p38 Activation Is Required Upstream of Potassium Current Enhancement and Caspase Cleavage in Thiol Oxidant-Induced Neuronal Apoptosis

Abstract

Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2Ј-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 M DTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDPinduced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death.

Apoptotic surface delivery of K+ channels

“…12 These cells are rendered highly susceptible to dithiodipyridine (DTDP)-induced apoptosis following expression of Kv2.1, but not other potassium channels. 10 As in neuronal models, 3,4 we observed that Kv2.1-expressing CHO cells undergoing apoptosis have a similar surge in channel activity. Currents were measured with standard patch clamp electrodes after evoking them with a series of depolarizing membrane potential steps.…”

“…27 This channel declustering appears to be correlated to calcium-dependent dephosphorylation of the channel and does not seem to affect surface expression. As the apoptotic current surge requires activation of the MAPK p38 pathway, 4 we believe that an initial phosphorylation event is responsible for the upregulation of Kv2.1 surface expression during apoptosis. Thus, it is unclear whether dispersion of surface clusters precedes or is related in any way to the apoptotic trafficking described in the present study.…”

“…CHO cells expressing Kv2.1(I379C) were treated with MTSET to block the pre-existing surface channels prior to induction of apoptosis with DTDP. At 3 h after DTDP treatment, the time it normally takes to observe a substantial current surge, 4 whole-cell recordings were performed. Vehicle-treated cells had very small currents, showing that few nascent channels arrived (Figure 1c, left).…”

“…9 Cortical neurons were treated with DTDP (100 mM) for 10 min at 371C 5% CO 2 , a condition that induces a well-characterized p38-dependent increase in K þ currents approximately 3 h later, followed by caspase activation at 5-7 h and subsequent cell death. 4 All data are expressed as mean7S.E.M. Current densities were analyzed using two-tailed t tests.…”

“…1,2 In cortical neurons, a delayed surge in voltage-gated potassium channel (Kv)-mediated electrical currents provides the likely exit route for this cation. 3 The potassium current enhancement, which is protein synthesis independent and precedes caspase activation, 4 can be triggered by classical apoptotic stimuli like staurosporine, 3 serum withdrawal, 3 and amyloid b, 5 or by oxidants such as 2,2 0 -dithiodipyridine (DTDP) 4,6 and peroxynitrite. 7,8 In oxidant-induced neuronal apoptosis, the enhancement of K þ currents is dependent upon the liberation of intracellular Zn 2 þ from metal-containing proteins 6,7 and requires the activation of the mitogen-activated protein kinase (MAPK) pathway involving apoptosis signal-regulating kinase-1 8 and p38.…”