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

Abstract

A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using alpha-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After alpha-toxin treatment the fibers developed tension as a function of Ca2+ concentration (EC50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5 X 10(-7) M Ca2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca2+ solution and a transient and maintained contraction in 5 X 10(-7) M Ca2+ solution. GTP-gamma-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca2+ and maintained tension revealed that NE and GTP-gamma-S cause increases in Ca2+ sensitivity of myofilament shifting the EC50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-gamma-S causes an increase in myofilament Ca2+ sensitivity and that G protein may be involved in receptor signal transduction system. alpha-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.