Importance of valine 567 in substrate recognition and oxidation by neuronal nitric oxide synthase

Nitric oxide (NO) is synthesised by a two-step oxidation of -arginine (L-Arg) in the active site of nitric oxide synthase (NOS) with formation of an intermediate, N omega-hydroxy-L-Arg (NOHA). Crystal structures of NOSs have shown the importance of an active-site Val567 residue (numbered for rat neuronal NOS, nNOS) interacting with non-amino acid substrates. To investigate the role of this Val residue in substrate recognition and NO-formation activity by nNOS, we generated and purified four Val567 mutants of nNOS, Val567Leu, Val567Phe, Val567Arg and Val567Glu. We characterized these proteins and tested their ability to generate NO from the oxidation of natural substrates L-Arg and NOHA, and from N-hydroxyguanidines previously identified as alternative substrates for nNOS. The Val567Leu mutant displayed lower NO formation activities than the wild type (WT) in the presence of all tested compounds. Surprisingly, the Val567Phe mutant formed low amounts of NO only from NOHA. These two mutants displayed lower affinity for L-Arg and NOHA than the WT protein. Val576Glu and Val567Arg mutants were much less stable and did not lead to any formation of NO. These results suggest that Val567 is an important residue for preserving the integrity of the active site, for substrate binding, and subsequently for NO-formation in nNOS.     

Structural basis for dipeptide amide isoform-selective inhibition of neuronal nitric oxide synthase

Three nitric oxide synthase (NOS) isoforms, eNOS, nNOS and iNOS, generate nitric oxide (NO) crucial to the cardiovascular, nervous and host defense systems, respectively. Development of isoform-selective NOS inhibitors is of considerable therapeutic importance. Crystal structures of nNOS-selective dipeptide inhibitors in complex with both nNOS and eNOS were solved and the inhibitors were found to adopt a curled conformation in nNOS but an extended conformation in eNOS. We hypothesized that a single-residue difference in the active site, Asp597 (nNOS) versus Asn368 (eNOS), is responsible for the favored binding in nNOS. In the D597N nNOS mutant crystal structure, a bound inhibitor switches to the extended conformation and its inhibition of nNOS decreases >200-fold. Therefore, a single-residue difference is responsible for more than two orders of magnitude selectivity in inhibition of nNOS over eNOS by L-N(omega)-nitroarginine-containing dipeptide inhibitors.     

Chimeras of nitric-oxide synthase types I and III establish fundamental correlates between heme reduction, heme-NO complex formation, and catalytic activity

Neuronal nitric-oxide synthase (nNOS or NOS I) and endothelial NOS (eNOS or NOS III) differ widely in their reductase and nitric oxide (NO) synthesis activities, electron transfer rates, and propensities to form a heme-NO complex during catalysis. We generated chimeras by swapping eNOS and nNOS oxygenase domains to understand the basis for these differences and to identify structural elements that determine their catalytic behaviors. Swapping oxygenase domains did not alter domain-specific catalytic functions (cytochrome c reduction or H(2)O(2)-supported N(omega)-hydroxy-l-arginine oxidation) but markedly affected steady-state NO synthesis and NADPH oxidation compared with native eNOS and nNOS. Stopped-flow analysis showed that reductase domains either maintained (nNOS) or slightly exceeded (eNOS) their native rates of heme reduction in each chimera. Heme reduction rates were found to correlate with the initial rates of NADPH oxidation and heme-NO complex formation, with the percentage of heme-NO complex attained during the steady state, and with NO synthesis activity. Oxygenase domain identity influenced these parameters to a lesser degree. We conclude: 1) Heme reduction rates in nNOS and eNOS are controlled primarily by their reductase domains and are almost independent of oxygenase domain identity. 2) Heme reduction rate is the dominant parameter controlling the kinetics and extent of heme-NO complex formation in both eNOS and nNOS, and thus it determines to what degree heme-NO complex formation influences their steady-state NO synthesis, whereas oxygenase domains provide minor but important influences. 3) General principles that relate heme reduction rate, heme-NO complex formation, and NO synthesis are not specific for nNOS but apply to eNOS as well.     

A novel neuroprotective agent with antioxidant and nitric oxide synthase inhibitory action

N(alpha)-vanillyl-N(omega)-nitroarginine (N - 1) that combines the active functions of natural antioxidant and nitric oxide synthase inhibitor was developed for its neuroprotective properties. N - 1 exhibited protective effects against hydrogen peroxide-induced cell damage and the inhibitory effect on nitric oxide 'NO' production induced by calcium ionophore in NG 108-15 cells. N - 1 inhibited the constitutive NOS isolated from rat cerebellar in a greater extent than constitutive NOS from human endothelial cells. Low binding energy (-10.2 kcal/mol) obtained from docking N - 1 to nNOS supported the additional mode of action of N - 1 as an nNOS inhibitor. The in vivo neuroprotective effect on kainic acid-induced nitric oxide production and neuronal cell death in rat brain was investigated via microdialysis. Rats were injected intra-peritonially with N - 1 at 75 micromol/kg before kainic acid injection (10 mg/kg). The significant suppression effect on kainic acid-induced NO and significant increase in surviving cells were observed in the hippocampus at 40 min after the induction.     

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.     

Neuronal nitric oxide synthase is resistant to ethanol.

To test for a possible role of nitric oxide (NO) in the neurotoxicity of ethanol, we studied the effects of ethanol on the neuronal NO synthase (nNOS) both in vitro and in vivo. Ethanol, up to 200 mM, did not change the NOS activity in the cerebellar homogenate or the production of NO by the cultured cerebellar granule cells. The number of NADPH diaphorase-positive cells in the culture did not change after the exposure to 200 mM ethanol in vitro. The NOS activity in the various brain regions of mice remained similar to the controls after the acute (3 g/kg) and the chronic (33 g/kg/day, 3.5 days) administration of ethanol. N(omega)-nitro-L-arginine, a NOS inhibitor, did not affect the ethanol-withdrawal behavior. These results indicate that nNOS is resistant to ethanol at clinically relevant concentrations and that ethanol affects the NO-operated system in the brain through a pathway other than that of nNOS.     

Effects of arginase isoforms on NO Production by nNOS.

Both arginase isoforms (AI and AII) regulate high-level NO production by the inducible NOS, but whether the arginase isoforms also regulate low-level NO production by neuronal NOS (nNOS) is not known. In this study, 293 cells that stably overexpress nNOS gene (293nNOS cells) were transfected with rat AI (pEGFP-AI) or AII (pcDNA-AII) plasmids, and nitrite production was measured with or without supplemental L-arginine. Transfection with pEGFP-AI increased AI expression and activity 10-fold and decreased intracellular l-arginine by 50%. Nitrite production was inhibited by >80% when no l-arginine was supplemented but not when 1 mM L-arginine was present. The inhibition was reversed by an arginase inhibitor, N(omega)-hydroxy-L-arginine. Transfection with pcDNA-AII increased AII expression and activity but had little effect on nitrite production even if no l-arginine was added. These results suggest that, in 293nNOS cells, AI was more effective in regulating NO production by nNOS, most likely by competing for L-arginine.     

Novel substrates for nitric oxide synthases

Enzymatic generation of nitric oxide (NO) by nitric oxide synthase (NOS) consists of two oxidation steps. The first step converts L-arginine to N(G)-hydroxy-L-arginine (NOHA), a key intermediate, and the second step converts NOHA to NO and L-citrulline. To fully probe the substrate specificity of the second enzymatic step, an extensive structural screening was carried out using a series of N-alkyl (and N-aryl) substituted-N'-hydroxyguanidines (1-14). Among the eleven N-alkyl-N'-hydroxyguanidines evaluated, N-n-propyl (2), N-iso-propyl (3), N-n-butyl (4), N-s-butyl (5), N-iso-butyl (6), N-pentyl (8) and N-iso-pentyl (9) derivatives were efficiently oxidized by the three isoenzymes of NOS (nNOS, iNOS and eNOS) to generate NO. N-Butyl-N'-hydroxyguanidine (4) was the best substrate for iNOS (K(m)=33 microM) and N-iso-propyl-N'-hydroxyguanidine (3) was the best substrate for nNOS (K(m)=56 microM). When the alkyl substituents were too small (such as ethyl 1) or too large (such as hexyl 10 and cyclohexyl 11), the activity decreased significantly. This suggests that the van der Waals interaction between the alkyl group and the hydrophobic cavity in the NOS active site contributes significantly to the relative reactivity of compounds 3-11. Moreover, five N-aryl-N'-hydroxyguanidines were found to be good substrates for iNOS, but not substrates for eNOS and nNOS. N-phenyl-N'-hydroxyguanidine was the best substrate among them (K(m)=243 microM). This work demonstrates that N-alkyl substituted hydroxyguanidine compounds are novel NOS substrates which 'short-circuit' the first oxidation step of NOS, and N-aryl substituted hydroxyguanidine compounds are isoform selective NOS substrate.     

Endothelial nitric oxide synthase is predominantly involved in angiotensin II modulation of renal vascular resistance and norepinephrine release

Nitric oxide (NO) is mainly generated by endothelial NO synthase (eNOS) or neuronal NOS (nNOS). Recent studies indicate that angiotensin II generates NO release, which modulates renal vascular resistance and sympathetic neurotransmission. Experiments in wild-type [eNOS(+/+) and nNOS(+/+)], eNOS-deficient [eNOS(-/-)], and nNOS-deficient [nNOS(-/-)] mice were performed to determine which NOS isoform is involved. Isolated mice kidneys were perfused with Krebs-Henseleit solution. Endogenous norepinephrine release was measured by HPLC. Angiotensin II dose dependently increased renal vascular resistance in all mice species. EC(50) and maximal pressor responses to angiotensin II were greater in eNOS(-/-) than in nNOS(-/-) and smaller in wild-type mice. The nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.3 mM) enhanced angiotensin II-induced pressor responses in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. In nNOS(+/+) mice, 7-nitroindazole monosodium salt (7-NINA; 0.3 mM), a selective nNOS inhibitor, enhanced angiotensin II-induced pressor responses slightly. Angiotensin II-enhanced renal nerve stimulation induced norepinephrine release in all species. L-NAME (0.3 mM) reduced angiotensin II-mediated facilitation of norepinephrine release in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. 7-NINA failed to modulate norepinephrine release in nNOS(+/+) mice. (4-Chlorophrnylthio)guanosine-3', 5'-cyclic monophosphate (0.1 nM) increased norepinephrine release. mRNA expression of eNOS, nNOS, and inducible NOS did not differ between mice strains. In conclusion, angiotensin II-mediated effects on renal vascular resistance and sympathetic neurotransmission are modulated by NO in mice. These effects are mediated by eNOS and nNOS, but NO derived from eNOS dominates. Only NO derived from eNOS seems to modulate angiotensin II-mediated renal norepinephrine release.     

Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase

We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0. 13 to 86 s(-1) upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N(omega)-hydroxy-l-arginine (0.005 and 0.014 s(-1), respectively, at 10 degrees C). O(2) binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved with N(omega)-hydroxy-l-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K(m) for O(2) to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology.     

Thermodynamic analysis of L-arginine and N omega-hydroxy-L-arginine binding to nitric oxide synthase

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS(oxy)) in the presence of the cofactor tetrahydrobiopterin. The intermediate N(omega)-hydroxy-L-arginine (NHA) has a larger affinity than L-Arginine (L-Arg) for nNOS(oxy), with K(d)=0.4+/-0.1 microM and 1.7+/-0.3 microM at 25 degrees C, respectively. nNOS(oxy) binds NHA and L-Arg with DeltaH -4.1+/-0.2 and -1.0+/-0.1 kcal/mol and DeltaS=15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to L-Arg. The changes in heat capacity (DeltaC(p)) are relatively small for binding of both NHA and L-Arg (-53+/-18 and -95+/-23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.     

Arginine conversion to nitroxide by tetrahydrobiopterin-free neuronal nitric-oxide synthase. Implications for mechanism

We studied catalysis by tetrahydrobiopterin (H4B)-free neuronal nitric-oxide synthase (nNOS) to understand how heme and H4B participate in nitric oxide (NO) synthesis. H4B-free nNOS catalyzed Arg oxidation to N(omega)-hydroxy-l-Arg (NOHA) and citrulline in both NADPH- and H(2)O(2)-driven reactions. Citrulline formation was time- and enzyme concentration-dependent but was uncoupled relative to NADPH oxidation, and generated nitrite and nitrate without forming NO. Similar results were observed when NOHA served as substrate. Steady-state and stopped-flow spectroscopy with the H4B-free enzyme revealed that a ferrous heme-NO complex built up after initiating catalysis in both NADPH- and H(2)O(2)-driven reactions, consistent with formation of nitroxyl as an immediate product. This differed from the H4B-replete enzyme, which formed a ferric heme-NO complex as an immediate product that could then release NO. We make the following conclusions. 1) H4B is not essential for Arg oxidation by nNOS, although it helps couple NADPH oxidation to product formation in both steps of NO synthesis. Thus, the NADPH- or H(2)O(2)-driven reactions form common heme-oxy species that can react with substrate in the presence or absence of H4B. 2) The sole essential role of H4B is to enable nNOS to generate NO instead of nitroxyl. On this basis we propose a new unified model for heme-dependent oxygen activation and H4B function in both steps of NO synthesis.     

Inhibitory effects of a series of 7-substituted-indazoles toward nitric oxide synthases: particular potency of 1H-indazole-7-carbonitrile

A series of new 7-monosubstituted and 3,7-disubstituted indazoles have been prepared and evaluated as inhibitors of nitric oxide synthases (NOS). 1H-indazole-7-carbonitrile (6) was found equipotent to 7-nitro-1H-indazole (1) and demonstrated preference for constitutive NOS over inducible NOS. By contrast, 1H-indazole-7-carboxamide (8) was slightly less potent but demonstrated a surprising selectivity for the neuronal NOS. Further substitution of 6 by a Br-atom at carbon-3 of the heterocycle enhanced 10-fold the inhibitory effects. Inhibition of NO formation by 6 appeared to be competitive versus both substrate and the cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B). In close analogies with 1, compound 6 strongly inhibited the NADPH oxidase activity of nNOS and induced a spin state transition of the heme-Fe(III). Our results are explained with the help of the X-ray structures that identified key-features for binding of 1 at the active site of NOS.     

Kinetics of CO and NO ligation with the Cys(331)-->Ala mutant of neuronal nitric-oxide synthase

Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX(4)C) that participates in a ZnS(4) center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys(331) --> Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of l-Arg and tetrahydrobiopterin (BH(4)). Because binding l-Arg and BH(4) to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH(4) has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.     

Comparison of wild type neuronal nitric oxide synthase and its Tyr588Phe mutant towards various l-arginine analogues

Crystal structures of nitric oxide synthases (NOS) isoforms have shown the presence of a strongly conserved heme active-site residue, Tyr588 (numbering for rat neuronal NOS, nNOS). Preliminary biochemical studies have highlighted its importance in the binding and oxidation to NO of natural substrates L-Arg and N^ω-hydroxy-l-arginine (NOHA) and suggested its involvement in mechanism. We have used UV-visible and EPR spectroscopy to investigate the effects of the Tyr588 to Phe mutation on the heme-distal environment, on the binding of a large series of guanidines and N-hydroxyguanidines that differ from L-Arg and NOHA by the nature of their alkyl- or aryl-side chain, and on the abilities of wild type (WT) and mutant to oxidize these analogues with formation of NO. Our EPR experiments show that the heme environment of the Tyr588Phe mutant differs from that of WT nNOS. However, the addition of L-Arg to this mutant results in EPR spectra similar to that of WT nNOS. Tyr588Phe mutant binds L-Arg and NOHA with much weaker affinities than WT nNOS but both proteins bind non α-amino acid guanidines and N-hydroxyguanidines with close affinities. WT nNOS and mutant do not form NO from the tested guanidines but oxidize several N-hydroxyguanidines with formation of NO in almost identical rates. Our results show that the Tyr588Phe mutation induces structural modifications of the H-bonds network in the heme-distal site that alter the reactivity of the heme. They support recent spectroscopic and mechanistic studies that involve two distinct heme-based active species in the two steps of NOS mechanism.     

Distinct influence of N-terminal elements on neuronal nitric-oxide synthase structure and catalysis

Nitric oxide (NO) is a signal molecule produced in animals by three different NO synthases. Of these, only NOS I (neuronal nitric-oxide synthase; nNOS) is expressed as catalytically active N-terminally truncated forms that are missing either an N-terminal leader sequence required for protein-protein interactions or are missing the leader sequence plus three core structural motifs that in other NOS are required for dimer assembly and catalysis. To understand how the N-terminal elements impact nNOS structure-function, we generated, purified, and extensively characterized variants that were missing the N-terminal leader sequence (Delta296nNOS) or missing the leader sequence plus the three core motifs (Delta349nNOS). Eliminating the leader sequence had no impact on nNOS structure or catalysis. In contrast, additional removal of the core elements weakened but did not destroy the dimer interaction, slowed ferric heme reduction and reactivity of a hemedioxy intermediate, and caused a 10-fold poorer affinity toward substrate l-arginine. This created an nNOS variant with slower and less coupled NO synthesis that is predisposed to generate reactive oxygen species along with NO. Our findings help justify the existence of nNOS N-terminal splice variants and identify specific catalytic changes that create functional differences among them.     

Acute and conditioned hypoxic tolerance augmented by endothelial nitric oxide synthase inhibition in mice.

To identify a possible role for nitric oxide (NO) in acute hypoxic tolerance (HT) we measured hypoxic survival time (HST), effect of hypoxic conditioning (HC), and survival following hypoxic conditioning while blocking or mimicking the action of nitric oxide synthase (NOS). To inhibit NOS, CD-1 mice were given supplemental endogenous NOS inhibitor asymmetrical dimethylarginine (ADMA) or a synthetic NOS inhibitor N(omega)-nitro-L-arginine (L-NNA), both of which nonselectively inhibit three of the isoforms of NOS [inducible (iNOS), neuronal (nNOS), and endothelial NOS (eNOS)]. ADMA (10 mg/kg i.p.) or saline vehicle was given 5 min before HST testing. L-NNA was given orally at 1 g/l in drinking water with tap water as the control for 48 h before testing. Both ADMA and L-NNA significantly increased HST and augmented the HC effect on HST. Neither the nNOS selective inhibitor 7-nitroindazole (7-NI) nor the iNOS selective inhibitor N-{[3-(aminomethyl)phenyl]methyl}-enthanimidamide (1400W) had a statistically significant effect on HST or HT. The NO donor, 3-morpholinosydnoeimine, when given alone did not significantly decrease HT, but it did mitigate the increased HT effect of L-NNA. These data confirm that acute hypoxic conditioning increases HT and that NOS inhibition by endogenous (ADMA) and a synthetic NOS inhibitor (L-NNA) further increases HT, whereas iNOS and nNOS inhibition does not, suggesting that it is the inhibition of eNOS that mediates enhancement of HT.     

Secretagogue-stimulated pancreatic secretion is differentially regulated by constitutive NOS isoforms in mice

Nitric oxide (NO) and NO synthase (NOS) play controversial roles in pancreatic secretion. NOS inhibition reduces CCK-stimulated in vivo pancreatic secretion, but it is unclear which NOS isoform is responsible, because NOS inhibitors lack specificity and three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Mice having individual NOS gene deletions were used to clarify the NOS species and cellular interactions influencing pancreatic secretion. In vivo secretion was performed in anesthetized mice by collecting extraduodenal pancreatic duct juice and measuring protein output. Nonselective NOS blockade was induced with N(omega)-nitro-L-arginine (L-NNA; 10 mg/kg). In vivo pancreatic secretion was maximal at 160 pmol.kg(-1).h(-1) CCK octapeptide (CCK-8) and was reduced by NOS blockade (45%) and eNOS deletion (44%). Secretion was unaffected by iNOS deletion but was increased by nNOS deletion (91%). To determine whether the influence of NOS on secretion involved nonacinar events, in vitro CCK-8-stimulated secretion of amylase from isolated acini was studied and found to be unaltered by NOS blockade and eNOS deletion. Influence of NOS on in vivo secretion was further examined with carbachol. Protein secretion, which was maximal at 100 nmol.kg(-1).h(-1) carbachol, was reduced by NOS blockade and eNOS deletion but unaffected by nNOS deletion. NOS blockade by L-NNA had no effect on carbachol-stimulated amylase secretion in vitro. Thus constitutive NOS isoforms can exert opposite effects on in vivo pancreatic secretion. eNOS likely plays a dominant role, because eNOS deletion mimics NOS blockade by inhibiting CCK-8 and carbachol-stimulated secretion, whereas nNOS deletion augments CCK-8 but not carbachol-stimulated secretion.     

Suppression of paraquat-induced wet dog shakes by nitric oxide synthase inhibitors in rats

We have found that paraquat (PQ), a widely used herbicide, causes wet dog shakes (WDS), which involve the central opioid system, in rats. A non-selective nitric oxide (NO) synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NA), but not its less active enantiomer, N(omega)-nitro-D-arginine, decreased the PQ-induced WDS in a dose-related manner. A selective neuronal NOS inhibitor in vivo, 7-nitroindazole, also decreased the PQ-induced WDS. Although an opioid receptor antagonist, naloxone, reversed the suppressive effect of these NOS inhibitors on the PQ-induced WDS, L-arginine, an NO precursor, had no effect on it. These findings suggest that the suppression of the PQ-induced WDS by NOS inhibition is associated with the central opioid system and is insusceptible to exogenous L-arginine.     

EPR studies on the photo-induced intermediates of ferric NO complexes of rat neuronal nitric oxide synthase trapped at low temperature.

Rat neuronal nitric oxide synthase (nNOS) was expressed in Escherichia coli and purified. Although the nitric oxide (NO) complex of the ferric heme was EPR-silent, photo-illumination at 5 K to the NO complex of the ferric nNOS in the substrate-free form produced a new high spin EPR signal similar to that of the ferric heme of N(omega)-nitro-L-arginine-bound nNOS, suggesting that the photo-dissociated NO might move away from the heme. Low photo-dissociability of NO in this complex indicated less restricted movement of the dissociated NO in the distal region of the heme, which might result in the rapid rebinding of the NO to the ferric heme at 5 K. In the presence of substrate L-arginine, derivatives, or product L-citrulline, the photo-products from the ferric NO complexes exhibited large novel EPR signals with a spin-coupled interaction between the ferric heme (S = 5/2) and the photolyzed NO (S = 1/2), suggesting a stereochemically restricted interaction between the photo-dissociated NO and the guanidino- or the ureido-group of the substrate analogues at the distal heme region of nNOS. The photo-product from the NO complex produced from citrulline-bound nNOS might be the same intermediate species as that formed in the last step of the catalytic cycle.