Characterization of Na/H exchange systems in immortalized rat caput epididymal (RCE) cells

Abstract

RCE cells are primary cultures of rat caput epididymal cells immortalized with SV40 large T antigens. The goals of the present studies were to characterize the endogenous Na/H exchange systems in these cells and to understand the regulation of endogenous Na/H exchangers by hyperosmolarity because epididymal fluids are hypertonic. By Western blotting, RCE cells were shown to have endogenous NHE1 (105kD protein), but not NHE2 and NHE3. For measurement of Na/H exchange with BCECF, RCE cells were grown on collagen coated glass cover slips. These cells exhibited Na dependent alkalinization which was completely inhibited by HOE694 (IC50 = 0.3μM), confirming the presence of NHE1. Kinetic analysis with Na showed a Km of 18±3mM with a Hill coefficient of 1.1±0.2. The acute effect of hyperosmolarity on NHE1 was studied at both steady-state pH and by initial rates of pH recovery after NH4Cl acidification. Addition of 100mM mannitol at steady-state pH of cells caused intracellular alkalization (ΔpH = 0.4±0.1) which was blocked by HOE694. Mannitol (100mM) also increased the initial pH recovery rates of cells. Kinetic analysis suggested that there was no change in Vmax but a change in Km(H) (Km(H): control; 6.51±0.01 vs mannitol; 6.75±0.01). Interestingly, mannitol stimulation of NHE1 was inhibited by BAPTA (50μM) and by the calmodulin (CaM) inhibitor, W13 (45μM), suggesting the involvement of Ca/CaM. The chronic effect of hyperosmolarity on NHE1 was also studied by culturing the cells in the presence of 100mM mannitol for 72h. Under this culturing condition, the amount of NHE1 protein was increased by 2.1 fold as determined by Western blotting. We conclude that RCE cells contain endogenous NHE1 which is stimulated by hyperosmolarity in a Ca/CaM dependent manner. Chronic exposure of RCE cells in hypertonic medium increases the amount of NHE1 protein.