Nitrergic Nerve Stimulation Research Articles

Activated Rho Kinase Mediates Diabetes‐Induced Elevation of Vascular Arginase Activation and Contributes to Impaired Corpora Cavernosa Relaxation: Possible Involvement of p38 MAPK Activation

IntroductionActivated RhoA/Rho kinase (ROCK) has been implicated in diabetes‐induced erectile dysfunction. Earlier studies have demonstrated involvement of ROCK pathway in the activation of arginase in endothelial cells. However, signaling pathways activated by ROCK in the penis remain unclear. AimWe tested whether ROCK and p38 MAPK are involved in the elevation of arginase activity and subsequent impairment of corpora cavernosal (CC) relaxation in diabetes. MethodsEight weeks after streptozotocin‐induced diabetes, vascular functional studies, arginase activity assay, and protein expression of RhoA, ROCK, phospho‐p38 MAPK, p38 MAPK, phospho‐MYPT‐1Thr850, MYPT‐1 and arginase levels were assessed in CC tissues from nondiabetic wild type (WT), diabetic (D) WT (WT + D), partial ROCK 2+/− knockout (KO), and ROCK 2+/− KO + D mice. Main Outcome MeasuresThe expression of RhoA, ROCK 1 and 2, phosphorylation of MYPT‐1Thr850 and p38 MAPK, arginase activity/expression, endothelial‐ and nitrergic‐dependent relaxation of CC was assayed. ResultsDiabetes significantly reduced maximum relaxation (Emax) to both endothelium‐dependent acetylcholine (WT + D: Emax; 61 ± 4% vs. WT: Emax; 75 ± 2%) and nitrergic nerve stimulation. These effects were associated with increased expression of active RhoA, ROCK 2, phospho‐MYPT‐1Thr850, phospho‐p38 MAPK, arginase II, and activity of corporal arginase (1.6‐fold) in WT diabetic CC. However, this impairment in CC of WT + D mice was absent in heterozygous ROCK 2+/− KO + D mice for acetylcholine (Emax: 80 ± 5%) and attenuated for nitrergic nerve‐induced relaxation. CC of ROCK 2+/− KO + D mice showed much less ROCK activity, did not exhibit p38 MAPK activation, and had reduced arginase activity and arginase II expression. These findings indicate that ROCK 2 mediates diabetes‐induced elevation of arginase activity. Additionally, pretreatment of WT diabetic CC with inhibitors of arginase (ABH) or p38 MAPK (SB203580) partially prevented impairment of ACh‐ and nitrergic nerve‐induced relaxation and elevation of arginase activity. ConclusionROCK 2, p38 MAPK and arginase play key roles in diabetes‐induced impairment of CC relaxation.

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

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. The effects of exogenous SOD, N,No-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. 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 hydroxocobalamin 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 hydroquinone 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. 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.

Do gap junctions play a role in nerve transmissions as well as pacing in mouse intestine?

Varicosities of nitrergic and other nerves end on deep muscular plexus interstitial cells of Cajal or on CD34-positive, c-kit-negative fibroblast-like cells. Both cell types connect to outer circular muscle by gap junctions, which may transmit nerve messages to muscle. We tested the hypotheses that gap junctions transmit pacing messages from interstitial cells of Cajal of the myenteric plexus. Effects of inhibitors of gap junction conductance were studied on paced contractions and nerve transmissions in small segments of circular muscle of mouse intestine. Using electrical field stimulation parameters (50 V/cm, 5 pps, and 0.5 ms) which evoke near maximal responses to nitrergic, cholinergic, and apamin-sensitive nerve stimulation, we isolated inhibitory responses to nitrergic nerves, inhibitory responses to apamin-sensitive nerves and excitatory responses to cholinergic nerves. 18beta-Glycyrrhetinic acid (10, 30, and 100 microM), octanol (0.1, 0.3, and 1 mM) and gap peptides (300 microM of (40)Gap27, (43)Gap26, (37,43)Gap27) all failed to abolish neurotransmission. 18beta-Glycyrrhetinic acid inhibited frequencies of paced contractions, likely owing to inhibition of l-type Ca(2+) channels in smooth muscle, but octanol or gap peptides did not. 18beta-Glycyrrhetinic acid and octanol, but not gap peptides, reduced the amplitudes of spontaneous and nerve-induced contractions. These reductions paralleled reductions in contractions to exogenous carbachol. Additional experiments with gap peptides in both longitudinal and circular muscle segments after N(G)-nitro-l-arginine and TTX revealed no effects on pacing frequencies. We conclude that gap junction coupling may not be necessary for pacing or nerve transmission to the circular muscle of the mouse intestine.

Interstitial Cells of Cajal Are Involved in Neurotransmission in the Gastrointestinal Tract

Interstitial cells of Cajal (ICC) are important cells which coordinate gastrointestinal motility. ICC express Kit receptor tyrosine kinase, and Kit immunohistochemistry reveals ICC morphology and distribution in the gastrointestinal musculature. ICC show a highly branched morphology and form unique networks. Myenteric ICC (ICC-MY) are located at the layer of the myenteric plexus and serve as electrical pacemakers. Intramuscular ICC (ICC-IM) and ICC in the deep muscular plexus (ICC-DMP) are distributed within the muscular layers, and are densely innervated by excitatory and inhibitory enteric motor neurons and in close contact with nerve terminals. Recent studies combined with morphological and functional techniques directly revealed that ICC-IM and ICC-DMP are mediators of enteric motor neurotransmission. These types of ICC express several receptors for neurotransmitters such as acetylcholine and substance P and show responses to excitatory nerve stimulations. ICC also express receptive mechanisms for nitric oxide, which is an inhibitory neurotransmitter in the gastrointestinal tract. They can respond to nitrergic nerve stimulation by cyclic GMP production. Kit mutant mice lack ICC-IM and show attenuated postsynaptic responses after intrinsic nerve stimulation. These findings indicate the importance for ICC in neurotransmission in the gastrointestinal tract.

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.

Enhanced responsiveness to nitric oxide, nitroxyl anions, and nitrergic transmitter by 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole in the rat anococcygeus muscle

The effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) on responses to sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP), the nitroxyl anion donor Angeli's salt, and nitrergic nerve stimulation, as well as the release of NO from nitrergic nerves, were studied in the rat isolated anococcygeus muscle. YC-1 (1-100 microM) produced concentration-dependent relaxations in contracted muscles, which were partially but significantly reduced by the inhibitor of soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 1 and 10 microM). At a concentration that did not affect tissue tension, YC-1 (1 microM) significantly enhanced relaxations to SNP, SNAP, and Angeli's salt but did not affect relaxations to papaverine (10 microM). Nitrergic relaxations elicited by short periods (1 Hz for 10 s, 15 V) and long periods of EFS (5 Hz for 5 min, 15 V) were also enhanced by YC-1. YC-1 (100 microM), in an l-NAME and tetrodotoxin-insensitive manner, also increased the amount of NO detected in the organ bath media after the tissue was field stimulated (5 Hz for 5 min), which may have resulted from the electrolytic degradation of YC-1, as this effect was also seen in the absence of tissue. In summary, YC-1 enhanced relaxations to donors of NO, Angeli's salt, and nitrergic nerve stimulation in the rat anococcygeus muscle; however, the enhanced release of NO by YC-1 following nitrergic nerve stimulation was not a tissue-dependent effect.

Effects Induced by Tityus serrulatus Scorpion Venom and Its Toxins TsTX-I and TsTX-V on the Rat Isolated Retractor Penis Muscle

The aims of the present study were to investigate the pharmacological effects induced by Tityus serrulatus venom (TsV) and its fractions and to compare with the effects induced by pure α (TsTX-V) and β (TsTX-I) toxins isolated from TsV on rat retractor penis muscle (RPM). TsV, fractions X, XI, XIIa, XIIb (0.01–100 µg/ml) and TsTX-V (1 nmol/l–10 µmol/l) induced concentration-dependent contractions. Prazosin and guanethidine or tetrodotoxin (TTX, 5 µmol/l, 30 min) completely abolished these contractions, suggesting complete dependence on sympathetic nerves. TsV or fractions X, XI, XIIa, XIIb (0.01– 100 µg/ml), TsTX-I and TsTX-V (1 nmol/l–10 µmol/l) induced concentration-dependent relaxations in the precontracted RPM. TTX or N^ω-nitro-L-arginine methyl ester (L-NAME, 100 µmol/l, 30 min) completely abolished the relaxations. Our results suggest that most of TsV-derivated toxins induce contraction and relaxation on RPM by sympathetic and NANC nitrergic nerve stimulation. Noteworthy, TsTX-I only induces relaxation on RPM suggesting that this protein selectively acts on inhibitory nerves.

Inhibition of l‐arginine transport by reactive oxygen species in rat anococcygeus muscle

The effect of L-arginine on nitrergic transmission and its alteration with reactive oxygen species (ROS) were investigated. L-arginine potentiated the relaxation response induced by electrical field stimulation in rat anococygeus muscle. This effect was inhibited by L-lysine, a cationic amino acid using y+ L and y+ transport systems in a similar way with L-arginine. The neutral amino acid L-leucine, which uses only y+ L system as a transport mechanism, inhibited this potentiation at only low frequency stimulation. Electrolysis of the physiological solution did not change the responses to electrical field stimulation, but inhibited the potentiation elicited by L-arginine that was prevented in the presence of mannitol and N-acetyl-L-cysteine. In conclusion, L-arginine is transported via y+ system predominantly to potentiate the relaxation response to nitrergic nerve stimulation in rat anococcygeus muscle. ROS, primarily hydroxyl radicals inhibited L-arginine-induced potentiation probably by interacting with the y+ amino acid transport system.

Interaction between excitatory and inhibitory metabotropic pathways in the guinea-pig antrum.

Intracellular recordings were made from isolated bundles of the circular muscle layer of guinea-pig gastric antrum and the responses evoked by stimulating nitrergic nerve fibres were examined. Nitrergic inhibitory junction potentials (nitrergic-IJPs), evoked by trains of stimuli, had small amplitudes and were associated with a reduction in the rate of occurrence and amplitude of spontaneously occurring depolarizing potentials, termed unitary potentials. Nitrergic-IJPs were abolished either by membrane hyperpolarization or by 4, 4'-diisothiocyano-2, 2'-stilbene disulfonic acid (DIDS); both of these abolished the discharge of unitary potentials. Membrane depolarization increased the rate of discharge of unitary potentials so that they summed to give rise to are generative potential. Nitrergic nerve stimulation abolished regenerative potentials; this inhibition did not result from a change in threshold for the initiation of regenerative potentials,rather it occurred at some stage after the gating process. Inhibitory nitrergic nerve responses were blocked by L-nitroarginine (NOLA) and oxadiazolo quinoxalin-l-one (ODQ), an inhibitor of soluble guanylate cyclase. The observations suggest that the inhibition of regenerative potentials results from an interaction between an inhibitory and an excitatory metabotropic pathway.

Effects of diabetes and evening primrose oil treatment on responses of aorta, corpus cavernosum and mesenteric vasculature in rats

Diabetes causes endothelial dysfunction, with deleterious effects on nitric oxide (NO) mediated vasodilatation. However, in many vessels other local vasodilators such as endothelium-derived hyperpolarizing factor (EDHF), prostacyclin, epoxides or endocannabinoids are also important. Several of these factors may be derived from ω-6 essential fatty acids via arachidonate metabolism. Diabetes inhibits this pathway, a defect that may be bypassed by diets enriched with ω-6 γ-linolenic acid-containing oils such as evening primrose oil (EPO). The aim was to examine the effects of preventive EPO treatment on endothelium-dependent and neurally mediated vasorelaxation. Diabetes was induced by streptozotocin in rats; duration was 8 weeks. Vascular responses were examined in vitro on thoracic aorta, corpus cavernosum and perfused mesenteric bed preparations. Diabetes caused 25% and 35% deficits, respectively, in aorta and corpus cavernosum NO-mediated endothelium-dependent relaxation to acetylcholine that were largely unaffected by EPO treatment. Moreover, a 44% reduction in maximum corpus cavernosum vasorelaxation to nitrergic nerve stimulation was not prevented by EPO. However, for the mesenteric vascular bed, a 29% diminution of responses to acetylcholine, mediated by both NO and EDHF, was 84% attenuated by EPO treatment. When the EDHF component was isolated during NO synthase inhibition, a 76% diabetic deficit was noted. This was completely prevented by EPO treatment, which also caused supernormal EDHF responses in nondiabetic rats. EPO treatment prevented the development of deficits in endothelium-dependent relaxation in diabetic rats. Effects were particularly marked on the resistance vessel EDHF system, which may have potential therapeutic relevance for diabetic microvascular complications.

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 ω-nitro- l-arginine inhibited relaxations induced by electrical field stimulation of nitrergic nerves, but not those with acidified NaNO 2. Furthermore, 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ) inhibited both types of relaxations while 2-amino-5,6-dihydro-6-methyl-4 H-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 NaNO 2. 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.

Influence of bilirubin and other antioxidants on nitrergic relaxation in the pig gastric fundus.

1. The influence of several antioxidants (bilirubin, urate, ascorbate, alpha-tocopherol, glutathione (GSH), Cu/Zn superoxide dismutase (SOD) and the manganese SOD mimic EUK-8) on nitrergic relaxations induced by either exogenous nitric oxide (NO; 10(-5) M) or electrical field stimulation (4 Hz; 10 s and 3 min) was studied in the pig gastric fundus. 2. Ascorbate (5x10(-4) M), alpha-tocopherol (4x10(-4) M), SOD (300 - 1000 u ml(-1)) and EUK-8 (3x10(-4) M) did not influence the relaxations to exogenous NO. In the presence of GSH (5x10(-4) M), the short-lasting relaxation to NO became biphasic, potentiated and prolonged. Urate (4x10(-4) M) and bilirubin (2x10(-4) M) also potentiated the relaxant effect of NO. None of the antioxidants influenced the electrically evoked relaxations. 3. 6-Anilino-5,8-quinolinedione (LY83583; 10(-5) M) had no influence on nitrergic nerve stimulation but nearly abolished the relaxant response to exogenous NO. Urate and GSH completely prevented this inhibitory effect, while it was partially reversed by SOD and bilirubin. Ascorbate, alpha-tocopherol and EUK-8 were without effect. 4. Hydroquinone (10(-4) M) did not affect the electrically induced nitrergic relaxations, but markedly reduced NO-induced relaxations. The inhibition of exogenous NO by hydroquinone was completely prevented by urate and GSH. SOD and ascorbate afforded partial protection, while bilirubin, EUK-8 and alpha-tocopherol were ineffective. 5. Hydroxocobalamin (10(-4) M) inhibited relaxations to NO by 50%, but not the electrically induced responses. Full protection versus this inhibitory effect was obtained with urate, GSH and alpha-tocopherol. 6. These results strengthen the hypothesis that several endogenous antioxidant defense mechanisms, enzymatic as well as non-enzymatic, might play a role in the nitrergic neurotransmission process.

Neocuproine potentiates the activity of the nitrergic neurotransmitter but inhibits that of S-nitrosothiols

In the present study, we investigated the cellular components that are involved in the release of nitric oxide (NO) from S-nitrosothiols and whether these components also modulate the activity of the nitrergic neurotransmitter in the rat gastric fundus. Electrical stimulation of nitrergic nerves induced frequency-dependent transient relaxations which were mimicked by exogenous NO. The S-nitrosothiols S-nitrosocysteine, S-nitrosoglutathione and S-nitroso- N-acetylpenicillamine induced concentration-dependent relaxations which were generally more sustained as compared to those to nitrergic nerve stimulation or NO. The relaxations to nitrergic nerve stimulation and those to NO were significantly enhanced by the copper(I) chelator neocuproine but not affected by the copper(II) chelator cuprizone. The relaxations to the S-nitrosothiols were significantly inhibited by neocuproine but not by cuprizone. The antioxidant ascorbate did not affect the tension of the muscle strip. However, in the presence of an S-nitrosothiol, ascorbate induced an immediate, sharp and transient relaxation that was significantly inhibited by a low concentration of neocuproine but not by cuprizone. Ascorbate did not induce a relaxation during short-train or prolonged nerve stimulation of the muscle strip. These results suggest that ascorbate interacts with copper to modulate the biological activity of S-nitrosothiols but not that of the nitrergic neurotransmitter. The differential effect of neocuproine indicates that S-nitrosothiols do not mediate the nitrergic neurotransmission of the rat gastric fundus. As neocuproine is to date the only compound that exerts an opposite effect on the biological activity of the nitrergic neurotransmitter and on that of S-nitrosothiols, it may be useful to elucidate the nature of the nitrergic neurotransmitter in the peripheral nervous system.

Insulin Reversal of Impaired Nitrergic Transmission in the Anococcygeus Muscle from Diabetic Rats

The aim of this study was to investigate whether insulin could reverse the impairment in nitrergic neurotransmission in the anococcygeus muscle from streptozotocin-induced diabetic rats. Relaxations to nitrergic nerve stimulation and sodium nitroprusside were significantly reduced in precontracted muscles from 8- and 4-week diabetic rats compared to the corresponding control rats. Treatment of diabetic rats with Lente insulin (1–12 units/day s.c.) for the final 4 weeks of the 8-week diabetes duration reversed the reductions, but treatment of diabetic rats with insulin for the last week only did not. The impairment of relaxations was also not altered by in vitro exposure of muscles from 8-week diabetic rats to soluble insulin (0.02 units/ml, 2 h). The findings suggest that in vivo insulin treatment can reverse the existing diabetes-induced impairment of nitrergic transmission in rat anococcygeus muscle. However, short-term treatment with insulin, either in vivo or in vitro, does not reverse the impairment.

Effects of endothelial impairment by saponin on the responses to vasodilators and nitrergic nerve stimulation in isolated canine corpus cavernosum.

1. Responsiveness to EDRF-releasing substances and inhibitory nerve stimulation of canine isolated penile corpus cavernosum with and without saponin treatment were investigated. 2. Histological studies demonstrated that saponin did not detach endothelial cells from underlying tissues, but induced degenerative changes in the endothelial cells selectively. 3. In the cavernous strips contracted with phenylephrine, addition of acetylcholine, sodium nitroprusside, ATP and Ca2+ ionophore A23187 induced relaxations, but substance P and bradykinin did not change the muscle tone. 4. Acetylcholine-induced relaxation was significantly attenuated but not abolished by NG-nitro-L-arginine (L-NOARG). L-arginine restored the response inhibited by L-NOARG. The L-NOARG resistant relaxation was not influenced by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) but was suppressed in the strips contracted with K+. Treatment with saponin abolished the relaxation elicited by acetylcholine and A23187 but did not influence the response to nitroprusside and ATP. The ATP-induced relaxation was attenuated by aminophylline. 5. Transmural electrical stimulation at 2-20 Hz produced endothelium-independent relaxations which were abolished by tetrodotoxin and L-NOARG but unaffected by treatment with saponin. In saponin-treated cavernous strips, the neurogenic relaxation was not affected by acetylcholine, physostigmine, atropine and vasoactive intestinal peptide (VIP) but was abolished by ODQ. 6. It is concluded that acetylcholine-induced relaxations are endothelium-dependent and mediated partly by NO and also by other substances from the endothelium. The endothelium-independent relaxation to ATP is likely to be mediated by P1 purinoceptors. The function of nitrergic nerve does not seem to be prejunctionally modulated by acetylcholine and VIP.

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.

Comparison of Relaxations Evoked by Photoactivation of NO-Containing Compounds and Nitrergic Nerve Stimulation in 5-Hydroxytryptamine- and Potassium-Contracted Rat Gastric Fundus

1. The aim of the present study was to further investigate our earlier proposal of liberation of nitric oxide (NO) by photoactivation of molecules containing NO or NO 2, which in turn relaxes gastric smooth muscle, and to determine whether presynaptic- and/or postsynaptic NO-mediated relaxation is affected differently by the degree of membrane depolarization in rat gastric fundus smooth muscle. 2. During contraction of rat gastric fundus with 5-hydroxytryptamine (5-HT, 10 μM), low (K +, 25 mM) and high potassium (K +, 65.4 mM), relaxation responses to nitrergic nerve stimulation, photoactivation of caged NO compounds (streptozotocin [STZ], N ω-nitro- L-arginine-methylacetate [ L-NAME], N ω-nitro- D-arginine-methylacetate [ D-NAME]), and sodium nitroprusside (SNP) were compared. 3. Nitrergic nerve (presynaptic) stimulation and photoactivation (postsynaptic) of all caged NO compounds produced rapid, transient and reversible relaxation of 5-HT and low-K +-contracted tissues. However, when contractions were induced by high K +, the relaxation induced by nerve stimulation was abolished, whereas relaxations induced by photoactivated NO compounds were significantly ( P<0.01) reduced. 4. The relaxation induced by sodium nitroprusside (SNP), but not papaverine, was also diminished in high-K +-contracted tissues. The magnitude of photoactivated NO-induced relaxation was related to the amount of NO release, light intensity and concentration of compounds. 5. The evidence that photoactivated NO-induced relaxation is mediated by cGMP comes from the observation that zaprinast, but not forskolin, potentiated the relaxation. 6. It is concluded that rat gastric smooth muscle relaxes to photoactivation of NO or NO 2-carrying molecules via NO, and it appears that degree of membrane depolarization may be a critical factor in dissociating the response to presynaptic- and postsynaptic NO-mediated relaxation in this muscle.

Role of potassium channels in the nitrergic nerve stimulation‐induced vasodilatation in the guinea‐pig isolated basilar artery

1. We studied the effects of various K+ channel blockers on the vasodilator responses of guinea-pig isolated basilar arteries to nitrergic nerve stimulation, the nitric oxide (NO) donor sodium nitroprusside (SNP), and the membrane permeable guanosine-3',5'-cyclic monophosphate (cyclic GMP) analogue 8-bromo-cyclic GMP (8-Br-cyclic GMP). 2. In endothelium-denuded preparations which were contracted with prostaglandin F2alpha (1 microM), electrical field stimulation (EFS, 10 Hz for 30 s) produced a vasodilatation which was totally blocked by the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester L-NAME; 100 microM) (n=3) and by the selective NO-sensitive guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; 1 microM) (n=4). The vasodilator response to SNP (100 nM) was not reduced by L-NAME but was abolished by ODQ (1 microM) (n=4). 3. EFS-elicited vasodilatation was partly but significantly reduced by the non-selective K+ channel blockers tetraethylammonium (TEA, 1 and 3 mM) and 4-aminopyridine (4-AP, 3 mM), and by the large-conductance calcium-activated K+ channel (K(Ca) channel) blockers charybdotoxin (ChTX, 150 nM) and iberiotoxin (IbTX, 30 and 100 nM). In contrast, the ATP-sensitive K+ channel (K(ATP) channel) blocker glibenclamide (1-10 microM) and the small-conductance K(Ca) channel blocker apamin (100-500 nM) did not affect EFS-induced vasodilatation. 4. The vasodilator response elicited by SNP (10-100 nM) was significantly reduced by TEA (3 mM) and ChTX (150 nM) but not by apamin (500 nM) or glibenclamide (1 microM). The vasodilatation elicited by 8-Br-cyclic GMP (100 microM) was also reduced by TEA (3 mM) and ChTX (150 nM). 5. The results indicate that the vasodilatations induced by nitrergic nerve stimulation and the NO donor SNP in endothelium-denuded guinea-pig basilar artery depend on the formation of intracellular cyclic GMP. The increased cyclic GMP level activates large-conductance K(Ca) channels which partly mediate the vasodilator response. Neither K(ATP) channels nor apamin-sensitive small-conductance K(Ca) channels are involved in nitrergic transmitter-mediated vasodilatation.

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.

Effect of estrogen on nitric oxide-induced relaxation of the rabbit urethra

We evaluated the effect of estrogen on nitric oxide (NO)-mediated urethral relaxation in rabbits. Female New Zealand white rabbits, 4–5 weeks old, were treated with 5 mg/kg estradiol dipropionate (estrogen group) or saline (control group) injected intramuscularly weekly for 2 weeks. Electrical field stimulation (supramaximum voltage, 2 ms pulse duration, 0.3–15 Hz and 3 s train) caused frequency-dependent relaxation of urethral strips in both groups, which was inhibited by N ω -nitro- l-arginine ( l-NNA). This inhibition was overcome by addition of l-arginine. The relaxation induced by nitrergic nerve stimulation was significantly lower in the estrogen group than in the control group. There was no significant difference in sodium nitroprusside-induced urethral relaxation between the two groups. The production of NO in urethral strips during nitrergic nerve stimulation was evaluated by measuring nitrite/nitrate (NO 2 −/NO 3 −) levels in both groups, using microdialysis. The NO 2 −/NO 3 − production during electrical field stimulation in the estrogen group was significantly less than that in the control group. The NADPH diaphorase-positive reaction in the control group was greater than that in the estrogen group. The results suggest that estrogen treatment may reduce NO synthase activity, and inhibit the relaxation induced by nitrergic nerve stimulation in rabbit urethral smooth muscle.