Lehman N L, Chang A T, Crook R B
Cellular Pharmacology Laboratory, Department of Ophthalmology, University of California, Box 0730, San Francisco, CA, 94143, USA.
Exp Eye Res. 1998 Nov;67(5):549-59. doi: 10.1006/exer.1998.0543.
Atrial natriuretic peptide (ANP) stimulates aqueous humor formation in primates, but the membrane-bound receptors which mediate this effect have not been well studied in the eye. Endocytosis of [125I]ANP bound to natriuretic peptide C receptors was characterized in fetal human nonpigmented ciliary epithelial (NPE) cells. [125I]ANP which bound to cells at 4 degreesC was detected in the cell interior after a temperature shift to 37 degreesC. Appearance of ligand within the cell peaked at 5 min, and then declined towards zero over 20 min. The endocytosis inhibitor phenylarsine oxide blocked the appearance of internalized ligand, whereas the lysosomotropic drug chloroquine had no effect on internalization but blocked subsequent loss of internalized ligand. Chloroquine also blocked the accumulation of degraded ligand in the extracellular medium. Treatment with phorbol 12-myristate, 13-acetate accelerated the loss of internalized ligand from cells and increased the accumulation of ligand in the extracellular medium. Ligand in the medium was also increased by dioctanoylglycerol but not by 4alpha phorbol didecanoate, an isomer which does not activate protein kinase C. The protein kinase inhibitors staurosporine and bisindolylymaleimide blocked the increase in ligand. Phorbol ester-stimulated loss of internalized ligand occurred in the presence of chloroquine. TCA precipitation of ligand in the extracellular medium showed that both degraded and undegraded [125I]ANP were present. However, in the presence of chloroquine only, undegraded ANP was detected in the medium, and phorbol esters stimulated its rate of appearance by approximately 2 fold. A similar stimulation occurred when cells containing internalized ligand, but stripped of membrane-bound ligand, were exposed to phorbol esters. The data suggest that ANP bound to natriuretic peptide C receptors on NPE cells is endocytosed, and that protein kinase C activates a non-lysosomal pathway for ANP retroendocytosis in these cells.
心房利钠肽（ANP）可刺激灵长类动物房水生成，但介导此效应的膜结合受体在眼部尚未得到充分研究。在人胎儿非色素睫状上皮（NPE）细胞中对与利钠肽C受体结合的[125I]ANP的内吞作用进行了表征。在4℃与细胞结合的[125I]ANP在温度升至37℃后在细胞内部被检测到。配体在细胞内的出现峰值在5分钟时，然后在20分钟内降至零。内吞作用抑制剂苯胂酸氧化物阻断了内化配体的出现，而溶酶体促渗药物氯喹对内化作用没有影响，但阻断了内化配体随后的丢失。氯喹还阻断了降解配体在细胞外介质中的积累。用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯处理可加速内化配体从细胞中的丢失，并增加配体在细胞外介质中的积累。二辛酰甘油也增加了介质中的配体，但4α佛波醇二癸酸酯（一种不激活蛋白激酶C的异构体）则没有。蛋白激酶抑制剂星形孢菌素和双吲哚马来酰亚胺阻断了配体的增加。在氯喹存在的情况下发生了佛波醇酯刺激的内化配体的丢失。细胞外介质中配体的TCA沉淀显示存在降解的和未降解的[125I]ANP。然而，仅在氯喹存在的情况下，在介质中检测到未降解的ANP，并且佛波醇酯刺激其出现速率增加约2倍。当含有内化配体但去除了膜结合配体的细胞暴露于佛波醇酯时，也发生了类似的刺激。数据表明，与NPE细胞上的利钠肽C受体结合的ANP被内吞，并且蛋白激酶C激活了这些细胞中ANP逆向内吞的非溶酶体途径。
