Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.

Förstermann, Ulrich; Closs, Ellen I.; Pollock, Jennifer S.; Nakane, Masaki; Schwarz, Petra; Gath, Ingolf; Kleinert, Hartmut

doi:10.1161/01.hyp.23.6.1121

Cited by 1,002 publications

(622 citation statements)

References 168 publications

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“…The enzymes also require bound FMN, FAD, (6R)-5,6,7,8-tetrahydro-L-biopterin (BH 4 ), and Ca 2ϩ /calmodulin for activity. Three main isoforms of NOS have been identified: isoform I or neuronal NOS (nNOS), which is constitutively expressed in a variety of neuronal cells as well as other cells; isoform II or inducible NOS, which is usually not constitutively expressed but can be induced by bacterial lipopolysaccharide and/or cytokines in macrophages and other cells; and isoform III or endothelial NOS, which is expressed in endothelial cells (5,6).…”

Section: Nitric-oxide Synthases (Nos)mentioning

confidence: 99%

Ubiquitination of Neuronal Nitric-oxide Synthase in Vitro and in Vivo

Bender¹,

Demady

Osawa

2000

Journal of Biological Chemistry

107

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It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) with guanidine compounds, or inhibition of the hsp90-based chaperone system with geldanamycin, leads to the enhanced proteolytic degradation of nNOS. This regulated proteolysis is mediated, in part, by the proteasome. We show here with the use of human embryonic kidney 293 cells transfected with nNOS that inhibition of the proteasome with lactacystin leads to the accumulation of immunodetectable higher molecular mass forms of nNOS. Some of these higher molecular mass forms were immunoprecipitated by an anti-ubiquitin antibody, indicating that they are nNOS-polyubiquitin conjugates. Moreover, the predominant nNOS-ubiquitin conjugate detected in human embryonic kidney 293 cells, as well as in rat brain cytosol, migrates on SDS-polyacrylamide gels with a mobility near that for the native monomer of nNOS and likely represents a conjugate containing a few or perhaps one ubiquitin. Studies in vitro with the use of 125 I-ubiquitin and reticulocyte extracts could mimic this ubiquitination reaction, which was dependent on ATP. The hemedeficient monomeric form of nNOS is preferentially ubiquitinated over that of the heme-sufficient functionally active homodimer. Thus, we have shown for the first time that ubiquitination of nNOS occurs and is likely involved in the regulated proteolytic removal of nonfunctional enzyme.

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Section: Nitric-oxide Synthases (Nos)mentioning

confidence: 99%

Ubiquitination of Neuronal Nitric-oxide Synthase in Vitro and in Vivo

Bender¹,

Demady

Osawa

2000

Journal of Biological Chemistry

107

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“…The constitutive forms of NOS, eNOS and nNOS are regulated by intracellular Ca 2¯u xes that permit calmodulin binding and enzyme activation. 5 In contrast, iNOS is transcriptionally regulated and is generally believed to be expressed in response to cellular stress and upregulated in pathophysiological conditions. 5,6 NO derived from nNOS is released from non-adrenergic, non-cholinergic (NANC) nerves, while NO from eNOS is released from the trabecular endothelium and diffuses into the smooth muscle cells where it directly interacts with the soluble form of guanylate cyclase to increase intracellular levels of cyclic GMP (cGMP) and cause penile smooth muscle relaxation.…”

Section: Introductionmentioning

confidence: 99%

“…5 In contrast, iNOS is transcriptionally regulated and is generally believed to be expressed in response to cellular stress and upregulated in pathophysiological conditions. 5,6 NO derived from nNOS is released from non-adrenergic, non-cholinergic (NANC) nerves, while NO from eNOS is released from the trabecular endothelium and diffuses into the smooth muscle cells where it directly interacts with the soluble form of guanylate cyclase to increase intracellular levels of cyclic GMP (cGMP) and cause penile smooth muscle relaxation. 7 A decrease in erectile function can be attributed to a decrease in NO synthesis in the NANC penile nerve ®bers or vascular endothelium of the penis.…”

Section: Introductionmentioning

confidence: 99%

Adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis improves age-related erectile dysfunction in the rat

et al. 2000

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Nitric oxide (NO) is the principal mediator of penile erection. NO is synthesized by a variety of nitric oxide synthases (NOS). It has been demonstrated that a decrease in NOS activity, as observed in aging, is associated with a diminished erectile response. The objective of this study was to determine if adenoviral-mediated gene transfer of eNOS could reverse age-related erectile dysfunction in the rat. Two groups of animals were transfected with adenoviruses: (1) aged rats (60 weeks) with AdRSVbgal; and (2) aged rats (60 weeks) with AdRSVeNOS. Five days after transfection, these study animals underwent cavernosal nerve stimulation (CNS) to assess erectile function and their responses were compared with young (20 weeks) control rats. Cross-sections of the rat penises transfected with AdRSVeNOS were examined after trichrome staining. Adenoviral transduction ef®ciency of b-galactosidase reporter gene was measured by a galacto-light chemiluminescent reporter gene assay in cavernosal tissues of rats administered AdRSVbgal. The transgene expression of eNOS was examined by RT-PCR in rats transfected with AdRSVbgal and AdRSVeNOS. eNOS and iNOS protein levels were measured by Western blot analysis, and cGMP levels were assessed in cavernosal tissue by enzyme immunoassay. Adenoviral expression of the b-galactosidase reporter gene was observed in cavernosal tissue for up to 30 days, with peak expression registered at 5 days after intracavernosal administration of AdRSVbgal. Cross-sections of the rat penises transfected with the AdRSVeNOS revealed no pathological (morphological or histological) changes. Five days after administration of AdRSVeNOS, eNOS protein, mRNA and cGMP levels in the corpora cavernosa were signi®cantly increased (P`0.05), while iNOS protein levels remained unchanged (P b 0.05). In conclusion, enhanced expression of eNOS employing an adenoviral vector signi®cantly increased the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the aged rat.

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“…NO is produced in biological systems via the enzymatic action of NO synthase (NOS) (32). NO generation by the constitutive NOS isoforms (neuronal NOS and endothelial NOS) is calcium dependent (16). NO production within skeletal muscle is enhanced during contractile activity (5).…”

mentioning

confidence: 99%

Effects of modulation of nitric oxide on rat diaphragm isotonic contractility during hypoxia

Zhu

Heunks

Machiels

et al. 2003

Journal of Applied Physiology

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Effects of modulation of nitric oxide on rat diaphragm isotonic contractility during hypoxia. J Appl Physiol 94: 612-620, 2003. First published October 18, 2002 10.1152/ japplphysiol.00441.2002 is essential for optimal myofilament function of the rat diaphragm in vitro during active shortening. Little is known about the role of NO in muscle contraction under hypoxic conditions. Hypoxia might increase the NO synthase (NOS) activity within the rat diaphragm. We hypothesized that NO plays a protective role in isotonic contractile and fatigue properties during hypoxia in vitro. The effects of the NOS inhibitor N G -monomethyl-L-arginine (L-NMMA), the NO scavenger hemoglobin, and the NO donor spermine NONOate on shortening velocity, power generation, and isotonic fatigability during hypoxia were evaluated (PO 2 ϳ 7 kPa). L-NMMA and hemoglobin slowed the shortening velocity, depressed power generation, and increased isotonic fatigability during hypoxia. The effects of L-NMMA were prevented by coadministration with the NOS substrate L-arginine. Spermine NONOate did not alter isotonic contractile and fatigue properties during hypoxia. These results indicate that endogenous NO is needed for optimal muscle contraction of the rat diaphragm in vitro during hypoxia.N G -monomethyl-L-arginine; hemoglobin; spermine NONOate SKELETAL MUSCLE, INCLUDING the diaphragm, continuously produces nitric oxide (NO) (3,26). NO is produced in biological systems via the enzymatic action of NO synthase (NOS) (32). NO generation by the constitutive NOS isoforms (neuronal NOS and endothelial NOS) is calcium dependent (16). NO production within skeletal muscle is enhanced during contractile activity (5).NO has been shown to modulate contractile properties of skeletal muscle in vitro. The overall effect depends on the experimental paradigm. Inhibition of NOS increases twitch and submaximal tetanic force of the rat diaphragm. These alterations are reversed by NO donors (26), suggesting that NO inhibits excitation-contraction coupling in unfatigued muscle. On the other hand, NO is essential for optimal myofilament function in the rat diaphragm during active shortening in vitro (36). Inhibition of NOS decreases the shortening velocity and power generation of the rat diaphragm under hyperoxic conditions (36). These findings of NOS inhibition on force generation and velocity of shortening indicate multiple possible targets for NO in skeletal muscle. In fatiguing contractions, supplementation with exogenous NO slowed the decline of maximal force in mouse soleus muscle (38). This finding suggests that NO preserves skeletal muscle function in vitro during strenuous contractile activity.Hypoxia impairs force generation and accelerates skeletal muscle fatigue in vitro (21, 48). To date, no study has investigated the role of NO in shortening velocity or isotonic fatigability during hypoxia. This is of particular interest because NO affects excitationcontraction coupling in striated muscle (26), and O 2 tension is an important factor in regulating NOS in an...

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Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.

Cited by 1,002 publications

References 168 publications

Ubiquitination of Neuronal Nitric-oxide Synthase in Vitro and in Vivo

Ubiquitination of Neuronal Nitric-oxide Synthase in Vitro and in Vivo

Adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis improves age-related erectile dysfunction in the rat

Effects of modulation of nitric oxide on rat diaphragm isotonic contractility during hypoxia

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