Inducible NO synthase: role in cellular signalling.
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The discovery of endothelium-derived relaxing factor and its identification as nitric oxide (NO) was one of the most exciting discoveries of biomedical research in the 1980s. Besides its potent vasodilatory effects, NO was found under certain circumstances to be responsible for the killing of microorganisms and tumour cells by activated macrophages and to act as a novel, unconventional type of neurotransmitter. In 1992, Science picked NO as the 'Molecule of the Year', and over the past years NO has become established as a universal intercellular messenger that acutely affects important signalling pathways and, on a more long-term scale, modulates gene expression in target cells. These actions will form the focus of the present review. (+info)
Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells.
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Under conditions associated with local and systemic inflammation, mesangial cells and invading immune cells are likely to be responsible for the release of large amounts of nitric oxide (NO) in the glomerulus. To further define the mechanisms of NO action in the glomerulus, we attempted to identify genes which are regulated by NO in rat glomerular mesangial cells. We identified vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase (FLT-1) to be under the regulatory control of exogenously applied NO in these cells. Using S-nitroso-glutathione (GSNO) as an NO-donating agent, VEGF expression was strongly induced, whereas expression of its FLT-1 receptor simultaneously decreased. Expressional regulation of VEGF and FLT-1 mRNA was transient and occurred rapidly within 1-3 h after GSNO treatment. Expression of a second VEGF-specific receptor, fetal liver kinase-1 (FLK-1/KDR), could not be detected. The inflammatory cytokine interleukin-1beta mediated a moderate increase in VEGF expression after 24 h and had no influence on FLT-1 expression. In contrast, platelet-derived growth factor-BB and basic fibroblast growth factor had no effect on VEGF expression, but strongly induced FLT-1 mRNA levels. Obviously, there is a differential regulation of VEGF and its receptor FLT-1 by NO, cytokines and growth factors in rat mesangial cells. (+info)
Identification of a domain in guanylyl cyclase-activating protein 1 that interacts with a complex of guanylyl cyclase and tubulin in photoreceptors.
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The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73-Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73-Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments. (+info)
Molecular characterization of a third member of the guanylyl cyclase-activating protein subfamily.
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The mammalian retina contains at least two guanylyl cyclases (GC1 and GC2) and two guanylyl cyclase-activating proteins (GCAP1 and GCAP2). Here we present evidence of the presence of a new photoreceptor-specific GCAP, termed GCAP3, which is closely related to GCAP1. The sequence similarity of GCAP3 with GCAP1 and GCAP2 is 57 and 49%, respectively. Recombinant GCAP3 and GCAP2 stimulate GC1 and GC2 in low [Ca2+]free and inhibit GCs when [Ca2+]free is elevated, unlike GCAP1, which only stimulates GC1. GCAP3 is encoded by a distinct gene present in other mammalian species but could not be detected by genomic Southern blotting in rodents, amphibians, and lower vertebrates. The intron/exon arrangement of the GCAP3 gene is identical to that of the other GCAP genes. While the GCAP1 and GCAP2 genes are arranged in a tail-to-tail array on chromosome 6p in human, the GCAP3 gene is located on 3q13.1, suggesting an ancestral gene duplication/translocation event. The identification of multiple Ca2+-binding proteins that interact with GC is suggestive of complex regulatory mechanisms for photoreceptor GC. (+info)
Sequence analysis of cDNA and genomic DNA, and mRNA expression of the medaka fish homolog of mammalian guanylyl cyclase C.
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We isolated the cDNA and genomic DNA encoding a membrane guanylyl cyclase of medaka fish (designated as OlGC6), and determined their complete nucleotide sequences. The open reading frame for OlGC6 cDNA predicted a protein of 1,075 amino acids. Phylogenetic analysis indicated that OlGC6 is a member of the enterotoxin/guanylin receptor family. We also determined the partial genomic structure of the gene of another membrane guanylyl cyclase of medaka fish, OlGC2, which is a member of the natriuretic peptide receptor family. The intron positions relative to the protein-coding sequence are highly conserved in the intracellular domains of OlGC6, OlGC2, mammalian GC-A, and GC-E. Despite their divergent primary structures, some intron positions also seem to be conserved in the extracellular domains of different membrane guanylyl cyclase genes. Northern blot analysis demonstrated that an OlGC6 transcript of 3.9 kb is only present in the intestine, while reverse transcription (RT)-PCR analysis demonstrated that the OlGC6 transcript is present in the kidney, spleen, liver, pancreas, gallbladder, ovary, testis, brain, and eye. RT-PCR also demonstrated that OlGC6 is only expressed zygotically and that transcripts are present from 1 day after fertilization, i.e. long before the intestinal tissues begin to develop. (+info)
Occupancy of the chromophore binding site of opsin activates visual transduction in rod photoreceptors.
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The retinal analogue beta-ionone was used to investigate possible physiological effects of the noncovalent interaction between rod opsin and its chromophore 11-cis retinal. Isolated salamander rod photoreceptors were exposed to bright light that bleached a significant fraction of their pigment, were allowed to recover to a steady state, and then were exposed to beta-ionone. Our experiments show that in bleach-adapted rods beta-ionone causes a decrease in light sensitivity and dark current and an acceleration of the dim flash photoresponse and the rate constants of guanylyl cyclase and cGMP phosphodiesterase. Together, these observations indicate that in bleach-adapted rods beta-ionone activates phototransduction in the dark. Control experiments showed no effect of beta-ionone in either fully dark-adapted or background light-adapted cells, indicating direct interaction of beta-ionone with the free opsin produced by bleaching. We speculate that beta-ionone binds specifically in the chromophore pocket of opsin to produce a complex that is more catalytically potent than free opsin alone. We hypothesize that a similar reaction may occur in the intact retina during pigment regeneration. We propose a model of rod pigment regeneration in which binding of 11-cis retinal to opsin leads to activation of the complex accompanied by a decrease in light sensitivity. The subsequent covalent attachment of retinal to opsin completely inactivates opsin and leads to the recovery of sensitivity. Our findings resolve the conflict between biochemical and physiological data concerning the effect of the occupancy of the chromophore binding site on the catalytic potency of opsin. We show that binding of beta-ionone to rod opsin produces effects opposite to its previously described effects on cone opsin. We propose that this distinction is due to a fundamental difference in the interaction of rod and cone opsins with retinal, which may have implications for the different physiology of the two types of photoreceptors. (+info)
Hepatocyte nuclear factor-4 regulates intestinal expression of the guanylin/heat-stable toxin receptor.
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We have investigated the regulation of gene transcription in the intestine using the guanylyl cyclase C (GCC) gene as a model. GCC is expressed in crypts and villi in the small intestine and in crypts and surface epithelium of the colon. DNase I footprint, electrophoretic mobility shift assay (EMSA), transient transfection assays, and mutagenesis experiments demonstrated that GCC transcription is regulated by a critical hepatocyte nuclear factor-4 (HNF-4) binding site between bp -46 and -29 and that bp -38 to -36 were essential for binding. Binding of HNF-4 to the GCC promoter was confirmed by competition EMSA and by supershift EMSA. In Caco-2 and T84 cells, which express both GCC and HNF-4, the activity of GCC promoter and/or luciferase reporter plasmids containing 128 or 1973 bp of 5'-flanking sequence was dependent on the HNF-4 binding site in the proximal promoter. In COLO-DM cells, which express neither GCC nor HNF-4, cotransfection of GCC promoter/luciferase reporter plasmids with an HNF-4 expression vector resulted in 23-fold stimulation of the GCC promoter. Mutation of the HNF-4 binding site abolished this transactivation. Transfection of COLO-DM cells with the HNF-4 expression vector stimulated transcription of the endogenous GCC gene as well. These results indicate that HNF-4 is a key regulator of GCC expression in the intestine. (+info)
Effect of nitric oxide donors on oxygen-dependent cytotoxic responses mediated by neutrophils.
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We analyzed the effect of nitric oxide (NO) on oxygen-dependent cytotoxic responses mediated by neutrophils against unopsonized erythrocytes using three NO donors: S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and sodium nitroprusside (SNP). Neutrophils were treated with these compounds for 1-2 min at 37 degrees C and cytotoxicity was then triggered in the presence of NO donors by precipitating immune complexes, aggregated IgG, the chemotactic peptide FMLP, or opsonized zymosan. GSNO induced, in all cases, a marked increase in cytotoxic responses, while SNAP moderately increased cytotoxicity triggered by immune complexes, aggregated IgG, or Z, opsonized zymosen, without modifying those responses induced by FMLP. By contrast, SNP dramatically suppressed cytotoxicity triggered by all of the stimuli assessed. The enhancing effects mediated by GSNO and SNAP did not depend on the stimulation of guanylyl cyclase and were prevented by the NO scavengers hemoglobin and PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide). The inhibitory activity of SNP, on the other hand, was not prevented by NO scavengers, suggesting that it cannot be ascribed to the release of NO. In another set of experiments, neutrophils were pretreated with GSNO or SNAP for different times. Then cells were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by different stimuli. It was found that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses, and pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Not only cytotoxic responses, but also the production of O2- and H2O2, and the release of myeloperoxidase were increased under these conditions. (+info)