G-ALPHA(I-2) IS REQUIRED FOR CARBACHOL-INDUCED STRESS FIBER FORMATIONIN HUMAN AIRWAY SMOOTH-MUSCLE CELLS

To determine which heterotrimeric G protein couples muscarinic recepto rs to stress fiber formation [measured by an increase in the filamento us (F)- to monomeric (G)-actin ratio] in human airway smooth muscle (A SM) cells, cultured human ASM cells expressing the M-2 muscarinic rece ptor were grown to confluence. Cells were exposed for 6 days to 10 M a ntisense oligonucleotides designed to specifically bind to the mRNA en coding G alpha(i-2), G alpha(i-3), or G(q)alpha. A randomly scrambled oligonucleotide served as a control. F- to G-actin ratios were measure d with dual-fluorescence labeling after 5 min of carbachol exposure, w hich is known to increase the F- to G-actin ratio. Cells in parallel w ells were harvested for immunoblot analysis of G protein alpha-subunit expression. Oligonucleotide antisense treatment decreased protein exp ression of the respective G protein alpha-subunit. Antisense depletion of the G alpha(i-2) protein but not of G alpha(i-3) or G(q)alpha prot ein blocked the carbachol-induced increase in the F- to G-actin ratio. These results show that the G alpha(i-2) protein couples muscarinic r eceptors to stress fiber formation in ASM.