Dual Activation of the Damage System that Causes the Release of Cytosolic Proteins in the Perfused Rat Heart

Abstract

Caffeine (10 m<i>M</i>) or 2, 4-dinitrophenol (DNP; 1 m<i>M</i>) caused release of creatine kinase (CK) in the perfused rat heart but only when previously activated by Ca²⁺o depletion. Simultaneous Ca²⁺&#8320; depletion and caffeine or DNP perfusion markedly reduced CK release. Activation by Ca²⁺&#8320; depletion was inhibited at 28°C. Both the Ca²⁺ paradox and caffeine-induced release of CK were inhibited by 1 <i>mM</i> amiloride. Perfusion of anoxic standard medium lacking glucose had no overt effects, but CK release began immediately on subsequent Ca²⁺ depletion. A small, but steady release of CK was observed after glucose-free, anoxic perfusion for 75 min. CK release produced by reoxygenation after anoxia (the O₂ paradox) is augmented if anoxic perfusion is lengthened from 20 to 45 min. The Ca²⁺ paradox is markedly inhibited by 2 m<i>M</i> iodoacetate (IAA), even when this is introduced after Ca²⁺o depletion. Since caffeine or DNP caused ultrastructural damage in the presence of extracellular Ca²⁺ (when no CK was released) it is concluded that the pathways of myofilament degradation and CK release can be independently activated. It is concluded that CK release is synergistically triggered by removal of extracellular Ca²⁺ and a rise in [Ca²⁺]i. It is suggested that Ca²⁺o depletion causes membrane perturbation that activates a trans-sarcolemma complex that is critically dependent on its mobility within the bilayer, is intimately linked to a Na⁺/H⁺ antiporter and is inhibited by IAA.