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

: Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have...

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Bibliographic Details
Published in:Journal of neurochemistry 1999-10, Vol.73 (4), p.1483-1491
Main Authors: Malo, M., Diebler, M.‐F., De Carvalho, L. Prado, Meunier, F.‐M., Dunant, Y., Bloc, A., Stinnakre, J., Tomasi, M., Tchélingérian, J., Couraud, P. O., Israël, M.
Format: Article
Language:English
Subjects:
Acetylcholine
Acetylcholine - metabolism
Acetylcholine release
Animals
Biological and medical sciences
Brain endothelial cells
Carrier Proteins
Carrier Proteins - genetics
Carrier Proteins - metabolism
Cell Line, Transformed
Cells, Cultured
Central nervous system
Central neurotransmission. Neuromudulation. Pathways and receptors
Cerebrovascular Circulation
Choline acetyltransferase
Choline O-Acetyltransferase
Choline O-Acetyltransferase - genetics
Choline O-Acetyltransferase - metabolism
Endothelium, Vascular
Endothelium, Vascular - cytology
Endothelium, Vascular - physiology
Fundamental and applied biological sciences. Psychology
Life Sciences
Membrane Potentials
Membrane Transport Proteins
Microcirculation
Muscle, Skeletal
Muscle, Skeletal - physiology
Myocyte
Neuromuscular Depolarizing Agents
Neuromuscular Depolarizing Agents - pharmacology
Neurons and Cognition
Patch-Clamp Techniques
Piperidines
Piperidines - pharmacology
Rats
Recombinant Proteins
Recombinant Proteins - metabolism
Transfection
Vertebrates: nervous system and sense organs
Vesicular Acetylcholine Transport Proteins
Vesicular acetylcholine transporter
Vesicular Transport Proteins
Xenopus laevis
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Description
Summary:: Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+‐dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT‐transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole‐cell voltageclamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh electrical stimulation elicited the release of ACh quanta detected as inward synaptic‐like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.
ISSN:0022-3042
1471-4159
DOI:10.1046/j.1471-4159.1999.0731483.x