Glutamate receptor antagonists inhibit calpain-mediated cytoskeletal proteolysis in focal cerebral ischemia

Excitatory amino acids may promote microtubular proteolysis observed in ischemic neuronal degeneration by calcium-mediated activation of calpain, a neutral protease. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. Spontaneously hypertensive rats were treated with 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)quinoxaline (NBQX), a competitive antagonist of the neuronal receptor for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or cis-4-[phosphono-methyl]-2-piperidine carboxylic acid (CGS 19755), a competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor. After treatment, all animals were subjected to permanent occlusion of the middle cerebral artery for 6 or 24 h. Infarct volumes measured in animals pretreated with CGS 19755 after 24 h of ischemia were significantly smaller than those quantified in ischemic controls. Rats pretreated with NBQX showed partial amelioration of cytoskeletal injury with preserved immunolabeling of microtubule-associated protein 2 (MAP 2) at 6 and 24 h and reduced accumulation of calpain-cleaved spectrin byproducts only at 6 h. Prevention of cytoskeletal damage was more effective after pretreatment with CGS 19755, as shown by retention of MAP 2 immunolabeling and significant restriction of calpain activity at both 6 and 24 h. Preserved immunolabeling of tau protein was observed at 6 and 24 h only in animals pretreated with CGS 19755. Western analysis performed on ischemic cortex taken from controls or rats pretreated with either NBQX or CGS 19755 suggested that loss of tau protein immunoreactivity was caused by dephosphorylation, rather than proteolysis. These results demonstrate a crucial link between excitotoxic neurotransmission, microtubular proteolysis, and neuronal degeneration in focal cerebral ischemia.     

Oxidative stress inhibits calpain activity in situ

In this study, the effects of oxidative stress on calpain-mediated proteolysis and calpain I autolysis in situ were examined. Calpain activity was stimulated in SH-SY5Y human neuroblastoma cells with the calcium ionophore, ionomycin. Calpain-mediated proteolysis of the membrane-permeable fluorescent substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcouma rin, as well as the endogenous protein substrates microtubule-associated protein 2, tau and spectrin, was measured. Oxidative stress, induced by addition of either doxorubicin or 2-mercaptopyridine N-oxide, resulted in a significant decrease in the extent of ionophore-stimulated calpain activity of both the fluorescent compound and the endogenous substrates compared with control, normoxic conditions. Addition of glutathione ethyl ester, as well as other antioxidants, resulted in the retention/recovery of calpain activity, indicating that oxidation-induced calpain inactivation was preventable/reversible. The rate of autolytic conversion of the large subunit of calpain I from 80 to 78 to 76 kDa was decreased during oxidative stress; however, the extent of calpain autolysis was not altered. These data indicate that oxidative stress may reversibly inactivate calpain I in vivo.     

Sandor Ferenczi, the grandfather of family therapy

Excessive mu-calpain activation has been linked to several cellular pathologies including excitotoxicity and ischemia. In erythrocytes and other non-central nervous system (CNS) cells, calpain activation is thought to occur following a Ca2+-induced translocation of inactive cytosolic enzyme to membranes and subsequent autolysis. In the present report, we show that transiently exposing primary rat cortical neurons to lethal (50 microM) N-methyl-D-aspartic acid (NMDA) caused protracted calpain activation, measured as increased spectrin hydrolysis, but this was independent of translocation or autolysis of the protease. An anti-mu-calpain antibody showed that calpain was largely membrane associated in cortical neurons, and, consequently, neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. By contrast, in rat erythrocytes, calpain was largely cytosolic and underwent rapid translocation and autolysis in response to ionomycin. Calpain-mediated spectrin hydrolysis was specifically coupled to Ca2+ entry through the NMDA receptor because nonspecific Ca2+ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. Thus, calpain appears selectively linked to glutamate receptors in cortical neurons and regulated by mechanisms distinct from that occurring in many non-CNS cells. The data suggest that intracellular signals coupled to the NMDA receptor are responsible for activating calpain already associated with cellular membranes in cortical cells.     

Role of calpain- and interleukin-1 beta converting enzyme-like proteases in the beta-amyloid-induced death of rat hippocampal neurons in culture

We investigated the potential role of different proteases in the death of cultured rat hippocampal pyramidal neurons induced by beta-amyloid (A beta) (25-35). Both A beta(25-35)- and staurosporine-induced death of these neurons appeared to involve apoptosis, as indicated using Hoechst 33342 and terminal dUDP nick end labeling staining, whereas NMDA-induced death appeared more complex. Two irreversible inhibitors of the interleukin-1 beta converting enzyme (ICE) and related proteases, Z-Val-Ala-Asp-CH2F and acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone, blocked neuronal death produced by A beta(25-35), staurosporine, and NMDA to differing extents. Furthermore, MDL 28,170, a selective inhibitor of the calcium-regulated protease calpain, also inhibited death induced by all agents. A beta(25-35) and staurosporine stimulated the breakdown of the protein spectrin, a calpain substrate. Spectrin breakdown was inhibited by MDL 28,170 but not by ICE inhibitors. Leupeptin was only effective in preventing NMDA-induced death. These results support the role of apoptosis in neuronal death due to A beta(25-35) treatment and also suggest a role for calcium-regulated proteases in this process.     

BTA stat and BTA TRAK: A comparative evaluation of urine testing for the diagnosis of transitional cell carcinoma of the bladder

The calcium-activated neutral protease calpain is activated in several pathological conditions. Calpain usually hydrolyses one or only a few peptide bonds in its substrate. One prominent substrate for calpain is spectrin and it has been shown that alpha-spectrin is the preferred substrate. We now show that the beta-chain of spectrin is also a substrate for calpain proteolysis, and that the cleavage site in each beta-subunit is located at the very C-terminal part of the molecule. Surprisingly, beta1sigma-spectrin is cleaved at a different site than betaIsigma2- and betaIIsigma1-spectrins despite their high degree of sequence identity.     

Calcium-activated neutral protease activity is decreased in PC12 cells after ethanol exposure

Calcium-activated neutral protease activity was determined in PC12 cells exposed to ethanol for 96 h using a fluorescence-based assay with N-succinyl-Leu-Tyr 7-amido-4-methylcoumarin as the substrate. Stimulated activity was measured at high (1,400 microM) or low (140 microM) Ca2+ concentrations in the presence of 20 microM ionomycin. Kinetic parameters were derived by fitting a model relating fluorescence intensity to time: F(t) = F(final)*(1 - e(-k(obs)t). Cell extracts were subjected to nondenaturing gel electrophoresis and casein zymography with quantification of the activity of the two calpain isoforms. Exposure to ethanol significantly decreased whole cell calpain activity measured by k(obs) beginning at 20 mM, to 27.8% of control at 1,400 microM Ca2+ and 29.2% of control at 140 microM Ca2+ in the presence of 20 microM ionomycin. No changes in mu-calpain or m-calpain activities were found in cell extracts from cells exposed to 20 mM ethanol, whereas at 40 and 80 mM ethanol, significant decreases in both mu-calpain and m-calpain activities were discovered.     

Alteration of lipid composition modulates Fc epsilon RI signaling in RBL-2H3 cells

We have used sonicated liposomes of phosphatidylcholine (PC), sphingomyelin (SM), or a mixture of cholesterol (chol) and PC to investigate the role of cellular lipid composition in Fc epsilon RI-mediated stimulation of RBL-2H3 cells. Overnight treatment with either PC or SM liposomes causes a substantial enhancement of antigen-stimulated degranulation and phospholipase A2 activity, whereas treatment with a PC/chol mixture results in partial inhibition of the antigen-stimulated response. The most consistent change in the cellular lipid composition that results from the PC and SM liposome treatments is an approximate 40% decrease in the chol/phospholipid (PL) ratio. The lipid treatments do not alter degranulation stimulated by AlF4- or by Ca2+ ionophore in the presence or absence of PMA, suggesting that lipid alteration affects a receptor-specific signaling process. The lipid treatments do not appear to alter antigen-stimulated tyrosine phosphorylation or Ca2+ mobilization. Possible involvement of protein kinase C (PKC) activation in the signal-enhancing effect of the PL treatments was investigated by using calphostin C and phorbol-12-myristol-13-acetate (PMA) to inhibit PKC activity and degranulation in RBL-2H3 cells. Both SM and PC treatment restore the antigen-mediated degranulation response that is inhibited by long-term treatment (> or = 16 h) with 100 nM PMA or short-term treatment (10 min) with 5 microM calphostin C. The results indicate that a decreased chol/PL ratio facilitates or enhances the receptor-mediated activation of a PKC-like pathway that plays an important role in Fc epsilon RI-stimulated degranulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

p75 and TrkA receptor signaling independently regulate amyloid precursor protein mRNA expression, isoform composition, and protein secretion in PC12 cells

The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75NTR-activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross-linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors--in contrast to p75NTR-associated signaling--stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.     

Activation of NMDA and non-NMDA receptors in the caudal ventrolateral medulla dilates the airways

Calpain is a ubiquitous calcium-dependent cysteine protease, whose cytoskeletal protein substrates suggest that it may be important in neuronal differentiation. Lead (Pb2+) is known to substitute for Ca2+ in a variety of intracellular processes, and interferes with the development of hippocampal neurons in vitro. We found that free Pb2+ at 1 nM does not activate calpain in the absence of Ca2+. Pb2+ inhibited the activity of calpain; the degree of calpain inhibition was dependent on an interaction between concentrations of both Ca2+ and Pb2+. In the presence of 1 microM free Ca2+, 10 pM free Pb2+ reduced calpain activity, but in the presence of 100 microM free Ca2+, 1 nM free Pb2+ failed to inhibit calpain. This provides evidence that Pb2+ competes for the Ca2+ binding sites on calpain. In the presence of 40 microM free Ca2+, 1 nM free Pb2+ significantly reduces Vmax without altering Km, suggesting that Pb2+ acts as a noncompetitive inhibitor of calpain. Inhibition of calpain is one mechanism by which Pb2+ may interfere with neuronal development.     

Disrupted [Ca2+]i homeostasis contributes to the toxicity of nitric oxide in cultured hippocampal neurons

Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca2+ concentration ([Ca2+]i) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca2+]i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S-nitrosocysteine (SNOC; 300 microM) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca2+]i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca2+]i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca2+ during the 20-min SNOC treatment failed to prevent the late [Ca2+]i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca2+ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca2+-activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca2+]i homeostatic mechanisms, and that the resulting persistent [Ca2+]i elevations contribute to the delayed neurotoxicity of NO.     

Involvement of sphingosine 1-phosphate in nerve growth factor-mediated neuronal survival and differentiation

Sphingolipid metabolites, such as ceramide and sphingosine-1-phosphate (SPP), are emerging as a new class of second messengers involved in cellular proliferation, differentiation, and apoptosis. Nerve growth factor (NGF), a neurotrophic factor for pheochromocytoma PC12 cells, induced a biphasic increase in the activity of sphingosine kinase, the enzyme that catalyzes the formation of SPP. This activation was blocked by K252a, an inhibitor of tyrosine kinase A (trkA). A rapid 1.7-fold increase was followed by a marked prolonged increase reaching a maximum of fourfold to fivefold stimulation with a concomitant increase in SPP levels and a corresponding decrease in endogenous sphingosine levels. Levels of ceramide, the precursor of sphingosine, were only slightly decreased by NGF in serum-containing medium. However, NGF decreased the elevation of ceramide induced by serum withdrawal. Treatment of PC12 cells with SPP did not induce neurite outgrowth or neurofilament expression, yet it enhanced neurofilament expression elicited by suboptimal doses of NGF. Moreover, SPP also protected PC12 cells from apoptosis induced by serum withdrawal. To further substantiate a role for SPP in the cytoprotective actions of NGF, we found that N, N-dimethylsphingosine, a competitive inhibitor of sphingosine kinase, also induced apoptosis and interfered with the survival effect of NGF. These effects were counteracted by exogenous SPP. Moreover, other structurally related compounds, such as dihydrosphingosine 1-phosphate and lysophosphatidic acid, had no significant protective effects. Our results suggest that activation of sphingosine kinase and subsequent formation of SPP may play an important role in the differentiation and survival effects induced by NGF.     

Bax cleavage is mediated by calpain during drug-induced apoptosis

The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.     

Reduced induction of HSP70 in PC12 cells during neuronal differentiation

Rat pheochromocytoma PC12 cells differentiate into nonreplicating neuronal cells with neurite extensions in response to nerve growth factor (NGF). To gain better understanding of the regulation of stress responses in neuronal cells, we examined the induction of HSP70, HSP70 mRNA, and heat shock factor 1 (HSF1) DNA-binding activity following treatment by heat shock or with sodium arsenite or amino acid analogue in PC12 cells treated with or without NGF. The induction of HSP70 and HSP70 mRNA following these stresses was diminished in the differentiated PC12 cells compared to the undifferentiated cells, whereas the HSF1 DNA-binding activity was enhanced in the differentiated PC12 cells. This phenomenon was characteristic of the differentiated neuronal cells rather than growth-arrested cells. Thus, neuronal cells appear to show an altered stress response depending on their differentiation state, and the diminished HSP70 expression in the differentiated neuronal cells may explain the sensitivity of neuronal cells to pathophysiological stressors.     

A comparison of the W-stapled ileal reservoir with hand-sewn reservoirs for orthotopic bladder replacement

Activated calpain I immunoreactivity (76 kDa band) was detected in membranes prepared from rat brain hippocampal slices using a polyclonal antiserum raised against an N-terminus peptide of the cleaved subunit of calpain I. While basal levels of activated calpain I were stable over incubation times, 1 nM dopamine (DA) produced an initial 32% increase (5 min) in the 76 kDa protein followed by a 53% decrease in this band at 20 min of incubation. The DA-induced changes in activated calpain I immunoreactivity were blocked by the calpain inhibitor peptide, N-acetyl-Leu-Leu-norleucinal(100 microM) or by EGTA. Basal levels of the 76 kDa band were not affected by the calpain inhibitor. These changes in activated calpain I, elicited by DA, are in accord with the DA-induced decreases in the levels of the calpain substrate, gamma PKC (Yurko-Mauro and Friedman; J Cell Biochem [Abstr] 180:80, 1994; J Neurochem 65: 1622-1630, 1995) and suggest that DA activates this Ca(++)-dependent protease in its regulation of neuronal signal transduction.     

Calmodulin modulates mitogen-activated protein kinase activation in response to membrane depolarization in PC12 cells

In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca2+ influx through voltage-gated Ca2+ channels, which is able to stimulate extracellular-regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca2+ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K+ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca2+ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K+ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca2+ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.     

Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis

Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early factors in the failure of the microtubule network that occurs during neuronal apoptosis. Furthermore, this study introduces new insights into the mechanism(s) that generate the truncated forms of tau present in Alzheimer's disease.