Molecular identification of P-glycoprotein: a role in lens circulation?
OBJECTIVE
To determine whether P-glycoprotein is expressed in the rat lens and to assess what type of damage occurs when P-glycoprotein inhibitors are applied to organ-cultured lenses.


METHODS
An initial screening for the P-glycoprotein isoforms multidrug resistance (mdr)1a, mdr1b, and mdr2 was performed by RT-PCR on RNA extracted from rat lens fiber cells. Northern blot analysis was used to determine whether transcript levels detected by RT-PCR were significant. The presence of P-glycoprotein in the lens was confirmed by Western blot analysis and immunocytochemistry. Organ-cultured lenses, maintained in isotonic artificial aqueous humor, were exposed to various concentrations of the P-glycoprotein inhibitor tamoxifen. Lens opacification was assessed by dark-field microscopy, and the underlying cellular changes were visualized by confocal microscopy of lens sections, using a fluorescent membrane marker. Initial cellular damage was assessed after a 6-hour exposure to 100 micro M tamoxifen. Other P-glycoprotein inhibitors, verapamil, and 1,9-dideoxyforskolin (DDFK) were assessed, and the damage phenotypes were compared with those seen for tamoxifen.


RESULTS
Transcript for all three P-glycoprotein isoforms was detected with RT-PCR, but only mdr1a and mdr2 could be detected by Northern blot analysis. P-glycoprotein was localized in the plasma membrane of lens epithelial and fiber cells. Treatment of organ-cultured lenses with increasing doses of the P-glycoprotein inhibitor tamoxifen for 18 hours showed that two distinct damage phenotypes were evident. At a dose of 20 micro M tamoxifen, tissue damage was found in a discrete zone that initially started approximately 100 micro m from the capsule, whereas at higher doses (60-100 micro M tamoxifen), extensive vesiculation of fiber cell membranes occurred throughout the entire lens cortex. Decreasing tamoxifen (100 micro M) exposure to 6 hours showed that the inner zone of damage was caused by the dilation of extracellular space between fiber cells. The extracellular space dilution and fiber cell vesiculation could be reproduced by varying the concentrations of other P-glycoprotein inhibitors, verapamil and DDKF.


CONCLUSIONS
The P-glycoproteins mdr1a and mdr2 are expressed in the lens and appear to be functional. The initial cellular damage phenotype of extracellular space dilations caused by the P-glycoprotein inhibitors was identical with that caused by chloride channel inhibitors, indicating that P-glycoprotein may play a role in regulating cell volume in the lens. Whether the secondary damage phenotype of fiber cell vesiculation, induced by high doses of P-glycoprotein inhibitors, was due to the inhibition of additional regulatory activities of P-glycoprotein or to nonspecific effects of the drugs remains to be determined. However, regardless of the precise mode of action, these results indicate that P-glycoprotein should be considered in the regulatory mechanisms associated with the control of lens volume and in the initiation of osmotic cataract.
Discipline of Physiology, Faculty of Medical and Health Sciences. School of Biological Sciences, University of Auckland, Auckland, New Zealand.
B Rachelle
BR
Merriman-Smith
Miriam A
MA
Young
Marc D
MD
Jacobs
Joerg
J
Kistler
Paul J
PJ
Donaldson
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018613:Microscopy, Confocal; D009924:Organ Culture Techniques; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013629:Tamoxifen / Q000494:pharmacology; D000097202:ATP-Binding Cassette Sub-Family B Member 4
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D020033:Protein Isoforms; D013629:Tamoxifen; D000097202:ATP-Binding Cassette Sub-Family B Member 4; C095122:multidrug resistance protein 3