Inducible nitric oxide synthase: identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

Mutagenesis of palmitoylation sites in endothelial nitric oxide synthase identifies a novel motif for dual acylation and subcellular targeting.

The endothelial nitric oxide synthase (ec-NOS) plays a key role in the transduction of signals from the bloodstream to the underlying smooth muscle. ecNOS undergoes a complex series of covalent modifications, including myristoylation and palmitoylation, which appear to play a role in ecNOS membrane association. Mutagenesis of the myristoylation site, which prevents both myristoylation and palmitoylation, blocks ecNOS targeting to cell membranes. Further, as described for some G-protein alpha subunits, both membrane association and palmitoylation of ecNOS are dynamically regulated: in response to agonists, the enzyme undergoes partial redistribution to the cell cytosol concomitant with depalmitoylation. To clarify the role of palmitoylation in determining ecNOS subcellular localization, we have constructed palmitoylation-deficient mutants of ecNOS. Serine was substituted for cysteine at two potential palmitoylation sites (Cys-15 and Cys-26) by site-directed mutagenesis. Immunoprecipitation of ecNOS mutants following cDNA transfection and biosynthetic labeling with [3H]palmitate revealed that mutagenesis of either cysteine residue attenuated palmitoylation, whereas replacement of both residues completely eliminated palmitoylation. Analysis of N-terminal deletion mutations of ecNOS demonstrated that the region containing these two cysteine residues is both necessary and sufficient for enzyme palmitoylation. The cysteines thus identified as the palmitoylation sites for ecNOS are separated by an unusual (Gly-Leu)5 sequence and appear to define a sequence motif for dual acylation. We analyzed the subcellular distribution of ecNOS mutants by differential ultracentrifugation and found that mutagenesis of the ecNOS palmitoylation sites markedly reduced membrane association of the enzyme. These results document that ecNOS palmitoylation is an important determinant for the subcellular distribution of ecNOS and identify a new motif for the reversible palmitoylation of signaling proteins.

Localization of choline acetyltransferase in rat peripheral sympathetic neurons and its coexistence with nitric oxide synthase and neuropeptides.

Indirect immunofluorescence methods using a mouse monoclonal antibody raised to rat choline acetyltransferase (ChAT) revealed dense networks of ChAT-immunoreactive fibers in the superior cervical ganglion, the stellate ganglion, and the celiac superior mesenteric ganglion of the rat. Numerous and single ChAT-immunoreactive cell bodies were observed in the stellate and superior cervical ganglia, respectively. The majority of ChAT-immunoreactive fibers in the stellate and superior cervical ganglia were nitric oxide synthase (NOS) positive. Some ChAT-immunoreactive fibers contained enkephalin-like immunoreactivity. Virtually all ChAT-positive cell bodies in the stellate ganglion were vasoactive intestinal polypeptide (VIP)-positive, and some were calcitonin gene-related peptide (CGRP)-positive. After transection of the cervical sympathetic trunk almost all ChAT- and NOS-positive fibers and most enkephalin- and CGRP-positive fibers disappeared in the superior cervical ganglion. The results suggest that most preganglionic fibers are cholinergic and that the majority of these in addition can release nitric oxide, some enkephalin, and a few CGRP. Acetylcholine, VIP, and CGRP are coexisting messenger molecules in some postganglionic sympathetic neurons.

Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death.

Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock. To further our understanding of the role of iNOS in normal biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon gamma, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shock.

Stimulation of ionotropic glutamate receptors activates transcription factor NF-kappa B in primary neurons.

L-Glutamate is the most common excitatory neurotransmitter in the brain and plays a crucial role in neuronal plasticity as well as in neurotoxicity. While a large body of literature describes the induction of immediate-early genes, including c-fos, fosB, c-jun, junB, zif/268, and krox genes by glutamate and agonists in neurons, very little is known about preexisting transcription factors controlling the induction of such genes. This prompted us to investigate whether stimulation of glutamate receptors can activate NF-kappa B, which is present in neurons in either inducible or constitutive form. Here we report that brief treatments with kainate or high potassium strongly activated NF-kappa B in granule cells from rat cerebellum. This was detected at the single cell level by immunostaining with a monoclonal antibody that selectively reacts with the transcriptionally active, nuclear form of NF-kappa B p65. The activation of NF-kappa B could be blocked with the antioxidant pyrrolidine dithiocarbamate, suggesting the involvement of reactive oxygen intermediates. The data may explain the kainate-induced cell surface expression of major histocompatibility complex class I molecules, which are encoded by genes known to be controlled by NF-kappa B. Moreover, NF-kappa B activity was found to change dramatically in neurons during development of the cerebellum between days 5 and 7 after birth.

Glutamate as a hippocampal neuron survival factor: an inherited defect in the trisomy 16 mouse.

The survival of cultured mouse hippocampal neurons was found to be greatly enhanced by micromolar concentrations of the excitatory neurotransmitter glutamate. Blockade of kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptors increased the rate of neuron death, suggesting that endogenous glutamate in the cultures promotes survival. Addition of glutamate (0.5-1 microM) further increased neuron survival, whereas glutamate in excess of 20 microM resulted in increased death. Thus, the survival vs. glutamate dose-response relation is bell-shaped with an optimal glutamate concentration near 1 microM. We found that hippocampal neurons from mice with the genetic defect trisomy 16 (Ts16) died 2-3 times faster than normal (euploid) neurons. Moreover, glutamate, at all concentrations tested, failed to increase survival of Ts16 neurons. In contrast, the neurotrophic polypeptide basic fibroblast growth factor did increase the survival of Ts16 and euploid neurons. Ts16 is a naturally occurring mouse genetic abnormality, the human analog of which (Down syndrome) leads to altered brain development and Alzheimer disease. These results demonstrate that the Ts16 genotype confers a defect in the glutamate-mediated survival response of hippocampal neurons and that this defect can contribute to their accelerated death.

Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B. The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.

Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro.

Neuronal proliferation, migration, and differentiation are regulated by the sequential expression of particular genes at specific stages of development. Such processes rely on differential gene expression modulated through second-messenger systems. Early postnatal mouse cerebellar granule cells migrate into the internal granular layer and acquire differentiated properties. The neurotransmitter glutamate has been shown to play an important role in this developmental process. We show here by immunohistochemistry that the RelA subunit of the transcription factor NF-kappa B is present in several areas of the mouse brain. Moreover, immunofluorescence microscopy and electrophoretic mobility-shift assay demonstrate that in cerebellar granule cell cultures derived from 3- to 7-day-old mice, glutamate specifically activates the transcription factor NF-kappa B, as shown by binding of nuclear extract proteins to a synthetic oligonucleotide reproducing the kappa B site of human immunodeficiency virus. The use of different antagonists of the glutamate recpetors indicates that the effect of glutamate occurs mainly via N-methyl-D-aspartate (NMDA)-receptor activation, possibly as a result of an increase in intracellular Ca2+. The synaptic specificity of the effect is strongly suggested by the observation that glutamate failed to activate NF-kappa B in astrocytes, while cytokines, such as interleukin 1 alpha and tumor necrosis factor alpha, did so. The effect of glutamate appears to be developmentally regulated. Indeed, NF-kappa B is found in an inducible form in the cytoplasm of neurons of 3- to 7-day-old mice but is constitutively activated in the nuclei of neurons derived from older pups (8-10 days postnatal). Overall, these observations suggest the existence of a new pathway of trans-synaptic regulation of gene expression.

A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants.

Sucrose synthase (SuSy; EC 2.4.1.13; sucrose + UDP reversible UDPglucose + fructose) has always been studied as a cytoplasmic enzyme in plant cells where it serves to degrade sucrose and provide carbon for respiration and synthesis of cell wall polysaccharides and starch. We report here that at least half of the total SuSy of developing cotton fibers (Gossypium hirsutum) is tightly associated with the plasma membrane. Therefore, this form of SuSy might serve to channel carbon directly from sucrose to cellulose and/or callose synthases in the plasma membrane. By using detached and permeabilized cotton fibers, we show that carbon from sucrose can be converted at high rates to both cellulose and callose. Synthesis of cellulose or callose is favored by addition of EGTA or calcium and cellobiose, respectively. These findings contrast with the traditional observation that when UDPglucose is used as substrate in vitro, callose is the major product synthesized. Immunolocalization studies show that SuSy can be localized at the fiber surface in patterns consistent with the deposition of cellulose or callose. Thus, these results support a model in which SuSy exists in a complex with the beta-glucan synthases and serves to channel carbon from sucrose to glucan.

Cone photoreceptors respond to their own glutamate release in the tiger salamander.

Pulse-like currents resembling miniature postsynaptic currents were recorded in patch-clamped isolated cones from the tiger salamander retina. The events were absent in isolated cones without synaptic terminals. The frequency of events was increased by either raising the osmotic pressure or depolarizing the cell. It was decreased by the application of either glutamate or the glutamate-transport blockers dihydrokainate and D,L-threo-3-hydroxyaspartate. The events required external Na+ for which Li+ could not substitute. The reversal potential of these currents followed the equilibrium potential for Cl- when internal Cl- concentration was changed. Thus, these miniature currents appear to represent the presynaptic activation of the glutamate receptor with glutamate transporter-like pharmacology, caused by the photoreceptor's own vesicular glutamate release. Using a noninvasive method to preserve the intracellular Cl- concentration, we showed that glutamate elicits an outward current in isolated cones. Fluorescence of the membrane-permeable form of fura-2 was used to monitor Ca2+ entry at the cone terminal as a measure of membrane depolarization. The increase in intracellular Ca2+ concentration, elicited by puff application of 30 mM KCl, was completely suppressed in the presence of 100 microM glutamate. Puff application of glutamate alone had no measurable depolarizing effect. These results suggest that the equilibrium potential for Cl-, ECl, was more negative than the activation range for Ca2+ channels and that glutamate elicited an outward current, hyperpolarizing the cones.

In vivo regulation of muscle glycogen synthase and the control of glycogen synthesis.

The activity of glycogen synthase (GSase; EC 2.4.1.11) is regulated by covalent phosphorylation. Because of this regulation, GSase has generally been considered to control the rate of glycogen synthesis. This hypothesis is examined in light of recent in vivo NMR experiments on rat and human muscle and is found to be quantitatively inconsistent with the data under conditions of glycogen synthesis. Our first experiments showed that muscle glycogen synthesis was slower in non-insulin-dependent diabetics compared to normals and that their defect was in the glucose transporter/hexokinase (GT/HK) part of the pathway. From these and other in vivo NMR results a quantitative model is proposed in which the GT/HK steps control the rate of glycogen synthesis in normal humans and rat muscle. The flux through GSase is regulated to match the proximal steps by "feed forward" to glucose 6-phosphate, which is a positive allosteric effector of all forms of GSase. Recent in vivo NMR experiments specifically designed to test the model are analyzed by metabolic control theory and it is shown quantitatively that the GT/HK step controls the rate of glycogen synthesis. Preliminary evidence favors the transporter step. Several conclusions are significant: (i) glucose transport/hexokinase controls the glycogen synthesis flux; (ii) the role of covalent phosphorylation of GSase is to adapt the activity of the enzyme to the flux and to control the metabolite levels not the flux; (iii) the quantitative data needed for inferring and testing the present model of flux control depended upon advances of in vivo NMR methods that accurately measured the concentration of glucose 6-phosphate and the rate of glycogen synthesis.

Human fatty acid synthase: properties and molecular cloning.

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.

High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli.

The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N omega-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos.

The dorsoventral axis is established early in Xenopus development and may involve signaling by Wnts, a family of Wnt1-protooncogene-related proteins. The protein kinase shaggy functions in the wingless/Wnt signaling pathway, which operates during Drosophila development. To assess the role of a closely related kinase, glycogen synthase kinase 3 beta (GSK-3 beta), in vertebrate embryogenesis, we cloned a cDNA encoding a Xenopus homolog of GSK-3 beta (XGSK-3 beta). XGSK-3 beta-specific transcripts were detected by Northern analysis in Xenopus eggs and early embryos. Microinjection of the mRNA encoding a catalytically inactive form of rat GSK-3 beta into a ventrovegetal blastomere of eight-cell embryos caused ectopic formation of a secondary body axis containing a complete set of dorsal and anterior structures. Furthermore, in isolated ectodermal explants, the mutant GSK-3 beta mRNA activated the expression of neural tissue markers. Wild-type XGSK-3 beta mRNA suppressed the dorsalizing effects of both the mutated GSK-3 beta and Xenopus dishevelled, a proposed upstream signaling component of the same pathway. These results strongly suggest that XGSK-3 beta functions to inhibit dorsoventral axis formation in the embryo and provide evidence for conservation of the Wnt signaling pathway in Drosophila and vertebrates.