CULTURE DENSITY AND THE ACTIVITY, ABUNDANCE AND PHOSPHORYLATION OF THE NA+ H+ EXCHANGER ISOFORM-1 IN HUMAN FIBROBLASTS/

The Na+/H+ exchanger isoform 1 (NHE-1) is a ubiquitous membrane glycop rotein present on most eukaryotic cells. Its activity, abundance and p hosphorylation are regulated by a variety of growth factors and agonis ts. Although cell contact and inhibition of proliferation may reduce i ts activity, little is known of the influence of cell culture density on these measurements. The effect of culture density on the intracellu lar pH (pH(i)) and activity of NHE-1 of human MRCS fibroblasts was thu s investigated using fluorometry with BCECF, NHE-1 abundance with west ern blotting and NHE-1 phosphorylation using specific polyclonal antib odies. Proliferating cells in low density cultures had lower pH(i) and NHE-1 activity (per litre of cell water) than contact inhibited confl uent cultures. Such cells in low density cultures were larger than tho se in very confluent cultures. NHE-1 activity per cell and NHE-1 prote in abundance also showed an increasing trend with culture density. How ever, the turnover number of NHE-1 remained at around 3000 s(-1) in lo w density and sub-confluent cultures, only decreasing in very confluen t cultures. Moreover, NHE-1 phosphorylation declined with increased cu lture density. Cell culture density has profound effects on NHE-1 acti vity, abundance and turnover number, with associated changes in NHE-1 phosphorylation. (C) 1995 Academic Press, Inc.