Background: Luminal growth factors are potent regulators of enterocyte nutrition. The purpose of this study was to investigate the effects of EGF on Na+-independent L-alanine absorption in differentiating intestinal epithelial cells. Hypothesis: Intestinal epithelial cell alanine absorption capacity is dually regulated by both differentiation state and EGF.
Methods: Human intestinal epithelial Caco-2 cells were grown in undifferentiated (2 day old) and differentiated (9 day old) states and exposed to EGF (0 - 100 ng/ml) for varying periods of time (1 min to 48 hrs). [3H] L-Alanine (5 mM - 10 mM) transport was measured in cell monolayers.
Results: Na+-independent L-alanine transport occurred via a single saturable system L in both undifferentiated and differentiated cells. System L activity decreased as the cells aged from the undifferentiated to the differentiated state, a response that was due to a decrease in transport maximal velocity (Vmax) (1.85 ± 0.25 nmole/mg protein/min vs. 0.38 ± 0.02 nmole/mg protein/min, p<0.01). Transport affinity was unchanged (Km = 1.10 ± 0.19 mM vs. Km = 1.02 ± 0.01 mM, p=NS). Prolong incubation with EGF (> 30 hrs) resulted in a 70 % increase in System L activity in both undifferentiated cells (0.064 ± 0.01 nmole/mg protein/min vs. 0.038 ± 0.005 nmole/mg protein/min, p<0.01) and differentiated cells (0.034 ± 0.005 nmole/mg protein/min vs. 0.022 ± 0.001 nmole/mg protein/min, p<0.01). EGF stimulated the System L Vmax in both undifferentiated (2.9 ± 0.3 nmole/mg protein/min vs. 1.85 ± 0.25 nmole/mg protein/min, p<0.01) and differentiated (0.65 ± 0.04 nmole/mg protein/min vs. 0.38 ± 0.02 nmole/mg protein/min, p<0.01) states, without affecting the transport affinity Km.
Conclusion: Intestinal epithelial cell differentiation is associated with a decrease in System L transport activity. EGF activates System L transport activity independent of cell differentiation state, but exerts greater effects on undifferentiated enterocytes. Both cell differentiation and EGF regulation of System L activity occur via mechanisms that alter functional copies of the System L transporter as opposed to modifying transport affinity.
