Activation of phospholipase A2 and phospholipase C endothelin-1 in human endometrium A.
Ahmed, I. T. Cameron, R. A. Ferriani and S. K. Smith
Department of Obstetrics and Gynaecology, University of Cambridge, Robinson Way, Cambridge CB2 2sw, U.K. REVISED MANUSCRIPT RECEIVED
4 June 1992
Human endometrium contains specific binding sites for iodinated endothelin (ET)-1, ET-2 and ET-3, and ET-1 stimulates prostaglandin (PG) F2\g=a\ synthesis from explants of proliferative endometrium in short\x=req-\ term culture. This study has investigated the cellular responses of normal proliferative endometrium to ET\x=req-\ 1. Radioimmunoassay was used to measure PG release and Dowex anion-exchange column chromatography was utilized to assess the accumulation of inositol phosphates. Endothelin-1 induced a significant increase in PGF2\g=a\ release (basal median: 1465 pg/mg per 60 min (range: 541\p=n-\3935 pg/mg 1813 pg/mg per per 60 min); ET-1-stimulated: 60 min (1021\p=n-\5714 pg/mg per 60 min); P < 0\m=.\04 using Wilcoxon signed rank test). The effect of ET-1 was attenuated in the presence of the phospholipase
A2 inhibitor quinacrine. Endothelin-1
rapid, hydrolysis a
induced transient and concentration-dependent of phosphatidylinositol 4,5-bisphosphate
Calcium-mobilizing agonists transmit their intracel¬ lular messages by binding to specific receptors on the cell surface. The ligand-bound receptors activate effector systems including phospholipase A2, phos¬ pholipase C and phospholipase D via a receptorcoupled G protein, to generate second messengers. The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by the PtdIns(4,5)P2-specific phospholipase C yields two second messengers, 1,2diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) (Berridge & Irvine, 1989). Ins(l,4,5)P3 stimulates the release of stored intracellu¬ lar calcium ([Ca2+]¡) (Berridge & Irvine, 1989), whilst DAG, in concert with Ca2+, activates the phosphatidylserine-dependent protein kinase C (Nishizuka, 1989).
(PtdIns(4,5)P2), measured by the accumulation of tritiated inositol phosphates. Following a 1-min stimulation with ET-1 (100 nmol/l), [3H]inositol mono-, bisand trisphosphate fractions increased from median values of 490\m=.\0 d.p.m./mg dry wt (range: 348\m=.\0\p=n-\ 807\m=.\0 d.p.m./mg dry wt), 120\m=.\0 d.p.m./mg dry wt (93\m=.\6\p=n-\144\m=.\1 d.p.m./mg dry wt) and 67\m=.\0 d.p.m./mg
dry wt (54\m=.\2\p=n-\85\m=.\0 d.p.m./mg dry wt) to 939\m=.\0 d.p.m./ mg dry wt (635\m=.\9\p=n-\1596\m=.\0 d.p.m./mg dry wt; P 0\m=.\03), 145\m=.\0 d.p.m./mg dry wt (127\m=.\0\p=n-\ 293\m=.\9 d.p.m./mg dry wt; P < 0\m=.\05) and 146\m=.\0 d.p.m./ mg dry wt (77\m=.\5\p=n-\187\m=.\0 d.p.m./mg dry wt; P 0\m=.\03) respectively. These results suggest that ET-1 activates the phospholipase A2 and PtdIns(4,5)P2-specific phospholipase C in human proliferative endometrium, resulting in the generation of PGF2\g=a\and second messengers respectively which are pivotal to endometrial