Nitric oxide synthesis in infantile hypertrophic pyloric stenosis.
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Amino Acid Oxidoreductases↗
Biomedical subjects
T Michel
Publications and source records attributed to T Michel.
Molecular cloning and characterization of human endothelial nitric oxide synthase.
The constitutive calcium/calmodulin-dependent nitric oxide (NO) synthase expressed in vascular endothelium shares common biochemical and pharmacologic properties with neuronal NO synthase. However, recent cloning and molecular characterization of NO synthase from bovine endothelial cells indicated the existence of a family of constitutive NO synthases. Accordingly, we undertook molecular cloning and sequence analysis of human endothelial NO synthase. Complementary DNA clones predict a protein of 1,203 amino acids sharing 94% identity with the bovine endothelial protein, but only 60% identity with the rat brain NO synthase isoform. Northern blot analysis with an endothelial-derived cDNA identified a 4.6-4.8 kb mRNA transcript in HUVEC and in situ hybridization localized transcripts to vascular endothelium but not neuronal tissue.
Amino Acid Oxidoreductases↗
Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform.
Nitric oxide (NO) is a ubiquitous intercellular messenger molecule synthesized from the amino acid L-arginine by NO synthases in diverse cells and tissues. NO is synthesized in vascular endothelial cells and appears to play an important role in the control of blood pressure and platelet aggregation. A detailed understanding of the regulation of NO synthesis by endothelial cells has been hampered by the lack of molecular clones for endothelial NO synthase; the isolation and characterization of such clones is reported herein. The constitutive NO synthases present in endothelial cells and in brain share common biochemical and pharmacologic features. We purified NO synthase from bovine brain and determined the amino acid sequence of several tryptic peptides. The sequence of the bovine brain peptides is nearly identical to the deduced amino acid sequence previously determined for the rat brain NO synthase. These sequence data were utilized to design PCR-generated NO synthase cDNA probes, which were used to isolate clones encoding NO synthase from a bovine aortic endothelial cell (BAEC) cDNA library. A full-length NO synthase cDNA clone was isolated, representing a protein of 1205 amino acids with a molecular mass of 133 kDa; transfection of this clone in a heterologous expression system demonstrated the expected enzymatic activity. The deduced amino acid sequence of the BAEC NO synthase cDNA differs at numerous residues from the sequence determined for the purified bovine brain protein and shows 50-60% sequence identity with recently isolated molecular clones for murine macrophage and rat brain NO synthase isoforms. Bovine genomic Southern blots probed with bovine brain and BAEC NO synthase cDNA probes identify distinct bands, indicating that these cDNAs are the products of different genes. Prolonged treatment of BAECs with the cytokine tumor necrosis factor alpha, which we have previously shown to result in a marked increase in NO synthase activity, is associated with a decrease in the abundance of the 4.8-kilobase BAEC NO synthase transcript. The increase in BAEC NO synthase activity induced by tumor necrosis factor alpha is thus likely to involve posttranscriptional mechanisms or the induction of a distinct endothelial NO synthase isoform.
Amino Acid Oxidoreductases↗
S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds.
Endothelium-derived relaxing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular milieux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhydryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism of NO and raise the possibility of an additional type of cellular regulatory mechanism.
Animals↗
Platelet alpha 2 adrenoceptor characteristics during the morning increase in platelet aggregability.
The incidence of myocardial infarction and sudden cardiac death increases in the morning, as do platelet aggregability and sympathetic activity. We considered the possibility that increased platelet aggregability in the morning was due to an increase in platelet alpha 2 adrenergic receptor number or agonist binding affinity, or to a temporal disparity between the increase in sympathetic activity that accompanies arising and the anticipated decrease in platelet high affinity alpha 2 adrenoceptors. To evaluate these possibilities, we studied eight healthy male volunteers (20-35 years) before and 1-1.5 and 3-4 h after they arose at 08.00 h and performed routine morning activities. Platelet aggregability was determined in platelet-rich plasma; alpha 2 adrenoceptor density and agonist binding affinity were determined in intact platelets and in membrane preparations using the alpha 2 selective ligand [3H]-yohimbine. The threshold concentration of epinephrine required to produce biphasic aggregation decreased (i.e. platelet aggregability increased) from 3.6 +/- 1.2 to 0.9 +/- 0.3 microM (P less than 0.05) after arising. However, alpha 2 adrenoceptor density (380 +/- 71 to 365 +/- 51 sites per platelet) and agonist binding affinity assessed simultaneously did not change after arising, suggesting that the increase in platelet aggregability is due to factors extrinsic to the platelets or to an intra-platelet mechanism distal to the receptor level.
Adenosine Diphosphate↗
Molecular cloning of constitutive endothelial nitric oxide synthase: evidence for a family of related genes.
Nitric oxide (NO) is synthesized in vascular endothelial cells, and appears to play an important role in the control of blood pressure and platelet aggregation. A detailed understanding of the regulation of NO synthesis by endothelial cells has been hampered by the lack of molecular clones for endothelial NO synthase; we now report the isolation and characterization of such clones. The constitutive NO synthases present in endothelial cells and in brain share common biochemical and pharmacologic features. We purified NO synthase from bovine brain, and determined the amino acid sequence of several tryptic peptides. These sequence data were utilized to design PCR-generated NO synthase cDNA probes, which were used to isolate clones encoding NO synthase from a bovine aortic endothelial cell (BAEC) cDNA library. A full-length NO synthase cDNA clone was isolated, representing a protein of 1,205 amino acids with a molecular mass of 133 kDa. The deduced amino acid sequence of the BAEC NO synthase cDNA differs at numerous residues from the sequence determined for the purified bovine brain protein, and shows 50-60% sequence identity with recently isolated molecular clones for murine macrophage and rat brain NO synthase isoforms. Bovine genomic Southern blots probed with bovine brain and BAEC NO synthase cDNA probes identify distinct bands, indicating that these cDNAs are the products of different genes. Prolonged treatment of BAEC with the cytokine TNF alpha, which results in a marked increase in NO synthase activity, is associated with a decrease in the abundance of the 4.8-kb BAEC NO synthase transcript.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Oxidoreductases↗
Culture and decontamination methods affecting enumeration of phages infecting Bacteroides fragilis in sewage.
A new medium has been adapted for the growth of Bacteroides fragilis so that its phages can be recovered from environmental samples, and its efficiency has been assessed. Polyvinylidene difluoride membranes allow significantly higher recoveries among different membrane filters used to decontaminate the samples. In all cases, a number of phages remain in the filters and a percentage of them can be recovered by treatment with an eluant.
Bacteriophages↗
Effects of interferon-gamma on nitric oxide synthase activity and endothelin-1 production by vascular endothelial cells.
Given the pivotal role suggested for IFN-gamma in immune diseases of the vascular wall, we investigated the effects of IFN-gamma on nitric oxide (NO) and endothelin-1 (ET-1) expression in bovine aortic endothelial cells (BAEC). We have previously reported that TNF-alpha enhanced NO synthase activity in BAEC as assessed by quantifying release of bioactive NO with reporter monolayers and measuring conversion of L-[14C]arginine to L-[14C] citrulline. In murine macrophages IFN-gamma synergizes with TNF-alpha or lipopolysaccharide to induce robust increases in calcium-independent NO synthase activity. In this study we have found that IFN-gamma alone failed to have a significant effect on NO synthase activity in BAEC. In contrast to murine macrophages, IFN-gamma inhibited TNF-alpha-stimulated induction of endothelial NO synthase activity in a concentration-dependent manner. This observation suggests that there is major difference in the response of BAEC and murine macrophages to IFN-gamma. A second major aim of this study was to determine the effect of IFN-gamma on preproET-1 mRNA expression and ET-1 secretion rates in BAEC. IFN-gamma alone had little or no effect on ET-1 mRNA levels and basal ET release when measured for 8 h. However, cotreatment with IFN-gamma potentiated the stimulatory effect of TNF-alpha on BAEC ET-1 mRNA transcript levels and ET release. In contrast, pretreatment of cells with IFN-gamma for 16-24 h blunted the stimulatory effect of TNF-alpha. These findings suggest that endothelial cell expression of vasoactive mediators is modified by the temporal interplay of at least two immune mediators, IFN-gamma and TNF-alpha.
Amino Acid Oxidoreductases↗
Expression of a G protein subunit, alpha i-1, in Balb/c 3T3 cells leads to agonist-specific changes in growth regulation.
Cellular receptors for many hormones, neurotransmitters, and growth factors are coupled to intracellular effector enzymes or ion channels through a set of heterotrimeric G proteins. In order to determine whether isoforms of G protein alpha subunits contribute differentially to mitogenic responses, we introduced an alpha subunit isoform, alpha i-1, into Balb/c 3T3 cells that normally lack this subtype. Balb/c 3T3 cells transfected with a plasmid containing cDNA encoding alpha i-1 expressed the alpha i-1 protein as judged both by the appearance of immunoreactive alpha i-1 protein on Western blots and by two-dimensional analysis of the proteins [32P]ADP-ribosylated by pertussis toxin. The amount of alpha i-1 expressed is less than the amount of alpha subunits endogenously present in these cells. Expression of alpha i-1 in the transfected cells slightly blunts stimulation of adenylylcyclase by GTP, guanosine 5'-3-O-(thio)triphosphate, or forskolin, but has no major effect on the ability of thrombin to inhibit the enzyme. In contrast, the expression of alpha i-1 has significant effects on cell growth and on the mitogenic response to thrombin. The alpha i-1-transfected cells have a doubling time that is twice as long as control cells transfected with the same plasmid without a cDNA insert. Despite their slower growth, thymidine incorporation in response to thrombin is greater in transfected than in control cells. Thrombin-stimulated DNA synthesis is sensitive to inhibition by pertussis toxin and is 5-fold more sensitive to inhibition by pertussis toxin in transfected cells than in control cells. The changes are receptor-specific since the mitogenic response to platelet-derived growth factor is indistinguishable between control and transfected cells. These studies suggest that the alpha i subunit composition of the cell may have profound effects on its growth and its response to stimulation through a specific cell surface receptor.
3T3 Cells↗
Nitric oxide synthesis in endothelial cells: evidence for a pathway inducible by TNF-alpha.
Nitric oxide (NO) release accounts for the biological activity of endothelium-derived relaxing factor. Given that tumor necrosis factor-alpha (TNF-alpha) has been implicated as an important mediator in septic shock, we explored whether TNF-alpha enhances L-arginine-dependent synthesis of NO and L-citrulline in endothelial cells. The release of NO was detected in a coincubation bioassay where measurement of guanosine 3',5'-cyclic monophosphate (cGMP) production in reporter monolayers, namely glomerular mesangial cells or fetal lung fibroblasts, reflected activation of soluble guanylate cyclase. Reporter monolayer cGMP content was greater in the presence of TNF-alpha-treated bovine aortic and renal artery endothelial cells than in the presence of vehicle-treated endothelial cells. TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were first evident at 8 h and maximal at 16-24 h. In addition, TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were abrogated by hemoglobin and methylene blue, blunted by N omega-nitro-L-arginine and augmented by superoxide dismutase and the calcium agonist bradykinin. These observations suggested that TNF-alpha enhanced release of NO. Furthermore, the formation of L-[14C]citrulline from L-[14C]arginine, as determined by quantitative cation-exchange chromatography and thin-layer chromatography, was enhanced by TNF-alpha in a time- and concentration-dependent manner. Thus it is evident that endothelial cells release NO for a prolonged period in response to TNF-alpha and transiently when stimulated with calcium agonists. The prolonged release of NO from TNF-alpha-stimulated endothelial cells may be implicated in the pathogenesis of septic shock.
Animals↗
Differential degradation rates of the G protein alpha o in cultured cardiac and pituitary cells.
Signal transduction in biological membranes is modulated by a family of GTP-binding proteins termed G proteins. Differences in the tissue-specific expression of G protein subtypes suggest that the levels of individual G proteins may be an important determinant of the hormonal response in a given cell type. We have used a polyclonal antibody raised against the purified G protein, alpha o to study alpha o in the rat pituitary cell line GH4 and in primary rat cardiocytes in culture by quantitative immunoprecipitation. Biosynthetic labeling and specific immunoprecipitation of alpha o in pulse-chase experiments demonstrated that the t1/2 for alpha o degradation is 28 +/- 7 h (n = 4) in GH4 pituitary cells and is greater than 72 h (n = 4) in cardiocytes. The steady-state level of alpha o protein is similar in both cell types as measured by Western blots. Northern blots of poly(A)-selected mRNA from these two cell types were probed with labeled alpha o cDNA and showed they have similar alpha o mRNA levels. The observation of different degradation rates, but similar steady-state protein levels, suggests that the rate of alpha o synthesis is different in GH4 cells and cardiocytes. Since mRNA levels are approximately equal in both, our studies imply that protein translation controls may be important determinants of G protein alpha subunit concentrations in biological membranes.
Animals↗
Streptokinase therapy and cholesterol embolization.
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Cholesterol↗
Genes for two homologous G-protein alpha subunits map to different human chromosomes.
Signal transduction across biological membranes is modulated by a family of related GTP-binding proteins termed G proteins. These G proteins have a heterotrimeric structure composed of alpha, beta, and gamma subunits. The alpha subunits of the G proteins bind GTP and appear to determine the biochemical specificity of the protein. We have recently cloned and characterized cDNA encoding two G-protein alpha subunits, alpha i and alpha h. The former is a substrate for ADP-ribosylation by pertussis toxin. The protein corresponding to alpha h has not yet been identified. These cDNAs encode proteins, which demonstrate 90% sequence identity to one another and also show marked similarity to other G proteins. The present studies were designed to determine whether the genes for these related proteins are clustered on a single human chromosome. Genomic DNA isolated from a panel of mouse-human hybrid cell lines was analyzed by hybridization to cDNAs for alpha i and alpha h. Based on the distribution patterns of alpha i and alpha h in cell hybrids, the gene for alpha i was assigned to human chromosome 7, and the gene for alpha h assigned to chromosome 12. These data suggest that the G-protein gene family may be distributed over at least two human chromosomes.
Animals↗
Molecular cloning and characterization of cDNA encoding the GTP-binding protein alpha i and identification of a related protein, alpha h.
We have cloned and characterized cDNA encoding alpha i, the GTP-binding subunit of Gi, a protein that mediates hormonal inhibition of adenylate cyclase and hormonal regulation of other membrane functions. We have also identified cDNA encoding a putative protein, which we have named alpha h, that is highly homologous to alpha i but different from other known GTP-binding proteins. Both cDNAs were isolated from a bovine pituitary library. The cDNA encoding alpha i was identified by finding that the amino acid sequence determined for two tryptic peptides from alpha i agreed exactly with amino acid sequences deduced from the cDNA. We also determined the amino acid sequence of peptides derived from alpha o, a related 39-kDa protein purified from bovine brain. These sequences are approximately 75% identical to the sequence determined for alpha i. Southern blot analysis of bovine genomic DNA, using as probes radiolabeled cDNAs for alpha i, alpha h, and the alpha subunit of a related protein, transducin, showed that each probe recognized different genomic DNA fragments. Our results suggest a further level of complexity in the organization of the G-protein gene family, with multiple G proteins of very similar structural properties likely to be identified as products of distinct genes.
Adenosine Diphosphate Ribose↗
Effect of pertussis toxin on alpha 2-adrenoceptors: decreased formation of the high-affinity state for agonists.
Administration of pertussis toxin to hamsters abolishes the alpha 2-adrenergic inhibition of adenylate cyclase in their adipocytes. The toxin did not modify the number of adipocyte alpha 2-adrenoceptors or their affinity for antagonists. In contrast, the affinity for agonists was significantly diminished in adipocyte membranes obtained from pertussis toxin-treated hamsters as compared to the controls. This decrease in affinity was due to a significant diminution in the proportion of sites displaying the high-affinity state for agonists. It is concluded that pertussis toxin induces a shift in the proportion of sites in high- and low-affinity states. for agonists towards the low-affinity conformation.
Adenylate Cyclase Toxin↗
Cell-free desensitization of catecholamine-sensitive adenylate cyclase. Agonist- and cAMP-promoted alterations in turkey erythrocyte beta-adrenergic receptors.
Conditions have been developed for desensitizing the beta-adrenergic receptor-coupled adenylate cyclase of turkey erythrocytes in a cell-free system. Desensitization is observed when cell lysates are incubated with isoproterenol or cAMP analogs for 30 min at 37 degrees C. Maximally effective concentrations of isoproterenol produce a 41.0 +/- 1.55% loss of iosproterenol-stimulated and a 15.0 +/- 2.35% loss of fluoride-stimulated enzyme activity. cAMP causes a 26.5 +/- 1.5% fall in isoproterenol-stimulated and a 21.5 +/- 4.4% fall in fluoride-sensitive activity. Desensitization by isoproterenol is dose-dependent, stereospecific, and blocked by the beta-adrenergic antagonist propranolol. Cell-free desensitization required ATP, Mg2+, and factor(s) present in the soluble fraction of the cell. Nonphosphorylating analogs of ATP did not support desensitization. Desensitization by agonist or cAMP in the cell-free system caused structural alterations in the beta-adrenergic receptor peptides apparent as an altered mobility of the photoaffinity labeled receptor peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As with the desensitization reaction, supernatant factors and ATP were also required for the agonist or cAMP-promoted receptor alterations. These data indicate that beta-adrenergic agonists promote a cAMP-mediated process which leads to receptor alterations and desensitization. The reactions involved in this process require ATP and soluble cellular factors. Additional processes must also occur to account for decreases in fluoride-sensitive enzyme activity. The availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.
8-Bromo Cyclic Adenosine Monophosphate↗
Plasma membrane receptors.
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Adenylyl Cyclases↗
Hormonal inhibition of adenylate cyclase. alpha 2 Adrenergic receptors promote release of [3H]guanylylimidodiphosphate from platelet membranes.
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Adenylyl Cyclase Inhibitors↗