Na(+) and Ca(2+) homeostasis pathways, cell death and protection after oxygen-glucose-deprivation in organotypic hippocampal slice cultures

Intracellular ATP supply and ion homeostasis determine neuronal survival and degeneration after ischemic stroke. The present study provides a systematic investigation in organotypic hippocampal slice cultures of the influence of experimental ischemia, induced by oxygen-glucose-deprivation (OGD). The pathways controlling intracellular Na(+) and Ca(2+) concentration ([Na(+)](i) and [Ca(2+)](i)) and their inhibition were correlated with delayed cell death or protection. OGD induced a marked decrease in the ATP level and a transient elevation of [Ca(2+)](i) and [Na(+)](i) in cell soma of pyramidal neurons. ATP level, [Na(+)](i) and [Ca(2+)](i) rapidly recovered after reintroduction of oxygen and glucose. Pharmacological analysis showed that the OGD-induced [Ca(2+)](i) elevation in neuronal cell soma resulted from activation of both N-methyl-d-aspartate (NMDA)-glutamate receptors and Na(+)/Ca(2+) exchangers, while the abnormal [Na(+)](i) elevation during OGD was due to Na(+) influx through voltage-dependent Na(+) channels. In hippocampal slices, cellular degeneration occurring 24 h after OGD, selectively affected the pyramidal cell population through apoptotic and non-apoptotic cell death. OGD-induced cell loss was mediated by activation of ionotropic glutamate receptors, voltage-dependent Na(+) channels, and both plasma membrane and mitochondrial Na(+)/Ca(2+) exchangers. Thus, we show that neuroprotection induced by blockade of NMDA receptors and plasma membrane Na(+)/Ca(2+) exchangers is mediated by reduction of Ca(2+) entry into neuronal soma, whereas neuroprotection induced by blockade of AMPA/kainate receptors and mitochondrial Na(+)/Ca(2+) exchangers might result from reduced Na(+) entry at dendrites level.     

Changes in mitochondrial function induced in isolated guinea-pig ventricular myocytes by calcium overload.

1. Changes in [Ca2+]i and pHi, mitochondrial membrane potential (psi m) and mitochondrial [NADH] have been measured independently using fluorescent techniques in single isolated guinea-pig ventricular myocytes subjected to Ca2+ overload. 2. The changes in NADH autofluorescence on the inhibition or uncoupling of respiration are consistent with the signal emanating from the mitochondrial NADH. 3. Removal of Ca2+ and Mg2+ from the bathing Tyrode solution induced a modest fall in both [Ca2+]i and pHi, a small slowly developing depolarization of psi m and an initial fall followed by a rise in mitochondrial [NADH]. 4. In myocytes that maintained an intact sarcolemma, return to Ca(2+)-containing fluid elicited a strong but brief intracellular acidification, a rise in [Ca2+]i which generally recovered more slowly to stabilize above the initial level in Tyrode solution, a steep fall in mitochondrial [NADH] and a brief transient recovery followed by a large sustained depolarization of psi m. NADH autofluorescence and mitochondrial depolarization often reached values that were not further increased by uncoupling respiration although recovery of NADH was elicited by inhibitors of respiration. 5. These changes were reduced when the Ca2+ overload was less severe as evidenced by a reduced hypercontracture upon Ca2+ repletion. A similar reduction could be routinely achieved by elevation of [Mg2+]o during the period of Ca2+ depletion. 6. These results suggest that the well-established depletion of energy-rich phosphates that occurs on Ca2+ overload is due to the combined effects of the failure of the citric acid cycle to provide sufficient mitochondrial NADH for the respiratory chain and an uncoupling of respiration from ATP production due to depolarization of psi m. The former effect could result from the depletion of sarcoplasmic amino acids and the latter from increased Ca2+ cycling across the mitochondrial wall provoked by the elevated [Na+]i and [Ca2+]i.     

Role of the mitochondrial sodium/calcium exchanger in neuronal physiology and in the pathogenesis of neurological diseases

In neurons, as in other excitable cells, mitochondria extrude Ca(2+) ions from their matrix in exchange with cytosolic Na(+) ions. This exchange is mediated by a specific transporter located in the inner mitochondrial membrane, the mitochondrial Na(+)/Ca(2+) exchanger (NCX(mito)). The stoichiometry of NCX(mito)-operated Na(+)/Ca(2+) exchange has been the subject of a long controversy, but evidence of an electrogenic 3 Na(+)/1 Ca(2+) exchange is increasing. Although the molecular identity of NCX(mito) is still undetermined, data obtained in our laboratory suggest that besides the long-sought and as yet unfound mitochondrial-specific NCX, the three isoforms of plasmamembrane NCX can contribute to NCX(mito) in neurons and astrocytes. NCX(mito) has a role in controlling neuronal Ca(2+) homeostasis and neuronal bioenergetics. Indeed, by cycling the Ca(2+) ions captured by mitochondria back to the cytosol, NCX(mito) determines a shoulder in neuronal [Ca(2+)](c) responses to neurotransmitters and depolarizing stimuli which may then outlast stimulus duration. This persistent NCX(mito)-dependent Ca(2+) release has a role in post-tetanic potentiation, a form of short-term synaptic plasticity. By controlling [Ca(2+)](m) NCX(mito) regulates the activity of the Ca(2+)-sensitive enzymes pyruvate-, alpha-ketoglutarate- and isocitrate-dehydrogenases and affects the activity of the respiratory chain. Convincing experimental evidence suggests that supraphysiological activation of NCX(mito) contributes to neuronal cell death in the ischemic brain and, in epileptic neurons coping with seizure-induced ion overload, reduces the ability to reestablish normal ionic homeostasis. These data suggest that NCX(mito) could represent an important target for the development of new neurological drugs.     

Induction of NMDA and GABAA receptor-mediated Ca2+ oscillations with KCC2 mRNA downregulation in injured facial motoneurons

To clarify the changes that occur in gamma-aminobutyric acid type A (GABA(A)) receptor-mediated effects and contribute to alterations in the network activities after neuronal injury, we studied intracellular Ca(2+) concentration ([Ca(2+)](i)) dynamics in a rat facial-nerve-transection model. In facial motoneurons, an elevation of the resting [Ca(2+)](i), GABA-mediated [Ca(2+)](i) transients, enhancement of the glutamate-evoked [Ca(2+)](i) increases, and spontaneous [Ca(2+)](i) oscillations were induced by axotomy. All these axotomy-induced modifications were abolished by the GABA(A)-receptor antagonist bicuculline and N-methyl-d-aspartate (NMDA)-receptor antagonist d(-)-2-amino-5-phosphonopentanoic acid. A downregulation of K(+)-Cl(-) cotransporter (KCC2) mRNA, an increase in intracellular Cl(-) concentration ([Cl(-)](i)), and transformation of GABAergic hyperpolarization to depolarization were also induced by axotomy. We suggest that in axotomized neurons KCC2 downregulation impairs Cl(-) homeostasis and makes GABA act depolarizing, resulting in endogenous GABA inducing [Ca(2+)](i) oscillations via facilitation of NMDA-receptor activation. Such GABA(A)-receptor-mediated [Ca(2+)](i) oscillations may play a role in neural survival and regeneration.     

3,4-dihydroxybenzoic acid from Smilacis chinae rhizome protects amyloid beta protein (25-35)-induced neurotoxicity in cultured rat cortical neurons

The neuroprotective effect of 3,4-dihydroxybenzoic acid (3,4-DHBA) isolated from Smilacis chinae rhizome against Abeta (25-35)-induced neurotoxicity on cultured rat cortical neurons was found in this study. The protective effect of 3,4-DHBA against Abeta (25-35)-induced neuronal cell death was investigated by measuring cell viability via a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. 3,4-DHBA (1 and 10 microM) concentration-dependently inhibited 10 microM Abeta (25-35)-induced neuronal apoptotic death. 3,4-DHBA (1 and 10 microM) inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)), which was measured by a fluorescent dye, Fluo-4 AM. 3,4-DHBA also inhibited glutamate release into medium, reactive oxygen species (ROS) generation, and caspase-3 activation, which were induced by 10 microM Abeta (25-35). These results suggest that 3,4-DHBA prevents Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of ROS and caspase-3 activity.     

Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat

During physiological activity neurons may experience localised energy demands which require intracellular signals for stimulation of mitochondrial NADH generation and subsequent delivery of ATP. To elucidate these mechanisms, we applied microfluorimetric monitoring of cytoplasmic (Fluo-3) and mitochondrial (Rhod-2) calcium concentration ([Ca(2+)](c), [Ca(2+)](m)), as well as of mitochondrial oxidative metabolism (NAD(P)H), whilst simultaneously measuring changes in extracellular potassium concentration ([K(+)](o)), as an indicator of neuronal activity in hippocampal slice cultures. Changes in neuronal activity were induced by repetitive stimulation at different frequencies (5, 20, 100 Hz) and intensities. Stimulation parameters were chosen to elicit rises in [K(+)](o) of less than 3 mM which is comparable to physiologically occurring rises in [K(+)](o).The mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) reduced stimulus-induced changes in Rhod-2 fluorescence by 79%, indicating that Rhod-2 signals were primarily of mitochondrial origin. Repetitive stimulation at 20 Hz applied to areas CA1 or CA3 resulted in moderate rises in [K(+)](o) which were associated with stimulus-dependent elevations in [Ca(2+)](c) and [Ca(2+)](m) of up to 15%. The same stimuli also elicited biphasic changes in NAD(P)H fluorescence characterised by an initial decline and a subsequent prolonged elevation of up to 10%. Variation of stimulus parameters revealed close correlations between rises in [K(+)](o), in [Ca(2+)](m) and changes in NAD(P)H fluorescence. To elucidate the role of intracellular Ca(2+) accumulation in induction of NAD(P)H fluorescence signals, the effect of application of Ca(2+)-free solution on these signals evoked by repetitive antidromic stimulation of the alveus during recordings in area CA1 was studied. Lowering extracellular Ca(2+) led to complete blockade of postsynaptic field potential components as well as of rises in [Ca(2+)](c) and [Ca(2+)](m). Amplitudes of NAD(P)H signals were reduced by 59%, though rises in [K(+)](o) were comparable in presence and absence of extracellular Ca(2+).The results suggest i) that mitochondrial oxidative metabolism is fine-tuned to graded physiological activity in neurons and ii) that rapid mitochondrial Ca(2+) signalling represents one of the main determinants for stimulation of oxidative metabolism under physiological conditions.     

Cytoplasmic and mitochondrial Ca levels in brown adipocytes

AIM: We elucidated the mitochondrial functions of brown adipocytes in intracellular signalling, paying attention to mitochondrial activity and noradrenaline- and forskolin-induced Ca(2+) mobilizations in cold-acclimated rats. METHODS: A confocal laser-scanning microscope of brown adipocytes from warm- or cold-acclimated rats was employed using probes rhodamine 123 which is a mitochondria-specific cationic dye, and the cytoplasmic and mitochondrial Ca(2+) probes fluo-3 and rhod-2. X-ray microanalysis was also studied. RESULTS: The signal of rhodamine 123 in the cells was decreased by antimycin A which effect was less in cold-acclimated cells than warm-acclimated cells. Cytoplasmic and mitochondrial Ca(2+) in cold-acclimated brown adipocytes double-loaded with fluo-3 and rhod-2 were measured. Noradrenaline induced the rise in cytoplasmic Ca(2+) ([Ca(2+)](cyto)) followed by mitochondrial Ca(2+) ([Ca(2+)](mito)), the effect being transformed into an increase in [Ca(2+)](cyto) whereas a decrease in [Ca(2+)](mito) by antimycin A or carbonyl cyanide m-chlorophenylhydrazone (CCCP). Antimycin A induced small Ca(2+) release from mitochondria. CCCP induced Ca(2+) release from mitochondria only after the cells were stimulated with noradrenaline. Further, forskolin also elicited an elevation in [Ca(2+)](cyto) followed by [Ca(2+)](mito) in the cells. The Ca measured by X-ray microanalysis was higher both in the cytoplasm and mitochondria whereas K was higher in the mitochondria of cold-acclimated cells in comparison to warm-acclimated cells. CONCLUSIONS: These results suggest that noradrenaline and forskolin evoked an elevation in [Ca(2+)](cyto) followed by [Ca(2+)](mito), in which H(+) gradient across the inner membrane is responsible for the accumulation of calcium on mitochondria. Moreover, cAMP also plays a role in intracellular and mitochondrial Ca(2+) signalling in cold-acclimated brown adipocytes.     

N-Acetylcysteine and ebselen but not nifedipine protected cerebellar granule neurons against 4-hydroxynonenal-induced neuronal death

4-Hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, has been shown to induce neurotoxicity in various types of neurons. To clarify the mechanisms underlying HNE-induced neurotoxicity, the effects of antioxidants (N-acetylcysteine (NAC) and ebselen with or without NAC pretreatment) and Ca(2+)-related reagents were examined in cerebellar granule neurons. The decreases in neuronal survival and mitochondrial membrane potential induced by HNE were suppressed by pretreatment with NAC at concentrations of 500 and 1000 microM. HNE-induced protein modification and reactive oxygen species generation were also suppressed by pretreatment with NAC at 1000 microM. Although simultaneous application of ebselen (10 microM) did not protect against HNE-induced neurotoxicity, it completely suppressed HNE-induced injury after pretreatment with NAC at 300 microM. HNE increased [Ca(2+)](i) levels, and this increase was significantly attenuated by simultaneous application of nifedipine (10 microM) or EGTA (1000 microM), but not by MK-801 or CNQX. However, none of these Ca(2+)-related reagents was able to prevent HNE-induced neuronal death or mitochondrial injury. These results suggest that pretreatment with a low concentration of NAC dramatically potentiates the neuroprotective activity of ebselen, and that HNE-induced increase in [Ca(2+)](i) is not involved in HNE-induced neuronal death in cerebellar granule neurons.     

Cellular and mitochondrial changes in glutamate-induced HT4 neuronal cell death

Elevated levels of extracellular glutamate are neurotoxic. The cytotoxic property of extracellular glutamate is known to mediate two primary mechanisms, excitotoxicity and excitotoxicity-independent processes. The excitotoxicity-independent pathway was investigated in the current study in a mouse hippocampal-derived HT4 cell line. Exposure of HT4 cells to glutamate for 12h induced loss of cell viability preceded by rapid loss of intracellular reduced glutathione followed by accumulation of intracellular reactive oxygen species, elevation of intracellular Ca(2+), progressive loss of mitochondrial membrane potential swelling and loss of mitochondrial outer membrane integrity. Glutamate-induced loss of DNA integrity has been detected. The antioxidants alpha-tocopherol and trolox, mitochondrial calcium uniporter inhibitor Ruthenium Red and protein synthesis inhibitor cycloheximide all showed protection against glutamate-induced toxicity. None of the protective agents except for alpha-tocopherol controlled the glutamate-induced reactive oxygen species build-up. However, these cell death regulators prevented the glutamate-induced mitochondrial damage and regulated glutamate-induced increase in intracellular Ca(2+). Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone, a mitochondrial uncoupler, partially protected against glutamate-induced cell death and mitochondrial damage, while the mitochondrial ribosomal inhibitor chloramphenicol and extracellular Ca(2+) chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not protect the cells against glutamate treatment.The results of this study demonstrated that mitochondrial dysfunction was a key event in the excitotoxicity-independent component of neuronal cell death. Reactive oxygen species accumulation and glutathione depletion were prominent in glutamate-treated cells; however, these events were not direct mediators of cell death.     

Striatal cells from mutant huntingtin knock-in mice are selectively vulnerable to mitochondrial complex II inhibitor-induced cell death through a non-apoptotic pathway

Extensive striatal neuronal loss occurs in Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt). Evidence suggests that mutant htt directly or indirectly compromises mitochondrial function, contributing to the neuronal loss. To determine the role of compromised mitochondrial function in the neuronal cell death in HD, immortalized striatal cells established from Hdh(Q7) (wild-type) and Hdh(Q111) (mutant) mouse knock-in embryos were treated with 3-nitropropionic acid (3-NP), a mitochondrial complex II toxin. 3-NP treatment caused significantly greater cell death in mutant striatal cells compared with wild-type cells. In contrast, the extent of cell death induced by rotenone, a complex I inhibitor, was similar in both cell lines. Although evidence of apoptosis was present in 3-NP-treated wild-type striatal cells, it was absent in 3-NP-treated mutant cells. 3-NP treatment caused a greater loss of mitochondrial membrane potential (deltapsim) in mutant striatal cells compared with wild-type cells. Cyclosporine A, an inhibitor of mitochondrial permeability transition pore (PTP), and ruthenium red, an inhibitor of the mitochondrial calcium uniporter, both rescued mutant striatal cells from 3-NP-induced cell death and prevented the loss of deltapsim. These data show that mutant htt specifically increases cell vulnerability to mitochondrial complex II inhibition and further switched the type of cell death induced by complex II inhibition from apoptosis to a non-apoptotic form, caused by mitochondrial membrane depolarization, probably initiated by mitochondrial calcium overload and subsequent PTP opening. These findings suggest that impaired mitochondrial complex II function in HD may contribute to non-apoptotic neuronal cell death.     

Ca(2+)- and metabolism-related changes of mitochondrial potential in voltage-clamped CA1 pyramidal neurons in situ

In hippocampal slices from rats, dialysis with rhodamine-123 (Rh-123) and/or fura-2 via the patch electrode allowed monitoring of mitochondrial potential (DeltaPsi) changes and intracellular Ca(2+) ([Ca(2+)](i)) of CA1 pyramidal neurons. Plasmalemmal depolarization to 0 mV caused a mean [Ca(2+)](i) rise of 300 nM and increased Rh-123 fluorescence signal (RFS) by 相似文献   

NMDA-induced calcium loads recycle across the mitochondrial inner membrane of hippocampal neurons in culture

Mitochondria sequester N-methyl-D-aspartate (NMDA)-induced Ca(2+) loads and regulate the shape of intracellular Ca(2+) concentration ([Ca(2+)](i)) responses in neurons. When isolated mitochondria are exposed to high [Ca(2+)](,) Ca(2+) enters the matrix via the uniporter and returns to the cytosol by Na(+)/Ca(2+) exchange. Released Ca(2+) may re-enter the mitochondrion recycling across the inner membrane dissipating respiratory energy. Ca(2+) recycling, the continuous uptake and release of Ca(2+) by mitochondria, has not been described in intact neurons. Here we used single-cell microfluorimetry to measure [Ca(2+)](i) and mitochondrially targeted aequorin to measure matrix Ca(2+) concentration ([Ca(2+)](mt)) to determine whether Ca(2+) recycles across the mitochondrial inner membrane in intact neurons following treatment with NMDA. We used ruthenium red and CGP 37157 to block uptake via the uniporter and release via Na(+)/Ca(2+) exchange, respectively. As predicted by the Ca(2+) recycling hypothesis, blocking the uniporter immediately following challenge with 200 microM NMDA produced a rapid and transient increase in cytosolic Ca(2+) without a corresponding increase in matrix Ca(2+). Blocking mitochondrial Ca(2+) release produced the opposite effect, depressing cytosolic Ca(2+) levels and prolonging the time for matrix Ca(2+) levels to recover. The Ca(2+) recycling hypothesis uniquely predicts these reciprocal changes in the Ca(2+) levels between the two compartments. Ca(2+) recycling was not detected following treatment with 20 microM NMDA. Thus Ca(2+) recycling across the inner membrane was more pronounced following treatment with a high relative to a low concentration of NMDA, consistent with a role in Ca(2+)-dependent neurotoxicity.     

Neuroprotective effects of the antifungal drug clotrimazole

Pretreatment with 10 microM of the antifungal drug clotrimazole potently reduced the death of cultured rat cerebellar granule cells induced by oxygen/glucose deprivation, and the excitotoxic effect of glutamate on cultured hippocampal neurons and cerebellar granule cells. In patch-clamped hippocampal pyramidal neurons, 10-50 microM clotrimazole caused a decrease in the amplitude of N-methyl-D-aspartate (NMDA) receptor-mediated currents. Glutamate induced intracellular Ca(2+) overload, as measured by Fluo-3 confocal fluorescence imaging, while clotrimazole reduced Ca(2+) overload and promoted the recovery of intracellular calcium homeostasis after glutamate treatment. Using tetramethylrhodamine ethyl ester fluorescence as a marker of mitochondrial membrane potential we found that clotrimazole prevented the glutamate-induced loss of mitochondrial membrane potential. Our data provide evidence that the protective effect of clotrimazole against oxygen/glucose deprivation and excitotoxicity is due to the ability of this drug to partially block NMDA receptor-gated channel, thus causing both reduced calcium overload and lower probability of the mitochondrial potential collapse.     

Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury

Mitochondria play an important role in cell death and cardioprotection. During ischemia, when ATP is progressively depleted, ion pumps cannot function resulting in a rise in calcium (Ca(2+)), which further accelerates ATP depletion. The rise in Ca(2+) during ischemia and reperfusion leads to mitochondrial Ca(2+) accumulation, particularly during reperfusion when oxygen is reintroduced. Reintroduction of oxygen allows generation of ATP; however, damage to the electron transport chain results in increased mitochondrial generation of reactive oxygen species (ROS). Mitochondrial Ca(2+) overload and increased ROS can result in opening of the mitochondrial permeability transition pore, which further compromises cellular energetics. The resultant low ATP and altered ion homeostasis result in rupture of the plasma membrane and cell death. Mitochondria have long been proposed as central players in cell death, since the mitochondria are central to synthesis of both ATP and ROS and since mitochondrial and cytosolic Ca(2+) overload are key components of cell death. Many cardioprotective mechanisms converge on the mitochondria to reduce cell death. Reducing Ca(2+) overload and reducing ROS have both been reported to reduce ischemic injury. Preconditioning activates a number of signaling pathways that reduce Ca(2+) overload and reduce activation of the mitochondrial permeability transition pore. The mitochondrial targets of cardioprotective signals are discussed in detail.     

Contribution of Ca(2+)-permeable AMPA/KA receptors to glutamate-induced Ca(2+) rise in embryonic lumbar motoneurons in situ

Intracellular Ca(2+) ([Ca(2+)](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a [Ca(2+)](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca(2+)](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca(2+)](i) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced [Ca(2+)](i) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca(2+)](i) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca(2+)](i) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca(2+)](i) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca(2+)](i) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.     

Oxytocin depolarizes mitochondria in isolated myometrial cells

Oxytocin is known to play important roles in uterine contractions, mediated at least in part by increasing intracellular Ca(2+) concentration ([Ca(2+)](i)), through enhancing extracellular Ca(2+) entry and Ca(2+) release from the sarcoplasmic reticulum, processes that are intimately linked with mitochondria. This study examined the effects of oxytocin on mitochondrial function. This was achieved by measuring the ratiometric JC-1 fluorescence signal in isolated myometrial cells, which provides a relative measure of the mitochondrial membrane potential (ψ(m)), and also by loading the cells with Oregon Green BAPTA-AM to examine changes in [Ca(2+)](i). Oxytocin (1 nm) depolarized the ψ(m) to 73.8 ± 3.7% of the control value (P?< 0.05; perfused for 11 min) and also caused a transient increase in [Ca(2+)](i). The depolarization of mitochondrial membrane potential was effectively reversed by 2-aminoethoxydiphenyl borate, nifedipine, Ca(2+)-free solution or oligomycin, with the ratiometric JC-1 fluorescence signal becoming no different from the control value in all cases (i.e.?P?> 0.05). The reduction in ψ(m) is likely to occur at least in part through the oxytocin-induced increase in [Ca(2+)](i), causing enhanced mitochondrial uptake of Ca(2+) and resultant dissipation of the mitochondrial electrochemical gradient. ATP synthase is also stimulated, which would further contribute to a decrease in ψ(m).     

Postnatal development of carotid body glomus cell response to hypoxia

This study examines developmental changes in CB glomus cell depolarization, intracellular calcium ([Ca(2+)](i)) and the magnitude of an O(2)-sensitive background ionic conductance that may play roles in the postnatal increase in oxygen sensitivity of glomus cells isolated from rats of 1-3 days and 11-14 days postnatal age. Using fura-2 and perforated patch whole cell recordings, we simultaneously measured [Ca(2+)](i) and membrane potential (E(m)) during normoxia and hypoxia. Resting E(m) in normoxia was similar at both ages. Hypoxia caused a larger E(m) depolarization and correspondingly larger [Ca(2+)](i) response in glomus cells from 11- to 14-day-old rats compared to 1-3-day-old rats. E(m) and [Ca(2+)](i) responses to 40mM K(+) were identical between the two age groups. Under normoxic conditions both age groups had similar background conductances. Under anoxic conditions (at resting membrane potential) background K(+) conductance decreased significantly more in cells from 11- to 14-day-old rats compared to cells from 1- to 3-day-old rats. Glomus cells from newborns therefore have less O(2)-sensitive background K(+) conductance. These results support the hypothesis that postnatal maturation of glomus cell O(2) sensitivity involves developmental regulation of the expression and/or O(2)-sensitivity of background ionic conductances.     

Electrophysiological mechanisms of delayed excitotoxicity: positive feedback loop between NMDA receptor current and depolarization-mediated glutamate release

Delayed excitotoxic neuronal death after insult from exposure to high glutamate concentrations appears important in several CNS disorders. Although delayed excitotoxicity is known to depend on NMDA receptor (NMDAR) activity and Ca(2+) elevation, the electrophysiological mechanisms underlying postinsult persistence of NMDAR activation are not well understood. Membrane depolarization and nonspecific cationic current in the postinsult period were reported previously, but were not sensitive to NMDAR antagonists. Here, we analyzed mechanisms of the postinsult period using parallel current- and voltage-clamp recording and Ca(2+) imaging in primary hippocampal cultured neurons. We also compared more vulnerable older neurons [about 22 days in vitro (DIV)] to more resistant younger (about 15 DIV) neurons, to identify processes selectively associated with cell death in older neurons. During exposure to a modest glutamate insult (20 microM, 5 min), similar degrees of Ca(2+) elevation, membrane depolarization, action potential block, and increased inward current occurred in younger and older neurons. However, after glutamate withdrawal, these processes recovered rapidly in younger but not in older neurons. The latter also exhibited a concurrent postinsult increase in spontaneous miniature excitatory postsynaptic currents, reflecting glutamate release. Importantly, postinsult NMDAR antagonist administration reversed all of these persisting responses in older cells. Conversely, repolarization of the membrane by voltage clamp immediately after glutamate exposure reversed the NMDAR-dependent Ca(2+) elevation. Together, these data suggest that, in vulnerable neurons, excitotoxic insult induces a sustained positive feedback loop between NMDAR-dependent current and depolarization-mediated glutamate release, which persists after withdrawal of exogenous glutamate and drives Ca(2+) elevation and delayed excitotoxicity.     

The mitochondria-regulated death pathway mediates asbestos-induced alveolar epithelial cell apoptosis

The mechanisms underlying asbestos-induced pulmonary toxicity are not fully understood. Alveolar epithelial cell (AEC) apoptosis by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. The two major pathways regulating apoptosis include (i) the mitochondrial death (intrinsic) pathway caused by DNA damage, and (ii) the plasma-membrane death receptor (extrinsic) pathway. However, it is unknown whether asbestos activates either death pathway in AEC. We determined whether asbestos triggers AEC mitochondrial dysfunction by exposing cells (A549 and rat alveolar type II) to amosite asbestos and assessing mitochondrial membrane potential changes (deltapsi(m)) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. Unlike inert particulates (titanium dioxide and glass beads), amosite asbestos caused dose- and time-dependent reductions in deltapsi(m). Asbestos-induced deltapsi(m) was associated with the release of cytochrome c from the mitochondria to the cytoplasm as well as activation of caspase 9, a mitochondrial-activated caspase. In contrast, a lower level of caspase 8, the death receptor-activated caspase, was detected in asbestos-exposed AEC. An iron chelator (phytic acid or deferoxamine) or a hydroxyl radical scavenger (sodium benzoate) each blocked asbestos-induced reductions in deltapsi(m) and caspase 9 activation, suggesting a role for iron-derived ROS. Finally, Bcl-X(L), a mitochondrial antiapoptotic protein that prevents cell death by preserving the outer mitochondrial membrane integrity, blocked asbestos-induced decreases in A549 cell deltapsi(m) and reduced apoptosis as assessed by DNA fragmentation. We conclude that asbestos-induced AEC apoptosis results from mitochondrial dysfunction, in part due to iron-derived ROS, which is followed by the release of cytochrome c and caspase 9 activation. Our findings suggest an important role for the mitochondria-regulated death pathway in the pathogenesis of asbestos-associated pulmonary toxicity.     

Alterations in neuronal calcium levels are associated with cognitive deficits after traumatic brain injury

Traumatic brain injury (TBI) survivors often suffer from a post-traumatic syndrome with deficits in learning and memory. Calcium (Ca(2+)) has been implicated in the pathophysiology of TBI-induced neuronal death. However, the role of long-term changes in neuronal Ca(2+) function in surviving neurons and the potential impact on TBI-induced cognitive impairments are less understood. Here we evaluated neuronal death and basal free intracellular Ca(2+) ([Ca(2+)](i)) in acutely isolated rat CA3 hippocampal neurons using the Ca(2+) indicator, Fura-2, at seven and thirty days after moderate central fluid percussion injury. In moderate TBI, cognitive deficits as evaluated by the Morris Water Maze (MWM), occur after injury but resolve after several weeks. Using MWM paradigm we compared alterations in [Ca(2+)](i) and cognitive deficits. Moderate TBI did not cause significant hippocampal neuronal death. However, basal [Ca(2+)](i) was significantly elevated when measured seven days post-TBI. At the same time, these animals exhibited significant cognitive impairment (F(2,25)=3.43, p<0.05). When measured 30 days post-TBI, both basal [Ca(2+)](i) and cognitive functions had returned to normal. Pretreatment with MK-801 blocked this elevation in [Ca(2+)](i) and also prevented MWM deficits. These studies provide evidence for a link between elevated [Ca(2+)](i) and altered cognition. Since no significant neuronal death was observed, the alterations in Ca(2+) homeostasis in the traumatized, but surviving neurons may play a role in the pathophysiology of cognitive deficits that manifest in the acute setting after TBI and represent a novel target for therapeutic intervention following TBI.