Ca2+ waves coordinate purinergic receptor–evoked integrin activation and polarization

Cells sense extracellular nucleotides through the P2Y class of purinergic G protein–coupled receptors (GPCRs), which stimulate integrin activation through signaling events, including intracellular Ca2+ mobilization. We investigated the relationship between P2Y-stimulated repetitive Ca2+ waves and fibrinogen binding to the platelet integrin αIIbβ3 (GPIIb/IIIa) through confocal fluorescence imaging of primary rat megakaryocytes. Costimulation of the receptors P2Y1 and P2Y12 generated a series of Ca2+ transients that each induced a rapid, discrete increase in fibrinogen binding. The peak and net increase of individual fibrinogen binding events correlated with the Ca2+ transient amplitude and frequency, respectively. Using BAPTA loading and selective receptor antagonists, we found that Ca2+ mobilization downstream of P2Y1 was essential for ADP-evoked fibrinogen binding, whereas P2Y12 and the kinase PI3K were also required for αIIbβ3 activation and enhanced the number of Ca2+ transients. ADP-evoked fibrinogen binding was initially uniform over the cell periphery but subsequently redistributed with a polarity that correlated with the direction of the Ca2+ waves. Polarization of αIIbβ3 may be mediated by the actin cytoskeleton, because surface-bound fibrinogen is highly immobile, and its motility was enhanced by cytoskeletal disruption. In conclusion, spatial and temporal patterns of Ca2+ increase enable fine control of αIIbβ3 activation after cellular stimulation. P2Y1-stimulated Ca2+ transients coupled to αIIbβ3 activation only in the context of P2Y12 coactivation, thereby providing an additional temporal mechanism of synergy between these Gq- and Gi-coupled GPCRs.

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Caveolin-1 regulates P2X7 receptor signaling in osteoblasts

AJP Cell Physiology ◽

10.1152/ajpcell.00037.2014 ◽

2015 ◽

Vol 308 (1) ◽

pp. C41-C50 ◽

Cited By ~ 13

Author(s):

Vimal Gangadharan ◽

Anja Nohe ◽

Jeffrey Caplan ◽

Kirk Czymmek ◽

Randall L. Duncan

Keyword(s):

Mechanical Load ◽

Purinergic Receptor ◽

Cell Types ◽

Signaling Proteins ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Lipid Microdomains ◽

Caveolin 1 ◽

Intracellular Ca ◽

Gradient Fractionation

The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X7R is central to this mechanotransduction signaling cascade. Recently, P2X7R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X7R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X7R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X7R agonist 2′(3′)- O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X7R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca2+ response to BzATP, suggesting that caveolae regulate P2X7R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X7R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X7R in osteoblasts.

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Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening

Leukemia & Lymphoma ◽

10.1080/10428190701225882 ◽

2007 ◽

Vol 48 (5) ◽

pp. 978-986 ◽

Cited By ~ 15

Author(s):

Shin-Ichiro Fujiwara ◽

Yoshihiro Yamashita ◽

Young Lim Choi ◽

Hideki Watanabe ◽

Kentaro Kurashina ◽

...

Keyword(s):

G Protein ◽

Purinergic Receptor ◽

Expression Screening ◽

Transforming Activity ◽

G Protein Coupled

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Simulation of P2X-mediated calcium signaling in microglia

10.1101/354142 ◽

2018 ◽

Author(s):

Ben Chun ◽

Bradley D. Stewart ◽

Darin Vaughan ◽

Adam D. Bachstetter ◽

Peter M. Kekenes-Huskey

Keyword(s):

Computational Model ◽

Purinergic Receptor ◽

Receptor Activation ◽

Quantitative Model ◽

Published Data ◽

Transcriptional Responses ◽

Extracellular Adenosine ◽

Wide Range ◽

Intracellular Ca

AbstractMicroglia function is orchestrated through highly-coupled signaling pathways that depend on calcium (Ca2+). In response to extracellular adenosine triphosphate (ATP), transient increases in intracellular Ca2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis and potentially their release. While steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic puriner-gic receptors, P2X4 and P2X7, with the graded production of a gene product, namely the tumor necrosis factor α (TNFα) cytokine. In addition to Ca2+ handling mechanisms common to eukaryotic cells, our model includes microglia-specific processes including ATP-dependent P2X4 and P2X7 activation, activation of NFAT transcription factors, and TNFα production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNFα production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNFα production. Our key findings are that TNFα production via P2X4 is maximized at low ATP when subject to high frequency ATP stimulation, whereas P2X7 contributes most significantly at millimolar ATPranges. Given that Ca2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.

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Abstract 340: Recombinant Soluble Apyrase Inhibits Vascular Smooth Muscle Cell Migration

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.340 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Oluwaseun Adeola ◽

Yan Ji ◽

Phillip Fish ◽

Tammy Strawn ◽

Gary A Weisman ◽

...

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Endothelial Cell ◽

Purinergic Receptor ◽

Neointimal Hyperplasia ◽

Receptor Activation ◽

Vehicle Control ◽

Extracellular Nucleotides ◽

Lower Chamber ◽

Upper Chamber

Background: Purinergic receptor activation by extracellular nucleotides is involved in thrombosis and neointimal hyperplasia that accompany atherosclerosis and postangioplasty restenosis. Human apyrases [ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases)] are membrane bound enzymes that hydrolyze extracellular nucleotides, thereby inhibiting purinergic receptor activation. CD39, the first identified human apyrase, is constitutively expressed on endothelial cell (EC) and vascular smooth muscle cell (VSMC) surfaces. APT102, a recombinant soluble form of CD39L3, has been shown to reduce platelet activation through its ADPase activity, but its effects on VSMC and EC function are yet to be established. We tested the hypothesis that APT102 will inhibit migration of VSMCs and ECs. Methods: We studied cell migration using a modified Boyden chamber assay in which 5x10 4 cells suspended in 0.2% FBS/DMEMF12 were added to the upper chamber of transwells separated from the lower chamber medium by a microporous membrane through which VSMCs and ECs can migrate. APT102 (100 nM) or vehicle control was added to the upper chamber; lower chamber contained 2.5% FBS/DMEMF12 and either ATP (10 μM) or vehicle control. Transwells were incubated at 37 0 C for 6 h, after which cells that migrated through pores and adhered to the lower chamber side of the membrane were fixed, stained and counted. Results: ATP (10μM) significantly enhanced migration of both VSMCs and ECs. APT102 significantly inhibited VSMC migration and completely abrogated the pro-migratory effect of ATP. In contrast, APT102 had no inhibitory effect on EC migration, either spontaneous or ATP-enhanced. Conclusion: APT102 inhibits VSMC but not EC migration. These results suggest that pharmacological targeting of extracellular nucleotides may provide a safe and effective therapeutic strategy to inhibit neointimal hyperplasia and restenosis after angioplasty, without delaying endothelial cell recovery, which is a significant limitation of drug-eluting stents. Further studies are needed to clarify the mechanism(s) underlying the differential effect of extracellular nucleotide degradation by APT102 on VSMC and EC migration.

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P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00237.2010 ◽

2011 ◽

Vol 300 (2) ◽

pp. C266-C275 ◽

Cited By ~ 32

Author(s):

Taras Lyubchenko ◽

Heather Woodward ◽

Kristopher D. Veo ◽

Nana Burns ◽

Hala Nijmeh ◽

...

Keyword(s):

Endothelial Cells ◽

Pulmonary Artery ◽

Purinergic Receptors ◽

Purinergic Receptor ◽

Extracellular Atp ◽

Vasa Vasorum ◽

Receptor Subtypes ◽

Extracellular Nucleotide ◽

Selective Antagonist ◽

Intracellular Ca

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca2+ concentration ([Ca2+]i) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca2+ signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca2+]i via Ca2+ influx through plasma membrane channels as well as Ca2+ mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca2+ responses in both cytosolic and nuclear compartments. An increase in [Ca2+]i was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPβS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αβMeATP, and βγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca2+ responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

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Blood Platelet Adenosine Receptors as Potential Targets for Anti-Platelet Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms20215475 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5475 ◽

Cited By ~ 6

Author(s):

Nina Wolska ◽

Marcin Rozalski

Keyword(s):

Adenosine Receptor ◽

Adenosine Receptors ◽

Purinergic Receptor ◽

Blood Platelets ◽

Blood Platelet ◽

Intracellular Camp ◽

Receptor Subtypes ◽

Adenosine Receptor Agonists ◽

Anti Platelet Therapy ◽

G Protein Coupled

Adenosine receptors are a subfamily of highly-conserved G-protein coupled receptors. They are found in the membranes of various human cells and play many physiological functions. Blood platelets express two (A2A and A2B) of the four known adenosine receptor subtypes (A1, A2A, A2B, and A3). Agonization of these receptors results in an enhanced intracellular cAMP and the inhibition of platelet activation and aggregation. Therefore, adenosine receptors A2A and A2B could be targets for anti-platelet therapy, especially under circumstances when classic therapy based on antagonizing the purinergic receptor P2Y12 is insufficient or problematic. Apart from adenosine, there is a group of synthetic, selective, longer-lasting agonists of A2A and A2B receptors reported in the literature. This group includes agonists with good selectivity for A2A or A2B receptors, as well as non-selective compounds that activate more than one type of adenosine receptor. Chemically, most A2A and A2B adenosine receptor agonists are adenosine analogues, with either adenine or ribose substituted by single or multiple foreign substituents. However, a group of non-adenosine derivative agonists has also been described. This review aims to systematically describe known agonists of A2A and A2B receptors and review the available literature data on their effects on platelet function.

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Mechanical strain-induced Ca2+waves are propagated via ATP release and purinergic receptor activation

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.2.c295 ◽

2000 ◽

Vol 279 (2) ◽

pp. C295-C307 ◽

Cited By ~ 104

Author(s):

H. Sauer ◽

J. Hescheler ◽

M. Wartenberg

Keyword(s):

Gap Junctions ◽

Purinergic Receptor ◽

Mechanical Strain ◽

Atp Release ◽

Anion Channel ◽

Receptor Activation ◽

Disulfonic Acid ◽

Channel Blockers ◽

Junctional Coupling ◽

Intracellular Ca

Mechanical strain applied to prostate cancer cells induced an intracellular Ca2+ (Cai 2+) wave spreading with a velocity of 15 μm/s. Cai 2+ waves were not dependent on extracellular Ca2+ and membrane potential because propagation was unaffected in high-K+ and Ca2+-free solution. Waves did not depend on the cytoskeleton or gap junctions because cytochalasin B and nocodazole, which disrupt microfilaments and microtubules, respectively, and 1-heptanol, which uncouples gap junctions, were without effects. Fluorescence recovery after photobleaching experiments revealed an absence of gap junctional coupling. Cai 2+ waves were inhibited by the purinergic receptor antagonists basilen blue and suramin; by pretreatment with ATP, UTP, ADP, UDP, 2-methylthio-ATP, and benzoylbenzoyl-ATP; after depletion of ATP by 2-deoxyglucose; and after ATP scavenging by apyrase. Waves were abolished by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid, tamoxifen, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, niflumic acid, and gadolinium. ATP release following strain was significantly inhibited by anion channel blockers. Hence, ATP is secreted via mechanosensitive anion channels and activates purinergic receptors on the same cell or neighboring cells in an autocrine and paracrine manner, thus leading to Cai 2+ wave propagation.

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Müller Cell Ca2+ Waves Evoked by Purinergic Receptor Agonists in Slices of Rat Retina

Journal of Neurophysiology ◽

10.1152/jn.2001.85.2.986 ◽

2001 ◽

Vol 85 (2) ◽

pp. 986-994 ◽

Cited By ~ 45

Author(s):

Yang Li ◽

Lynne A. Holtzclaw ◽

James T. Russell

Keyword(s):

Glial Cells ◽

Purinergic Receptor ◽

Rank Order ◽

Müller Cells ◽

Uridine Diphosphate ◽

Muller Cells ◽

Wave Amplification ◽

Dye Loading ◽

Intracellular Ca ◽

Retinal Slices

We have measured agonist evoked Ca2+ waves in Müller cells in situ within freshly isolated retinal slices. Using an eye cup dye loading procedure we were able to preferentially fill Müller glial cells in retinal slices with calcium green. Fluorescence microscopy revealed that bath perfusion of slices with purinergic agonists elicits Ca2+ waves in Müller cells, which propagate along their processes. These Ca2+ signals were insensitive to tetrodotoxin (TTX, 1.0 μM) pretreatment. Cells were readily identified as Müller cells by their unique morphology and by subsequent immunocytochemical labeling with glial fibrillary acidic protein antibodies. While cells never exhibited spontaneous Ca2+ oscillations, purinoreceptor agonists, ATP, 2 MeSATP, ADP, 2 MeSADP, and adenosine readily elicited Ca2+ waves. These waves persisted in the absence of [Ca2+]o but were abolished by thapsigargin pretreatment, suggesting that the purinergic agonists tested act by releasing Ca2+ from intracellular Ca2+ stores. The rank order of potency of different purines and pyrimidines for inducing Ca2+ signals was 2 MeSATP = 2MeSADP > ADP > ATP ≫ αβmeATP = uridine triphosphate (UTP) > uridine diphosphate (UDP). The Ca2+signals evoked by ATP, ADP, and 2 MeSATP were inhibited by reactive blue (100 μM) and suramin (200 μM), and the adenosine induced signals were abolished only by 3,7-dimethyl-1-propargylxanthine (200 μM) and not by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine) or 8-cyclopentyl-1,3-dipropylxanthine at the same concentration. Based on these pharmacological characteristics and the dose-response relationships for ATP, 2 MeSATP, 2 MeSADP, ADP, and adenosine, we concluded that Müller cells express the P1A2 and P2Y1 subtypes of purinoceptors. Analysis of Ca2+ responses showed that, similar to glial cells in culture, wave propagation occurred by regenerative amplification at specialized Ca2+ release sites (wave amplification sites), where the rate of Ca2+ release was significantly enhanced. These data suggest that Müller cells in the retina may participate in signaling, and this may serve as an extra-neuronal signaling pathway.

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Human dipeptidyl peptidase III regulates G-protein coupled receptor-dependent Ca2+ concentration in human embryonic kidney 293T cells

Biological Chemistry ◽

10.1515/hsz-2016-0117 ◽

2016 ◽

Vol 397 (6) ◽

pp. 563-569 ◽

Cited By ~ 3

Author(s):

Subhash C. Prajapati ◽

Ratnakar Singh ◽

Shyam S. Chauhan

Keyword(s):

Angiotensin Ii ◽

G Protein ◽

Biological Function ◽

Dipeptidyl Peptidase ◽

Luciferase Reporter ◽

G Protein Coupled Receptor ◽

Intracellular Ca ◽

First Time ◽

G Protein Coupled

Abstract The precise biological function of human dipeptidyl peptidase III (hDPP III) is poorly understood. Using luciferase reporter constructs responsive to change in Ca2+ and/or cAMP and Fura 2-AM fluorometric assay, we show a significant decrease in intracellular Ca2+ following hDPP III overexpression and angiotensin II stimulation in angiotensin II type 1 receptor (G-protein coupled receptor, GPCR) expressing HEK293T cells. Silencing the expression of hDPP III by siRNA reversed the effect of hDPP III overexpression with a concomitant increase in Ca2+. These results, for the first time, show involvement of hDPP III in GPCR dependent Ca2+ regulation in HEK293T cells.

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Testosterone Signaling through Internalizable Surface Receptors in Androgen Receptor-free Macrophages

Molecular Biology of the Cell ◽

10.1091/mbc.10.10.3113 ◽

1999 ◽

Vol 10 (10) ◽

pp. 3113-3123 ◽

Cited By ~ 167

Author(s):

W. Peter M. Benten ◽

Michèle Lieberherr ◽

Olaf Stamm ◽

Christian Wrehlke ◽

Zhiyong Guo ◽

...

Keyword(s):

Phospholipase C ◽

Binding Sites ◽

Plasma Membranes ◽

Androgen Receptors ◽

Surface Receptors ◽

Toxin Binding ◽

Agonist Stimulation ◽

Intracellular Ca ◽

Cytoskeletal Elements ◽

G Protein Coupled

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca2+], which is due to release of Ca2+from intracellular Ca2+stores. This Ca2+mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

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