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 glycoprotein present on most eukaryotic cells. Its activity, abundance and phosphorylation are regulated by a variety of growth factors and agonists. Although cell contact and inhibition of proliferation may reduce its activity, little is known of the influence of cell culture density on these measurements. The effect of culture density on the intracellular pH (pHi) and activity of NHE-1 of human MRC5 fibroblasts was thus investigated using fluorometry with BCECF, NHE-1 abundance with western blotting and NHE-1 phosphorylation using specific polyclonal antibodies. Proliferating cells in low density cultures had lower pHi and NHE-1 activity (per litre of cell water) than contact inhibited confluent cultures. Such cells in low density cultures were larger than those in very confluent cultures. NHE-1 activity per cell and NHE-1 protein abundance also showed an increasing trend with culture density. However, the turnover number of NHE-1 remained at around 3000 s-1 in low density and sub-confluent cultures, only decreasing in very confluent cultures. Moreover, NHE-1 phosphorylation declined with increased culture density. Cell culture density has profound effects on NHE-1 activity, abundance and turnover number, with associated changes in NHE-1 phosphorylation.
Siczkowski
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M
Department of Medicine and Therapeutics, Leicester Royal Infirmary, United Kingdom.
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D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010766:Phosphorylation; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1