Actions of endothelin-1 on calcium homeostasis in Madin-Darby canine kidney tubule cells.
BACKGROUND
In both ischaemic and nephrotoxic models, renal failure is associated with increased endothelin-1 (ET-1) and cell calcium overload, and ET receptor antagonists are protective. Vascular and tubular actions of endothelins appear to be involved. This study examines the actions of ET-1 on intracellular Ca ([Ca2+]i) in the tubule model cell line MDCK (Madin-Darby canine kidney).


METHODS
Single-cell [Ca2+]i was measured using fura-2 and actions of ET-1 were compared with thapsigargin, which empties IP3-sensitive intracellular Ca stores.


RESULTS
Mean resting [Ca2+]i was 84 nM (s.e.m. 6, n = 87). 1 microM thapsigargin and 100 nM ET-1 each caused a transient increase in [Ca2+]i by 696 nM (s.e.m. 160, n = 9) and 727 nM (s.e.m. 121, n = 5) respectively. After 1 microM thapsigargin, 100 nM ET-1 had no effect on [Ca2+]i. Oscillations in [Ca2+]i were frequently observed following 100 nM ET-1. In Ca(2+)-free extracellular solution, mean resting [Ca2+]i was reduced by 37 nM (s.e.m. 5, n = 11) and the mean transient increase in [Ca2+]i in response to ET-1 was 419 nM (s.e.m. 97, n = 5). Inhibition of the plasma membrane Ca-ATPase with La3+ halved the rate of [Ca2+]i removal from the cytoplasm following ET-1. The PKC inhibitor, chelerythrine (1 microM), reduced the ET-1 induced increase in [Ca2+]i to 349 nM (s.e.m. 97, n = 5) and also reduced the rate of removal of [Ca2+]i. Ligand binding studies demonstrated ETA receptor expression in MDCK cells sensitive to ET-1.


CONCLUSIONS
ET-1 releases Ca2+ from IP3-sensitive stores in MDCK cells as well as stimulating extracellular Ca2+ entry leading to oscillations of [Ca2+]i. Ca2+ responses to ET-1 are potentiated by PKC; the plasma membrane Ca-ATPase contributes to removal of Ca2+ from the cytoplasm.
Parkinson
N A
NA
Department of Medicine, Kings College School of Medicine & Dentistry, London, UK.
James
A F
AF
Hendry
B M
BM
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D019332:Endothelin-1 / Q000494:pharmacology*; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D016257:Fura-2; D006706:Homeostasis / Q000187:drug effects*; D007424:Intracellular Fluid / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D019332:Endothelin-1; D005456:Fluorescent Dyes; D012965:Sodium Chloride; D019284:Thapsigargin; D011493:Protein Kinase C; D002118:Calcium; D016257:Fura-2