Expression of G protein alpha subunits in the lateral wall of the rat cochlea.

Abstract

Expression of five G protein alpha subunits was investigated in the rat cochlea by reverse transcription-polymerase chain reaction (RT-PCR) in order to understand their role in the cochlear signal transduction mechanisms. Immunohistochemical techniques were employed to study their distribution in the lateral wall of the cochlea. Total RNA was extracted with guanidine thiocyanate from cochleas and brains of 14-21-day-old rats. The extract was treated with DNase to degrade genomic DNA. After RT, the resulting cDNA was amplified by PCR using primers specific for the nucleotide sequences representing alpha subunits of heterotrimeric G proteins. The results indicated that mRNA for all five alpha subunits was expressed in the brain and cochlear samples. For immunohistochemical localization, temporal bones of 6-week-old rats were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for embedding in paraffin wax. The dewaxed, midmodiolar sections of the cochlea were incubated with subunit-specific polyclonal antibodies. The pattern of immunoreactivity varied for the five G protein alpha subunits studied in the stria vascularis and spiral ligament. The significance of these findings and the role of G protein alpha subunits in cochlear fluid homeostasis are discussed.