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

<p>Abstract</p> <p>Background</p> <p>Purinergic 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).</p> <p>Methods</p> <p>TIC 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.</p> <p>Results</p> <p>RT-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.</p> <p>Conclusion</p> <p>Here, 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.</p

Granulosa cells express three inositol 1,4,5-trisphosphate receptor isoforms: cytoplasmic and nuclear Ca2+ mobilization

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P2X7 Receptors as a Therapeutic Target in Cerebrovascular Diseases

Shortage of oxygen and nutrients in the brain induces the release of glutamate and ATP that can cause excitotoxicity and contribute to neuronal and glial damage. Our understanding of the mechanisms of ATP release and toxicity in cerebrovascular diseases is incomplete. This review aims at summarizing current knowledge about the participation of key elements in the ATP-mediated deleterious effects in these pathologies. This includes pannexin-1 hemichannels, calcium homeostasis modulator-1 (CALHM1), purinergic P2X7 receptors, and other intermediaries of CNS injury downstream of ATP release. Available data together with recent pharmacological developments in purinergic signaling may constitute a new opportunity to translate preclinical findings into more effective therapies in cerebrovascular diseases.This study was supported by grants from CONACYT-Mexico No. 252121 and PAPIITUNAM-Mexico No. IN203519 to ROA laboratory; by Spanish Ministry of Education and Science/FEDER (SAF2016-75292-R), Basque Government (IT1203/19), CIBERNED, Eranet-Neuron and Universidad del Pais Vasco to CM's laboratory. AC-M is a researcher from Catedras-CONACYT commissioned at Instituto de Neurobiologia at Universidad Nacional Autonoma de Mexico (UNAM)

Expression and Function of GABA Receptors in Myelinating Cells

Myelin facilitates the fast transmission of nerve impulses and provides metabolic support to axons. Differentiation of oligodendrocyte progenitor cells (OPCs) and Schwann cell (SC) precursors is critical for myelination during development and myelin repair in demyelinating disorders. Myelination is tightly controlled by neuron-glia communication and requires the participation of a wide repertoire of signals, including neurotransmitters such as glutamate, ATP, adenosine, or gamma-aminobutyric acid (GABA). GABA is the main inhibitory neurotransmitter in the central nervous system (CNS) and it is also present in the peripheral nervous system (PNS). The composition and function of GABA receptors (GABARs) are well studied in neurons, while their nature and role in glial cells are still incipient. Recent studies demonstrate that GABA-mediated signaling mechanisms play relevant roles in OPC and SC precursor development and function, and stand out the implication of GABARs in oligodendrocyte (OL) and SC maturation and myelination. In this review, we highlight the evidence supporting the novel role of GABA with an emphasis on the molecular identity of the receptors expressed in these glial cells and the possible signaling pathways involved in their actions. GABAergic signaling in myelinating cells may have potential implications for developing novel reparative therapies in demyelinating diseases.This work was supported by CIBERNED (CB06/05/0076; CM) and by grants from the Ministry of Economy and Competitiveness, Government of Spain (SAF2016-75292-R and PID2019-109724RB-I00; CM), Basque Government (IT1203-19; CM), CONACYT-Mexico (No. 252121; RA), PAPIIT-UNAM-Mexico (IN203519; RA) and NIH (R21AG053740 and R21MH113177; AL). MS-R was hired thanks to the Gangoiti Foundation (Bilbao). LB-C and RO hold fellowships from Basque Government and CONACYT-Mexico, respectively

Activation of volume-regulated Cl− channels by ACh and ATP in Xenopus follicles

Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique.In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application.In follicles, the permeability ratios for different anions with respect to Cl− were similar for both ICl,swell and Sin, with a sequence of: SCN− > I− > Br−≥ NO3−≥ Cl− > gluconate ≥ cyclamate > acetate > SO42−.Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was ∼100% slower at pH 8.0 compared with that at pH 6.0.Lanthanides inhibited ICl,swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 ± 1.9 μm, while Sin was blocked up to 55% with an apparent IC50 of 36 ± 2.6 μm.Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 ± 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to −20 mV. This single-channel activity was increased by application of ACh or ATP.The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling.All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity, strongly suggest that ICl,swell and Sin currents depend on the opening of the same type or a closely related class of volume-regulated chloride channels