibited by lower concentrations of ACh, carbachol, tetraethylammonium (TEA), compared with uptake of [ H]choline. Uptake of [ H]ACh was also inhibited by several organic cations, including choline, hemicholinium-3 (HC-3), quinidine, decynium 22, clonidine, diphenhydramine, but was little affected by some amino acids and biogenic amines, corticosterone, spermine, atropine, and tetrodotoxin. Unlike diisopropylfluorophosphate, several ACh esterase inhibitors, including drugs for Alzheimer's disease, such as donepezil, galantamine, and rivastigmine, also suppressed the uptake of [ H]ACh, but not [ H]choline. These results indicate that in the brain, ACh is specifically taken up through a unique transport system with different pharmacological properties from known organic cation transporters (OCTs), and suggest that this mechanism may be involved in intracellular cholinergic transmission in the brain.

Regulation of synaptic acetylcholine concentrations by acetylcholine transport in rat striatal cholinergic transmission

“…In fact, the concentrations of HC‐3 and TEA used were high enough such that both drugs could act on presynaptic mAChRs (Muramatsu et al . ). Figure (a and b) show the representative results of the experiments with HC‐3 and TEA, in which not only AChE but also presynaptic mAChRs were suppressed by DFP‐pretreatment and persistent perfusion of atropine, respectively.…”

“…; Kuhar and Simon ; Muramatsu et al . ). In subsequent superfusion experiments, however, we showed that the accumulated [ 3 H]ACh was not liberated in response to electrical stimulation (Fig.…”

“…Nonspecific tissue accumulation of both radioligands was determined after incubation in the presence of 3 mM tetraethylammonium (TEA) for [ 3 H]ACh uptake or 3 mM hemicholinium‐3 (HC‐3) for [ 3 H]choline uptake (Muramatsu et al . ).…”

“…Then the striatal segments were prepared according to a previously described method (Muramatsu et al . , ; Yoshiki et al . ).…”

“…In addition to postsynaptic intracellular AChRs, it has been suggested that ACh may be directly taken up by specific ACh transporters (AChTs) into postsynaptic neurons, although the molecular entity of AChT has not been identified (Muramatsu et al . ). Thus, synaptic ACh levels in the central nervous system (CNS) may be regulated by ACh transport in addition to AChE hydrolysis and subsequent uptake of choline by the CHT1, as well as by modulation of ACh release from nerve terminals by presynaptic mAChRs (Starke et al .…”