IMMUNOLOCALIZATION OF PLASMA MEMBRANE CA2+-ATPASE IN NATIVE HUMAN RETINAL PIGMENT EPITHELIUM

97th DOG Annual Meeting 1999

K428
IMMUNOLOCALIZATION OF PLASMA MEMBRANE CA2+-ATPASE IN NATIVE HUMAN RETINAL PIGMENT EPITHELIUM

K. U. Loeffler¹, B. G. Kennedy² , N. J. Mangini³

The regulation of calcium is of major importance for normal retinal function, and disturbances in Ca²⁺homeostasis have been associated with various di-seases. For the outer retina, the retinal pigment epithelium (RPE) plays a key role in maintaining the extracellular Ca²⁺ equilibrium during phototransduction. The plasma membrane Ca²⁺-ATPase (PMCA) is one of the 2 active mechanisms involved in transcellular Ca²⁺ regulation. Four isoforms of PMCA with tissue and cell specific patterns have been identified, all of which appear to be present in cultured human RPE. To further study the precise mechanism of Ca²⁺ regulation in vivo, we have investigated the subcellular localization of PMCA in native human RPE.

Material and Methods: Frozen sections of RPE/choroid were obtained from 2 apparently normal post mortem eyes, and paraffin sections were taken from 3 eyes enucleated for malignant melanoma. The tissues were labeled with a monoclonal antibody (mAb) 5F10 (Affinity BioReagents, dil.1:500) that recognizes all 4 isoforms of PMCA, and with 2 mAbs (JA3 and JA9, J.Penniston, dil.1:500) that recognize isoform 4b and 4a+b, re-spectively. Prior to the regular staining procedure, lipofuscin autofluorescence was reduced (acc. to Bouras et al.), and melanin was bleached. Labeling was visualized with 2 different fluorescent secondary antibodies (FITC & CY-5) and analyzed with a confocal microscope. Immunoelectron microscopy (IEM) using Lowicryl and post embedding gold labeling with 5F10 was performed on tissues from 2 additional healthy donor eyes.

Results: On frozen sections incubated with mAb 5F10, a distinct linear pattern of labeling was seen along the RPE cell membrane, particularly at the apical surface and - in oblique sections - at the circumference. Using CY-5, staining could also be distinguished at the basal aspect of the RPE. With mAbs JA3 and JA9, this pattern was much less obvious. Only little labeling was seen in RPE cyto-plasm with any of the mAbs used. - On paraffin sections, no specific labeling pattern of the RPE was obvious but by IEM with 5F10, labeling was also distinctly localized to the plasma membrane and was detected both apically and basolaterally.

Conclusions: The RPE PMCA appears localized predominantly at the (latero-)apical surface of the cell but is also present at the basal infoldings. Further studies will concentrate on the distribution of specific isoforms. (NIH RO1 EY-11308, core grant EY-1792, & Lions of Ill. Fdtn.)

¹Universitäts-Augenklinik Bonn,
²Northwest Center for Med. Educ., Indiana Univ. School of Medicine, Gary, IN, USA,
³Dept. of Ophthalmology & Vis. Sciences, UIC College of Medicine, Chicago, USA

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