Interaction between neuronal nitric oxide synthase and inhibitory G protein activity in heart rate regulation in conscious mice

Nitric oxide (NO) synthesized within mammalian sinoatrial cells has been shown to participate in cholinergic control of heart rate (HR). However, it is not known whether NO synthesized within neurons plays a role in HR regulation. HR dynamics were measured in 24 wild-type (WT) mice and 24 mice in which the gene for neuronal NO synthase (nNOS) was absent (nNOS-/- mice). Mean HR and HR variability were compared in subsets of these animals at baseline, after parasympathetic blockade with atropine (0.5 mg/kg i.p.), after beta-adrenergic blockade with propranolol (1 mg/kg i.p.), and after combined autonomic blockade. Other animals underwent pressor challenge with phenylephrine (3 mg/kg i.p.) after beta-adrenergic blockade to test for a baroreflex-mediated cardioinhibitory response. The latter experiments were then repeated after inactivation of inhibitory G proteins with pertussis toxin (PTX) (30 microgram/kg i.p.). At baseline, nNOS-/- mice had higher mean HR (711+/-8 vs. 650+/-8 bpm, P = 0.0004) and lower HR variance (424+/-70 vs. 1,112+/-174 bpm2, P = 0.001) compared with WT mice. In nNOS-/- mice, atropine administration led to a much smaller change in mean HR (-2+/-9 vs. 49+/-5 bpm, P = 0.0008) and in HR variance (64+/-24 vs. -903+/-295 bpm2, P = 0.02) than in WT mice. In contrast, propranolol administration and combined autonomic blockade led to similar changes in mean HR between the two groups. After beta-adrenergic blockade, phenylephrine injection elicited a fall in mean HR and rise in HR variance in WT mice that was partially attenuated after treatment with PTX. The response to pressor challenge in nNOS-/- mice before PTX administration was similar to that in WT mice. However, PTX-treated nNOS-/- mice had a dramatically attenuated response to phenylephrine. These findings suggest that the absence of nNOS activity leads to reduced baseline parasympathetic tone, but does not prevent baroreflex-mediated cardioinhibition unless inhibitory G proteins are also inactivated. Thus, neuronally derived NO and cardiac inhibitory G protein activity serve as parallel pathways to mediate autonomic slowing of heart rate in the mouse.     

The effect of ketotifen on nitric oxide synthase activity

1. We studied the effect of ketotifen, a second generation H1-receptor antagonist on nitric oxide synthase (NOS) activity in colonic mucosa and in renal tissues, and on rat renal haemodynamics in vivo. 2. Ketotifen (100 micrograms ml-1) increased human colonic NOS activity from 3.7 +/- 0.6 to 14.5 +/- 1.3 nmol g-1 min-1 (P < 0.005, ANOVA). In rat renal cortical and medullary tissues ketotifen increased NOS activity by 55% and 86%, respectively (P < 0.001). The stimulation of NOS activity was attenuated by NADPH deletion and by the addition of N omega nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, but not by [Ca2+] deprivation. NOS activity was unaffected by two other H1-antagonists, diphenhydramine and astemizole, or by the structurally related cyproheptadine. Renal cortical NOS activity was also significantly stimulated 90 min after intravenous administration of ketotifen to anaesthetized rats. 3. Ketotifen administration to anaesthetized rats induced modest declines in blood pressure and reduced total renal, cortical and outer medullary vascular resistance. This is in contrast to diphenhydramine, which did not induce renal vasodilatation. 4. We conclude that ketotifen stimulates NOS activity by mechanisms other than H1-receptor antagonism. The association of this effect with therapeutic characteristics of ketotifen and the clinical implications of these findings are yet to be defined.     

Modulation of sulfur mustard toxicity by arginine analogues and related nitric oxide synthase inhibitors in vitro

Obstructive sleep apnea is a common medical disorder with significant adverse health consequences. The pathogenesis of pharyngeal obstruction during sleep, however, remains elusive. This article addresses the key mechanisms of upper airway (UA) obstruction including the role of transmural pressure, pharyngeal compliance, pharyngeal dilating muscle activity and non-neuromuscular factors. A proposed scheme of the pathophysiology of UA obstruction is outlined.     

Inhibition of neuronal nitric oxide synthase by antipsychotic drugs

Hydralazine was administered at cardiac catheterization to eight children with a ventricular septal defect (age: 2.2-8.8 years), and the extent of afterload reduction was determined using aortic input impedance and wall stress. The pulmonary to systemic blood flow ratio decreased from 2.2 +/- 0.8 to 1.8 +/- 0.4 (p < 0.05) and the pulmonary systemic resistance ratio increased from 0.11 +/- 0.08 to 0.13 +/- 0.10 (p < 0.05) after hydralazine administration. Hydralazine reduced mean aortic pressure and the amplitude of the late systolic peak of the aortic pressure wave. Peak flow velocity in the descending aorta increased from 62 +/- 14 to 81 +/- 24 cm/sec (p < 0.05). Peripheral resistance decreased significantly from 13.3 +/- 5.9 to 6.6 +/- 3.7 10(3) dyn sec/cm3 (p < 0.05). The modulus of the first harmonic, indicating pulse wave reflection, decreased from 1196 +/- 575 to 815 +/- 382 dyn sec/cm3 (p < 0.05). The characteristic impedance, indicating aortic stiffness, did not change. End-systolic wall stress decreased significantly from 54.4 +/- 16.7 to 34.8 +/- 10.2 g/cm2 (p < 0.01). Hydralazine acutely achieved afterload reduction by reducing both peripheral resistance and pulse wave reflection, and increased stroke volume.     

Characterization of C415 mutants of neuronal nitric oxide synthase

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.     

Peroxynitrite reduction of calmodulin stimulation of neuronal nitric oxide synthase

The Ca(2+)-dependent binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) stimulates the catalytic oxidation of L-arginine to nitric oxide. The CaM-dependent increase in catalytic activity is associated with an increase in the flow of electrons from the flavoprotein to the heme domain. In the presence of suboptimal arginine concentrations, uncoupled turnover of nNOS produces both nitric oxide and superoxide, reactive species which combine to form peroxynitrite. We demonstrate here that peroxynitrite and other oxidants produced by nNOS oxidize the methionine residues of CaM and show that the ability of CaM to stimulate nNOS is impaired by this oxidative modification. Of the nine Met residues, those at the C-terminus (Met-144, -145, -124, -109) are most sensitive to oxidation. Correlation of the Met oxidation pattern with ability to stimulate nNOS suggests that oxidation of Met-36 is particularly important for the stimulation of nNOS. Incubation of nNOS with suboptimal concentrations of arginine results in sulfoxidation of the CaM methionine residues. Although nitration of the tyrosine residues in CaM could also occur, this does not occur to a significant extent in the present system. The results suggest that peroxynitrite may exert a feedback effect on its own formation by oxidizing CaM and thereby decreasing its ability to stimulate the turnover of nNOS.     

Non-anticoagulant heparin increases endothelial nitric oxide synthase activity: role of inhibitory guanine nucleotide proteins

Heparin, which is widely used clinically, has recently been shown to have specific properties affecting the vascular endothelium. We hypothesized that heparin stimulates endothelial nitric oxide synthase (eNOS) activity by a mechanism independent of its anticoagulant properties and dependent on an inhibitory guanine nucleotide regulatory protein (Gi). We determined the effect of both heparin and N-acetyl heparin (Non-Hep), a heparin derivative without anticoagulant properties, on eNOS activity in cultured bovine aortic endothelial cells and on endothelium-dependent relaxation in isolated vascular rings. The eNOS activity was determined by measuring both citrulline and nitric oxide (NO) metabolite formation. Heparin and Non-Hep dose-dependently increased basal eNOS activity (ED50 1.0 microgram/ml or 0.15 U/ml), an effect that was significantly inhibited by pertussis toxin (100 ng/ml), a Gi-protein inhibitor. Agonist-stimulated (acetylcholine, 10 microM) eNOS activity was potentiated following pre-treatment with both heparin and Non-Hep and reversed by pertussis toxin. Heparin and Non-Hep induced a dose-dependent relaxation in preconstricted thoracic aortic rings, an effect that was significantly inhibited by pertussis toxin, endothelial inactivation (following treatment with sodium deoxycholate) and NG-nitro-L-arginine-methyl ester (L-NAME). We conclude that heparin and non-anticoagulant heparin induce endothelium-dependent relaxation following activation of eNOS by a mechanism involving a Gi-protein. Administration of heparin derivatives without anticoagulant properties may have therapeutic implications for the preservation of eNOS in conditions characterized by endothelial dysfunction.     

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K(m) for L-arginine 2.8, 2.0 and 2.3 microM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.     

Nitroaromatic amino acids as inhibitors of neuronal nitric oxide synthase

Nitric oxide (NO.) is an important biomodulator of many physiological processes. The inhibition of inappropriate production of NO. by the isoforms of nitric oxide synthase (NOS) has been proposed as a therapeutic approach for the treatment of stroke, inflammation, and other processes. In this study, certain 2-nitroaryl-substituted amino acid analogues were discovered to inhibit NOS. Analogues bearing a 5-methyl substituent on the aromatic ring demonstrated maximal inhibitory potency. For two selected inhibitors, investigation of the kinetics of the enzyme showed the inhibition to be competitive with l-arginine. Additionally, functional NOS inhibition in tissue preparations was demonstrated.     

Down-regulation of neuronal nitric oxide synthase by nitric oxide after oxygen-glucose deprivation in rat forebrain slices

The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-iu m-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

The role of neuronal and endothelial nitric oxide synthase in retinal excitotoxicity

PURPOSE: Nitric oxide synthase (NOS) plays an essential role in neuronal function and is critical in the brain for normal and pathologic responses to glutamate. The role of NOS in the retina is less well understood. The retina provides an experimental system in which the intrinsic circuitry is well defined; retinal excitotoxic damage has been well characterized. METHODS: To determine whether neuronal NOS (nNOS) and endothelial NOS (eNOS) are critical in excitotoxic damage in the retina, nNOS- and eNOS-deficient mice were subjected to intravitreal injections of N-methyl-D-aspartate (NMDA) or to arterial occlusions. RESULTS: Retinal ganglion cells in the nNOS-deficient mouse were relatively resistant to NMDA and to arterial occlusion. In contrast, the damage in the eNOS-deficient mouse retina was not distinguishable from that in control animals. Preinjection with an NOS inhibitor was partially protective. CONCLUSIONS: The presence of nNOS is a prerequisite for the full expression of excitotoxicity in the retina; eNOS does not appear to play a significant role.     

Increased neuronal nitric oxide synthase expression in brain following portacaval anastomosis

The long-sleep (LS) and short-sleep (SS) mice were selected for differences in sensitivity to ethanol but also differ in response to propofol and some neurosteroids. To determine the role of strychnine-sensitive glycine receptors in genetic differences between these mice, effects of propofol, ethanol and pregnenolone sulfate on glycine responses were compared in Xenopus oocytes expressing mRNA extracted from spinal cord of LS and SS mice. The two lines of mice did not differ in sensitivity to glycine, ethanol or pregnenolone sulfate. However, receptors expressed from LS mRNA were more sensitive to the potentiation induced by propofol than those from SS. Binding of [3H]strychnine to spinal cord membranes demonstrated a similar affinity and density of receptors in LS and SS. These results suggest that glycine receptor function could account for differences in propofol sensitivity between LS and SS mice, but may not be responsible for the differences in behavioral sensitivity to ethanol or steroids previously reported.     

Analysis of neuronal nitric oxide synthase isoform expression and identification of human nNOS-mu

Basonuclin was first described as a human keratinocyte zinc finger protein present in the nuclei of proliferative basal keratinocytes in the epidermis. It disappears from keratinocytes that have lost their proliferative ability and have entered terminal differentiation. We now report that basonuclin is present also in the germ cells of the mouse testis and ovary. Immunocytochemical staining detected basonuclin in the nuclei of spermatogonia and spermatocytes at various developmental stages. During spermiogenesis, it relocated from the nucleus to the midpiece of the flagellum of the spermatozoa. In the ovary, basonuclin was found mainly in the nuclei of developing oocytes. The dual presence of basonuclin in differentiated spermatozoa and oocytes suggests that it may play a role in their differentiation and the early development of an embryo.     

HMN-1180, a small molecule inhibitor of neuronal nitric oxide synthase

A newly synthesized isoquinolinesulfonamide, HMN-1180 (1-(5-isoquinolinylsulfonyl)-7-methylhomopiperazine), was shown to have selective inhibitory action against rat neuronal nitric oxide synthase (nNOS) with a Ki value of 5.4 microM. Kinetic analysis indicated that the inhibition was competitive with respect to L-arginine but not to calmodulin (CaM). However HMN-1180 exhibited no significant influence up to a concentration of 1 mM on activity of endothelial NOS (eNOS) and it was less active toward inducible NOS (iNOS) (IC50 > 100 microM). Moreover, nNOS bound to a HMN-1180-coupled Sepharose column, but eNOS and iNOS did not. These results suggest that inhibition of nNOS activity is due to direct binding of the compound to the L-arginine binding site of the synthase. Several HMN-1180 derivatives were synthesized and analyzed for their inhibitory actions against nNOS, eNOS and iNOS to cast light on its structure-activity relationships. The potency of inhibition proved dependent on the position of methyl group in the homopiperazine molecule. HMN-1180 was also found to inhibit glutamate stimulated NO production generated by nNOS in the human neuroblastoma cell line SK-N-MC, thus indicating that it is useful tool for elucidating the physiological role of nNOS in neuronal function.     

Isolation and characterization of a novel, human neuronal nitric oxide synthase cDNA

The G protein beta subunit G beta 5 deviates significantly from the four other members of the G beta family in amino acid sequence, unique expression pattern (only in the CNS), and cytosolic localization. To identify the members of the G beta 5-mediated signaling pathway, we purified the native protein complex containing G beta 5 from the cytosolic fraction of bovine retina. Analysis of the isolated complex revealed that G beta 5 is tightly associated with RGS7, a member of the superfamily of negative regulators of G protein signaling. This finding, for the first time, demonstrates an interaction between a G beta subunit and an RGS protein. G beta 5 was not detected in the outer segments of photoreceptor cells, suggesting that the cytosolic G beta 5-RGS7 complex is not directly involved in phototransduction.     

Relationship of structure and biological activity of monosaccharide lipid A analogues to induction of nitric oxide production by murine macrophage RAW264.7 cells

Lipid A is the active center of bacterial lipopolysaccharide (LPS), which exhibits diverse biological activities via the production of various mediators. We investigated the production of nitric oxide (NO), one of the mediators, by a murine macrophage cell line, RAW264. 7, upon stimulation with a series of monosaccharide lipid A analogues to elucidate the relationship of structure and activity in NO production. The production of other representative mediators, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), was also investigated to compare the structural requirements for the production of these cytokines with those for the production of NO. Structure-activity relationships in NO production correlated well with those in the production of TNF-alpha and IL-6. Among the lipid A analogues possessing different numbers of acyl groups on a 4-O-phosphono-D-glucosamine backbone, compounds like GLA-60 that possess three tetradecanoyl (C14) groups exhibited stronger activities in the production of the mediators than compounds possessing four or two C14 groups. Time course study of the production of these mediators showed that production of NO started and peaked later than those of TNF-alpha and IL-6. Neither neutralization of TNF-alpha activity by antibody nor suppression of TNF-alpha production by pentoxifylline showed a significant suppressive effect on production of NO and IL-6 upon stimulation with LPS or lipid A analogues. Neutralization of IL-6 activity by antibody showed no significant suppressive effect on production of NO and TNF-alpha. A monosaccharide lipid A analogue (GLA-58) which exhibited no detectable agonistic activity showed a suppressive effect on the production of all three mediators upon stimulation with LPS or lipid A analogues. These results indicate that signals for NO production by LPS agonists in murine macrophages are transduced in good correlation with those for production of TNF-alpha and IL-6, although they are not transduced via production of those cytokines.     

N-Phenylamidines as selective inhibitors of human neuronal nitric oxide synthase: structure-activity studies and demonstration of in vivo activity

Selective inhibition of the neuronal isoform of nitric oxide synthase (NOS) compared to the endothelial and inducible isoforms may be required for treatment of neurological disorders caused by excessive production of nitric oxide. Recently, we described N-(3-(aminomethyl)benzyl)acetamidine (13) as a slow, tight-binding inhibitor, highly selective for human inducible nitric oxide synthase (iNOS). Removal of a single methylene bridge between the amidine nitrogen and phenyl ring to give N-(3-(aminomethyl)phenyl)acetamidine (14) dramatically altered the selectivity to give a neuronal selective nitric oxide synthase (nNOS) inhibitor. Part of this large shift in selectivity was due to 14 being a rapidly reversible inhibitor of iNOS in contrast to the essentially irreversible inhibition of iNOS observed with 13. Structure-activity studies revealed that a basic amine functionality tethered to an aromatic ring and a sterically compact amidine are key pharmacophores for this class of NOS inhibitors. Maximal nNOS inhibition potency was achieved with N-(3-(aminomethyl)phenyl)-2-furanylamidine (77) (Ki-nNOS = 0.006 microM; Ki-eNOS = 0.35 microM; Ki-iNOS = 0.16 microM). Finally, alpha-fluoro-N-(3-(aminomethyl)phenyl)acetamidine (74) (Ki-nNOS = 0. 011 microM; Ki-eNOS = 1.1 microM; Ki-iNOS = 0.48 microM) had excellent brain penetration and inhibited nNOS in a rat brain slice assay as well as in the rat brain (cerebellum) in vivo. Thus, N-phenylamidines should be useful in validating the role of nNOS in neurological disorders.     

Expression and regulation of protein inhibitor of neuronal nitric oxide synthase in ventilatory muscles

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.