Expression of vasopressin receptors (V2-subtype) on LLC-PK1 cells during cell culture.

Vasopressin receptor expression on LLC-PKrcells (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 ([I][8-/?-hydroxy-phenylpropionyl]-lys-vasopressin, 74EBq/mol (2000 Ci/mmol)). cells were grown in monolayers for 1 to 6 days. Scatchard analysis performed with membranes of cells revealed a single binding site with a binding constant (Kd) of 0.46 ± 0.04 nmol/1. During cell culture, the binding constant (Kd) was not altered, but receptor density increased significantly (21 115 ± 645 receptors per cell, day 2; 42315 ± 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 [I][8-/?-hydroxyphenylpropionyl]-lys-vasopressin-binding sites are oxytocin receptors. For Iys-vasopressin receptor studies, great care has to be taken to examine cells in identical culture phases.