Norepinephrine and GTP-gamma-S increase myofilament Ca2+ sensitivity in alpha-toxin permeabilized arterial smooth muscle.
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.
Nishimura
J
J
Department of Pharmacology, University of Miami School of Medicine, Florida 33101.
Kolber
M
M
van Breemen
C
C
D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D019204:GTP-Binding Proteins / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006460:Hemolysin Proteins; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D009638:Norepinephrine / Q000494:pharmacology*; D011817:Rabbits; D011942:Receptors, Adrenergic, alpha / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology*
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D011942:Receptors, Adrenergic, alpha; D013873:Thionucleotides; C021091:staphylococcal alpha-toxin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D002118:Calcium; D009638:Norepinephrine