Non-adrenergic Non-cholinergic Nerve Stimulation Research Articles

BDNF augments rat internal anal sphincter smooth muscle tone via RhoA/ROCK signaling and nonadrenergic noncholinergic relaxation via increased NO release.

Presently, there are no studies examining the neuromodulatory effects of brain-derived neurotropic factor (BDNF) on the basal internal anal sphincter (IAS) tone and nonadrenergic noncholinergic (NANC) relaxation. To examine this, we determined the neuromuscular effects of BDNF on basal IAS smooth muscle tone and the smooth muscle cells (SMCs) and the effects of NANC nerve stimulation before and after high-affinity receptor tyrosine kinase receptor B (TrkB) antagonist K252a. We also investigated the mechanisms underlying BDNF-augmented increase in the IAS tone and NANC relaxation. We found that BDNF-increased IAS tone and SMC contractility were TTX resistant and attenuated by K252a. TrkB-specific agonist 7,8-dihydroxyflavone, similar to BDNF, also produced a concentration-dependent increase in the basal tone, whereas TrkB inhibitors K252a and ANA-12 produced a decrease in the tone. In addition, BDNF produced leftward shifts in the concentration-response curves with U46619 and ANG II (but not with bethanechol and K+ depolarization), and these shifts were reversed by K252a. Effects of Y27632 and Western blot data indicated that the BDNF-induced increase in IAS tone was mediated via RhoA/ROCK. BDNF-augmented NANC relaxation by electrical field stimulation was found to be mediated via the nitric oxide (NO)/soluble guanylate cyclase (sGC) pathway rather than via increased sensitivity to NO. In conclusion, the net effect of BDNF was that it caused an increase in the basal IAS tone via RhoA/ROCK signaling. BDNF also augmented NANC relaxation via NO/sGC. These findings may have relevance to the role of BDNF in the pathophysiology and therapeutic targeting of the IAS-associated rectoanal motility disorders.NEW & NOTEWORTHY These studies for the first time to our knowledge demonstrate that increased levels of brain-derived neurotrophic factor (BDNF; conceivably released from smooth muscle cells and/or the enteric neurons) has two major effects. First, BDNF augments the internal anal sphincter (IAS) tone via tyrosine kinase receptor B/thromboxane A2-receptor, angiotensin II receptor type 1/RhoA/ROCK signaling; and second, it increases nonadrenergic noncholinergic relaxation via nitric oxide/soluble guanylate cyclase. These studies may have relevance in therapeutic targeting in the anorectal motility disorders associated with the IAS.

The effect of clonidine on the response to stimulation of non-adrenergic non-cholinergic nerves in the guinea-pig urinary bladder in-vitro.

The effects of clonidine on the response of the guinea-pig urinary bladder detrusor muscle to stimulation of non-adrenergic non-cholinergic (NANC) nerves were investigated in-vitro. In tissues from both treated and chemically sympathectomized animals, in the presence of atropine (10(-5) M) to inhibit cholinergic responses, clonidine (10(-10)-10(-6) M) invariably enhanced the contractile response to NANC nerve stimulation at 2, 5, 10 and 20 Hz. The enhancement was not inhibited by yohimbine (10(-5) M), phentolamine (10(-5) M), or the histamine H1- or H2-receptor antagonists mepyramine (10(-6) M) or cimetidine (10(-5) M) respectively. Phentolamine (10(-5) M), like clonidine, enhanced the response to stimulation of NANC nerves at 2, 5, 10 and 20 Hz. Xylazine, another alpha 2-agonist, which, unlike both clonidine and phentolamine, is not a substituted 2-imidazoline compound, failed to enhance the response in the bladder to NANC nerve stimulation. The results suggest that clonidine enhances the response to NANC nerve stimulation, independently of its effects on alpha-adrenoceptors, by increasing the amount of NANC transmitter available.

Dopamine depresses non-adrenergic, non-cholinergic neurotransmission in the rat bladder.

The effects of dopamine on non-adrenergic, non-cholinergic (NANC) neurotransmission have been investigated using rat bladder strips in-vitro. Dopamine administered alone did not produce any effect on the bladder but it depressed responses to NANC nerve stimulation in a dose-related fashion. All doses of dopamine employed depressed KCl-evoked contractions of the bladder to similar extents. The depressant action of dopamine on NANC transmission was not mediated by blockade of purinergic receptors, but was partially reversed by haloperidol. It is suggested that depression of NANC neurotransmission induced by dopamine in the rat bladder is partly mediated by dopaminergic receptors.

Involvement of soluble guanylate cyclase α1 and α2, and SKCa channels in NANC relaxation of mouse distal colon

In distal colon, both nitric oxide (NO) and ATP are involved in non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission. The role of the soluble guanylate cyclase (sGC) isoforms α1β1 and α2β1, and of the small conductance Ca2+-dependent K+ channels (SKCa channels) in the relaxation of distal colon by exogenous NO and by NANC nerve stimulation was investigated, comparing wild type (WT) and sGCα1 knockout (KO) mice. In WT strips, the relaxation induced by electrical field stimulation (EFS) at 1 Hz but not at 2–8 Hz was significantly reduced by the NO-synthase inhibitor l-NAME or the sGC inhibitor ODQ. In sGCα1 KO strips, the EFS-induced relaxation at 1 Hz was significantly reduced and no longer influenced by l-NAME or ODQ. The SKCa channel blocker apamin alone had no inhibitory effect on EFS-induced relaxation, but combined with ODQ or l-NAME, apamin inhibited the relaxation induced by EFS at 2–8 Hz in WT strips and at 8 Hz in sGCα1 KO strips. Relaxation by exogenous NO was significantly attenuated in sGCα1 KO strips, but could still be reduced further by ODQ. Basal cGMP levels were lower in sGCα1 KO strips but NO still significantly increased cGMP levels versus basal. In conclusion, in the absence of sGCα1β1, exogenous NO is able to partially act through sGCα2β1. NO, acting via sGCα1β1, is the principal neurotransmitter in EFS-evoked responses at 1 Hz. At higher stimulation frequencies, NO, acting at sGCα1β1 and/or sGCα2β1, functions together with another transmitter, probably ATP acting via SKCa channels, with some degree of redundancy.

Mechanism of internal anal sphincter relaxation by CORM-1, authentic CO, and NANC nerve stimulation.

The present studies compared the effects of CO-releasing molecule (CORM-1), authentic CO, and nonadrenergic noncholinergic (NANC) nerve stimulation in the internal anal sphincter (IAS). Functional in vitro experiments and Western blot studies were conducted in rat IAS smooth muscle. We examined the effects of CORM-1 (50-600 microM) and authentic CO (5-100 microM) and NANC nerve stimulation by electrical field stimulation (EFS; 0.5-20 Hz, 0.5-ms pulse, 12 V, 4-s train). The experiments were repeated after preincubation of the tissues with the neurotoxin TTX, the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), the selective heme oxygenase (HO) inhibitor tin protoporphyrin IX (SnPP-IX), the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA), and SnPP-IX + L-NNA. We also investigated the effects of the HO substrate hematin (100 microM). CORM-1, as well as CO, produced concentration-dependent IAS relaxation, whereas hematin had no effect. TTX abolished and L-NNA significantly blocked IAS relaxation by EFS without any effect on CORM-1 and CO. ODQ blocked IAS relaxation by CORM-1, authentic CO, and EFS. SnPP-IX had no significant effect on IAS relaxation by CORM-1, CO, or EFS. The presence of neuronal nitric oxide synthase, HO-1, and HO-2 in IAS smooth muscle was confirmed by Western blot studies. CORM-1 and CO, as well as NANC nerve stimulation, produced IAS relaxation via guanylate cyclase/cGMP-dependent protein kinase activation. The advent of CORM-1 with potent effects in the IAS has significant implications in anorectal motility disorders with regard to pathophysiology and therapeutic potentials.

Involvement of nitric oxide in non-adrenergic non-cholinergic relaxation and action of vasoactive intestinal polypeptide in circular muscle strips of the rat gastric fundus

We examined the characteristics of the non-adrenergic non-cholinergic (NANC) nerve induced relaxation and the possible interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) on the basal tone of the circular muscle of the rat gastric fundus. Electrically induced NANC relaxations were partly inhibited by Nω-nitro- l -arginine (100 μ M), whereas sodium nitroprusside (SNP; 10 μ M) and VIP (5 nM) induced relaxations were not affected. 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; 5 μ M) also inhibited the responses to electrical stimuli to a similar extent as Nω-nitro- l -arginine but not VIP. However, AMT plus Nω-nitro- l -arginine did not give an additional inhibition above that of each drug alone on NANC relaxations, and dexamethasone (10 μ M) had no effect on NANC nerve induced relaxations. 1H-[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μ M), a selective inhibitor of guanylate cyclase, abolished the responses to NANC nerve stimulation and SNP, while VIP responses were not influenced. N-ethylmaleimide (100μ M), an adenylate cyclase inhibitor, attenuated relaxations to NANC nerve stimulation, VIP and isoproterenol (1 nM), while having no effect on those to SNP, but in combination with Nω-nitro- l -arginine, there was no additional inhibition on the responses to nerve stimulation. Alpha-chymotrypsin (10 u ml−1) severely diminished VIP induced relaxations, but did not reduce electrical responses. In conclusion, these results suggest that NO is involved in the relaxations induced by short-term electrical stimulation. However, another possible unidentified transmitter that can trigger the accumulation of cyclic GMP is not entirely ruled out and there is no interaction between NO and VIP in the circular muscle strip of the rat gastric fundus, even in the basal state of the tissue.

Altered non-adrenergic non-cholinergic neurotransmission in gastric fundus from streptozotocin-diabetic rats

The influence of streptozotocin-induced diabetes has been investigated on responses to non-adrenergic, non-cholinergic (NANC) nerve stimulation in rat gastric fundus. NANC relaxations in precontracted muscle strips from diabetic rats were smaller than those from control rats. In addition, the relaxations in diabetic but not control rats were followed by rapidly-developing frequency-dependent contractions. In the presence of α-chymotrypsin and N g -nitro- l-arginine methyl ester ( l-NAME), the NANC contractions were markedly enhanced in diabetic rats. Treatment with the aldose reductase inhibitor, sorbinil, did not affect NANC relaxations or contractions in tissues from diabetic rats, and responses remained significantly different from those from control rats. The findings suggest that diabetes impairs relaxations to NANC nerve stimulation in the rat gastric fundus, and that a contractile NANC neurotransmitter(s) is released in diabetic rats. The results also suggest that diabetes-induced alterations in the NANC nerve response are not caused by increased activity of the aldose reductase pathway.

Heme oxygenase activity in the internal anal sphincter: Effects of nonadrenergic, noncholinergic nerve stimulation

Background & Aims: To date, the exact role of carbon monoxide (CO) in the nonadrenergic, noncholinergic (NANC) relaxation is not known. This is partly related to the lack of an appropriate method to measure heme oxygenase (HO) activity in the gastrointestinal tissues. Methods: HO activity of the opossum internal anal sphincter (IAS) smooth muscle was determined using a newly developed assay system that used radiolabeled hemin as a substrate. Enzyme activity of the IAS tissues was measured in the basal state, after electric field stimulation (EFS), ganglionic stimulant dimethyl diphenyl piperazinium iodide (DMPP), and neuropeptide vasoactive intestinal polypeptide (VIP). The presence and localization of HO was examined by Western blot analysis and immunocytochemistry. Results: NANC nerve stimulation of the IAS smooth muscle by EFS (0.25–5 Hz), DMPP, and VIP caused a significant increase in the HO activity of the IAS. The increase in HO activity by EFS was inhibited by the HO inhibitor Tin protoporphyrin (1 × 10 −4 mol/L). Both HO-1 and HO-2 were present in the IAS tissue extracts, and both enzymes were localized in the neurons of the myenteric plexus. The method for HO activity determination used in the present study was found to be reliable and reproducible. Conclusions: The data suggest that the HO pathway may have a role in neurally mediated relaxation of the IAS. The exact site of involvement and the source of HO activity, however, remains to be determined. GASTROENTEROLOGY 2000;118:477-486

ALTERATIONS IN THE NITRIC OXIDE SYNTHASE BINDING SITES AND NON-ADRENERGIC, NON-CHOLINERGIC MEDIATED SMOOTH MUSCLE RELAXATION IN THE DIABETIC RABBIT BLADDER OUTLET: POSSIBLE RELEVANCE TO THE PATHOGENESIS OF DIABETIC CYSTOPATHY

Purpose To investigate the effect of diabetes mellitus (DM) on the density and distribution of nitric oxide synthase (NOS) and the smooth muscle responses to non-adrenergic, non-cholinergic (NANC) nerve stimulation and exogenous nitric oxide (NO) in the rabbit lower urinary tract. Materials and Methods Transverse sections of detrusor, bladder neck and urethra, from control and six months alloxan-induced DM New Zealand White rabbits were incubated with a radioligand for NOS ([( 3) H]-L-N G-nitroarginine). Densitometric analysis was performed on the autoradiographs. NADPH diaphorase histochemistry was also used as a marker for NOS activity. Responses to NANC nerve stimulation (5 to 20 Hz) and to NO (10 -6 to 3 x 10 -4 M.) on smooth muscle strips from detrusor, bladder neck and urethra were measured in organ baths. Results NOS binding sites were significantly (p <0.03) more dense in the bladder neck than in the detrusor in both DM and control groups. In DM bladder neck, NOS binding sites were significantly (p <0.04) increased compared with the controls. NADPH diaphorase activity appeared markedly increased in the detrusor, bladder neck and urethra of DM animals compared with controls. The mean IC 50 for exogenous NO in control versus DM were not statistically different in the bladder neck (1.03 x 10 -4 M versus 9.8 x 10 -5 M) and urethra (8.1 x 10 -5 M versus 8.8 x 10 -5 M), but the relaxations to 5 x 10 -6 M of NO were significantly impaired (p <0.04) in the DM urethral smooth muscle. NANC nerve-mediated relaxations were significantly impaired (p <0.001) in the DM urethral smooth muscle. Conclusions Alterations of both the NOS binding sites and functional responses to NANC nerve stimulation suggest that NO may have a pathophysiological role in the urinary bladder dysfunction associated with DM.

Involvement of pituitary adenylate cyclase-activating peptide in opossum internal anal sphincter relaxation

Despite its widespread distribution and significance in the gut, the role of pituitary adenylate cyclase-activating peptide (PACAP) in internal anal sphincter (IAS) relaxation has not been examined. This study examined the role of PACAP in nonadrenergic noncholinergic (NANC) nerve-mediated relaxation of IAS smooth muscle. Circular smooth muscle strips from the opossum IAS were prepared for measurement of isometric tension. The influence of PACAP and vasoactive intestinal peptide (VIP) antagonists and tachyphylaxis on the neurally mediated IAS relaxation was examined either separately or in combination. The release of these neuropeptides in response to NANC nerve stimulation before and after the nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine and NO was also investigated. Both PACAP and VIP antagonists caused significant attenuation of IAS relaxation by NANC nerve stimulation. The combination of the antagonists, however, did not have an additive effect on IAS relaxation. VIP tachyphylaxis caused significant suppression of IAS relaxation by NANC nerve stimulation. PACAP and VIP were found to be released by NANC nerve stimulation and exogenous NO. The data suggest the involvement of PACAP in IAS relaxation primarily by the activation of PACAP1/VIP receptor and lack of its independent role in the relaxation. Furthermore, NO may regulate the presynaptic release of PACAP and VIP.

Halothane and Isoflurane Attenuate the Relaxant Response to Nonadrenergic and Noncholinergic Nerve Stimulation of Isolated Canine Cerebral Arteries

Stimulation of nonadrenergic noncholinergic (NANC) nerves elicits relaxation of canine cerebral arteries via the nitric oxide (NO)-cGMP pathway. The purpose of this study was to investigate the effects of halothane and isoflurane on the relaxant response of isolated canine cerebral arteries to NANC nerve stimulation. The isometric tension of isolated canine cerebral arteries, which had been denuded of endothelium, was measured in a tissue bath. The application of transmural electrical stimulation (TES) at a frequency of 5 Hz elicited a transient relaxation of arteries partially contracted with prostaglandin F2alpha. This effect was abolished by treatment with N(G)-nitro-L-arginine (3 x 10[-5] M), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10[-5] M), or tetrodotoxin (10[-6] M). Treatment with halothane (2.3%) or isoflurane (2.3% and 3.5%) attenuated the relaxant response to TES (P < 0.05). Halothane (2.3%) but not isoflurane (2.3% and 3.5%) attenuated relaxation induced by s-nitro-N-acetylpenicillamine. We suggest that halothane and isoflurane inhibit cerebroarterial vasodilation mediated via NO-cGMP pathway activated by stimulation of the NANC nerves. The sites of action of halothane and isoflurane on the NO-cGMP pathway may differ. Nonadrenergic noncholinergic nerves play a role in the regulation of vascular tone in cerebral arteries via the nitric oxide-cGMP pathway. This study showed that, in isolated canine cerebral arteries, halothane and isoflurane inhibit the relaxation caused by nonadrenergic noncholinergic nerve stimulation, but their sites of action may differ.

Evidence that NO acts as a redundant NANC inhibitory neurotransmitter in the guinea-pig isolated taenia coli.

1. The relative contribution of the putative transmitters, nitric oxide (NO) and an apamin-sensitive factor, possibly ATP, to inhibitory responses evoked by electrical field stimulation (EFS; 0.2-5 Hz, 0.2 ms duration, supra-maximal voltage for 10 s) of non-adrenergic, non-cholinergic (NANC) nerves was investigated in the guinea-pig isolated taenia coli contracted with histamine (1 microM). 2. Peak relaxations to EFS (0.2-5 Hz) were tetrodotoxin (1 microM)-sensitive, maximal at 0.2 Hz and completely resistant to the nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NOARG; 100 microM) in either the presence or absence of atropine (1 microM). Furthermore, the specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ; 10 microM), the cytochrome P450 inhibitor and free radical generator, 7-ethoxyresorufin (7-ER; 10 microM) and the NO scavenger, oxyhaemoglobin (HbO; 30 microM) had no effect on EFS-induced relaxations alone and in combination with L-NOARG (100 microM). 3. Maximum relaxation to the NO donor, sodium nitroprusside (SNP; 1 microM) was significantly reduced by HbO (30 microM), abolished by 7-ER (10 microM) and ODQ (10 microM) but was unaffected by apamin (0.1 microM), an inhibitor of small conductance Ca(2+)-activated K+ channels. 4. The relaxation to EFS at 0.2 Hz was resistant to apamin but those to 0.5 and 5 Hz were significantly reduced. EFS (0.2-5 Hz)-evoked relaxations that persisted in the presence of apamin were further significantly inhibited by L-NOARG (100 microM) or ODQ (10 microM), but not by HbO (30 microM) or 7-ER (10 microM). 5. ATP (1-30 microM) produced concentration-dependent relaxations that were abolished by apamin (0.1 microM), unaffected by ODQ (10 microM) but only significantly reduced by L-NOARG (100 microM) at the lowest concentration of ATP (1 microM) used. 6. Nifedipine (0.3 microM), abolished contractions to 67 mM KCl, histamine (10 microM), endothelin-1 (0.03 microM), 5-hydroxytryptamine (5-HT; 10 microM) and the thromboxane-mimetic, 9-11-dideoxy-9 alpha, 11 alpha-methano-epoxy-prostaglandin F2 alpha (U46619; 0.1 microM). 7. The findings of the present study suggest that NO is released during NANC nerve stimulation, but plays no role in NANC relaxations in the guinea-pig taenia coli unless the effects of another apamin-sensitive, nerve-derived hyperpolarizing factor (NDHF) are blocked. Thus, we propose that in this tissue, NO acts as a 'backup' or redundant NANC nerve inhibitory transmitter and like NDHF mediates relaxation via hyperpolarization.

Pharmacological properties of non-adrenergic, non-cholinergic inhibitory transmission in chicken gizzard.

1. Non-adrenergic, non-cholinergic (NANC) inhibitory transmission in chicken gizzard was studied by use of intracellular microelectrode techniques. Changes in membrane potential in response to NANC nerve stimulation were recorded in the gizzard smooth muscle pretreated with atropine (1 microM) and guanethidine (1 microM). 2. Field stimulation of the intramural nerves (FS) evoked inhibitory junction potentials (i.j.ps) which were abolished by tetrodotoxin (1 microM), but not inhibited at all by K+ channel blockers including apamin (0.5 microM), tetraethylammonium (TEA, 10 mM), charybdotoxin (0.2 microM) and glibenclamide (10 microM). 3. NG-nitro-L-arginine (3 mM), an inhibitor of nitric oxide (NO) synthase, inhibited i.j.ps. The effect was reversed by L-arginine (3 mM), but not by D-arginine (3 mM). 4. 8-Bromo cyclic GMP (100 microM), a membrane permeable analogue of cyclic GMP, produced a membrane hyperpolarization which was blocked by TEA (10 mM) or glibenclamide (10 microM). 5. NO at concentrations of up to 400 microM affected neither i.j.ps nor resting membrane potential. On the other hand, NO (80 microM) caused the membrane to hyperpolarize in the smooth muscle of guinea-pig ileum. 6. These results suggest that in the chicken gizzard, NANC i.j.ps may not arise from opening of conventional types of K+ channel and that NO seems unlikely to be involved in the generation of i.j.ps. A possible mechanism by which the inhibitory effect of NG-nitro-L-arginine on i.j.ps was brought about will be discussed.

L-citrulline recycling in opossum internal anal sphincter relaxation by nonadrenergic, noncholinergic nerve stimulation

BACKGROUND & AIMS: L-citrulline formed stoichiometrically along with nitric oxide (1:1) from L-arginine may be enzymatically converted to L- arginine. The possibility of L-citrulline recycling in the maintenance of nitrergic neurotransmission in the opossum internal anal sphincter (IAS) smooth muscle strips was investigated. METHODS: Responses to nonadrenergic, noncholinergic (NANC) nerve stimulation by electrical field stimulation (EFS) (either short-train or continuous stimulation) on the basal IAS tension were recorded before and after the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA), L-NNA plus L-citrulline, or L-arginine. During continuous EFS, when the basal IAS tone after the initial relaxation had recovered to almost pre-EFS levels, the effects of L-citrulline or L-arginine were examined before and after L- glutamine, which is a putative blocker of L-citrulline uptake. RESULTS: Inhibition of NANC nerve-mediated IAS relaxation by L-NNA was reversed by L-citrulline as well as L-arginine. L-Citrulline and L-arginine caused concentration-dependent relaxation of the IAS tone recovered during the prolonged EFS. L-Glutamine blocked the responses of L- citrulline but not of L-arginine. Furthermore, L-glutamine increased the speed of recovery of IAS tone during continuous EFS. CONCLUSIONS: L- citrulline recycling may be responsible for the maintenance of IAS relaxation during frequent short-train and prolonged NANC nerve stimulation. (Gastroenterology 1997 Apr;112(4):1250-9)

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.

Characterization of the membrane conductance changes underlying the apamin-resistant NANC inhibitory junction potential in the guinea-pig proximal and distal colon

The nature of the electrically- or stretch-evoked nonadrenergic, noncholinergic (NANC) inhibitory junction potentials (UPs) in circular smooth muscle cells of the guinea-pig proximal and distal colon were investigated using standard intracellular microelectrode recording techniques. We have confirmed that the NANC UP, recorded in the presence of hyoscine (1 μM) and nifedipine (1 μM), can be divided into two components with apamin (250 nM), a blocker of the small conductance Ca 2+-activated K + channels. Both the apamin-sensitive and the apamin-resistant components of the UP were blocked by tetrodotoxin (1.6 μM) or by lowering the external Ca 2+ concentration (to 0.25 mM). The apamin-sensitive UP was also blocked by ω-conotoxin GVIA (100 nM), a blocker of ‘N-type’ Ca 2+ channels. The apamin-resistant UP and rebound post-stimulus depolarization (PSD) were reduced upon exposure to either N G- l-arginine (NOLA), an inhibitor of nitric oxide synthase (NOS), or the nitric oxide (NO) scavenger, haemoglobin. The effects of NOLA were partially reversed in the presence of excess l-arginine, a substrate for NOS, suggesting that NO, or a related NO-donor compound, is likely to be the apamin-resistant inhibitory transmitter. Blockade of either the apamin-sensitive or apamin-resistant UP was associated with membrane depolarization and a decrease in the membrane conductance in the absence of nerve stimulation. In the proximal colon, the apamin-resistant UP and PSD could both be demonstrated to arise from an increase in the membrane conductance after subtraction of a non-linear background conductance. The hyperpolarization upon repetitive NANC nerve stimulation was mimicked by the NO donor, S-nitroso- l-cysteine (2.5–25 RM), which evoked a transient apamin-sensitive, but w-conotoxin GVIA resistant, component followed by a slower apamin-resistant component. These results suggest that neurally-released NO has a number of actions in the guinea-pig colon, causing apamin-resistant hyperpolarization and depolarization, as well as directly opening apamin-sensitive K + channels.

Neuronal mediators of inhibitory junction potentials and relaxation in the guinea-pig internal anal sphincter.

1. Inhibitory junction potentials (IJPs) and relaxations evoked in response to field stimulation (supramaximal voltage, 0.1 ms, single stimulus and 5 stimuli at 5-40 Hz) of non-adrenergic non-cholinergic (NANC) nerves with atropine and phentolamine (each 1 microM) were measured in the guinea-pig internal anal sphincter (gpIAS). The mean resting membrane potential was -44.2 +/- 0.2 mV (n = 1119 cells from 260 preparations). 2. NANC nerve stimulation evoked frequency-dependent IJPs (19.7 +/- 1.1 mV, n = 165, 33 tissues to a single stimulus) and relaxations. IJPs consisted of two tetrodotoxin (1 microM)-sensitive components: one was abolished by apamin (0.3 microM) and the P2-purinoceptor antagonist suramin (100 microM); the other, smaller in amplitude, was sensitive to inhibitors of nitric oxide synthase (NOS, e.g. L-NAME, 100 microM) and the nitric oxide (NO) scavenger oxyhaemoglobin (HbO, 10 microM). 3. ATP (1 mM), vasoactive intestinal polypeptide (VIP, 0.01-0.25 microM) and pituitary adenylate cyclase-activating peptide (PACAP(1-27), 0.84 microM) each hyperpolarized and relaxed the gpIAS; only ATP responses resembled the evoked IJPs in time course. 4. The guanylyl cyclase inhibitor LY83583 (10 microM) abolished apamin-insensitive IJPs and relaxations. The cGMP phosphodiesterase inhibitor M&B 22948 (30 microM) and 8-Br-cGMP (100 microM) each hyperpolarized the gpIAS. 5. Two components comprise the IJP and relaxation evoked in response to NANC nerve stimulation in the gpIAS. One, sensitive to apamin, resembles the response to ATP and is modulated by purinoceptor antagonists; the other, apamin and suramin insensitive, is inhibited by NO antagonists.

Involvement of nitric oxide in the non-adrenergic non-cholinergic neurotransmission of horse deep penile arteries: role of charybdotoxin-sensitive K(+)-channels.

1. The involvement of nitric oxide (NO) and the signal transduction mechanisms mediating neurogenic relaxations were investigated in deep intracavernous penile arteries with an internal lumen diameter of 600-900 microns, isolated from the corpus cavernosum of young horses. 2. The presence of nitric oxide synthase (NOS)-positive nerves was examined in cross and longitudinal sections of isolated penile arteries processed for NADPH-diaphorase (NADPH-d) histochemistry. NADPH-d-positive nerve fibres were observed in the adventitia-media junction of deep penile arteries and in relation to the trabecular smooth muscle. 3. Electrical field stimulation (EFS) evoked frequency-dependent relaxations of both endothelium-intact and denuded arterial preparations treated with guanethidine (10(-5) M) and atropine (10(-7) M), and contracted with 10(-6) M phenylephrine. These EFS-induced relaxations were tetrodotoxin-sensitive indicating their non-adrenergic non-cholinergic (NANC) neurogenic origin. 4. EFS-evoked relaxations were abolished at the lowest frequency (0.5-2 Hz) and attenuated at higher frequencies (4-32 Hz) by the NOS inhibitor, NG-nitro-L-arginine (L-NOARG, 3 x 10(-3) M). This inhibitory effect was antagonized by the NO precursor, L-arginine (3 x 10(-3) M). NG-nitro-D-arginine (10(-4) M) did not affect the relaxations to EFS. 5. Incubation with either the NO scavenger, oxyhaemoglobin (10(-5) M), or methylene blue (10(-5) M), an inhibitor of guanylate cyclase activation by NO, caused significant inhibitions of the EFS-evoked relaxations, and while oxyhaemoglobin abolished the relaxations to exogenously added NO (acidified sodium nitrite, 10(-6) - 10(-3) M), there still persisted a relaxation to NO of 24.4 +/- 5.1% (n = 6) in the presence of methylene blue. 6. Glibenclamide (3 x 10(-6) M), an inhibitor of ATP-activated K(+)-channels, did not alter the relaxations to either EFS-stimulation or NO, while the blocker of Ca(2+)-activated K(+)-channels, charybdotoxin (3 x 10(-8) M), caused a significant inhibition of both the electrically-induced relaxations and the relaxations to exogenously added NO. Furthermore, charybdotoxin blocked relaxations induced by the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (8 Br-cyclic GMP). 7. These results suggest that relaxations of horse deep penile arteries induced by NANC nerve stimulation involve mainly NO or a NO-like substance from nitrergic nerves. NO would stimulate the accumulation of cyclic GMP followed by increases in the open probability of Ca(2+)-activated K(+)-channels and hyperpolarization leading to relaxation of horse penile arteries.

Effect of diabetes on relaxations to non‐adrenergic, non‐cholinergic nerve stimulation in longitudinal muscle of the rat gastric fundus

1. The effect of 8-week streptozotocin-induced diabetes has been examined on relaxations to non-adrenergic, non-cholinergic (NANC) nerve stimulation in longitudinal strips of rat gastric fundus. 2. In the presence of noradrenergic and cholinergic blockade and raised tissue tone, electrical field stimulation (0.5-4 Hz, 30 s trains) induced frequency-dependent relaxations that were significantly smaller in gastric fundus strips from diabetic rats than in strips from control rats. 3. NG-nitro-L-arginine methyl ester (NAME, 100 microM) significantly reduced NANC relaxations in muscle strips from both control and diabetic rats, but the reduction was greater in muscle strips from diabetic rats than in those from control rats at frequencies of 2 and 4 Hz. alpha-Chymotrypsin (1 u ml-1) slightly reduced relaxations to nerve stimulation in muscle strips from both control and diabetic rats. 4. The duration of NANC nerve relaxations (1-4 Hz, 30 s trains) was smaller in muscle strips from diabetic rats than in those from control rats. The duration of NANC relaxations was reduced by alpha-chymotrypsin (1 u ml-1) in muscle strips from control rats but not in muscle strips from diabetic rats. 5. Relaxations to both nitric oxide (NO; 1-30 microM) and vasoactive intestinal polypeptide (VIP; 0.1-30 microM) were concentration-dependent and did not differ between muscle strips from control and diabetic rats. 6. The results suggest that streptozotocin-induced diabetes impairs relaxations to NANC nerve stimulation in the rat gastric fundus, which are largely mediated by NO and to a lesser extent by VIP. The impairment appears to occur at the prejunctional level, as smooth muscle reactivity to NO and VIP is not altered.