The use of GABA(A) receptors expressed in neural precursor cells for cell-based assays.
GABA(A) receptors are known targets for certain classes of environmental neurotoxins and pharmaceutical compounds. Since few neural cell lines express functional GABA(A) receptors, the capacity to rapidly screen for compounds that affect GABA(A) receptor function is presently limited. Previous work has demonstrated that rat neural precursor cells express functional GABA(A) receptors that can be monitored via Ca(2+) imaging. This study examined GABA(A) receptor subunit expression to determine whether GABA(A) receptor function and its interactions with neurotoxins is preserved after passaging. Neural precursor cells isolated from embryonic day 13 rat brain were expanded in serum-free medium containing basic fibroblast growth factor and passaged three times. Reverse transcription-polymerase chain reaction analysis demonstrated early expression of abundant mRNAs encoding various GABA(A) receptor subunits. Ca(2+) imaging showed that the highly proliferating precursor cells in passaged cultures maintained expression of functional GABA(A) receptors. In addition, we showed that trimethylolpropane phosphate, a neurotoxin generated during partial pyrolysis of a synthetic ester turbine engine lubricant, potently inhibited muscimol (GABA(A) receptor agonist) but not depolarization-induced cytosolic Ca(2+) increase. The findings of this study suggest that neural precursor cells may be well suited for the evaluation of certain environmental neurotoxins with convulsant activity. The potential use of neural precursor cells in high-throughput screens for compounds acting on GABA(A) receptors is discussed.
Shaffer
K M
KM
Center for Bio/Molecular Science and Engineering, Code 6900, Naval Research Laboratory, Washington, DC 20375, USA.
Lin
H J
HJ
Maric
D
D
Pancrazio
J J
JJ
Stenger
D A
DA
Barker
J L
JL
Ma
W
W
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000633:toxicity; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D015870:Gene Expression; D009474:Neurons / Q000166:cytology; D009498:Neurotoxins / Q000633:toxicity; D021122:Protein Subunits; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000378:metabolism
D019086:Bridged Bicyclo Compounds, Heterocyclic; D009498:Neurotoxins; D021122:Protein Subunits; D012333:RNA, Messenger; D011963:Receptors, GABA-A; C065518:4-ethyl-1-phospha-2,6,7 trioxabicyclo(2.2.2)octane-1-oxide; D002118:Calcium