Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells

BACKGROUND: Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) via activation of Ca(2+)-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca(2+)](i), vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells. RESULTS: Maitotoxin produced a concentration-dependent increase in [Ca(2+)](i) followed by a biphasic uptake of ethidium. Comparison of ethidium (M(w) 314 Da), YO-PRO-1 (M(w) 375 Da), and POPO-3 (M(w) 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation. CONCLUSIONS: These results suggest that maitotoxin-induced Ca(2+) influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death

Blockade of maitotoxin-induced oncotic cell death reveals zeiosis

BACKGROUND: Maitotoxin (MTX) initiates cell death by sequentially activating 1) Ca(2+) 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 (IC(50) ≈ 1.9 and 0.66 μM, 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

Paroxysmal extreme pain disorder M1627K mutation in human Nav1.7 renders DRG neurons hyperexcitable

Background: Paroxysmal extreme pain disorder (PEPD) is an autosomal dominant painful neuropathy with many, but not all, cases linked to gain-of-function mutations in SCN9A which encodes voltage-gated sodium channel Nav1.7. Severe pain episodes and skin flushing start in infancy and are induced by perianal probing or bowl movement, and pain progresses to ocular and mandibular areas with age. Carbamazepine has been effective in relieving symptoms, while other drugs including other anti-epileptics are less effective. Results: Sequencing of SCN9A coding exons from an English patient, diagnosed with PEPD, has identified a methionine 1627 to lysine (M1627K) substitution in the linker joining segments S4 and S5 in domain IV. We confirm that M1627K depolarizes the voltage-dependence of fast-inactivation without substantially altering activation or slow-inactivation, and inactivates from the open state with slower kinetics. We show here that M1627K does not alter development of closed-state inactivation, and that M1627K channels recover from fast-inactivation faster than wild type channels, and produce larger currents in response to a slow ramp stimulus. Using current-clamp recordings, we also show that the M1627K mutant channel reduces the threshold for single action potentials in DRG neurons and increases the number of action potentials in response to graded stimuli. Conclusion: M1627K mutation was previously identified in a sporadic case of PEPD from France, and we now report it in an English family. We confirm the initial characterization of mutant M1627K effect on fast-inactivation of Nav1.7 and extend the analysis to other gating properties of the channel. We also show that M1627K mutant channels render DRG neurons hyperexcitable. Our new data provide a link between altered channel biophysics and pain in PEPD patients