Cell Death and Differentiation (2002) 9, 734 ± 741 ã 2002 Nature Publishing Group All rights reserved 1350-9047/02 $25.00 www.nature.com/cdd Endoplasmic reticulum Ca2+ signaling and calpains mediate renal cell death 1,4 1 2 3 JF Harriman , XL Liu , MD Aleo , K Machaca and Introduction RG Schnellmann*,1,5 An initial effect of ischemia, hypoxia, or mitochondrial 1 Department of Pharmacology and Toxicology, University of Arkansas for inhibition is the prevention of ATP production. When ATP Medical Sciences, 4301 West Markham Street, Slot 638, Little Rock, Arkansas, production falls, protein kinases are unable to phosphorylate AR 72205-7199, USA phosphoproteins and ATP-dependent ion transporters are 2 Drug Safety Evaluation, P®zer, Inc., Eastern Point Road, Groton, Connecticut, inhibited. Further, the loss of oxidative phosphorylation and CT 06340-4947, USA ATP depletion results in either oncotic (necrotic, rapid) or 3 Department of Physiology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 505, Little Rock, Arkansas, AR 72205-7199, USA apoptotic (delayed) cell death, depending on the level of 4 Current address: Wil Research, Inc., 1407 George Road, Ashland, Ohio, OH glycolytic ATP production. For example, in cultured mouse 1 44805-9281, USA renal proximal tubules (RPT), Lieberthal et al. determined 5 Current address: Department of Pharmaceutical Sciences, Medical University that an 85% reduction in ATP levels was required to switch the of South Carolina, 280 Calhoun St., POB 250140, Charleston, SC 29425, USA mechanism of cell death from apoptosis to oncosis. RPT have * Corresponding author: Rick G Schnellmann, Department of Pharmaceutical low glycolytic activity and are very susceptible to ischemia- Sciences, Medical University of South Carolina, 280 Calhoun St., POB 250140, and toxicant-induced oncosis. Charleston, SC 29425, USA. Tel: (843) 792-3754; Fax: (843) 792-2620; E-mail: [email protected] Many studies suggest that increases in cytosolic free 2+ 2+ Ca (Ca f) mediate oncosis following anoxia/hypoxia and Received 5.12.01; revised 8.1.02; accepted 16.1.02 chemical exposure. For example, studies using concurrent 2+ Edited by T Ferguson measurement of Ca f and cell viability revealed that 2+ increases in Ca f occur prior to plasma membrane damage.2,3 In addition, decreasing the extracellular Ca2+ Abstract concentration reduced the release of lactate dehydrogen- The goal of the current study was to determine the roles of ATP ase (LDH), a marker of cell death and membrane damage, content, endoplasmic reticulum (ER) Ca2+ stores, cytosolic from rabbit RPT subjected to anoxia or mitochondrial inhibition and rat RPT subjected to hypoxia.4±6 Further, free Ca2+ (Ca2+ ) and calpain activity in the signaling of rabbit f chelation of intracellular Ca2+ prevented extracellular Ca2+ renal proximal tubular (RPT) cell death (oncosis). Increasing uptake and cell death in mitochondrial inhibitor-exposed concentrations (0.3 ± 10 mM) of the mitochondrial inhibitor RPT.7 Finally, evidence supporting a role for Ca2+ in cell antimycin A produced rapid ATP depletion that correlated to a injury comes from the observed cytoprotection of Ca2+ 2+ rapid and sustained increase in Ca f, but not phospholipase channel blockers in renal cell models subjected to anoxia or C activation. The ER Ca2+-ATPase inhibitors thapsigargin hypoxia.6±8 These studies support an important role for 2+ 2+ (5 mM) or cyclopiazonic acid (100 mM) alone produced similar Ca in cell death and suggest that a rise in Ca f occurs 2+ before extracellular Ca2+ influx. but transient increases in Ca f. Pretreatment with thapsi- gargin prevented antimycin A-induced increases in Ca2+ and The endoplasmic reticulum (ER) contains the largest f 2+ antimycin A pretreatment prevented thapsigargin-induced intracellular store of Ca and is replenished by a high- 2+ increases in Ca2+ . Calpain activity increased in conjunction affinity, low-capacity Ca -ATPase uptake system (SER- f CA).9,10 Physiological release of ER Ca2+ results from with ER Ca2+ release. Pretreatment, but not post-treatment, phospholipase C-mediated inositol trisphosphate (IP3) with thapsigargin or cyclopiazonic acid prevented antimycin formation and its subsequent binding to IP3 receptors on A-induced cell death. These data demonstrate that extensive the ER.10 In many cells, depletion of ER Ca2+ stores 2+ ATP depletion signals oncosis through ER Ca release, a 2+ 2+ triggers extracellular Ca entry, resulting in a rise in Ca F 2+ 2+ sustained increase in Ca f and calpain activation. Depletion and the refilling of the ER Ca store. This influx pathway is of ER Ca2+ stores prior to toxicant exposure prevents termed capacitative Ca2+ entry or store-operated Ca2+ 2+ 11 12 2+ increases in Ca f and oncosis. entry. Waters et al. suggested that ER Ca release Cell Death and Differentiation (2002) 9, 734 ± 741. doi:10.1038/ played a role in RPT cell death by demonstrating that sj.cdd.4401029 depletion of ER Ca2+ stores prior to hypoxia or mitochon- drial inhibitor exposure prevented cell death. 2+ Keywords: cell death; oncosis; renal proximal tubule; endoplasmic Supraphysiological and/or prolonged increases in Ca f reticulum; calpain; calcium are thought to activate degradative enzymes such as calpains, Ca2+-activated cysteine proteases. Ischemic/ 2+ Abbreviations: ER, endoplasmic reticulum; Ca f, cytosolic free hypoxic injury in brain, kidney, liver and myocardium is Ca2+; RPT, renal proximal tubule; SERCA, smooth endoplasmic thought to be mediated by calpains.13±19 Using the RPT 2+ reticulum Ca -ATPase; LDH, lactate dehydrogenase; IP3, inositiol model, we demonstrated that dissimilar calpain inhibitors trisphosphate; PLC, phospholipase C decrease RPT cell death produced by a variety of Calcium release mediates cell death JF Harriman et al 735 toxicants.7,20,21 However, there is limited data demonstrat- 292+13 nM, respectively) before returning to the baseline ing increased calpain activity during cell injury and death. level (30 s after addition) (Figure 2A,B). Both agents 16 2+ Edelstein et al. reported an increase in calpain activity in produced similar rises and decays in Ca f (Figure 2A,B). rat RPT subjected to anoxia and Bronk and Gores13 To test whether the ER Ca2+ store was completely depleted reported an increase in calpain-like protease activity in rat by 5 mM thapsigargin or 100 mM cyclopiazonic acid, two hepatocytes subjected to anoxia. However, increases in experiments were performed. In the first, the agents were calpain activity have not been examined in relation to added again 1 min following the first exposure to thapsigargin. 2+ increases in Ca f during cell injury and death. The The second addition of thapsigargin or cyclopiazonic acid did pathway in Figure 1 illustrates our current hypothesis for not result in a second peak, indicating ER Ca2+ depletion the role of ATP content, ER Ca2+ stores, cytosolic free Ca2+ (data not shown). The addition of ionomycin in the absence of and calpains in cell death. However, several critical extracellular Ca2+ non-specifically releases internal Ca2+ questions remain. Is ER Ca2+ released during cell injury stores. In the second experiment, pretreatment with thapsi- 2+ and death? What is the signal that results in the release of gargin deceased Ca f increases due to ionomycin (10 mM) ER Ca2+? Is calpain activity increased as a result of the ER by 89%. To determine whether extracellular Ca2+ influx 2+ 2+ 2+ Ca release? The goal of the studies in this manuscript is contributed to the rise or decay of Ca f, extracellular Ca to answer these questions. was chelated with EGTA. Decreasing extracellular Ca2+ levels to 100 mM immediately prior to thapsigargin or cyclopiazonic acid did not significantly alter the peak or decay 2+ 2+ Results of Ca f, suggesting little or no extracellular Ca influx during or immediately after ER Ca2+ depletion with these agents Characterization of ER Ca2+ release with ER (Figure 2). Ca2+-ATPase inhibitors To determine the kinetics of ER Ca2+ release with smooth endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, thapsigargin (5 mM) or cyclopiazonic acid (100 mM) was 2+ added to RPT suspensions and Ca f monitored. Thapsigar- gin is an irreversible inhibitor and cyclopiazonic acid is a reversible inhibitor of SERCAs.22,23 Inhibition of SERCAs 2+ 2+ quickly results in increases in Ca f through the ER Ca leak. 2+ Increases in Ca f occurred within 10 s after exposure to thapsigargin or cyclopiazonic acid, resulting in similar peak 2+ Ca f levels (184+7 nM baseline to 275+20 and 2+ Figure 1 Hypothesis of Ca f increases and calpain activation in RPT during antimycin A treatment. ER Ca2+ release may occur by either phospholipase C (PLC)-generated inositol trisphosphate (IP3) binding to the IP3 receptor on the ER or by inhibition of the ER Ca2+-ATPase by ATP depletion. The early events Figure 2 The effect of thapsigargin (Thaps) (A) and cyclopiazonic acid (CPA) 2+ 2+ 2+ upon antimycin A exposure include increases in Ca f levels due to ER Ca (B) on cytosolic Ca f levels in RPT. Thaps (5 mM) or CPA (100 mM) were release and extracellular Ca2+ influx and resulting increase in calpain activity. added with or without addition of EGTA (900 mM) and readings obtained for an Calpain activation leads to substrate hydrolysis, late stage Ca2+ influx and cell additional 2 min. Shown are representative traces (n=4). Closed circles are death without EGTA, open circles are with EGTA Cell Death and Differentiation Calcium release mediates cell death JF Harriman et al 736 2+ 2+ ATPase and ER Ca release may account for the increases Effect of antimycin A exposure on Ca f levels 2+ in Ca f. Antimycin A blocks the electron transport chain and has been used as a model of anoxia (i.e. chemical anoxia).24 Antimycin Effect of ER Ca2+ depletion on antimycin A caused a concentration-dependent (0.3 ± 10 mM) increase 2+ 2+ A-induced Ca f increases in Ca f levels, resulting in a sustained peak by 90 s (Figure 2+ 3A,B).