Amiloride inhibits constitutive internalization and increases the surface number of epidermal growth factor receptors in intact rat hepatocytes.

In previous experiments the surface expression of epidermal growth factor (EGF) receptors in freshly isolated rat hepatocytes varied temperature- and time-dependently and was depleted by monensin and cycloheximide in a way suggesting that a subpopulation of these receptors are subject to constitutive cycling (Gladhaug and Christoffersen; 1988). We here report the finding that pretreatment of the hepatocytes with amiloride exerts marked effects on cellular EGF receptor movements. After 2 h incubation with 1 mM amiloride, the receptor level was approximately 270,000 sites/cell surface vs. 140,000 in the untreated cell, with no change in receptor affinity. Amiloride thus stabilized the surface EGF receptor pool at an elevated level. In cells pretreated with amiloride for 60 min, the relative endocytosis decreased from about 2.6 EGF molecules internalized per receptor during 15 min endocytosis in untreated cells to about 1.5 molecules/receptor in amiloride-treated cells. These results suggest that amiloride causes an accumulation of EGF receptors at the hepatocyte surface due to inhibition of constitutive receptor internalization. In addition, it was found that in amiloride-treated hepatocytes the phorbol ester TPA strongly inhibited high-affinity EGF binding without affecting the total surface receptor number. In control cells, TPA did not consistently affect binding. Pretreatment with amiloride prevented surface EGF receptor depletion induced by cycloheximide and puromycin, but it did not significantly inhibit surface receptor depletion caused by monensin. Although the underlying mechanism of the amiloride effect on intracellular receptor trafficking is not clear, the results provide further evidence for a continuous, ligand-independent EGF receptor cycling pathway in hepatocytes.