Inhibition of apoptosis and reduction of intracellular pH decrease in retinal neural cell cultures by a blocker of carbonic anhydrase.
OBJECTIVE
Methylglyoxal and glyoxal are intermediates of advanced glycation end products (AGEs). These substances, as well as hydrogen peroxide, induce retinal neurons to reduce their intracellular pH and augment their production of reactive oxygen species, leading to apoptosis. Because these processes may play a role in diabetic retinopathy, the authors undertook this study to investigate the protective action of dorzolamide, an inhibitor of carbonic anhydrase, on retinal neural cells.


METHODS
E1A-NR3 cells were incubated with varying concentrations of glyoxal, methylglyoxal, and H2O2 for different periods of time in the presence or absence of dorzolamide. Apoptotic changes were determined by cytofluorometry after the cells were incubated with appropriate dyes and antibodies. The parameters studied were DNA strand breaks (TUNEL assay), subdiploid DNA content (sub-G1 assay), annexin V binding, reactive oxygen species intermediates production, active caspase-3, N(epsilon)-(carboxymethyl)lysine (a glycation product), and intracellular pH.


RESULTS
Optimal conditions for detection of the cell-protecting effect of dorzolamide were incubation with 0.6 to 0.8 mM glyoxal or methylglyoxal for 5 hours or with 0.1 mM H2O2 for 30 minutes, respectively, followed by 20-hour incubation with fresh medium. All apoptotic changes were reduced in the assays in which dorzolamide was included.


CONCLUSIONS
Dorzolamide reduced the damage inflicted on retinal neural cells by agents that induced apoptosis and, therefore, can be considered a neuroprotectant.
Kniep
Eva M
EM
Institute of Anatomy, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden, Germany. eva-maria.kniep@mailbox.tu-dresden.de
Roehlecke
Cora
C
Ozkucur
Nurdan
N
Steinberg
Alexander
A
Reber
Friedemann
F
Knels
Lilla
L
Funk
Richard H W
RH
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques; D002470:Cell Survival; D005434:Flow Cytometry; D006037:Glyoxal / Q000633:toxicity; D006861:Hydrogen Peroxide / Q000633:toxicity; D006863:Hydrogen-Ion Concentration; D020287:In Situ Nick-End Labeling; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D011765:Pyruvaldehyde / Q000633:toxicity; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012160:Retina / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*; D013876:Thiophenes / Q000494:pharmacology*
D017304:Annexin A5; D002257:Carbonic Anhydrase Inhibitors; D018696:Neuroprotective Agents; D017382:Reactive Oxygen Species; D013449:Sulfonamides; D013876:Thiophenes; D006037:Glyoxal; C048496:N(6)-carboxymethyllysine; D011765:Pyruvaldehyde; C062765:dorzolamide; D006861:Hydrogen Peroxide; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D008239:Lysine