Expression of vasopressin receptors (V2-subtype) on LLC-PK1 cells during cell culture.
Vasopressin receptor expression on LLC-PK1-cells (a porcine renal tubular cell line) during cell culture is still not fully understood. We studied receptor expression using a novel vasopressin analogue with high specific radioactivity ([125I][8-p-hydroxy-phenylpropionyl]-lys8-vasopressin, 74EBq/mol (2000 Ci/mmol)). LLC-PK1 cells were grown in monolayers for 1 to 6 days. Scatchard analysis performed with membranes of LLC-PK1 cells revealed a single binding site with a binding constant (Kd) of 0.46 +/- 0.04 nmol/l. During cell culture, the binding constant (Kd) was not altered, but receptor density increased significantly (21,115 +/- 645 receptors per cell, day 2; 42,315 +/- 1512 receptors per cell, day 6). A receptor occupancy of about 30% was found to be associated with a cAMP stimulation of 50%. The receptor reserve might be even higher because, by using a highly specific oxytocin antagonist, we found that 20% of the occupied [125I][8-p-hydroxy-phenylpropionyl]-lys8-vasopressin-binding sites are oxytocin receptors. For lys8-vasopressin receptor studies, great care has to be taken to examine cells in identical culture phases.
Ehnis
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Abteilung für Innere Medizin, Klinikum Steglitz, Freie Universität Berlin, Germany.
Hocher
B
B
Abou-Rebyeh
H
H
Oelkers
W
W
Hensen
J
J
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D008236:Lypressin / Q000031:analogs & derivatives / Q000378:metabolism; D017483:Receptors, Vasopressin / Q000378:metabolism*; D013552:Swine
D017483:Receptors, Vasopressin; C082916:vasopressin, 8-(4-hydroxyphenylpropionyl)-Lys(8)-; D008236:Lypressin; D000242:Cyclic AMP