Evoked Acetylcholine Release by Immortalized Brain Endothelial Cells Genetically Modified...

Evoked Acetylcholine Release by Immortalized Brain Endothelial Cells Genetically Modified to Express Choline Acetyltransferase and/or the Vesicular Acetylcholine Transporter

M. Malo, M.-F. Diebler, L. Prado de Carvalho, F.-M. Meunier, *Y. Dunant, *A. Bloc, J. Stinnakre, M. Tomasi, †J. Tche´linge´rian, †P. O. Couraud, and M. Israe¨l

The goal of the study was to genetically modify RBE4 cells, rat brain endothelial cells, so that not only do they produce ACh but package it into an internal store, as to mimic the vesicular store if neurotransmitters in neurons. RBE4 cells can’t accumulate ACh but can release it upon calcium entry. RBE4 were used because they already normally have machinery specific to the release of ACh (and no other neurotransmitter) and they are convenient for gene manipulations.

In this experiment, two things were done:
(1) The RBE4 cells were engineered to express ChAT, choline acetyltransferase. (Those RBE4 cells that expressed ChAT produced endogenous Ach).
(2) The experimenters attempted to modify the RBE4 cells expressing ChAT further to express the vesicular Ach transporter (VAChT).

Gene modification was done with the RBE4 cells in suspension with a transfection reagent, a selection vector, a ChAT expression vector and a VAChT expression vector. ChAT activity was determined by radiolabeling the ACh and was quantified using the Bradford assay. VAChT expression was assessed via a binding assay with vesamicol as specific ligand. An immunodetection of ChAT and VAChT was done and visualized with a chemiluminescent system. Results of these tests showed that stable transfected RBE4 cells only expressed either ChAT or VAChT, not both (but transfection overall was high). Thus, the effects of VAChT on ACh release could only be tested through evoked ACh release after passive loading of the VAChT-expressing cells with ACh in comparison to the ChAT-expressing cells and no significant difference was seen.

A chemiluminescent procedure was also used to quantify ACh release. Electrophysiologically, ACh release was measured by measuring the membrane current of Xenopus myocytes (used as detector cells). It was seen that with calcium addition, ACh was released. Synaptic-like currents were also observed. Electrophysiological testing of VAChT expressing cells suggested that temporal pattern of ACh release was controlled by VAChT. Why this is was not answered in this experiment.

I chose this paper because I'm particularly interested in neurons and the experiment includes techniques that we have learned in class. It is important because the researchers were able to induce synthesis and accumulation of ACh in non-neuronal cells. This may be a great treatment for neurodegenerative diseases that lead to cholinergic deficits.