Functional expression and intracellular signaling of UTP-sensitive P2Y receptors in theca-interstitial cells

Reproductive Biology and Endocrinology

Research

Edith Garay¹  Francisco G Vázquez-Cuevas¹  Rogelio O Arellano¹  Erika P Zárate-Díaz¹ 

[1] Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México., Boulevard Juriquilla 3001, 76230, Juriquilla Querétaro, CP, México;

关键词: P2X7 Receptor;    Suramin;    cAMP Response Element Binding;    Purinergic Receptor;    Theca Cell;   

DOI  :  10.1186/1477-7827-8-88

 received in 2010-03-22, accepted in 2010-07-14,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundPurinergic receptors are expressed in the ovary of different species; their physiological roles remain to be elucidated. UTP-sensitive P2Y receptor activity may regulate cell proliferation. The aim of the present work was to study the functional expression of these receptors in theca/interstitial cells (TIC).MethodsTIC were isolated by centrifugation in a Percoll gradient. P2Y receptors and cellular markers in TIC were detected by RT-PCR and Western blot. Intracellular calcium mobilization induced by purinergic drugs was evaluated by fluorescence microscopy, phosphorylation of MAPK p44/p42 and of cAMP response element binding protein (CREB) was determined by Western blot and proliferation was quantified by [3H]-thymidine incorporation into DNA.ResultsRT-PCR showed expression of p2y2r and p2y6r transcripts, expression of the corresponding proteins was confirmed. UTP and UDP, agonists for P2Y2 and P2Y6 receptors, induced an intracellular calcium increase with a maximum of more than 400% and 200% of basal level, respectively. The response elicited by UTP had an EC50 of 3.5 +/- 1.01 μM, while that for UDP was 3.24 +/- 0.82 μM. To explore components of the pathway activated by these receptors, we evaluated the phosphorylation induced by UTP or UDP of MAPK p44 and p42. It was found that UTP increased MAPK phosphorylation by up to 550% with an EC50 of 3.34 +/- 0.92 and 1.41 +/- 0.67 μM, for p44 and p42, respectively; these increases were blocked by suramin. UDP also induced p44/p42 phosphorylation, but at high concentrations. Phosphorylation of p44/p42 was dependent on PKC and intracellular calcium. To explore possible roles of this pathway in cell physiology, cell proliferation and hCG-induced CREB-phosphorylation assays were performed; results showed that agonists increased cell proliferation and prevented CREB-phosphorylation.ConclusionHere, it is shown that UTP-sensitive P2Y receptors are expressed in cultured TIC and that these receptors had the ability to activate mitogenic signaling pathways and to promote cell proliferation, as well as to prevent CREB-phosphorylation by hCG. Regulation of TIC proliferation and steroidogenesis is relevant in ovarian pathophysiology since theca hyperplasia is involved in polycystic ovarian syndrome. Purinergic receptors described might represent an important new set of molecular therapeutic targets.

【 授权许可】

Unknown   
© Vázquez-Cuevas et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.