The effect of culture density and proliferation rate on the expression of ouabain-sensitive [formula omitted] ATPase pumps in cultured human retinal pigment epithelium

Abstract

The number and activity of ouabain-sensitive Na K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [ 3H]thymidine incorporation and for Na K ATPase pump number by measuring specific [ 3H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [ 3H]thymidine and [ 3H]ouabain. Cycling cells which had [ 3H]thymidine-labeled nuclei did not have notably higher labeling with [ 3H]ouabain. However, [ 3H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation.