maitotoxin and Necrosis

MS-based proteomics has been the method of choice for biomarker discovery in the field of traumatic brain injury (TBI). Due to its high sensitivity and specificity, MS is now being explored for biomarker quantitative validation in tissue and biofluids. In this study, we demonstrate the use of MS in both qualitative protein identification and targeted detection of acute TBI biomarkers released from degenerating cultured rat cortical mixed neuronal cells, mimicking intracellular fluid in the central nervous system after TBI. Calpain activation was induced by cell treatment with maitotoxin (MTX), a known calcium channel opener. Separate plates of mixed neuronal-glial culture were subjected to excitotoxin N-methyl-D-aspartate (NMDA) and apoptotic inducer staurosporine. Acute TBI biomarkers, GFAP and UCH-L1, were first detected and assessed in the culture media by Western blot. The cell-conditioned media were then trypsinized and subjected to bottom up proteomic analysis. GFAP was readily detected by data-dependent scanning but not UCH-L1. As a proof-of-principle study, rat glia-enriched cell cultures treated with MTX were used to investigate the time-dependent release of GFAP breakdown product by Western blot and for isotope dilution MS absolute quantitation method development. Absolute quantitation of the GFAP release was conducted using the three cortical mixed neuronal cell cultures treated with different agents. Other differentially expressed proteins identified in the glial-enriched and cortical mixed neuronal cell culture models were further analyzed by bioinformatic tools. In summary, this study demonstrates the use of MS in both protein identification and targeted quantitation of acute TBI biomarkers and is the preliminary step toward development of TBI biomarker validation by targeted MS.

Topics: Animals; Apoptosis; Biomarkers; Brain Injuries; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Glial Fibrillary Acidic Protein; Marine Toxins; Mass Spectrometry; N-Methylaspartate; Necrosis; Neuroglia; Neurons; Oxocins; Protein Interaction Maps; Proteomics; Rats; Rats, Sprague-Dawley; Signal Transduction; Staurosporine; Ubiquitin Thiolesterase

Maitotoxin (MTX) is one of the most potent toxins known to date. It causes massive calcium (Ca(2+)) influx and necrotic cell death in various tissues. However, the exact mechanism(s) underlying its cellular toxicity is not fully understood. In the present study, the role of the sodium hydrogen exchanger (NHE) in MTX-induced increases in intracellular Ca(2+) and subsequent cell death were investigated in cultured rat cortical neurons. Intracellular Ca(2+) concentrations ([Ca(2+)](i)) were measured fluorimetrically using FURA-2 as the fluorescence indicator. Cell death was measured with the alamarBlue cell viability assay and the vital dye ethidium bromide (EB) uptake assay. Results showed that MTX increased, in a concentration dependent manner, both [Ca(2+)](i) and cell death in cortical neurons. Decreasing the pH of the treatment medium from 7.5 to 6.0 diminished MTX-induced cell death. The protection offered by lowering extracellular pH was not due to MTX degradation, because it was still effective even if the cells were treated with MTX in normal pH and then switched to a lower pH. Pretreatment of cells with the specific NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), prevented MTX-induced increases in [Ca(2+)](i), as well as cell death in a concentration dependent manner. Furthermore, knockdown of NHE1 by SiRNA transfection suppressed MTX-induced cell death in human embryonic kidney (HEK) cells. Together, these results suggest that NHE1 plays a major role in MTX-induced neurotoxicity.

Topics: Acidosis; Amiloride; Animals; Antimetabolites, Antineoplastic; Calcium; Cell Death; Cell Survival; Cells, Cultured; Cerebral Cortex; Female; Fluorometry; Marine Toxins; Methotrexate; Necrosis; Neurons; Neuroprotective Agents; Oxocins; Pregnancy; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Sodium-Hydrogen Exchangers

Apoptosis and oncotic necrosis in neuronal and glial cells have been documented in many neurological diseases. Distinguishing between these two major types of cell death in different neurological diseases is needed in order to better reveal the injury mechanisms so as to open up opportunities for therapy development. Accumulating evidence suggests apoptosis and oncosis epitomize the extreme ends of a broad spectrum of morphological and biochemical events. Biochemical markers that can distinguish between the calpain and caspase dominated types of cell death would help in this process. In this study, three chemical agents, maitotoxin (MTX), staurosporine (STS) and thylenediaminetetraacetic acid (EDTA), were used to induce different types of cell death in PC12 neuronal-like cells. MTX-induced necrosis, as determined by the increased levels of calpain-specific cleaved fragments of spectrin by antibodies specific to the calpain-cleaved 150 kDa alphaII-spectrin breakdown product (SBDP150) and 145 kDa alphaII-spectrin breakdown product (SBDP145). In this paradigm, there were no detectable SBDP150i and SBDP120 fragments as determined by antibodies specific to the caspase-cleaved specific fragments similar to those seen in the EDTA-mediated apoptotic PC-12 cells. In contrast to the calpain specific MTX necrosis treatment and the caspase EDTA apoptotic treatment is the STS treatment which induced both proteases as shown by the increase in all the SBDP fragments. Furthermore, compared to SBDP150, SBDP145 appears to be a more specific and sensitive biomarker for calpain activation. Taken together, our results suggested calpains and caspases which dominate the two major types of cell death could be independently discriminated by specifically examining the multiple alphaII-spectrin cleavage breakdown products.

Topics: Amino Acid Sequence; Animals; Apoptosis; Calpain; Caspases; Cell Death; Edetic Acid; Marine Toxins; Necrosis; Oxocins; PC12 Cells; Rats; Spectrin; Staurosporine

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca(2+) overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca(2+)-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules <800 Da to enter the cell. The cells then lyse not by rupture of the plasmalemma but through the activation of a "death" channel that lets large proteins (e.g., 140-160 kDa) leave the cell. These changes in permeability are accompanied by the formation of membrane blebs. In this study, we took advantage of the well-known differences in affinity of various Ca(2+)-binding proteins for Ca(2+) and Sr(2+) vs. Ba(2+) to probe their involvement in each phase of the cell death cascade. Using fluorescence techniques at the cell population level (cuvette-based) and at the single-cell level (time-lapse videomicroscopy), we found that the replacement of Ca(2+) with either Sr(2+) or Ba(2+) delayed both MTX-induced activation of COP, as indicated by the uptake of ethidium bromide, and subsequent cell lysis, as indicated by the uptake of propidium iodide or the release of cell-associated green fluorescent protein. MTX-induced responses were mimicked by ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba(2+) not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca(2+)-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba(2+). Taken together, these results suggest that intracellular high-affinity Ca(2+)-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise.

Topics: Animals; Aorta; Barium; Biological Transport; Calcium; Cations, Divalent; Cattle; Cell Death; Cell Membrane; Cells, Cultured; Endothelium, Vascular; Marine Toxins; Microscopy, Video; Necrosis; Oxocins; Strontium

Maitotoxin (MTX) initiates cell death by sequentially activating 1) Ca2+ influx via non-selective cation channels, 2) uptake of vital dyes via formation of large pores, and 3) release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC) in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells.. Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC50 asymptotically equal to 1.9 and 0.66 microM, respectively) suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program.. Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms.

Topics: Animals; Apoptosis; Calcium; Cattle; Cell Membrane; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Estrenes; Ethidium; Fluorescent Dyes; Marine Toxins; Necrosis; Oxocins; Pyrrolidinones; Type C Phospholipases