Abstract:We studied the novel hypothesis that an upmodulation of channels for outward delayed rectifier K ϩ current (I K ) plays a key role in ceramide-induced neuronal apoptosis. Exposure for 6 -10 h to the membrane-permeable C 2 -ceramide (25 M) or to sphingomyelinase (0.2 unit/ml), but not to the inactive ceramide analogue C 2 -dihydroceramide (25 M), enhanced the whole-cell I K current without affecting the transient A-type K ϩ current and increased caspase activity, followed by neuronal apoptosis 24 h after exposure onset. Tetraethylammonium (TEA) or 4-chloro-N,N-diethyl-N-heptylbenzenebutanaminium tosylate (clofilium), at concentrations inhibiting I K , attenuated the C 2 -ceramide-induced caspase-3-like activation as well as neuronal apoptosis. Raising extracellular K ϩ to 25 mM similarly blocked the C 2 -ceramideinduced cell death; the neuroprotection by 25 mM K ϩ or TEA was not eliminated by blocking voltage-gated Ca 2ϩ channels. An inhibitor of tyrosine kinases, herbimycin A (10 nM) or lavendustin A (0.1-1 M), suppressed I K enhancement and/or apoptosis induced by C 2 -ceramide. It is suggested that ceramide-induced I K current enhancement is mediated by tyrosine phosphorylation and plays a critical role in neuronal apoptosis. Key Words: Outward delayed rectifier I K channel-Ceramide-Neuronal apoptosis-Tyrosine phosphorylation. J. Neurochem. 73, 933-941 (1999).Ceramide, a long-chain sphingolipid generated intracellularly on hydrolysis of sphingomyelin, has been implicated as a second messenger molecule involved in several forms of intracellular signaling, including those triggered by cytokines, growth factors, and stress (Obeid and

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Background and Purpose-In light of recent evidence suggesting that an upregulation of K ϩ efflux mediated by outward delayed rectifier (I K ) channels promotes central neuronal apoptosis, we sought to test the possibility that blockers of I K channels might be neuroprotective against hypoxia/ischemia-induced neuronal death. Methods-Membrane currents were recorded with the use of patch clamp recordings in cultured murine cortical neurons.Protective effects of K ϩ channel blockers were examined in rats subjected to transient middle cerebral artery occlusion followed by 14-day reperfusion. Results-The K ϩ channel blocker tetraethylammonium (TEA) (5 mmol/L) selectively blocked I K without affecting N-methyl-D-aspartate receptor-mediated current or voltage-gated Ca 2ϩ currents. Both TEA and a lipophilic K ϩ channel blocker, clofilium, attenuated neuronal apoptosis induced by hypoxia in vitro and infarct volume induced by ischemia in vivo. Conclusions-These data are consistent with the idea that K ϩ channel-mediated K ϩ efflux may contribute to ischemia-triggered apoptosis and suggest that preventing excessive K ϩ efflux through K ϩ channels may constitute a therapeutic approach for the treatment of stroke.

Neuronal death following ischemic insults has been thought to reflect necrosis. However, recent evidence from several labs suggests that programmed cell death, leading to apoptosis, might additionally contribute to this death. We have used both in vitro and in vivo models to study the role of apoptosis in ischemic cell death. Some features of apoptosis (TUNEL staining, internucleosomal DNA fragmentation, sensitivity to cycloheximide) were observed following transient focal ischemia in rats. Brief transient focal ischemia was followed by delayed infarction more than 3 days later; this delayed infarction was sensitive to cycloheximide. A cycloheximide-sensitive component of neuronal cell death was also observed in cultured murine neocortical neurons deprived of oxygen-glucose in the presence of glutamate receptor antagonists. This presumed ischemic apoptosis was attenuated by caspase inhibitors, or by homozygous deletion of the bax gene. Neurons may undergo both apoptosis and necrosis after ischemic insults, and thus it may be therapeutically desirable to block both processes.

Four commonly used methods for the assessment of neuronal (or glial) cell viability are described in this unit. The MTT assay is sensitive to the function of labile mitochondrial enzymes, which typically lose activity early in the progression towards death. The lactate dehydrogenase (LDH) assay measures the appearance of this cytosolic enzyme in the bathing medium, providing a measure of plasma membrane integrity. Loss of plasma membrane integrity is also the basis of the trypan blue dye assay and the propidium iodide assay. Trypan blue staining is assessed by cell counts; propidium iodide labeling can be assessed either by cell counts, typically in conjunction with fluorescein diacetate counterstaining to identify intact cells containing adequate levels of functional esterases, or with a fluorescence plate reader.