CAS:7440-70-2 - FACTA Search

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Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.
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PMID:Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2. 869 78

During inflammation neutrophils receive multiple signals that are integrated, allowing a single modified response. One mechanism for this discrimination is receptor desensitization, a process whereby ligand-receptor binding is disassociated from cell activation. We examined the effect of heterologous receptor desensitization on neutrophil chemotaxis, calcium mobilization, and arachidonic acid production, using interleukin-8 (IL-8), C5a, and N-formyl-methionyl-leucyl-phenylalanine (fMLP). We observed reciprocal inhibition with respect to chemotaxis. We demonstrated that homologous desensitization, with respect to the mobilization of intracellular calcium stores, lasted approximately 15 min. Heterologous desensitization between the fMLP receptor and the C5a receptor was reciprocal; either stimulant would diminish the cells' response to stimulation by the other for approximately 3-5 min. However, we observed a unidirectional heterologous desensitization of the IL-8 receptor by both the fMLP and the C5a receptor. This unidirectional heterologous desensitization was observed with respect to both calcium mobilization and arachidonic acid production (i.e., prestimulation of the IL-8 receptor had no effect on subsequent stimulation by either fMLP or C5a).
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PMID:Unidirectional heterologous receptor desensitization between both the fMLP and C5a receptor and the IL-8 receptor. 869 29

Interleukin 8 (IL-8) is a potent neutrophil chemoattractant and activator. Two IL-8 receptor subtypes, A and B, are expressed in neutrophils. In this work, we analyzed the role of the C terminus domain of the IL-8 receptor on the signal transduction and receptor internalization mechanisms. The IL-8 receptor A was tagged with an epitope corresponding to the monoclonal antibody 1D4 to monitor the localization of the IL-8 receptor. We demonstrated IL-8-dependent receptor internalization by monitoring the density of surface 125I-labeled IL-8 binding sites and by immunofluorescence microscopy. Truncation of the last 27 amino acids of the IL-8 receptor A severely impaired the IL-8-induced internalization of the receptor. Of importance was the observation that binding of IL-8 to receptors A and B triggered a dramatically faster rate of internalization of receptor B than receptor A, suggesting that the heterologous C termini among receptor subtypes modulate the rate of internalization of IL-8 receptors. However, substitution of the C terminus of the receptor subtype A for the C terminus of receptor B reduced the internalization rate of receptor A. Furthermore, we found that the rate of internalization of IL-8 receptor B triggered by IL-8 was faster than the one induced by the IL-8-related peptide, melanoma growth stimulatory activity. Studies with human neutrophils pretreated with 100 nM IL-8 for 5 min revealed a positive and a negative calcium response mediated by receptors A and B, respectively. In contrast, neutrophils pretreated with melanoma growth stimulatory activity showed positive calcium responses to both receptors A and B. These data suggest that the neutrophil responses mediated by IL-8 are modulated by the rate of internalization of receptors.
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PMID:Role of the C terminus of the interleukin 8 receptor in signal transduction and internalization. 870 97

Interleukin 8 (IL-8) and Gro-alpha are members of the CXC branch of a family of cytokines recently designated the "chemokine" superfamily. Recent evidence indicates that, contrary to previously held beliefs, IL-8 and Gro-alpha may not be perceived equivalently by neutrophils. In this study, we have evaluated the effects of IL-8 and Gro-alpha on the rate of calcium influx in human neutrophils and in 293 cells transfected with type A or type B IL-8 receptors. Of these two chemokines, only Gro-alpha induced an influx of calcium in neutrophils as judged by the sensitivity of the mobilization of calcium to the extracellular calcium chelator EGTA and to the nonselective divalent cation channel inhibitor SK&F 96365, as well as by manganese quenching experiments. IL-8 was similarly without effect on the rate of Mn2+ influx in 293 cells transfected with IL-8 receptor A (IL-8RA) or IL-8RB. On the other hand, Gro-alpha induced an SK&F 96365-sensitive increase of the rate of Mn+2 influx in IL-8RB-, but not in IL-8RA-transfected 293 cells. These results indicate not only that neutrophils respond differently to IL-8 than they do to Gro-alpha but, furthermore, that the consequences of the binding of IL-8 and Gro-alpha to IL-8RB are distinct.
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PMID:Diverging signal transduction pathways activated by interleukin 8 (IL-8) and related chemokines in human neutrophils. IL-8 and Gro-alpha differentially stimulate calcium influx through IL-8 receptors A and B. 870 97

cDNA cloning has revealed the presence of at least three distinct human receptors for macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES: C-C chemokine receptor (CCR) 1, 4, and 5. To clarify the physiological role of CCR1, we prepared specific antibodies to CCR1 by immunizing rabbits with recombinant glutathione-S-transferase (GST) fused with its NH2-terminal portion. The resultant antibodies stained positively 293 cells transfected with CCR1 cDNA but neither parental cells nor cells transfected with CXCR1 [interleukin-8 (IL-8) receptor type A] cDNA, confirming its specificity. Immunofluorescence analysis revealed that peripheral blood lymphocytes and monocytes but not neutrophils express CCR1. Positive staining of transfectants, monocytes, and lymphocytes was inhibited by the GST protein fused with the NH2-terminal portion of CCR1, further indicating that this antibody recognized the NH2-terminal portion of CC CKR1. A majority of CD3+, CD4+, CD8+, or CD16+ peripheral blood lymphocytes but not CD19+ lymphocytes expressed CCR1. Among CD4+ peripheral blood lymphocytes, CD45RO+ cells expressed a larger number of CCR1 compared with CD45RO-. Moreover, CD34+ cells in human bone marrow as well as cord blood were uniformly stained with this antibody. Furthermore, the antibody inhibited calcium mobilization in CCR1 transfectants stimulated with human rMIP-1alpha, suggesting that its NH2-terminal portion is critically involved in ligand binding or signaling. Finally, the antibody partially inhibited monocyte chemotactic activities of human rMIP-1alpha, suggesting that CCR1 is a functional receptor for MIP-1alpha on human peripheral blood monocytes.
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PMID:Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leukocytes. 892 58

The genes encoding two functional human interleukin-8 (IL-8) receptors have been identified by molecular cloning techniques and they are members of the rhodopsin G-protein coupled receptor (GCR) superfamily. We report the molecular cloning of two rat GCR genes (rat CXCR1-like and rat CXCR2) whose conceptualized amino acid sequences are approximately 70% identical to the human IL-8 A and B receptor subtypes. The murine GRO-like peptide, macrophage inflammatory peptide-2 (MIP-2), elevates intracellular calcium levels in HEK293 cells expressing the rat CXCR2 receptor. Southern blot analysis of restriction-digested rodent and human genomic DNAs indicate that rat CXCR1-like and CXCR2 are: 1) each single copy genes in the rat genome; 2) most closely related to the human IL-8 receptor genes; and 3) orthologous to two previously identified murine genes. CXCR2 mRNA is detected in adult rat lung, spleen, and neutrophils. CXCR1-like mRNA can be detected in adult rat lung, native rat macrophages, and a rat alveolar macrophage cell line (NR8383). These data identify the rat orthologs of the human IL-8 receptors, and describe cellular and tissue targets of rat C-X-C chemokine peptides.
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PMID:Identification of two rat genes orthologous to the human interleukin-8 receptors. 895 12

Human neutrophils undergo rapid homologous receptor desensitization following repeated stimulation with chemoattractants such as IL-8, C5a, and FMLP. It has also been demonstrated that cross-desensitization among these chemoattractant receptors occurs. We investigated the mechanisms underlying the cross-desensitization of responses to IL-8 induced by pretreatment with FMLP or C5a. In [125I]-labeled IL-8 binding studies we found that the cross-desensitization induced by FMLP or C5a was associated with a subsequent reduction in IL-8 binding to neutrophils. There was no recovery of [125I]-labeled IL-8 binding on removal of the C5a or FMLP pretreatment. FACS analysis using mAbs specific for the two IL-8R subtypes showed differential regulation of IL-8R A and IL-8R B cell surface expression after chemoattractant pretreatment. Homologous desensitization by IL-8 resulted in internalization of IL-8R A and IL-8R B, but only IL-8R A was completely re-expressed after removal of agonist. FMLP stimulation led to a substantial loss of IL-8R B from the cell surface, whereas C5a stimulation induced only a partial loss. In both cases there was no re-expression of IL-8R B on removal of the chemoattractant stimulation. C5a and FMLP did not affect IL-8R A expression. Calcium mobilization studies using melanoma growth stimulatory activity and IL-8 suggest that a sustained loss of IL-8R B may play a part in maintaining FMLP-induced IL-8R cross-desensitization. Chemoattractant-induced cross-desensitization of neutrophils may be of importance in regulating neutrophil accumulation during the inflammatory response in vivo.
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PMID:Chemoattractant cross-desensitization of the human neutrophil IL-8 receptor involves receptor internalization and differential receptor subtype regulation. 901 80

Human granulocyte chemotactic protein 2 (GCP-2) has originally been isolated from cytokine-stimulated osteosarcoma cells as a chemokine coproduced in minute amounts together with interleukin 8. Human GCP-2 (75 residues) was synthesized on a 0.25-mmol scale using Fmoc chemistry. After disulfide bridge formation and purification, monomeric GCP-2 was recovered as a 6-kDa protein; the pure synthetic protein showed a molecular mass of 8076 Da as determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The exact amino acid sequence of synthetic GCP-2 was confirmed by Edman degradation. Synthetic GCP-2 was an equally active (minimal effective concentration of 1-3 nM) chemoattractant for neutrophilic granulocytes as was natural 75-residue GCP-2. At concentrations up to 30 nM, synthetic GCP-2 did not stimulate eosinophil, monocyte, or lymphocyte chemotaxis. GCP-2 induced a dose-dependent increase in [Ca2+]i in neutrophils, 1 nM being the minimal effective concentration. The GCP-2-induced [Ca2+]i increase was completely prevented by pertussis toxin. Prestimulation of neutrophils with equimolar concentrations of purified natural IL-8, GROalpha, GROgamma and ENA-78 abolished the [Ca2+]i increase in response to 1 nM GCP-2. Alternatively, the [Ca2+]i rise induced by these CXC chemokines was inhibited by pretreatment of neutrophils with GCP-2. GCP-2 stimulated [Ca2+]i increases in CXCR1- and CXCR2-transfected cells, demonstrating that GCP-2 binds to both IL-8 receptors. Intradermal injection of synthetic GCP-2 resulted in a dose-dependent neutrophil accumulation and plasma extravasation in rabbit skin. To provoke this skin reaction, GCP-2 (10 pmol/site) was nearly as effective as IL-8, indicating that it is an important complementary mediator of the inflammatory response.
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PMID:Characterization of synthetic human granulocyte chemotactic protein 2: usage of chemokine receptors CXCR1 and CXCR2 and in vivo inflammatory properties. 905 80

Human neutrophils express two interleukin (IL)-8 receptors, CXC chemokine receptor (CXCR) 1 and CXCR2. IL-8 with changes to the NH2-terminal ELR motif can block IL-8-induced neutrophil functions (Moser, B., Dewald, B., Barella, L., Schumacher, C., Baggiolini, M., and Clark-Lewis, I. (1993) J. Biol. Chem. 268, 7125-7128). We have now examined the effect of NH2-terminally modified analogs of IL-8, GROalpha, and PF4 on CXCR1 and CXCR2 independently. Using stable Jurkat transfectants expressing either CXCR1 or CXCR2, it was shown that analogs derived from IL-8 bound both IL-8 receptors with similar affinity and could block IL-8-induced Ca2+ mobilization. By contrast, analogs of GROalpha and PF4, (R)GROalpha and (R)PF4, bound only CXCR2 with high affinity and blocked Ca2+ mobilization induced only via CXCR2. The differential effect on CXCR1 and CXCR2 was also demonstrated in studies with isolated neutrophils. Thus (R)GROalpha and (R)PF4 inhibited only the GROalpha but not the IL-8-stimulated elastase release, and these two analogs had no effect on IL-8-elicited superoxide generation, a response that is mediated by CXCR1 but not by CXCR2. These results show that CXCR2 selective receptor antagonists can be generated based upon the secondary binding determinants of GROalpha and PF4. They also highlight the primary importance of CXCR1 in chemokine-mediated release of granule enzymes and superoxide generation. The selective antagonists described may be used in future studies on IL-8 receptor signaling to define distinct steps leading to various functional responses induced in neutrophils via CXCR1 and CXCR2.
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PMID:Chemokine antagonists that discriminate between interleukin-8 receptors. Selective blockers of CXCR2. 919 14

The nucleotide sequence for a putative chemokine receptor, termed TER1, ChemR1, or CKR-L1, was recently obtained by a polymerase chain reaction-based cloning technique. It encodes a protein of 355 amino acids that shows 32-45% sequence identity with human chemokine receptors. The gene was localized on human chromosome 3p21-24, the site for the genes for the five known CC chemokine receptors, suggesting that the natural ligand may be a CC chemokine. We have stably expressed this receptor in murine pre-B cells 300-19 and have tested their responsiveness to 20 human chemokines and some other potential agonists. The CC chemokine I-309 was the only agonist that selectively induced intracellular Ca2+ mobilization and chemotaxis in receptor-transfected 300-19 cells. Stromal cell-derived factor 1, which binds to murine CXCR4 expressed in parental as well as transfected 300-19 cells, served as positive control in the functional screening. The interaction of I-309 with TER1 was of high affinity as shown by 125I-I-309 binding (Kd of 1.2 nM) and transient [Ca2+]i changes at subnanomolar concentrations of agonist. Migration responses in receptor-transfected 300-19 cells was typically bimodal with maximal activity at 10 nM of I-309. These data demonstrate that TER1 (ChemR1 or CKR-L1) is the receptor for I-309, and we propose to call this receptor CCR8 in agreement with the current nomenclature for chemokine receptors. The expression of CCR8 in blood leukocytes and lymphocytes was analyzed by Northern blot. No transcripts were found in RNA from freshly isolated blood neutrophils, monocytes, cultured macrophages, and phytohemagglutinin-stimulated T lymphocytes, and a faint hybridization signal corresponding to the RNA species of 4 kb was obtained only with RNA from interleukin-2-treated T lymphocytes. CCR8 is unusual for its selectivity for a single chemokine, previously shown only for CXCR1 and CXCR4, which bind interleukin-8 and stromal cell-derived factor 1, respectively. Identification of the receptor for I-309 represents a significant progress in determining the function of I-309 in inflammation and disease.
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PMID:Identification of CCR8, the receptor for the human CC chemokine I-309. 921 59

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