Control of cyclic AMP concentration in bovine endometrial stromal cells by arachidonic acid

Z Cheng,E Marshall,E L Sheldrick,D C Wathes,D R E Abayasekara,A P F Flint

doi:10.1530/rep-06-0220

Abstract

Second messenger signalling through cyclic AMP (cAMP) plays an important role in the response of the endometrium to prostaglandin (PG) E(2) during early pregnancy. Arachidonic acid, which is a by-product of the luteolytic cascade in ruminants, is a potential paracrine signal from the epithelium to the stroma. We investigated the effects of arachidonic acid on the response of the stroma to PGE(2). cAMP was measured in bovine endometrial stromal cells treated with agents known to activate or inhibit adenylyl cyclase, protein kinase C (PKC) or phosphodiesterase (PDE). PGE(2) increased the intracellular cAMP concentration within 10 min, and this effect was attenuated by arachidonic acid and the PKC activator, 4beta-phorbol myristate acetate (PMA). The inhibitory effect of arachidonic acid on PGE(2)-induced cAMP accumulation was prevented by the PKC inhibitor, RO318425, and was absent in cells in which PKC had been downregulated by exposure to PMA for 24 h. The effect of arachidonic acid was also prevented by the PDE inhibitor, 3-isobutyl-1-methylxanthine. Arachidonic acid was shown by immunoblotting to prevent induction of cyclooxygenase-2 by PGE(2), forskolin or dibutyryl cAMP. The results indicate that arachidonic acid activates PDE through a mechanism involving PKC, counteracting a rise in intracellular cAMP in response to PGE(2). The data suggest that arachidonic acid antagonizes PGE(2) signalling through cAMP in the bovine endometrium, possibly acting to ensure a rapid return to oestrus in the case of failure of the maternal recognition of pregnancy.

Highlights

Intracellular signalling through adenylyl cyclase and 30,50-cyclic AMP plays an important role in the preparation of the uterus for implantation
Bovine uterine stromal (BST) cells isolated from one uterine horn of a non-pregnant cyclic cow on day 16 post-oestrus (Flint et al 2002) were maintained in Dulbecco’s modified Eagles medium (DMEM; Sigma) with 1% antibiotic– antimycotic (ABAM; Sigma) and 10% foetal bovine serum at 37 8C, 95% humidityand 5% CO2.They were passaged at intervals of 3–4 days when about 80% confluent
To confirm that PGE2 activated adenylyl cyclase in BST cells, cyclic AMP (cAMP) was measured in cells exposed to PGE2 for varying times

Summary

Introduction

Intracellular signalling through adenylyl cyclase and 30,50-cyclic AMP (cAMP) plays an important role in the preparation of the uterus for implantation. The role of PGE2 in activating adenylyl cyclase at implantation has been well defined in species with endometrial decidualization. Rat, rabbit and man, PGE2 is involved in endometrial angiogenesis (Jabbour & Sales 2004), vasodilatation and vascular permeability (Kennedy 1983), stromal cell prolactin expression (Frank et al 1994) and decidualization. These processes involve signalling through the PGE2 receptors EP2 and EP4, which are linked to adenylyl cyclase (Fujino et al 2005) and are expressed by endometrial epithelial and stromal cells

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Results

Conclusion