Relationship between ATPase activity, Ca²⁺, and force in α-toxin- and β-escin-treated smooth muscle

Smooth muscle was made permeable with α-toxin and β-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. α-Toxin-and β-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 GTPγS was added to the solutions and was of much greater magnitude than the Ca²⁺-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 α-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 β-escin-treated cells showed the cells to be much more permeable to Ca EGTA. These experiments indicate that great care must be taken in α-toxin- and β-escin-treated fibers to make sure that the intracellular ATP, ADP, and Ca²⁺ are held constant.