Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes.

Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production.

Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism by which this protection is mediated is unclear. Since animal studies strongly suggest that production of endothelium-derived relaxing factor is enhanced by estradiol, we have examined the effect of estrogens on nitric oxide (NO) synthase (NOS) activity, protein, and mRNA in cultured bovine aortic endothelial cells. In reporter cells rich in guanylate cyclase, it has been observed that long-term treatment (> or = 24 hr) with ethinylestradiol (EE2) dose-dependently increased guanylate cyclase-activating factor activity in the conditioned medium of endothelial cells. However, conversion of L-[14C]arginine to L-[14C]citrulline by endothelial cell homogenate or quantification of nitrite and nitrate released by intact cells in the conditioned medium did not reveal any change in NOS activity induced by EE2 treatment. Similarly, Western and Northern blot analyses did not reveal any change in the endothelial NOS protein and mRNA content in response to EE2. However, EE2 dose- and time-dependently decreased superoxide anion production in the conditioned medium of endothelial cells with an EC50 value (0.1 nM) close to that which increased guanylate cyclase-activating factor activity (0.5 nM). Both of these effects were completely prevented by the antiestrogens tamoxifen and RU54876. Thus, endothelium exposure to estrogens appears to induce a receptor-mediated antioxidant effect that enhances the biological activity of endothelium-derived NO. These effects could account at least in part for the vascular protective properties of these hormones.

Nitric oxide synthase content of hypothalamic explants: increase by norepinephrine and inactivated by NO and cGMP.

Release of luteinizing hormone (LH)-releasing hormone (LHRH), the hypothalamic peptide that controls release of LH from the adenohypophysis, is controlled by NO. There is a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers in the lateral median eminence, intermingled with terminals of the LHRH neurons. To study relations between NOS and LHRH in this brain region, we measured NOS activity in incubated medial basal hypothalamus (MBH). NOS converts [14C]arginine to equimolar quantities of [14C]citrulline plus NO, which rapidly decomposes. The [14C]citrulline serves as an index of the NO produced. NOS basal activity was suppressed by incubation of the tissue with an inhibitor of NOS, nitroarginine methyl ester (NAME) (10(-5) M). Furthermore, incubation of MBH explants for 30 min with norepinephrine (NE) increased NOS activity and the increase was prevented by prazosine (10(-5) M), an alpha 1-adrenergic receptor blocker; however, direct addition of NE to the tissue homogenate or to a preparation of MBH synaptosomes did not alter enzyme activity, which suggested that NE increased the content of NOS during incubation with the tissue. After purification of NOS, the increase in enzyme content induced by NE was still measurable. This indicates that within 30 min NE increased the synthesis of NOS in vitro. Incubation of MBH or the MBH homogenate with various concentrations of sodium nitroprusside (NP), a releaser of NO, reduced NOS activity at high concentrations (> or = 0.9 mM), which were associated with either a reduction of stimulation or a plateau of LHRH release. Finally, incubation of either MBH or the homogenate with cGMP, a major mediatior of NO action, at concentrations that increased LHRH release also reduced NOS activity. These results indicate that NO at high concentrations can inactivate NOS and that cGMP can also inhibit the enzyme directly. Therefore, the increased NOS activity induced by activation of alpha 1 receptors by NE is inhibited by NO itself and a principal product of its activity, cGMP, providing negative feedback on NOS. In central nervous system (CNS) infections with high concentrations of inducible NOS produced by glial elements, the high concentrations of NO and cGMP produced may suppress LHRH release, resulting in decreased gonadotropin and gonadal steroid release.

Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B.

The objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation products of NO (NO2- + N03-, designated as NOX-), cellular RNA was extracted for determination of iNOS mRNA levels by Northern blot analysis, and nuclear extracts were prepared for determination of NF-kappa B by electrophoretic mobility-shift assay. Inhibitors of calpain (alpha-N-acetyl-Leu-Leu-norleucinal; N-benzyloxycarbonyl-Leu-leucinal) and the proteasome (N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-leucinal) markedly inhibited or abolished the induction of iNOS in macrophages. The proteinase inhibitors interfered with lipopolysaccharide-induced NOX- production by macrophages, and this effect was accompanied by comparable interference with the appearance of both iNOS mRNA and NF-kappa B. Calpain inhibitors elicited effects at concentrations of 1-100 microM, whereas the proteasome inhibitor was 1000-fold more potent, producing significant inhibitory effects at 1 nM. The present findings indicate that the proteasome pathway is essential for lipopolysaccharide-induced expression of the iNOS gene in rat alveolar macrophages. Furthermore, the data support the view that the proteasome pathway is directly involved in promoting the activation of NF-kappa B and that the induction of iNOS by lipopolysaccharide involves the transcriptional action of NF-kappaB.

P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R.J., Derkach, V. & Surprenant, A. (1992) Nature (London) 357, 503-505; Edwards, F.A., Gigg, A.J. & Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, we report the cloning and characterization of complementary DNA from rat brain, encoding an additional member (P2X4) of the emerging multigenic family of ligand-gated ATP channels, the P2X receptors. Expression in Xenopus oocytes gives an ATP-activated cation-selective channel that is highly permeable to Ca2+ and whose sensitivity is modulated by extracellular Zn2+. Surprisingly, the current elicited by ATP is almost insensitive to the common P2X antagonist suramin. In situ hybridization reveals the expression of P2X4 mRNA in central nervous system neurons. Northern blot and reverse transcription-PCR (RT-PCR) analysis demonstrate a wide distribution of P2X4 transcripts in various tissues, including blood vessels and leukocytes. This suggests that the P2X4 receptor might mediate not only ATP-dependent synaptic transmission in the central nervous system but also a wide repertoire of biological responses in diverse tissues.

Increased mRNA levels for components of the lysosomal, Ca2+-activated, and ATP-ubiquitin-dependent proteolytic pathways in skeletal muscle from head trauma patients.

The cellular mechanisms responsible for enhanced muscle protein breakdown in hospitalized patients, which frequently results in lean body wasting, are unknown. To determine whether the lysosomal, Ca2+-activated, and ubiquitin-proteasome proteolytic pathways are activated, we measured mRNA levels for components of these processes in muscle biopsies from severe head trauma patients. These patients exhibited negative nitrogen balance and increased rates of whole-body protein breakdown (assessed by [13C]leucine infusion) and of myofibrillar protein breakdown (assessed by 3-methylhistidine urinary excretion). Increased muscle mRNA levels for cathepsin D, m-calpain, and critical components of the ubiquitin proteolytic pathway (i.e., ubiquitin, the 14-kDa ubiquitin-conjugating enzyme E2, and proteasome subunits) paralleled these metabolic adaptations. The data clearly support a role for multiple proteolytic processes in increased muscle proteolysis. The ubiquitin proteolytic pathway could be activated by altered glucocorticoid production and/or increased circulating levels of interleukin 1beta and interleukin 6 observed in head trauma patients and account for the breakdown of myofibrillar proteins, as was recently reported in animal studies.

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain.

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

An ATP-activated, ligand-gated ion channel on a cholinergic presynaptic nerve terminal.

ATP has recently been identified as a fast neurotransmitter in both the central and peripheral nervous systems. Several studies have suggested that ATP can also affect the release of classical neurotransmitters, including acetylcholine with which it is co-released. We have searched for ATP receptors on a cholinergic presynaptic nerve terminal using the calyx-type synapse of the chicken ciliary ganglion. ATP was pulsed onto the terminals under voltage clamp and induced a short latency cation current that exhibited inward rectification and marked desensitization. This current was not seen with adenosine but was mimicked by several sterically restricted ATP analogs and was blocked by suramin. ATP-activated single ion channels exhibited prominent flickering and had a conductance of approximately 17 pS. Our results demonstrate a ligand-gated P2X-like purinergic receptor on a cholinergic presynaptic nerve terminal.

Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) catalyzes the first step in biosynthesis of lysine in plants and bacteria. DHPS in plants is highly sensitive to end-product inhibition by lysine and, therefore, has an important role in regulating metabolite flux into lysine. To better understand the feedback inhibition properties of the plant enzyme, we transformed a maize cDNA for lysine-sensitive DHPS into an Escherichia coli strain lacking DHPS activity. Cells were mutagenized with ethylmethanesulfonate, and potential DHPS mutants were selected by growth on minimal medium containing the inhibitory lysine analogue S-2-aminoethyl-L-cysteine. DHPS assays identified surviving colonies expressing lysine-insensitive DHPS activity. Ten single-base-pair mutations were identified in the maize DHPS cDNA sequence; these mutations were specific to one of three amino acid residues (amino acids 157, 162, and 166) localized within a short region of the polypeptide. No other mutations were present in the remaining DHPS cDNA sequence, indicating that altering only one of the three residues suffices to eliminate lysine inhibition of maize DHPS. Identification of these specific mutations that change the highly sensitive maize DHPS to a lysine-insensitive isoform will help resolve the lysine-binding mechanism and the resultant conformational changes involved in inhibition of DHPS activity. The plant-derived mutant DHPS genes may also be used to improve nutritional quality of maize or other cereal grains that have inadequate lysine content when fed to animals such as poultry, swine, or humans.

An ATP-dependent As(III)-glutathione transport system in membrane vesicles of Leishmania tarentolae.

Membrane preparations enriched in plasma membrane vesicles prepared from promastigotes of Leishmania tarentolae were shown to accumulate thiolate derivatives of 73As(III). Free arsenite was transported at a low rate, but rapid accumulation was observed after reaction with reduced glutathione (GSH) conditions that favor the formation of As(GS)3. Accumulation required ATP but not electrochemical energy, indicating that As(GS)3 is transported by an ATP-coupled pump. Pentostam, a Sb(V)-containing drug that is one of the first-line therapeutic agents for treatment of leishmaniasis, inhibited uptake after reaction with GSH. Vesicles prepared from a strain in which both copies of the pgpA genes were disrupted accumulated As(GS)3 at wild-type levels, demonstrating that the PgpA protein is not the As(GS)3 pump. These results have important implications for the mechanism of drug resistance in the trypanosomatidae, suggesting that a plasma membrane As(GS)3 pump catalyzes active extrusion of metal thiolates, including the Pentostam-glutathione conjugate.

Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene.

Electrical currents associated with nucleotide transport by the reconstituted mitochondrial ADP/ATP carrier.

The electrophoretic export of ATP against the import of ADP in mitochondria bridges the intra- versus extramitochondrial ATP potential gap. Here we report that the electrical nature of the ADP/ATP exchange by the mitochondrial ADP/ATP carrier (AAC) can be directly studied by measuring the electrical currents via capacitive coupling of AAC-containing vesicles on a planar lipid membrane. The currents were induced by the rapid liberation of ATP or ADP with UV flash photolysis from caged nucleotides. Six different transport modes of the AAC were studied: heteroexchange with either ADP or ATP inside the vesicles, initiated by photolysis of caged ATP or ADP; homoexchange with ADPex/ADPin or ATPex/ATPin; and caged ADP or ATP with unloaded vesicles. The heteroexchange produced the largest currents with the longest duration in line with the electrical charge difference ATP4- versus ADP3-. Surprisingly, also in the homoexchange and with unloaded vesicles, small currents were measured with shorter duration. In all three modes with caged ATP, a negative charge moved into the vesicles and with caged ADP it moved out of the vesicles. All currents were completely inhibited by a mixture of the inhibitors of the AAC, carboxyatractyloside and hongkrekate, which proves that the currents are exclusively due to AAC function. The observed charge movements in the heteroexchange system agree with the prediction from transport studies in mitochondria and reconstituted vesicles. The unexpected charge movements in the homoexchange or unloaded systems are interpreted to reveal transmembrane rearrangements of charged sites in the AAC when occupied with ADP or ATP. The results also indicate that not only ATP4- but also ADP3- contribute, albeit in opposite direction, to the electrical nature of the ADP/ATP exchange, which is at variance with former conclusions from biochemical transport studies. These measurements open up new avenues of studying the electrical interactions of ADP and ATP with the AAC.

Intracerebral injection of human immunodeficiency virus type 1 coat protein gp120 differentially affects the expression of nerve growth factor and nitric oxide synthase in the hippocampus of rat.

We have studied the neuropathological characteristics of the brain of rats receiving daily intracerebroventricular administration of freshly dissolved human immunodeficiency virus type 1 recombinant protein gp120 (100 ng per rat per day) given for up to 14 days. Histological examination of serial brain sections revealed no apparent gross damage to the cortex or hippocampus, nor did cell counting yield significant neuronal cell loss. However, the viral protein caused after 7 and 14 days of treatment DNA fragmentation in 10% of brain cortical neurons. Interestingly, reduced neuronal nitric oxide synthase (NOS) expression along with significant increases in nerve growth factor (NGF) were observed in the hippocampus, where gp120 did not cause neuronal damage. No changes in NGF and NOS expression were seen in the cortex, where cell death is likely to be of the apoptotic type. The present data demonstrate that gp120-induced cortical cell death is associated with the lack of increase of NGF in the cerebral cortex and suggest that the latter may be important for the expression of neuropathology in the rat brain. By contrast, enhanced levels of NGF may prevent or delay neuronal death in the hippocampus, where reduced NOS expression may be a reflection of a subcellular insult inflicted by the viral protein.

Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene.

Sterol-regulated transcription of the gene for rat farnesyl diphosphate (FPP) synthase (geranyl-diphosphate:isopentenyl-diphosphate geranyltranstransferase, EC 2.5.1.10) is dependent in part on the binding of the ubiquitous transcription factor NF-Y to a 6-bp element within the proximal promoter. Current studies identify a second element in this promoter that is also required for sterol-regulated transcription in vivo. Mutation of three nucleotides (CAC) within this element blocks the 8-fold induction of FPP synthase promoter-reporter genes that normally occurs when the transfected cells are incubated in medium deprived of sterols. Gel mobility-shift assays demonstrate that the transcriptionally active 68-kDa fragment of the sterol regulatory element (SRE-1)-binding protein assays (SREBP-1) binds to an oligonucleotide containing the wild-type sequence but not to an oligonucleotide in which the CAC has been mutated. DNase 1 protection pattern (footprint) analysis indicates that SREBP-1 binds to nucleotides that include the CAC. Both the in vivo and in vitro assays are affected by mutagenesis of nucleotides adjacent to the CAC. Coexpression of SREBP with a wild-type FPP synthase promoter-reporter gene in CV-1 cells results in very high levels of reporter activity that is sterol-independent. In contrast, the reporter activity remained low when the promoter contained a mutation in the CAC trinucleotide. We conclude that sterol-regulated transcription of FPP synthase is controlled in part by the interaction of SREBP with a binding site that we have termed SRE-3. Identification of this element may prove useful in the identification of other genes that are both regulated by SREBP and involved in lipid biosynthesis.