Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons.

K+ and Cl- homeostasis have been implicated in cell volume regulation and apoptosis. We addressed the hypothesis that K+ and Cl- efflux may contribute to apoptotic cell shrinkage and apoptotic death in cultured cortical neurons. CLC-2 and CLC-3 chloride channels were detected in cultured cortical neurons. The Cl- channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the outwardly rectifying Cl- current, prevented apoptotic cell shrinkage, and mildly attenuated cell death induced by staurosporine, C2-ceramide, or serum deprivation. Cl- channel blockers, however, at concentrations that prevented cell shrinkage had no significant effects on caspase activation and/or DNA fragmentation. Cell death in the presence of a Cl- channel blocker was still sensitive to blockade by the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk). Electron microscopy revealed that, although DIDS prevented apoptotic cell shrinkage, certain apoptotic ultrastructural alterations still took place in injured neurons. On the other hand, the K+ channel blocker tetraethylammonium (TEA), clofilium, or the caspase inhibitor z-VAD-fmk prevented cell shrinkage as well as caspase activation and/or DNA damage, and showed stronger neuroprotection against apoptotic alterations and cell death. The results indicate that neurons may undergo apoptotic process without cell shrinkage and imply distinct roles for Cl- and K+ homeostasis in regulating different apoptotic events.

Apoptotic insults impair Na+, K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress.

The Na+, K+-ATPase (Na+, K+-pump) plays critical roles in maintaining ion homeostasis. Blocking the Na+, K+-pump may lead to apoptosis. By contrast, whether an apoptotic insult may affect the Na+, K+-pump activity is largely undefined. In cultured cortical neurons, the Na+, K+-pump activity measured as a membrane current Ipump was time-dependently suppressed by apoptotic insults including serum deprivation, staurosporine, and C2-ceramide, concomitant with depletion of intracellular ATP and production of reactive oxygen species. Signifying a putative relationship among these events, Ipump was highly sensitive to changes in ATP and reactive oxygen species levels. Moreover, the apoptosis-associated Na+, K+-pump failure and serum deprivation-induced neuronal death were antagonized by pyruvate and succinate in ATP- and reactive-oxygen-species-dependent manners. We suggest that failure of the Na+, K+-pump as a result of a combination of energy deficiency and production of reactive oxygen species is a common event in the apoptotic cascade; preserving the pump activity provides a neuroprotective strategy in certain pathological conditions.

Block of Na+,K+-ATPase and induction of hybrid death by 4-aminopyridine in cultured cortical neurons.

K(+) channel blockers such as 4-aminopyridine (4-AP) can be toxic to neurons; the cellular mechanism underlying the toxicity, however, is obscure. In cultured mouse cortical neurons, we tested the hypothesis that the toxic effect of 4-AP might result from inhibiting the Na(+),K(+)-ATPase (Na(+),K(+)-pump) and thereafter induction of a hybrid death of concomitant apoptosis and necrosis. The Na(+),K(+)-pump activity, monitored as whole-cell membrane currents, was markedly blocked by 4-AP in concentration- and voltage-dependent manners in low millimolar ranges. At similar concentrations, 4-AP induced a neuronal death sensitive to attenuation by the caspase inhibitor Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-fluoromethyl ketone) or Ca(2+) chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Electron microscopy confirmed hybrid ultrastructural features of coexisting apoptotic and necrotic components in same cells. We suggest that 4-AP is a potent antagonist of the Na(+),K(+)-ATPase and an inducer of the hybrid death of central neurons.

Slight impairment of Na+,K+-ATPase synergistically aggravates ceramide- and beta-amyloid-induced apoptosis in cortical neurons.

Dysfunction of the Na(+),K(+)-ATPase (Na(+),K(+)-pump), due to reduced energy supply or increased endogenous ouabain-like inhibitors, likely occurs under pathological conditions in the central nervous system. In cultured mouse cortical neurons, we examined the hypothesis that a mild non-toxic inhibition of the Na(+),K(+)-ATPase could synergistically sensitize the vulnerability of neurons to normally non-lethal apoptotic signals. Ouabain at a low concentration of 0.1 microM slightly lessened the Na(+),K(+)-pump activity measured as an ouabain-sensitive current, yet did not affect K(+) homeostasis and viability of cortical neurons. Co-exposure to 0.1 microM ouabain plus non-lethal C(2)-ceramide (5 microM) or beta-amyloid 1-42 (5 microM), however, induced marked intracellular K(+) loss, caspase-3 cleavage, DNA laddering, and synergistically triggered neuronal death. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) predominantly blocked the caspase activation and neuronal death. These results suggest that slight impairment of Na(+),K(+)-pump activity may amplify the disruption of K(+) homeostasis in the presence of a non-lethal apoptotic insult, leading to activation of apoptotic cascade and substantial neuronal injury.

Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons.

Energy deficiency and dysfunction of the Na+, K+-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na+, K+-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na+, K+-ATPase induces apoptosis by depleting cellular K+ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca2+ and Na+. The Na+, K+-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca2+ and Na+ increase, K+ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K+ medium, but not Ca2+ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K+, Ca2+, or Na+ channels or high K+ medium each attenuated the ouabain-induced cell death; combined inhibition of K+ channels and Ca2+ or Na+ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifedipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na+, K+-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K+ and accumulation of Ca2+ and Na+, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.