Pharmacological strategies to block rod photoreceptor apoptosis caused by calcium overload: a mechanistic target-site approach to neuroprotection.
OBJECTIVE
Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited, and disease-, injury- and chemical-induced retinal degeneration. Our aims were three-fold: 1) to determine the kinetics of rod apoptosis and Ca2+ overload in Pde6b9rd1) mice and developmentally lead-exposed rats, 2) to establish a pathophysiologically-relevant model of Ca2+ overload/rod-selective apoptosis in isolated rat retina and 3) to examine different mechanistic based neuroprotective strategies that would abrogate or mollify rod Ca2+ overload/apoptosis.


METHODS
Retinal morphometry and elemental calcium content ([Ca]) determined the kinetics of rod apoptosis and Ca2+ overload. A multiparametric analysis of apoptosis including rod [Ca], a live/dead assay, rod oxygen consumption, cytochrome c immunoblots and caspase assays was combined with pharmacological studies of an isolated rat retinal model of rod-selective Ca2+ overload/apoptosis.


RESULTS
Ca2+ overload preceded rod apoptosis in mice and rats, although the extent and kinetics in each differed significantly. The isolated rat model of rod Ca2+ overload/apoptosis showed that blockade of Ca2+ entry through rod cGMP-activated channels with L-cis diltiazem was partially neuroprotective, whereas blockade of Ca2+ entry into rods through L-type Ca2+ channels with D-cis diltiazem or verapamil provided no protection. Inhibition of the mitochondrial Na+/Ca2+ exchanger with D-cis diltiazem provided no protection. CsA and NIM811, mitochondrial permeability transition pore (mPTP) inhibitors, blocked all Ca(2+)-induced apoptosis, whereas the caspase-3 inhibitor DEVD-fmk only blocked the downstream cytochrome c-induced apoptosis.


CONCLUSIONS
The successful pharmacological neuroprotective strategies for rod Ca2+ overload/apoptosis targeted the rod cGMP-activated channels or mPTP, but not the rod L-type Ca2+ channels.
College of Optometry , University of Houston, Houston, Texas 77204-2020, USA. dafox@uh.edu
D A
DA
Fox
A T
AT
Poblenz
L
L
He
J B
JB
Harris
C J
CJ
Medrano
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D019610:Cytoprotection / Q000187:drug effects; D004110:Diltiazem / Q000494:pharmacology; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009942:Organometallic Compounds / Q000633:toxicity; D010101:Oxygen Consumption; D051381:Rats; D020318:Rats, Long-Evans; D012162:Retinal Degeneration / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D017948:Retinal Rod Photoreceptor Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D003574:Cytochrome c Group; D009942:Organometallic Compounds; D019831:Sodium-Calcium Exchanger; D016572:Cyclosporine; D014700:Verapamil; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D004110:Diltiazem; C008261:lead acetate; D002118:Calcium