Galphai-2 is required for carbachol-induced stress fiber formation in human airway smooth muscle cells.
To determine which heterotrimeric G protein couples muscarinic receptors to stress fiber formation [measured by an increase in the filamentous (F)- to monomeric (G)-actin ratio] in human airway smooth muscle (ASM) cells, cultured human ASM cells expressing the M2 muscarinic receptor were grown to confluence. Cells were exposed for 6 days to 10 microM antisense oligonucleotides designed to specifically bind to the mRNA encoding Galphai-2, Galphai-3, or Gqalpha. A randomly scrambled oligonucleotide served as a control. F- to G-actin ratios were measured with dual-fluorescence labeling after 5 min of carbachol exposure, which is known to increase the F- to G-actin ratio. Cells in parallel wells were harvested for immunoblot analysis of G protein alpha-subunit expression. Oligonucleotide antisense treatment decreased protein expression of the respective G protein alpha-subunit. Antisense depletion of the Galphai-2 protein but not of Galphai-3 or Gqalpha protein blocked the carbachol-induced increase in the F- to G-actin ratio. These results show that the Galphai-2 protein couples muscarinic receptors to stress fiber formation in ASM.
Hirshman
C A
CA
Department of Anesthesiology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA.
Togashi
H
H
Shao
D
D
Emala
C W
CW
D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014132:Trachea / Q000187:drug effects / Q000502:physiology*; D015293:Transducin / Q000235:genetics / Q000502:physiology*
D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D002217:Carbachol; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D015293:Transducin