Effect of Na/H exchanger 3 (NHE3) silencing on Na-glucose cotransporter 1 (SGLT1) in small intestinal epithelial cells (IEC-18). A: in NHE3-silenced cells, NHE3 activity was significantly reduced compared with IEC-18 cells transfected with negative control small interfering RNA (siRNA). B: However, in NHE3-silenced cells, SGLT1 activity was increased significantly compared with IEC-18 cells transfected with negative control siRNA. Thus, silencing NHE3 stimulates SGLT1 activity. C: kinetics of SGLT1 stimulation of Na-dependent [3H]-3-O-methyl-d-glucose (OMG) uptake is shown as a function of varying concentrations of extracellular glucose in NHE3-silenced cells. Uptake for all concentrations was determined at 30 s. A representative kinetics plot shows that as the concentration of extracellular glucose was increased, uptake of glucose was stimulated and subsequently became saturated in IEC-18 cells in the negative control siRNA and NHE3 siRNA-transfected cells. Analysis of the data provided kinetic parameters. The affinity for glucose was not affected in NHE3 siRNA-treated cells (Km was 12.4 ± 2.3 mM in control and 11.6 ± 1.3 in NHE3 siRNA-transfected cells; n = 5). However, the Vmax or maximal rate of uptake of glucose was increased in cells transfected with NHE3 siRNA (Vmax was 4.2 ± 0.2 nmol/mg protein for 30 s in control and 10.3 ± 0.3 in NHE3 siRNA-transfected cells, P < 0.01, n = 5). Thus, the mechanism of stimulation of SGLT1 when NHE3 is silenced is due to increased transporter numbers.