scholarly journals Ionomycin induces prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells via mechanisms independent of its ionophoric nature

2016 ◽  
Vol 94 (3) ◽  
pp. 236-240
Author(s):  
Hans Jörg Leis ◽  
Werner Windischhofer
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Divalent Cation ◽  
Specific Binding ◽  
Prostaglandin E ◽  
Cellular Events ◽  
Marked Preference ◽  
Arachidonate Release ◽  
G Protein Coupled ◽  
Calcium Elevation

Ionomycin and A23187 are divalent cation ionophores with a marked preference for calcium. Studies using these ionophores have almost exclusively interpreted their results in the light of calcium elevation. It was the aim of this study to investigate the effects of ionomycin in osteoblatic MC3T3-E1 cells that are not attributable to its ionophoric properties. Thus, we have found that in contrast to A23187, ionomycin shows similar effects on prostaglandin E2 formation as bradykinin and endothelin-1, being potentiated by extracellular nickel and inhibited by cholera toxin and pertussis toxin. Our data strongly suggest that inomycin, at least in part, exerts its effects via specific binding to a G-protein coupled receptor, thereby evoking downstream cellular events like arachidonate release with subsequent prostaglandin formation.

Recent Advances of Small Molecular Regulators Targeting G Protein- Coupled Receptors Family for Oncology Immunotherapy

2019 ◽  
Vol 19 (16) ◽  
pp. 1464-1483 ◽  
Author(s):  
Peng He ◽  
Wenbo Zhou ◽  
Mingyao Liu ◽  
Yihua Chen
Keyword(s):  
G Protein ◽  
Cell Biology ◽  
Immune Cell ◽  
G Protein Coupled Receptors ◽  
Treatment Modalities ◽  
Immune Checkpoints ◽  
Considerable Proportion ◽  
Prostaglandin E ◽  
Recent Advances ◽  
G Protein Coupled

The great clinical success of chimeric antigen receptor T cell (CAR-T) and PD-1/PDL-1 inhibitor therapies suggests the drawing of a cancer immunotherapy age. However, a considerable proportion of cancer patients currently receive little benefit from these treatment modalities, indicating that multiple immunosuppressive mechanisms exist in the tumor microenvironment. In this review, we mainly discuss recent advances in small molecular regulators targeting G Protein-Coupled Receptors (GPCRs) that are associated with oncology immunomodulation, including chemokine receptors, purinergic receptors, prostaglandin E receptor EP4 and opioid receptors. Moreover, we outline how they affect tumor immunity and neoplasia by regulating immune cell recruitment and modulating tumor stromal cell biology. We also summarize the data from recent clinical advances in small molecular regulators targeting these GPCRs, in combination with immune checkpoints blockers, such as PD-1/PDL-1 and CTLA4 inhibitors, for cancer treatments.

Prostaglandin E2 receptors in the heart are coupled to inhibition of adenylyl cyclase via a pertussis toxin sensitive G protein

1992 ◽  
Vol 70 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Richard W. Lerner ◽  
Gary D. Lopaschuk ◽  
Peter M. Olley
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Pertussis Toxin ◽  
Binding Sites ◽  
Cyclase Activity ◽  
Prostaglandin E ◽  
Adenylyl Cyclase Activity ◽  
Adp Ribosylation ◽  
Number Of Binding Sites ◽  
Guanine Nucleotide Binding

In previous studies we have identified and isolated a prostaglandin E2 (PGE2) receptor from cardiac sarcolemmal (SL) membranes. Binding of PGE2 to this receptor in permeabilized SL vesicles inhibits adenylyl cyclase activity. The purpose of this study was to determine if the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive guanine nucleotide binding inhibitory (Gi) protein. Incubation of permeabilized SL vesicles in the presence of 100 μM 5′-guanylamidiophosphate, Gpp(NH)p, a nonhydrolyzable analogue of GTP, resulted in a shift in [3H]PGE2 binding from two sites, one of high affinity (KD = 0.018 ± 0.003 nM) comprising 7.7% of the total available binding sites and one of lower affinity (KD = 1.9 ± 0.7 nM) to one site of intermediate affinity (KD = 0.52 ± 0.01 nM) without a significant change in the total number of PGE2 binding sites. A shift from two binding sites to one binding site in the presence of Gpp(NH)p was also observed for [3H]dihydroalprenolol binding to permeabilized cardiac SL. When permeabilized SL vesicles were pretreated with activated pertussis toxin, ADP-ribosylation of a 40- to 41-kDa protein corresponding to Gi was observed. ADP-ribosylation of SL resulted in a shift in [3H]PGE2 binding to one site of intermediate affinity without significantly changing the number of binding sites. In alamethicin permeabilized SL vesicles, 1 nM PGE2 significantly decreased (30%) adenylyl cyclase activity. Pretreatment with activated pertussis toxin overcame the inhibitory effects of PGE2. These results demonstrate that the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive Gi protein. They also demonstrate that the interaction of this Gi protein with the PGE2 receptor is important in the regulation of PGE2 binding to its receptor.Key words: prostaglandin E2, sarcolemma, heart, adenylyl cyclase, G protein.

scholarly journals Structure of the Mouse Sex Peptide Pheromone ESP1 Reveals a Molecular Basis for Specific Binding to the Class C G-protein-coupled Vomeronasal Receptor

2013 ◽  
Vol 288 (22) ◽  
pp. 16064-16072 ◽  
Author(s):  
Sosuke Yoshinaga ◽  
Toru Sato ◽  
Makoto Hirakane ◽  
Kaori Esaki ◽  
Takashi Hamaguchi ◽  
...  
Keyword(s):  
G Protein ◽  
Molecular Basis ◽  
Specific Binding ◽  
Sex Peptide ◽  
Vomeronasal Receptor ◽  
Peptide Pheromone ◽  
Class C ◽  
G Protein Coupled

scholarly journals The neurobiology and evolution of cannabinoid signalling

2001 ◽  
Vol 356 (1407) ◽  
pp. 381-408 ◽  
Author(s):  
Maurice R. Elphick ◽  
Michaelà Egertova
Keyword(s):  
G Protein ◽  
Cannabinoid Receptors ◽  
Cannabis Sativa ◽  
Specific Binding ◽  
Acid Amide ◽  
Presynaptic Terminals ◽  
Smooth Muscle Contractility ◽  
Signalling System ◽  
G Protein Coupled ◽  
The Brain

The plant Cannabis sativa has been used by humans for thousands of years because of its psychoactivity. The major psychoactive ingredient of cannabis is δ 9 –tetrahydrocannabinol, which exerts effects in the brain by binding to a G–protein–coupled receptor known as the CB 1 cannabinoid receptor. The discovery of this receptor indicated that endogenous cannabinoids may occur in the brain, which act as physiological ligands for CB 1 . Two putative endocannabinoid ligands, arachidonylethanolamide (‘anandamide’) and 2–arachidonylglycerol, have been identified, giving rise to the concept of a cannabinoid signalling system. Little is known about how or where these compounds are synthesized in the brain and how this relates to CB 1 expression. However, detailed neuroanatomical and electrophysiological analysis of mammalian nervous systems has revealed that the CB 1 receptor is targeted to the presynaptic terminals of neurons where it acts to inhibit release of ‘classical’ neurotransmitters. Moreover, an enzyme that inactivates endocannabinoids, fatty acid amide hydrolase, appears to be preferentially targeted to the somatodendritic compartment of neurons that are postsynaptic to CB 1 –expressing axon terminals. Based on these findings, we present here a model of cannabinoid signalling in which anandamide is synthesized by postsynaptic cells and acts as a retrograde messenger molecule to modulate neurotransmitter release from presynaptic terminals. Using this model as a framework, we discuss the role of cannabinoid signalling in different regions of the nervous system in relation to the characteristic physiological actions of cannabinoids in mammals, which include effects on movement, memory, pain and smooth muscle contractility. The discovery of the cannabinoid signalling system in mammals has prompted investigation of the occurrence of this pathway in non–mammalian animals. Here we review the evidence for the existence of cannabinoid receptors in non–mammalian vertebrates and invertebrates and discuss the evolution of the cannabinoid signalling system. Genes encoding orthologues of the mammalian CB 1 receptor have been identified in a fish, an amphibian and a bird, indicating that CB 1 receptors may occur throughout the vertebrates. Pharmacological actions of cannabinoids and specific binding sites for cannabinoids have been reported in several invertebrate species, but the molecular basis for these effects is not known. Importantly, however, the genomes of the protostomian invertebrates Drosophila melanogaster and Caenorhabditis elegans do not contain CB 1 orthologues, indicating that CB 1 –like cannabinoid receptors may have evolved after the divergence of deuterostomes (e.g. vertebrates and echinoderms) and protostomes. Phylogenetic analysis of the relationship of vertebrate CB 1 receptors with other G–protein–coupled receptors reveals that the paralogues that appear to share the most recent common evolutionary origin with CB 1 are lysophospholipid receptors, melanocortin receptors and adenosine receptors. Interestingly, as with CB 1 , each of these receptor types does not appear to have Drosophila orthologues , indicating that this group of receptors may not occur in protostomian invertebrates. We conclude that the cannabinoid signalling system may be quite restricted in its phylogenetic distribution, probably occurring only in the deuterostomian clade of the animal kingdom and possibly only in vertebrates.

scholarly journals G-Protein Coupled Receptor-Evoked Glutamate Exocytosis from Astrocytes: Role of Prostaglandins

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Corrado Cali ◽  
Jan Lopatar ◽  
Francesco Petrelli ◽  
Luca Pucci ◽  
Paola Bezzi
Keyword(s):  
G Protein ◽  
Neutralizing Antibody ◽  
Secretory Cells ◽  
Prostaglandin E ◽  
Specific Population ◽  
Extracellular Release ◽  
Pharmacological Blockade ◽  
G Protein Coupled ◽  
Cell Medium

Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca2+-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca2+elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2(PGE2). Interestingly, administration of exogenous PGE2producedper serapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2.

scholarly journals Sphingosine 1-phosphate receptor 2 antagonist JTE-013 increases the excitability of sensory neurons independently of the receptor

2012 ◽  
Vol 108 (5) ◽  
pp. 1473-1483 ◽  
Author(s):  
Chao Li ◽  
Xian Xuan Chi ◽  
Wenrui Xie ◽  
J. A. Strong ◽  
J.-M. Zhang ◽  
...  
Keyword(s):  
G Protein ◽  
Sensory Neurons ◽  
Neuronal Excitability ◽  
Small Diameter ◽  
G Protein Coupled Receptors ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Sphingosine 1 Phosphate ◽  
G Protein Coupled

Previously we demonstrated that sphingosine 1-phosphate receptor 1 (S1PR1) played a prominent, but not exclusive, role in enhancing the excitability of small-diameter sensory neurons, suggesting that other S1PRs can modulate neuronal excitability. To examine the potential role of S1PR2 in regulating neuronal excitability we used the established selective antagonist of S1PR2, JTE-013. Here we report that exposure to JTE-013 alone produced a significant increase in excitability in a time- and concentration-dependent manner in 70–80% of recorded neurons. Internal perfusion of sensory neurons with guanosine 5′- O-(2-thiodiphosphate) (GDP-β-S) via the recording pipette inhibited the sensitization produced by JTE-013 as well as prostaglandin E2. Pretreatment with pertussis toxin or the selective S1PR1 antagonist W146 blocked the sensitization produced by JTE-013. These results indicate that JTE-013 might act as an agonist at other G protein-coupled receptors. In neurons that were sensitized by JTE-013, single-cell RT-PCR studies demonstrated that these neurons did not express the mRNA for S1PR2. In behavioral studies, injection of JTE-013 into the rat's hindpaw produced a significant increase in the mechanical sensitivity in the ipsilateral, but not contralateral, paw. Injection of JTE-013 did not affect the withdrawal latency to thermal stimulation. Thus JTE-013 augments neuronal excitability independently of S1PR2 by unknown mechanisms that may involve activation of other G protein-coupled receptors such as S1PR1. Clearly, further studies are warranted to establish the causal nature of this increased sensitivity, and future studies of neuronal function using JTE-013 should be interpreted with caution.

scholarly journals Hematopoietic Stem and Progenitor Cells Manifest Direct Migration Towards Secreted DEK in a CXCR2- and G Protein Signaling-Dependent Manner

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1196-1196
Author(s):  
Maegan L. Capitano ◽  
Yasser Sammour ◽  
Maureen Legendre ◽  
Scott Cooper ◽  
David Markovitz ◽  
...  
Keyword(s):  
Cell Migration ◽  
G Protein ◽  
Pertussis Toxin ◽  
Conflicts Of Interest ◽  
Neutrophil Migration ◽  
Regulation Of Transcription ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
G Protein Coupled

DEK, a nuclear DNA-binding protein implicated in the regulation of transcription, chromatin architecture, and mRNA processing, is secreted by macrophages and acts as a proinflammatory molecule (Mor-Vanknin et al., 2006, Mol. Cell. Bio., 26: 9484). Recombinant (r)DEK functions as a chemotactic factor attracting neutrophils, CD8+ T lymphocytes and natural killer cells. Few cytokines/growth modulating proteins are known to be chemoattractants for hematopoietic stem (HSC) and progenitor (HPC) cells; stromal cell-derived factor-1 (SDF-1/CXCL12) being the most potent known protein with this capability. To test whether rDEK can serve as a chemotactic agent, transwell assays were performed utilizing lineage negative mouse bone marrow (BM) cells with neutrophils (Ly6G+ cells) as a positive control. Both SDF-1 and DEK induced directed migration of Lin-Sca1+cKit+ (LSK) BM cells at a dose of 100ng/mL, as determined by flow cytometry of input and migrated cells, with no significant migration occurring towards 100ng/mL of IL-8 or MIP-2. All four cytokines induced migration of Ly6G+ neutrophils. After examining the ability of LSK cells to migrate towards various doses of rDEK (0-200ng/mL), it was determined that LSK cells can migrate towards rDEK in a dose dependent manner with maximum chemoattraction potential (~20%) occurring at a dose of DEK equal to or greater than 50ng/mL. A checkerboard assay using LSK cells was performed to determine whether rDEK acted more as a chemotactic (directed cell movement) or a chemokinetic (random migration) agent. Checkerboard analysis demonstrated that DEK acted as a chemotactic molecule. Upon our recent discovery and report that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8 and binds to the chemokine receptor CXCR2 to regulate hematopoiesis (Capitano et al., 2019, J.C.I. 130: 2555-2570), we hypothesized that DEK may manifest its chemotactic actions through CXCR2, known previously to only bind and mediate the actions of the chemokines IL-8 and MIP-2. To examine this, we first confirmed expression of CXCR2 on the surface of HSC and HPC. Next, to determine if LSK migration towards DEK is dependent upon its ability to signal through CXCR2, LSK cells were pretreated with a neutralizing monoclonal antibody for CXCR2 immediately prior to being placed in a transwell chemotaxis assay utilizing 100ng/mL of rDEK in the bottom chamber. Neutralizing anti-CXCR2 antibodies inhibited migration of both LSK and Ly6G+ cells toward DEK; however, if LSK cells were pretreated with an isotype control or a neutralizing antibody towards CXCR4, migration towards DEK still occurred. To confirm that the neutralizing CXCR2 antibody did not inhibit migration in a non-specific manner, transwell assays were performed examining LSK cell migration towards SDF-1, IL-8, and MIP-2. LSK cells were still able to migrate towards SDF-1 except when CXCR4 was neutralized. No migration of LSK cells was observed when IL-8 or MIP-2 was utilized. When Ly6G+ neutrophils were used, CXCR2 neutralizing antibodies blocked migration of the Ly6G+ neutrophils towards DEK, IL-8 and MIP-2. Neutralizing CXCR4 only blocked Ly6G+ neutrophil migration towards SDF-1. CXCR2 is a G protein-coupled receptor and this interaction can be blocked using pertussis toxin which prevents G proteins from interacting with G protein-coupled receptors thus interfering with receptor signaling. Pretreatment of LSK cells with pertussis toxin significantly inhibited the migration of LSK cells towards DEK and SDF-1. To determine if DEK and SDF-1 could inhibit one another, checkboard assays were performed where either different concentrations of DEK was used in the top well and different concentrations of SDF-1 was used on the bottom. Starting at 100ng/mL, DEK in the top well inhibited LSK cell migration towards SDF-1. However, when SDF-1 was in the top well, regardless of the SDF-1 dose, SDF-1 always inhibited LSK cell migration towards DEK. These data suggest that DEK acts as a chemotactic agent for HSC and HPC in vitro but is not as strong of a signaling protein for migration when competing against SDF-1. Thus, DEK may be involved as a compensatory chemotactic agent for HSCs and HPCs, especially under certain inflammatory conditions and when SDF-1 signaling is reduced. Disclosures No relevant conflicts of interest to declare.

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