BINDING AND INTERNALIZATION OF VIP IN RAT INTESTINAL EPITHELIAL-CELLS

We have prepared villous cells from the jejunum of the rat small intestine and studied the effects of divalent cations and bacitracin on the binding and internalization of VIP. Villous epithelial cells (4.10(6) cells/ml) were suspended in a Hepes-NaCl buffer with 1.0% BSA, (pH 7.4) and the cells were incubated for varying periods of time with I-125-VIP at 24-degrees-C. Specific binding of radiolabeled VIP was maximal within 10 min (10%) and slowly declined to 9.0 percent after 30 min. In the presence of 1.0 mg/ml bacitracin, however, maximal specific binding of VIP was only 2.7 percent (P less-than-or-equal-to 0.001). The addition of CA2+ or Mg2+ to the buffer significantly decreased binding of VIP in a concentration dependent manner. At 8.0, 4.0, 2.0 and 1.0 mM Ca2+, binding of I-125-VIP decreased by 70, 60, 40 and 25 percent, whereas in the presence of the same concentrations of Mg2+ binding was decreased to 50, 38, 25 and 10 percent (P less-than-or-equal-to 0.01). To determine if epithelial cells internalize VIP, we bound I-125-VIP to villous cells and then differentiated surface-bound and internalized radioactivity by treating with trypsin (150-mu-g/ml). Surface bound radioligand was the same at both 24 and 4-degrees-C (5.3%), while internalized I-125-VIP was 4.0% at 24-degrees-C compared to only 1.0% at 4-degrees-C (P less-than-or-equal-to 0.001). At 24 and 4-degrees-C, both Ca2+ (4.0 mM) and Mg2+ (8.0 mM) decreased surface bound radioligand by 60 percent (P less-than-or-equal-to 0.01) and lowered internalized radioactivity. These data demonstrate that (1) bacitracin decreases the binding of VIP to small intestinal epithelial cells, (2) both Ca2+ and Mg2+ affect the binding of VIP to its surface receptor and (3) VIP is internalized into epithelial cells.