Neuronal Nitric Oxide Synthase is Refractory to Mechanism-Based Inactivation in GH3Pituitary Cells

doi:10.1006/abbi.1998.0828

Archives of Biochemistry and Biophysics

Volume 357, Issue 2, 15 September 1998, Pages 195-206

https://doi.org/10.1006/abbi.1998.0828 Get rights and content

Abstract

Nitric oxide formation by GH₃pituitary cells is stimulated by depolarizing concentrations of K⁺and by the L-channel Ca²⁺agonist Bay k 8644 in an additive manner that depends on extracellular Ca²⁺. Ca²⁺-dependent NO formation at 100 μM arginine was inhibited 50% over a 30-min period by 5 μM N^G-amino-l-arginine, 30 μMN⁶-iminoethyl-l-ornithine (NIO), and 520 μMN⁵-iminoethyl-l-lysine (NIL) but required concentrations of aminoguanidine (AG) greater than 3 mM. As measured at 100 μM extracellular arginine, intracellular neuronal nitric oxide synthase (nNOS) was inactivated 50% over a 30-min period by 150 μMN^G-amino-l-arginine and 1500 μM NIO, but required concentrations of NIL or AG greater than 5 mM. The inactivation of nNOS by these agents occurred only under conditions that mobilized extracellular Ca²⁺and was inhibited by increased extracellular arginine. Thus these agents inhibit cellular Ca²⁺-dependent NO formation at concentrations far lower than those required to inactivate the cellular nNOS. Inhibition of NO formation by these agents was not attributable to effects on cellular arginine uptake. In contrast diphenyliodonium chloride produced a rapid and complete inactivation of cellular NO formation and nNOS activity. These inactivations produced by diphenyliodonium chloride occurred with identical kinetics but displayed no requirement for Ca²⁺. These data support the assertion that neuronal NO synthase is refractory to mechanism-based inactivation in GH₃pituitary cells.

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A homogeneous fluorescent cell-based assay for detection of heterologously expressed nitric oxide synthase activity
2005, Journal of Biomolecular Screening
Arhodamine-derived, membrane-permeable fluorophore (DAR-4MAM) sensitive to nitric oxide production has been developed recently. The authors evaluated this reagent in both 96 and 384-well formats using heterologously expressed neuronal nitric oxide synthase (nNOS). nNOS transfected into HEK-293T cellswas stimulated by the addition of ionomycin. The calcium mobilization resulting from ionomycin treatment of nNOS-expressing 293T cells induced a robust increase in emission intensity, as measured using a standard rhodamine filter set. The effect was time dependent, and a 3 to 4-fold stimulation could be achieved in a 2-h time period. Ionomycin-dependent nitric oxide (NO) production was completely inhibited by several arginine analogs at micromolar concentrations (e.g., L-NAME IC 50= 3.0 μM). Several arginine analog inhibitors of nNOS were revealed to be differentially reversible over increasing substrate concentrations. The assay is a facile method for characterizing inhibitors of nNOS in a relatively unperturbed cell environment. (Journal of Biomolecular Screening 2005:849-855)
Trisamine C<inf>60</inf>-fullerene adducts inhibit neuronal nitric oxide synthase by acting as highly potent calmodulin antagonists
2002, Archives of Biochemistry and Biophysics
C₆₀-Fullerene trisamine adducts inhibit neuronal nitric oxide synthase and calcineurin phosphatase activities in a manner completely reversible by calmodulin. As measured by difference spectroscopy, D₃-trisamine and C₃-semiamine fullerene adducts displace trifluoperazine bound to calmodulin coincident with their binding. These binding events are complete at a molar ratio of 4 mol added fullerene per mole calmodulin. Trisamine fullerene adducts alter the native electrophoretic mobility of calmodulin, producing a heterogeneity of bands with associated fullerene. D₃- and C₃-trisamine fullerene adducts interact with dansylated calmodulin, producing a 50% loss of maximal fluorescence at concentrations of 30 nM. At higher concentrations than those required to inhibit neuronal nitric oxide synthase, trisamine fullerene adducts inhibit nitric oxide formation by the cytokine-inducible nitric oxide synthase isoform. These inhibitions are fully reversible by calmodulin and skeletal muscle troponin C but not by skeletal muscle parvalbumin. Of the trisamine fullerene adducts tested only the C₃- and D₃-semiamine adducts inhibit Ca²⁺-dependent nitric oxide production in GH₃ pituitary cells. These observations support the proposal that trisamine C₆₀-fullerene adducts are potent calmodulin antagonists, some of which display activity in intact cellular systems.
The antithyroid agent 6-n-propyl-2-thiouracil is a mechanism-based inactivator of the neuronal nitric oxide synthase isoform
2002, Archives of Biochemistry and Biophysics
Citation Excerpt :
The concentration of the ferrous–CO complex of nNOS could be calculated using an extinction coefficient of 121 mM−1 cm−1. Adherent, confluent GH3 monolayers prepared as described previously [8] were incubated at 37 °C in 2 ml of Ham’s F-10 medium, 5 μM oxyhemoglobin without or containing additional additives as indicated in the appropriate figure legend. The release of NO from the cells was assessed by measuring the formation of methemoglobin as the absorbance difference at 401 nm (absorbance maximum) and 411 nm (isosbestic point) over time as described by Noack et al. [25].
6-n-Propyl-2-thiouracil (6-PTU), the antithyroid agent, produces a time-, concentration-, and turnover-dependent inactivation of the NO synthetic capability of the neuronal nitric oxide synthase isoform irreversible by either arginine or (6R)-5,6,7,8-tetrahydro-l-biopterin. By contrast 6-PTU produces an inhibition of the cytokine-inducible and endothelial nitric oxide synthases fully reversible by arginine. The inactivation of neuronal nitric oxide synthase by 6-PTU follows first order kinetics, and is inhibited competitively by both arginine and (6R)-5,6,7,8-tetrahydro-l-biopterin, but is not accompanied by either a loss of heme-CO binding, heme fluorescence, or disassembly of dimeric structure. 2-Thiouracil behaves qualitatively identically to 6-PTU. Turnover-dependent inactivation of neuronal nitric oxide synthase by [2-¹⁴C]-2-thiouracil is accompanied by incorporation of radioactivity into the polypeptide chain. Ca²⁺-dependent NO formation by GH₃ pituitary cells is inhibited by 6-PTU in a manner enhanced by depletion of either extracellular arginine or intracellular (6R)-5,6,7,8-tetrahydro-l-biopterin. These observations establish that 6-PTU is an alternate substrate, mechanism-based inactivator of the neuronal nitric oxide synthase isoform with the ability to suppress cellular NO formation.
Minimal amidine structure for inhibition of nitric oxide biosynthesis
2001, Biochemical Pharmacology
Citation Excerpt :
Culture medium for primary hepatocytes was also supplemented with 26 U/mL of insulin. Nitric oxide biosynthesis was induced in GH3 cells following treatment with the potassium ionophore Bay K-8644 (2.8 μM, 8–24 hr) [14]. Induction of nitric oxide biosynthesis by all other cells was achieved following treatment with γ-interferon (100 U/mL, 24–48 hr).
Pharmacological modulation of nitric oxide synthase activity has been achieved using structural analogs of arginine. In the present studies, we demonstrated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cultures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a concentration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expressing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidine had no effect on γ-interferon-induced expression of nitric oxide synthase protein, its enzymatic activity was blocked. Kinetic analysis revealed that formamidine acts as a simple competitive inhibitor with respect to arginine (K_i formamidine ∼ 800 μM). Using a polarographic microsensor to measure real-time flux of nitric oxide release from RAW 264.7 macrophages, formamidine was found to require 30–90 min to inhibit enzyme activity, suggesting that cellular uptake of the drug may limit its biological activity. Our data indicate that formamidine is an effective inhibitor of nitric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased production of nitric oxide.
Cellular and enzymatic studies of N(ω)-propyl-L-arginine and S-ethyl-N- [4-(trifluoromethyl)phenyl]isothiourea as reversible, slowly dissociating inhibitors selective for the neuronal nitric oxide synthase isoform
2000, Archives of Biochemistry and Biophysics
N^ωpropyl- $l$ -arginine (NPA) and S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea (TFMPITU) inhibit selectively the neuronal nitric oxide (NO) synthase (nNOS) isoform. In the presence of Ca²⁺ and calmodulin (CaM), NPA and TFMPITU produce a time- and concentration-dependent suppression of nNOS catalyzed NO formation. This suppression of activity occurs by a first order kinetic process as revealed from linear Kitz–Wilson plots but does not depend on catalytic turnover since it occurs in the absence of NADPH. Following full suppression of NO synthetic activity by either NPA or TFMPITU, NO synthesis can be restored slowly by excess arginine or by dilution, indicating that the effects of these agents are reversible. This behavior is consistent with a dissociation of NPA and TFMPITU from nNOS slowed by a conformational transition produced by Ca²⁺ CaM-binding. NPA and TFMPITU bind to nNOS rapidly producing a heme–substrate interaction as revealed by difference spectrophotometry. At physiological conditions (100 μM extracellular arginine), NPA and TFMPITU inhibit Ca²⁺-dependent NO formation by GH₃ pituitary cells with IC₅₀ values of 19 and 47 μM, respectively, but require millimolar concentrations to inhibit NO formation by cytokine-induced RAW 264.7 murine macrophages. The inhibition of NO formation by these agents in GH₃ cells is rapidly reversible and not due to suppression of cellular arginine uptake.
Pharmacological modulation of nitric oxide synthesis by mechanism-based inactivators and related inhibitors
1999, Pharmacology and Therapeutics
Nitric oxide synthase (NOS) (EC 1.14.13.39) is a homodimeric cytochrome P450 monooxygenase analog that generates nitric oxide (NO) from the amino acid l-arginine. Enzymatically produced NO acts as an intracellular messenger in neuronal networks, blood pressure regulatory mechanisms, and immune responses. Isoform-selective pharmacological modulation of NO synthesis has emerged as a new therapeutic strategy for the treatment of diverse clinical conditions associated with NO overproduction. Mechanism-based inactivators (MBIs) represent a class of NOS mechanistic inhibitors that require catalytic turnover to produce irreversible inactivation of the ability of NOS to generate NO. Diverse isoform-selective NOS MBIs have been characterized with respect to their kinetic parameters and chemical mechanisms of inactivation. In studies with isolated and purified NOS isoforms, MBIs produce irreversible inactivation of NOS enzymatic activities. The inactivation process is associated with covalent modification of the NOS active site and proceeds either through heme destruction, its structural alteration, or covalent modification of the NOS protein chain. The behavior of NOS MBIs in intact cells is different from their behavior observed with the isolated NOS isoforms. In cytokine-induced RAW 264.7 macrophages, treatment with MBIs produces a complete loss of cellular NOS synthetic competence and inducible NOS activity. However, following drug removal, cells can recover at least partially in the absence of protein synthesis. In GH₃ cells containing the neuronal NOS isoform, calcium transients are too low and abbreviated to allow significant NOS inactivation; hence, the cellular effects of MBIs on the neuronal isoform are almost completely and immediately reversible.

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^☆: This work was supported by National Institutes of Health Grants HL 54768 and ES 06897.

^☆☆: Colowick, S. P.Kaplan, N. O.

²: To whom correspondence should be addressed. Fax: (732) 235-4073.

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Regular ArticleNeuronal Nitric Oxide Synthase is Refractory to Mechanism-Based Inactivation in GH3Pituitary Cells☆,☆☆

Abstract

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Neuroprotocols

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

FASEB J.

Annu. Rev. Biochem.

Br. J. Pharmacol.

Regular Article
Neuronal Nitric Oxide Synthase is Refractory to Mechanism-Based Inactivation in GH₃Pituitary Cells☆,☆☆