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 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 voltage-clamped 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 interrupted 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.
Malo
M
M
Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS, Gif sur Yvette, France.
Diebler
M F
MF
Prado de Carvalho
L
L
Meunier
F M
FM
Dunant
Y
Y
Bloc
A
A
Stinnakre
J
J
Tomasi
M
M
Tchélingérian
J
J
Couraud
P O
PO
Israël
M
M
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation; D002795:Choline O-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D008564:Membrane Potentials; D026901:Membrane Transport Proteins; D008833:Microcirculation; D018482:Muscle, Skeletal / Q000502:physiology*; D009467:Neuromuscular Depolarizing Agents / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D014982:Xenopus laevis
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents; D010880:Piperidines; D011994:Recombinant Proteins; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; C006189:vesamicol; D002795:Choline O-Acetyltransferase; D000109:Acetylcholine