calcimycin and Nerve-Degeneration

Agmatine is an endogenous guanido amine and has been shown to be neuroprotective in vitro and in vivo. The aims of this study are to investigate whether agmatine is protective against cell death induced by different agents in cultured neurons and PC12 cells.. Cell death in neurons, cultured from neonatal rat cortex, was induced by incubating with (a) NMDA (100 microM) for 10 min, (b) staurosporine (protein kinase inhibitor, 100 nM) for 24 h, and (c) calcimycin (calcium ionophore, 100 nM) for 24 h in the presence and absence of agmatine (1 micro M to 1 mM). Cell death in PC12 cells was induced by exposure to glutamate (10 mM), staurosporine (100 nM), and calcimycin (100 nM). The activity of lactate dehydrogenase (LDH) in the medium was measured as the marker of cell death and normalized to cellular LDH activity.. Agmatine significantly reduced the medium LDH in NMDA-treated neurons but failed to reduce the release of LDH induced by staurosporin or calcimycin. In PC12 cells, agmatine significantly reduced LDH release induced by glutamate exposure, but not by staurosporine or calcimycin. Agmatine itself neither increased LDH release nor directly inhibited the enzyme activity.. We conclude that agmatine protects against NMDA excitotoxicity in neurons and PC12 cells but not the cell death induced by protein kinase blockade or increase in cellular calcium.

Topics: Agmatine; Animals; Calcimycin; Cell Death; Cerebral Cortex; Enzyme Inhibitors; Fetus; Glutamic Acid; Ionophores; L-Lactate Dehydrogenase; N-Methylaspartate; Nerve Degeneration; Neurons; Neuroprotective Agents; Neurotoxins; PC12 Cells; Protein Kinase Inhibitors; Protein Kinases; Rats; Staurosporine

P35 or its truncated fragment p25 is required for cyclin dependent kinase (Cdk)5 activation. It has been reported that p25 is accumulated in the brain of Alzheimer's disease (AD) patients and that p25/Cdk5 induces high phosphorylation of tau and apoptosis in cultured neurons (Nature 402 (1999) 615). Our investigation of AD brain did not show specific accumulation of p25. Exposure to Ca ionophore (A23187) at 10(-6) M induced p25 accumulation in rat primary hippocampal neurons, causing neuronal death without showing hyperphosphorylation of tau. Transgenic mice expressing p25 showed the accumulation of p25 but neither hyperphosphorylation of tau nor neuronal death was shown in these mice. The feature of these mice was the progression of cell growth in pituitary gland. These results suggest that overexpression of p25 lead to the activation of cell cycle but not to the direct phosphorylation of tau.

Topics: Aged; Alzheimer Disease; Animals; Brain; Calcimycin; Cells, Cultured; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Gene Expression Regulation; Hippocampus; Humans; Ionophores; Mice; Mice, Transgenic; Nerve Degeneration; Nerve Tissue Proteins; Neurofibrillary Tangles; Neurons; Phosphorylation; Pituitary Diseases; tau Proteins

Ca2+ influx is one of the main causative events in hypoxic PC12 cell death, because an extracellular Ca2+ chelator, ethylene glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited and Ca2+ ionophore A23187 mimicked the hypoxic cell death. The hypoxic cell death was markedly prevented by a broad spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) as well as a calpain inhibitor, calpeptin, as assessed by nuclear staining with Hoechst 33258 and lactate dehydrogenase release. The processing of procaspase-3 was inhibited by z-VAD-FMK, but not by calpeptin. In contrast, z-VAD-FMK failed to block the proteolytic cleavage of fodrin-alpha, a preferential substrate for calpain. On the other hand, degradation of actin and fodrin-alpha was prevented by calpeptin but not by z-VAD-FMK. In addition, not only protein kinase C (PKC)-alpha but also PKC-delta were cleaved to generate approximately 46 kDa fragments. The PKC fragmentation was inhibited by calpeptin but not by z-VAD-FMK. These findings suggest that the extracellular Ca2+ influx induced by hypoxic stress activates calpain, resulting in the degradation of cytoskeletal proteins and generation of PKC fragments almost independently of caspase activation. Therefore, calpain may play an important role in hypoxic PC12 cell death.

Topics: Animals; Calcimycin; Calcium; Calpain; Caspases; Cell Death; Chelating Agents; Cysteine Proteinase Inhibitors; Cytoskeleton; Dipeptides; Egtazic Acid; Hypoxia-Ischemia, Brain; Ionophores; Isoenzymes; Nerve Degeneration; PC12 Cells; Peptide Hydrolases; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Rats

The calcium-binding protein calbindin-D28k (CB) has been hypothesized to function, in part, as a neuroprotective protein. CB is localized within nerve cells that are often less vulnerable to degeneration in patients with Alzheimer's disease and Parkinson's disease, and cells containing CB can buffer intracellular calcium concentrations ([Ca2+]i). The present study was designed to directly test the hypothesis that CB can protect cells from degeneration by reducing [Ca2+]i. PC12 cells, transfected to express different levels of CB, were found to be significantly less vulnerable to degeneration caused by serum withdrawal, glutamate, and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). However, CB did not protect cells from degeneration caused by the calcium ionophore A23187. CB-transfected cells exhibited reduced elevations in [Ca2+]i following treatment with bradykinin, or ATP compared to non-CB-containing cells. These data indicate that CB can protect cells from degeneration caused by certain conditions, and it reduces elevations in [Ca2+]i caused by influx from extracellular sources.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Apoptosis; Calbindin 1; Calbindins; Calcimycin; Calcium; Culture Media, Serum-Free; Drug Resistance, Neoplasm; Glutamic Acid; Intracellular Fluid; Nerve Degeneration; Nerve Tissue Proteins; PC12 Cells; Rats; S100 Calcium Binding Protein G; Time Factors

We have studied in living chick embryos the effects of an extracellular calcium load on the induction of apoptosis in spinal cord motoneurons. The action of a calcium ionophore, A23187, that does not raise extracellular calcium was also evaluated in order to explore the role of endogenous calcium in determining developmentally-regulated cell death of motoneurons. The application of a single dose of 50 microliters of 1.8 M CaCl2 onto the chorioallantoic membrane of E7 chick embryos produce a transient elevation of intraembryonic calcium concentration that was followed by a transitory rise in the number of apoptotic cells in the lateral motor column. Administration of 250 microM of the ionophore A23187 (100 microliters), also results in an increase in apoptosis of motoneurons in the lateral motor column on E6 and E7 but this effect is progressively lost following treatment at more advanced stages of development. Neither of these effects can be explained by unspecific calcium cytotoxicity since they can be inhibited by prior administration of the protein synthesis inhibitor cycloheximide or the neuromuscular blocking agent (+)-tubocurarine. After calcium loading, degenerating cells display similar ultrastructural characteristics as during physiologically occurring motoneuron death and exhibit histochemically detectable DNA fragmentation. Chronic administration of CaCl2 or A23187 does not reduce the total number of surviving motoneurons at the end of the normal period of naturally occurring motoneuron death (E10). It is suggested that calcium loading stimulates and accelerates the physiological degeneration of a restricted subpopulation of motoneurons which will undergo the process of natural cell death.

Topics: Animals; Apoptosis; Calcimycin; Calcium; Calcium Chloride; Chick Embryo; Motor Neurons; Nerve Degeneration; Spinal Cord

Both calcium and aluminum have been implicated in the cell damage and death that occurs in several neurodegenerative disorders including Alzheimer's disease (AD). We examined the effects of experimentally elevated intraneuronal levels of aluminum ([Al]i) and/or calcium ([Ca2+]i) on neuronal degeneration and antigenic alterations in the microtubule-associated protein tau in cell cultures of rat hippocampus and human cerebral cortex. Exposure of cultures to Al3+ alone (200 microM) for up to 6 d did not result in neuronal degeneration. Neurons exposed to the divalent cation ionophore A23187 degenerated within 4 h when Ca2+ was present in the culture medium whether or not Al3+ was present. Measurements of [Ca2+]i using the calcium indicator dye fura-2 demonstrated a direct relationship between increased [Ca2+]i and neuronal degeneration. In contrast, neurons did not degenerate when exposed to A23187 in the presence of Al3+ and the absence of Ca2+, despite a 10-fold elevation in [Al]i as measured by laser microprobe mass spectrometry. Calcium influx, but not aluminum influx, elicited antigenic changes in tau similar to those seen in AD neurofibrillary tangles. Neurons exposed to glutamate in the presence of Al3+ but in the absence of Ca2+ were not vulnerable to injury. Finally, increased [Al]i occurred in neurons that degenerated as the result of exposure to glutamate indicating that aluminum associates with degenerating neurons. Taken together these data indicate that, in contrast to increased [Ca2+]i, elevated [Al]i may not induce degeneration or antigenic changes in tau.

Topics: Aluminum; Animals; Calcimycin; Calcium; Cell Survival; Cells, Cultured; Fura-2; Hippocampus; Humans; Immunohistochemistry; Lasers; Mass Spectrometry; Nerve Degeneration; Neurofibrillary Tangles; Neurons; Rats; tau Proteins

In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta-amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1-38 or 25-35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Calcimycin; Calcium; Cell Survival; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Fetus; Glutamates; Glutamic Acid; Homeostasis; Humans; Indicators and Reagents; Kinetics; Molecular Sequence Data; Nerve Degeneration; Neurons; Peptide Fragments; Structure-Activity Relationship

The causal role of Ca2+ in neuronal necrosis is controversial and it has been suggested that neuronal Ca2+ uptake is only a secondary effect to cell death. Here, I address this question directly by studying the morphological effects of calcium ionophore A23187 on immature cerebellar slices. Parasagittal slices were prepared and incubated for 30, 90 or 120 min in physiological saline with or without A23187. In some cases Ca2+ was omitted from the incubation medium. Slices were processed for light microscopy. A23187 produced nuclear changes indicative of cell death that encompassed cells of the external granule cell layer at short incubation times (30 min) and more deeply situated cells at longer times (120 min). This indicates that A23187 diffusion is limited in the slice. The histological changes produced by 30 min exposure to the ionophore could not be reversed by incubation for 90 min in normal medium. Necrosis was never observed when slices were exposed to A23187 in Ca2+-free medium. The results demonstrate that influx of excessive amounts of Ca2+ kills cells of the central nervous system.

Topics: Animals; Calcimycin; Calcium; Cerebellum; Female; In Vitro Techniques; Male; Nerve Degeneration; Rats; Rats, Inbred Strains