Alteration of the intracellular pH and apoptosis induction in a retinal cell line by the AGE-inducing agent glyoxal.
BACKGROUND
Methylglyoxal and glyoxal, intermediate products of glycation, are known to accelerate glycation and the formation of advanced glycation endproducts (AGEs). These mechanisms may play a role in the degenerative progression of diabetic retinopathy and macular degeneration. The present study was undertaken to elucidate the retinal neurotoxicity of the Maillard reaction intermediate alpha-oxoaldehyde glyoxal.


METHODS
E1A-NR3 is an immortalized retinal cell line that manifests specific phenotypes of retinal neurons. These cells were incubated with 0 microM (control), 200 microM, 400 microM, and 800 microM glyoxal for different lengths of time. For intracellular pH measurements, cells were incubated for 30 min with BCECF-AM prior to measurement of the emission ratio at two wavelengths. For semiquantitative analysis of the mitochondrial potential, cells were incubated for 15 min with 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). For detection of apoptotic cells, cultures were incubated for 15 min with YO-PRO-1 iodide. Immunohistochemical labeling of cell components, which are indicative for apoptosis like active caspase-3 and the caspase cleavage product fractin, was performed after fixation. DNA gel electrophoresis was carried out to detect apoptotic cell death at the DNA level.


RESULTS
Morphological changes in living cells after glyoxal incubation showed signs of cell damage, including cell membrane blebbing and aggregation of intracellular organelles. Glyoxal produced a dose- and time-dependent acidification from physiological pH to pH 7.2. The semiquantitative analysis of mitochondrial membrane potential in living control cells showed nearly all mitochondria with intact hyperpolarized membranes. One hour after the beginning of incubation with 200 microM glyoxal we found most mitochondria depolarized but still with an elongated shape and only regional swelling. With higher glyoxal concentrations more depolarized mitochondria developed blebs and were located around the nucleus. The membrane permeability assay with the nucleic acid stain YO-PRO-1 showed increasing cell membrane permeability with time and glyoxal dose and, finally, nuclear fragmentation. By means of immunohistochemistry we found a glyoxal-induced accumulation of the AGE N(epsilon)-(carboxymethyl) lysine in the cytoplasm and nuclei. The cells were immunoreactive for active caspase-3 after glyoxal in a dose-dependent manner. Neurofilament protein was strongly expressed in controls and disappeared gradually with increasing glyoxal concentration. The immunoreactivity for the caspase cleavage product fractin was found in most of the cells after 800 microM glyoxal. DNA gel electrophoresis showed DNA ladder formation.


CONCLUSIONS
The ability of glyoxal to induce apoptosis was confirmed by our findings demonstrating the time- and dose-dependent acidification of retinal cells. The onset of acidification is a hallmark because acidification is a measurable cytosolic event that follows the mitochondrial change but precedes caspase activation. Therefore, monitoring of pHi can allow one to assess cell stresses such as hypoxia, and metabolic stress (AGEs), and to test whether these stresses have a cumulative effect on apoptosis induction. Our study showed that intracellular pH and mitochondrial potential in living retinal cells are useful parameters for monitoring metabolic status in retinal tissue. These findings may be relevant in the study of retinal cell death mechanisms associated with age-related diabetic retinopathy and macular degeneration.
Reber
Friedemann
F
Institute of Anatomy, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, 01307 Dresden, Germany.
Kasper
Michael
M
Siegner
Axel
A
Kniep
Eva
E
Seigel
Gail
G
Funk
Richard H W
RH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D004247:DNA / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006037:Glyoxal / Q000633:toxicity*; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D007425:Intracellular Membranes / Q000502:physiology; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology; D016900:Neurofilament Proteins / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000378:metabolism / Q000473:pathology*; D013997:Time Factors
D001562:Benzimidazoles; D002232:Carbocyanines; D005452:Fluoresceins; D017127:Glycation End Products, Advanced; D016900:Neurofilament Proteins; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D006037:Glyoxal; D004247:DNA; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases