|
|
| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
|
MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 627 |
Rapid
Ca21 Entry through Ca21-Permeable AMPA/Kainate Channels Triggers Marked Intracellular Ca21 Rises
and Consequent
Oxygen Radical Production |
Sean
G. Carriedo, Hong Zhen Yin, Stefano L. Sensi, John H. Weiss |
cell
culture, glutamate, AMPA, kainate, NMDA, cobalt, hydroethidine, calcium imaging, fura-2, fura-2FF,
free radicals, superoxide, tetramethylrhodamine ethylester |
Cultures were plated on glass-bottomed
dishes (Mattek Cultureware, Ashland, MA) and mounted to a stage adapter on an
inverted microscope (Nikon Diaphot, Tokyo, Japan).
|
inverted
microscopy |
mouse |
neuronal |
mixed
neocortical |
| |
The widespread neuronal injury that results after
brief activation of highly Ca21-permeable NMDA channels may, in large part, reflect mitochondrial
Ca21 overload and the consequent production of injurious oxygen radicals. In contrast, AMPA/kainate
receptor activation generally causes slower toxicity, and most studies have not found evidence
of comparable oxygen radical production. Subsets of central neurons, composed mainly of GABAergic
inhibitory interneurons, express AMPA/kainate channels that are directly permeable to Ca21 ions.
Microfluorometric techniques were performed by using the oxidationsensitive dye hydroethidine
(HEt) to determine whether the relatively rapid Ca21 flux through AMPA/kainate channels expressed
on GABAergic neurons results in oxygen radical production comparable to that triggered by NMDA.
Consistent with previous studies, NMDA exposures triggered increases in fluorescence in most
cultured cortical neurons, whereas high K1 (50 mM) exposures (causing depolarization-induced
Ca21 influx through voltage-sensitive Ca21 channels) caused little fluorescence change. In contrast,
kainate exposure caused fluorescence increases in a distinct subpopulation of neurons; immunostaining
for glutamate decarboxylase revealed the responding neurons to constitute mainly the GABAergic
population. The effect of NMDA, kainate, and high K1 exposures on oxygen radical production
paralleled the effect of these exposures on intracellular Ca21 levels when they were monitored
with the low-affinity Ca21-sensitive dye fura-2FF, but not with the high-affinity dye fura-2.
Inhibition of mitochondrial electron transport with CN2 or rotenone almost completely blocked
kainate-triggered oxygen radical production. Furthermore, antioxidants attenuated neuronal injury
resulting from brief exposures of NMDA or kainate. Thus, as with NMDA receptor activation, rapid
Ca21 influx through Ca21-permeable AMPA/ kainate channels also may result in mitochondrial Ca21
overload and consequent injurious oxygen radical production. |
|
|
|
|
