Ca2+ release and InsP3 production in avian uterine cells: effects of PGF2 alpha and AVT

Abstract

Primary cultures of smooth muscle cells isolated from the shell gland ("uterus") of the domestic hen were permeabilized with digitonin and loaded with 45Ca2+ in the presence of ATP. When these cells were stimulated with prostaglandin F2 alpha (PGF2 alpha), arginine vasotocin (AVT), or D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], there was a rapid, biphasic, and dose-related release of 45Ca2+ from nonmitochondrial pools. 2-Nitro-4-carboxyphenyl-N,N-diphenylcarbamate, an inhibitor of phospholipase C, had no effect on PGF2 alpha - and Ins(1,4,5)P3-promoted 45Ca2+ efflux, whereas it significantly inhibited AVT-stimulated and a stable analogue of GTP-stimulated 45Ca2+ release. In fura-2-loaded intact cells, both PGF2 alpha and AVT increased intracellular Ca2+ levels [( Ca2+]i) in a dose-related manner in the presence of extracellular Ca2+. However, omission of extracellular Ca2+ prevented a PGF2 alpha, but not AVT-induced, rise in [Ca2+]i In D-myo-[3H]inositol-labeled cells, 10 nM AVT caused a rapid, two- to threefold increase in [3H]-Insp3, whereas PGF2 alpha up to 1 microM was infective. Raising PGF2 alpha to 10 microM increased total inositol phosphates by 22% over controls (P less than 0.05). These results point to marked differences in the mechanisms by which AVT and PGF2 alpha regulate [Ca2+]i in uterine smooth muscle cells. It is suggested that the two agonists act in concert to initiate oviposition.