Adenovirus-mediated Gα-protein antisense transfer in neurons replicates Gα 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 α antisense into rat superior cervical ganglion (SCG) neurons and neuronal cell lines to dissect Gα-mediated signaling pathways in these cells. This approach was compared with other Gα gene knockdown strategies, namely, antisense plasmid and knockout mice. Infection with adenovirus expressing Gα antisense (GαAS AdV) selectively decreased immunoreactivity for the Gα protein. Expression of other Gα protein subunits, such as Gα was unaltered. Consistent with this, modulation of Ca currents by the Gα-coupled M muscarinic receptor was severely impaired in neurons infected with GαAS AdV whereas modulation via the Gα-coupled M muscarinic receptor was unchanged. In agreement, activation of phospholipase C and consequent mobilization of intracellular Ca by UTP receptors was lost in NG108-15 cells infected with GαAS 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 Gα antisense plasmids or SCG neurons from Gα knockout mice. This combined antisense/recombinant adenoviral approach can therefore be useful for dissecting signal transduction mechanisms in SCG and other neurons.