Isolated permeabilized rat heart cells as a model to study heart bioenergetics in situ

Isolated rat heart cells permeabilized by digitonin were examined as an experimental model to study heart bioenergetics. The cells displayed good parameters of oxidative phosphorylation (acceptor control ratio ca. 8 with pyruvate + malate). High ATPase activity detected in the cells was characterized. The ATPase activity was Ca-dependent (optimum [free Ca] ca. 400 nM), and Mg was necessary for its full activity. Double reciprocal plot l/v vs. 1/[free Ca] at physiological [free Ca] was linear, thus showing free Ca to be a substrate for the ATPase (Km for Ca ca. 149 nM). Double reciprocal plot 1/v vs. 1/[ATP] was also liner, thus showing the ATPase activity could be ascribed to a single enzyme. The ATPase is supposed to represent Ca, Mg-ATPase of sarcoplasmic reticulum. The ATPase activity appeared to be functionally coupled to oxidative phosphorylation of the cells by apparently preferring ATP supplied by mitochondria (KmATP = 74 microM) to external ATP (KmATP = 169 microM; P less than 0.05). Apparent preference by oxidative phosphorylation for ADP supplied by the ATPase (KmATP = 45 microM) to external ADP (KmATP = 152 microM) was also manifested by significant difference (P less than 0.02) in Km.