Nephrol Dial Transplant (1996) 11: 1532-1537
© 1996 European Renal Association-European Dialysis and Transplant Association
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Actions of endothelin-1 on calcium homeostasis in Madin—Darby canine kidney tubule cells
Renal Group, Department of Medicine, Kings College School of Medicine & Dentistry London, UK
Correspondence and offprint requests to: Correspondence and offprint requests to: BM Hendry, Renal Group, Department of Medicine, Kings College School of Medicine & Dentistry, Bessemer Road, London SE5 9PJ, UK
BACKGROUND.: In both ischaemic and nephrotoxic models, renal failure is associated with increased endothelin-l (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 µM 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 µM thapsigargin, 100 nM ET-1 had no effect on [Ca2+]i Oscillations in [Ca2+]i were frequently observed following 100 nM ET-1. In Ca2+-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 µM), 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.
Keywords: calcium; endothelin-1; epithelial cells; fura-2; MDCK; protein kinase C
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