AJP - Renal Physiology, Vol 258, Issue 4 963-F972, Copyright © 1990 by American Physiological Society
Oxytocin receptors from LLC-PK1 cells: expression in Xenopus oocytes
B. Cantau, J. N. Barjon, D. Chicot, P. P. Baskevitch and S. Jard
Centre National de la Recherche Scientifique, Institut National de la Sante et de la Recherche Medicale de Pharmacologie-Endocrinologie, Montpellier, France.
Two selective radioligands for oxytocin receptors,
[3H]-[4-threonine,7-glycine]oxytocin [( 3H]-[Thr4,Gly7]OT) and
125I-[1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),
2-(O-methyl)tyrosine, 4-threonine, 8-ornithine, 9-tyrosine amide]-oxytocin
(125I-OTA), were used to characterize oxytocin receptors from two pig
kidney-derived cell lines, LLC-PK1 and LLC-PK1L. [3H]-[Thr4,Gly7]OT and
125I-OTA bind with high affinity (mean Kd values of 14 and 0.06 nM,
respectively) to the same population of sites on LLC-PK1 cell membranes
[maximum binding (Bmax) of 100 fmol/mg membrane protein]. These sites had
the expected ligand selectivity of oxytocin receptors. [3H]-[Thr4,Gly7]OT
and 125I-OTA binding sites could be distinguished from V2 vasopressin
receptors present on LLC-PK1 and LLC-PK1L cells on the basis of clearly
different maximal capacities and ligand selectivities, different
sensitivities to insulin and serum, and absence of heterologous
downregulation. Oxytocin receptors from LLC-PK1 cells have no functional
relationship with adenylate cyclase. [Thr4,Gly7]OT affected neither the
basal adenosine 3',5'-cyclic monophosphate (cAMP) content nor the
vasopressin-induced cAMP accumulation by LLC-PK1 cells. Xenopus laevis
oocytes injected with LLC-PK1 cell mRNA responded to [Thr4,Gly7]OT by an
increase in 45Ca2+ outflux; this effect is antagonized by a highly
selective oxytocin antagonist.
