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.