Relationship between ATPase activity, Ca2+, and force in alpha-toxin- and beta-escin-treated smooth muscle.
Smooth muscle was made permeable with alpha-toxin and beta-escin. ATPase activity was measured using a phosphoenolpyruvate-pyruvate kinase regenerating system for ATP that was monitored by NADH fluorescence changes, and Ca2+ was measured using fura 2 fluorescence. alpha-Toxin-and beta-escin-treated bundles of cells had a high ATPase activity, which was reduced 80% when exposed to 1% Triton X-100. This Triton-sensitive ATPase activity was increased by approximately 20% when GTP or GTP gamma S was added to the solutions and was of much greater magnitude than the Ca(2+)-activated ATPase associated with contraction. This high membrane ATPase activity will cause a gradient of ATP into and ADP out of the bundle of cells. Thus modulation of this ATPase by G-protein-receptor mechanisms could alter the force at a constant Ca2+ concentration by changing the ADP/ATP ratio within the cells. Measurements of the fura 2 fluorescence ratio (340/380) in alpha-toxin-treated bundles of cells following sudden changes in extracellular Ca2+ showed that the cells were not freely permeable to Ca EGTA. Similar experiments in beta-escin-treated cells showed the cells to be much more permeable to Ca EGTA. These experiments indicate that great care must be taken in alpha-toxin- and beta-escin-treated fibers to make sure that the intracellular ATP, ADP, and Ca2+ are held constant.
Kerrick
W G
WG
Department of Physiology and Biophysics, University of Miami, School of Medicine, FL 33101, USA.
Hoar
P E
PE
D000251:Adenosine Triphosphatases / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology*; D004533:Egtazic Acid / Q000378:metabolism; D004928:Escin / Q000494:pharmacology*; D016257:Fura-2; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D010725:Phosphocreatine / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D010738:Type C Phospholipases / Q000494:pharmacology*
D010725:Phosphocreatine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D004928:Escin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D000251:Adenosine Triphosphatases; D002118:Calcium; D016257:Fura-2