Tseng Y L, Burman K D, Lahiri S, Abdelrahim M M, D'Avis J C, Wartofsky L
Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, D.C. 20307-5001.
J Clin Endocrinol Metab. 1991 Mar;72(3):669-74. doi: 10.1210/jcem-72-3-669.
In a prior study of atrial natriuretic peptide (ANP) binding to cultured thyroid cells, we reported that at 4 C, more than 95% of bound ANP is recovered on cell membranes, with negligible ANP internalization observed. Since ANP binding was inhibited by TSH, we have further studied TSH effects on postbinding ANP processing to determine whether this phenomenon reflects enhanced endocytosis of the ANP-receptor complex. An ANP chase study was initiated by binding [125I] ANP to thyroid cells at 4 C for 2 h, followed by incubation at 37 C. ANP processing was then traced by following 125I activity at various time intervals in three fractions: cell surface membranes, incubation medium, and inside the cells. Radioactivity released into medium represented processed ANP rather than ANP dissociated from surface membranes, since prebound [125I]ANP could not be competitively dissociated by a high concentration of ANP (1 mumol/L) at 37 C. Chase study results showed that prebound ANP quickly disappeared from cell membranes down to 34% by 30 min. Internalized ANP peaked at 10 min, with 21% of initial prebound ANP found inside the cells. At the same time, radioactivity recovered in incubation medium sharply increased between 10-30 min from 8% to 52%. Preincubation of cells with chloroquine (which blocks degradation of the ANP-receptor complex by inhibiting lysosomal hydrolase) caused a 146% increase in internalized [125I]ANP by 30 min (39% compared to 15% control), while medium radioactivity decreased from 52% to 16%, suggesting that processing of the receptor complex is mediated via lysosomal enzymes. In chase studies employing cells pretreated with chloroquine, TSH stimulated the internalization rate of ANP-receptor complex. By 30 min, TSH significantly reduced the membrane-bound ANP, and the decrease was inversely correlated to the increase in internalized radioactivity. In the absence of ANP ligand, TSH also modulated receptor translocation, resulting in decreased receptor number on surface membrane. However, TSH did not affect ANP degradation on the basis of analysis by immunoprecipitation and high performance liquid chromatography. Taken together, these data suggest that TSH downregulates ANP receptor on thyroid cell surface membranes, and that an ANP degradative pathway exists which is not mediated through receptor binding.
在先前一项关于心房利钠肽（ANP）与培养的甲状腺细胞结合的研究中，我们报道在4℃时，超过95%的结合型ANP可在细胞膜上回收，观察到的ANP内化可忽略不计。由于TSH抑制ANP结合，我们进一步研究了TSH对结合后ANP加工过程的影响，以确定这种现象是否反映了ANP - 受体复合物内吞作用的增强。通过在4℃将[125I]ANP与甲状腺细胞结合2小时启动ANP追踪研究，随后在37℃孵育。然后通过在三个部分（细胞表面膜、孵育培养基和细胞内）的不同时间间隔追踪125I活性来追踪ANP的加工过程。释放到培养基中的放射性代表加工后的ANP，而不是从表面膜解离的ANP，因为预结合的[125I]ANP在37℃不能被高浓度的ANP（1μmol/L）竞争性解离。追踪研究结果表明，预结合的ANP在30分钟内迅速从细胞膜上消失，降至34%。内化的ANP在10分钟时达到峰值，细胞内发现的初始预结合ANP的21%存在于细胞内。与此同时，孵育培养基中回收的放射性在10 - 30分钟内从8%急剧增加到52%。用氯喹（通过抑制溶酶体水解酶来阻断ANP - 受体复合物的降解）预孵育细胞导致30分钟内内化的[125I]ANP增加146%（与15%的对照相比为39%），而培养基放射性从52%降至16%，表明受体复合物的加工是通过溶酶体酶介导的。在使用氯喹预处理细胞的追踪研究中，TSH刺激了ANP - 受体复合物的内化速率。到30分钟时，TSH显著降低了膜结合的ANP，并且这种降低与内化放射性的增加呈负相关。在没有ANP配体的情况下，TSH也调节受体易位，导致表面膜上的受体数量减少。然而，基于免疫沉淀和高效液相色谱分析，TSH不影响ANP的降解。综上所述，这些数据表明TSH下调甲状腺细胞表面膜上的ANP受体，并且存在一条不通过受体结合介导的ANP降解途径。
