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Blockade of maitotoxin-induced oncotic cell death reveals zeiosis.

Estacion M, Schilling WP - BMC Physiol. (2002)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rammelkamp Center for Education and Research, MetroHealth Medical Center, Cleveland, OH, USA. mestacion@metrohealth.org

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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BAECs rescued from MTX-induced lytic cell death have elevated caspase-3 activity. BAECs were treated with 0.3 nM MTX for 2.5 min at which time the action of MTX was stopped by addition of 5 μM U73343. Cells were harvested after 48 hr and specific caspase-3 activity was determined using the fluorescence substrate assay (M/U). For control, caspase-3 activity was determined in cells treated with U73343 alone (IU) or vehicle (DMSO). Values represent the mean ± se (n = 3) relative to no-treatment controls.
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Figure 7: BAECs rescued from MTX-induced lytic cell death have elevated caspase-3 activity. BAECs were treated with 0.3 nM MTX for 2.5 min at which time the action of MTX was stopped by addition of 5 μM U73343. Cells were harvested after 48 hr and specific caspase-3 activity was determined using the fluorescence substrate assay (M/U). For control, caspase-3 activity was determined in cells treated with U73343 alone (IU) or vehicle (DMSO). Values represent the mean ± se (n = 3) relative to no-treatment controls.

Mentions: Zeiosis is a type of membrane blebbing associated with apoptosis [22-24]. Since U73343 protects cells from MTX-induced oncosis and causes zeiosis, we considered the possibility that the cells surviving the initial insult may ultimately die by apoptosis. To test this hypothesis, BAECs were treated with vehicle alone (DMSO), U73343 alone, or MTX followed by U73343, and specific caspase-3 activity was determined after 48 hrs by the fluorescence substrate assay (Fig 7). A significant increase in caspase-3 activity (p < 0.035; n = 3) was observed in cells challenged with MTX and subsequently rescued by U73343, consistent with increased apoptotic cell death.


Blockade of maitotoxin-induced oncotic cell death reveals zeiosis.

Estacion M, Schilling WP - BMC Physiol. (2002)

BAECs rescued from MTX-induced lytic cell death have elevated caspase-3 activity. BAECs were treated with 0.3 nM MTX for 2.5 min at which time the action of MTX was stopped by addition of 5 μM U73343. Cells were harvested after 48 hr and specific caspase-3 activity was determined using the fluorescence substrate assay (M/U). For control, caspase-3 activity was determined in cells treated with U73343 alone (IU) or vehicle (DMSO). Values represent the mean ± se (n = 3) relative to no-treatment controls.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC65053&req=5

Figure 7: BAECs rescued from MTX-induced lytic cell death have elevated caspase-3 activity. BAECs were treated with 0.3 nM MTX for 2.5 min at which time the action of MTX was stopped by addition of 5 μM U73343. Cells were harvested after 48 hr and specific caspase-3 activity was determined using the fluorescence substrate assay (M/U). For control, caspase-3 activity was determined in cells treated with U73343 alone (IU) or vehicle (DMSO). Values represent the mean ± se (n = 3) relative to no-treatment controls.
Mentions: Zeiosis is a type of membrane blebbing associated with apoptosis [22-24]. Since U73343 protects cells from MTX-induced oncosis and causes zeiosis, we considered the possibility that the cells surviving the initial insult may ultimately die by apoptosis. To test this hypothesis, BAECs were treated with vehicle alone (DMSO), U73343 alone, or MTX followed by U73343, and specific caspase-3 activity was determined after 48 hrs by the fluorescence substrate assay (Fig 7). A significant increase in caspase-3 activity (p < 0.035; n = 3) was observed in cells challenged with MTX and subsequently rescued by U73343, consistent with increased apoptotic cell death.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rammelkamp Center for Education and Research, MetroHealth Medical Center, Cleveland, OH, USA. mestacion@metrohealth.org

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus