Down-regulation of cell surface insulin receptors by sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor in adrenal chromaffin cells.
Long-term (> or =12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), caused a time (t(1/2)=16.3 h)- and concentration (IC50=37.8 nM)-dependent decrease of cell surface 125I-insulin binding by 35%, but did not change the Kd value. TG caused a sustained increase of cytoplasmic concentration of Ca2+ ([Ca2+]c) in a biphasic manner, and the effect of TG on 125I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) beta-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca2+]c down-regulates cell surface IR by retarding externalization of IR from the TGN.
Shiraishi
S
S
Department of Pharmacology, Miyazaki Medical College, 889-1692, Miyazaki, Japan.
Yamamoto
R
R
Yanagita
T
T
Yokoo
H
H
Kobayashi
H
H
Uezono
Y
Y
Wada
A
A
D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D019439:Chromaffin Cells / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism; D009994:Osmolar Concentration; D011972:Receptor, Insulin / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D019284:Thapsigargin / Q000494:pharmacology*; D013997:Time Factors
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007328:Insulin; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D020126:Brefeldin A; D004533:Egtazic Acid; D019284:Thapsigargin; D011972:Receptor, Insulin; D000252:Calcium-Transporting ATPases; D002118:Calcium