Izzo R S, Scipione R A, Pellecchia C, Lokchander R S
Department of Medicine, Nassau County Medical Center, East Meadow, NY 11554.
Regul Pept. 1991 Mar 26;33(1):21-30. doi: 10.1016/0167-0115(91)90011-5.
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 x 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 125I-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 125I-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 125I-VIP to villous cells and then differentiated surface-bound and internalized radioactivity by treating with trypsin (150 micrograms/ml). Surface bound radioligand was the same at both 24 and 4 degrees C (5.3%), while internalized 125I-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.
我们从大鼠小肠空肠制备了绒毛细胞，并研究了二价阳离子和杆菌肽对血管活性肠肽（VIP）结合与内化的影响。绒毛上皮细胞（4×10⁶个细胞/毫升）悬浮于含1.0%牛血清白蛋白的Hepes - NaCl缓冲液（pH 7.4）中，在24℃下将细胞与¹²⁵I - VIP孵育不同时间。放射性标记的VIP特异性结合在10分钟内达到最大值（10%），30分钟后缓慢降至9.0%。然而，在存在1.0毫克/毫升杆菌肽的情况下，VIP的最大特异性结合仅为2.7%（P≤0.001）。向缓冲液中添加Ca²⁺或Mg²⁺以浓度依赖方式显著降低了VIP的结合。在8.0、4.0、2.0和1.0毫摩尔/升Ca²⁺时，¹²⁵I - VIP的结合分别降低了70%、60%、40%和25%，而在相同浓度的Mg²⁺存在下，结合分别降至50%、38%、25%和10%（P≤0.01）。为了确定上皮细胞是否内化VIP，我们将¹²⁵I - VIP与绒毛细胞结合，然后用胰蛋白酶（150微克/毫升）处理以区分表面结合和内化的放射性。在24℃和4℃时表面结合的放射性配体相同（5.3%），而在24℃时内化的¹²⁵I - VIP为4.0%，相比之下在4℃时仅为1.0%（P≤0.001）。在24℃和4℃时，Ca²⁺（4.0毫摩尔/升）和Mg²⁺（8.0毫摩尔/升）均使表面结合的放射性配体减少60%（P≤0.01）并降低内化放射性。这些数据表明：（1）杆菌肽降低VIP与小肠上皮细胞的结合；（2）Ca²⁺和Mg²⁺均影响VIP与其表面受体的结合；（3）VIP被内化进入上皮细胞。
