title: Phosphate release and force generation in cardiac myocytes investigated with caged phosphate and caged calcium.
abstract: The phosphate (P(i)) dissociation step of the cross-bridge cycle was investigated in skinned rat ventricular myocytes to examine its role in force generation and Ca(2+) regulation in cardiac muscle. Pulse photolysis of caged P(i) (alpha-carboxyl-2-nitrobenzyl phosphate) produced up to 3 mM P(i) within the filament lattice, resulting in an approximately exponential decline in steady-state tension. The apparent rate constant, k (rho i), increased linearly with total P(i) concentration (initial plus photoreleased), giving an apparent second-order rate constant for P(i) binding of 3100 M(-1) s(-1), which is intermediate in value between fast and slow skeletal muscles. A decrease in the level of Ca(2+) activation to 20% of maximum tension reduced k (rho i) by twofold and increased the relative amplitude by threefold, consistent with modulation of P(i) release by Ca2+. A three-state model, with separate but coupled transitions for force generation and P(i) dissociation, and a Ca(2+)-sensitive forward rate constant for force generation, was compatible with the data. There was no evidence for a slow phase of tension decline observed previously in fast skeletal fibers at low Ca(2+), suggesting differences in cooperative mechanisms in cardiac and skeletal muscle. In separate experiments, tension development was initiated from a relaxed state by photolysis of caged Ca(2+). The apparent rate constant, k(Ca), was accelerated in the presence of high P(i) consistent with close coupling between force generation and P(i) dissociation, even when force development was initiated from a relaxed state. k(Ca) was also dependent on the level of Ca(2+) activation. However, significant quantitative differences between k (rho i) and k(Ca), including different sensitivities to Ca(2+) and P(i) indicate that caged Ca(2+) tension transients are influenced by additional Ca(2+)-dependent but P i-independent steps that occur before P(i) release. Data from both types of measurements suggest that kinetic transitions associated with P(i) dissociation are modulated by the Ca(2+) regulatory system and partially limit the physiological rate of tension development in cardiac muscle.
authors.affiliation: Department of Physiology, University of Wisconsin, Madison 53706, USA.
authors.forename: A
authors.initials: A
authors.lastname: Araujo
authors.forename: J W
authors.initials: JW
authors.lastname: Walker
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002448:Cell Adhesion; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D006321:Heart / Q000502:physiology; D006352:Heart Ventricles; D007700:Kinetics; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D010782:Photolysis; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D009943:Organophosphorus Compounds; D010710:Phosphates; C085112:2-nitrophenyl-EGTA; D004533:Egtazic Acid; D002118:Calcium