Non-adrenergic Non-cholinergic Nerves Research Articles

Mechanical activity of frog esophagus muscle in response to electrical stimulation of intramural nerves

Microscopic observation of intramural nerves in the frog esophagus, fixed and stained with OsO(4) and ZnI(2), revealed that nerve cell bodies and bundles connecting the nerve cell bodies formed loose and irregular networks. The nerve cell bodies were mostly lying singly in the nerve bundles, with occasional observations of two closely linked nerve cell bodies. Isolated circular and longitudinal segments of esophageal muscle were spontaneously rhythmically contractile, with a frequency of 2.2-3.0 per min. This was not altered by tetrodotoxin (TTX). In longitudinal muscle segments, transmurally applied electrical stimulation produced contractile responses which were not inhibited by atropine or guanethidine, but were reduced in amplitude by TTX, suggesting a nonadrenergic-noncholinergic (NANC) excitatory innervation in the esophagus muscle. In circular muscle segments, transmural application of brief electrical stimulation evoked two types of mechanical response: a biphasic response consisting of an initial relaxation and a following contraction (type I) and a contraction alone (type II). These mechanical responses were not modulated by either atropine or guanethidine. In the type I response, TTX abolished the relaxation component, suggesting that this was produced by non-adrenergic non-cholinergic (NANC) inhibitory nerve excitation. In about half of the type II responses, the amplitude of the contraction was significantly reduced by TTX, suggesting that a part of the contraction was produced by activation of NANC excitatory nerves. Thus, the esophageal smooth muscle of the frog demonstrates myogenic activity, and is innervated by both excitatory and inhibitory NANC nerves.

The lung in the balance: arginine, methylated arginines, and nitric oxide

the lung is a major source of nitric oxide (NO), be it from nitric oxide synthase (NOS) III in the endothelium of the vast pulmonary circulation, NOS II in the epithelium of the large surface area of the airways, or NOS I in the nonadrenergic noncholinergic nerves ([6][1]). NO produced in the lung

Role of the l-citrulline/ l-arginine cycle in iNANC nerve-mediated nitric oxide production and airway smooth muscle relaxation in allergic asthma

Nitric oxide synthase (NOS) converts l-arginine into nitric oxide (NO) and l-citrulline. In NO-producing cells, l-citrulline can be recycled to l-arginine in a two-step reaction involving argininosuccinate synthase (ASS) and -lyase (ASL). In guinea pig trachea, l-arginine is a limiting factor in neuronal nNOS-mediated airway smooth muscle relaxation upon inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation. Moreover, in a guinea pig model of asthma iNANC nerve-induced NO production and airway smooth muscle relaxation are impaired after the allergen-induced early asthmatic reaction, due to limitation of l-arginine. Using guinea pig tracheal preparations, we now investigated whether (i) the l-citrulline/ l-arginine cycle is active in airway iNANC nerves and (ii) the NO deficiency after the early asthmatic reaction involves impaired l-citrulline recycling. Electrical field stimulation-induced relaxation was measured in tracheal open-rings precontracted with histamine. l-citrulline as well as the ASL inhibitor succinate did not affect electrical field stimulation-induced relaxation under basal conditions. However, reduced relaxation induced by a submaximal concentration of the NOS inhibitor N ω-nitro- l-arginine was restored by l-citrulline, which was prevented by the additional presence of succinate or the ASS inhibitor α-methyl- d, l-aspartate. Remarkably, the impaired iNANC relaxation after the early asthmatic reaction was restored by l-citrulline. In conclusion, the l-citrulline/ l-arginine cycle is operative in guinea pig iNANC nerves in the airways and may be effective under conditions of low l-arginine utilization by nNOS (caused by NOS inhibitors), and during reduced l-arginine availability after allergen challenge. Enzymatic dysfunction in the l-citrulline/ l-arginine cycle appears not to be involved in the l-arginine limitation and reduced iNANC activity after the early asthmatic reaction.

Neuropeptides and nitric oxide synthase in the gill and the air‐breathing organs of fishes

Anatomical and histochemical studies have demonstrated that the bulk of autonomic neurotransmission in fish gill is attributed to cholinergic and adrenergic mechanisms (Nilsson. 1984. In: Hoar WS, Randall DJ, editors. Fish physiology, Vol. XA. Orlando: Academic Press. p 185-227; Donald. 1998. In: Evans DH, editor. The physiology of fishes, 2nd edition. Boca Raton: CRC Press. p 407-439). In many tissues, blockade of adrenergic and cholinergic transmission results in residual responses to nerve stimulation, which are termed NonAdrenergic, NonCholinergic (NANC). The discovery of nitric oxide (NO) has provided a basis for explaining many examples of NANC transmissions with accumulated physiological and pharmacological data indicating its function as a primary NANC transmitter. Little is known about the NANC neurotransmission, and studies on neuropeptides and NOS (Nitric Oxide Synthase) are very fragmentary in the gill and the air-breathing organs of fishes. Knowledge of the distribution of nerves and effects of perfusing agonists may help to understand the mechanisms of perfusion regulation in the gill (Olson. 2002. J Exp Zool 293:214-231). Air breathing as a mechanism for acquiring oxygen has evolved independently in several groups of fishes, necessitating modifications of the organs responsible for the exchange of gases. Aquatic hypoxia in freshwaters has been probably the more important selective force in the evolution of air breathing in vertebrates. Fishes respire with gills that are complex structures with many different effectors and potential control systems. Autonomic innervation of the gill has received considerable attention. An excellent review on branchial innervation includes Sundin and Nilsson's (2002. J Exp Zool 293:232-248) with an emphasis on the anatomy and basic functioning of afferent and efferent fibers of the branchial nerves. The chapters by Evans (2002. J Exp Zool 293:336-347) and Olson (2002) provide new challenges about a variety of neurocrine, endocrine, paracrine and autocrine signals that modulate gill perfusion and ionic transport. The development of the immunohistochemical techniques has led to a new phase of experimentation and to information mainly related to gills rather than air-breathing organs of fishes. During the last few years, identification of new molecules as autonomic neurotransmitters, monoamines and NO, and of their multiple roles as cotransmitters, has reshaped our knowledge of the mechanisms of autonomic regulation of various functions in the organs of teleosts (Donald, '98).NO acts as neurotransmitter and is widely distributed in the nerves and the neuroepithelial cells of the gill, the nerves of visceral muscles of the lung of polypterids, the vascular endothelial cells in the air sac of Heteropneustes fossilis and the respiratory epithelium in the swimbladder of the catfish Pangasius hypophthalmus. In addition, 5-HT, enkephalins and some neuropeptides, such as VIP and PACAP, seem to be NANC transmitter candidates in the fish gill and polypterid lung. The origin and function of NANC nerves in the lung of air-breathing fishes await investigation. Several mechanisms have developed in the Vertebrates to control the flow of blood to respiratory organs. These mechanisms include a local production of vasoactive substances, a release of endocrine hormones into the circulation and neuronal mechanisms. Air breathers may be expected to have different control mechanisms compared with fully aquatic fishes. Therefore, we need to know the distribution and function of autonomic nerves in the air-breathing organs of the fishes.

Lead effects on non-adrenergic non-cholinergic relaxations in the rat gastric fundus

The effect of lead exposure on non-adrenergic non-cholinergic (NANC) relaxations in rat gastric fundus was evaluated in this work. Wistar rats were divided into four groups: The control group received tap water and the three other received 0.008% of lead acetate in their drinking water for 15, 30 and 120 days. NANC relaxations induced by electrical field stimulation (0.5–8Hz, 1ms, 60V) of gastric fundus strips was inhibited in all groups treated with lead. The strips from groups, control and 120 days of lead treatment (LEAD 120), were incubated with l-NOARG (100μM). The presence of this blocker did not produce any additional inhibition. Sodium nitroprusside (10−10–10−6M) and 8-Br-GMPc (3×10−8–3×10−4M) produced dose-dependent relaxations in strips of both groups control and LEAD 120, however, in the LEAD 120, the potencies were significantly reduced from 7.32±0.05 to 6.40±0.09 (n=5) and 4.26±0.06 to 3.69±0.05 (n=5), respectively. Our data suggest that the chronic exposure to lead inhibits NANC relaxations probably by modulating NO release from NANC nerves and/or by interacting with intracellular transducer mechanisms in rat gastric fundus.

Functional characterisation of tachykinin receptors in the circular muscle layer of the mouse ileum

Objectives Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum. Results Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK 1, NK 2 or NK 3 receptors significantly reduced contractions to EFS. The NK 1 receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK 2 and NK 3 receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK 1 and NK 2 receptor agonists, septide and [β-ala 8]-NKA 4-10 (β-A-NKA), respectively, but not the NK 3 receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to β-A-NKA were insensitive to atropine but augmented by l-NNA. Conclusions Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK 1 receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK 2 and NK 3 receptors, most likely located presynaptically.

The internal anal sphincter: regulation of smooth muscle tone and relaxation

Basal tone in the internal anal sphincter (IAS) is primarily myogenic. Neurohumoral substances like angiotensin II may partially provide external signal for the basal tone in the IAS. The sphincteric relaxation on the contrary is neurogenic by activation of non-adrenergic non-cholinergic (NANC) nerves that release nitric oxide (NO), vasoactive intestinal polypeptide (VIP) and perhaps carbon monoxide. Because of the presence of spontaneous tone, the IAS offers an excellent model to investigate the nature of the inhibitory neurotransmission for NANC relaxation. Work from different laboratories in different species concludes that NO is the major contributor in the NANC relaxation. This may invoke the role of other inhibitory neurotransmitters such as VIP, working partly via NO. An understanding of the basic regulation of basal tone in the IAS and nature of inhibitory neurotransmission are critical in the pathophysiology and therapeutic potentials in the anorectal motility disorders.

Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

BackgroundRecent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS)-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation.MethodsElectrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz)-induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM). Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine.ResultsEFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz (P < 0.01 all). In contrast, the arginase inhibitor nor-NOHA increased EFS-induced relaxation by 3.3 ± 1.2-fold at 0.5 Hz to 1.2 ± 0.1-fold at 4 Hz (P < 0.05 all), which was reversed by L-NNA to the level of control airways in the presence of L-NNA (P < 0.01 all). Similar to nor-NOHA, exogenous L-arginine increased EFS-induced airway relaxation (P < 0.05 all).ConclusionThe results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS.

Relationship between airway responsiveness to neurokinin A and methacholine in asthma

Non-adrenergic non-cholinergic (NANC) nerves release bronchoactive tachykinins such as substance P (SP) and neurokinin A (NKA) that can induce features of asthma. The airway response to NKA in humans closely resembles that of methacholine (M). Hence, we investigated the relationship between airway responsiveness to NKA and M in subjects with asthma. To this end, we analyzed baseline data of 27 subjects with mild persistent asthma (20F/7M) 19–46 y; FEV 1 81–136% pred.; PC 20FEV 1 (M)<80 μmol/mL) participating in a proof-of-concept study. All subjects were non-smokers and asthma was controlled by on demand short-acting β 2-agonists only. Dose-response curves to M (0.15–80 μmol/mL) and NKA (3.4 (10 −3)–0.88 μmol/mL) were performed on two separate days, and airway response was measured by FEV 1 until a ≥20% fall from baseline (PC 20FEV 1). Twenty-two subjects reached a PC 20FEV 1 on both occasions. The PC 20FEV 1 values of both agonists correlated significantly (Spearman's r=−0.721; p=0.0002), and the relationship was given by 10log(PC 20FEV 1(NKA))=−1.36+ (0.60× 10log(PC 20FEV 1(M)). We have demonstrated a significant relationship between airway responsiveness to NKA and methacholine in asthma. This suggests that both agonists may share common final pathways in causing bronchoconstriction in patients with mild persistent asthma. Based on our data and previous studies in asthma, it can be hypothesized that this direct NKA-induced bronchoconstrictor response may be mediated by predominant stimulation of the tachykinin NK-2 receptors on airway smooth muscle cells.

Electrodynamic smooth muscle sphincter

Many authors have suggested that the activity of the enteric inhibitory nerves is important in regulating normal gastrointestinal motility and inducing smooth muscle relaxation. Hitherto, no experimental or clinical models exist that transfer these physiological aspects to creating an autologous artificial sphincter for the treatment of major incontinence. Therefore, this study was performed to determine the contractile and relaxant capacity of gastrointestinal muscle types and to investigate the efficiency of a novel smooth muscle sphincter, based on the non-adrenergic, non-cholinergic (NANC) receptive relaxation under electrical field stimulation (EFS). For the first step, the isometric tension from isolated circular porcine fundus and colon muscle strips was recorded during pharmacological stimulation (TTX, L-NNA and atropine) and EFS. As a result, a continent electrodynamic smooth muscle sphincter (ESMS) was created by wrapping a fundus muscle flap around an isolated segment of porcine distal colon. The EFS of the free nerve fibers of the flap was realized using a circular platinum wire electrode. Parameters such as threshold of continence, intra/preluminal pressure and fluid passage were analyzed in a newly designed in vitro stoma simulator. Electrical field stimulation produced a maximal and voltage-dependent fundus relaxation to --12.4 mN/mm(2) (frequency of 40 Hz, pulse duration, train duration and voltage of 5 ms, 1 s and 60 mA respectively), which were abolished by N-nitro-L -arginine (L-NNA; 10(-4) M) in a dose-dependent manner, confirming that relaxant responses were mediated by NANC nerves. The results of eight ESMS showed that circular electrical stimulation of the muscle flap caused muscle relaxation with a concomitant and effective reduction in the occlusion pressure. The NANC-induced relaxation mechanism of porcine fundus preparations could be transferred to an efficient smooth muscle sphincter with a high threshold of continence and electrically controlled defecation.

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.

Functional evidence that ATP or a related purine is an inhibitory NANC neurotransmitter in the mouse jejunum: study on the identity of P2X and P2Y purinoceptors involved.

1. Conflicting views exist on whether ATP is a neurotransmitter in the enteric nervous system. We investigated the role of ATP in enteric transmission in circular muscle strips of the mouse jejunum. 2. On PGF2alpha-precontracted muscle strips and in the presence of atropine and guanethidine, electrical field stimulation (EFS, 1-8 Hz) of nonadrenergic noncholinergic (NANC) nerves induced transient relaxations that were abolished by the nerve-conductance blocker tetrodotoxin. The NO synthase blocker l-nitroarginine (l-NOARG) partially inhibited the NANC relaxations to EFS, but fast-twitch relaxations to EFS were still observed in the presence of l-NOARG. 3. In the presence of l-NOARG, ATP, the P2X receptor agonist alphabetaMeATP and the P2Y receptor agonist ADPbetaS relaxed jejunal muscle strips. Tetrodotoxin did not affect the relaxation to ATP and ADPbetaS, but inhibited that to alphabetaMeATP. 4. The l-NOARG-resistant NANC relaxations to EFS were almost abolished by apamin, a blocker of small-conductance Ca2+ activated K+ channels, and by suramin and PPADS, blockers of P2 purinoceptors. Relaxations to ATP were almost abolished by apamin and suramin but not affected by PPADS. 5. Desensitisation of alphabetaMeATP-sensitive P2X receptors, the P2X receptor blocker Evans blue and the P2X1,2,3 receptor blocker NF 279 inhibited the l-NOARG-resistant NANC relaxations to EFS and that to alphabetaMeATP without affecting the relaxation to ADPbetaS. Brilliant blue G, a P2X2,5,7 receptor blocker, did not affect the relaxations to EFS. 6. Desensitisation of P2Y receptors and MRS 2179, a P2Y1 receptor blocker, virtually abolished the l-NOARG-resistant NANC relaxations to EFS and the relaxation to ADPbetaS without affecting the relaxation to alphabetaMeATP. 7. Dipyridamole, an adenosine uptake inhibitor, or theophylline and 8-phenyltheophylline, blockers of P1 and A1 purinoceptors, respectively, did not affect the purinergic NANC relaxations to EFS. 8. Our results suggest that ATP or a related purine acts as an inhibitory NANC neurotransmitter in the mouse jejunum, activating P2 but not P1 purinoceptors. Relaxations to the purinergic NANC neurotransmitter mainly involve P2Y receptors of the P2Y1 subtype that are located postjunctionally. Purinergic NANC neurotransmission also involves P2X receptors, most likely of the P2X1 and P2X3 subtype, located pre- and/or postjunctionally.

Therapeutic potential of phosphodiesterase 5 inhibition for cardiovascular disease.

Several families of phosphodiesterases (PDE), the enzymes catalyzing hydrolysis of cyclic (c) nucleoside monophosphates, namely, 3′5′-cAMP (cAMP) and 3′5′-cGMP (cGMP), have been identified and characterized in recent years.1 Since selective pharmacological inhibitors of isoform 5 (a cGMP-specific PDE), such as sildenafil, tadalafil, or vardenafil, have become available, the physiological function and interaction of different PDE isoforms,2 their tissue distribution,2 and the therapeutic potential of PDE 5 inhibition have attracted increasing interest.3 The discovery in 1989 of sildenafil, a highly selective inhibitor of PDE 5, was the result of extensive research on chemical agents targeting PDE 5 that might potentially be useful in the treatment of coronary heart disease. Initial clinical studies on sildenafil in the early 1990s were not promising with respect to its antianginal potential. However, a remarkable side effect was reported by a number of volunteers participating in these investigations; sildenafil seemed to enhance penile erections, which soon thereafter became the main focus of further studies. PDE 5 is found in high concentration in smooth muscle cells of the corpora cavernosa.2 Relaxation of smooth muscle cells of penile arteries, arterioles, and sinusoids in response to sexual stimulation results in an increase in blood volume within the rigid tunica albuginea and compression of draining venules, and hence a penile erection. Relaxation of the arterial smooth muscles occurs after stimulation of the enzyme guanylate cyclase by nitric oxide released from nonadrenergic-noncholinergic nerves and endothelial cells, with subsequent formation of cGMP4 (Figure 1). cGMP activates a cGMP-dependent protein kinase, which leads to phosphorylation of ion channels with the final consequence of a reduced cytosolic calcium concentration.5 Thus, sildenafil inhibiting the breakdown of cGMP was found to be effective in a high percentage of male patients suffering from erectile dysfunction.3,6 Synthesis and breakdown of …

Prejunctional modulation of non-adrenergic non-cholinergic (NANC) inhibitory responses in the isolated guinea-pig gastric fundus.

The inhibitory neurotransmission of the stomach was investigated in isolated guinea-pig gastric fundus. In preparations treated with guanethidine (1 micro mol L-1) and p-fluoro-hexahydro-sila-difenidol (1 micro mol L-1), electrical stimulation evoked neurogenic inhibitory responses not modified by hexamethonium (100 micro mol L-1), suggesting that inhibitory postganglionic non-adrenergic non-cholinergic (NANC) nerve fibres are involved. The nitric oxide (NO)-synthase inhibitor Nomega-nitro-l-argininine-methyl-ester hydrochloride (1-100 micro mol L-1) and the soluble guanylyl cyclase inhibitor ODQ (0.1-3 micro mol L-1) also abolished such relaxant response, suggesting the involvement of NO/Cyclic Guanosine 3',5' monophosphate (cGMP) system as the final mechanism of muscle relaxation. The alpha2-adrenoceptor agonist, UK 14 304 (10 nmol L-1-10 micro mol L-1) did not influence the electrical field stimulation (EFS)-evoked NANC responses. These latter responses were also refractory to a variety of receptor agonists and antagonists, acting at Gamma Aminobutyric Acid (GABA), serotonin 5HT1a, opioid micro , delta and kappa, muscarinic M1 and M2, histamine H2 and H3 and cannabinoid receptors. The NANC response was insensitive to the P/Q-type Ca2+-channel blocker omega-agatoxin TK (1 nmol L-1-0.1 micro mol L-1), but partially inhibited by the N-type Ca2+-channel blocker omega-conotoxin GVIA (0.1 nmol L-1-0.1 micro mol L-1), and by the L-type Ca2+-channel blockers nifedipine and calcicludine (0.1 nmol L-1-0.1 micro mol L-1). These data suggest that the NANC relaxation of the isolated guinea-pig gastric fundus is mediated by NO as the final inhibitory (neuro)transmitter at the longitudinal smooth muscle cells. The mechanism(s) promoting NO production is/are Ca2+-dependent, but apparently insensitive to presynaptic modulation. Both N- and L-type channels seem to occur in nitrergic nerve endings, where they contribute to trigger NO diffusion at the synaptic cleft.

Mexiletine inhibits nonadrenergic noncholinergic lower oesophageal sphincter relaxation in rabbits

Nonadrenergic noncholinergic (NANC) nerves are known to be nitrergic and to have an important role in the regulation of gastrointestinal motility and function. Cardiac antiarrhythmic therapy in humans is accompanied by a high incidence of gastrointestinal side–effects. We investigated the effect of mexiletine, a class Ib antiarrhythmic drug, on NANC lower oesophageal sphincter relaxation. Mexiletine concentration dependently inhibited the NANC relaxation induced by 30 mM KCl (EC 50=4.4×10 −6 M); the production of 3′,5′-cyclic guanosine monophosphate (cGMP) after KCl stimulation was concentration dependently decreased. The relaxation induced by the exogenous nitric oxide (NO) donor, diethylamine NONOate (10 −5 M), was not inhibited by mexiletine, and the cGMP production after diethylamine NONOate application was not altered. Mexiletine did not alter the activity of NO synthase. These findings suggest that mexiletine inhibits NANC relaxation via NO-cGMP pathway modulation, possibly by inhibiting myenteric nitrergic neurotransmission in the lower oesophageal sphincter in rabbits.

Inhibitory effect of beta3-adrenoceptor agonist in lower esophageal sphincter smooth muscle: in vitro studies.

We investigated the effects of (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl] - amino]propyl] - 1,3 - benzodioxole - 2, 2 - dicarboxylate (CL 316243) (a typical beta3-agonist) on the spontaneously tonic smooth muscle of the lower esophageal sphincter (LES). Studies were carried out in smooth muscle strips and smooth muscle cells (SMCs) of opossum LES. Isometric tension was recorded in the basal state and after CL 316243, and before and after beta3-antagonist (S)-N-[4-[2-[[3-[-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L 748337) and nonselective antagonist propranolol. In some experiments, the effects of nonadrenergic noncholinergic (NANC) nerve activation by electrical field stimulation (EFS) were also examined. The effects of CL 316243 were compared with those of nonselective beta-agonist isoproterenol. CL 316243 caused a concentration-dependent relaxation of the LES smooth muscle. The relaxant action of CL 316243 was determined to be directly at the smooth muscle because it remained unmodified by the neurotoxin tetrodotoxin and other neurohumoral antagonists, and also was observed in the SMCs. L 748337 selectively antagonized the relaxant effect of CL 316243 and, conversely, had no significant effect on the inhibitory actions of isoproterenol. CL 316243 (1 x 10(-8) M) caused an augmentation of NANC relaxation in the LES. Another beta3-agonist, (S)-4-[hydroxy-3-phenoxy-propylamino-ethoxy]-N-(2-methoxyethyl)-phenoxyacetamide (ZD 7114), also caused concentration-dependent full relaxation of the LES that was selectively antagonized by beta3-anatagonist 3-(2-ethylphenoxy)-1-[(1S)1,2,3,4-tetrahydronaphth-1-ylaminol]-(2S)-2-propanol oxalate (SR 59230A). These studies defined the effects of characteristic inhibitory beta3-adrenoceptors in the spontaneously tonic LES smooth muscle and suggested a potential therapeutic role in the esophageal motility disorders characterized by hypertensive LES.

Morphological and functional characterization of a rat vaginal smooth muscle sphincter.

The aim of the present study was to gain information about adrenergic-, cholinergic- and non-adrenergic, non-cholinergic (NANC)- transmitter systems/mediators in the rat vagina, and to characterize its smooth muscles functionally. Tissue sections from vagina of Sprague Dawley rats were immunolabelled with antibodies against protein gene product 9.5 (PGP), synaptophysin (Syn), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Circularly cut vaginal smooth muscle preparations from the distal vagina were studied in organ baths. In the paravaginal tissue, a large number of PGP-, NOS-, TH-, VIP-immunoreactive (IR) and few CGRP-IR nerve trunks were observed, giving off branches to the smooth muscle wall. The smooth muscle wall was supplied by a large number of PGP-, Syn-, VAChT-, NPY-, NOS- and TH- IR nerve terminals, whilst only a moderate to few numbers of CGRP-, VIP- and PACAP-IR terminals were identified. Especially the distal part of the vaginal wall, where the circularly running smooth muscle was thickened into a distinct sphincter structure, was very richly innervated, predominantly by PGP- and NOS-IR terminals. Below and within the basal parts of the epithelium in the distal half of the vagina, a large number of PGP- and few NOS- and PACAP-IR varicose terminals were observed. The vaginal arteries were encircled by plexuses of nerve terminals. A large number of these were PGP-, Syn-, VAChT-, NOS-, TH-, NPY- and VIP-IR, and few were CGRP- and PACAP-IR. In isolated preparations of the distal vagina, electrical field stimulation (EFS) caused frequency-dependent contractions, which were reduced by sildenafil, tetrodotoxin (TTX) and phentolamine. In preparations contracted by norepinephrine (NA), EFS produced frequency-dependent relaxations. Pretreatment with the NOS-inhibitor N(G)-nitro-L-arginine, TTX, or the inhibitor of soluble guanylate cyclase, ODQ, abolished the EFS relaxations. In NE precontracted preparations, cumulative addition of sildenafil caused concentration-dependent relaxation. Carbachol contracted the strips concentration-dependently from baseline. It can be concluded that the distal part of the rat vagina forms a distinct smooth muscle sphincter, which is richly innervated by adrenergic, cholinergic and NANC nerves. The present studies suggest that in the rat the L-arginine/NO-system not only plays an important role in the regulation of vaginal smooth muscle tone, but also affects blood flow, and may have sensory functions.

Potassium channels and erectile dysfunction

The incidence of erectile dysfunction (ED), defined as the persistent inability to achieve or maintain an erection sufficient for satisfactory sexual performance, increases with age and with risk factors for vascular disease, including smoking, diabetes and hypertension. Penile erection results from an arousal-induced synthesis of nitric oxide (NO) in nonadrenergic-noncholinergic nerves (NANC), endothelial cells and cavernosal smooth muscle cells (SMCs). Vasodilation and relaxation of cavernosal SMCs engorges the corpora cavernosa with blood at arterial pressure. The subcellular mechanism by which tumescence occurs involves NO-induced activation of soluble guanylate cyclase, increased cyclic guanosine monophosphate (cGMP) levels and activation of cGMP-dependent protein kinase (PKG). PKG phosphorylates numerous ion channels and pumps, each promoting a reduction in cytosolic calcium. In particular, PKG activates high-conductance Ca2+(-)sensitive K+ (BKCa) channels, which hyperpolarize the arterial and cavernosal SMC membranes, causing relaxation. This mechanism appears to be compromised with age and with vascular disease, leading to ED. Thus, increasing cavernosal nitric oxide synthase (NOS) expression, cGMP levels and/or BKCa channel expression is an effective therapy for experimental ED. Future therapies may involve augmenting K+ channel expression by gene transfer or increasing channel function through the use of Type 5 phosphodiesterase (Type 5 PDE) inhibitors or phosphatase inhibitors.

Potassium channels and erectile dysfunction

The incidence of erectile dysfunction (ED), defined as the persistent inability to achieve or maintain an erection sufficient for satisfactory sexual performance, increases with age and with risk factors for vascular disease, including smoking, diabetes and hypertension. Penile erection results from an arousal-induced synthesis of nitric oxide (NO) in nonadrenergic–noncholinergic nerves (NANC), endothelial cells and cavernosal smooth muscle cells (SMCs). Vasodilatation and relaxation of cavernosal SMCs engorges the corpora cavernosa with blood at arterial pressure. The subcellular mechanism by which tumescence occurs involves NO-induced activation of soluble guanylate cyclase, increased cyclic guanosine monophosphate (cGMP) levels and activation of cGMP-dependent protein kinase (PKG). PKG phosphorylates numerous ion channels and pumps, each promoting a reduction in cytosolic calcium. In particular, PKG activates high-conductance Ca 2+-sensitive K + (BK Ca) channels, which hyperpolarize the arterial and cavernosal SMC membranes, causing relaxation. This mechanism appears to be compromised with age and with vascular disease, leading to ED. Thus, increasing cavernosal nitric oxide synthase (NOS) expression, cGMP levels and/or BK Ca channel expression is an effective therapy for experimental ED. Future therapies may involve augmenting K + channel expression by gene transfer or increasing channel function through the use of Type 5 phosphodiesterase (Type 5 PDE) inhibitors or phosphatase inhibitors.

Nitroxidergic (nitrergic) nerve and erectile dysfunction

In vascular tissues including the corpus cavernosum, the organ function is reciprocally regulated by noradrenergic and non-adrenergic, non-cholinergic (NANC) nerves. NANC nerves innervating the corpus cavernosum is thought to be nitroxidergic (nitrergic) nerves which liberate nitric oxide (NO) produced by neuronal NO synthase, and liberated NO activates soluble guanylate cyclase (sGC) in cavernous smooth muscle cells. Intracellular increase in cyclic (c) GMP by activation of sGC dilates cavernous smooth muscle and then induces penile erection. Nitroxidergic (nitrergic) vasodilator nerves also innervate cavernous arteries and veins which regulate the blood volume in the corpus cavernosum. The order of potency of nitroxidergic nerve functions in these tissues (cavernosum > artery >> vein) may be suitable for producing the erection. Therefore, obstruction of the arteries and impairment of nitroxidergic (nitrergic) nerve function are speculated to be one of the causes for erectile dysfunction (ED). On the other hand, NO derived from the cavernous endothelium may partly contribute to erectile function. Sildenafil (Viagra) is one of the potent therapeutics for ED. The agent is a selective phosphodiesterase type 5 (PDE-V) inhibitor that inhibits degradation of cGMP elevated by NO mainly derived from the nerves. To develop more selective and safer therapeutics for ED, further systematic investigations are required.