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

Caffeine (10 mM) or 2, 4-dinitrophenol (DNP; 1 mM) caused release of creatine kinase (CK) in the perfused rat heart but only when previously activated by Ca2+o depletion. Simultaneous Ca2+₀ depletion and caffeine or DNP perfusion markedly reduced CK release. Activation by Ca2+₀ depletion was inhibited at 28°C. Both the Ca2+ paradox and caffeine-induced release of CK were inhibited by 1 mM 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 mM 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.