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Characterization of purinergic P2X4 receptor channels expressed in anterior pituitary cells.

ABSTRACT: Anterior pituitary cells express cation-conducting P2X receptor channels (P2XRs), but their molecular identity, electrophysiological properties, cell-specific expression pattern, and physiological roles have been only partially characterized. In this study, we show by quantitative RT-PCR that mRNA transcripts for the P2X(4) subunit are the most abundant in rat anterior pituitary tissue and confirm the P2X(4)R protein expression by Western blot analysis. Single-cell patch-clamp recordings show that extracellular ATP induced an inward depolarizing current in a majority of thyrotropin-releasing hormone-responsive pituitary cells, which resembled the current profile generated by recombinant P2X(4)R. The channels were activated and desensitized in a dose-dependent manner and deactivated rapidly. Activation of these channels led to stimulation of electrical activity and promotion of voltage-gated and voltage-insensitive Ca(2+) influx. In the presence of ivermectin, a specific allosteric modulator of P2X(4)Rs, there was an approximately fourfold increase in the maximum amplitude of the ATP-induced inward current, accompanied by an increase in the sensitivity of receptors for ATP, slowed deactivation of receptors, and enhanced ATP-induced prolactin release. These results indicate that thyrotropin-releasing hormone-responsive cells, including lactotrophs, express homomeric and/or heteromeric P2X(4)Rs, which facilitate Ca(2+) influx and hormone secretion.

SUBMITTER: Zemkova H

PROVIDER: S-EPMC2838522 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Characterization of purinergic P2X4 receptor channels expressed in anterior pituitary cells.

Zemkova Hana H Kucka Marek M Li Shuo S Gonzalez-Iglesias Arturo E AE Tomic Melanija M Stojilkovic Stanko S SS

American journal of physiology. Endocrinology and metabolism 20091215 3

Anterior pituitary cells express cation-conducting P2X receptor channels (P2XRs), but their molecular identity, electrophysiological properties, cell-specific expression pattern, and physiological roles have been only partially characterized. In this study, we show by quantitative RT-PCR that mRNA transcripts for the P2X(4) subunit are the most abundant in rat anterior pituitary tissue and confirm the P2X(4)R protein expression by Western blot analysis. Single-cell patch-clamp recordings show th ...[more]

PMID: 20009029

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Project description:Extracellular nucleotides have been recognized as important mediators of activation, triggering multiple responses via plasma membrane receptors known as P2 receptors. P2 receptors comprise P2X ionotropic receptors and G protein-coupled P2Y receptors. P2X receptors are expressed in many tissues, where they are involved in a number of functions including synaptic transmission, muscle contraction, platelet aggregation, inflammation, macrophage activation, differentiation and proliferation, neuropathic and inflammatory pain. P2X4 is one of the most sensitive purinergic receptors (at nanomolar ATP concentrations), about one thousand times more than the archetypal P2X7. P2X4 is widely expressed in central and peripheral neurons, in microglia, and also found in various epithelial tissues and endothelial cells. It localizes on the plasma membrane, but also in intracellular compartments. P2X4 is preferentially localized in lysosomes, where it is protected from proteolysis by its glycosylation. High ATP concentration in the lysosomes does not activate P2X4 at low pH; P2X4 gets activated by intra-lysosomal ATP only in its fully dissociated tetra-anionic form, when the pH increases to 7.4. Thus, P2X4 is functioning as a Ca2+-channel after the fusion of late endosomes and lysosomes. P2X4 modulates major neurotransmitter systems and regulates alcohol-induced responses in microglia. P2X4 is one of the key receptors mediating neuropathic pain. However, injury-induced upregulation of P2X4 expression is gender dependent and plays a key role in pain difference between males and females. P2X4 is also involved in inflammation. Extracellular ATP being a pro-inflammatory molecule, P2X4 can trigger inflammation in response to high ATP release. It is therefore involved in multiple pathologies, like post-ischemic inflammation, rheumatoid arthritis, airways inflammation in asthma, neurodegenerative diseases and even metabolic syndrome. Although P2X4 remains poorly characterized, more studies are needed as it is likely to be a potential therapeutic target in these multiple pathologies.

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Project description:Stroke remains a leading cause of disability in the United States. Despite recent advances, interventions to reduce damage and enhance recovery after stroke are lacking. P2X4R, a receptor for adenosine triphosphate (ATP), regulates activation of myeloid immune cells (infiltrating monocytes/macrophages and brain-resident microglia) after stroke injury. However, over-stimulation of P2X4Rs due to excessive ATP release from dying or damaged neuronal cells can contribute to ischemic injury. Therefore, we pharmacologically inhibited P2X4R to limit the over-stimulated myeloid cell immune response and improve both acute and chronic stroke recovery. We subjected 8-12-week-old male and female wild type mice to a 60 min right middle cerebral artery occlusion (MCAo) followed by 3 or 30 days of reperfusion. We performed histological, RNA sequencing, behavioral (sensorimotor, anxiety, and depressive), and biochemical (Evans blue dye extravasation, western blot, quantitative PCR, and flow cytometry) analyses to determine the acute (3 days after MCAo) and chronic (30 days after MCAo) effects of P2X4R antagonist 5-BDBD (1 mg/kg P.O. daily x 3 days post 4 h of MCAo) treatment. 5-BDBD treatment significantly (p < .05) reduced infarct volume, neurological deficit (ND) score, levels of cytokine interleukin-1 beta (IL-1β) and blood brain barrier (BBB) permeability in the 3-day group. Chronically, 5-BDBD treatment also conferred progressive recovery (p < .05) of motor balance and coordination using a rotarod test, as well as reduced anxiety-like behavior over 30 days. Interestingly, depressive-type behavior was not observed in mice treated with 5-BDBD for 3 days. In addition, flow cytometric analysis revealed that 5-BDBD treatment decreased the total number of infiltrated leukocytes, and among those infiltrated leukocytes, pro-inflammatory cells of myeloid origin were specifically reduced. 5-BDBD treatment reduced the cell surface expression of P2X4R in flow cytometry-sorted monocytes and microglia without reducing the total P2X4R level in brain tissue. In summary, acute P2X4R inhibition protects against ischemic injury at both acute and chronic time-points after stroke. Reduced numbers of infiltrating pro-inflammatory myeloid cells, decreased surface P2X4R expression, and reduced BBB disruption are likely its mechanism of neuroprotection and neuro-rehabilitation.

Dataset Information

Characterization of purinergic P2X4 receptor channels expressed in anterior pituitary cells.

Publications

Characterization of purinergic P2X4 receptor channels expressed in anterior pituitary cells.

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