L-ß-N-oxalyl-α,ß-diaminopropionic acid toxicity in motor neurons.

The excitatory amino acid L-ß-N-oxalyl-α,ß-diaminopropionic acid (L-ß-ODAP) in Lathyrus sativus L. is proposed as the causative agent of the neurodegenerative disease neurolathyrism. We investigated the effect of L-ß-ODAP on [Ca2+]i handling, redox homeostasis, and cell death in rat spinal motor neurons. L-ß-ODAP and L-glutamate triggered [Ca2+]i transients, which were inhibited by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor blockers; 2,3-dioxo-6-nitro-1,2,3, 4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide and 1-naphthyl acetylspermine, the latter specifically blocking Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. In addition, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, and to a lesser extent 1-naphthyl acetylspermine, protected the neurons against cell death induced by L-ß-ODAP or L-glutamate. Methionine and cysteine were also protective against neuronal cell death. We conclude that deregulation of [Ca2+]i homeostasis and oxidative stress contribute to motor neuron cell death in neurolathyrism.

L-beta-ODAP alters mitochondrial Ca2+ handling as an early event in excitotoxicity.

The neurotoxin beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (L-beta-ODAP) is an L-glutamate analogue at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors in neurons and therefore acts as an excitotoxic substance. Chronic exposure to L-beta-ODAP present in Lathyrus sativus L. (L. sativus) seeds is proposed as the cause of the neurodegenerative disease neurolathyrism, but the mechanism of its action has not been conclusively identified. A key factor in excitotoxic neuronal cell death is a disturbance of the intracellular Ca2+ homeostasis, including changes in the capacity of intracellular Ca2+ stores like the endoplasmic reticulum (ER) or mitochondria. In this study, aequorin and other Ca2+ indicators were used in N2a neuroblastoma cells to investigate alterations of cellular Ca2+ handling after 24 h exposure to L-beta-ODAP. Our data demonstrate increased mitochondrial Ca2+ loading and hyperpolarization of the mitochondrial membrane potential (Psi(m)), which was specific for L-beta-ODAP and not observed with L-glutamate. We conclude that L-beta-ODAP disturbs the ER-mitochondrial Ca2+ signaling axis and thereby renders the cells more vulnerable to its excitotoxic effects that ultimately will lead to cell death.

Ca(2+) regulation of connexin 43 hemichannels in C6 glioma and glial cells.

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca(2+) ([Ca(2+)](i)) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca(2+)](i) was in the 500nM range but the responses disappeared with larger [Ca(2+)](i) transients. Ca(2+)-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca(2+)-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca(2+)](i) triggers hemichannel opening, not by a direct action of Ca(2+) on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca(2+)](i) and acting to restrain cellular ATP loss.

In situ bipolar electroporation for localized cell loading with reporter dyes and investigating gap junctional coupling.

Electroporation is generally used to transfect cells in suspension, but the technique can also be applied to load a defined zone of adherent cells with substances that normally do not permeate the plasma membrane. In this case a pulsed high-frequency oscillating electric field is applied over a small two-wire electrode positioned close to the cells. We compared unipolar with bipolar electroporation pulse protocols and found that the latter were ideally suited to efficiently load a narrow longitudinal strip of cells in monolayer cultures. We further explored this property to determine whether electroporation loading was useful to investigate the extent of dye spread between cells coupled by gap junctions, using wild-type and stably transfected C6 glioma cells expressing connexin 32 or 43. Our investigations show that the spatial spread of electroporation-loaded 6-carboxyfluorescein, as quantified by the standard deviation of Gaussian dye spread or the spatial constant of exponential dye spread, was a reliable approach to investigate the degree of cell-cell coupling. The spread of reporter dye between coupled cells was significantly larger with electroporation loading than with scrape loading, a widely used method for dye-coupling studies. We conclude that electroporation loading and dye transfer is a robust technique to investigate gap-junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-cell communication.

Neurobarrier coupling in the brain: adjusting glucose entry with demand.

Glucose transport over the blood-brain barrier (BBB) is a nonrate-limiting step and has therefore received little attention as a possible adjustment point within the transport reaction cascade from blood glucose to brain cell glycolysis. Considerations of the normal working point of facilitated BBB glucose shuttling via the GLUT-1 protein indicate that the transport is working at about one-third of T(max) under basal conditions. Substitution of T(max) estimates indicates that the transport is then just enough to keep up with glucose consumption, maintaining the steady state. After brain activation, glucose transport has to be stimulated, and this can be accomplished by increasing the driving force or changing the T(max) and/or K(t) parameters of BBB transport. The first possibility involves a decrease of brain interstitial glucose with subsequent flow stimulation according to the law of mass action (LMA), whereas the second possibility involves signaling from activated neurons to the BBB, a regulation loop that we propose to be called "neurobarrier coupling" (NBC). Theoretical analysis of the LMA effect and comparison with data on glucose dynamics during brain activation suggest that this factor alone only covers about half of the stimulation necessary to bring glucose delivery into line with the elevated glucose consumption during activation. Adjusting glucose entry with demand thus probably involves both LMA and NBC effects, depending on the degree of brain activation. Further work is needed to demonstrate NBC effects following physiological brain activation in vivo and to identify the signals that lead to NBC in in vitro experiments.