title: Oxytocin induces a transient increase in cytosolic free [Ca2+] in renal tubular epithelial cells: evidence for oxytocin receptors on LLC-PK1 cells.
abstract: We examined the effects of oxytocin on renal tubular epithelial LLC-PK1 cells. In cells loaded with Fura 2, we found that 1 microM oxytocin induced a rapid increase in cytosolic free [Ca2+]i from 120 nM to 250 nM within 12 sec. [Ca2+]i then decreased and leveled at 148 nM. Calcium was mobilized from intra- and extra-cellular sources. Oxytocin-induced calcium mobilization was dose dependent (EC50 between 5 and 30 nM). Oxytocin also stimulated calcium efflux which was blocked by the selective oxytocin antagonist KB-5-21. Calcium mobilization was a likely consequence of enhanced phosphatidylinositol turnover, because oxytocin rapidly increased the formation of inositol phosphates including Ins1,4,5P3. Calcium transients were induced by oxytocin and the oxytocin selective analog AM-2-40 and blocked by the oxytocin-selective antagonist KB-5-21. Lysine vasopressin, the selective V2 agonist dDAVP, and the V1-selective agonist SK&F 105349 were at least 10- to 100-fold less potent than oxytocin and exhibited only partial agonist activity. Using peptide analogs, a poor correlation was found between antagonism of oxytocin-induced calcium transients of LLC-PK1 cells and pig kidney V2 and rat liver V1 receptor affinity. These data indicate that oxytocin-induced calcium transients in LLC-PK1 cells were not mediated by V1 or V2 vasopressin receptors, but by oxytocin receptors. However, the poor correlation between antagonism at the LLC-PK1 receptors and the rat uterus oxytocin receptors suggests marked differences in antagonist recognition. We have also identified specific, saturable, high affinity oxytocin-binding sites of low density on intact LLC-PK1 cells (KD = 1.9 nM; Bmax = 3.2 fmol/10(6) cells). The relative analog affinities for these binding sites correlated well with their effects on oxytocin-induced calcium transients. We conclude that in LLC-PK1 cells, oxytocin stimulates a transient rise in cytosolic free [Ca2+]i and the formation of inositol phosphates, including Ins1,4,5P3. The effects on [Ca2+]i probably are not mediated by V1 and V2 vasopressin receptors, but by putative oxytocin receptors.
authors.affiliation: Department of Molecular Pharmacology, Smith Kline & French Laboratories, Philadelphia, Pennsylvania 19101.
authors.forename: F L
authors.initials: FL
authors.lastname: Stassen
authors.forename: G
authors.initials: G
authors.lastname: Heckman
authors.forename: D
authors.initials: D
authors.lastname: Schmidt
authors.forename: M T
authors.initials: MT
authors.lastname: Papadopoulos
authors.forename: P
authors.initials: P
authors.lastname: Nambi
authors.forename: H
authors.initials: H
authors.lastname: Sarau
authors.forename: N
authors.initials: N
authors.lastname: Aiyar
authors.forename: M
authors.initials: M
authors.lastname: Gellai
authors.forename: L
authors.initials: L
authors.lastname: Kinter
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000032:analysis; D004848:Epithelium / Q000032:analysis; D007295:Inositol Phosphates / Q000378:metabolism; D007684:Kidney Tubules / Q000032:analysis / Q000187:drug effects*; D008236:Lypressin / Q000494:pharmacology; D010121:Oxytocin / Q000494:pharmacology*; D051381:Rats; D011945:Receptors, Angiotensin / Q000032:analysis* / Q000187:drug effects / Q000502:physiology; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin
chemical_list: D007295:Inositol Phosphates; D011945:Receptors, Angiotensin; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin; D010121:Oxytocin; D008236:Lypressin; D002118:Calcium