Soluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway

Zygote ◽  
2014 ◽  
Vol 23 (6) ◽  
pp. 821-835 ◽  
Author(s):  
Takeshi Nakano ◽  
Keiichiro Kyozuka
Keyword(s):  
G Protein ◽  
Mature Oocyte ◽  
Second Step ◽  
Second Phase ◽  
Entire Surface ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Sperm Extract ◽  
Cytoplasmic Protrusion ◽  
G Protein Coupled

SummaryMatured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation.

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

2016 ◽  
Vol 397 (6) ◽  
pp. 563-569 ◽  
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.

scholarly journals Purinergic Receptors Crosstalk with CCR5 to Amplify Ca2+ Signaling

2020 ◽  
Author(s):  
Mizuho Horioka ◽  
Emilie Ceraudo ◽  
Emily Lorenzen ◽  
Thomas P. Sakmar ◽  
Thomas Huber
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Purinergic Receptors ◽  
P2y Receptors ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Extracellular Milieu ◽  
Immunodeficiency Virus ◽  
Intracellular Ca ◽  
G Protein Coupled

AbstractMany G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C–C chemokine receptor type 5 (CCR5), which serves as a co-receptor to facilitate cellular entry of human immunodeficiency virus 1 (HIV-1), normally signals through the heterotrimeric G protein, Gi. However, CCR5 also exhibits G protein signaling bias and certain chemokine analogs can cause a switch to Gq pathways to induce Ca2+ signaling. We want to understand how much of the Ca2+ signaling from Gi-coupled receptors is due to G protein promiscuity and how much is due to transactivation and crosstalk with other receptors. We propose a possible mechanism underlying the apparent switching between different G protein signaling pathways. We show that chemokine-mediated Ca2+ flux in HEK293T cells expressing CCR5 can be primed and enhanced by ATP pretreatment. In addition, agonist-dependent lysosomal exocytosis results in the release of ATP to the extracellular milieu, which amplifies cellular signaling networks. ATP is quickly degraded via ADP and AMP to adenosine. ATP, ADP and adenosine activate different cell surface purinergic receptors. Endogenous Gq-coupled purinergic P2Y receptors amplify Ca2+ signaling and allow for Gi- and Gq-coupled receptor signaling pathways to converge. Associated secretory release of GPCR ligands, such as chemokines, opioids, and monoamines, should also lead to concomitant release of ATP with a synergistic effect on Ca2+ signaling. Our results suggest that crosstalk between ATP-activated purinergic receptors and other Gi-coupled GPCRs is an important cooperative mechanism to amplify the intracellular Ca2+ signaling response.

scholarly journals Comparative Analysis of Functional Assays for Characterization of Agonist Ligands at G Protein-Coupled Receptors

2003 ◽  
Vol 8 (5) ◽  
pp. 500-510 ◽  
Author(s):  
Anke Niedernberg ◽  
Sorin Tunaru ◽  
Andree Blaukat ◽  
Bruce Harris ◽  
Evi Kostenis
Keyword(s):  
G Protein ◽  
Partial Agonist ◽  
A Priori ◽  
G Protein Coupled Receptors ◽  
Binding Assays ◽  
Functional Assays ◽  
Gtpγs Binding ◽  
Sphingosine 1 Phosphate ◽  
Intracellular Ca ◽  
G Protein Coupled

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P5 receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPγS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca2+ via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC50 values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPγS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPγS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays. ( Journal of Biomolecular Screening 2003:500-510)

scholarly journals Role of Ca2+Feedback on Single Cell Inositol 1,4,5-Trisphosphate Oscillations Mediated by G-protein-coupled Receptors

2003 ◽  
Vol 278 (23) ◽  
pp. 20753-20760 ◽  
Author(s):  
Kenneth W. Young ◽  
Mark S. Nash ◽  
R. A. John Challiss ◽  
Stefan R. Nahorski
Keyword(s):  
Single Cell ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Inositol 1,4,5 Trisphosphate ◽  
G Protein Coupled

17β-Estradiol induces nongenomic effects in renal intercalated cells through G protein-coupled estrogen receptor 1

2012 ◽  
Vol 302 (3) ◽  
pp. F358-F368 ◽  
Author(s):  
Marlene Vind Hofmeister ◽  
Helle Hasager Damkier ◽  
Birgitte Mønster Christensen ◽  
Björn Olde ◽  
L. M. Fredrik Leeb-Lundberg ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Atpase Activity ◽  
Steroid Hormones ◽  
G Protein ◽  
Intercalated Cells ◽  
Ici 182,780 ◽  
Intracellular Ca ◽  
17Β Estradiol ◽  
G Protein Coupled ◽  
Estrogen Receptor 1

Steroid hormones such as 17β-estradiol (E2) are known to modulate ion transporter expression in the kidney through classic intracellular receptors. Steroid hormones are also known to cause rapid nongenomic responses in a variety of nonrenal tissues. However, little is known about renal short-term effects of steroid hormones. Here, we studied the acute actions of E2 on intracellular Ca2+ signaling in isolated distal convoluted tubules (DCT2), connecting tubules (CNT), and initial cortical collecting ducts (iCCD) by fluo 4 fluorometry. Physiological concentrations of E2 induced transient increases in intracellular Ca2+ concentration ([Ca2+]i) in a subpopulation of cells. The [Ca2+]i increases required extracellular Ca2+ and were inhibited by Gd3+. Strikingly, the classic E2 receptor antagonist ICI 182,780 also increased [Ca2+]i, which is inconsistent with the activation of classic E2 receptors. G protein-coupled estrogen receptor 1 (GPER1 or GPR30) was detected in microdissected DCT2/CNT/iCCD by RT-PCR. Stimulation with the specific GPER1 agonist G-1 induced similar [Ca2+]i increases as E2, and in tubules from GPER1 knockout mice, E2, G-1, and ICI 182,780 failed to induce [Ca2+]i elevations. The intercalated cells showed both E2-induced concanamycin-sensitive H+-ATPase activity by BCECF fluorometry and the E2-mediated [Ca2+]i increment. We propose that E2 via GPER1 evokes [Ca2+]i transients and increases H+-ATPase activity in intercalated cells in mouse DCT2/CNT/iCCD.

Identification of a protein hydrolysate responsive G protein-coupled receptor in enterocytes

2007 ◽  
Vol 292 (1) ◽  
pp. G98-G112 ◽  
Author(s):  
Sungwon Choi ◽  
Mike Lee ◽  
Amy L. Shiu ◽  
Sek Jin Yo ◽  
Gregory W. Aponte
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Protein Hydrolysate ◽  
Mucosal Cell ◽  
Activated T Cells ◽  
Molecular Sensors ◽  
Synergistic Enhancement ◽  
Cell Proliferation And Differentiation ◽  
Intracellular Ca ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) have the potential to play a role as molecular sensors responsive to luminal dietary contents. Although such a role for GPCRs has been implicated in the intestinal response to protein hydrolysate, no GPCR directly involved in this process has been previously identified. In the present study, for the first time, we identified GPR93 expression in enterocytes and demonstrated its activation in these cells by protein hydrolysate with EC50 of 10.6 mg/ml as determined by the induction of intracellular free Ca2+. In enterocytes, GPR93 was synergistically activated by protein hydrolysate in combination with an agonist, oleoyl-l-α-lysophosphatidic acid (LPA), which activated the receptor in these enterocytes with EC50 of 7.9 nM. The increased intracellular Ca2+ by GPR93 activation was observed without the addition of a promiscuous Gα protein and was pertussis toxin sensitive, which suggests Gαq- and Gαi-mediated pathways. Activated GPR93 also induced pertussis toxin-sensitive ERK1/2 phosphorylation. Both nuclear factor of activated T cells and 12- O-tetradecanoylphorbol 13-acetate responsive elements reporter activities were induced by protein hydrolysate in cells exogenously expressing GPR93. The peptidomimetic cefaclor by itself did not activate GPR93 but potentiated the protein hydrolysate response and further amplified the synergistic enhancement of GPR93 activation by protein hydrolysate and LPA. These data suggest that, physiologically, the composition of stimuli might determine GPR93 activity or its sensitivity toward a given activator and suggest a new mechanism of the regulation of mucosal cell proliferation and differentiation and hormonal secretion by dietary products in the lumen.

GPCR Screening via ERK 1/2: A Novel Platform for Screening G Protein–Coupled Receptors

2005 ◽  
Vol 10 (7) ◽  
pp. 730-737 ◽  
Author(s):  
Ronald I. W. Osmond ◽  
Antony Sheehan ◽  
Romana Borowicz ◽  
Emma Barnett ◽  
Georgina Harvey ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
Measurement Techniques ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
G Protein Coupled Receptors ◽  
Drug Candidates ◽  
Gpcr Activation ◽  
Intracellular Ca ◽  
G Protein Coupled

Discovery of novel agonists and antagonists for G protein–coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.

Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells

2000 ◽  
Vol 278 (4) ◽  
pp. L726-L736 ◽  
Author(s):  
Dong-Mei Zhao ◽  
Hai-Hui Xue ◽  
Kingo Chida ◽  
Takafumi Suda ◽  
Yutaka Oki ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Cell Line ◽  
G Protein ◽  
Extracellular Space ◽  
A549 Cells ◽  
Rt Pcr ◽  
Calcium Response ◽  
Store Depletion ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca

ATP induced a biphasic increase in the intracellular Ca2+concentration ([Ca2+]i), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca2+]i spike but only in the presence of extracellular calcium ([Formula: see text]). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3] formation and UTP-induced [Ca2+]i spike, implying that EM perturbs Ca2+ influx from the extracellular space rather than Ca2+release from intracellular Ca2+ stores via the G protein-phospholipase C-Ins(1,4,5) P 3 pathway. A verapamil-sensitive, KCl-induced increase in [Ca2+]i and the Ca2+ influx activated by Ca2+ store depletion were insensitive to EM. 3′- O-(4-benzoylbenzoyl)-ATP evoked an[Formula: see text]-dependent [Ca2+]i response even in the presence of verapamil or the absence of extracellular Na+, and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X4 as well as P2Y2, P2Y4, and P2Y6 are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X4 and P2Y2/P2Y4 subtypes contribute to the ATP-induced [Ca2+]i spike and 2) EM selectively inhibits Ca2+ influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.

scholarly journals Ascaris suum-Derived Products Induce Human Neutrophil Activation via a G Protein-Coupled Receptor That Interacts with the Interleukin-8 Receptor Pathway

2001 ◽  
Vol 69 (6) ◽  
pp. 4007-4018 ◽  
Author(s):  
Franco H. Falcone ◽  
Adriano G. Rossi ◽  
Rose Sharkey ◽  
Alan P. Brown ◽  
David I. Pritchard ◽  
...  
Keyword(s):  
G Protein ◽  
Shape Change ◽  
Ascaris Suum ◽  
Cell Polarization ◽  
Interleukin 8 ◽  
Human Neutrophils ◽  
G Protein Coupled Receptor ◽  
Superoxide Anion Production ◽  
Intracellular Ca ◽  
G Protein Coupled

ABSTRACT Infection with tissue-migrating helminths is frequently associated with intense granulocyte infiltrations. Several host-derived factors are known to mediate granulocyte recruitment to the tissues, but less attention has been paid to how parasite-derived products trigger this process. Parasite-derived chemotactic factors which selectively recruit granulocytes have been described, but nothing is known about which cellular receptors respond to these agents. The effect of products from the nematodes Ascaris suum, Toxocara canis, andAnisakis simplex on human neutrophils were studied. We monitored four parameters of activation: chemotaxis, cell polarization, intracellular Ca2+ transients, and priming of superoxide anion production. Body fluids of A. suum (ABF) and T. canis (TcBF) induced strong directional migration, shape change, and intracellular Ca2+ transients. ABF also primed neutrophils for production of superoxide anions. Calcium mobilization in response to A. suum-derived products was completely abrogated by pretreatment with pertussis toxin, implicating a classical G protein-coupled receptor mechanism in the response to ABF. Moreover, pretreatment with interleukin-8 (IL-8) completely abrogated the response to ABF, demonstrating desensitization of a common pathway. However, ABF was unable to fully desensitize the response to IL-8, and binding to CXCR1 or CXCR2 was excluded in experiments using RBL-2H3 cells transfected with the two human IL-8 receptors. Our results provide the first evidence for a direct interaction between a parasite-derived chemotactic factor and the host's chemotactic network, via a novel G protein-coupled receptor which interacts with the IL-8 receptor pathway.

scholarly journals Gpr158 mediates osteocalcin’s regulation of cognition

2017 ◽  
Vol 214 (10) ◽  
pp. 2859-2873 ◽  
Author(s):  
Lori Khrimian ◽  
Arnaud Obri ◽  
Mariana Ramos-Brossier ◽  
Audrey Rousseaud ◽  
Stéphanie Moriceau ◽  
...  
Keyword(s):  
G Protein ◽  
Neurotrophic Factor ◽  
Molecular Tools ◽  
Wild Type ◽  
Inositol 1,4,5 Trisphosphate ◽  
Regulation Of Cognition ◽  
G Protein Coupled ◽  
Old Mice ◽  
Circulating Levels ◽  
Behavioral Assays

That osteocalcin (OCN) is necessary for hippocampal-dependent memory and to prevent anxiety-like behaviors raises novel questions. One question is to determine whether OCN is also sufficient to improve these behaviors in wild-type mice, when circulating levels of OCN decline as they do with age. Here we show that the presence of OCN is necessary for the beneficial influence of plasma from young mice when injected into older mice on memory and that peripheral delivery of OCN is sufficient to improve memory and decrease anxiety-like behaviors in 16-mo-old mice. A second question is to identify a receptor transducing OCN signal in neurons. Genetic, electrophysiological, molecular, and behavioral assays identify Gpr158, an orphan G protein–coupled receptor expressed in neurons of the CA3 region of the hippocampus, as transducing OCN’s regulation of hippocampal-dependent memory in part through inositol 1,4,5-trisphosphate and brain-derived neurotrophic factor. These results indicate that exogenous OCN can improve hippocampal-dependent memory in mice and identify molecular tools to harness this pathway for therapeutic purposes.

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