RELATIONSHIP BETWEEN ATPASE ACTIVITY, CA2-TOXIN-TREATED AND BETA-ESCIN-TREATED SMOOTH-MUSCLE(, AND FORCE IN ALPHA)

Smooth muscle was made permeable with alpha-toxin and beta-escin. ATPa se activity was measured using a phosphoenolpyruvate - pyruvate kinase regenerating system for ATP that was monitored by NADH fluorescence c hanges, and Ca2+ was measured using fura 2 fluorescence. alpha-Toxin-a nd beta-escin-treated bundles of cells had a high ATPase activity, whi ch was reduced 80% when exposed to 1% Triton X-100. This Triton-sensit ive 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 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-r eceptor mechanisms could alter the force at a constant Ca2+ concentrat ion by changing the ADP/ATP ratio within the cells. Measurements of th e fura 2 fluorescence ratio (340/380) in alpha-toxin-treated bundles o f cells following sudden changes in extracellular Ca2+ showed that the cells were not freely permeable to Ca EGTA. Similar experiments in be ta-escin-treated cells showed the cells to be much more permeable to C a EGTA. These experiments indicate that great care must be taken in al pha-toxin- and beta-escin-treated fibers to make sure that the intrace llular ATP, ADP, and Ca2+ are held constant.