Adenovirus-mediated G(alpha)(q)-protein antisense transfer in neurons replicates G(alpha)(q) gene knockout strategies.
Antisense approaches are increasingly used to dissect signaling pathways linking cell surface receptors to intracellular effectors. Here we used a recombinant adenovirus to deliver G-protein alpha(q) antisense into rat superior cervical ganglion (SCG) neurons and neuronal cell lines to dissect G(alpha)(q)-mediated signaling pathways in these cells. This approach was compared with other G(alpha)(q) gene knockdown strategies, namely, antisense plasmid and knockout mice. Infection with adenovirus expressing G(alpha)(q) antisense (G(alpha)(q)AS AdV) selectively decreased immunoreactivity for the G(alpha)(q) protein. Expression of other G(alpha) protein subunits, such as G(alpha)(oA/B,) was unaltered. Consistent with this, modulation of Ca(2+) currents by the G(alpha)(q)-coupled M(1) muscarinic receptor was severely impaired in neurons infected with G(alpha)(q)AS AdV whereas modulation via the G(alpha)(oA)-coupled M(4) muscarinic receptor was unchanged. In agreement, activation of phospholipase C and consequent mobilization of intracellular Ca(2+) by UTP receptors was lost in NG108-15 cells infected with G(alpha)(q)AS AdV but not in cells infected with the control GFP-expressing adenovirus. Results obtained with this recombinant AdV strategy qualitatively and quantitatively replicated results obtained using SCG neurons microinjected with G(alpha)(q) antisense plasmids or SCG neurons from G(alpha)(q) knockout mice. This combined antisense/recombinant adenoviral approach can therefore be useful for dissecting signal transduction mechanisms in SCG and other neurons.
Abogadie
F C
FC
Wellcome Laboratory for Molecular Pharmacology, University College London, UK. f.abogadie@ucl.ac.uk
Bron
R
R
Marsh
S J
SJ
Drew
L J
LJ
Haley
J E
JE
Buckley
N J
NJ
Brown
D A
DA
Delmas
P
P
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D005822:Genetic Vectors; D008959:Models, Neurological; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D051381:Rats; D011922:Rats, Mutant Strains; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology; D017783:Superior Cervical Ganglion / Q000502:physiology*
D020319:Oligodeoxyribonucleotides, Antisense; D011976:Receptors, Muscarinic