Abstract
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.
Highlights
Purinergic receptors are expressed in the ovary of different species; their physiological roles remain to be elucidated
We found that uridine-sensitive P2Y2 and P2Y6 receptors are expressed in the theca/interstitial cells (TIC) membrane and that P2Y activation promoted three important responses in these cells: 1) elicited Ca2+ mobilization from intracellular reservoirs, increasing the concentration of this important second messenger in the cytoplasm; 2) increased cell proliferation through a mechanism dependent on the activation of protein kinase C (PKC) as well as MAPK p44 and p42, and; 3) down regulated hCG-dependent phosphorylation of cAMP response element binding protein (CREB), an important element in steroidogenesis cascade control
Theca cell identity and expression of P2Y2, P2Y4, and P2Y6 receptors TIC were isolated, and their identity was confirmed by RT-PCR amplification of cyp11A, cyp17A, and star transcripts as specific markers for theca cells, and of follicle stimulating hormone (FSH) receptor transcripts as indicator of a possible contamination with granulosa cells; the β-actin transcript was used as a control housekeeping gene (Figure 1A)
Summary
Purinergic receptors are expressed in the ovary of different species; their physiological roles remain to be elucidated. The main physiological roles recognized for theca cells are the initial steps in the steroidogenic process; these cells convert acetate or cholesterol to androgens [1], which are secreted into the intra-follicular medium and Several studies in recent years indicate that the purinergic signaling system is functionally expressed in the ovary of several species [3] and represents another regulatory element in ovarian physiology; the physiological role of ATP in this context and its membrane receptors is unknown. The ovary is innervated by sympathetic terminals through the superior ovarian nerve and ovarian plexus [2,4] It has been shown in other tissues that ATP is co-released with noradrenaline by sympathetic terminals and that it participates in several physiological events such as the induction and regulation of smooth muscle contraction [5] and the modulation of cardiac muscle excitation [6]. Several cell types are able to release ATP in a basal manner and/or in response to different stimuli, such as mechanical stimulation, changes in pH, or hypotonic stress [7,8,9]
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