Effects of elevated cytoplasmic calcium and protein kinase C on endoplasmic reticulum structure and function in HEK293 cells.
In human embryonic kidney (HEK) cells stably transfected with green fluorescent protein targeted to the endoplasmic reticulum (ER), elevation of intracellular Ca2+ ([Ca2+]i) altered ER morphology, making it appear punctate. Electron microscopy revealed that these punctate structures represented circular and branched rearrangements of the endoplasmic reticulum, but did not involve obvious swelling or pathological fragmentation. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA), prevented the effects of ionomycin on ER structure without affecting the elevation of [Ca2+]i. These results suggest that protein kinase C activation alters cytoplasmic or ER components underlying the effects of high [Ca2+]i on ER structure. Treatment of HEK cells with PMA also reduced the size of the thapsigargin-sensitive Ca2+ pool and inhibited Ca2+ entry in response to thapsigargin. Thus, protein kinase C activation has multiple actions on the calcium storage and signalling function of the endoplasmic reticulum in HEK cells: (1) reduced intracellular Ca2+ storage capacity, (2) inhibition of capacitative Ca2+ entry, and (3) protection of the endoplasmic reticulum against the effects of high [Ca2+]i.
Ribeiro
C M
CM
National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709, USA.
McKay
R R
RR
Hosoki
E
E
Bird
G S
GS
Putney
J W
JW
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D003592:Cytophotometry; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007668:Kidney; D016210:Methacholine Chloride / Q000494:pharmacology; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D018721:Muscarinic Agonists / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007476:Ionophores; D018721:Muscarinic Agonists; D011993:Recombinant Fusion Proteins; D016210:Methacholine Chloride; D015759:Ionomycin; D019284:Thapsigargin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2