Effects of hydroxocobalamin and carboxy-PTIO on nitrergic transmission in porcine anococcygeus and retractor penis muscles.

The effects of carboxy-PTIO and hydroxocobalamin were studied on nitrergic transmission in anococcygeus and retractor penis muscles taken during post mortem examination from young male pigs. In both muscles under resting conditions, electrical field stimulation (EFS) caused contractions that were sensitive to tetrodotoxin (1 microM) and were greatly inhibited by prazosin (1 microM) and guanethidine (10-30 microM), but were not significantly affected by atropine (1 microM). In the anococcygeus muscle, but not in the retractor penis muscle, guanethidine produced a prolonged contraction. After tone was raised by guanethidine in the anococcygeus or by phenylephrine (1 microM) in the presence of guanethidine in the retractor penis, EFS caused tetrodotoxin-sensitive relaxations. The EFS-induced relaxations were abolished by the NO synthase inhibitor N(G)-L-nitro-arginine methyl ester (L-NAME; 100 microM) and its effect was partly overcome by L-arginine (1 mM), indicating it was mediated by nitrergic nerves. Carboxy-PTIO (0.1-1 mM) had no significant effect in reducing stimulation-induced nitrergic relaxations in either muscle. However, hydroxocobalamin (0.1-1 mM) caused concentration-dependent reductions of nitrergic relaxations in both muscles. Relaxations to exogenous nitric oxide (1 microM) in both muscles were abolished by carboxy-PTIO (0.3 mM) and hydroxocobalamin (0.1 mM). There were no differences in reactivity to carboxy-PTIO or hydroxocobalamin between anococcygeus and retractor penis muscles from the same species (pig). The finding also confirms earlier observations that the nitrergic transmitter is generally resistant to the NO-scavenger carboxy-PTIO.     

Blockade of nitrergic transmission by hydroquinone, hydroxocobalamin and carboxy-PTIO in bovine retractor penis: role of superoxide anion.

1. The effects of inhibiting endogenous Cu/Zn superoxide dismutase (SOD) with diethyldithiocarbamate (DETCA) were examined on the ability of hydroquinone, hydroxocobalamin and carboxy-PTIO to block nitrergic relaxation in the bovine retractor penis (BRP) muscle. 2. Incubation of strips of BRP with DETCA (3 mM) for 2 h reduced SOD activity from 73.1 +/- 15.7 to 8.2 +/- 1.9 units mg-1 protein. 3. Hydroquinone (10 microM--1 mM) produced weak inhibition of nitrergic (4 Hz, 10 s) relaxation in control strips of BRP, but powerful inhibition in strips treated with DETCA (3 mM, 2 h). Exogenous SOD (250 units ml--1) produced a partial blockade of the ability of hydroquinone to inhibit nitrergic relaxation in DETCA-treated strips. 4. In an assay of SOD-inhibitable reduction of cytochrome C, hypoxanthine (0.1 mM)/xanthine oxidase (16 munits ml-1) and pyrogallol (10 microM), led to the rapid generation of superoxide anion. Hydroquinone (10 microM) also led to the generation of the free radical, although the rate of generation was slower. 5. Two NO-scavenging agents, hydroxocobalamin (0.1 microM--1 mM) and carboxy-PTIO (0.1-1 mM), produced concentration-dependent blockade of nitrergic relaxation of the BRP. The magnitude of the blockade induced by these agents was unaffected following treatment with DETCA or SOD. 6. The findings with hydroquinone support our previous proposal that endogenous Cu/Zn SOD plays a vital role in protecting nitrergic neurotransmission from inactivation by superoxide anion. Results with hydroxocobalamin and carboxy-PTIO are consistent with the known ability of these agents to scavenge NO. The nitrergic neurotransmitter in the BRP thus appears to have the properties of NO.     

Discrimination by the NO-trapping agent, carboxy-PTIO, between NO and the nitrergic transmitter but not between NO and EDRF.

1. The effects of carboxy-PTIO, a scavenger of free radical nitric oxide (NO), were studied on endothelium-dependent relaxations of rat aorta and nitrergic nerve stimulation-induced relaxations of anococcygeus muscle and gastric fundus strips to test the hypothesis that endothelium-derived relaxing factor (EDRF) and the transmitter released by nitrergic nerves is free radical NO. 2. Carboxy-PTIO (10-300 microM) produced concentration-dependent reductions of relaxations elicited by exogenous NO, and relaxations mediated by EDRF released by acetylcholine and ATP in rings of rat aorta. The inhibitory effect of carboxy-PTIO was removed by washing the tissues. 3. In the rat anococcygeus muscle, carboxy-PTIO (10-300 microM) produced concentration-dependent reductions of relaxations to exogenous NO; however, in concentrations up to 2000 microM it did not reduce relaxations elicited by nitrergic nerve stimulation (1-2 Hz), in fact, concentrations of 300 microM or more slightly enhanced them. 4. In rat gastric fundus strips, carboxy-PTIO (100 and 300 microM) reduced relaxations to exogenous NO, but relaxations elicited by stimulation of the nitrergic component of non-adrenergic, non-cholinergic nerves were not affected. 5. These results suggest that EDRF is free radical NO and may be designated EDNO, but the transmitter released from nitrergic nerves does not appear to be identical to EDNO and may not be free radical NO.     

Superoxide anions,free-radical scavengers,and nitrergic neurotransmission

• 1.1. There is now strong evidence that the L-arginine/nitric oxide (NO) pathway generates the transmitter released from certain nonadrenergic, noncholinergic nerves that mediate smooth-muscle relaxation in the respiratory, gastrointestinal, and urogenital tracts. In particular, nitric oxide synthase (NOS) has been detected in these nitrergic nerves, and nerve-induced relaxation can be prevented by NOS inhibitors. Thus, free-radical NO has been considered the putative transmitter candidate.
• 2.2. Despite such evidence, a number of superoxide anion-generating compounds and direct NO scavengers have been found to abolish relaxations to exogenous NO, but to have very little effect on relaxations in response to nitrergic field stimulation. A number of hypotheses have been put forward to explain this paradox: first, that the NO generated within the nerve is attached to a carrier molecule (such as a thiol) to form an adduct, that is released into the junctional gap and that is resistant to superoxide anions and other scavengers; second, that over short distances (up to 200 μm) the rapid diffusion characteristics of NO render it resistant to inhibition by scavengers; third, that NO is indeed released as a free radical, but that it is protected from radical scavengers by other substances present in the junctional region.
• 3.3. Recent experimental evidence supports the third explanation, because nitrergic relaxations, normally resistant to inhibition by superoxide anions, become sensitive following inactivation of copper/ zinc superoxide dismutase (Cu/Zn SOD); the inhibition can be reversed by adding exogenous Cu/Zn SOD (or ascorbate). In addition, the ability of two NO-scavenger compounds, hydroquinone and carboxy-PTIO, to inhibit relaxations to exogenous NO is prevented by certain physiological antioxidants (ascorbate and reduced glutathione in the case of hydroquinone, and ascorbate and α-tocopherol in the case of carboxy-PTIO).
• 4.4. Thus, it is possible that the presence of integrated antioxidant mechanisms within the tissue protects neuronally- released NO from attack by scavenging molecules; exogenous NO would be vulnerable before reaching the protection of the tissue, thus explaining the paradoxical effects mentioned. Organ antioxidant status may therefore be very important in preserving the potency of nitrergic transmission and in preventing NO from reacting with other compounds to produce cytotoxic metabolites (eg., with superoxide anions to form peroxynitrite).

     

Comparison of the pharmacological profile of S-nitrosothiols, nitric oxide and the nitrergic neurotransmitter in the canine ileocolonic junction.

1. In organ bath experiments, hydroquinone (30-100 microM) and hydroxocobalamin (30-100 microM) concentration-dependently inhibited the relaxations induced by NO (0.3-30 microM) but not those by nitroglycerin (GTN, 1 microM) in the canine ileocolonic junction (ICJ). Hydroxocobalamin reduced the relaxation to low frequency (2 Hz) stimulation of the non-adrenergic, non-cholinergic (NANC) nerves, whereas hydroquinone only reduced the NANC nerve-mediated relaxations to electrical stimulation at 16 Hz, 0.5 ms. 2. Relaxations to S-nitroso-L-cysteine (CysNO, 1-30 microM), or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1-30 microM) were not inhibited by hydroquinone (30-100 microM), hydroxocobalamin (30-100 microM), pyrogallol (30-100 microM) or L-cysteine (1-3 microM). Hydroquinone (100 microM) only reduced the relaxation to 10 microM CysNO. Hydroxocobalamin, but not hydroquinone, pyrogallol or L-cysteine, potentiated the relaxations to the lowest concentration (1 microM) of S-nitrosoglutathione (GSNO, 1-30 microM). 3. In the superfusion bioassay, hydroquinone (100 microM) and hydroxocobalamin (1 microM) concentration-dependently inhibited the biological activity of authentic NO (1-4 pmol) to the same extent as that of the transferable nitrergic factor, released from the canine ICJ in response to NANC nerve stimulation (8-16 Hz, 2 ms). Responses to GTN (10 pmol) or adenosine 5'-triphosphate (10 nmol) were not affected. 4. In conclusion, the nitrosothiols CysNO, SNAP and GSNO relax the canine ileocolonic junction, but these relaxations, pharmacologically, behave differently from the NANC nerve-mediated relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of superoxide dismutase inhibition on the discrimination between NO and the nitrergic neurotransmitter in the rat gastric fundus.

1. The influence of diethyldithiocarbamate (DETCA), that irreversibly inhibits Cu/Zn-containing superoxide dismutase, on the inability of 6-anilino-5,8-quinolinedione (LY83583), hypoxanthine/xanthine oxidase, hydroquinone and hydroxocobalamin to reduce electrically-induced NANC relaxations in the rat gastric fundus was investigated. 2. Longitudinal muscle strips of the rat gastric fundus were mounted for auxotonic recording in the presence of atropine and guanethidine and tone was raised by administration of prostaglandin F2 alpha DETCA (3 x 10(-3) M) slightly reduced the short-lasting relaxations induced by 10(-5) M exogenous nitric oxide (NO) and transmural electrical stimulation for 10 s at 4 Hz but this effect was not influenced by 1000 u ml-1 superoxide dismutase (SOD). 3. DETCA (3 x 10(-5) -3 x 10(-3) M) concentration-dependently potentiated the inhibitory effect of LY83583 upon the electrically-induced relaxations, although this was less pronounced than the inhibition of the NO-induced relaxations. The inhibition of the electrically-induced non-adrenergic non-cholinergic (NANC) relaxations was not reversed by SOD while that of the NO-induced relaxations was partially reversed. 4. The inhibitory effect of hypoxanthine/xanthine oxidase, hydroquinone and hydroxocobalamin on the electrically-induced NANC relaxations in the presence of DETCA (3 x 10(-3) M) was not different from the inhibitory effect of DETCA alone. 5. It was concluded that the differentiating effect of LY83583 between exogenous NO and the endogenous nitrergic neurotransmitter is partially related to protection of the endogenous nitrergic neurotransmitter by high levels of intracellular superoxide dismutase. This mechanism does not hold for hydroquinone and hydroxocobalamin, as they still discriminate between exogenous NO and the endogenous nitrergic neurotransmitter in the presence of DETCA. The possibility that the endogenous nitrergic neurotransmitter is not free NO in the rat gastric fundus therefore remains open.     

Effects of superoxide anion generators and thiol modulators on nitrergic transmission and relaxation to exogenous nitric oxide in the sheep urethra

The effects of superoxide anion generators, the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoine-1-oxyl 3-oxide (carboxy-PTIO), the specific guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ), and thiol modulating agents were investigated on relaxations induced by nitrergic stimulation and exogenous NO addition in the sheep urethra. Methylene blue (MB, 10 microM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml(-1)) inhibited NO-mediated relaxations, without affecting those induced by nitrergic stimulation. This resistance was not diminished following inhibition of endogenous Cu/Zn superoxide dismutase (Cu/Zn SOD) with diethyldithiocarbamic acid (DETCA, 3 mM), which almost abolished tissue SOD activity. Carboxy-PTIO (0.1 - 0.5 mM) inhibited NO-mediated relaxations but had no effect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml(-1)). Relaxations to NO were reduced, but not abolished, by ODQ (10 microM), while nitrergic responses were completely blocked. The thiol modulators, ethacrynic acid (0.1 mM), diamide (1.5 mM), or 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB, 0. 5 mM), and subsequent treatment with dithiothreitol (DTT, 2 mM) had no effect on responses to nitrergic stimulation or NO. In contrast, N-ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. L-cysteine (L-cys, 0.1 mM) had no effect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD-independent manner. Our results do not support the view that the urethral nitrergic transmitter is free NO, and the possibility that another compound is acting as mediator still remains open. British Journal of Pharmacology (2000) 129, 53 - 62     

Effect of thiol modulators and Cu/Zn superoxide dismutase inhibition on nitrergic relaxations in the rat gastric fundus.

1. The effects of superoxide anion generators before and after treatment with inhibitors of Cu/Zn superoxide dismutase (Cu/Zn SOD) and the effects of thiol-modulating agents were investigated on nitrergic relaxations to electrical stimulation of non-adrenergic non-cholinergic (NANC) nerves of the rat gastric fundus and on relaxations to authentic nitric oxide (NO) and nitroglycerin. 2. The superoxide anion generators, pyrogallol (30 microM) and duroquinone (30-60 microM), significantly inhibited the relaxations to NO (0.03-3 microM) but not nitrergic relaxations to NANC nerve stimulation (0.5-8 Hz) or those to ATP (10 microM). Treatment of the rat gastric fundus with the inhibitors of Cu/Zn SOD, diethyldithiocarbamate (DETC, 1 mM for 2 h) or triethylenetetramine (TETA, 100 microM for 2 h) had no effect on the relaxations to NANC nerve stimulation (1-8 Hz), NO (0.03-3 microM) or on those to ATP (10 microM). 3. After treatment of the rat gastric fundus with DETC (1 mM) but not after treatment with TETA (100 microM), pyrogallol (30 microM) and duroquinone (30-60 microM) significantly inhibited the nitrergic relaxations to electrical stimulation (0.5-8 Hz) and those to NO (0.03-3 microM). This inhibitory effect of pyrogallol and duroquinone was prevented by addition of exogenous SOD (250 units ml-1). Pyrogallol but not duroquinone also inhibited the NO-independent relaxations to ATP (10 microM). 4. The thiol modulators, buthionine sulphoximine (1 mM for 2 h) and ethacrynic acid (30 microM for 2 h), significantly inhibited the relaxations to nitroglycerin (0.03-3 microM) but had no effect on the nitrergic relaxations to electrical stimulation (0.5-8 Hz) or on those to NO (0.03-10 microM) and ATP (10 microM). The thiol modulators, sulphobromophthalein (100 microM for 2 h) and diamide (30-100 microM for 2 h) did not affect the relaxations to nitroglycerin, or those to NANC nerve stimulation and NO. 5. In summary, thiol modulators significantly inhibited the thiol-dependent relaxations to nitroglycerin but not those to NANC nerve stimulation or NO. Relaxations to nitrergic stimulation were decreased by superoxide anion generators only after inhibition of Cu/Zn SOD. These results suggest that the nitrergic NANC neurotransmitter in the rat gastric fundus is not a nitrosothiol but more likely free NO, which is protected from breakdown by tissue SOD.     

The influence of nitric oxide donors on the responses to nitrergic nerve stimulation in the mouse duodenum

We investigated whether exogenous nitric oxide (NO) donors have a prejunctional and/or postjunctional inhibitory effect on the nitrergic responses and whether this inhibitory effect was mediated by NO itself and in part, by cyclic GMP in mouse duodenal strips. N(omega)-nitro-L-arginine inhibited relaxations induced by electrical field stimulation of nitrergic nerves, but not those with acidified NaNO2. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) inhibited both types of relaxations while 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) and N-ethylmaleimide were ineffective. NO donors, nitroglycerin and sodium nitroprusside, inhibited relaxations induced by nitrergic nerve stimulation, but not those with acidified NaNO2. Hemoglobin, exogenous Cu(2+)/Zn(2+) superoxide dismutase, diethyldithiocarbamic acid and pyrogallol did not influence the relaxation with nitrergic nerve stimulation. However, hemoglobin, diethyldithiocarbamic acid, pyrogallol and diethyldithiocarbamic acid plus pyrogallol attenuated the inhibitory effect of NO donors on relaxation with nitrergic nerve stimulation, and exogenous superoxide dismutase potentiated this inhibitory effect. Moreover, nitrergic nerve-mediated relaxations were inhibited by 8-bromo-cyclic GMP, but not by 8-bromo-cyclic AMP. These results suggest that exogenous NO donors have a prejunctional inhibitory effect on the nerve-mediated nitrergic relaxation and that the inhibitory effects of nitroglycerin and sodium nitroprusside are NO-dependent, but not related to NO metabolites such as peroxynitrite or a nitrosothiol intermediate. However, a contribution of S-nitrosothiol formed intracellularly cannot be entirely ruled out. Also, this prejunctional inhibition is mediated, at least in part, by the cyclic GMP, but not the cyclic AMP, pathway.     

Pre- and postjunctional protective effect of neocuproine on the nitrergic neurotransmitter in the mouse gastric fundus

1. Electrical field stimulation (EFS) of non-adrenergic non-cholinergic nerves of the mouse gastric fundus induced frequency-dependent transient relaxations which were mimicked by nitric oxide (NO), added as acidified NaNO(2). The NO donors S-nitrosocysteine, S-nitrosoglutathione, SIN-1 and hydroxylamine induced sustained concentration-dependent relaxations. The NO synthase blocker L-nitro arginine (L-NOARG; 300 microM) abolished the relaxations to EFS without affecting the relaxations to NO. 2. The copper(I) chelator neocuproine (10 microM) enhanced the relaxations to EFS and NO but inhibited those to S-nitrosocysteine and S-nitrosoglutathione. Neocuproine potentiated the relaxations to SIN-1, which releases NO extracellularly, without affecting the relaxations to hydroxylamine, which releases NO intracellularly. 3. The potentiating effect of neocuproine on the relaxations to EFS was more pronounced after inhibition of catalase with 3-amino-1,2,4-triazole (1 mM) but not after inhibition of Cu/Zn superoxide dismutase (SOD) with diethyl dithiocarbamic acid (DETCA, 1 mM). The potentiating effect of neocuproine on relaxations to NO was not altered by 3-amino-1,2,4-triazole or DETCA treatment. 4. The relaxations to EFS were significantly inhibited by the oxidants hydrogen peroxide (70 microM) and duroquinone (10 microM) but only after inhibition of catalase with 3-amino-1,2,4-triazole or after inhibition of Cu/ZnSOD with DETCA respectively. 5. Our results suggest that neocuproine can act as an antioxidant in the mouse gastric fundus and that both catalase and Cu/ZnSOD protect the nitrergic neurotransmitter from oxidative breakdown. Since inhibition of catalase but not inhibition of Cu/ZnSOD potentiated the effect of neocuproine on relaxations to EFS without affecting the relaxations to NO, catalase may protect the nitrergic neurotransmitter mainly at a prejunctional site whereas Cu/ZnSOD protects at a postjunctional site.     

Effects of pyrogallol, hydroquinone and duroquinone on responses to nitrergic nerve stimulation and NO in the rat anococcygeus muscle

1. The hypothesis that endogenous superoxide dismutase (SOD) protects the nitrergic transmitter from inactivation by superoxide and that this explains the lack of sensitivity of the transmitter to superoxide generators was tested in the rat isolated anococcygeus muscle. 2. Responses to nitrergic nerve stimulation or to NO were not significantly affected by exogenous SOD or by the Cu/Zn SOD inhibitor diethyldithiocarbamic acid (DETCA). 3. Hydroquinone produced a concentration-dependent reduction of responses to NO with an IC50 of 27 microM, and higher concentrations reduced relaxant responses to nitrergic nerve stimulation with an IC50 of 612 microM. The effects of hydroquinone were only slightly reversed by SOD, so it does not appear to be acting as a superoxide generator. 4. Pyrogallol produced a concentration-dependent reduction in responses to NO with an IC50 value of 39 microM and this effect was reversed by SOD (100-1000 u ml(-1)). Pyrogallol did not affect responses to nitrergic nerve stimulation. Treatment with DETCA did not alter the differentiating action of pyrogallol. 5. Duroquinone produced a concentration-dependent reduction of relaxations to NO with an IC50 value of 240 microM and 100 microM slightly decreased nitrergic relaxations. After treatment with DETCA, duroquinone produced greater reductions of relaxant responses to NO and to nitrergic stimulation, the IC50 values being 8.5 microM for NO and 40 microM for nitrergic nerve stimulation: these reductions were reversed by SOD. 6. The findings do not support the hypothesis that the presence of Cu/Zn SOD explains the greater susceptibility of NO than the nitrergic transmitter to the superoxide generator pyrogallol, but suggest that it may play a role in the effects of duroquinone.     

EFFECTS OF HYDROXOCOBALAMIN AND HAEMOGLOBIN ON NO-MEDIATED RELAXATIONS IN THE RAT ANOCOCCYGEUS MUSCLE

1. The effects of hydroxocobalamin (Vitamin B_12a) on relaxations produced by nitric oxide (NO), some NO-donating compounds and nitrergic nerve stimulation in isolated preparations of the rat anococcygeus muscle were compared with the effects of haemoglobin. 2. Hydroxocobalamin (30 μmol/L) significantly reduced relaxations induced by NO (0.1–3 μmol/L) and sodium nitroprusside (SNP; 0.01–0.3 μmol/L) but did not affect relaxations induced by glyceryl trinitrate (GTN; 0.01–1 μmol/L), S-nitrosocysteine (0.1–0.3 μmol/L) or stimulation of nitrergic nerves. A higher concentration of hydroxocobalamin (100 μmol/L) slightly reduced nitrergic nerve stimulation-induced relaxations. 3. Haemoglobin (10 μmol/L) blocked relaxation induced by NO and reduced relaxations induced by SNP, GTN, S-nitrosocysteine and nitrergic nerve stimulation. 4. When nitrergic nerve stimulation-induced relaxations had been partially reduced by the NO synthase inhibitor l-NAME (5–10 μmol/L), hydroxocobalamin had only a weak and transient inhibitory effect. 5. Noradrenergic contractions induced by field stimulation were not affected by hydroxocobalamin (30 μmol/L), but were enhanced by haemoglobin (10 μmol/L). 6. The results suggest that the transmitter released from nitrergic nerves in anococcygeus muscles resembles NO-releasing compounds such as S-nitrosocysteine and GTN but not SNP or free NO.     

Role of superoxide dismutase enzymes and ascorbate in protection of nitrergic relaxation against superoxide anions in mouse duodenum

Aim: The aim of this study was to investigate whether superoxide dismutase (SOD) enzymes and ascorbate play a role in the protection of the nitrergic relaxation against superoxide anion inhibition in the mouse duodenum. Methods: The effects of exogenous SOD, N , N '-bis(salicylidene) ethylenediamine chloride (EUK-8; a synthetic cell-permeable mimetic of the manganese SOD [Mn SOD] and ascorbate on relaxant responses induced by nitrergic nerve stimulation), exogenous nitric oxide (NO), and nitroglycerin were investigated in isolated mouse duodenum tissues. Results: Diethyldithiocarbamate (DETCA) inhibited the relaxation to exogenous NO and nitroglycerin, but not relaxation to electrical field stimulation (EFS). SOD and ascorbate partially prevented the inhibitory effect of DETCA on relaxation to NO, abut not to nitroglycerin. The DETCA-induced inhibition on nitroglycerin was prevented by EUK-8. Hemoglobin, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolinel-oxyl-3-oxide, and hydroxo-cobalamin inhibited the relaxation to NO, but not to EFS and nitroglycerin in the presence of DETCA. Pyrogallol and hydroquinone inhibited the relaxation to NO, but not to EFS and nitroglycerin. This inhibition was prevented by exogenous SOD and ascorbate, but was not prevented by EUK-8. Pyrogallol and hy-droquinone did not inhibit the EFS-induced relaxation in the presence of DETCA. Duroquinone and 6-anilino-5.8-quinolinedione inhibited the relaxation to EFS, NO, and nitroglycerin, and this inhibition was prevented by EUK-8. Conclusion: These results suggest that the nitrergic neurotransmission in the mouse duodenum is protected by endogenous tissue antioxidants against superoxide anions, and Mn SOD, in addition to copper/zinc SOD, can protect NO from attack from superoxide anion generators intracellularly. Also, the possibility that the endogenous neurotransmitter may not be the free NO but a NO-containing or NO-generating molecule in the mouse duodenum remains open.     

Comparison of the redox forms of nitrogen monoxide with the nitrergic transmitter in the rat anococcygeus muscle.

1. A sustained tone was produced in rat isolated anococcygeus muscles with guanethidine and clonidine and relaxant responses were elicited by electrical stimulation of its nitrergic nerves and by the three redox forms of nitrogen monoxide. 2. The nitroxyl anion (NO ) was donated by dissociation of Angeli's salt; the free radical (NO*) was from an aqueous solution of nitric oxide gas; the nitrosonium cation (NO+) was donated by dissociation of nitrosonium tetrafluoroborate. 3. The concentrations producing approximately 50% relaxations of the anococcygeus muscle were 0.3 microM for Angeli's salt (nitroxyl), 0.5 microM for NO* and 100 microM for nitrosonium tetrafluoroborate. Nitrergic nerve stimulation at 1 Hz for 10 s produced equivalent relaxant responses. 4. The superoxide generator pyrogallol (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 5. The NO* scavenger carboxy-PTIO (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 6. Hydroxocobalamin (30 microM) had no significant effect on responses to the nitrergic transmitter, enhanced the response to Angeli's salt, and significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 7. The findings suggest that the nitroxyl anion donated by Angeli's salt is a better candidate than NO* to serve as the nitrergic transmitter in the rat anococcygeus muscle, although it still does not behave exactly like the transmitter.     

Influence of polyethylene-glycol-superoxide dismutase and combined depletion and repletion of antioxidants on nitrergic relaxation in the pig gastric fundus

In circular smooth muscle strips of porcine gastric fundus, polyethylene-glycol-superoxide dismutase, a membrane-permeable analogue of endogenous copper/zinc (Cu/Zn) superoxide dismutase, reversed the inhibitory effect of the superoxide anion generator 6-anilino-5,8-quinolinedione (LY83583) on electrically induced nitrergic relaxations of fundic tissues which are depleted of the endogenous antioxidant Cu/Zn superoxide dismutase by diethyldithiocarbamate, to the same extent as exogenously added Cu/Zn superoxide dismutase. Addition of a second antioxidant together with Cu/Zn superoxide dismutase does not result in a higher degree of reversal of the inhibitory effect of LY83583. Depletion of either tissue glutathione or tissue catalase in combination with diethyldithiocarbamate does not increase the inhibitory action of LY83583 or the nitric oxide (NO)-scavenger hydroxocobalamin upon nitrergic relaxations (electrically induced or by exogenous NO) when compared to their action in the presence of diethyldithiocarbamate alone. In conclusion, these results demonstrate that endogenous Cu/Zn superoxide dismutase is the essential antioxidant responsible for safeguarding peripheral nitrergic neurotransmission, whereby extracellular protection of endogenous NO is most important.     

Influence of antioxidant depletion on nitrergic relaxation in the pig gastric fundus

1. The hypothesis that endogenous tissue antioxidants might explain the inability of the superoxide generators 6-anilino-5,8-quinolinedione (LY83583) and hydroquinone (HQ) and of the NO-scavengers hydroxocobalamin (HC) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) to affect nitrergic neurotransmission in the porcine gastric fundus was tested by selective pharmacological depletion of respectively Cu/Zn superoxide dismutase (Cu/Zn SOD) and reduced glutathione (GSH) in circular smooth muscle preparations. 2. Diethyldithiocarbamate (DETCA; 3x10(-3) M), which almost completely abolished tissue Cu/Zn SOD activity, had no effect per se on nitrergic relaxations induced by either electrical field stimulation (EFS; 4 Hz, 10 s) or exogenous nitric oxide (NO; 10(-5) M). In these DETCA-treated tissues however, electrically-induced nitrergic relaxations became sensitive to inhibition by LY83583 (10(-5) M) or HC (10(-4) M), but not by HQ (10(-4) M) or c-PTIO (10(-4) M); only for the combination of DETCA plus LY83583, this inhibition was partially reversed by exogenous Cu/Zn SOD (1000 u ml(-1)). 3. Immunohistochemical analysis of porcine gastric fundus revealed a 100% colocalization of Cu/Zn SOD and neuronal nitric oxide synthase (nNOS) in the intrinsic neurons of the myenteric plexus. 4. Buthionine sulphoximine (BSO; 10(-3) M) in the absence or presence of LY83583 (10(-5) M) or HC (10(-4) M) did not alter nitrergic relaxations, although it reduced per se the tissue GSH content to 62% of control. 5. Pharmacological depletion studies, corroborated by immunohistochemical data, thus suggest a role for Cu/Zn SOD but not for GSH in nitrergic neurotransmission in the porcine gastric fundus.     

THE INHIBITION OF NITRIC OXIDE-MEDIATED RELAXATIONS IN RAT AORTA AND ANOCOCCYGEUS MUSCLE BY DIPHENYLENE IODONIUM

1. The effects of diphenylene iodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate-dependent oxidases (which generate superoxide anions), were studied on nitric oxide (NO)-mediated responses in isolated preparations of the rat aorta and anococcygeus muscle. 2. In aortic rings, the endothelium-dependent relaxant action of acetylcholine was reduced by DPI (0.3–10 μmol/L) in a concentration-dependent manner and abolished by the NO synthase (NOS) inhibitor L-nitro-N^G-arginine methylester (l-NAME; 100 μmol/L). Relaxations induced by sodium nitroprusside (SNP) or NO were not affected by DPI or l-NAME. 3. In anococcygeus muscles, DPI (0.3–10 μmol/ L) as well as l-NAME (5–100 μmol/L) produced concentration-dependent reductions of relaxations produced by nitrergic nerve stimulation. Relaxations induced by NO and SNP were not affected by either DPI or l-NAME. l-Arginine (1 mmol/L) prevented the reduction of nitrergic relaxations by l-NAME but not by DPI. 4. Contractions of anococcygeus muscles elicited by exogenous noradrenaline (1 μmol/ L) were not affected or were inhibited by DPI (0.3–10 μmol/L), but the contractions elicited by noradrenergic nerve stimulation were significantly enhanced by DPI and l-NAME. When noradrenergic contractions had already been maximally enhanced by l-NAME (100 μmol/L), DPI produced no further enhancement. l-Arginine (1 mmol/L) prevented the enhancement of noradrenergic contractions by l-NAME but not by DPI. 5. The efflux of radioactivity induced by field stimulation from anococcygeus muscles previously incubated with [³H]-noradrenaline was not affected by either DPI or l-NAME. 6. Superoxide dismutase (SOD, 100 U/mL) had no significant effects on noradrenergic contractions, nitrergic relaxations, relaxations induced by NO or the actions of DPI in the rat anococcygeus muscle. 7. The results suggest that the effects of DPI in reducing the NO-mediated relaxations produced by acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle are due to the inhibition of NOS in these tissues. The effects of DPI were not sensitive to l-arginine, and thus the mechanism of inhibition of NOS differs from that of l-NAME.     

The effects of superoxide anion generators on responses to exogenous nitric oxide and non-adrenergic, non-cholinergic nerve stimulation in rat isolated penile bulb

The effects of the superoxide anion generators, pyrogallol and hydroquinone on relaxations induced by electrical field stimulation (70 V, 0.7 msec., 0.5-8 Hz for 5 sec.) and exogenous nitric oxide donor sodium nitroprusside, were investigated in rat penile bulb precontracted with phenylephrine (10(-4) M). Pyrogallol (10(-4) M, 3 x 10(-4) M) and hydroquinone (3 x 10(-4) M) reduced the relaxations induced by sodium nitroprusside, but had no effect on relaxations elicited by nitrergic nerve stimulation. After treatment with diethyldithiocarbamate (3 x 10(-3) M), an inhibitor of Cu/Zn superoxide dismutase, both agents reduced the relaxations induced by electrical field stimulation. Superoxide dismutase, at 300 U/ml, significantly reversed the inhibitory action of pyrogallol and hydroquinone on responses to sodium nitroprusside. This concentration of superoxide dismutase failed to reverse the inhibitory action of pyrogallol on responses to electrical field stimulation observed in the presence of diethyldithiocarbamate, while at 600 U/ml it significantly prevented the reduction in relaxations. However, even at 600 U/ml, superoxide dismutase did not alter the decrease in responses to electrical field stimulation evoked by hydroquinone in tissues pretreated with diethyldithiocarbamate. These results suggest that the nitrergic transmitter in rat penile bulb is protected against superoxide anions by endogenous Cu/Zn superoxide dismutase in a manner similar to gastric fundus and anococcygeus muscles.     

Effect of hydroxocobalamin on vasodilatations to nitrergic transmitter, nitric oxide and endothelium-derived relaxing factor in guinea-pig basilar artery

In endothelium-denuded guinea-pig isolated basilar artery preparations, hydroxocobalamin (30, 100 and 300 μM) concentration-dependently inhibited the vasodilator responses to exogenous nitric oxide (NO), whereas the vasodilator responses to nitrergic nerve stimulation were slightly reduced by high (100 and 300 μM) but not by the low (30 μM) concentration of hydroxocobalamin. Vasodilatation in response to sodium nitroprusside (10–100 nM) was totally abolished by 300 μM hydroxocobalamin. In endothelium-intact preparations, vasodilator responses to acetylcholine (0.3–3 μM) were significantly reduced or abolished by hydroxocobalamin (30–300 μM). The mean reduction by hydroxocobalamin of relaxations to acetylcholine was significantly greater than that of the equivalent response evoked by nitrergic nerve stimulation. The findings suggest that the nitrergic transmitter in the guinea-pig basilar artery may be quantitatively less susceptible than the endothelium-derived relaxing factor to the NO scavenger hydroxocobalamin.     

Comparative study of the quercetin,ascorbic acid,glutathione and superoxide dismutase for nitric oxide protecting effects in mouse gastric fundus

The aim of this work was to compare the preventing capacity of quercetin with Cu/Zn superoxide dismutase (Cu/Zn SOD), ascorbic acid and glutathione on nitric oxide (NO)-induced relaxation in mouse gastric fundus. Furthermore, the effects of the quercetin on the tissue level of total oxidant and antioxidant was investigated. Nitrergic stimulation (4 Hz, 25 V, 0.1 ms, 10 s-train) and exogenous NO (10 μM) induced relaxation. Pyrogallol (10 μM), hydroquinone (100 μM) and LY83583 (6-Anilino-quinolin-5,8-quinone, 5 μM) inhibited nitrergic relaxations. The inhibition observed with pyrogallol, hydroquinone and LY83583 was prevented by quercetin (0.1 μM). Also, ascorbic acid (500 μM), glutathione (100 μM) and Cu/Zn SOD (100 U/ml) prevented the inhibitory effect of superoxide anion generators on the relaxation to nitrergic stimulation and NO. Diethyldithiocarbamic acid (DETCA; 8 mM) inhibited nitrergic relaxations. DETCA-induced inhibition on nitrergic stimulation and NO-induced relaxation was prevented by quercetin, ascorbic acid, glutathione or Cu/Zn SOD. DETCA plus pyrogallol, hydroquinone or LY83583 strengthened the inhibition on the relaxations. Also, pre-treatment with quercetin, ascorbic acid and glutathione prevented the inhibitory effect of DETCA plus LY-83583 on the relaxation to nitrergic stimulation and NO but Cu/Zn SOD did not prevent this inhibition. Also, quercetin increased tissue total antioxidant capacity and decreased tissue oxidant level and oxidative stress index in DETCA-treatment group. These results indicate that quercetin has antioxidant effect and protects NO from endogenous superoxide anion-driven inactivation and enhances its biological activity, suggesting that quercetin may scavenge superoxide anion in a Cu/Zn SOD, glutathione or ascorbic acid-inhibitable manner.