Luteinizing hormone increases inositol trisphosphate and cytosolic free Ca2+ in isolated bovine luteal cells

Abstract

The present studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases intracellular cAMP, also increases "second messengers" derived from inositol phospholipid hydrolysis in isolated bovine luteal cells. In luteal cells prelabeled with 32PO4, LH provoked increases in labeling of phosphatidic acid, phosphatidylinositol, and polyphosphatidylinositol (PIP). No reductions in 32P-prelabeled PIP and PIP2 were observed in LH-treated cells. In luteal cells prelabeled with myo-[2-3H]inositol, LH provoked rapid (10-30 s) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, and IP3, respectively. IP3 was formed more rapidly than IP2 or IP following LH treatment. In addition, LH increased (50%) levels of [3H]inositol phospholipids in 30-min incubations. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH. Maximal increases in IP3 occurred at 1-10 micrograms/ml of LH. Similar temporal and dose-response relationships were observed for LH-stimulated IP3 and cAMP accumulation. However, exogenous cAMP (8-bromo-cAMP, 5 mM) and forskolin (10 microM) had no effect on inositol phosphate synthesis. The initial (1 min) effects of LH on IP3 and cAMP were independent of extracellular calcium concentrations, whereas the sustained (5 min) effect of LH on IP3, but not cAMP, was dependent on a source of extracellular calcium. LH-stimulated progesterone synthesis was also dependent on the presence of extracellular calcium. LH induced rapid and concentration-dependent increases in [Ca2+]i as measured by Quin 2 fluorescence. The LH-induced increases in [Ca2+]i were maximal within 30 s (approximately 2-fold) and remained elevated for at least 10 min. In Ca2+-free media containing 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, LH was still able to increase [Ca2+]i, but the increase was slightly less in magnitude and of shorter duration (2-4 min). These findings demonstrate that LH can rapidly raise levels of IP3 and [Ca2+]i, as well as, cAMP in bovine luteal cells. These findings suggest that at least two second messenger systems exist to mediate the action of LH in the corpus luteum.