Structural basis for pterin antagonism in nitric-oxide synthase. Development of novel 4-oxo-pteridine antagonists of (6R)-5,6,7,8-tetrahydrobiopterin

Pathological nitric oxide (NO) generation in sepsis, inflammation, and stroke may be therapeutically controlled by inhibiting NO synthases (NOS). Here we targeted the (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)Bip)-binding site of NOS, which, upon cofactor binding, maximally increases enzyme activity and NO production from substrate l-arginine. The first generation of H(4)Bip-based NOS inhibitors employed a 4-amino pharmacophore of H(4)Bip analogous to antifolates such as methotrexate. We developed a novel series of 4-oxo-pteridine derivatives that were screened for inhibition against neuronal NOS (NOS-I) and a structure-activity relationship was determined. To understand the structural basis for pterin antagonism, selected derivatives were docked into the NOS pterin binding cavity. Using a reduced 4-oxo-pteridine scaffold, derivatives with certain modifications such as electron-rich aromatic phenyl or benzoyl groups at the 5- and 6-positions, were discovered to markedly inhibit NOS-I, possibly due to hydrophobic and electrostatic interactions with Phe(462) and Ser(104), respectively, within the pterin binding pocket. One of the most effective 4-oxo compounds and, for comparisons an active 4-amino derivative, were then co-crystallized with the endothelial NOS (NOS-III) oxygenase domain and this structure solved to confirm the hypothetical binding modes. Collectively, these findings suggest (i) that, unlike the antifolate principle, the 4-amino substituent is not essential for developing pterin-based NOS inhibitors and (ii), provide a steric and electrostatic basis for their rational design.     

Nitric oxide synthase (NOS) in mouse skeletal muscle development and differentiated myoblasts

The neuronal isoform of nitric oxide synthase (nNOS, termed also NOS-I) is expressed in normal adult skeletal muscle, suggesting important functions for NO in muscle biology. However, the expression and subcellular localization of NOS in muscle development and myoblast differentiation are largely unknown. In the present study, NOS was immunolocalized with isoform-specific antibodies in developing muscle and in differentiated myoblast cultures (mouse C2C12) together with histochemical NADPH-dependent diaphorase activity that is blocked by specific NOS inhibitors and therefore designated as NOS-associated diaphorase activity (NOSaD). Western blot analysis revealed immunoreactive bands for NOS-I-III in lysates from perinatal and adult muscle tissue and C2C12-myotubes that comigrated with prototypical proteins. In embryonic skeletal muscle, but not in adult myofibers, diffuse cytosolic staining and lack of sarcolemmal NOSaD activity and NOS-I immunoreaction were evident. In both myoblasts and fusioned myotubes, NOSaD and NOS isoforms I-III colocalize in the cytosol. Additionally, members of the sarcolemmal dystrophin-glycoprotein complex (i.e., dystrophin, adhalin, β1-dystroglycan) immunolocalize in the cytosol of differentiating myoblasts, whereas anti-dystrophin and anti-β1-dystroglycan clearly delineate the sarcolemma in myotubes. Thus, expression of NOS isoforms I-III and NOSaD is cytosolic in fusion-competent myoblasts during myotube formation in vitro. Interaction of NOSaD/NOS-I with the sarcolemmal dystrophin-complex known from mature myofibers is apparently lacking in prenatal muscle development and differentiating myoblasts. Localization of NOS isoforms thus characterized in myogenic cultures may help further to investigate regulated NO formation in muscle cells in vitro.     

Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction.

Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker.     

Local ascorbate administration augments NO- and non-NO-dependent reflex cutaneous vasodilation in hypertensive humans

Full expression of reflex cutaneous vasodilation (VD) is dependent on nitric oxide (NO) and is attenuated with essential hypertension. Decreased NO-dependent VD may be due to 1) increased oxidant stress and/or 2) decreased L-arginine availability through upregulated arginase activity, potentially leading to increased superoxide production through uncoupled NO synthase (NOS). The purpose of this study was to determine the effect of antioxidant supplementation (alone and combined with arginase inhibition) on attenuated NO-dependent reflex cutaneous VD in hypertensive subjects. Nine unmedicated hypertensive [HT; mean arterial pressure (MAP) = 112 +/- 1 mmHg] and nine age-matched normotensive (NT; MAP = 81 +/- 10 mmHg) men and women were instrumented with four intradermal microdialysis (MD) fibers: control (Ringer), NOS inhibited (NOS-I; 10 mM N(G)-nitro-L-arginine), L-ascorbate supplemented (Asc; 10 mM L-ascorbate), and Asc + arginase inhibited [Asc+A-I; 10 mM L-ascorbate + 5 mM (S)-(2-boronoethyl)-L-cysteine-HCl + 5 mM N(omega)-hydroxy-nor-L-arginine]. Oral temperature was increased by 0.8 degrees C via a water-perfused suit. N(G)-nitro-L-arginine was then ultimately perfused through all MD sites to quantify the change in VD due to NO. Red blood cell flux was measured by laser-Doppler flowmetry over each skin MD site, and cutaneous vascular conductance (CVC) was calculated (CVC = flux/MAP) and normalized to maximal CVC (%CVC(max); 28 mM sodium nitroprusside + local heating to 43 degrees C). During the plateau in skin blood flow (Delta T(or) = 0.8 degrees C), cutaneous VD was attenuated in HT skin (NT: 42 +/- 4, HT: 35 +/- 3 %CVC(max); P < 0.05). Asc and Asc+A-I augmented cutaneous VD in HT (Asc: 57 +/- 5, Asc+A-I: 53 +/- 6 %CVC(max); P < 0.05 vs. control) but not in NT. %CVC(max) after NOS-I in the Asc- and Asc+A-I-treated sites was increased in HT (Asc: 41 +/- 4, Asc+A-I: 40 +/- 4, control: 29 +/- 4; P < 0.05). Compared with the control site, the change in %CVC(max) within each site after NOS-I was greater in HT (Asc: -19 +/- 4, Asc+A-I: -17 +/- 4, control: -9 +/- 2; P < 0.05) than in NT. Antioxidant supplementation alone or combined with arginase inhibition augments attenuated reflex cutaneous VD in hypertensive skin through NO- and non-NO-dependent mechanisms.     

The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I

The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.     

A tryptophan that modulates tetrahydrobiopterin-dependent electron transfer in nitric oxide synthase regulates enzyme catalysis by additional mechanisms

Nitric oxide synthases (NOSs) are flavo-heme enzymes that require (6R)-tetrahydrobiopterin (H(4)B) for activity. Our single-catalytic turnover study with the inducible NOS oxygenase domain showed that a conserved Trp that interacts with H(4)B (Trp457 in mouse inducible NOS) regulates the kinetics of electron transfer between H(4)B and an enzyme heme-dioxy intermediate, and this in turn alters the kinetics and extent of Arg hydroxylation [Wang, Z.-Q., et al. (2001) Biochemistry 40, 12819-12825]. To investigate the impact of these effects on NADPH-driven NO synthesis by NOS, we generated and characterized the W457A mutant of inducible NOS and the corresponding W678A and W678F mutants of neuronal NOS. Mutant defects in protein solubility and dimerization were overcome by purifying them in the presence of sufficient Arg and H(4)B, enabling us to study their physical and catalytic profiles. Optical spectra of the ferric, ferrous, heme-dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similar to that of the wild type. However, the mutants had higher apparent K(m) values for H(4)B and in one mutant for Arg (W457A). They all had lower NO synthesis activities, uncoupled NADPH consumption, and a slower and less prominent buildup of enzyme heme-NO complex during steady-state catalysis. Further analyses showed the mutants had normal or near-normal heme midpoint potential and heme-NO complex reactivity with O(2), but had somewhat slower ferric heme reduction rates and markedly slower reactivities of their heme-dioxy intermediate. We conclude that the conserved Trp (1) has similar roles in two different NOS isozymes and (2) regulates delivery of both electrons required for O(2) activation (i.e., kinetics of ferric heme reduction by the NOS flavoprotein domain and reduction of the heme-dioxy intermediate by H(4)B). However, its regulation of H(4)B electron transfer is most important because this ensures efficient coupling of NADPH oxidation and NO synthesis by NOS.     

Role of bound zinc in dimer stabilization but not enzyme activity of neuronal nitric-oxide synthase

Nitric-oxide synthases (NOS) are homodimeric proteins and can form an intersubunit Zn(4S) cluster. We have measured zinc bound to NOS purified from pig brain (0.6 mol/mol of NOS) and baculovirus-expressed rat neuronal NOS (nNOS) (0.49 +/- 0.13 mol/mol of NOS), by on-line gel-filtration/inductively coupled plasma mass spectrometry. Cobalt, manganese, molybdenum, nickel, and vanadium were all undetectable. Baculovirus-expressed nNOS also bound up to 2. 00 +/- 0.58 mol of copper/mol of NOS. Diethylenetriaminepentaacetic acid (DTPA) reduced the bound zinc to 0.28 +/- 0.07 and the copper to 0.97 +/- 0.24 mol/mol of NOS. Desalting of samples into thiol-free buffer did not affect the zinc content but completely eliminated the bound copper ( or =75%) of the bound zinc was released from baculovirus-expressed rat nNOS by p-chloromercuriphenylsulfonic acid (PMPS). PMPS-treated nNOS was strongly (90 +/- 5%) inactivated. To isolate functional effects of zinc release from other effects of PMPS, PMPS-substituted thiols were unblocked by excess reduced thiol in the presence of DTPA, which hindered reincorporation of zinc. The resulting enzyme contained 0.12 +/- 0.05 mol of zinc but had a specific activity of 426 +/- 46 nmol of citrulline.mg(-1).min(-1), corresponding to 93 +/- 10% of non-PMPS-treated controls. PMPS also caused dissociation of nNOS dimers under native conditions, an effect that was blocked by the pteridine cofactor tetrahydrobiopterin (H(4)biopterin). H(4)biopterin did not affect zinc release. Even in the presence of H(4)biopterin, PMPS prevented conversion of NOS dimers to an SDS-resistant form. We conclude that zinc binding is a prerequisite for formation of SDS-resistant NOS dimers but is not essential for catalysis.     

NOS3 is involved in the increased protein and arginine metabolic response in muscle during early endotoxemia in mice

Sepsis is a severe catabolic condition. The loss of skeletal muscle protein mass is characterized by enhanced release of the amino acids glutamine and arginine, which (in)directly affects interorgan arginine and the related nitric oxide (NO) synthesis. To establish whether changes in muscle amino acid and protein kinetics are regulated by NO synthesized by nitric oxide synthase-2 or -3 (NOS2 or NOS3), we studied C57BL6/J wild-type (WT), NOS2-deficient (NOS2-/-), and NOS3-deficient (NOS3-/-) mice under control (unstimulated) and lipopolysaccharide (LPS)-treated conditions. Muscle amino acid metabolism was studied across the hindquarter by infusing the stable isotopes L-[ring-2H5]phenylalanine, L-[ring-2H2]tyrosine, L-[guanidino-15N2]arginine, and L-[ureido-13C,2H2]citrulline. Muscle blood flow was measured using radioactive p-aminohippuric acid dilution. Under baseline conditions, muscle blood flow was halved in NOS2-/- mice (P < 0.1), with simultaneous reductions in muscle glutamine, glycine, alanine, arginine release and glutamic acid, citrulline, valine, and leucine uptake (P < 0.1). After LPS treatment, (net) muscle protein synthesis increased in WT and NOS2-/- mice [LPS vs. control: 13 +/- 3 vs. 8 +/- 1 (SE) nmol.10 g(-1).min(-1) (WT), 18 +/- 5 vs. 7 +/- 2 nmol.10 g(-1).min(-1) (NOS2-/-); P < 0.05 for LPS vs. control]. This response was absent in NOS3-/- mice (LPS vs. control: 11 +/- 4 vs. 10 +/- 2 nmol.10 g(-1).min(-1)). In agreement, the increase in muscle arginine turnover after LPS was also absent in NOS3-/- mice. In conclusion, disruption of the NOS2 gene compromises muscle glutamine release and muscle blood flow in control mice, but had only minor effects after LPS. NOS3 activity is crucial for the increase in muscle arginine and protein turnover during early endotoxemia.     

Catalytic reduction of a tetrahydrobiopterin radical within nitric-oxide synthase

Nitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H(4)B) cofactor. During Arg hydroxylation, H(4)B acts as a one-electron donor and is then presumed to redox cycle (i.e. be reduced back to H(4)B) within NOS before further catalysis can proceed. Whereas H(4)B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H(4)B, and a method that could separately quantify the flavin and pterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical following Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H(4)B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a "through-heme" mechanism may operate for H(4)B radical reduction in NOS.     

Palmitate increases nitric oxide synthase activity that is involved in palmitate-induced cell death in cardiomyocytes.

The objective of this study was to test the hypothesis that nitric oxide synthase (NOS) is subjected to regulatory control by palmitate, and that nitric oxide (NO) is operative in palmitate-induced cell death. Palmitate induced a significant ( p<0.05 ) concentration-dependent increase in NOS activity measured by the conversion of [(3)H]arginine to [3H]citrulline in embryonic chick cardiomyocytes. Cellular eNOS and iNOS, determined by immunocytochemistry, were increased by palmitate. Western blotting also showed that palmitate, 500 microM for 4h, significantly increased the amount of cellular of eNOS and iNOS by 36.2+/-6.5% ( p<0.001 ) and 38.4+/-14.4% ( p<0.05 ), respectively. The NOS inhibitor L-NAME significantly ( p<0.05 ) accentuated palmitate-induced cell death These data suggest that palmitate has a bifunctional effect on cell viability--in addition to loss of cell viability, palmitate stimulates NOS activity by inducing an increase in cellular eNOS and iNOS with the resultant NO production serving to protect cardiomyocytes from palmitate-induced cell death.     

A role for nitric oxide in muscle repair: nitric oxide-mediated activation of muscle satellite cells

Muscle satellite cells are quiescent precursors interposed between myofibers and a sheath of external lamina. Although their activation and recruitment to cycle enable muscle repair and adaptation, the activation signal is not known. Evidence is presented that nitric oxide (NO) mediates satellite cell activation, including morphological hypertrophy and decreased adhesion in the fiber-lamina complex. Activation in vivo occurred within 1 min after injury. Cell isolation and histology showed that pharmacological inhibition of nitric oxide synthase (NOS) activity prevented the immediate injury-induced myogenic cell release and delayed the hypertrophy of satellite cells in that muscle. Transient activation of satellite cells in contralateral muscles 10 min later suggested that a circulating factor may interact with NO-mediated signaling. Interestingly, satellite cell activation in muscles of mdx dystrophic mice and NOS-I knockout mice quantitatively resembled NOS-inhibited release of normal cells, in agreement with reports of displaced and reduced NOS expression in dystrophin-deficient mdx muscle and the complete loss of NOS-I expression in knockout mice. Brief NOS inhibition in normal and mdx mice during injury produced subtle alterations in subsequent repair, including apoptosis in myotube nuclei and myotube formation inside laminar sheaths. Longer NOS inhibition delayed and restricted the extent of repair and resulted in fiber branching. A model proposes the hypothesis that NO release mediates satellite cell activation, possibly via shear-induced rapid increases in NOS activity that produce "NO transients."     

NOS3 deficiency augments hypoxic pulmonary vasoconstriction and enhances systemic oxygenation during one-lung ventilation in mice.

Nitric oxide (NO), synthesized by NO synthases (NOS), plays a pivotal role in regulation of pulmonary vascular tone. To examine the role of endothelial NOS (NOS3) in hypoxic pulmonary vasoconstriction (HPV), we measured left lung pulmonary vascular resistance (LPVR), intrapulmonary shunting, and arterial PO2 (PaO2) before and during left mainstem bronchus occlusion (LMBO) in mice with and without a deletion of the gene encoding NOS3. The increase of LPVR induced by LMBO was greater in NOS3-deficient mice than in wild-type mice (151 +/- 39% vs. 109 +/- 36%, mean +/- SD; P < 0.05). NOS3-deficient mice had a lower intrapulmonary shunt fraction than wild-type mice (17.1 +/- 3.6% vs. 21.7 +/- 2.4%, P < 0.05) during LMBO. Both real-time PaO2 monitoring with an intra-arterial probe and arterial blood-gas analysis during LMBO showed higher PaO2 in NOS3-deficient mice than in wild-type mice (P < 0.05). Inhibition of all three NOS isoforms with Nomega-nitro-L-arginine methyl ester (L-NAME) augmented the increase of LPVR induced by LMBO in wild-type mice (183 +/- 67% in L-NAME treated vs. 109 +/- 36% in saline treated, P < 0.01) but not in NOS3-deficient mice. Similarly, systemic oxygenation during one-lung ventilation was augmented by L-NAME in wild-type mice but not in NOS3-deficient mice. These findings indicate that NO derived from NOS3 modulates HPV in vivo and that inhibition of NOS3 improves systemic oxygenation during acute unilateral lung hypoxia.     

Interaction of nitric oxide, 20-HETE, and EETs during functional hyperemia in whisker barrel cortex

Nitric oxide (NO) modulates vasodilation in cerebral cortex during sensory activation. NO is known to inhibit the synthesis of 20-HETE, which has been implicated in arteriolar constriction during astrocyte activation in brain slices. We tested the hypothesis that the attenuated cerebral blood flow (CBF) response to whisker stimulation seen after NO synthase (NOS) inhibition requires 20-HETE synthesis and that the ability of an epoxyeicosatrienoic acids (EETs) antagonist to reduce the CBF response is blunted after NOS inhibition but restored with simultaneous blockade of 20-HETE synthesis. In anesthetized rats, the increase in CBF during whisker stimulation was attenuated after the blockade of neuronal NOS with 7-nitroindazole. Subsequent administration of the 20-HETE synthesis inhibitor N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016) restored the CBF response to control levels. After the administration of 7-nitroindazole, the inhibitory effect of an EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) on the CBF response was lost, whereas the simultaneous administration of 7-nitroindazole and HET0016 restored the inhibitory effect of 14,15-EEZE. The administration of HET0016 alone had only a small effect on the evoked CBF response in rats. Furthermore, in neuronal NOS(+/+) and NOS(-/-) mice, HET0016 administration did not increase the CBF response to whisker stimulation. In neuronal NOS(+/+) mice, HET0016 also blocked the reduction in the response seen with acute NOS inhibition. These results indicate that 20-HETE synthesis normally does not substantially restrict functional hyperemia. Increased NO production during functional activation may act dynamically to suppress 20-HETE synthesis or downstream signaling and permit EETs-dependent vasodilation. With the chronic loss of neuronal NOS in mice, other mechanisms apparently suppress 20-HETE synthesis or signaling.     

Nitric oxide nerves in the uterus are parasympathetic,sensory, and contain neuropeptides

Nitric oxide (NO) is synthesized in neurons and is a potent relaxor of vascular and nonvascular smooth muscle. The uterus contains abundant NO-synthesizing nerves which could be autonomic and/or sensory. This study was undertaken to determine: 1) the source(s) of NO-synthesizing nerves in the rat uterus and 2) what other neuropeptides or transmitter markers might coexist with NO in these nerves. Retrograde axonal tracing, utilizing Fluorogold injected into the uterine cervix, was employed for identifying sources of uterine-projecting neurons. NO-synthesizing nerves were visualized by staining for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and immunostaining with an antibody against neuronal/type I NO synthase (NOS). NADPH-d-positive perikarya and terminal fibers were NOS-immunoreactive (-I). Some NOS-I/NADPH-d-positive nerves in the uterus are parasympathetic and originate from neurons in the pelvic paracervical ganglia (PG) and some are sensory and originate from neurons in thoracic, lumbar, and sacral dorsal root ganglia. No evidence for NOS-I/NADPH-d-positive sympathetic nerves in the uterus was obtained. Furthermore, double immunostaining revealed that in parasympathetic neurons, NO-I/NADPH-d-reactivity coexists with vasoactive intestinal polypeptide, neuropeptide Y, and acetylcholinesterase and in sensory nerves, NOS-I/NADPH-d-reactivity coexists with calcitonin generelated peptide and substance P. In addition, tyrosine hydroxylase(TH)-I neurons of the PG do not contain NOS-I/NADPH-d-reactivity, but some TH-I neurons are apposed by NOS-I varicosities. These results suggest NO-synthesizing nerves in the uterus are autonomic and sensory, and could play significant roles, possibly in conjunction with other putative transmitter agents, in the control of uterine myometrium and vasculature.     

Transient expression of NOS-II during development of the murine enteric nervous system

In the enteric nervous system, nitric oxide (NO) is regarded as an important messenger for the non-adrenergic and non-cholinergic neurotransmission. Synthesized mainly by the constitutive nitric oxide synthase (NOS) isoforms NOS I and NOS III, this molecule exerts prejunctional inhibitory effects in the submucosal plexus as well as relaxation of enteric smooth muscles. In order to elucidate the role for NO during enteric development, we looked for the expression of all three NOS-isoforms in the enteric nervous system during mouse development from E8 to E20 using immunohistochemistry. Starting around midgestation, a transient expression of the NOS-II isoform during the very early development of enteric neurones was detected in parallel to that of HNK-1 exclusively in the myenteric plexus. Similar to findings for other neuronal systems, NOS-I and NOS III isoforms could be traced starting significantly later to increase toward the end of embryonic development when NOS II immunoreactivity faded and a strong expression of the vasointestinal peptide could be detected. In contrast to the NOSII expression, the constitutive isoforms can also be detected in the submucosal plexus. Altogether, these findings suggest NOS-II to be exclusively involved during early steps of enteric nervous system development. Absence of downstream signalling elements, such as sGC and cGMP both in neurons and in enteric muscle until the end of the second third of gestation, may indicate different effects executed by NO during development, expressed by Ca²⁺ -dependent and Ca²⁺ -independent NOS isoforms.     

Nitric oxide drives embryonic myogenesis in chicken through the upregulation of myogenic differentiation factors

The muscle-specific variant of neuronal nitric oxide (NO) synthase (NOS-I), is developmentally regulated in mouse suggesting a role of NO during myogenesis. In chick embryo, a good model of development, we found that the expression of NOS-I is up-regulated, but only in the early phase of development. Through a pharmacological intervention in ovo we found that NO signalling plays a relevant role during embryonic development. The inhibition of NOS-I decreased the growth of embryo, in particular of muscle tissue, while the restoring of physiological NO levels, via administration of a NO donor, reversed this effect. We found a selective action of NO, produced by NOS-I, on regulatory factors involved in myogenic differentiation in the early phase of chick embryo development: inhibition of NO generation leads to a decreased expression of the Myocyte enhancer factor 2a (Mef2a), Mef2c, Myogenin and Myosin, which was reversed by the administration of a NO donor. NO had no effects on Myf5 and MyoD, the myogenic regulatory factors necessary for myogenic determination. The action of NO on the myogenic regulatory factors was mediated via generation of cyclic GMP (cGMP) and activation of the cGMP-dependent protein kinase G (PKG). Finally we found in myoblasts in vitro that the activation of Mef2c was the key event mediating the NO-induced modulation of myogenesis.     

Cutaneous neuronal nitric oxide is specifically decreased in postural tachycardia syndrome

Low flow postural tachycardia syndrome (POTS), is associated with reduced nitric oxide (NO) activity assumed to be of endothelial origin. We tested the hypothesis that cutaneous microvascular neuronal NO (nNO) is impaired, rather than endothelial NO (eNO), in POTS. We performed three sets of experiments on subjects aged 22.5 +/- 2 yr. We used laser-Doppler flowmetry response to sequentially increase acetylcholine (ACh) doses and the local cutaneous heating response of the calf as bioassays for NO. During local heating we showed that when the selective neuronal nNO synthase (nNOS) inhibitor N(omega)-nitro-L-arginine-2,4-L-diaminobutyric amide (N(omega), 10 mM) was delivered by intradermal microdialysis, cutaneous vascular conductance (CVC) decreased by an amount equivalent to the largest reduction produced by the nonselective NO synthase (NOS) inhibitor nitro-L-arginine (NLA, 10 mM). We demonstrated that the response to ACh was minimally attenuated by nNOS blockade using N(omega) but markedly attenuated by NLA, indicating that eNO largely comprises the receptor-mediated NO release by ACh. We further demonstrated that the ACh dose response was minimally reduced, whereas local heat-mediated NO-dependent responses were markedly reduced in POTS compared with control subjects. This is consistent with intact endothelial function and reduced NO of neuronal origin in POTS. The local heating response was highly attenuated in POTS [60 +/- 6 percent maximum CVC(%CVC(max))] compared with control (90 +/- 4 %CVC(max)), but the plateau response decreased to the same level with nNOS inhibition (50 +/- 3 %CVC(max) in POTS compared with 47 +/- 2 %CVC(max)), indicating reduced nNO bioavailability in POTS patients. The data suggest that nNO activity but not NO of endothelial NOS origin is reduced in low-flow POTS.     

Inhibition of nitric-oxide synthase-I (NOS-I)-dependent nitric oxide production by lipopolysaccharide plus interferon-gamma is mediated by arachidonic acid. Effects on NFkappaB activation and late inducible NOS expression

Previous results have indicated that lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) inhibits nitric-oxide synthase (NOS)-I activity in glial cells. We report here that arachidonic acid (AA) plays a pivotal role in this response, which was consistently reproduced in different glial cell lines and in primary rat astrocytes. This notion was established using pharmacological inhibitors of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2) antisense oligonucleotides, and AA add-back experiments. This approach not only allowed the demonstration that AA promotes inhibition of NOS-I activity but also produced novel experimental evidence that LPS/IFNgamma itself is a potential stimulus for NOS-I. Indeed, LPS/IFNgamma fails to generate nitric oxide (NO) via NOS-I activation simply because it activates the AA-dependent signal that impedes NOS-I activity. Otherwise, LPS/IFNgamma promotes NO formation, sensitive to exogenous AA, in cells in which cPLA2 is pharmacologically inhibited or genetically depleted. Because NO suppresses the NFkappaB-dependent NOS-II expression, inactivation of NOS-I by the LPS/IFNgamma-induced AA pathway provides optimal conditions for NFkappaB activation and subsequent NOS-II expression. Inhibition of cPLA2 activity, while reducing the availability of AA, consistently inhibited NFkappaB activation and NOS-II mRNA induction and delayed NO formation. These responses were promptly reestablished by addition of exogenous AA. Finally, we have demonstrated that the LPS/IFNgamma-dependent tyrosine phosphorylation of NOS-I and inhibition of its activity are mediated by endogenous AA.