Sodium-calcium exchanger in cultured human retinal pigment epithelium.
Regulation of intracellular free Ca2+ concentration ([Ca2+]i) by an Na+/Ca2+ exchanger was studied in cultures of human retinal pigment epithelial cells using Ca(2+)-indicator dyes (fura-2 and fluo-3) and digital fluorescence imaging. Mean resting [Ca2+]i of cultured RPE in a control Ringer solution was 189 +/- 16 nM. Replacing extracellular Na+ with N-methyl-D-glucamine elicited a two-fold rise in [Ca2+]i; the magnitude of the [Na+]o-free-induced rise in [Ca2+]i varied as a function of extracellular [Ca2+]. The [Na+]o-free response was not significantly affected by the Ca2+ channel blocker nifedipine, or by pretreatment with thapsigargin which depletes intracellular Ca2+ stores. By contrast, the [Na+]o-free-induced rise in [Ca2+]i was significantly reduced by CBDMB, an amiloride derivative that is highly selective for Na+/Ca2+ exchange inhibition. These findings indicate that removal of extracellular Na+ promotes net [Ca2+]i gain via Na+/Ca2+ exchange. Western and Northern blot analyses, respectively, confirmed the presence of Na+/Ca2+ exchanger protein and mRNA in cultures of human RPE. Specifically, Western blot analysis of whole cell lysates of cultured RPE using a polyclonal antibody made against the canine cardiac exchanger identified a major band at approximately 126 kD. Northern blot analysis of total human RPE RNA using a restriction fragment cRNA probe coding for the canine cardiac Na+/Ca2+ exchanger showed that the major exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-type Na+/Ca2+ exchanger in cultures of human RPE.
Mangini
N J
NJ
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine 60612, USA.
Haugh-Scheidt
L
L
Valle
J E
JE
Cragoe
E J
EJ
Ripps
H
H
Kennedy
B G
BG
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000814:Aniline Compounds; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism*; D046508:Culture Techniques; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012313:RNA / Q000032:analysis; D012815:Signal Processing, Computer-Assisted; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D019831:Sodium-Calcium Exchanger; D014966:Xanthenes; C059715:Fluo-3; D012313:RNA; D012964:Sodium; D002118:Calcium; D016257:Fura-2