Endocrine Abstracts (2004) 7 P191

Protein kinase (PKA)-independent but cAMP-dependent regulation of progesterone secretion by luteinizing human granulosa cells

EC Chin & DRE Abayasekara

Department of Veterinary Basic Sciences, Royal Veternary College, London, UK.

The mammalian corpus luteum secretes progesterone under the influence of luteinizing hormone (LH). LH acts by binding to its G-protein coupled receptor and elevating intracellular cAMP thus activating protein kinase A (PKA). However, the exact role of PKA in the regulation of progesterone secretion remains unelucidated. Thus in this study we have investigated the relationship between PKA expression/activity and progesterone secretion during luteinization. Human granulosa cells were obtained from women undergoing oocyte retrieval for in vitro fertilization. All samples were obtained with informed patient consent (in accordance with the Declaration of Helsinki) and with the approval of the local ethics committee. Luteinizing human granulosa cells were isolated by percoll gradient centrifugation and cultured in serum-supplemented medium for up to 3 days. On each day of culture, cells were incubated in the presence or absence of hLH (100ng/ml) and assessed for PKA protein expression with spent media being assessed for progesterone (P4). Cells were also incubated in the presence or absence of hLH in the additional presence of increasing concentrations of two structurally dissimilar PKA inhibitors, H89 and myristoylated PKI. The results showed that hLH induced a dose-dependent increase in P4 secretion (Day 3: 252.1 plus/minus 26.9% vs control) coincident with a dose-dependent inhibition of PKA expression (Day 3: 66.7 plus/minus 4.1% vs control). This was also associated with an increase in expression of cAMP-guanine nucleotide exchange factor (GEF) I (Day 3, 128.8 plus/minus 8.9% vs control) the activation of which is cAMP-dependent. H89 and myristoylated PKI induced dose-dependent inhibition of both basal and hLH-induced progesterone secretion (H89, day 3: 62.1 plus/minus 3.8% vs control; PKI day 3: 74.1 plus/minus 2.6 % vs control). These results suggest that during luteinization progesterone secretion becomes progressively less dependent on PKA and thus may be regulated through cAMP-GEF I.

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