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This Article Full Text Full Text (PDF) All Versions of this Article: 285/6/C1367    most recent 00217.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (21) Citing Articles via Google Scholar Google Scholar Articles by Erfurt, C. Articles by Thévenod, F. Search for Related Content PubMed PubMed Citation Articles by Erfurt, C. Articles by Thévenod, F.

PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Apoptosis by Cd²⁺ or CdMT in proximal tubule cells: different uptake routes and permissive role of endo/lysosomal CdMT uptake

¹Department of Physiology, University of Saarland, D-66421 Homburg; ²Department of Neuroanatomy, University of Göttingen, D-37075 Göttingen; and ³Department of Physiology & Pathophysiology, University of Witten/Herdecke, D-58448 Witten, Germany

Submitted 27 May 2003 ; accepted in final form 21 July 2003

The mechanisms of cadmium-metallothionein (CdMT) uptake and toxicity in proximal tubule (PT) cells are not well understood. The effects of 10 µM CdCl₂ or Cd₇MT-1 (MT-1 saturated with 10 µM CdCl₂) on ¹⁰⁹Cd²⁺ uptake, viability, and MT levels of cultured rat PT cells were investigated. Apical ¹⁰⁹Cd²⁺ uptake was measured in confluent monolayers, apoptosis was assessed with Hoechst 33342, and intracellular MT levels were monitored by immunofluorescence and quantitative morphometry. ¹⁰⁹Cd²⁺ uptake into PTC increased over time and plateaued at 24 h. ¹⁰⁹Cd₇MT-1 uptake was delayed but reached a similar magnitude after 40 h. With Cd²⁺, apoptosis occurred within 4 h, peaked at 24 h, and declined at 48-72 h. Cd₇MT-1 induced apoptosis after 24-36 h, reaching similar levels as with Cd²⁺ after 48 h. Cd²⁺ and Cd₇MT-1 significantly increased intracellular MT immunoreactivity after 20 and 4 h, respectively. The weak base chloroquine and the inhibitor of phosphatidylinositol 3-kinases, LY-294002, selectively inhibited the effects of Cd₇MT-1 on MT immunoreactivity and apoptosis. PT cells accumulated ¹⁰⁹Cd₇MT-1 in membrane vesicles associated with the late endo/lysosomal marker LAMP1 but less with the early endosomal marker Rab5a, which was abolished by chloroquine or LY-294002. Thus development of apoptosis followed the uptake kinetics of Cd²⁺ and Cd₇MT-1. Endo/lysosomal inhibitors prevented uptake of Cd₇MT-1 into endo/lysosomes and apoptosis but had no effect on these parameters with Cd²⁺, suggesting that apoptosis of PT cells is triggered by free cytosolic Cd²⁺, either by direct apical transport or by translocation of free Cd²⁺ from endo/lysosomes after endocytosis of Cd₇MT-1.

trafficking; endocytosis; necrosis; chloroquine; LY-294002

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