Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The effect of activation of nicotinic cholinoceptors in rat duodenal segments following electrical field stimulation (EFS) was investigated. 2. Electrical field stimulation elicited a two-component response: transient relaxation followed by contraction. The EFS-evoked response was tetrodotoxin (TTX; 1 mumol/L) sensitive. The relaxation component was NG-nitro-L-arginine (L-NNA; 100 mumol/L) sensitive, while the contractile response was atropine (1 mumol/L) sensitive. 3. 1,1-Dimethyl-4-phenyl-piperazinium iodide (DMPP; 20 mumol/L) induced relaxation of spontaneously active preparations that was L-NNA sensitive. L-Arginine (1 mmol/L) reversed the effects of L-NNA on DMPP-induced relaxation. 4. When EFS was applied, DMPP increased the amplitude of the relaxation component of the response and reduced the contractile component. 5. In the presence of L-NNA, the effect of DMPP on the relaxation component of the response to EFS was reduced, but the contractile response was not affected. L-Arginine partly reduced this effect of L-NNA. 6. Neither propranolol (1 mumol/L) nor yohimbine (1 mumol/L) had any effect on the actions of DMPP on EFS-evoked responses, but prazosin (1 mumol/L) strongly reduced the effect of DMPP on the contractile component of the response to EFS and slightly reduced the effect of DMPP on the relaxation response. 7. Histochemical studies demonstrated that, in the myenteric plexus of the rat duodenum, there are many reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons and that their number decreased after treatment with L-NNA. In the presence of L-arginine and L-NNA, the number of NADPH-d-positive neurons was similar to that found in control samples. 8. The data suggest that activation of nicotinic cholinoceptors modulates EFS-evoked responses in the rat duodenum as a result of the potentiation of nitrergic and adrenergic neurotransmission.
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PMID:Modulation of electrically evoked responses in rat duodenum by activation of nicotinic cholinoceptors. 961 59

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774-777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC50 for nNOS was 18 +/- 6 microM GST-PIN with 63 nM nNOS after 30 min at 37 degrees C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K(M) for L-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.
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PMID:The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases. 968 79

1. The distribution and localization of nitric oxide synthase (NOS) immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the bovine oesophageal groove were investigated by immnunohistochemical and histochemical staining techniques. Functional in vitro studies were performed to correlate the presence of NOS-immunoreactivity (IR) and NADPH-d staining with smooth muscle relaxations involving the L-arginine/nitric oxide neural pathway in the bovine oesophageal groove activity. 2. NOS-IR and NADPH-d were expressed in nerve cell bodies of the myenteric, submucosal and intramuscular ganglia, and in nerve fibres distributed around blood vessels and throughout the different muscular layers of the bovine oesophageal groove. 3. In oesophageal groove strips treated with guanethidine (10(-5) M) and atropine (10(-7) M) to block noradrenergic neurotransmission and muscarinic receptors, respectively, electrical field stimulation (EFS, 0.5-32 Hz, 1 ms duration, 20-s trains) induced relaxations which were practically abolished by tetrodotoxin (TTX, 10(-6) M). 4. Incubation with an inhibitor of nitric oxide synthesis, NG-nitro-L-arginine (L-NOARG, 3 x 10(-5) M), significantly inhibited relaxations induced by EFS. This inhibition was partially reversed by L-arginine (L-arg, 5 x 10(-3) M). D-NOARG (3 x 10(-5) M) had no effect on EFS-induced relaxations. 5. NO added as an acidified solution of NaNO2 (10(-6) - 10(-3) M) and S-nitroso-L-cysteine (10(-7) - 10(-4) M) caused concentration-dependent relaxations of the bovine oesophageal groove. These relaxations were unaffected by L-NOARG (3 x 10(-5) M). 6. The presence of NO-synthesizing enzyme in nerves and ganglia, and the pharmacological evidence for NO-mediated smooth muscle relaxation suggested that the L-arg/NO neuronal pathway is involved in the inhibitory neurotransmission of the bovine oesophageal groove.
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PMID:Involvement of the L-arginine/nitric oxide neural pathway in non-adrenergic, non-cholinergic relaxation of the bovine oesophageal groove. 973 Feb 60

The in vitro effects of the nitric oxide (NO) substrate L-arginine on ciliary beat frequency and the in vivo effects of the NO donor sodium nitroprusside (SNP) on mucociliary activity were investigated in the rabbit maxillary sinus mucosa with photoelectric techniques. L-Arginine increased ciliary beat frequency in vitro with a maximum response of 27.1% +/- 6.4% at 10(-3) mol/L, and this effect was reversibly blocked by pretreatment with the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine, whereas D-arginine had no such effect. SNP increased mucociliary activity in vivo, the peak response of 36.8% +/- 4.2% being obtained at the dose of 30.0 microg/kg. No tachyphylaxis was observed after repeat challenge with SNP. The increase in mucociliary activity caused by SNP was largely unaffected by pretreatment with the calcium channel blocker nifedipine, the cyclooxygenase inhibitor diclofenac, and the cholinergic antagonist atropine. The nonselective beta-blocker propranolol delayed the peak response of SNP to 7 to 8 minutes after challenge, compared with 1 to 2 minutes after challenge in animals without pretreatment. The results show the NO substrate L-arginine and the NO donor SNP to have ciliostimulatory effects in vitro and in vivo, respectively. The occurrence of NOS production in the sphenopalatine ganglion and sinus mucosa of the rabbit was studied by immunohistochemistry for NOS activity or nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. The latter is an indirect sign of neuronal NOS activity. Numerous NOS-containing cell bodies were seen in the sphenopalatine ganglion; in the sinus mucosa a moderate supply of thin NOS-immunoreactive nerve fibers was seen. Taken together, the morphologic findings and the functional results indicate NO to be a regulator of mucociliary activity in upper airways.
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PMID:Nitric oxide is a regulator of mucociliary activity in the upper respiratory tract. 974 84

Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR), indicate that the positive charge of Lys75 might be directly involved in the interaction between FNR and its protein partners, ferredoxin (Fd) and flavodoxin (Fld). To assess this possibility, this residue has been replaced by another positively charged residue, Arg, by two uncharged residues, Gln and Ser, and by a negatively charged residue, Glu. UV-vis absorption, fluorescence, and CD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Lys75Ser, and Lys75Glu) indicate that all the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Steady-state kinetic parameters for these FNR mutants, utilizing the diaphorase activity with DCPIP, indicate that Lys75 is not a critical residue for complex formation and electron transfer (ET) between FNR and NADP+ or NADPH. However, steady-state kinetic activities requiring complex formation and ET between FNR and Fd or Fld were appreciably affected when the positive charge at position of Lys75 was removed, and the ET reaction was not even measurable if a negatively charged residue was placed at this position. These kinetic parameters also suggest that it is complex formation that is affected by mutation. Consistent with this, when dissociation constants (Kd) for FNRox-Fdox (differential spectroscopy) and FNRox-Fdrd (laser flash photolysis) were measured, it was found that neutralization of the positive charge at position 75 increased the Kd values by 50-100-fold, and that no complex formation could be detected upon introduction of a negative charge at this position. Fast transient kinetic studies also corroborated the fact that removal of the positive charge at position 75 of FNR appreciably affects the complex formation process with its protein partners but indicates that ET is still achieved in all the reactions. This study thus clearly establishes the requirement of a positive charge at position Lys75 for complex formation during ET between FNR and its physiological protein partners. The results also suggest that the interaction of this residue with its protein partners is not structurally specific, since Lys75 can still be efficiently substituted by an arginine, but is definitely charge specific.
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PMID:Lys75 of Anabaena ferredoxin-NADP+ reductase is a critical residue for binding ferredoxin and flavodoxin during electron transfer. 975 47

The genes encoding the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli as the major cell proteins (at least 13.5% and 11.4% of total soluble protein, respectively) and the gene products purified to homogeneity. The FLDR reduces potassium ferricyanide with a kcat of 1610.3 min(-1) and a Km of 23.6 microM, and cytochrome c with a kcat of 141.3 min(-1) and a Km of 17.6 microM. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent Km of 6.84 microM was measured for the affinity of the two flavoproteins. The molecular masses of FLDR and FLD apoproteins were determined as 27648 Da and 19606 Da and the isoelectric points as 4.8 and 3.5, respectively. The mass of the FLDR is precisely that predicted from the atomic structure and indicates that residue 126 is arginine, not glutamine as predicted from the gene sequence. FLDR and FLD were covalently crosslinked using 1-ethyl-3(dimethylamino-propyl) carbodiimide to generate a catalytically active heterodimer. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (-308 mV and -268 mV, respectively) and FLD (-254 mV and -433 mV, respectively) were measured using redox potentiometry. This confirms the electron-transfer route as NADPH-->FLDR-->FLD. Binding of 2' adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone (absorption coefficient calculated as 4933 M(-1) cm(-1) at 583 nm) with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions, and to facilitate catalytic activity of microsomal cytochromes P-450 heterologously expressed in E. coli. Kinetic studies of electron transfer from FLDR/FLD to the fatty acid oxidase P-450 BM3 support this conclusion, indicating a ping-pong mechanism. This is the first report of the potentiometric analysis of the full E. coli NAD(P)H/FLDR/FLD electron-transfer chain; a complex critical to the function of a large number of E. coli redox systems.
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PMID:Characterisation of flavodoxin NADP+ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli. 983 46

The present study was designed to examine the role of nitric oxide (NO) in quinolinic acid (QUIN)-induced depletion of rat striatal nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase and enkephalinergic neurons. Intrastriatal injection of QUIN produced a dose-dependent decrease in NADPH diaphorase and enkephalin positive cells, with cell loss being evident following the injection of 6 and 18 nmol QUIN, respectively. To evaluate the role of NO in QUIN-induced toxicity, animals were pretreated with the non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine (l-NAME) or the selective neuronal NOS inhibitor, 7-nitro indazole (7-NI). l-NAME (2x250 mg/kg, i.p. 8 h apart) maximally inhibited striatal NOS activity by 85%, while 7-NI (50 mg/kg, i.p.) maximally inhibited striatal NOS activity by 60%. Pretreatment with l-NAME or 7-NI potentiated the loss of NADPH diaphorase neurons resulting from intrastriatal injection of low doses of QUIN (18 nmol). Neither NOS inhibitor had any effect on the loss of striatal NADPH diaphorase neurons induced by a higher dose of QUIN (24 nmol). In contrast, 7-NI partially prevented the QUIN (18 and 24 nmol)-induced loss of enkephalinergic neurons, while l-NAME had no effect. These results indicate that NO formation may play a role in QUIN-induced loss of enkephalinergic neurons, but not in the loss of NADPH diaphorase neurons.
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PMID:Modulation of quinolinic acid-induced depletion of striatal NADPH diaphorase and enkephalinergic neurons by inhibition of nitric oxide synthase. 988 56

Nitric oxide (NO) has been implicated as a retrograde signal in the process of refining axonal pathways during brain development. To determine some of the factors involved in this process, we have used two model pathway systems in the rat and mouse superior colliculus (SC). The first, the patch-cluster system, consists of clusters of neurons in the intermediate gray layer (igl) which transiently express NO during development and which receive input from a cholinergic pathway from the parabrachial brainstem as well as from other pathways containing different transmitters. The second system, the retinocollicular pathway, consists of glutamatergic fibers that project to the superficial gray layer. We have used both nitric oxide synthase inhibition (nw-nitro-L-arginine, NoArg) and single (nNOS) and double (nNOS and eNOS) gene knockout mice to examine the effect that reduction in NOS has upon the development of these two systems. The onset of NOS expression in rat, as revealed by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) labeling, occurred in igl cells as early as postnatal day P5, with clusters being well-established by P14. Cholinergic fibers were first visible at P10 and formed obvious patches and tiers by P14. Intraperitoneal injections of NoArg from P1-P22 had no effect upon the development of these cholinergic patches. The pathway also developed normally in both single and double-knockout mice. In contrast, the ipsilateral retinocollicular pathway was altered in the double, but not in the single knockout mouse. This pathway is exuberant during the first week of life, being distributed across much of the mediolateral axis of the rostral SC. By P8-P15, this pathway has retracted to the most mediorostral SC. This refinement was delayed substantially in the double NOS gene knockout mouse. Ipsilateral fibers were found within 3-5 separate medio-lateral patches within the rostral 600 microns of SC at P15, and patches of abnormal size and extent were also seen at P18. We conclude from these results that NO plays a role in pathway development in the rodent SC, but only in glutamatergic pathways and only when both endothelial and neuronal forms of NOS have been deleted. The mechanism of this effect must involve pathway elimination in situations where there is non-correlated electrical activity. It is likely that NO promotes fiber retraction rather than fiber stabilization in these developing nerve fibers.
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PMID:The role of nitric oxide in development of the patch-cluster system and retinocollicular pathways in the rodent superior colliculus. 993 39

Aims of this study were to functionally and histologically determine the localization of ganglia that distribute inhibitory nerves to the penile corpus cavernosum in dogs. In isolated corpus cavernosa from seven control dogs contracted with endothelin-1, transmural electrical stimulation (5 Hz for 40 s) elicited contractions which were reversed to relaxations by prazosin. The relaxation was abolished by NG-nitro-l-arginine (l-NNA), a nitric oxide (NO) synthase inhibitor, and restored by l-arginine. Parts of bilateral pelvic nerve plexuses running to the penis were surgically denervated in anesthetized three dogs, or the bilateral neuronal tissues close to the corpus cavernosum were removed for denervation in seven dogs. One week after the operation, the dogs were sacrificed. Denervation of pelvic plexus did not attenuate neurogenic relaxations, whereas denervation of the distal portion abolished the responses. In the tissues close to the corpus cavernosum excised for denervation, ganglia containing abundant nerve cells and fibers stained by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase method were histochemically detected. One week after the denervation, there were no NADPH diaphorase-positive nerve fibers in the trabecula of corpus cavernosum. It is concluded that neurogenic relaxations of canine corpus cavernosum are mediated by NO synthesized from l-arginine in nerve terminals, and this nerve is originated from ganglia located close to the corpus cavernosum but not directly from the pelvic nerve plexus.
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PMID:Influence of denervation on neurogenic inhibitory response of corpus cavernosum and nitric oxide synthase histochemistry. 1021 69

Tissue injury initiates a temporally ordered sequence of local cellular and metabolic responses presumably necessary for successful repair. Previous investigations demonstrated that metabolic evidence for nitric oxide synthase (NOS) activity is detectable in wounds only during the initial 48 to 72 hours of the repair process. Present results identify the cell types contributing inducible NOS (iNOS) to experimental wounds in rats. iNOS antigen was expressed in most macrophages present in wounds 6 to 24 hours after injury, and these cells exhibited NAPDH diaphorase and NOS activity. Polymorphonuclear leukocytes contained little iNOS antigen and no NADPH diaphorase activity and were minimally able to convert L-arginine to L-citrulline. The frequency of iNOS-positive macrophages declined on days 3 and 5 after wounding. By day 10, most macrophages in the wound were negative for iNOS. These cells, however, acquired iNOS antigen and activity in culture. Wound fluids, but not normal rat serum, suppressed the induction of iNOS during culture. Findings indicate that the expression of iNOS in healing wounds is restricted to macrophages present during the early phases of repair and that components of wound fluid suppress the induction of iNOS in macrophages in late wounds. Polymorphonuclear leukocytes contribute little iNOS activity to the healing wound.
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PMID:Molecular and metabolic evidence for the restricted expression of inducible nitric oxide synthase in healing wounds. 1023 48


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