Norepinephrine and GTP-γ-S increase myofilament Ca2+ sensitivity in α-toxin permeabilized arterial smooth muscle

Summary A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using α-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After α-toxin treatment the fibers developed tension as a function of Ca 2+ concentration (EC 50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5×10 −7 M Ca 2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca 2+ solution and a transient and maintained contraction in 5×10 −7 M Ca 2+ solution. GTP-γ-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca 2+ and maintained tension revealed that NE and GTP-γ-S cause increases in Ca 2+ sensitivity of myofilament shifting the EC 50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-γ-S causes an increase in myofilament Ca 2+ sensitivity and that G protein may be involved in receptor signal transduction system. α-Toxin is a useful tool to permeabilize the smooth muscle tissue to ions and small molecules without any damage of receptor and signal transduction system.