dizocilpine-maleate and cobaltous-chloride

Protracted elevation in intracellular calcium caused by the activation of the N-methyl-d-aspartate receptor is the main cause of glutamate excitotoxic injury in stroke. However, upon excitotoxic injury, despite the presence of calcium entry antagonists, calcium unexpectedly continues to enter the neuron, causing extended neuronal depolarization and culminating in neuronal death. This phenomenon is known as the calcium paradox of neuronal death in stroke, and it represents a major problem in developing effective therapies for the treatment of stroke. To investigate this calcium paradox and to determine the source of this unexpected calcium entry after neuronal injury, we evaluated whether glutamate excitotoxicity activates an injury-induced calcium-permeable channel responsible for conducting a calcium current that underlies neuronal death. We used a combination of whole-cell and single-channel patch-clamp recordings, fluorescent calcium imaging, and neuronal cell death assays in a well characterized primary hippocampal neuronal culture model of glutamate excitotoxicity/stroke. Here, we report activation of a novel calcium-permeable channel upon excitotoxic glutamate injury that carries calcium current even in the presence of calcium entry inhibitors. Blocking this injury-induced calcium-permeable channel for a significant time period after the initial injury is still effective in preventing calcium entry, extended neuronal depolarization, and delayed neuronal death, thereby accounting for the calcium paradox. This injury-induced calcium-permeable channel represents a major source for the initial calcium entry following stroke, and it offers a new target for extending the therapeutic window for preventing neuronal death after the initial excitotoxic (stroke) injury.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Apoptosis; Calcium; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Chlorides; Cobalt; Dizocilpine Maleate; Dose-Response Relationship, Drug; Electric Impedance; Ethosuximide; Gadolinium; Glutamic Acid; Membrane Potentials; Neurons; Nifedipine; omega-Conotoxins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sodium; Stroke; Zinc Compounds

Topics: 2-Amino-5-phosphonovalerate; Animals; Calcium; Cells, Cultured; Cerebellum; Cobalt; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Membrane Potentials; Mitochondria; Neurons; Osmosis; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Sodium; Solutions; Sucrose

Rhodamine 123 staining, light and electron microscopy were used to evaluate the ultrastructural and functional state of cultured cerebellar granule cells after short treatment with the solution where NaCl was substituted by sucrose (sucrose balance salt medium, SBSM). Cell exposure to SBSM for 20 min resulted in the fact that mitochondria in the neurons lost their ability to sequester rhodamine 123. This effect could be prevented by: (i) non-competitive N-methyl-D-aspartate (NMDA) receptor channel blocker, 10(-5) M MK-801; (ii) a competitive specific antagonist of NMDA glutamate receptors, 0.25 x 10(-3) M D,L-2-amino-7-phosphonoheptanoate (APH); (iii) 10(-3) M cobalt chloride; (iv) removal of Ca2+ from the medium. Low Na+ in the Ca2+-containing medium caused considerable mitochondrial swelling in granule cells. However, the same treatment in the absence of calcium ions in the medium abolished the deleterious effect of SBSM on the neuronal mitochondrial structure and functions. It is suggested that (i) the exposure of cultured cerebellar granule cells to SBSM leads to a release of endogenous glutamate from cells; (ii) Ca2+ ions potentially de-energizing neuronal mitochondria enter the neuron preferentially through the NMDA channels rather than through the Na+/Ca2+ exchanger; (iii) mitochondrial swelling in granule cells is highly Ca2+-dependent; (iv) cellular overload with sodium ions can activate mitochondrial Na+/Ca2+ exchanger and thus prevent permeability transition pore opening in mitochondria.

Topics: Animals; Calcium; Cells, Cultured; Cerebellum; Cobalt; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Microscopy, Electron; Rats; Rats, Wistar; Rhodamine 123; Rhodamines; Sodium Chloride