Nitrergic Stimulation Research Articles

Inhibition of the left medial prefrontal cortex (mPFC) prolongs the social defeat-induced anxiogenesis in mice: Attenuation by NMDA receptor blockade in the right mPFC

Chemical inhibition and nitrergic stimulation of the left and right medial prefrontal cortex (L and RmPFC), respectively, provoke anxiety in mice. Moreover, LmPFC inhibition immediately followed by a single social defeat stress (SDS) led to anxiogenesis in mice exposed to the elevated plus maze (EPM) 24 h later. Given that glutamate NMDA (N-methyl-D-aspartate) receptors are densely present in the mPFC, we investigated (i) the time course of LmPFC inhibition + SDS-induced anxiogenesis and (ii) the effects of intra-RmPFC injection of AP-7 (a NMDA receptor antagonist) on this long-lasting anxiety. Male Swiss mice received intra-LmPFC injection of CoCl2 (1 mM) and 10 min later were subjected to a single SDS episode and then (i) exposed to the EPM 2, 5, or 10 days later or (ii) 2 days later, received intra-RmPFC injection of AP-7 (0.05 nmol) and were exposed to the EPM to observe the percentage of open arm entries and time (%OE; %OT) and frequency of closed arm entries (CE). Dorsal but not ventral LmPFC inhibition + SDS reduced open arm exploration 2, 5, and 10 days later relative to that of saline-treated or non-defeated mice. Moreover, this effect is not due to locomotor impairment as assessed using the general activity. Intra-RmPFC AP-7 injection 2 days after LmPFC inhibition + SDS prevented this type of anxiogenesis. These results suggest that the integrity of the LmPFC is important for mice to properly cope with SDS, and that NMDA receptor blockade in the RmPFC facilitates resilience to SDS-induced anxiogenesis in mice.

Diet-induced obesity in young mice: Consequences on the pancreatic intrinsic nervous system control of insulin secretion.

IntroductionObesity has become a pandaemic even in children. We aimed to investigate the impact of obesity in youth on later pancreatic intrinsic nervous system (PINS) phenotype and control of insulin secretion.MethodsYoung mice (5‐week‐old, T0 group) were fed either a normal diet (ND group) or a Western diet (WD group) for 12 weeks. Pancreas nervous system density, PINS phenotype and pancreas anatomy were analysed by immunohistochemistry at T0 and in adulthood (ND and WD groups). Insulin secretion was also studied in these 3 groups using a new model of ex vivo pancreatic culture, where PINS was stimulated by nicotinic and nitrergic agonists with and without antagonists. Insulin was assayed in supernatants by ELISA.ResultsPancreas nervous system density decreased with age in ND (P < .01) but not in WD mice (P = .08). Western diet decreased the PINS nitrergic component as compared to normal diet (P < .01) but it did not modify its cholinergic component (P = .50). Nicotinic PINS stimulation induced greater insulin secretion in ND compared to WD mice (P < .001) whereas nitrergic stimulation significantly decreased insulin secretion in ND mice (P < .001) and tended to increase insulin secretion in WD mice (P = .08). Endocrine pancreas anatomy was not modified by the Western diet as compared to the normal diet (P = .93).ConclusionsEarly Western diet induced neuronal density and phenotype changes in PINS that might be involved in the pancreas insulin secretion dysfunctions associated with obesity.

Alterations of colonic function in the Winnie mouse model of spontaneous chronic colitis.

This is the first study to provide analyses of intestinal transit and whole colon motility in an animal model of spontaneous chronic colitis. We found that cholinergic and purinergic neuromuscular transmission, as well as the smooth muscle cell responses to cholinergic and nitrergic stimulation, is altered in the chronically inflamed Winnie mouse colon. The changes to intestinal transit and colonic function we identified in the Winnie mouse are similar to those seen in inflammatory bowel disease patients.

Pharmacological characterisation of the relaxation induced by the soluble guanylate cyclase activator, BAY 60-2770 in rabbit corpus cavernosum.

To characterise the relaxation induced by the soluble guanylate cyclase (sGC) activator, BAY 60-2770 (4-({(4-carboxybutyl) [2- (5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl]methoxy}phenyl)ethyl] amino}methyl)benzoic acid) in rabbit corpus cavernosum (CC). The penis from male New Zealand rabbits was removed and fours strips of CC were obtained. Concentration-response curves to BAY 60-2770 were constructed in the absence and presence of inhibitors of nitric oxide synthase, N (G)-nitro-L- arginine methyl ester (L-NAME, 100 μm), sGC, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μm) and phosphodiesterase type 5 (PDE-5), tadalafil (0.1 μm). The potency (pEC50 ) and maximal response (Emax ) values were determined. Then, electrical-field stimulation (EFS)-induced contraction or relaxation was tested in the absence and presence of BAY 60-2770 (0.1 or 1 μm) alone or combined with ODQ (10 μm). For EFS-induced relaxation two protocols were used: (i) ODQ (10 μm) was first incubated for 20 min and then BAY 60-2770 (1 μm) was added for another 20 min (ODQ + BAY 60-2770); (ii) in different CC strips, BAY 60-2770 was incubated for 20 min followed by another 20 min with ODQ (BAY 60-2770 + ODQ). The intracellular levels of cyclic guanosine monophosphate (cGMP) were also determined. BAY 60-2770 potently relaxed rabbit CC with mean (sem) pEC50 and Emax values of 7.58 (0.19) and 81 (4)%, respectively. The inhibitors ODQ (n = 7) or tadalafil (n = 7) produced 4.2- and 6.3-leftward shifts, respectively in BAY 60-2770-induced relaxation without interfering with the Emax values. The intracellular levels of cGMP were augmented after stimulation with BAY 60-2770 (1 μm) alone, whereas its co-incubation with ODQ produced even higher levels of cGMP. The EFS-induced contraction was reduced in the presence of BAY 60-2770 (1 μm) and this inhibition was even greater when BAY 60-2770 was co-incubated with ODQ. The nitrergic stimulation induced CC relaxation, which was abolished in the presence of ODQ. BAY 60-2770 alone increased the amplitude of relaxation. Co-incubation of ODQ and BAY 60-2770 did not alter the relaxation in comparison with ODQ alone. Interestingly, when BAY 60-2770 was incubated before ODQ, EFS-induced relaxation was partly restored in comparison with ODQ alone or ODQ + BAY 60-2770. The relaxation induced by the sGC activator, BAY 60-2770 was increased after sGC oxidation and unaltered in the absence of nitric oxide. Thus, this class of substances may have advantages over sGC stimulators or PDE-5 inhibitors for treating patients with erectile dysfunction and extensive endothelial damage.

The effects of thiol modulators on nitrergic nerve- and S-nitrosothiols-induced relaxation in duodenum

The aim of this study was to investigate whether thiols are involved in the nitrergic neurotransmission in mouse duodenum. The effects of thiol-modulating agents, ethacrynic acid (100 μM), a non-specific sulfhydryl alkylator, and diamide (100 μM), an alkylating agent that oxidizes protein sulfhydryl groups and depletes intracellular glutathione, on relaxations to nitrergic stimulation (electrical field stimulation, EFS;10 Hz, 25 V, 1 ms, 15 s-train), S-nitrosoglutathione (GSNO; 5 μM), S-nitroso-acetylpenicillamine (SNAP; 5 μM), and S-nitrosocysteine (CysNO; 10 μM) were investigated. Moreover, the effects of buthionine sulfoximine (100 μM), an inhibitor of γ-glutamylcysteine synthetase, and sulfobromophthalein (100 μM), an inhibitor of glutathione-S-transferase, were studied on relaxant responses to EFS and S-nitrosothiols in mouse duodenum. Exogenous free thiol, glutathione (GSH, 100 μM) did not influence relaxation to EFS, GSNO, SNAP, and CysNO. Ethacrynic acid and diamide significantly decreased relaxation of duodenum to EFS, GSNO, SNAP, and CysNO. This inhibition was prevented by addition of GSH. Buthionine sulfoximine and sulfobromophthalein significantly decreased relaxation to EFS and GSNO but did not influence relaxation to SNAP and CysNO. The inhibitory effect of buthionine sulfoximine and sulfobromophthalein on the relaxant response to EFS and GSNO was prevented by addition of GSH. These results suggest that relaxation to nitrergic stimulation is thiol-dependent, and nitrosothiols, possibly S-nitrosoglutathione may play a role, as an intermediate compound in nitrergic neurotransmission in mouse duodenum.

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 (4Hz, 25V, 0.1ms, 10s-train) and exogenous NO (10μM) induced relaxation. Pyrogallol (10μM), hydroquinone (100μM) and LY83583 (6-Anilino-quinolin-5,8-quinone, 5μM) inhibited nitrergic relaxations. The inhibition observed with pyrogallol, hydroquinone and LY83583 was prevented by quercetin (0.1μM). Also, ascorbic acid (500μM), glutathione (100μM) and Cu/Zn SOD (100U/ml) prevented the inhibitory effect of superoxide anion generators on the relaxation to nitrergic stimulation and NO. Diethyldithiocarbamic acid (DETCA; 8mM) 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.

Extracellular Signal‐Regulated Kinase (ERK) Inhibition Decreases Arginase Activity and Improves Corpora Cavernosal Relaxation in Streptozotocin (STZ)‐Induced Diabetic Mice

Increased arginase activity (AA) has been implicated in hypertension and diabetes-induced endothelial dysfunction by reducing L-arginine availability and nitric oxide production. Higher levels of active extracellular signal-regulated kinase (ERK) have been found in patients with erectile dysfunction (ED) compared to patients without it. Both ERK and arginase have been reported to affect the expression and activity of nitric oxide synthase (NOS) and consequently penile erection. Nevertheless, signaling pathways activated by ERK in the penis are not well known. We hypothesized that inhibition of ERK by ERK inhibitor PD98059 decreases AA and thus improves cavernosal relaxation in streptozotocin (STZ)-diabetic mice. The AA, ERK, eNOS, and arginase I and II expressions were examined through Western blot, and functional response of cavernosal tissue were determined. Control and diabetic cavernosal tissues were pretreated with PD98059 (10(-5) M) and arginase inhibitor ((S)-(2-boronoethyl)-L-cysteine hydrochloride, [BEC]10(-4) M]). Diabetes increased AA significantly (twofold) over control mice and this effect was blocked by acute treatment with PD98059. Cavernosal strips from diabetic mice exhibited decreased relaxation (STZ-diabetic vs. control, respectively) to both the endothelium-dependent agonist acetylcholine (38.0 ± 5% vs. 82.5 ± 7%) and nitrergic stimulation (27 ± 2% vs. 76 ± 6%) by electrical field stimulation (EFS, 1-32 Hz). However, this impairment in cavernosal relaxation from diabetic mice was attenuated by treatment with PD98059 in nitrergic (27 ± 2% vs. 60 ± 4%) and endothelium-dependent relaxation responses (38.0 ± 5% vs. 67.5 ± 6%). Acute treatment with the arginase inhibitor BEC (10(-4) M) also improves EFS-induced relaxation in diabetic mice (31 ± 3% vs. 49 ± 2%). Moreover, vascular expression of activated ERK was increased in diabetic over control mice. These data suggest that ERK inhibition prevents elevation of penile AA and protects against ED caused by diabetes.

Amelioration of portal hypertension and the hyperdynamic circulatory syndrome in cirrhotic rats by neuropeptide Y via pronounced splanchnic vasoaction

BackgroundSplanchnic vasodilation triggers the development of the hyperdynamic circulatory syndrome in portal hypertension. Neuropeptide Y (NPY), a sympathetic co-transmitter of norepinephrine, improves contractility in mesenteric arteries of pre-hepatic portal hypertensive...

Blockade of CNG channels abrogates urethral relaxation induced by soluble guanylate cyclase activation

In the present study, we have characterized the presence and distribution of cGMP-gated cationic channels (CNG) in the rat urethra as well as its putative role in the mediation of the nitrergic relaxation. Previous studies have shown the inhibition of the sheep urethral nitrergic relaxations by the CNG's inhibitor L-cis-diltiazem [1]. Also in the rat urethra, L-cis-diltiazem (50 μM) inhibited nitrergic relaxations elicited by electrical field stimulation (EFS) of arginine-vasopresin (AVP)-precontracted urethral preparations (Figure ​(Figure1A1A). Figure 1 The presence of L-cis-diltiazem (50 μM) rapidly and effectively inhibited relaxation in arginine-vasopresin (AVP)-precontracted urethral preparations induced by both A) EFS (0.8 ms, 10 Hz, 5 s at 2 min-intervals) and B) guanylate cyclase activation ... This effect was stereoselective since the isomer D-cis-diltiazem did not have any effect on urethral relaxations. Similarly, L-cis-diltiazem (50 μM) showed a complete blockade of the relaxation induced by the addition of YC-1 (50 μM), an specific activator of the soluble guanylate cyclase (sGC) (Figure ​(Figure1B),1B), thus reinforcing that L-cis-diltiazem would be acting as specific inhibitor of CNG channels in this tissue. The presence of these channels in urethral tissue was tested by carrying out conventional RT-PCR studies using retina as a positive control. A band of the predicted size showing after sequencing, a 99% identity with the rod-type CNGA1, together to a second band with a similar size to that ofthe CNGB1 fragment, were obtained. These results suggest that they are heteromeric retina-like CNG channels. Immunofluorescence studies were performed to analyze the distribution of CNG immunoreactivity (-ir) in sections of the urethral wall. As can be seen in Figure ​Figure2,2, a strong CNG-ir was present in a subpopulation of vimentin-ir cells known as Interstitial Cells of Cajal (ICC), while a fainter labeling was observed in smooth muscle cells (identified by actin-ir). CNG-ir was never observed in neural structures, nerve terminals, nerve trunks or ganglia. This pattern of CNG labeling and co-localization is similar to that described for the cGMP-ir found in the urethra upon functional nitrergic stimulation [2]. We suggest that CNG channels could be an essential ionic mechanism that would link activation of the NOS-GC pathway to relaxation. In addition, these results point to ICC as key elements in the mediation of the nitrergic urethral relaxation, although its precise role remains to be elucidated. Figure 2 Section of rat urethra showing immunofluorescence for CNG1 channels (green) and vimentin (red) and its co-localization in a intramuscular Interstitial cell [nuclei are stained with DAPI (blue)]. Scale bar: 20 μm.

Interstitial cells of Cajal contain signalling molecules for transduction of nitrergic stimulation in guinea pig caecum

Nitric oxide (NO) is an inhibitory signalling molecule in the gastrointestinal (GI) tract that is released from neurons and from leucocytes during inflammation. NO stimulates soluble guanylate cyclase (sGC), elevates cyclic guanosine 3',5'-monophospate (cGMP), and subsequently activates cGMP-dependent protein kinase (PKG). Targets for NO in the guinea pig caecum were investigated by characterizing the cellular distribution of sGC, cGMP and PKG. Immunoreactivity for both isoforms of sGC, sGCalpha1 and sGCbeta1, was observed in the interstitial cells of Cajal (ICC) and enteric neurons in the tunica muscularis. Double labelling with anti-Kit and anti-sGC antibodies showed sGCalpha1 and sGCbeta1-like immunoreactivity (LI) in almost all intramuscular (IM) and myenteric ICC. Neuronal processes with neuronal NO synthase were closely apposed to ICC expressing sGC-LI. Cells with sGC-LI possessed ultrastructural features of ICC-IM: caveolae, close association with nerve bundles and contacts with smooth muscle cells (SMC). Sodium nitroprusside, added with the phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine and zaprinast), enhanced cGMP-LI in almost all ICC and in some enteric neurons. Nerve stimulation also increased cGMP-LI in ICC and enteric neurons. In contrast, no resolvable increase in cGMP-LI was observed in any cells when the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was present. ICC and SMC also expressed PKG type I-LI. These data show that ICC express the downstream signalling molecules necessary to transduce nitrergic signals and activate inhibitory pathways and thus are primary targets for NO released from neurons and other cells in the GI tract.

Modulation of the activity of two pacemakers by transmural nerve stimulation in circular smooth muscle preparations isolated from the rat proximal colon

Circular smooth muscle preparations isolated from the rat proximal colon periodically generated two different amplitudes and frequencies of phasic contractions: large phasic contractions (LPC) with a frequency of about 1.5 times/min and small phasic contractions (SPC) with a frequency of about 9 times/min. Preparations with no attached longitudinal smooth muscle layer (and also myenteric layer) generated SPC alone, while those with no attached submucosal layer generated only LPC, indicating that the pacemakers of the LPC and SPC are distributed in the myenteric and submucosal layers, respectively. In intact preparations, transmural nerve stimulation (TNS) applied for 1-2 min with different frequencies (0.2-2 Hz) inhibited the phasic contractions. The amplitude of LPC was reduced at >0.25 Hz and abolished at >0.3 Hz, while the amplitude but not the frequency of SPC was reduced at >0.5 Hz (in a frequency-dependent way). The TNS-induced inhibitory responses were augmented by atropine and attenuated by N(omega)-nitro-L-arginine (L-NA). In the presence of L-NA and atropine, TNS elicited biphasic (inhibitory and following excitatory) responses. The former were not antagonized by apamin, guanethidine or suramin, while the latter were antagonized by capsaicin, suggesting an innervation by non-adrenergic non-cholinergic non-nitrergic (NANCNN) inhibitory and peptidergic excitatory nerves, respectively. In preparations with the longitudinal muscle layer removed, TNS inhibited only the amplitude of SPC, which was augmented by atropine and antagonized by L-NA. In intact preparations, muscarinic stimulation with acetylcholine increased the frequency of LPC, while nitrergic stimulation with sodium nitroprusside reduced the amplitude and frequency of LPC, and also the amplitude but not the frequency of SPC. These results indicate that the rat proximal colon has two types of pacemaker cells. Myenteric pacemaker cells which receive predominantly nitrergic, but also cholinergic, peptidergic and NANCNN innervation, and submucosal pacemaker cells that are not markedly influenced by intramural nerves.

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

Nitrergic stimulation of the locus coeruleus modulates blood pressure and heart rate in the anaesthetized rat

To investigate whether nitric oxide is involved in the cardiovascular responses mediated via the locus coeruleus, the effects of microinjections of l-arginine and l-glutamate into the locus coeruleus on blood pressure and heart rate were investigated in sodium pentobarbitone-anaesthetized rats. Unilateral microinjection of l-arginine (25, 50 nmol) elicited dose-related depressor (−17±4, −25±4 mmHg) and bradycardic (13±3, 24±6 b.p.m.) effects. Furthermore, these effects were attenuated by prior local microinjection of N G-nitro- l-arginine (40 nmol). Peripheral muscarinic receptor blockade with atropine methyl nitrate (1 mg/kg, i.v.) attenuated the bradycardic but not the depressor responses to l-arginine. l-Glutamate (2 nmol) microinjections also mediated depressor (−27±6 mmHg) and bradycardic (53±23 b.p.m.) effects that were attenuated by microinjections of dizocilpine maleate (1 nmol) into the locus coeruleus. In addition, pretreatment with N G-nitro- l-arginine (40 nmol) also significantly attenuated the depressor response elicited by l-glutamate. These results suggest that nitrergic and glutamatergic pathways are operative within the locus coeruleus to modulate cardiovascular function, and also that a functional interaction may exist between the nitrergic and glutamatergic systems within the rat locus coeruleus.

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.

Dissociation between electrical and mechanical responses to nitrergic stimulation in the canine gastric fundus

1. We examined the relationships between membrane potential, intracellular [Ca2+] ([Ca2+]i), and tension in muscles of the canine gastric fundus in response to nitrergic stimulation by NO donors and electrical field stimulation (EFS) of intrinsic enteric inhibitory neurons when adrenergic and cholinergic responses were blocked. 2. NO donors reduced [Ca2+]i and tension in a concentration-dependent manner. A close relationship was noted between these parameters. 3. In terms of the [Ca2+] vs. force relationship, relaxation responses to EFS differed from responses to NO donors. EFS resulted in smaller decreases in [Ca2+]i to produce a given relaxation compared with responses to NO donors. Thus, muscles stimulated with EFS were less sensitive to [Ca2+]i than muscles stimulated with exogenous NO. 4. When membrane potential, [Ca2+]i and tension were monitored simultaneously in the same muscles, a temporal dissociation was noted between the electrical responses and changes in [Ca2+]i and tension. Brief electrical responses were associated with more sustained changes in [Ca2+]i and tension. 5. Further dissociation between electrical and mechanical effects was noted. Changes in [Ca2+]i and tension caused by sodium nitroprusside and EFS were blocked by arginine analogues and by oxyhaemoglobin, but electrical responses were unaffected. 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, also blocked the effects of nitrergic stimulation on [Ca2+]i and tension, without affecting hyperpolarization. Thus, in the presence of continued hyperpolarization, the reductions in [Ca2+]i and tension caused by nitrergic stimulation were blocked. 6. Block of hyperpolarization in response to nitrergic stimulation with tetrapentylammonium chloride (TPEA) had relatively little effect on the [Ca2+]i and tension responses. Thus, hyperpolarization is not required for nitrergic effects on [Ca2+]i and tension. 7. In summary, reduction in [Ca2+]i and tension in response to nitrergic stimulation of the canine gastric fundus does not depend upon electrical hyperpolarization. Non-electrical mechanisms such as enhanced uptake of Ca2+ by the sarcoplasmic reticulum or reduction in the Ca2+ sensitivity of the contractile apparatus may be the primary mechanisms mediating nitrergic responses in these muscles.

S‐nitrosothiols and the nitrergic neurotransmitter in the rat gastric fundus: effect of antioxidants and metal chelation

1. The effects of the antioxidants ascorbic acid and alpha-tocopherol and of the metal chelator ethylenediaminetetraacetic acid (EDTA) were studied on relaxations in response to S-nitrosothiols, authentic nitric oxide (NO) and nitrergic non-adrenergic non-cholinergic stimulation of the rat gastric fundus. 2. The S-nitrosothiols S-nitrosocysteine (1-100 nM), S-nitrosoglutathione (0.01-3 microM) and S-nitroso-N-acetylpenicillamine (0.01-3 microM) induced concentration-dependent relaxations of the rat gastric fundus muscle strips, which were precontracted with prostaglandin F2alpha. The relaxations to all S-nitrosothiols were concentration-dependently enhanced by the antioxidants ascorbic acid (0.1-3 microM) and alpha-tocopherol (3-30 microM) and inhibited by the metal chelator EDTA (26 microM). 3. Ascorbic acid and alpha-tocopherol alone did not induce a relaxation of the precontracted rat gastric fundus muscle strip. However, when ascorbic acid (1 microM) or alpha-tocopherol (1 microM) were injected in the organ bath 1 minute after S-nitrosoglutathione (0.1 microM) or after S-nitroso-N-acetylpenicillamine (0.1 microM), they induced an immediate, sharp and transient relaxation. This relaxation was inhibited by the superoxide generator pyrogallol (2 microM). Such a relaxation to ascorbic acid or alpha-tocopherol was not observed in the presence of S-nitrosocysteine (10 nM). 4. Electrical field stimulation (0.5-4 Hz) of the precontracted rat gastric fundus strips induced frequency-dependent nitrergic relaxations which were mimicked by authentic NO (3-300 nM) and by acidified sodium nitrite NaNO2 (0.3-10 microM). Ascorbic acid (0.33-3 microM), alpha-tocopherol (3-30 microM) or EDTA (26 microM) did not affect the relaxations to nitrergic stimulation, NO or NaNO2. 5. In summary, relaxations to S-nitrosothiols in the rat gastric fundus are enhanced by the antioxidants ascorbic acid and alpha-tocopherol and inhibited by the metal chelator EDTA. However, relaxations to nitrergic stimulation of the rat gastric fundus or those to authentic NO were not affected by the antioxidants or by the metal chelator. These results indicate that antioxidants and metal chelators have a different effect on the biological activity of S-nitrosothiols and on that of the nitrergic neurotransmitter. Therefore, our results suggest that S-nitrosothiols do not act as intermediate compounds in nitrergic neurotransmission in the rat gastric fundus.

Nitrergic and cholinergic innervation of the rat lower urinary tract after pelvic ganglionectomy.

The possible coexistence of nitric oxide (NO) and acetylcholine in the rat major pelvic ganglion (MPG) was examined by double immunohistochemistry using antisera raised against NO synthase (NOS) and choline acetyltransferase (ChAT). The smooth muscle responses of the isolated bladder and urethra were recorded after bilateral cryoganglionectomy of the MPG, focusing on the possible development of denervation supersensitivity. In the MPG, NOS immunoreactivity (ir) was seen in a large number of cell bodies, but it was not as abundant as ChAT-ir cell bodies. Double immunolabeling showed that all NOS-ir cell bodies also displayed ChAT-ir. In ganglionectomized bladders, the electrical field stimulation (EFS)-evoked contractile response was markedly reduced. When compared with control bladders, detrusor strips from ganglionectomized rats were more sensitive to carbachol as revealed by a lower negative logarithm of the drug concentration eliciting 50% relaxation (6.5 +/- 0.04 vs. 5.9 +/- 0.07). In the urethra, the NO-mediated relaxant response to EFS was practically abolished by ganglionectomy, whereas no difference was found in sensitivity to 3-morpholinosydnonimine hydrochloride (SIN-1). SIN-1 produced an equal increase in tissue levels of guanosine 3',5'-cyclic monophosphate in urethral preparations from control and ganglionectomized rats. The results suggest that the NOS-ir nerves that mediate inhibition of rat urethral smooth muscle tone originate from the MPG and contain ChAT. No denervation supersensitivity to nitrergic stimulation was observed in the urethra after ganglionectomy.

Effect of Cu2+ on relaxations to the nitrergic neurotransmitter, NO and S-nitrosothiols in the rat gastric fundus.

1. The effects of addition of Cu2+ and chelation of Cu2+ were studied on relaxations in response to S-nitrosothiols and on relaxations to non-adrenergic non-cholinergic (NANC) nerve stimulation, nitric oxide (NO) and glyceryl trinitrate (GTN) in the rat gastric fundus. 2. The S-nitrosothiols S-nitroso-L-cysteine (NOCys, 1-300 nM), S-nitrosoglutathione (GSNO, 0.01-3 microM) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.01-3 microM) induced concentration-dependent relaxations of the rat gastric fundus muscle strip. The relaxant potencies of the S-nitrosothiols were NOCys > SNAP > GSNO. Relaxations to NOCys were transient and comparable to those to NANC nerve stimulation and NO whereas relaxations to GSNO and SNAP were sustained. The relaxations to NOCys, GSNO and SNAP were significantly and concentration-dependently enhanced by CuSO4 (3-30 microM). The order of relaxant potency in the presence of CuSO4 was reversed to GSNO approximately SNAP > NOCys. 3. In the presence but not in the absence of 0.1 microM GSNO, CuSO4 (1 microM) induced a rapid and transient relaxation which was inhibited by the superoxide radical generator, pyrogallol (30 microM). CuCl2 but not FeSO4 mimicked the effect of CuSO4. 4. Electrical stimulation (0.5-8 Hz) of the rat gastric fundus strips induced frequency-dependent relaxations which were previously shown to be nitrergic in nature and which were not affected by CuSO4 (3-30 microM). Relaxations to NO (3-100 nM) and GTN (0.01-1 microM) were not affected by 3 and 10 microM CuSO4 but were inhibited by 30 microM CuSO4. 5. The Cu2+ chelator, bathocuproine (3-30 microM) significantly and concentration-dependently inhibited the relaxations to NOCys (0.01-3 microM), GSNO (0.01-10 microM) and SNAP (0.01-3 microM). The inhibitory effect of 10 microM bathocuproine was reversed by 3 microM CuSO4. 6. Bathocuproine (3-30 microM) had no effect on the relaxations to NANC nerve stimulation (0.5-8 Hz) or on the concentration-response curve to NO (0.01-0.3 microM), whereas relaxations to GTN (0.01-1 microM) were significantly inhibited by 30 microM bathocuproine. 7. From these results we conclude that relaxations to S-nitrosothiols and to nitrergic stimulation of the rat gastric fundus are differentially affected by addition and chelation of Cu2+, suggesting that the nitrergic NANC neurotransmitter in the rat gastric fundus is not an S-nitrosothiol but is more likely to be free nitric oxide.

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.

Antioxidant protection of NO-induced relaxations of the mouse anococcygeus against inhibition by superoxide anions, hydroquinone and carboxy-PTIO.

1. The potential protective effect of several antioxidants [Cu/Zn superoxide dismutase (Cu/Zn SOD), ascorbate, reduced glutathione (GSH), and alpha-tocopherol (alpha-TOC)] on relaxations of the mouse anococcygeus muscle to nitric oxide (NO; 15 microM) and, where appropriate, nitrergic field stimulation (10 Hz; 10 s trains) was investigated. 2. The superoxide anion generating drug duroquinone (100 microM) reduced relaxations to exogenous NO by 54 +/- 6%; this inhibition was partially reversed by Cu/Zn SOD (250 u ml-1), and by ascorbate (500 microM). Following inhibition of endogenous Cu/Zn SOD activity with diethyldithiocarbamate (DETCA), duroquinone (50 microM) also reduced relaxations to nitrergic field stimulation (by 53 +/- 6%) and this effect was again reversed by Cu/Zn SOD and by ascorbate. Neither GSH (500 microM) nor alpha-TOC (400 microM) afforded any protection against duroquinone. 3. Xanthine (20 mu ml-1); xanthine oxidase (100 microM) inhibited NO-induced relaxations by 73 +/- 14%, but had no effect on those to nitrergic field stimulation, even after DETCA treatment. The inhibition of exogenous NO was reduced by Cu/Zn SOD (250 u ml-1) and ascorbate (400 microM), but was unaffected by GSH or alpha-TOC (both 400 microM). 4. Hydroquinone (100 microM) also inhibited relaxations to NO (by 52 +/- 10%), but not nitrergic stimulation. In this case, however, the inhibition was reversed by GSH (5-100 microM) and ascorbate (100-400 microM), although Cu/Zn SOD and alpha-TOC were ineffective. 5. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO, 50 microM) inhibited NO-induced relaxations by 50 +/- 4%, but had no effect on nitrergic responses; the inhibition was reduced by ascorbate (2-200 microM) and alpha-TOC (10-200 microM), but not by Cu/Zn SOD or GSH. 6. Hydroxocobalamin (5-100 microM) inhibited, equally, relaxations to both NO (-logIC40 3.14 +/- 0.33) and nitrergic stimulation (-logIC40 3.17 +/- 0.22). 7. Thus, a number of physiological antioxidants protected NO from superoxide anions, and from direct NO-scavengers. The possibility that the presence of these antioxidants within nitrergically-innervated tissues might explain the lack of effect of the NO inhibitors on nerve-induced relaxation, without the need to invoke a transmitter other than free radical NO, is discussed.