ROLE OF CALCIUM-ACTIVATED NEUTRAL PROTEASE (CALPAIN) IN CELL-DEATH INCULTURED NEONATAL RAT CARDIOMYOCYTES DURING METABOLIC INHIBITION

Calcium-activated neutral protease (CANP), also known as calpain, has been implicated in the development of cell death in ischemic hearts. C ANP is thought to be activated by the calcium overload that develops d uring ischemia. We studied the involvement of CANP in cell death in cu ltured neonatal rat cardiomyocytes during metabolic inhibition (5 mmol /L NaCN+10 mmol/L 2-deoxyglucose). First, we isolated CANP using ion e xchange and affinity chromatography. Then the efficacy of the CANP inh ibitors calpain I inhibitor, leupeptin, and E64 to inhibit isolated CA NP activity was tested with the use of fluorescently labeled beta-case in as a substrate. The IC50 for the inhibitors was between 2.1 and 56 mu mol/L. Uptake of the inhibitors by intact cells was assessed with t he use of Tc-99m-radiolabeled inhibitors. The calculated intracellular inhibitor concentrations were sufficiently high to yield substantial inhibition of intracellular CANP activity. Intracellular CANP activity was measured directly with the use of the cell-permeant fluorogenic C ANP-specific substrate uccinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumar in. During metabolic inhibition, intracellular CANP activity was incre ased compared with control incubation. The time course of CANP activat ion was compatible with that of the rise in [Ca2+](i), as measured by fura 2 and digital imaging fluorescence microscopy. Calpain I inhibito r and leupeptin inhibited intracellular CANP activity both during meta bolic inhibition and control incubation, whereas E64 did not. Despite their substantial inhibition of intracellular CANP activity, calpain I inhibitor and leupeptin did not attenuate cell death during metabolic inhibition. We therefore conclude that intracellular CANP in cardiomy ocytes is activated during metabolic inhibition, but it does not play a major role in the development of cell death.