Dual Insult Research Articles

Nitric oxide donors rescue metabolic and mitochondrial dysfunction in obese Alzheimer’s model

Reduced nitric oxide (NO) bioavailability is a pathological link between obesity and Alzheimer’s disease (AD). Obesity-associated metabolic and mitochondrial bioenergetic dysfunction are key drivers of AD pathology. The hypothalamus is a critical brain region during the development of obesity and dysfunction is an area implicated in the development of AD. NO is an essential mediator of blood flow and mitochondrial bioenergetic function, but the role of NO in obesity-AD is not entirely clear. We investigated diet-induced obesity in female APPswe/PS1dE9 (APP) mouse model of AD, which we treated with two different NO donors (sodium nitrite or L-citrulline). After 26 weeks of a high-fat diet, female APP mice had higher adiposity, insulin resistance, and mitochondrial dysfunction (hypothalamus) than non-transgenic littermate (wild type) controls. Treatment with either sodium nitrite or L-citrulline did not reduce adiposity but improved whole-body energy expenditure, substrate oxidation, and insulin sensitivity. Notably, both NO donors restored hypothalamic mitochondrial respiration in APP mice. Our findings suggest that NO is an essential mediator of whole-body metabolism and hypothalamic mitochondrial function, which are severely impacted by the dual insults of obesity and AD pathology.

Levels of IGFBP-1, 3 and 7 in human serum induced by alcohol consumption, NAFLD and dual insult

Levels of IGFBP-1, 3 and 7 in human serum induced by alcohol consumption, NAFLD and dual insult

Cognition and Behavior in the Aging Brain Following TBI: Surveying the Preclinical Evidence.

Elderly individuals (65years and older) represent the fastest-growing demographic of new clinical traumatic brain injury (TBI) cases, yet there is a paucity of preclinical research in aged animals. The limited preclinical work available aligns with the clinical literature in that there appear to be significant differences in pathophysiology, recovery potential, and response to medications between animals at different points across the age spectrum. The aim of this review is to discuss the limited studies and highlight critical age-related differences in affective, cognitive, and neurobehavioral deficits, to discuss factors that influence functional outcomes, and to identify targets for future research. The consensus is that aged rodents face challenges related to dysregulated inflammation, reduced neuroplasticity, and age-related cellular changes, which hinder their recovery after TBI. The most successful intervention studies in animal models to date, of the limited array available, have explored interventions targeting inflammatory downregulation. Current standards of neuropsychopharmacology for post-TBI neurocognitive recovery have not been investigated in a significant capacity. In addition, currently available animal models do not sufficiently account for important age-related comorbidities, dual insults, or differences in TBI mechanism of injury in elderly individuals. TBI in the aged population is more likely to lead to additional neurodegenerative diseases that further complicate recovery. The findings underscore the need for tailored therapeutic interventions to improve outcomes in both adult and elderly populations.

Myocardial Composition in Light-Chain Cardiac Amyloidosis More Than 1 Year After Successful Therapy

ObjectivesThe goals of this study were to characterize myocardial composition during the active and remission phases of light-chain (AL) cardiac amyloidosis. BackgroundCardiac dysfunction in AL amyloidosis is characterized by dual insults to the myocardium from infiltration and toxicity from light chains during the active phase and by infiltration alone in the remission phase. MethodsProspectively enrolled subjects with cardiac AL amyloidosis (21 remission AL amyloidosis; age: 63.4 ± 7.3 years; 47.6% male; and 48 active AL amyloidosis; age: 62.5 ± 7.4 years; 60.4% male) underwent contrast-enhanced cardiac magnetic resonance with T1 and T2 mapping and measurement of extracellular volume (ECV). By definition, serum free light-chain levels were normal for at least 1 year following successful AL therapy in the remission group and abnormal in the active group. ResultsMyocardial ECV was similarly expanded in the remission and active AL amyloidosis groups (0.488 ± 0.082 vs 0.519 ± 0.083, respectively; P = 0.15). However, myocardial T2 relaxation times (47.7 ± 3.2 ms vs 45.5 ± 3.0 ms; P = 0.008) as well as native T1 times (1,368 ms [IQR: 1,290-1,422 ms] vs 1,264 ms [IQR: 1,203-1,380 ms]; P = 0.024) were significantly higher in the remission compared to the active AL amyloidosis group. ConclusionsMyocardial ECV is substantially expanded in the active AL and remission AL cardiac amyloidosis groups, but native T1 values were higher, suggesting a different myocardial composition. There is no evidence of myocardial edema in active AL cardiac amyloidosis. Future phenotyping studies of AL cardiac amyloidosis need to consider complementary myocardial markers that define the interstitial milieu in addition to changes in extracellular volume. (Molecular Imaging of Primary Amyloid Cardiomyopathy; NCT02641145)

PSMD9 ribosomal protein network maintains nucleolar architecture and WT p53 levels

Capitalizing on an unexpected observation that multiple free ribosomal proteins co-purify/pull-down with PSMD9, we report here for the first time that PSMD9 is necessary to maintain the morphology and integrity of the nucleolus. As seen by NPM1 immunofluorescence and electron microscopy, the nucleolar structure is clearly disrupted in PSMD9 null MCF7 breast cancer cells. The resultant stress is pronounced leading to the accumulation of WT p53 and slow growth. A dual insult with Actinomycin D exasperates the nucleolar stress in these cells which fail to recover in stipulated time. This double insult in the WT cells enhances the interaction of PSMD9 with ribosomal subunits. Our data also reveals that in PSMD9 null cells, ribosomal proteins RPS25 and RPL15 fail to localise in the nucleolus. We speculate that the interaction of PSMD9 with multiple free ribosome subunits has at least two important implications: a) PSMD9 plays a role in trafficking of ribosomal proteins into the nucleolus, therefore contributing to the maintenance of structural and morphological organization of the membrane-less nucleolar compartment; b) under conditions that induce nucleolar stress, PSMD9-Ribosomal Protein interaction protects WT MCF7 breast cancer cells from slow growth and eventual death. This possibility renders the domains of PSMD9 to be attractive drug targets in the context of cancer and other multiple ribosome-associated disorders.

In Situ Cold Perfusion of the Liver on Cardiopulmonary Bypass: Coagulopathy and Its Correction: A Case Report

We report a case of profound coagulopathy after the dual insult of cold in-situ perfusion of the liver and cardiopulmonary bypass in a patient undergoing complex hepatobiliary and cardiovascular surgery. Management of this coagulopathy with conventional blood products was hindered by elevated venous pressures, thought to contribute to persistent bleeding and risked liver and right ventricular dysfunction, necessitating a change in strategy. Anesthesiologists should consider fluid-restrictive strategies to correct coagulopathy in combined liver and cardiac surgery.

PAD4 Deficiency Leads to Decreased Organ Dysfunction and Improved Survival in a Dual Insult Model of Hemorrhagic Shock and Sepsis.

Indirect acute respiratory distress syndrome (iARDS) is caused by a nonpulmonary inflammatory process resulting from insults such as nonpulmonary sepsis. Neutrophils are thought to play a significant role in mediating ARDS, with the development of iARDS being characterized by dysregulation and recruitment of activated neutrophils into the lung. Recently, a novel mechanism of microbial killing by neutrophils was identified through the formation of neutrophil extracellular traps (NETs). NETs are composed of large webs of decondensed chromatin released from activated neutrophils into the extracellular space; they are regulated by the enzyme peptidylarginine deiminase 4 (PAD4) through mediation of chromatin decondensation via citrullination of target histones. Components of NETs have been implicated in ARDS. However, it is unknown whether there is any pathological significance of NET formation in ARDS caused indirectly by nonpulmonary insult. We subjected PAD4-/- mice and wild-type mice to a "two-hit" model of hypovolemic shock (fixed-pressure hemorrhage [Hem]) followed by septic cecal ligation and puncture (CLP) insult (Hem/CLP). Mice were hemorrhaged and resuscitated; 24 h after Hem, mice were then subjected to CLP. Overall, PAD4 deletion led to an improved survival as compared with wild-type mice. PAD4-/- mice displayed a marked decrease in neutrophil influx into the lung, as well decreased presence of proinflammatory mediators. PAD4-/- mice were also able to maintain baseline kidney function after Hem/CLP. These data taken together suggest PAD4-mediated NET formation contributes to the mortality associated with shock/sepsis and may play a role in the pathobiology of end organ injury in response to combined hemorrhage plus sepsis.

Sonic Hedgehog Agonist Protects Against Complex Neonatal Cerebellar Injury

The cerebellum undergoes rapid growth during the third trimester and is vulnerable to injury and deficient growth in infants born prematurely. Factors associated with preterm cerebellar hypoplasia include chronic lung disease and postnatal glucocorticoid administration. We modeled chronic hypoxemia and glucocorticoid administration in neonatal mice to study whole cerebellar and cell type-specific effects of dual exposure. Chronic neonatal hypoxia resulted in permanent cerebellar hypoplasia. This was compounded by administration of prednisolone as shown by greater volume loss and Purkinje cell death. In the setting of hypoxia and prednisolone, administration of a small molecule Smoothened-Hedgehog agonist (SAG) preserved cerebellar volume and protected against Purkinje cell death. Such protective effects were observed even when SAG was given as a one-time dose after dual insult. To model complex injury and determine cell type-specific roles for the hypoxia inducible factor (HIF) pathway, we performed conditional knockout of von Hippel Lindau (VHL) to hyperactivate HIF1α in cerebellar granule neuron precursors (CGNP) or Purkinje cells. Surprisingly, HIF activation in either cell type resulted in no cerebellar deficit. However, in mice administered prednisolone, HIF overactivation in CGNPs resulted in significant cerebellar hypoplasia, whereas HIF overactivation in Purkinje cells caused cell death. Together, these findings indicate that HIF primes both cell types for injury via glucocorticoids, and that hypoxia/HIF + postnatal glucocorticoid administration act on distinct cellular pathways to cause cerebellar injury. They further suggest that SAG is neuroprotective in the setting of complex neonatal cerebellar injury.

Enhanced Innate Inflammation Induced by Anti-BTLA Antibody in Dual Insult Model of Hemorrhagic Shock/Sepsis.

Sepsis following hemorrhagic shock is a common clinical condition, in which innate immune system suffers from severe suppression. B and T lymphocyte attenuator (BTLA) is an immune-regulatory coinhibitory receptor expressed not only on adaptive, but also on innate immune cells. Our previous data showed that BTLA gene deficient mice were protected from septic mortality when compared with wild-type control C57BL/6 mice. Here, we extended our study by treating C57BL/6 mice with an anti-BTLA monoclonal antibody (clone 6A6; reported to have the ability to neutralize or agonize/potentiate BTLA signaling) in a mouse model of hemorrhagic shock (Hem) followed by sepsis induced by cecal ligation and puncture (CLP); positing initially that if BTLA engagement was neutralized, like gene deficiency, an anti-BTLA mAb would have the similar effects on the inflammatory response/morbidity in these mice after such insults. Here, we report that BTLA expression is elevated on innate immune cells after Hem/CLP. However, anti-BTLA antibody treatment increased cytokine (TNF-α, IL-12, IL-10)/chemokine (KC, MIP-2, MCP-1) levels and inflammatory cells (neutrophils, macrophages, dendritic cells) recruitment in the peritoneal cavity, which in turn aggravated organ injury and elevated these animals' mortality in Hem/CLP. When compared with the protective effects of our previous study using BTLA gene deficient mice in a model of lethal septic challenge, we further confirmed BTLA's contribution to enhanced innate cell recruitment, elevated IL-10 levels, and reduced survival, and that engagement of antibody with BTLA potentiates/exacerbates the pathophysiology in Hem/sepsis.

Cognitive deficits associated with combined HIV gp120 expression and chronic methamphetamine exposure in mice

Methamphetamine abuse is common among individuals infected by human immunodeficiency virus (HIV). Neurocognitive outcomes tend to be worse in methamphetamine users with HIV. However, it is unclear whether discrete cognitive domains are susceptible to impairment after combined HIV infection and methamphetamine abuse. The expression of HIV/gp120 protein induces neuropathology in mice similar to HIV-induced pathology in humans. We investigated the separate and combined effects of methamphetamine exposure and gp120 expression on cognitive function in transgenic (gp120-tg) and control mice. The mice underwent an escalating methamphetamine binge regimen and were tested in novel object/location recognition, object-in-place recognition, and Barnes maze tests. gp120 expression disrupted performance in the object-in-place test (i.e. similar time spent with all objects, regardless of location), indicating deficits in associative recognition memory. gp120 expression also altered reversal learning in the Barnes maze, suggesting impairments in executive function. Methamphetamine exposure impaired spatial strategy in the Barnes maze, indicating deficits in spatial learning. Methamphetamine-exposed gp120-tg mice had the lowest spatial strategy scores in the final acquisition trials in the Barnes maze, suggesting greater deficits in spatial learning than all of the other groups. Although HIV infection involves interactions between multiple proteins and processes, in addition to gp120, our findings in gp120-tg mice suggest that humans with the dual insult of HIV infection and methamphetamine abuse may exhibit a broader spectrum of cognitive deficits than those with either factor alone. Depending on the cognitive domain, the combination of both insults may exacerbate deficits in cognitive performance compared with each individual insult.

Depressed by Dual Insults

Two neuroinflammatory insults lead to microglial-mediated long-term depression of hippocampal neurons.

Etiology, clinical profile, and inhospital mortality of acute-on-chronic liver failure: a prospective study

The causes of acute injury in acute-on-chronic liver failure (ACLF) are variable. There may be simultaneous presence of more than one acute insult. We describe the clinical profile of ACLF and the effect of dual acute insult on the natural history. Patients with jaundice diagnosed to have ACLF were prospectively enrolled. Patients were evaluated for the clinical presentation, etiology of acute decompensation and underlying chronic liver disease, and inhospital mortality. We compared the clinical profile of patients who had dual acute insult with those of single/unknown insult. Fifty-two patients with ACLF (mean age 38.6 ± 16.7 years; M/F 41:11) were included. Hepatitis virus infection (46.1 %) and bacterial infection (36.5 %) were the most common acute insults. Hepatitis virus infections were the sole acute insult in 34.6 % and associated with another injury in 11.5 %. Bacterial infections were identified as acute insult in 19 patients (sole acute insult in 13). Drugs, autoimmune disease, surgery, malaria, and dengue were other acute injuries identified. The cause of acute decompensation was unknown in 11.5 %. Mortality (66.6 % vs. 51.1 %) was higher in patients with dual insult (n=9) as compared with single/unknown insult (n=43). Hepatitis virus and bacterial infection/sepsis were the common acute insults in ACLF. Dual acute insult is not uncommon, poses diagnostic dilemma, and may increase mortality in these patients. Plasmodium falciparum infection and dengue fever may be associated with ACLF.

Liver histological parameters correlate with clinical severity in patients with acute-on-chronic liver failure (ACLF)

S 21 ANNUAL CONFERENCE—2013 A cu te o n C h ro n ic Lver Fa ilu re Conclusions: Dual acute insult is not uncommon, poses diagnostic dilemma, and may increase mortality in these patients. The P falciparum and dengue fever may be responsible for decompensation of stable CLD patients. Corresponding Address. Sandeep Nijhawan. E-mail: dr_nijhawan@hotmail.com

A variant conferring cofactor-dependent assembly of Escherichia coli dimethylsulfoxide reductase

We have investigated the final steps of complex iron–sulfur molybdoenzyme (CISM) maturation using Escherichia coli DMSO reductase (DmsABC) as a model system. The catalytic subunit of this enzyme, DmsA, contains an iron–sulfur cluster (FS0) and a molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD). We have identified a variant of DmsA (Cys59Ser) that renders enzyme maturation sensitive to molybdenum cofactor availability. DmsA-Cys59 is a ligand to the FS0 [4Fe–4S] cluster. In the presence of trace amounts of molybdate, the Cys59Ser variant assembles normally to the cytoplasmic membrane and supports respiratory growth on DMSO, although the ground state of FS0 as determined by EPR is converted from high-spin (S=3/2) to low-spin (S=1/2). In the presence of the molybdenum antagonist tungstate, wild-type DmsABC lacks Mo-bisPGD, but is translocated via the Tat translocon and assembles on the periplasmic side of the membrane as an apoenzyme. The Cys59Ser variant cannot overcome the dual insults of amino acid substitution plus lack of Mo-bisPGD, leading to degradation of the DmsABC subunits. This indicates that the cofactor can serve as a chemical chaperone to mitigate the destabilizing effects of alteration of the FS0 cluster. These results provide insights into the role of the Mo–bisPGD–protein interaction in stabilizing the tertiary structure of DmsA during enzyme maturation.

Local Tissue Expression of the Cell Death Ligand, Fas Ligand, Plays a Central Role in the Development of Extrapulmonary Acute Lung Injury

Indirect acute lung injury (ALI) is a common manifestation in critically ill patients. Using a model of indirect ALI in mice, our laboratory has shown that local/pulmonary inhibition of extrinsic death receptor protein (Fas) leads to a decrease in lung inflammation and improved survival. However, it is unknown if local, i.e., autocrine/paracrine, inhibition of Fas ligand (FasL) affects Fas-expressing target cells itself or blockade of the actions of a more distal/endocrine source of FasL that accounts for these findings. To examine this, we used a model of indirect ALI in mice (dual insult of hemorrhagic shock followed 24 h later by cecal ligation and puncture, in which animals received FasL small interfering RNA (siRNA) intratracheally (local silencing) or intravenously (systemic/distal delivery) after hemorrhage. After intratracheal delivery of FasL siRNA, there was a significant decrease in inflammatory cytokines, myeloperoxidase activity, and caspase 3 activity in lung tissue along with protein leak as compared with controls. There was no difference found in these various outcome markers between those treated with intravenously administered FasL siRNA versus controls. The observation that local silencing of FasL, as opposed to distal/systemic silencing, ameliorates the effects of indirect ALI suggests not only that FasL produced in an autocrine/paracrine fashion in local tissues has pathological consequences within the lungs, but also that FasL might be a valuable pulmonary therapeutic target.

Exogenous Neonatal Erythropoietin Mitigates Brain Damage After a Combination of Prenatal Hypoxic-Ischemia and Lipopolysaccharide Inflammation in Rats

Introduction Many infants born very preterm who suffer brain damage most likely experienced a combined insult from intrauterine infection and placental insufficiency. Damage is thought to be synergistic rather than additive but the mechanisms of combined injury remain elusive. A combination of lipopolysaccharide-induced inflammation and hypoxia-ischemia has been used in rats to model the dual insult that occurs in human infants prenatally. Erythropoietin, a pleiotrophic cytokine that is essential for central nervous system development, ameliorates brain injury after isolated hypoxic-ischemic or inflammatory insults through different intracellular signaling pathways. We hypothesized that exogenous neonatal EPO administration would lessen the damage of a combined prenatal insult in rats. Methods On embryonic Day 18 fetal rats experienced 60 minutes of transient uterine artery occlusion with or without intracervical LPS administration with sham controls receiving surgery but no occlusion and saline for LPS. Survival was recorded and histological biochemical and functional assays were performed. Means were compared with ANOVA with Tukey HSD post hoc analysis. Results After a combined insult of HI and 0.15-mg/kg LPS on E18 the survival of pups by postnatal Day 1 (P1) decreased from 77% with HI alone to 22% for LPS plus HI. When exogenous systemic EPO was administered P1–P3 survival to P9 improved markedly from 40% (2 of 5) for saline-treated insult pups to 100% (6 of 6) for EPO-treated. Initial histological analyses show EPO decreases the number of brain activated caspase 3 and activated microglia by P9. Additional analyses will be presented. Conclusion As at least 60% of placentas from infants born pre-term show evidence of chorioamnionitis, assessment of the impact of exogenous EPO on a model of a combination injury is essential prior to proceeding with a clinical trial. Initial results indicate neonatal exogenous EPO mitigates damage from the combined insult.

Dendritic cells as anti‐inflammatory regulators of extra‐pulmonary acute lung injury

Acute Lung injury (ALI) is one of the most common organ dysfunctions in critically ill patients and is associated with lung inflammation and apoptosis. However, the cellular interactions in the lung contributing to its pathophysiology are unclear. The objective of this study was to determine the contribution of dendritic cells (DCs) to lung apoptosis, inflammation and neutrophil recruitment during ALI resultant from hemorrhagic shock (Hem)/sepsis (CLP).Using a dual insult model of Hem followed 24h later by CLP, we measured, 24h after CLP, caspase 3 activity, lung myeloperoxidase (MPO) and pro‐inflammatory cytokine production in the lung and plasma of CD11c‐Diphteria Toxin (DT) Receptor conjugated vs background mice injected intraperitoneally (IP) or intratracheally (IT) with DT prior to shock.Systemic DC depletion (IP DT) was associated with a marked increase in IL‐6, TNF‐α and MCP‐1 concentrations after Hem/CLP, whereas lung apoptosis and neutrophil recruitment (MPO) were not modified in comparison with background mice. Similar results were observed after local lung DCs depletion (IT DT). Preliminary experiments suggest that this DC effect is mediated through the recruitment and/or activation of macrophage to/in the lung. Thus the resident and possibly recruited DCs may play here‐to‐for unappreciated role in suppressing lung inflammation and promoting the resolution of ALI. (NIH‐HL73525)

Pregabalin in the management of central neuropathic pain

Central neuropathic pain is a painful condition, often severe, that occurs in a person who is already affected by an injury or disease of the brain or spinal cord. This dual insult is especially threatening to the quality of life of a person and their ability to perform even the most basic of tasks. Despite this high level of suffering there are relatively few trials investigating the management of central neuropathic pain. However, two randomised placebo-controlled studies have recently emerged demonstrating efficacy of pregabalin in reducing central neuropathic pain due to spinal cord injury and central poststroke pain. Pregabalin, an anticonvulsant, has been shown to be efficacious in the management of peripheral neuropathic pain of various causes and now may have a role to play in central neuropathic pain.

INFLAMMATORY/CARDIOVASCULAR-METABOLIC RESPONSES IN A RAT MODEL OF BURN INJURY WITH SUPERIMPOSED INFECTION

Infection remains the major cause of morbidity and mortality in burn patients. Furthermore, the use of antibiotics in such patients has led to the prevalence of antibiotic-resistant microbial infections; one such infection in intensive care unit turns out to be caused by the enterococcal organisms. Our laboratory studies have used a rat model of bum injury and Enterococcus faecalis infection. Sprague-Dawley male rats ( approximately 250 g) were initially given an intragastric gavage of the antibiotic ciprofloxacin for 3 days. This procedure allowed for decontamination of intestine of gram-negative and some gram-positive organisms. The remainders of the gram-positive organisms were, to a large extent, Enterococci. After the decontamination procedure, rats were intra-abdominally inoculated with E. faecalis; inoculation involved preparation of sterilized rat fecal pellets impregnated with E. faecalis (10 colony-forming units) and their implants through a midline abdominal wall incision. Some of the rats that were implanted with the fecal pellets were subject to full-thickness skin bums ( approximately 30% total body surface area; 95 degrees C water for 7 s). Sham abdominal infection rats received a sterile pellet only; sham bum procedure consisted of exposing the skin to room temperature water. All sham and bum and/or E. faecalis infection procedures were carried out on rats under pentobarbital anesthesia. Inflammation and innate host defense-related responses were assessed via measurements of neutrophil effector responses, i.e., oxygen anion free radical (O2)/eIastase production, CD11b/CD18 expression, apoptosis, and tissue infiltration. Determining epithelial lactulose permeability, microvascular albumin leakage, and epithelial tight junction integrity assessed the status of intestinal function/structural derangements. The animals' metabolic and cardiovascular integrity was evaluated determining blood pH, p02, pC02, heart rate, respiratory rate, blood pressure, and cardiac output. Whereas the aforementioned measurements were carried out at 24 to 48 h postbum injury with and without the Enterococcal infection, animal mortality was determined for up to 5 days after the experimental injuries. The results of the studies indicated that whereas bum or E. faecalis infection alone did not produce significant mortality, the dual insult with bum and E. faecalis infection resulted in significant animal death accompanied by relatively more profound metabolic and cardiovascular derangements. Inappropriately heightened neutrophil effector responses were present with bum alone as well as with the dual bum and infection complications. These studies suggest that animal models of bum injury with Enterococcal infection complications simulate the adverse outcomes bum patients infected with Enterococcal organisms.

Characterization of Calcium Release-activated Apoptosis of LNCaP Prostate Cancer Cells

Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize residual apoptotic pathways in CaP cells. When intracellular Ca(2+) stores are released and plasma membrane "store-operated" Ca(2+) entry channels subsequently open, cytosolic [Ca(2+)] increases and is thought to induce apoptosis. However, cells incapable of releasing Ca(2+) stores are resistant to apoptotic stimuli, indicating that Ca(2+) store release is also important. We investigated whether release of intracellular Ca(2+) stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca(2+) without elevating cytosolic [Ca(2+)]; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca(2+) store release with the dual insults of store release and cytosolic [Ca(2+)] elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca(2+) perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins Bclx(L) and catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca(2+) store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic [Ca(2+)] elevation and intracellular store release.