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PubMed1990

Structural similarities among enzyme pterin binding sites as demonstrated by a monoclonal anti-idiotypic antibody.

BALB/c mice were immunized with a synthetic co-factor of the aromatic amino acid hydroxylases, 6,7-dimethyl-5,6,7,8-tetrahydropterin, conjugated to albumin. Hybridoma cell lines isolated from the immunized mice secreted monoclonal antibodies reacting specifically with the pterin molecule and monoclonal antibodies which were found to bind phenylalanine hydroxylase. Several lines of evidence were consistent with the anti-phenylalanine hydroxylase antibodies being anti-idiotype antibodies mimicking the pterin molecule and binding to the pterin binding site of phenylalanine hydroxylase. (a) An anti-idiotype monoclonal antibody, NS7, when reimmunized into mice produced anti-pterin antibodies consistent with NS7 being an internal image anti-idiotypic antibody. (b) NS7 antibody was prevented from binding to phenylalanine hydroxylase when a competitive inhibitor of phenylalanine hydroxylase enzyme activity, 6,7-dimethyl-7,8-dihydropterin, was bound to phenylalanine hydroxylase. (c) NS7 antibody was shown to bind to a wide range of pterin-requiring enzymes: phenylalanine, tyrosine and tryptophan hydroxylases, dihydropteridine reductase, dihydrofolate reductase, and sepiapterin reductase. Thus the NS7 antibody has successfully mimicked a common portion of the pterin cofactors utilized by these enzymes and demonstrated structure homology in their pterin binding sites despite their diverse function and little amino acid sequence homology except among the three aromatic amino acid hydroxylases.

Adrenal Glands↗

PubMed1990

Control of tetrahydrobiopterin synthesis in T lymphocytes by synergistic action of interferon-gamma and interleukin-2.

The control of (6R)-5,6,7,8-tetrahydrobiopterin (H4biopterin) synthesis in primed T cells was analyzed by using the human T cell leukemia virus type I (HTLV-I)-transformed T cell line MT-2. In contrast to the slowly progressing induction of H4biopterin synthesis during activation of resting T cells, it is completed during a 59-h period and is directed by a synergism of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Both GTP cyclohydrolase and (6R)-(1',2'-dioxopropyl)-5,6,7,8-tetrahydropterin synthase activities are induced by IFN-gamma. They are further enhanced by combined treatment with IL-2, which per se is ineffective. Furthermore, the combined treatment synchronizes the time periods of both maximum activities, now extending from 33 to 44 h. This period correlates with high cellular H4biopterin levels. It is preceded by a fast and transient period of H4biopterin increase which depends on the synergistic action of both IFN-gamma and IL-2. It coincides with a transient increase in sepiapterin reductase activity. In contrast to MT-2 cells, HTLV-I-transformed HUT 102 cells constitutively secrete IFN-gamma and express IFN-gamma mRNA. The accumulation of H4biopterin is suppressed by anti-IFN-gamma polyclonal antibody and correlates with constitutive expression of all H4 biopterin-synthesizing enzymes.

Alcohol Oxidoreductases↗

PubMed1985

Evaluation of neurotropic drug actions on tyrosine hydroxylase activity and dopamine metabolism in clonal cell lines.

Two clonal cell lines (the pheochromocytoma clone PC-12 and the neuroblastoma clone N1E-115) were used to compare direct and indirect drug effects on tyrosine hydroxylase and dopamine turnover. Both clones contain the cofactor of tyrosine hydroxylase, tetrahydrobiopterin, in sufficient concentrations. 2,4-Diamino-6-hydroxy-pyrimidine (DAO-Pyr), an inhibitor of GTP cyclohydrolase, which is the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, lowers DOPA production indicating that cofactor supply is a limiting factor for catecholamine synthesis. DOPA synthesis in the PC-12 cells can be stimulated by incubation with the natural cofactor tetrahydrobiopterin, but also by its possible precursors sepiapterin and dihydrobiopterin or the analogs methyl-tetrahydropterin and dihydropterin. The regulating enzyme for DOPA synthesis, tyrosine hydroxylase, can be inhibited by certain drugs either directly or indirectly by increasing dopamine concentrations in the cytoplasm after release from its vesicular stores. Using the neuroblastoma clone N1E-115 which lacks DOPA decarboxylase and thus contains only low levels of dopamine the site of action of certain drugs could be determined. Drugs affecting the tyrosine hydroxylase directly (alpha-methyl-para-tyrosine, apomorphine) decreased DOPA production in both clones, while drugs acting via interference with the vesicular stores (reserpine, amphetamine, nigericin) were effective only in the PC-12 cells. After total depletion of dopamine by nigericin at high concentrations or long-term incubation with 3-hydroxybenzyl-hydrazine (NSD 1015), DOPA production increased in the PC-12 cells indicating a usually occurring regulation of tyrosine hydroxylase by cytoplasmic dopamine. Dopamine concentration in the cytoplasm was calculated to be in the range of 1 X 10(-6) mol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

The C-6 proton of tetrahydrobiopterin is acquired from water, not NADPH, during de novo biosynthesis.

Tetrahydrobiopterin, the cofactor for the aromatic amino acid hydroxylases, is synthesized in mammals from GTP via a pathway involving both dihydropterin and tetrahydropterin intermediates. In this work, we have investigated the mechanism of conversion of the product formed from GTP, 7,8-dihydroneopterin triphosphate, into the tetrahydropterin intermediates. Tetrahydrobiopterin can be oxidized under conditions which yield pterin or pterin 6-carboxylate without exchange of the C-6 and C-7 protons. Using these techniques, a gas chromatography/mass spectrometry method was developed to determine that in the biosynthesis of tetrahydrobiopterin de novo, in preparations of bovine adrenal medulla, the C-6 proton of tetrahydrobiopterin is derived from water and not from NADPH. In contrast, the C-6 proton of tetrahydrobiopterin produced from sepiapterin (6-lactoyl-7,8-dihydropterin) comes from NADPH. The results are consistent with evidence for the formation of the first tetrahydropterin intermediate by a tautomerization without any requirement for NADPH.

Animals↗

PubMed1987

Preclinical biochemical pharmacology and toxicology of piritrexim, a lipophilic inhibitor of dihydrofolate reductase.

Piritrexim (PTX), 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidin e, formerly called BW 301U, is a potent small-molecule inhibitor of dihydrofolate reductase (DHFR) that enters cells rapidly by passive diffusion and thus does not depend upon the transport-mediated uptake that can limit cell entry of methotrexate (MTX). PTX is as active as MTX in inhibiting DHFR and mammalian cell growth. In vivo, PTX is active against Walker 256, L1210, P388, Sarcoma 180, and Ehrlich ascites tumors. After iv administration of [14C]PTX to rats, the elimination profile of intact drug from plasma was first order with a half-life (t1/2) of 38 minutes. PTX penetrates extensively into tissues and its tissue:plasma concentration ratios are generally 10-fold higher than those reported for MTX. When administered systemically, PTX inhibits the DHFR-dependent conversion of sepiapterin or 7,8-dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), demonstrating that PTX enters brain at pharmacologically relevant concentrations. Pharmacokinetic studies in the dog indicated a mean plasma t1/2 (after iv dose) of 2.15 hours, total body clearance of 0.625 liters/hr/kg and steady-state volume of distribution of 1.82 liters/kg; the absolute bioavailability was 0.64. Toxicologic studies were conducted in rats and dogs that received daily doses for 1, 5, or 90 days. In dogs, oral doses of 480 (single dose), 25 (5 daily doses), and 2.5 mg/kg (90 daily doses) were lethal, whereas 240 (single dose), 2.5 (5 daily doses), and 0.5 mg/kg (90 daily doses) produced reversible alterations in clinical toxicity and histopathologic parameters. The lethal toxicity of PTX in dogs given 25 mg/kg/day for 5 days is prevented by oral calcium leucovorin rescue with either 0.75 or 3.0 mg/kg every hour for 4 hours on any of the 5 treatment days. The general pharmacologic profile indicates that PTX should be free of CNS, cardiovascular, and respiratory side effects at clinically useful doses.

Animals↗

PubMed1979

Pterin deaminase from Bacillus megaterium. Purification and properties.

A pterin deaminase catalyzing the hydrolytic deamination of various pteridines was found in the bacterium, Bacillus megaterium, and partially purified from bacterial extract. The specific activity was raised 90-fold over that of the crude extract. The pH optimum is around 7.3, and the Km value for 6-carboxypterin is 1.3 mM. The molecular weight of the enzyme was estimated by gel filtration to be about 110,000. The enzyme deaminated pterin, 6-carboxypterin, biopterin, 6-methylpterin, 7-methylpterin, xanthopterin, 6-hydroxymethylpterin, sepiapterin, isosepiapterin, folic acid, and 6,7-dimethylpterin to their corresponding lumazines, whereas guanine, 7-carboxypterin, leucopterin, isoxanthopterin, and 6-methylisoxanthopterin did not serve as substrates. The enzyme was inhibited by PCMB and 8-azaguanine.

Aminohydrolases↗

PubMed1986

The pigmentary system of developing axolotls. III. An analysis of the albino phenotype.

The albino mutant in the Mexican axolotl (Ambystoma mexicanum) is analysed with respect to the differentiation of pigment cells. Pigment cells were observed with the transmission electron microscope in order to determine any unusual structural characteristics and to determine what happens to each of the cell types as development proceeds. Chemical analyses of pteridine pigments were also carried out, and the pattern of pteridines in albino animals was found to be more complex than, and quantitatively enhanced (at all developmental stages examined) over, the pattern observed in comparable wild-type axolotls. The golden colour of albino axolotls is due primarily to sepiapterin (a yellow pteridine) and secondarily to riboflavin (and other flavins). Coincident with enhanced levels of yellow pigments, xanthophore pigment organelles (pterinosomes) in albino skin reach a mature state earlier than they do in wild-type axolotl skin. This morphology is conserved throughout development in albino animals whereas it is gradually lost in the wild type. Unpigmented melanophores from albino axolotls are illustrated for the first time, and in larval albino axolotls the morphology of these cells is shown to be very similar to xanthophore morphology. In older animals xanthophores are easily distinguished from unpigmented melanophores. Iridophores seem to appear in albino skin at an earlier stage than they have been observed in wild-type skin. Morphologically, wild-type and albino iridophores are identical.

Albinism↗

PubMed1985

The enzymatic conversion of dihydroneopterin triphosphate to tripolyphosphate and 6-pyruvoyl-tetrahydropterin, an intermediate in the biosynthesis of other pterins in Drosophila melanogaster.

The enzyme, previously called "sepiapterin synthase A," has been purified by approximately 700-fold from the heads of Drosophila melanogaster. This enzyme catalyzes the Mg2+-dependent conversion of 2-amino-4-oxo-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydrop teridine triphosphate (dihydroneopterin triphosphate or H2-NTP) to two products, one of which we have identified as tripolyphosphate. The other product is a phosphate-free, unstable compound which is an intermediate in the biosynthesis of several other naturally occurring pterins in Drosophila. This product is stable enough under anaerobic conditions to allow it to be characterized as 6-pyruvoyl-5,6,7,8-tetrahydropterin (6-pyruvoyl-H4-pterin). The 3-carbon side chain was identified as a pyruvoyl group on the basis of the susceptibility of the enzymatic product to reduction with tritiated sodium borohydride and the determination of the amounts and the sites of incorporation of tritium resulting from this reduction. From these observations, we suggest that this enzyme be renamed "6-pyruvoyl-H4-pterin synthase."

Alcohol Oxidoreductases↗

PubMed1986

Synthesis, utilization, and structure of the tetrahydropterin intermediates in the bovine adrenal medullary de novo biosynthesis of tetrahydrobiopterin.

The biosynthesis of two tetrahydropterin intermediates (H4pterin-1 and H4pterin-2), their conversion to tetrahydrobiopterin, and their overall chemical structures are described. A new high performance liquid chromatographic separation of these and other tetrahydropterins is also described. The biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in the presence of the bovine adrenal medullary biosynthetic enzymes, Mg2+, NADPH. The biosynthesis of H4pterin-2 occurs under identical conditions, and the compound accumulates in the presence of 1 to 10 microM of N-acetylserotonin, an inhibitor of sepiapterin reductase. At higher concentrations of the inhibitor, the synthesis of H4pterin-2 is also inhibited, and H4pterin-1 accumulates. H4pterin-1 also accumulates in the absence of NADPH. In the presence of NADPH the biosynthetic enzymes convert both intermediates to tetrahydrobiopterin at rates which are greater than the rate of conversion of dihydroneopterin triphosphate to tetrahydrobiopterin. Electrochemical, UV/VIS, oxidation, and ionization properties identify the compounds as tetrahydropterins. The side chain structures of the compounds were determined by a combination of chemical means. The structures of the compounds are 6R-(1',2'-dioxopropyl)-tetrahydropterin (H4pterin-1) and 6R-(L-1'-hydroxy-2'-oxopropyl)-tetrahydropterin (H4pterin-2). The data indicate that the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in three steps: 1) formation of H4pterin-1 in the presence of Mg2+, 2) NADPH-dependent conversion of H4pterin-1 to H4pterin-2, and 3) NADPH-dependent conversion of H4pterin-2 to tetrahydrobiopterin.

Adrenal Medulla↗

PubMed1984

Enzymatic conversion of dihydroneopterin triphosphate to the pyrimidodiazepine intermediate involved in the biosynthesis of the drosopterins in Drosophila melanogaster.

The compound 2-amino-4-oxo-6-acetyl-7,8-dihydro-3H,9H-pyrimido[4,5-b]-[1,4]diazepine (pyrimidodiazepine or PDA, for short) is a precursor of the red eye pigments called the drosopterins in Drosophila melanogaster. The precursor of PDA is 2-amino-4-oxo-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydrop teridine triphosphate (dihydroneopterin triphosphate or H2-NTP). The synthesis of of PDA from H2-NTP requires reduced glutathione, another thiol such as 2-mercaptoethanol, Mg2+, and at least three enzymes: one that is missing in the eye color mutant, sepia; one that is present only in limited quantities in the mutant, clot; and a third one that has been described as sepiapterin synthase A. The last enzyme is present only in relatively small quantities in the mutant, purple. Because PDA is two electrons more reduced than H2-NTP, it would appear that the reducing power needed for this transformation is probably supplied by glutathione. Oxidized glutathione cannot replace reduced glutathione in the system. The yield of PDA produced enzymatically from H2-NTP can be as high as 40% under optimal conditions.

Animals↗

PubMed1984

Purification and properties of the enzymes from Drosophila melanogaster that catalyze the conversion of dihydroneopterin triphosphate to the pyrimidodiazepine precursor of the drosopterins.

The enzyme system responsible for the conversion of 2-amino-4-oxo-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihyd roptridine triphosphate (dihydroneopterin triphosphate or H2-NTP) to 2-amino-4-oxo-6-acetyl-7,8-dihydro-3H,9H-pyrimido[4,5-b]-[1,4]diazepine (pyrimidodiazepine or PDA), a precursor to the red eye pigments, he drosopterins, has been purified from the heads of Drosophila melanogaster. The PDA-synthesizing system consists of two components, a heat-stable enzyme and a heat-labile enzyme. The heat-stable enzyme can be replaced by sepiapterin synthase A, a previously purified enzyme required for the Mg2+-dependent conversion of H2-NTP to an unstable compound that appears to be 6-pyruvoyltetrahydropterin (pyruvoyl-H4-pterin). The heat-labile enzyme, purified to near-homogeneity and termed PDA synthase (Mr = 48,000), catalyzes the conversion of pyruvoyl-H4-pterin to PDA in a reaction requiring the presence of reduced glutathione. Because PDA is two electrons more reduced than pyruvoyl-H4-pterin, the reducing power required for this transformation is probably supplied by glutathione. The PDA-synthesizing system requires the presence of another thiol-containing compound such as 2-mercaptoethanol when incubation conditions 2-mercaptoethanol is no longer required. Evidence is presented to indicate that the Drosophila eye color mutant, sepia, is missing PDA synthase.

Alcohol Oxidoreductases↗

PubMed1984

The pigmentary system of developing axolotls. I. A biochemical and structural analysis of chromatophores in wild-type axolotls.

A biochemical and transmission electron microscopic description of the wild-type pigment phenotype in developing Mexican axolotls (Ambystoma mexicanum) is presented. There are three pigment cell types found in adult axolotl skin - melanophores, xanthophores and iridophores. Both pigments and pigment cells undergo specific developmental changes in axolotls. Melanophores are the predominant pigment cell type throughout development; xanthophores occur secondarily and in fewer numbers than melanophores; iridophores do not appear until well into the larval stage and remain thereafter as the least frequently encountered pigment cell type. Ultrastructural differences in xanthophore organelle (pterinosome) structure at different developmental stages correlate with changes in the pattern of pteridine biosynthesis. Sepiapterin, a yellow pteridine, is present in larval axolotl skin but not in adults. Riboflavin (also yellow) is present in minimal quantities in larval skin and large quantities in adult axolotl skin. Pterinosomes undergo a morphological "reversion" at some point prior to or shortly after axolotls attain sexual maturity. Correlated with the neotenic state of the axolotl, certain larval pigmentary features are retained throughout development. Notably, the pigment cells remain scattered in the dermis such that no two pigment cell bodies overlap, although cell processes may overlap. This study forms the basis for comparison of the wild type pigment phenotype to the three mutant phenotypes-melanoid, axanthic and albino-found in the axolotl.

Ambystoma↗

PubMed1983

Participation of pterins in the control of lymphocyte stimulation and lymphoblast proliferation.

Biopterin accumulation had been demonstrated as the result of normal and, especially, of malignant hemopoietic cell proliferation (Ziegler, I. et al. Blut, 44: 231-240, and 261-270, 1982). Among 13 major intermediates of pterin metabolism and two lumazines, xanthopterin (but not dihydroxanthopterin) was found to inhibit cell proliferation (half-maximum inhibition at 1.8 X 10(-5) M) during concanavalin A-induced lymphocyte activation in pre-stimulated lymphocytes and in a lymphoid cell line grown in continuous culture (LS-2). LS-2 cells exposed to maximum inhibitor concentrations largely maintained the initial thymidine incorporation rate for about 40 hr but failed to enter logarithmic growth. Isoxanthopterin inhibition was found only in serum-free medium, since it is trapped by the alpha-acid glycoprotein present in the serum. The reduced biopterin derivatives, sepiapterin, dihydrobiopterin, and tetrahydrobiopterin, are costimulators during concanavalin A-induced lymphocyte activation. Their costimulatory effect follows an optimum curve and peaks at 1.5 to 3 X 10(-5) M. It is highest at the suboptimal and supraoptimal concanavalin A concentration. The D-erythro isomer dihydroneopterin was inactive. The results indicate that the anabolic-reduced biopterin derivatives are not simply lymphocytic products, but, in combination with the catabolites xanthopterin and isoxanthopterin, they also participate in the regulation of lymphocyte activation. Hence, they fulfill the criteria for lymphokines.

Animals↗

PubMed1995

Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.

Amino Acid Oxidoreductases↗

PubMed1993

Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW 264).

The murine macrophage cell line RAW 264 constitutively synthesizes tetrahydrobiopterin (BH4), the cofactor required for the hydroxylation of the aromatic amino acids and for the production of nitric oxide. Stimulation of the cells with interferon-gamma and lipopolysaccharide induced the production of nitric oxide and increased BH4 levels further. When the cells were stimulated in the presence of 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of BH4 biosynthesis, biopterin levels decreased by 90% within 6 hr, whereas nitrite production was essentially unaffected. Pretreatment of the cells for 12 hr with DAHP decreased intracellular BH4 concentrations by > 95% yet inhibited the cytokine-stimulated production of nitric oxide by only 50%. However, pretreatment with DAHP plus N-acetylserotonin, an inhibitor of sepiapterin reductase, the terminal enzyme of the BH4 biosynthetic pathway, decreased biopterin levels by > 99% and inhibited nitric oxide synthesis by 90%. This inhibition could be reversed by loading the cells with dihydrobiopterin, a precursor of BH4 via the dihydrofolate reductase salvage pathway. In addition, these studies revealed that N-acetylserotonin has a direct inhibitory effect on nitric oxide synthesis, acting in a BH4-independent manner. The results presented here support previous suggestions, based on experiments with isolated enzymes, that BH4 is absolutely required for cytokine-stimulated nitric oxide production in macrophages and they suggest that only a small fraction of the total intracellular BH4 pool in macrophages is utilized in the production of fully active nitric oxide synthase.

Amino Acid Oxidoreductases↗

PubMed1993

In a concerted action kit ligand and interleukin 3 control the synthesis of serotonin in murine bone marrow-derived mast cells. Up-regulation of GTP cyclohydrolase I and tryptophan 5-monooxygenase activity by the kit ligand.

Mouse bone marrow-derived mast cells (BMMC) store and release serotonin whose synthesis is initiated by tryptophan 5-monooxygenase. (6R)-H4biopterin serves as the natural cofactor for this reaction. GTP cyclohydrolase I catalyzes the first and rate-limiting step of its synthesis. In this study we demonstrate that among a panel of growth-promoting cytokines including kit ligand (KL), interleukin 3 (IL-3), IL-4, IL-9, and nerve growth factor, KL selectively enhances the synthesis of H4biopterin through up-regulation of GTP cyclohydrolase I activity to 6.2-fold levels. The activities of the subsequent enzymes 6-pyruvoyl-H4pterin synthase and sepiapterin reductase remain unaffected. The activity of tryptophan 5-monooxygenase was selectively enhanced 4.5-fold by the combination of IL-3 with KL. All other factors could not substitute for KL. The constitutive high activity of aromatic L-amino acid decarboxylase is not different in cells cultured in IL-3 and/or KL. In consequence, the concerted action of IL-3 and KL on the GTP cyclohydrolase I and the tryptophan 5-monooxygenase reaction enhances the production of serotonin to about 20-fold levels. Additionally, KL specifically causes the release of about half of total serotonin produced. Hence, our data demonstrate a novel role of these cytokines for the function of mouse BMMC and provide a coherent view of the regulation of serotonin synthesis in this cell type.

5-Hydroxytryptophan↗

PubMed1996

Genetically engineered smooth muscle cells as linings to improve the biocompatibility of cardiovascular prostheses.

BACKGROUND: The seeding of the blood-contacting surfaces of cardiovascular prostheses with autologous endothelial cells to improve their biocompatibility has had little success. In most instances, cells have sloughed off under flow conditions. The performance of left ventricular assist devices (LVADs) designed to stabilize patients awaiting donor hearts for transplantation has been remarkably good. After prolonged implantation, pump surfaces become covered with a pannus of smooth muscle-like cells (myofibroblasts). Occasional islands of endothelial cells have been identified on top of such cell layers. Therefore, in an attempt to accelerate the beneficial conditioning and improve biomaterial-blood compatibility of LVAD internal surfaces, their seeding with autologous, genetically engineered smooth muscle cells (SMCs) was investigated. METHODS AND RESULTS: Since routine testing of the Thermocardiosystems HeartMate LVAD is carried out in calves, SMCs were isolated from calves, propagated in culture, and transduced with NO synthase genes to yield stable production of NO. Previous studies had demonstrated that SMCs attached strongly to the biomaterials that compose the internal surfaces of LVADs. Transduction of NO synthase gene expression in the SMCs was achieved by electroporation and antibiotic (G418) selection. Inhibition of smooth muscle cell proliferation by NO has been documented, and the same molecule has been shown to inhibit platelet adhesion to cell surfaces. Cells transduced with NO synthase expressed enzyme protein at consistently high levels for several passages in culture; however, NO production was dependent on the supplementation of culture medium with a source of tetrahydrobiopterin (sepiapterin). Under such conditions, transduced cells were growth-inhibited compared with mock-transfected controls. Induction of GTP cyclohydrolase (the rate-limiting enzyme for the production of tetrahydrobiopterin) expression also resulted in NO production by NO synthase-transduced cells. CONCLUSIONS: Preliminary studies have shown that SMCs form strong attachments to the surface materials of LVADs and that their proliferation rates could be controlled after transformation with NO synthase under conditions that support production of NO. Therefore, genetically engineered SMCs may provide an improved blood biomaterial interface for cardiovascular prostheses.

Animals↗

PubMed1996

Adenoviral gene transfer of nitric oxide synthase: high level expression in human vascular cells.

OBJECTIVES: Nitric oxide synthases (NOS) generate nitric oxide (NO), a second messenger with key regulatory roles. In the cardiovascular system, deficient endothelial NO production is an early, persistent feature of atherosclerosis and vascular injury. Accordingly, the NOS isoforms represent attractive targets for vascular gene therapy. We aimed to generate and evaluate an adenoviral vector for gene transfer of an NOS isoform to vascular cells. METHODS: We constructed a recombinant adenovirus, Ad.nNOS, for gene transfer of the neuronal isoform of NOS (nNOS) and characterized its expression in 293 cells, human vascular smooth muscle cells (hVSMC) and human umbilical vein endothelial cells (HUVEC). NOS expression was analyzed by Western immunoblotting, and NOS enzyme activity in response to receptor-dependent and receptor-independent agonists was determined by Griess assay or by NO chemiluminescence. RESULTS: Ad.nNOS-infected 293 cells expressed high levels of functional nNOS enzyme, even higher than in 293.NOS cells (a cell line that expresses supraphysiologic levels of nNOS). In hVSMC, nNOS activity reached levels 50% of those seen in 293.NOS cells. nNOS expression and activity in hVSMC increased linearly with titer of Ad.nNOS. NO production in hVSMC was stimulated both by calcium ionophore and by physiologic agonists such as acetylcholine or bradykinin. In HUVEC, endogenous NOS activity was significantly augmented by Ad.nNOS infection. Supplementation with the tetrahydrobiopterin precursor sepiapterin enhanced NOS activity in all cells. CONCLUSIONS: Ad.nNOS, a novel adenoviral vector for gene transfer of NOS, generates high-level nNOS expression in a variety of vascular cell types. nNOS activity in hVSMC is physiologically regulated and of a magnitude comparable to native eNOS activity in HUVEC. Our findings demonstrate Ad.nNOS to be a versatile and efficient tool for nNOS gene transfer, with widespread potential applications in cell culture and for gene therapy.

Adenoviridae↗