Calpain activation contributes to oxidative stress-induced pancreatic acinar cell injury.

Oxygen radicals have been implicated as mediators in the pathogenesis of pancreatic acinar cell necrosis. However, the sequence of events between the oxidative insult and cell damage remains unclear. In the current study, we investigated whether the Ca(2+)-regulated cytosolic cysteine protease calpain is activated by oxidative stress and contributes to oxidant-induced acinar cell damage. Isolated rat pancreatic acinar cells were exposed to hydrogen peroxide (H(2)O(2))-generated oxidative stress in the presence or absence of the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and different calpain inhibitors including benzyloxycarbonyl-valyl-phenylalanine methyl ester. Calpain activation was studied by fluorescence spectrophotometry and immunoblotting. Cell injury was assessed by lactate dehydrogenase (LDH) release and characterization of the cellular ultrastructure including fluorescence-labeled actin filaments. Exposure of acinar cells to H(2)O(2) provoked a time- and dose-dependent increase in calpain proteolytic activity involving the ubiquitous isoforms mu- and m-calpain. The activation of calpain reflected the time course of developing cytotoxicity as demonstrated by increased LDH release. Inhibition of oxidant-induced calpain activity by BAPTA-AM and various calpain inhibitors provoked a decline in oxidant-induced cell injury. In particular, changes in the actin filament organization characterized by an increase in the basolateral actin and by a detachment of actin from the cell membrane in the region of membrane blebs were clearly reduced. In summary, our findings suggest that acinar cell damage through oxidative stress requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease. The results support the hypothesis that calpain activation may play a role in the development of acute pancreatitis.

5-Fluorouracil/irinotecan induced lethal toxicity as a result of a combined pharmacogenetic syndrome: report of a case.

Combination cancer chemotherapy induced toxicity can be associated with combined pharmacogenetic syndromes. Dihydropyrimidine dehydrogenase (DPD) is the principal enzyme involved in the catabolic detoxification of 5-fluorouracil (5FU). A heterozygous G > A transition at the 5' splicing donor consensus sequence in intron 14 leading to exon 14 skipping (IVS14+1 G > A, DPYD*2A) with partial loss of enzyme activity may be partly responsible for 5FU induced toxicity, whereas irinotecan associated toxicity may in part be explained by an aberrant UGT1A1 promoter (TA)(n) genotype underlying Gilbert's syndrome with reduced liver glucuronidation activity. This report describes a 44 year old white woman who suffered from severe gastrointestinal and haematological toxicity while undergoing 5FU(24h)/folinic acid/irinotecan treatment for adenocarcinoma of the sigmoid colon. Despite appropriate supportive treatment, her condition rapidly deteriorated and led to death. Molecular analysis revealed a hitherto undescribed combined pharmacogenetic syndrome, consisting of heterozygosity for the DPD IVS14+1 G > A mutation and UGT1A1 (TA)(6/7) heterozygosity, which probably contributed to the fatal outcome in this patient.

Resistance of tumour cells to chemotherapy: importance of host defence factors.

The arguments favouring the hypothesis that chemotherapeutic agents might act in cooperation with host defence mechanisms are reviewed briefly. In patients with far advanced solid tumours plasma factors blocking in vitro immune reactions have been identified and successfully removed by immune adsorption or plasma exchange. By plasmapheresis performed in patients with metastatic malignancies resistant to chemotherapy it was possible to induce tumour regressions. In 25/28 patients responding to the combined plasmapheresis/chemotherapy procedure a positive correlation was found to clinical results and patterns of plasma-blocking factor activities.

Enhanced procoagulatory activity (PCA) of human monocytes/macrophages after in vitro stimulation with chemically modified LDL.

In cultured human monocytes/macrophages, surface expression of procoagulatory activity (PCA) was induced by chemically modified LDL (acetyl-LDL and MDA-LDL) in a dose- and time-dependent manner. Maximum PCA (30-fold increase) was detected after 24 h of culture with modified LDL at doses of 25-750 micrograms protein/ml. Using factor VII deficient human plasma and phospholipase C this PCA was identified as tissue thromboplastin activity (factor III). These results suggest a further atherogenic potential for modified LDL through stimulation of the conversion of fibrinogen to fibrin in the atheromatous lesion.

Apolipoprotein A-I and apolipoprotein B containing lipoprotein particles in coronary patients treated with extracorporal low density lipoprotein precipitation (HELP).

Evidence for chemical and biological heterogeneity of human plasma lipoprotein density classes has been steadily accumulating over the last 15 years. Furthermore, several recent reports have indicated potential clinical significance of certain lipoprotein subspecies as either atherogenic or antiatherogenic. It is generally accepted that lipid lowering treatments can retard or even reverse development of atherosclerotic lesions. However, very little is known about effects of various lipid lowering treatments on specific lipoprotein particles. The purpose of this study was to explore the effects of heparin induced extracorporal low density lipoprotein precipitation (HELP) on various subspecies of plasma lipoprotein particles defined primarily by their apolipoprotein composition. Using particle specific enzyme immunoassays, the immediate changes in lipoprotein particle profiles were analyzed after a single HELP treatment in 12 patients with angiographically documented coronary artery disease. In a separate group of 6 patients, particles were repeatedly measured over a period of 96 h following a HELP treatment. Single HELP treatment caused an immediate and highly significant decrease (67%) in the concentration of simple lipoprotein particles containing apolipoprotein B (apo B) as a sole apolipoprotein (LP-B). Various subspecies of complex particles containing apo B and other apolipoproteins (Lp-B-complex) were also decreased although to a lesser degree (44-53%). HELP treatment caused an insignificant, 3% decrease of lipoprotein particles containing apo A-I but no apo A-II (Lp-A-I) and a 6% decrease in the concentration of particles containing both apo A-I and apo A-II (Lp-A-I:A-II). During the 96-h period following HELP treatment various apo B containing particles recovered at different rates in different patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Acceleration of hepatocellular energy by idebenone during early reperfusion after cold preservation ameliorates heat shock protein 70 gene expression in a pig liver model.

BACKGROUND: Heat shock proteins (HSPs) are induced in the liver after warm ischemia/reperfusion and are thought to be markers of hepatocellular injury and oxidative stress. METHODS: The influence of variable periods of cold storage followed by reperfusion on the expression of HSP70 was studied in the isolated perfused pig liver. Organs were harvested and stored in histidine-tryptophan-ketoglutarate solution at 4 degrees C and then perfused (210 min) in a closed water bath (38 degrees C), which subjects the liver to fluctuating outer pressure. The role of energy depletion, reactive oxygen intermediates, Kupffer cells, and circulating leukocytes in HSP70 expression was determined. RESULTS: HSP70 expression was not detectable in liver tissue before explantation or before reperfusion by Northern blot analysis using a pig HSP70 gene probe. HSP70 expression was observed after reperfusion depending on cold storage time. Kinetics of HSP70 expression monitored by reverse transcriptase polymerase chain reaction showed a rapid increase of mRNA within 1 hr, which was closely associated with delayed recovery of hepatocellular energy charge, as assessed by the ketone body ratio. The inactivation of Kupffer cells, the presence or absence of leukocytes, and the suppression of oxidative stress with the antioxidant idebenone, given during reperfusion, had no influence. However, feeding the animals with idebenone over 7 days before explantation led to a faster recovery of ketone body ratio, paralleled by a substantial suppression of HSP70 expression. CONCLUSIONS: Our data show that HSP70 expression during reperfusion is mainly dependent on the preceding cold storage time and the consecutive delayed recovery of the hepatocellular energy charge.

609 C --> T polymorphism in NAD(P)H:quinone oxidoreductase gene in patients with prostatic adenocarcinoma or benign prostatic hyperplasia.

Allelic variations at the NQO1 locus encoding for NAD(P)H:quinone oxidoreductase have recently been implicated in carcinogenesis, cancer chemoprevention and chemotherapy. Two naturally occurring alleles differ at nucleotide position 609 with the variant allele leading to diminished or absent enzyme activity. Using polymerase chain reaction-restriction fragment length polymorphic analysis, NQO1 genotyping was performed in DNA from blood cells from 54 patients with prostatic adenocarcinoma, 49 patients with benign prostatic hyperplasia and 100 healthy control subjects. Prostatic adenocarcinoma patients and healthy controls demonstrated almost identical genotype distribution and frequencies of the variant allele (17.6 versus 17.5%). The variant allele was slightly more frequent in benign prostatic hyperplasia patients (23.5%). Established prostate cancer-derived cell lines LnCAP, DU-145, and PC-3 demonstrated NQO1 wild-type genotype. Our study does not support the hypothesis that the variant NQO1 allele is a risk modifier for prostatic adenocarcinoma and/or benign prostatic hyperplasia in the Caucasian population.

Severe hypophosphatemia during hematopoietic reconstitution after allogeneic peripheral blood stem cell transplantation.

A patient suffering from acute myeloid leukemia (FAB M5a) received a PBSC allograft from a matched, related donor. On day 13 after transplantation severe hypophosphatemia (0.21 mmol/l) was first noted which persisted irrespective of intravenous phosphate administration, and within 2 days reached concentrations below 0.13 mmol/l. After repeated phosphate substitution serum phosphate returned to 1.40 mmol/l on day 17. Phosphate in urine, and calcium in serum were recorded as unchanged throughout. Clinical signs and symptoms due to severe hypophosphatemia were not observed except for paresthesia in the lower extremities. The precipitous fall in serum phosphate coincided with hematopoietic reconstitution as reflected by a steep rise in leukocyte count from 0.08 x 109/l on day 10 to 5. 94 x 109/l on day 15 after transplantation. Thus, isolated hypophosphatemia was likely the result of excessive cellular phosphate uptake during hematopoietic reconstitution. Electrolyte monitoring after PBSCT should include serum phosphate to identify the hypophosphatemia associated with hematopoietic recovery.

Suppression of leukocyte-enhanced cold ischemia/reperfusion injury of liver endothelium with the benzoquinone antioxidant idebenone.

OBJECTIVE: Despite the large body of evidence for a major role of neutrophils and oxidant stress, the exact pathogenesis of the early ischemia/reperfusion injury after cold preservation of the liver is not well understood. The potential benefit of an antioxidant on metabolic liver function during reperfusion has been demonstrated in several studies. MATERIALS AND METHODS: We describe a cold storage/reperfusion damage model with isolated perfused pig livers, where the effects of neutrophils and idebenone, a recently developed benzoquinone antioxidant were studied. The integrity of sinusoidal endothelial cells (SEC) was estimated by hyaluronic acid concentration in perfusate and the expression of endothelial constitutive nitric oxide synthase (ecNOS) after reperfusion and compared to lipid peroxidation and antioxidant content. RESULTS: Hyaluronic acid displayed the highest levels and ecNOS mRNA was most depressed in livers reperfused with neutrophils after 20 h cold storage; this was accompanied by an increase in lipid peroxidation (TBARS) and a breakdown of endogenous lipophilic antioxidants (alpha-tocopherol and coenzyme Q-10). These effects were attenuated, when neutrophils were excluded from reperfusion and almost completely abolished by the addition of 200 mumol/L idebenone. CONCLUSIONS: These data suggest that a leukocyte-mediated damage based on reactive oxygen species markedly contributes to the reperfusion injury of SEC after cold preservation of the liver. Therefore, the presence of effective antioxidants in the early reperfusion phase may be beneficial for liver graft integrity.

Diagnostic value of urinary alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase in comparison to alpha 1-microglobulin as a marker in evaluating tubular dysfunction in glomerulonephritis patients.

To estimate the diagnostic value of tubular parameters, the urinary alanine aminopeptidase (AAP), N-acetyl-beta-D-glucosaminidase (NAG) and the alpha(1)-microglobulin (a1M) of 150 patients with histologically proven glomerulonephritis (GN) were determined. In addition, the reabsorption rate of the proximal tubule and the fractional excretion of sodium, the free water clearance and the renal function were assessed by inulin and p-aminohippurate (PAH) clearance. Compared to healthy controls, urinary AAP, NAG and a1M were found significantly elevated in GN patients. Morphological tubular changes were confirmed by significant differences in urinary laboratory parameters. In patients with tubular atrophy, the diagnostic sensitivity and specificity were calculated as follows: AAP (0.94/0.35), NAG (0.75/0.59) and a1M (0.73/0.52). In patients showing tubular protein droplets, the values were 0.90/0.17 for AAP, 0.78/0.76 for NAG and 0.84/0.74 for a1M and in patients with interstitial fibrosis, the values were AAP (0.95/0.35), NAG (0.75/0. 46) and a1M (68/0.38). Urinary AAP, NAG and a1M reflect histologically proven tubulus alteration in GN, although in most cases, the renal function is still intact. AAP indicates very early tubular impairment and, in some cases, AAP is elevated although NAG and a1M are still within normal ranges. We suggest that the enzyme activities are useful in the diagnostics of early stages of the disease.

Oxidation of the indole nucleus of 5-hydroxytryptamine and formation of dimers in the presence of peroxidase and H2O2.

5-Hydroxytryptamine (5-HT) is rapidly oxidized in the presence of peroxidase and H2O2. The major reaction product was isolated by gel chromatography and analyzed by mass spectroscopy. It is a 5-HT dimer formed under abstraction of two protons, most likely by the reaction at the C(4) position of two phenoxyradicals of 5-HT. In the presence of 5-HT an increased H2O2-consumption and a dose dependent reduction of the formation of reactive oxygen metabolites during H2O2 degradation was observed. The pattern of 5-HT reaction products separated by TLC was dependent on the H2O2 concentrations used. In the presence of albumen, plasma or tissue homogenates, massive binding of the 5-HT oxidation products to proteins was observed.

Heparin extracorporeal LDL precipitation (HELP): an effective apheresis procedure for lowering Lp(a) levels.

Heparin-induced extracorporeal low-density lipoprotein precipitation (HELP) is based on the precipitation of apolipoprotein B (apo B) containing lipoproteins with heparin at low pH (4.85). In in vitro experiments we could show that Lp(a) is quantitatively (> 99%) precipitated from plasma by heparin in the pH range 4.6-5.2. The acute changes in Lp(a) after a single HELP-LDL apheresis were investigated in twelve patients with Lp(a) concentrations > 30 mg/dl. A single treatment caused a highly significant decrease (62%) in the concentration of Lp(a), similar to the decrease (60%) observed for LDL-cholesterol. Analysis of the data from ten patients with different apo(a) phenotypes indicated that Lp(a) is eliminated with almost 100% efficiency in the extracorporeal circulation, irrespective of apo(a) phenotype and plasma concentration. The mean rate of recovery of Lp(a) following HELP-LDL apheresis was slightly slower than that of LDL-cholesterol. Plasma Lp(a) concentrations were monitored in seven patients over 2 years. Mean Lp(a) concentrations after 2 years were lower than pre-treatment levels, indicating that repeated elimination of the lipoprotein does not lead to an induction in its synthesis. HELP-LDL apheresis should be particularly suitable for treatment of patients with elevated LDL-cholesterol levels who are also at increased coronary risk because of high Lp(a) concentrations.

Serum-to-urinary prostate-specific antigen ratio in patients with benign prostatic hyperplasia and prostate cancer.

BACKGROUND: Prostate-specific antigen (PSA) is the single most important tumor marker in early detection and monitoring of prostate cancer (CaP). However, routine analysis of serum PSA concentrations does not allow differentiation between CaP and prostatic diseases. The aim of the present study was to evaluate the usefulness of the serum-to-urinary PSA ratio in a clinical setting. MATERIALS AND METHODS: In a retrospective clinical study, we determined serum and urine PSA concentrations in 48 patients with benign prostatic hyperplasia (BPH) and 57 patients with histologically confirmed CaP. RESULTS: The serum-to-urinary PSA ratio is able to discriminate BPH from CaP. CONCLUSIONS: Determination of the serum-to-urinary PSA ratio enhances the specificity of PSA in screening for CaP and monitoring of patients with CaP under androgen deprivation therapy.

The effects of Trolox, a water-soluble vitamin E analogue, in regionally ischemic, reperfused porcine hearts.

Myocardial protection by the water-soluble vitamin E analogue, Trolox, was investigated in 18 regionally ischemic, reperfused porcine hearts. The left anterior descending coronary artery was distally ligated for 45 min and was reperfused for three days. Five grams of Trolox (n = 9) were infused intravenously before coronary occlusion. Treatment was continued with an intravenous dose of 5 grams Trolox/24 hours until the end of the experiment. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was assessed by sonomicrometry. Generation of free radicals by stimulated neutrophils was evaluated by luminol-enhanced chemiluminescence. Plasma concentrations of Trolox were measured by high-performance liquid chromatography. Aside from heart rate before ischemia, global hemodynamic values including calculated left ventricular oxygen consumption did not differ significantly between the two groups. Plasma concentrations of Trolox measured 1.8 +/- 0.3 mmol/l (before ischemia), 0.96 +/- 0.13 mmol/l (before reperfusion), 0.77 +/- 0.1 mmol/l (40 min of reperfusion), and 0.08 mmol/l (end of the experiment). Generation of free radicals by stimulated neutrophils was reduced by about 30% in the treatment group before ischemia and immediately before reperfusion, but was not reduced at the end of the experiment. Risk regions (control group 19.4 +/- 6 g, treatment group 19.3 +/- 7 g) and infarct sizes (control group 69.3 +/- 8%, treatment group 69.3 +/- 12%) were almost identical. Regional systolic shortening of a control segment and of the risk region were similar in both groups before ischemia, before reperfusion, and after 45 min of reperfusion. After 3 days of reperfusion, regional systolic shortening of the reperfused myocardium of the treated group had recovered to a significantly greater extent (P = 0.027). This parameter amounted to 9 +/- 6% in the treated group and to 3 +/- 3% in the control group. Improved functional recovery was not accompanied by higher tissue concentrations of adenosine triphosphate. It is concluded that the chosen treatment with Trolox does not reduce infarct size but accelerates functional recovery. This finding suggests that the mechanisms resulting in myocardial necrosis during ischemia/reperfusion and in post-ischemic myocardial dysfunction may differ.

Immediate and late effects of coronary angiography on soluble endothelial cell markers and P-selectin in patients with and without coronary artery disease.

Endothelial cell injury and platelet activation are considered primary events in the pathogenesis of coronary artery disease (CAD) and are marked by plasma concentrations of von Willebrand factor (vWF) and soluble thrombomodulin, and by soluble P-selectin, respectively. Because both endothelial cells and platelets interact with contrast media, we aimed to detect immediate and 24-h changes in these markers following coronary angiography in patients with and without CAD. Sixteen patients with angiographically proven CAD and 14 patients without significant coronary stenosis were investigated. Blood samples were obtained from an antecubital vein before and 24 h after cardiac catheterization, and from the coronary sinus before and immediately after angiography. Concentrations of the markers were determined using enzyme-linked immunosorbent assays. In the coronary sinus samples, the only significant finding was an increase in levels of soluble P-selectin in the patients with CAD (P < 0.038). In the post-catheterization peripheral blood samples, concentrations of soluble P-selectin (P = 0.004), vWF (P = 0.0007) and soluble thrombomodulin (P = 0.0013) were all increased in patients with CAD. In contrast, patients without CAD demonstrated increased levels of vWF only (P = 0.0015) in peripheral blood samples obtained 24 h after angiography. We conclude that both immediate and 24-h changes take place in endothelial cells and platelet markers in response to cardiac catheterization, and that these changes are different in patients with angiographically proven CAD and in patients free of disease. These differences may reflect alterations in endothelial cell or platelet reactivity in patients with CAD.

Antioxidative properties of serotonin and the bactericidal function of polymorphonuclear phagocytes.

We investigated the antioxidative properties of platelet-released serotonin on the bactericidal function of polymorphonuclear neutrophils (PMN) since there is a surprising co-incidence of low blood serotonin and an increased rate of infections. The antioxidative properties of serotonin were demonstrated by its suppressive effects on phagocytosis-associated, luminol-enhanced chemiluminescence (CL). The bactericidal activity of PMN was determined by a microbiological assay using opsonized Staphylococcus aureus. Serotonin suppresses luminol-enhanced chemiluminescence in a dose-dependent manner indicating an interaction with reactive oxygen species, which are responsible for effective bacterial killing during the phagocytosis-associated "respiratory burst". The modulation of the bactericidal function of PMN by serotonin is complex and depends upon the amount of serotonin: at concentrations normally present at sites of tissue injury and consecutive thrombus formation (10(-6) to 10(-5) M) bacterial killing increases by about 50%. In contrast, at pharmacological concentrations (10(-3) to 10(-2) M) an adverse effect can be observed: the elimination of opsonized S. aureus is reduced by 30 to 90%. Exogenous serotonin is capable of modulating important biological functions of human PMN in vitro. At appropriate concentrations, the antibacterial defence improves significantly probably due to reduced autooxidation, whereas higher concentrations counteract an efficient bacterial killing.

[Maximal therapy of hypercholesterolemia in coronary heart disease]. / Maximaltherapie der Hypercholesterinämie bei koronarer Herzerkrankung.

Extracorporeal procedures to eliminate LDL from plasma now allow us to drastically lower the plasma LDL-concentrations to virtually every desired level. In a new therapeutic approach the combination of HMG-CoA reductase inhibitors with an LDL/fibrinogen apheresis procedure (the HELP-system) was evaluated in hypercholesterolemic CAD-patients. HELP treatment alone can lower the mean plasma LDL-cholesterol by about 50-60%, HMG-CoA reductase inhibitor therapy reduces the LDL-cholesterol by about 40%. The combination of both treatments resulted in a lowering of mean LDL-cholesterol to about 80% of baseline values. Improvement of blood rheology by lowering plasma viscosity and inhibiting erythrocyte aggregation became apparent. No relevant adverse effects were noted over a period of two years. This therapeutic strategy for maximal LDL-cholesterol lowering may be useful in secondary prevention of coronary heart disease in hypercholesterolemic patients if plasma LDL-C cannot be reduced by diet and drug treatment to desirable plasma levels (LDL-cholesterol less than 120 mg/dl). Preliminary data show an improvement in the symptoms of our CAD-patients treated with this combined therapy. Furthermore, within the near future a combined HELP and dialysis unit will be available for patients with terminal renal insufficiency and progressive atherosclerosis.

[Current therapy of hypercholesterolemia. How much statin does your patient need?]. / Aktuelle Therapie der Hypercholesterinämie. Wie viel Statin braucht Ihr Patient?

Today, statins play an important role in the treatment of hypercholesterolemia. They have two effects on the metabolism of cholesterol: firstly, they reduce the synthesis of cholesterol and secondly they stimulate the expression of LDL receptors. LDL is reduced via both of the mechanisms. Various studies (the 4S, LIPID and CARE studies) have demonstrated the efficacy of statins in secondary prevention, that is, in patients with hypercholesterolemia and CAD. In the CARE study, for example, the statins reduced the incidence of fatal and non-fatal myocardial infarctions by 24%. A number of studies show that although primary prevention is effective, long-term tolerability is still a matter of controversy. A relatively frequent, dose-dependent side effect is myopathy, which has a reported incidence of 0.1-0.5%. In combination with fibrates, the incidence increases, and cases of rhabdomyolysis, some fatal, have been described. To minimize the side effects of statin treatment, therefore, target levels--which must be derived on the basis of the results of large studies--must be established for the individual patient.

Calpain mediates caspase-dependent apoptosis initiated by hydrogen peroxide in pancreatic acinar AR42J cells.

Several studies have shown that oxidative stress induces apoptosis in many cellular systems including pancreatic acinar cells. However, the exact molecular mechanisms leading to apoptosis remain partially understood. This study aimed to investigate the role of the cytosolic cysteine protease calpain in H2O2-induced apoptosis in pancreatic AR42J cells. Apoptosis was evaluated using flow cytometric analysis of sub-G1 DNA populations, electron-microscopic analysis, caspase-3-specific αII-spectrin breakdown, and measuring the proteolytic activities of the initiator caspase-12 and caspase-8, and the executioner caspase-3. H2O2 induced an increase in the calpain proteolytic activity immediately after starting the experiments that tended to return to a nearly normal level after 8 h and could be attributed to m-calpain. Whereas no caspase-12, caspase-8 and caspase-3 activations could be detected within the first 0.5 h, significantly increased proteolytic activities were observed after 8 h compared with the control. At the same time, the cells showed first ultrastructural hallmarks of apoptosis and a decreased viability. In addition, αII-spectrin fragmentation was identified using immunoblotting that could be attributed to both calpain and caspase-3. Calpain inhibition reduced the activities of caspase-12, caspase-8, and caspase-3 leading to a decrease in the number of apoptotic cells. Immunoblotting analyses of caspase-12 and caspase-8 indicate that calpain may be involved in the activation process of both proteases. The results suggest that H2O2-induced apoptosis of AR42J cells requires activation of m-calpain initiating the endoplasmic reticulum stress-induced caspase-12 pathway and a caspase-8-dependent pathway. The findings also suggest that calpain may be involved in the execution phase of apoptosis.