Mitochondrial but not plasmalemmal BK channels are hypoxia-sensitive in human glioma.

Tumor cells are resistant to hypoxia but the underlying mechanism(s) of this tolerance remain poorly understood. In healthy brain cells, plasmalemmal Ca(2+)-activated K(+) channels ((plasma)BK) function as oxygen sensors and close under hypoxic conditions. Similarly, BK channels in the mitochondrial inner membrane ((mito)BK) are also hypoxia sensitive and regulate reactive oxygen species production and also permeability transition pore formation. Both channel populations are therefore well situated to mediate cellular responses to hypoxia. In tumors, BK channel expression increases with malignancy, suggesting these channels contribute to tumor growth; therefore, we hypothesized that the sensitivity of (plasma)BK and/or (mito)BK to hypoxia differs between glioma and healthy brain cells. To test this, we examined the electrophysiological properties of (plasma)BK and (mito)BK from a human glioma cell line during normoxia and hypoxia. We observed single channel activities in whole cells and isolated mitoplasts with slope conductance of 199 ± 8 and 278 ± 10 pA, respectively. These currents were Ca(2+)- and voltage-dependent, and were inhibited by the BK channel antagonist charybdotoxin (0.1 µM). (plasma)BK could only be activated at membrane potentials >+40 mV and had a low open probability (NPo) that was unchanged by hypoxia. Conversely, (mito)BK were active across a range of membrane potentials (-40 to +40 mV) and their NPo increased during hypoxia. Activating (plasma)BK, but not (mito)BK induced cell death and this effect was enhanced during hypoxia. We conclude that unlike in healthy brain cells, glioma (mito)BK channels, but not (plasma)BK channels are oxygen sensitive.

DIDS (4,4-diisothiocyanatostilbenedisulphonic acid) induces apoptotic cell death in a hippocampal neuronal cell line and is not neuroprotective against ischemic stress.

DIDS is a commonly used anion channel antagonist that is putatively cytoprotective against ischemic insult. However, recent reports indicate potentially deleterious secondary effects of DIDS. To assess the impact of DIDS on cellular viability comprehensively we examined neuronal morphology and function through 24 hours treatment with ACSF ± DIDS (40 or 400 µM). Control cells were unchanged, whereas DIDS induced an apoptotic phenotype (chromatin condensation, nuclear fragmentation and cleavage of the nuclear membrane protein lamin A, expression of pro-apoptotic proteins c-Jun N-terminal kinase 3, caspase 3, and cytochrome C, Annexin V staining, RNA degradation, and oligonucleosomal DNA cleavage). These deleterious effects were mediated by DIDS in a dose- and time-dependant manner, such that higher [DIDS] induced apoptosis more rapidly while apoptosis was observed at lower [DIDS] with prolonged exposure. In an apparent paradox, despite a clear overall apoptotic phenotype, certain hallmarks of apoptosis were not present in DIDS treated cells, including mitochondrial fission and loss of plasma membrane integrity. We conclude that DIDS induces apoptosis in cultured hippocampal neurons, in spite of the fact that some common hallmarks of cell death pathways are prevented. These contradictory effects may cause false-positive results in certain assays and future evaluations of DIDS as a neuroprotective agent should incorporate multiple viability assays.

Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra.

The development of clinical stroke therapies remains elusive. The neuroprotective efficacies of thousands of molecules and compounds have not yet been determined; however, screening large volumes of potential targets in vivo is severely rate limiting. High throughput screens (HTS) may be used to discover promising candidates, but this approach has been hindered by the lack of a simple in vitro model of the ischemic penumbra, a clinically relevant region of stroke-afflicted brain. Recently, our laboratory developed such a mimic (ischemic solution: IS) suitable for HTS, but the etiology of stress pathways activated by this model are poorly understood. The aim of the present study was to determine if the cell death phenotype induced by IS accurately mimics the in vivo penumbra and thus whether our model system is suitable for use in HTS. We treated cultured neuron and astrocyte cell lines with IS for up to 48 hrs and examined cellular energy state ([ATP]), cell and organelle morphology, and gene and molecular profiles related to stress pathways. We found that IS-treated cells exhibited a phenotype of mixed apoptosis/autophagy characteristic of the in vivo penumbra, including: (1) short-term elevation of [ATP] followed by progressive ATP depletion and Poly ADP Ribose Polymerase cleavage, (2) increased vacuole number in the cytoplasm, (3) mitochondrial rupture, decreased mitochondrial and cristae density, release of cytochrome C and apoptosis inducing factor, (4) chromatin condensation, nuclear lamin A and DNA cleavage, fragmentation of the nuclear envelope, and (5) altered expression of mRNA and proteins consistent with autophagy and apoptosis. We conclude that our in vitro model of the ischemic penumbra induces autophagy and apoptosis in cultured neuron and astrocyte cell lines and that this mimic solution is suitable for use in HTS to elucidate neuroprotective candidates against ischemic penumbral cell death.

DIDS prevents ischemic membrane degradation in cultured hippocampal neurons by inhibiting matrix metalloproteinase release.

During stroke, cells in the infarct core exhibit rapid failure of their permeability barriers, which releases ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra). Plasma membrane degradation is key to penumbral spread and is mediated by matrix metalloproteinases (MMPs), which are released via vesicular exocytosis into the extracellular fluid in response to stress. DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic solution: IS) and we asked whether this action was mediated via inhibition of MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular proMMP-2 and -9 expression increased 4-10 fold and extracellular latent and active MMP isoform expression increased 2-22 fold. MMP-mediated extracellular gelatinolytic activity increased ∼20-50 fold, causing detachment of 32.1±4.5% of cells from the matrix and extensive plasma membrane degradation (>60% of cells took up vital dyes and >60% of plasma membranes were fragmented or blebbed). DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active MMPs. DIDS similarly inhibited extracellular MMP expression and cellular detachment induced by the pro-apoptotic agent staurosporine or the general proteinase agonist 4-aminophenylmercuric acetate (APMA). Conversely, DIDS-treatment did not impair stress-induced intracellular proMMP production, nor the intracellular cleavage of proMMP-2 to the active form, suggesting DIDS interferes with the vesicular extrusion of MMPs rather than directly inhibiting proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular ATPase also inhibited extracellular MMP expression to a similar degree as DIDS. In addition, in a proteinase-independent model of vesicular exocytosis, DIDS prevented stimulus-evoked release of von Willebrand Factor from human umbilical vein endothelial cells. We conclude that DIDS inhibits MMP exocytosis and through this mechanism preserves neuronal membrane integrity during pathological stress.

An in vitro ischemic penumbral mimic perfusate increases NADPH oxidase-mediated superoxide production in cultured hippocampal neurons.

The currently accepted scheme for reactive oxygen species production during ischemia/reperfusion injury is characterized by a deleterious mitochondria-derived burst of radical generation during reperfusion; however, recent examination of the penumbra suggests a central role for NADPH-oxidase (Nox)-mediated radical generation during the ischemic period. Therefore, we utilized a novel in vitro model of the penumbra to examine the free radical profile of ischemic murine hippocampal neurons using electron paramagnetic resonance spectroscopy, and also the role of Nox in this generation and in cell fate. We report that free radical production increased ~75% at 2 h of ischemia, and this increase was abolished by: (1) scavenging of extracellular free radicals with superoxide dismutase (SOD), (2) a general anion channel antagonist, or (3) the Nox inhibitor apocynin. Similarly, at 24 h of ischemia, [ATP] decreased >95% and vital dye uptake increased 6-fold relative to controls; whereas apocynin, the Cl(-) channel antagonist 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), or the free radical scavenger N-acetyl cysteine (NAC) each provided moderate neuroprotection, ameliorating 13-32% of [ATP]-depletion and 19-56% of vital dye uptake at 24 h. Our results support a cytotoxic role for Nox-mediated free radical production from penumbral neurons during the ischemic period.

Hypoxia induces Kv channel current inhibition by increased NADPH oxidase-derived reactive oxygen species.

There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates K(v) channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pulmonary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypoxia. Our data indicate colocalization of K(v)1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in K(v) current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated K(v)1.5 channel oxidation. We conclude that sustained hypoxia decreases K(v) channel currents by a direct effect of a NOX4-derived increase in ROS.

Insulin/PI3K signaling protects dentate neurons from oxygen-glucose deprivation in organotypic slice cultures.

It is known that ischemia/reperfusion induces neurodegeneration in the hippocampus in a subregion-dependent manner. This study investigated the mechanism of selective resistance/vulnerability to oxygen-glucose deprivation (OGD) using mouse organotypic hippocampal cultures. Analysis of propidium iodide uptake showed that OGD-induced duration- and subregion-dependent neuronal injury. When compared with the CA1-3 subregions, dentate neuronal survival was more sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling under basal conditions. Dentate neuronal sensitivity to PI3K/Akt signaling activation was inversely related to its vulnerability to OGD-induced injury; insulin/insulin-like growth factor 1 pre-treatment conferred neuroprotection to dentate neurons via activation of PI3K/Akt signaling. In contrast, CA1 and CA3 neurons were less sensitive to disruptions of endogenous PI3K/Akt signaling and protective effects of insulin/insulin-like growth factor 1, but more vulnerable to OGD. OGD-induced injury in CA1 was reduced by inhibition of NMDA receptor or mitogen-activated protein kinase signaling, and was prevented by blocking NMDA receptor in the presence of insulin. The CA2 subregion was distinctive in its response to glutamate, OGD, and insulin, compared with other CA subregions. CA2 neurons were sensitive to the protective effects of insulin against OGD-induced injury, but more resistant to glutamate. Distinctive distribution of insulin receptor beta and basal phospho-Akt was detected in our slice cultures. Our results suggest a role for insulin signaling in subregional resistance/vulnerability to cerebral ischemia.

Hypoxia increases activity of the BK-channel in the inner mitochondrial membrane and reduces activity of the permeability transition pore.

Hypoxia can cause severe damage to cells by initiating signaling cascades that lead to cell death. A cellular oxygen sensor, other than the respiratory chain, might exist in sensitive components of these signaling cascades. Recently, we found evidence that mitochondrial ion channels are sensitive to low levels of oxygen. We therefore studied the effects of hypoxia on the mitochondrial BK-channel (mtBK), on the mitochondrial permeability transition pore (PTP), and on their possible interaction. Using single-channel patch-clamp techniques we found that hypoxia inhibited the PTP but substantially increased the mtBK activity of mitoplasts from rat liver and astrocytes. Experiments measuring the mitochondrial membrane potential of intact rat brain mitochondria (using the fluorescence dye safranine O) during hypoxia exhibited an increased Ca(2+)-retention capacity implying an impaired opening of the PTP. We also found a reduced Ca(2+)-retention capacity with 100 nM iberiotoxin, a selective inhibitor of BK-channels. We therefore conclude that there is interaction between the mtBK and the PTP in a way that an open mtBK keeps the PTP closed. Thus, the response of mitochondrial ion channels to hypoxia could be interpreted as anti-apoptotic.

Hypoxia increases BK channel activity in the inner mitochondrial membrane.

To explore the potential function of the BK channel in the inner mitochondrial membrane under physiological and hypoxic conditions, we used on-mitoplast and whole-mitoplast patches. Single BK channels had a conductance of 276+/-9 pS under symmetrical K(+) solutions, were Ca(2+)- and voltage-dependent and were inhibited by 0.1 microM charybdotoxin. In response to hypoxia, BK increased open probability, shifted its reversal potential (9.3+/-2.4 mV) in the positive direction and did not change its conductance. We conclude that (1) the properties at rest of this mitoplast K(+) channel are similar to those of BK channels in the plasma membrane; (2) hypoxia induces an increase, rather than a decrease (as in the plasmalemma), in the open probability of this K(+) channel, leading to K(+) efflux from the mitochondrial matrix to the outside. We speculate that this increase in K(+) efflux from mitochondria into the cytosol is important during hypoxia in maintaining cytosolic K(+).

Chronic high-inspired CO2 decreases excitability of mouse hippocampal neurons.

To examine the effect of chronically elevated CO(2) on excitability and function of neurons, we exposed mice to 8 and 12% CO(2) for 4 wk (starting at 2 days of age), and examined the properties of freshly dissociated hippocampal neurons obtained from slices. Chronic CO(2)-treated neurons (CC) had a similar input resistance (R(m)) and resting membrane potential (V(m)) as control (CON). Although treatment with 8% CO(2) did not change the rheobase (64 +/- 11 pA, n = 9 vs. 47 +/- 12 pA, n = 8 for CC 8% vs. CON; means +/- SE), 12% CO(2) treatment increased it significantly (73 +/- 8 pA, n = 9, P = 0.05). Furthermore, the 12% CO(2) but not the 8% CO(2) treatment decreased the Na(+) channel current density (244 +/- 36 pA/pF, n = 17, vs. 436 +/- 56 pA/pF, n = 18, for CC vs. CON, P = 0.005). Recovery from inactivation was also lowered by 12% but not 8% CO(2). Other gating properties of Na(+) current, such as voltage-conductance curve, steady-state inactivation, and time constant for deactivation, were not modified by either treatment. Western blot analysis showed that the expression of Na(+) channel types I-III was not changed by 8% CO(2) treatment, but their expression was significantly decreased by 20-30% (P = 0.03) by the 12% treatment. We conclude from these data and others that neuronal excitability and Na(+) channel expression depend on the duration and level of CO(2) exposure and maturational changes occur in early life regarding neuronal responsiveness to CO(2).

Effect of chronically elevated CO2 on CA1 neuronal excitability.

To study the effect of chronically elevated CO(2) on the excitability and function of neurons, we exposed mice to 7.5-8% CO(2) for approximately 2 wk (starting at 2 days of age) and examined the properties of freshly dissociated hippocampal neurons. Neurons from control mice (CON) and from mice exposed to chronically elevated CO(2) had similar resting membrane potentials and input resistances. CO(2)-exposed neurons, however, had a lower rheobase and a higher Na(+) current density (580 +/- 73 pA/pF; n = 27 neurons studied) than did CON neurons (280 +/- 51 pA/pF, n = 34; P < 0.01). In addition, the conductance-voltage curve was shifted in a more negative direction in CO(2)-exposed than in CON neurons (midpoint of the curve was -46 +/- 3 mV for CO(2) exposed and -34 +/- 3 mV for CON, P < 0.01), while the steady-state inactivation curve was shifted in a more positive direction in CO(2)-exposed than in CON neurons (midpoint of the curve was -59 +/- 2 mV for CO(2) exposed and -68 +/- 3 mV for CON, P < 0.01). The time constant for deactivation at -100 mV was much smaller in CO(2)-exposed than in CON neurons (0.8 +/- 0.1 ms for CO(2) exposed and 1.9 +/- 0.3 ms for CON, P < 0.01). Immunoblotting for Na(+) channel proteins (subtypes I, II, and III) was performed on the hippocampus. Our data indicate that Na(+) channel subtype I, rather than subtype II or III, was significantly increased (43%, n = 4; P < 0.05) in the hippocampi of CO(2)-exposed mice. We conclude that in mice exposed to elevated CO(2), 1) increased neuronal excitability is due to alterations in Na(+) current and Na(+) channel characteristics, and 2) the upregulation of Na(+) channel subtype I contributes, at least in part, to the increase in Na(+) current density.

Calcium influx via L- and N-type calcium channels activates a transient large-conductance Ca2+-activated K+ current in mouse neocortical pyramidal neurons.

Ca2+-activated K+ currents and their Ca2+ sources through high-threshold voltage-activated Ca2+ channels were studied using whole-cell patch-clamp recordings from freshly dissociated mouse neocortical pyramidal neurons. In the presence of 4-aminopyridine, depolarizing pulses evoked transient outward currents and several components of sustained currents in a subgroup of cells. The fast transient current and a component of the sustained currents were Ca2+ dependent and sensitive to charybdotoxin and iberiotoxin but not to apamin, suggesting that they were mediated by large-conductance Ca2+-activated K+ (BK) channels. Thus, mouse neocortical neurons contain both inactivating and noninactivating populations of BK channels. Blockade of either L-type Ca2+ channels by nifedipine or N-type Ca2+ channels by omega-conotoxin GVIA reduced the fast transient BK current. These data suggest that the transient BK current is activated by Ca2+ entry through both N- and L-type Ca2+ channels. The physiological role of the fast transient BK current was also examined using current-clamp techniques. Iberiotoxin broadened action potentials (APs), indicating a role of BK current in AP repolarization. Similarly, both the extracellular Ca2+ channel blocker Cd2+ and the intracellular Ca2+ chelator BAPTA blocked the transient component of the outward current and broadened APs in a subgroup of cells. Our results indicate that the outward current in pyramidal mouse neurons is composed of multiple components. A fast transient BK current is activated by Ca2+ entry through high-threshold voltage-activated Ca2+ channels (L- and N-type), and together with other voltage-gated K+ currents, this transient BK current plays a role in AP repolarization.

Maturation of neuronal excitability in hippocampal neurons of mice chronically exposed to cyclic hypoxia.

To examine the effects of chronic cyclic hypoxia on neuronal excitability and function in mice, we exposed mice to cyclic hypoxia for 8 h daily (9 cycles/h) for approximately 2 wk (starting at 2-3 days of age) and examined the properties of freshly dissociated hippocampal neurons obtained from slices. Compared with control (Con) hippocampal CA1 neurons, exposed neurons (CYC) had similar resting membrane potentials (V(m)) and action potentials (AP). CYC neurons, however, had a lower rheobase than Con neurons. There was also an upregulation of the Na(+) current density (333 +/- 84 pA/pF, n = 18) in CYC compared with that of Con neurons (193 +/- 20 pA/pF, n = 27, P < 0.03). Na(+) channel characteristics were significantly altered by hypoxia. For example, the steady-state inactivation curve was significantly more positive in CYC than in Con (-60 +/- 6 mV, n = 8, for CYC and -71 +/- 3 mV, n = 14, for Con, P < 0.04). The time constant for deactivation (tau(d)) was much shorter in CYC than in Con (at -100 mV, tau(d)=0.83 +/- 0.23 ms in CYC neurons and 2.29 +/- 0.38 ms in Con neurons, P = 0.004). We conclude that the increased neuronal excitability in mice neurons treated with cyclic hypoxia is due to alterations in Na(+) channel characteristics and/or Na(+) channel expression. We hypothesize from these and previous data from our laboratory (Gu XQ and Haddad GG. J Appl Physiol 91: 1245-1250, 2001) that this increased excitability is a reflection of an enhanced central nervous system maturation when exposed to low O(2) conditions in early postnatal life.

Na+ channel expression and neuronal function in the Na+/H+ exchanger 1 null mutant mouse.

Mice lacking Na(+)/H(+) exchanger 1 (NHE1) suffer from recurrent seizures and die early postnatally. Although the mechanisms for seizures are not well established, our previous electrophysiological work has shown that neuronal excitability and Na(+) current density are increased in hippocampal CA1 neurons of these mutant mice. However, it is unknown whether this increased density is related to altered expression or functional regulation of Na(+) channels. In this work, we asked three questions: is the increased excitability limited to CA1 neurons, is the increased Na(+) current density related to an increased Na(+) channel expression, and, if so, which Na(+) channel subtype(s) is upregulated? Using neurophysiological, autoradiographic, and immunoblotting techniques, we showed that both CA1 and cortical neurons have an increase in membrane excitability and Na(+) current density; Na(+) channel density is selectively upregulated in the hippocampus and cortex (P < 0.05); and Na(+) channel subtype I is significantly increased in the hippocampus and Na(+) channel subtype II is increased in the cortex. Our results demonstrate that mice lacking NHE1 upregulate their Na(+) channel expression in the hippocampal and cortical regions selectively; this leads to an increase in Na(+) current density and membrane excitability. We speculate that neuronal overexcitability due to Na(+) channel upregulation in the hippocampus and cortex forms the basis of epileptic seizures in NHE1 mutant mice.