Induction of apoptosis by the calcium antagonist mibefradil correlates with depolarization of the membrane potential and decreased integrin expression in human lens epithelial cells.
BACKGROUND
Posterior capsule opacification is still the major complication in cataract surgery and is caused by migration and proliferation of residual lens epithelial cells. The challenge of a suitable therapy to inhibit capsule opacification is to specifically interfere with cellular mechanisms. Our approach using the T-calcium channel antagonist mibefradil is based on the hypothesis that this drug inhibits the signaling pathways mediated by cell adhesion.


METHODS
The influence of mibefradil dihydrochloride was investigated on primary human lens epithelial cells (hLEC) from cataract surgery and on the human lens cell line HLE-B3. Apoptosis was quantitatively analyzed by flow cytometry (% increase of the sub-G1 peak), and verified by confocal microscopy (annexin V-biotin, TUNEL reaction). The membrane potential was detected by a membrane potential-sensitive dye. Integrin expression and proliferation were measured by flow cytometry. T-calcium channels in hLEC were verified by the whole-cell configuration of the patch-clamp technique.


RESULTS
Mibefradil induced apoptosis in hLEC. Early signs of apoptosis were observed after only 4 h of incubation with mibefradil, accompanied by a significantly reduced cell area. Apoptosis correlated with inhibited integrin expression, reduced proliferation and the depolarization of the membrane potential. We could identify calcium channels of the T-type in our primary hLEC.


CONCLUSIONS
We suggest that depolarization of the membrane potential and the inhibition of integrin expression leads to the loss of cell adhesion, which is the reason for the induction of apoptosis. Thus, mibefradil seems to be a suitable drug to prevent cell adhesion, migration and proliferation.
Nebe
Barbara
B
Department of Internal Medicine, University of Rostock, Ernst-Heydemann-Strasse 6, 18055 Rostock, Germany. barbara.nebe@med.uni-rostock.de
Kunz
Friederike
F
Peters
Annelie
A
Rychly
Joachim
J
Noack
Thomas
T
Beck
Ria
R
D000199:Actins / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D020748:Mibefradil / Q000494:pharmacology*; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques
D000199:Actins; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D016023:Integrins; D020748:Mibefradil