Non-adrenergic Non-cholinergic Conditions Research Articles

NNOS phosphorylatability Presages NANC Inhibition: Investigation with a Novel Phosphorylation-deficient nNOS Mutant Mouse

Phosphorylation of the C-terminal Akt/PKA consensus site activates endothelial NOS (eNOS) and decreases its calcium/calmodulin requirement, but no definite physiological role for phosphorylation of the homologous site in neuronal NOS (nNOS) has been described. Therefore, we created a knock-in mouse in which nNOS Serine-1412 (S1412) is mutated to non-phosphorylatable alanine (nNOSS1412A). We tested whether nNOSS1412A ileal rings have altered nitrergic inhibitory non-adrenergic, non-cholinergic (NANC) signaling in a physiological organ bath. Under NANC conditions, electrical field stimulation (EFS; 20V, 2msec, 1Hz, 30sec train) relaxed WT to a greater degree than nNOSS1412A ileum (73 ± 20% inhibition vs 31 ± 17%; N=3). Similarly, pharmacological treatment with forskolin (FSK) also relaxed WT more effectively than nNOSS1412A ileum (IC50 95% CI 35-44nM; N=42 vs 78-107nM; N=29). Application of selective nNOS (NANT) or pan-NOS antagonists (L-NAME) reduced FSK dependent relaxation identically in WT and nNOSS1412A ilea (IC50 ~150nM for both genotypes), confirming that a portion of FSK relaxation was due to nNOS activity. Akt and PKA are putative nNOS S1412 kinases. While there was no difference between WT and nNOSS1412A relaxation with FSK using the Akt inhibitor MK-2206, PKA inhibitors (H-89, Myr-PKI, and Rp-cAMPs) only decreased FSK relaxation in WT ileum (~3-fold increase in IC50) but not in nNOSS1412A ileum, suggesting a critical link between PKA and nNOS. Together, our data identify a novel NANC neuroinhibitory mechanism in which PKA phosphorylation of nNOS S1412 leads to calcium independent neuronal NO production and smooth muscle relaxation. This previously unrecognized NO physiology might be important in other cell types and organ systems as well.

Pre- and post-junctional bradykinin B2 receptors regulate smooth muscle tension to the pig intravesical ureter.

Neuronal and non-neuronal bradykinin (BK) receptors regulate the contractility of the bladder urine outflow region. The current study investigates the role of BK receptors in the regulation of the smooth muscle contractility of the pig intravesical ureter. Western blot and immunohistochemistry were used to show the expression of BK B1 and B2 receptors and myographs for isometric force recordings. B2 receptor expression was consistently detected in the intravesical ureter urothelium and smooth muscle layer, B1 expression was not detected where a strong B2 immunoreactivity was observed within nerve fibers among smooth muscle bundles. On ureteral strips basal tone, BK induced concentration-dependent contractions, were potently reduced by extracellular Ca(2+) removal and by B2 receptor and voltage-gated Ca(2+) (VOC) channel blockade. BK contraction did not change as a consequence of urothelium mechanical removal or cyclooxygenase and Rho-associated protein kinase inhibition. On 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2α (U46619)-precontracted samples, under non-adrenergic non-cholinergic (NANC) and nitric oxide (NO)-independent NANC conditions, electrical field stimulation-elicited frequency-dependent relaxations which were reduced by B2 receptor blockade. Kallidin, a B1 receptor agonist, failed to increase preparation basal tension or to induce relaxation on U46619-induced tone. The present results suggest that BK produces contraction of pig intravesical ureter via smooth muscle B2 receptors coupled to extracellular Ca(2+) entry mainly via VOC (L-type) channels. Facilitatory neuronal B2 receptors modulating NO-dependent or independent NANC inhibitory neurotransmission are also demonstrated.

Evidence that Nitric Oxide Is a Non-Adrenergic Non-Cholinergic Inhibitory Neurotransmitter in the Circular Muscle of the Mouse Distal Colon: A Study on the Mechanism of Nitric Oxide-Induced Relaxation

The gastrointestinal tract is composed of outer longitudinal muscle layers and inner circular muscle layers. Nitric oxide (NO), carbon monoxide (CO), and ATP play major roles as non-adrenergic non-cholinergic (NANC) inhibitory neurotransmitters in the longitudinal muscle of the mouse distal colon, whereas it is unclear which NANC inhibitory neurotransmitters are in its circular muscle. We investigated the electric field stimulation (EFS)-induced relaxations in the circular smooth muscle of the distal colon under NANC conditions. In the experiments in which N^ω-nitro-<smlcap>L</smlcap>-arginine, an inhibitor of NO synthase, was added, the EFS-induced relaxation decreased in a concentration-dependent manner and finally vanished. In contrast, CO, purinergic receptor ligands, and peptidergic substances do not play major roles as NANC neurotransmitters in the circular muscle of the mouse distal colon. ODQ, an inhibitor of soluble guanylate cyclase, strongly attenuated EFS-induced relaxation. Ryanodine, a Ca²⁺ release modulator at the sarcoplasmic reticulum, strongly attenuated EFS-induced relaxation as well. Relaxation induced by NOR-1, which generates NO, was inhibited by ODQ and ryanodine. Next, we performed experiments that simultaneously measured tension and the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt). NOR-1 decreased the tension and [Ca²⁺]_cyt levels in the circular muscle. ODQ and ryanodine strongly attenuated the NOR-1-induced change in both tension and [Ca²⁺]_cyt levels. In this study, we demonstrate that NO functions as a NANC inhibitory neurotransmitter in the circular muscle obtained from the mouse distal colon.

Accommodation and peristalsis are functional responses to obstruction in rat hypertrophic ileum

To investigate the effects of chronic obstruction on enteric reflexes evoked by electrical stimulation (EFS) or intraluminal distension of the rat hypertrophic ileum. Motor responses to EFS and to intraluminal distension were studied in the absence and in the presence of various inhibitors of enteric mediators. Ileum segments from operated (chronic ileal obstruction), sham-operated (control) and normal rats were horizontally mounted, connected to a pressure transducer and intraluminally perfused. The effects of selective serotonin receptor (5-HTR) blockers were investigated on distension-induced responses. The cellular localization of 5-HT3Rs was also examined in control and hypertrophic tissues through confocal microscopy. In non-obstructed segments, EFS elicited tetrodotoxin (TTX)-sensitive responses with high amplitude contraction followed by weak relaxation. In hypertrophic tissues, EFS lowered the baseline pressure and evoked TTX-sensitive contractions significantly larger than normal (P < 0.01) or control (P < 0.05), and devoid of any relaxation phase (P < 0.01 vs normal). Incubation with atropine and guanethidine [non-adrenergic non-cholinergic (NANC) conditions] did not modify intestinal tone in normal and control preparations, but reversed the accommodation produced by EFS in hypertrophic tissues, and depressed the amplitude of contractions in all types of tissues. L-NAME and α-chymotrypsin blocked residual NANC motility in all tissues and augmented intraluminal pressure in hypertrophic segments (P < 0.05 vs NANC conditions). Intraluminal distension of the intestinal wall evoked non-propulsive cycles of contractions and relaxations in non-obstructed tissues. In all hypertrophic segments, strong propulsive strokes, markedly wider (P < 0.001), and larger than normal (P < 0.001) or control (P < 0.05) were elicited. Both motor patterns were blocked under NANC conditions and with simultaneous incubation with L-NAME and α-chymotrypsin. In all types of tissues, incubation with ketanserin or GR125487 did not modify distension-induced motility. In contrast, blockade of 5-HT3Rs by ondansetron concentration-dependently inhibited motor responses in normal and control tissues, but only slightly impaired enteric reflexes in the hypertrophic preparations. Finally, confocal microscopy did not reveal a different cellular distribution of 5-HT3Rs in control and hypertrophic ileum. Accommodation and distension-induced peristalsis of rat hypertrophic ileum are controlled by cholinergic and peptidergic transmission and are negligibly affected by 5-HT3Rs, which modulate distension-induced motility in non-obstructed tissues.

Na+/Ca2+ Exchanger 1/2 Double-Heterozygote Knockout Mice Display Increased Nitric Oxide Component and Altered Colonic Motility

The Na+/Ca2+ exchanger (NCX) is a plasma membrane transporter involved in regulating intracellular Ca2+ concentrations. NCX is critical for Ca2+ regulation in cardiac muscle, vascular smooth muscle, and nerve fibers. To determine the role of NCX1 and NCX2 in gastrointestinal tissues, we examined electric field stimulation (EFS)-induced responses in the longitudinal smooth muscle of the distal colon in NCX1 and NCX2 double-heterozygote knockout mice (Double HET). We found that the amplitudes of EFS-induced relaxation that persisted during EFS were greater in Double HET than in wild-type mice (WT). Under the non-adrenergic, non-cholinergic (NANC) condition, EFS-induced relaxation in Double HET was similar in amplitude to that of WT. In the experiments in which l-NNA was added under NANC conditions following the EFS, the magnitudes of EFS-induced relaxation were smaller in Double HET than those in WT. In addition, an NCX inhibitor, SN-6, enhanced EFS-induced relaxation but did not affect EFS-induced relaxation under NANC condition, as in Double HET. Moreover, the magnitudes of relaxation induced by NOR-1, which generates NO, were greater in Double HET compared with WT. Similarly, SN-6 potentiated the magnitudes of NOR-1–induced relaxation. In this study, we demonstrate that NCX regulate colonic motility by altering the sensitivity of the inhibitory component.

Role of myosin Va in purinergic vesicular neurotransmission in the gut

We examined the hypothesis that myosin Va, by transporting purinergic vesicles to the varicosity membrane for exocytosis, plays a key role in purinergic vesicular neurotransmission. Studies were performed in wild-type (WT) and myosin Va-deficient dilute, brown, nonagouti (DBA) mice. Intracellular microelectrode recordings were made in mouse antral muscle strips. Purinergic inhibitory junction potential (pIJP) was recorded under nonadrenergic noncholinergic conditions after masking the nitrergic junction potentials. DBA mice showed reduced pIJP but normal hyperpolarizing response to P2Y1 receptor agonist MRS-2365. To investigate the mechanism of reduced purinergic transmission in DBA mice, studies were performed in isolated varicosities obtained from homogenates of whole gut tissues by ultracentrifugation and sucrose cushion purification. Purinergic varicosities were identified in tissue sections and in isolated varicosities by immunostaining for the vesicular ATP transporter, the solute carrier protein SLC17A9. The varicosities were similar in WT and DBA mice. Myosin Va was markedly reduced in DBA varicosities compared with the WT varicosities. Proximity ligation assay showed that myosin Va was closely associated with SLC17A9. Vesicular exoendocytosis was examined by FM1–43 staining of varicosities, which showed that exoendocytosis after KCl stimulation was impaired in DBA varicosities compared with WT varicosities. These studies show that SLC17A9 identifies ATP-containing purinergic varicosities. Myosin Va associates with SLC17A9-stained vesicles and possibly transports them to varicosity membrane for exocytosis. In myosin Va-deficient mice, purinergic inhibitory neurotransmission is impaired.

K V7 channels regulate muscle tone and nonadrenergic noncholinergic relaxation of the rat gastric fundus

Voltage-dependent type 7 K + (K V7) channels play important physiological roles in neurons and muscle cells. The aims of the present study were to investigate the motor effects of K V7 channel modulators in the rat gastric fundus and the expression of K V7 channels in this tissue. Muscle tone and electrical field stimulation (EFS)-evoked relaxations of precontracted longitudinal muscle strips of the rat gastric fundus were investigated under nonadrenergic noncholinergic conditions by organ bath studies. Gene expression was studied by real-time PCR and tissue localization of channels was investigated by immunohistochemistry. The K V7 channel blocker XE-991 induced concentration-dependent contractions, with mean pD 2 and E max of 5.4 and 48% of the maximal U46619-induced contraction, respectively. The K V7 channel activators retigabine and flupirtine concentration-dependently relaxed U46619-precontracted strips, with pD 2s of 4.7 and 4.4 and E max of 93% and 91% of the maximal relaxation induced by papaverine, respectively. XE-991 concentration-dependently inhibited retigabine-induced relaxation with a pIC 50 of 6.2. XE-991 and DMP-543, another K V7 channel blocker, increased by 13–25% or reduced by 11–21% the relaxations evoked by low- or high-frequency EFS, respectively. XE-991 also reduced the relaxation induced by vasoactive intestinal polypeptide (VIP) by 33% of controls. Transcripts encoded by all K V7 genes were detected in the fundus, with 7.4 and 7.5 showing the highest expression levels. K V7.4 and 7.5 channels were visualized by confocal immunofluorescence in both circular and longitudinal muscle layers. In conclusion, in the rat proximal stomach, K V7 channels appear to contribute to the resting muscle tone and to VIP- and high-frequency EFS-induced relaxation. K V7 channel activators could be useful relaxant agents of the gastric smooth muscle.

Voltage-gated calcium channels involved in the inhibitory motor responses and vasoactive intestinal polypeptide release in the rat gastric fundus

Ca 2+ inflow responsible for neurotransmitter release at most peripheral junctions is mainly mediated by activation of Ca V2.2 and Ca V2.1 channels. The aim of the present study was to characterize the voltage-gated Ca 2+ channels (VGCCs) responsible for the non-adrenergic non-cholinergic (NANC) relaxation and vasoactive intestinal polypeptide (VIP)-like immunoreactivity release in the rat gastric fundus. Precontracted longitudinal muscle strips of the rat gastric fundus were subjected to electrical field stimulation (EFS) under NANC conditions to evoke the relaxation and VIP-like immunoreactivity release. Nifedipine (1 µM) completely relaxed the preparations, so that its effects on EFS-induced NANC relaxations could not be investigated. ω-Conotoxin GVIA (0.3–100 nM) concentration-dependently reduced the amplitude of low frequency and the area under the curve (AUC) of high-frequency EFS-evoked relaxations (maximal reductions: approximately 55% and 42% of controls, respectively). The ω-conotoxin GVIA-resistant component of relaxation was not affected by ω-agatoxin IVA (300 nM), ω-conotoxin MVIIC (100 nM), SNX-482 (100 nM) or flunarizine (1 µM). ω-Conotoxin GVIA (30 nM), ω-agatoxin IVA (30 nM) and ω-conotoxin MVIIC (100 nM) reduced high-frequency EFS-evoked VIP-like immunoreactivity release by approximately 70%, 27% and 35% of controls, respectively. ω-Conotoxin GVIA (30 nM) plus ω-conotoxin MVIIC (100 nM) almost abolished the EFS-induced VIP-like immunoreactivity outflow. In the rat gastric fundus, the activation of Ca V2.2 and P-type of Ca V2.1 channels is responsible for the EFS-induced VIP-like immunoreactivity release. In contrast, Ca V1 channels, novel VGCCs and/or molecular variants of VGCCs cloned to date may mediate a substantial component of the NANC relaxation.

Selective stimulation of intrinsic excitatory and inhibitory motor pathways in porcine lower oesophageal sphincter

The mechanisms of stimulation of inhibitory and excitatory motor neurons (MNs) in the lower oesophageal sphincter (LOS) are not fully understood. The aim of this study was to assess the effect of selective stimulation of inhibitory and excitatory MNs in porcine LOS through nicotinic acetylcholine receptors (nAChRs), 5-HT(3) and P2X receptors. Circular LOS strips from adult pigs were studied in organ baths. We compared the effects of stimulation of MNs by electrical field stimulation (26 V, 0.3-20 Hz); nicotine (1-300 micromol L(-1)); 5-HT and 2-Me-5-HT (1 nmol(-1)-30 micromol L(-1)); and alpha,beta-methylene ATP (alpha,beta-meATP 1-100 micromol L(-1)); in standard Krebs solution; a non-adrenergic non-nitrergic non-purinergic (NANNNP) solution; and a non-adrenergic non-cholinergic (NANC) solution. Electrical stimulation of inhibitory MNs caused an intense LOS relaxation (-78.94 +/- 4.50% of LOS tone); and of excitatory MNs, a strong contraction (17.89 +/- 1.96 g). Nicotine 100 micromol L(-1) relaxed LOS (-84.67 +/- 3.98%) in standard Krebs solution, an effect reduced by Tetrodotoxin (TTX) 1 micromol L(-1). Nicotine induced a weak TTX-sensitive contraction (1.64 +/- 0.4 g) in NANNNP solution. 5-HT 10 micromol L(-1) and 2-Me-5-HT 30 micromol L(-1) contracted LOS in standard, NANC and NANNNP conditions, maximal responses (7.30 +/- 1.52 g, 3.50 +/- 0.18 g respectively) being reduced by TTX. alpha,beta-meATP 100 micromol L(-1) caused a LOS relaxation (-17.45 +/- 6.62%) unaffected by TTX in NANC solution, and a contraction (6.7 +/- 0.85 g) antagonized by TTX in NANNNP solution. Our results suggest selective mechanisms for stimulation of intrinsic excitatory and inhibitory motor pathways in porcine LOS. Inhibitory MNs are strongly stimulated by nAChRs and do not respond to stimulation of 5-HT(3) and P2X receptors. By contrast, excitatory MNs are stimulated through 5-HT(3) and P2X receptors, stimulation through nACRs being difficult and causing a weak response.

Cell‐free supernatants of Escherichia coli Nissle 1917 modulate human colonic motility: evidence from an in vitro organ bath study

Abstract Clinical studies have shown that probiotics influence gastrointestinal motility, e.g. Escherichia coli Nissle 1917 (EcN) (Mutaflor) proved to be at least as efficacious as lactulose and more potent than placebo in constipated patients. As the underlying mechanisms are not clarified, the effects of EcN culture supernatants on human colonic motility were assessed in vitro. Human colonic circular smooth muscle strips (n = 94, 17 patients) were isometrically examined in an organ bath and exposed to different concentrations of EcN supernatants. Contractility responses were recorded under (i) native conditions, (ii) electrical field stimulation (EFS), (iii) non-adrenergic non-cholinergic conditions, and (iv) enteric nerve blockade by tetrodotoxin (TTX). As concentrations of acetic acid were increased in EcN supernatants, contractility responses to acetic acid were additionally tested. EcN supernatants significantly increased the maximal tension forces both at low and high concentrations. Neither blockade of both adrenergic and cholinergic nerves nor application of TTX abolished these effects. EFS-induced contractility responses were not altered after exposure to EcN supernatants. Acetic acid elicited effects comparable to EcN supernatants only under TTX conditions. EcN supernatants modulate in vitro contractility of the human colon. As neither partial nor TTX blockade of enteric nerves abolished these effects, EcN supernatants appear to enhance colonic contractility by direct stimulation of smooth muscle cells. Active metabolites may include other substances than acetic acid, as acetic acid only partially resembled the effects elicited by EcN supernatants. The data provide a rationale for therapeutical application of probiotics in gastrointestinal motility disorders.

Milrinone and theophylline act as lower oesophageal sphincter relaxing agents: a comparative pharmacodynamic study in the rabbit

This study demonstrates that the inotropic agent milrinone and the bronchodilator drug theophylline exert a relaxing effect on the rabbit lower oesophageal sphincter in vitro. The relaxing effect of milrinone and theophylline, which is concentration-dependent, involves a second messenger 3',5'-cyclic adenosine monophosphate pathway and most probably it is accomplished through inhibition of phosphodiesterase (PDE) type III, as according to the obtained results it is not significantly modified either by nicotinic acid, an inhibitor of adenylate cyclase, or by the inhibitor of nitric oxide-synthetase N(omega)-nitro-L-arginine methylester and the purinergic antagonist suramin; moreover, it persists under non-adrenergic non-cholinergic conditions and it is both hexamethonium- and tetrodotoxin-insensitive. Both milrinone and theophylline display equal efficacy, comparable to that of the calcium blocker verapamil and the non-selective PDE inhibitor papaverine, but milrinone appears 50 times more potent than theophylline and three times less potent than verapamil, as, according to the pIC(50) values the potency rank of order is found to be verapamil (5.56) > milrinone (5.12) > theophylline (3.42). The here obtained pharmacodynamic profiles of the drugs suggest that both milrinone and theophylline may be considered as potent relaxing agents of the lower oesophageal sphincter.

Involvement of CB1 and CB2 receptors in the modulation of cholinergic neurotransmission in mouse gastric preparations

While most of the studies concerning the role of cannabinoids on gastric motility have focused the attention on the gastric emptying in in vivo animal models, there is little information about the cannabinoid peripheral influence in the stomach. In addition, the functional features of CB2 receptors in the gastrointestinal tract have been poorly characterized. The purpose of the present study was to investigate the effects of cannabinoid drugs on the excitatory cholinergic and inhibitory non-adrenergic non-cholinergic (NANC) neurotransmission in mouse isolated gastric preparations. Intraluminal pressure from isolated whole stomach was recorded and mechanical responses induced by electrical field stimulation (EFS) were analyzed in different experimental conditions. EFS (0.5 ms duration, supramaximal voltage, in trains of 5 s, 2–16 Hz) caused a cholinergic contraction, which was abolished by atropine or tetrodotoxin (TTX). The cannabinoid receptor agonist, WIN 55,212-2, the endogenous ligand, anandamide, the selective CB1 receptor agonist ACEA, and the selective CB2 receptor agonists, JWH015 and JWH133, produced a concentration-dependent reduction of the EFS-evoked cholinergic contractions. SR141716A, CB1 receptor antagonist, significantly attenuated the inhibitory effects induced by WIN 55,212-2, anandamide or ACEA, without affecting those caused by JWH133. AM630, CB2 receptor antagonist, reduced the inhibitory effects induced by WIN 55,212-2, anandamide, JWH015 or JWH133, without affecting those caused by ACEA. The joint application of SR141716A and AM630 was able of fully preventing the WIN 55,212-2 and anandamide actions. The cannabinoid antagonists failed per se to affect the neurally evoked responses. Cannabinoids did not modify the contractions produced by exogenous carbachol. In the presence of atropine and guanethidine (NANC conditions) EFS-induced TTX-sensitive relaxation consisting in an early and rapid component followed by a second slow phase, which were unaffected by cannabinoid drugs. In conclusion, the present results suggest that cannabinoids play a prejunctional modulatory role on the cholinergic excitatory transmission without affecting the NANC inhibitory transmission. In addition, this study provides experimental evidence that also the activation of CB2 receptors is able to reduce cholinergic neurotransmission in the mouse stomach.

Nitrergic and purinergic interplay in inhibitory transmission in rat gastric fundus

1 This study was undertaken to analyse the involvement of ATP in non-adrenergic non- cholinergic (NANC) relaxation and possible interplay between nitrergic and purinergic systems in rat gastric fundus. 2 Experiments were performed in vitro on strips of longitudinal muscle from rat gastric fundus, recording the mechanical activity as changes in isometric force. In addition, NO release induced by different experimental conditions was assayed. 3 Under NANC conditions in serotonin-precontracted strips, electrical field stimulation (EFS) elicited a tetrodotoxin (TTX)-sensitive relaxation accompanied by nitric oxide (NO) release. This effect was antagonized by pretreatment with the NO synthase antagonist Nomega-nitro-L-arginine (L-NA) or by desensitization of purinergic receptors. Purinergic desensitization was also able to further antagonize the residual EFS-induced relaxation remaining after L-NA treatment. Exogenously applied NO [delivered as sodium nitroprusside (SNP)] or ATP (and related purines) induced concentration-dependent, TTX-insensitive relaxant responses. ATP also induced the release of NO. A reduction in the responses to ATP was observed in the presence of L-NA. In contrast, SNP-induced relaxation remained unchanged after desensitization of purinergic receptors. Finally, apamin, a blocker of the small conductance Ca2+ -dependent K+ channels, reduced the amplitude of the muscular relaxation evoked by either EFS, ATP or SNP. 4 In conclusion, this study provides evidence that in rat gastric fundus, ATP is one of the inhibitory transmitters released from NANC intramural neurones acting directly on the muscle, through receptors coupled to apamin-sensitive Ca2+ -dependent K+ channels and, indirectly, through the stimulation of NO production.

Evidence for a modulatory role of cannabinoids on the excitatory NANC neurotransmission in mouse colon

It is well accepted that endogenous cannabinoids and CB1 receptors are involved in the regulation of smooth muscle contractility and intestinal motility, through a mechanism mainly related to reduction of acetylcholine release from cholinergic nerve endings. Because, few data exist on a possible modulatory action of the cannabinoid agents on the non-adrenergic non-cholinergic (NANC) excitatory and inhibitory neurotransmission, the aim of the present study was to investigate the effects of cannabinoid drugs on the NANC responses elicited by electrical field stimulation (EFS) in the circular muscle of mouse proximal colon. Colonic contractions were monitored as changes in endoluminal pressure. In NANC conditions, EFS evoked TTX-sensitive responses, characterized by a relaxation, nitrergic in origin, followed by a contraction. The EFS-evoked contraction was significantly reduced by SR48968, NK2 receptor antagonist, and abolished by co-administration of SR48968 and SR140333, NK1 receptor antagonist, suggesting that it was due to release of tachykinins. The cannabinoid receptor synthetic agonist, WIN55,212-2, the putative endogenous ligand, anandamide, the selective CB1 receptor agonist ACEA, but not the selective CB2 receptor agonist JWH-015, produced a concentration-dependent reduction of the NANC contractile responses, without affecting the NANC relaxation. ACEA or anandamide did not modify the contractions induced by exogenous [β-Ala 8]-NKA(4–10), agonist of NK2 receptors. The selective antagonist of CB1 receptors, SR141716A, per se failed to affect the EFS-evoked responses, but antagonized the inhibitory effects of WIN55,212-2, anandamide and ACEA on NANC contractile responses. AM630, CB2 receptor antagonist, did not modify the inhibitory effects of WIN55,212-2 or anandamide. URB597, inhibitor of the fatty acid amide hydrolase, enzyme which catalyze the hydrolysis of anandamide, was without any effect on the NANC evoked responses. We conclude that the activation of prejunctional CB1 receptors produces inhibition of NANC contractile responses in mouse colonic preparations. However, endogenous ligands do not seem to modulate tonically the NANC transmission in mouse colon.

Caveolin‐1 gene knockout impairs nitrergic function in mouse small intestine

Caveolin-1 is a plasma membrane-associated protein that is responsible for caveolae formation. It plays an important role in the regulation of the function of different signaling molecules, among which are the different isoforms of nitric oxide synthase (NOS). Nitric oxide (NO) is known to be an important inhibitory mediator in the mouse gut. Caveolin-1 knockout mice (Cav1(-/-)) were used to examine the effect of caveolin-1 absence on the NO function in the mouse small intestine (ileum and jejunum) compared to their genetic controls and BALB/c controls. Immunohistochemical staining showed loss of caveolin-1 and NOS in the jejunal smooth muscles and myenteric plexus interstitial cells of Cajal (ICC) of Cav1(-/-) mice; however, nNOS immunoreactive nerves were still present in myenteric ganglia. Under nonadrenergic noncholinergic (NANC) conditions, small intestinal tissues from Cav1(-/-) mice relaxed to electrical field stimulation (EFS), as did tissues from control mice. Relaxation of tissues from control mice was markedly reduced by N-omega-nitro-L-arginine (10(-4) M), but relaxation of Cav1(-/-) animals was affected much less. Also, Cav1(-/-) mice tissues showed reduced relaxation responses to sodium nitroprusside (100 microM) compared to controls; yet there were no significant differences in the relaxation responses to 8-bromoguanosine-3': 5'-cyclic monophosphate (100 microM). Apamin (10(-6) M) significantly reduced relaxations to EFS in NANC conditions in Cav1(-/-) mice, but not in controls. The data from this study suggest that caveolin-1 gene knockout causes alterations in the smooth muscles and the ICC, leading to an impaired NO function in the mouse small intestine that could possibly be compensated by apamin-sensitive inhibitory mediators.

Evidence for an apamin‐sensitive, but not purinergic, component in the nonadrenergic noncholinergic relaxation of the rat gastric fundus

The involvement of adenosine triphosphate (ATP) and carbon monoxide (CO) in the non-nitrergic nonpeptidergic component of high-frequency electrical field stimulation (EFS)-induced nonadrenergic noncholinergic (NANC) relaxation of longitudinal muscle strips from the rat gastric fundus was investigated. Under NANC conditions (1 microM atropine + 5 microM guanethidine), N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mM) slightly reduced the amplitude, but did not affect the area under the curve (AUC) of EFS (13 Hz, 2 min)-induced relaxation of 9,11-dideoxy-9alpha,11alpha-methanoepoxy prostaglandin F(2alpha) (U46619, 0.1 microM)-precontracted strips. With L-NAME (1 mM) plus alpha-chymotrypsin (1 U ml(-1)), the amplitude and the AUC of relaxation were reduced to approximately two-third and one-third of controls, respectively. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (100 microM), apamin (0.3 microM), desensitization to ATP, suramin (100 microM), zinc protoporphyrin IX (300 microM) or ferrous haemoglobin (100 microM) did not inhibit the component of relaxation resistant to L-NAME plus alpha-chymotrypsin. L-NAME (1 mM) plus anti-vasoactive intestinal peptide (VIP) serum (1 : 100) reduced the amplitude and the AUC of relaxation to a similar extent as L-NAME (1 mM) plus alpha-chymotrypsin (1 U ml(-1)). Adding apamin (0.1 microM) to L-NAME (1 mM) plus anti-VIP serum (1 : 100) further reduced the amplitude and the AUC of relaxation. These findings suggest that the non-nitrergic nonpeptidergic component of NANC relaxation of the rat gastric fundus induced by high-frequency stimulation is mediated by a neurotransmitter that acts through apamin-sensitive mechanisms, that is neither ATP nor CO.

The effects of selective phosphodiesterase III and V inhibitors on adrenergic and non-adrenergic, non-cholinergic relaxation responses of guinea-pig pulmonary arteries.

1. The aim of the present study was to investigate the role of several possible neurotransmitters in mediating non-adrenergic, non-cholinergic (NANC) relaxation, and the effects of phosphodiesterase (PDE) III and V inhibitors on adrenergic and NANC relaxation in branch pulmonary artery (PA) of guinea-pig. 2. Under the NANC conditions, electrical field stimulation (EFS, 60 V, 0.2 ms, 20 Hz) induced a tetrodotoxin-sensitive relaxation of the histamine-precontracted PA rings. The nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 10(-4) m) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10(-5) m) partially inhibited the EFS-induced relaxation. The inhibitory effect of l-NAME was reversed completely by l-arginine (10(-3) m), but not d-arginine (10(-3) m). 3. This NANC relaxation was attenuated by 8-phenyltheophylline (10(-5) m), a P1-purinoceptor antagonist. 4. The NANC response was potentiated by 10-6 m zaprinast, a type V PDE inhibitor, but was unaffected by 3 x 10-6 m milrinone, a type III PDE inhibitor. 5. Sodium nitroprusside (SNP) caused a concentration-dependent vasodilator effect which was potentiated by zaprinast, but unaffected by milrinone. Moreover, the effect of combination of zaprinast with milrinone was not significantly different from that observed with zaprinast alone. 6. Isoprenaline produced a concentration-dependent vasodilatation in branch PA of guinea-pig which was potentiated by both zaprinast and milrinone, the efficacy of milrinone being greater than zaprinast. 7. These results suggest that both nitrergic and purinergic pathways are involved in mediating the NANC relaxation in branch PA of guinea-pig. The combination of PDE III or V inhibitors with vasorelaxant drugs may be a hopeful approach for the treatment of pulmonary hypertension.

Interaction of the Growth Hormone-Releasing Peptides Ghrelin and Growth Hormone-Releasing Peptide-6 with the Motilin Receptor in the Rabbit Gastric Antrum

The structural relationship between the motilin and the growth hormone secretagogue receptor (GHS-R), and between their respective ligands, motilin and ghrelin, prompted us to investigate whether ghrelin and the GHS-R agonist growth hormone-releasing peptide-6 (GHRP-6), could interact with the motilin receptor. The interaction was evaluated in the rabbit gastric antrum with binding studies on membrane preparations and with contraction studies on muscle strips in the presence of selective antagonists under conditions of electrical field stimulation (EFS) or not. Binding studies indicated that the affinity (pK(d)) for the motilin receptor was in the order of ghrelin (4.23 +/- 0.07) < GHRP-6 (5.54 +/- 0.08) < motilin (9.13 +/- 0.03). The interaction of ghrelin with the motilin receptor requires the octanoyl group. Motilin induced smooth muscle contractile responses but ghrelin and GHRP-6 were ineffective. EFS elicited on- and off-responses that were increased by motilin already at 10(-9) M, but not by 10(-5) M ghrelin. In contrast, GHRP-6 also enhanced the on- and off-responses. The motilin antagonist Phe-cyclo[Lys-Tyr(3-tBu)-betaAla-] trifluoroacetate (GM-109) blocked the effect of GHRP-6 on the off-responses but not on the on-responses. Under nonadrenergic noncholinergic conditions, the effects of motilin and GHRP-6 on the on-responses were abolished; those on the off-responses were preserved. All responses were blocked by neurokinin (NK)(1) and NK(2) antagonists. In conclusion, ghrelin is unable to induce contractions via the motilin receptor. However, GHRP-6 enhances neural contractile responses, partially via interaction with the motilin receptor on noncholinergic nerves with tachykinins as mediator, and partially via another receptor that may be a GHS-R subtype on cholinergic nerves that corelease tachykinins.

Nitric oxide induces muscular relaxation via cyclic GMP-dependent and -independent mechanisms in the longitudinal muscle of the mouse duodenum

The aim of this study was to investigate, in mouse duodenum, the role of nitric oxide (NO) in the relaxation of longitudinal muscle evoked by nerve activation and the coupled action mechanism. Electrical field stimulation (EFS; 0.5 ms, 10-s train duration, supramaximal voltage, at various frequencies) under nonadrenergic noncholinergic conditions evoked muscular relaxation occasionally followed, at the higher stimulus frequencies, by rebound contractions. Inhibition of the synthesis of NO by N ω -nitro- l-arginine methyl ester ( l-NAME; 100 μM) virtually abolished the evoked relaxation. The relaxation was reduced also by apamin (0.1 μM) and by 1H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ; 1 μM), a guanylyl cyclase inhibitor. The coadministration of apamin and ODQ produced additive effects on the responses to EFS. Sodium nitroprusside (0.1–100 μM) produced a concentration-dependent reduction of the phasic spontaneous activity and at the highest dose used suppressed phasic activity and induced muscular relaxation. These effects were tetrodotoxin and l-NAME resistant and were antagonized both by apamin and by ODQ. 8-Bromoguanosine 3 ′,5 ′-cyclic monophosphate (0.1–100 μM) reduced in a concentration-dependent manner the spontaneous mechanical activity and at 100 μM suppressed the phasic activity and induced muscular relaxation, not antagonized by apamin. This study indicates that NO is the primary transmitter released by inhibitory nerves supplying the longitudinal muscle of mouse duodenum and that guanylate cyclase stimulation and opening of Ca 2+-dependent K + channels are independent mechanisms working in parallel to mediate NO action.

Involvement of peptide histidine isoleucine in non-adrenergic non-cholinergic relaxation of the rat gastric fundus induced by high-frequency neuronal firing.

The role of peptide histidine isoleucine (PHI) as a neurotransmitter of the inhibitory motor neurones, the physiological role of PHI and vasoactive intestinal polypeptide (VIP) in the non-adrenergic non-cholinergic (NANC) relaxation and the relative amounts of VIP- and PHI-like immunoreactivity (LI) co-released by neuronal activation were investigated in the rat gastric fundus. Longitudinal muscle strips from the rat gastric fundus precontracted by the thromboxane receptor agonist U46619 (0.1 micro M) were studied in organ baths under conditions of muscarinic receptor blockade by atropine (1 micro M) and adrenergic neurone blockade by guanethidine (5 micro M) ("NANC conditions"). Concentration-response curves were plotted for both amplitude and area under the curve (AUC) of the relaxant responses induced by VIP (0.3 nM-0.3 micro M), PHI (0.3 nM-1 micro M) and peptide histidine valine [PHV(1-42); 0.3 nM-1 micro M]. All three peptides were more potent in the curve based on amplitude than in that based on the AUC of relaxation. In addition, VIP was 5.3 and 7 times more potent than PHI and PHV(1-42), respectively, in producing relaxation expressed as amplitude, and 2.7 and 2.8 times, respectively, in producing relaxation expressed as AUC. PHI and PHV(1-42) behaved as partial agonists with respect to VIP in producing relaxation expressed as AUC. Electrical field stimulation (EFS; 120 mA, 1 ms, 4-32 Hz, pulse trains of 2 min) evoked frequency-dependent relaxant responses. Alpha-chymotrypsin (1 u/ml or 3 u/ml), an anti-VIP serum (1:100 or 1:50) and an anti-PHI serum (1:25), slightly reduced the amplitude, but greatly inhibited the AUC of the NANC relaxation induced by EFS (13 Hz) [approximately 72%, 47% and 28% less than that seen in time controls or with normal rabbit serum (1:100 or 1:25), respectively]. EFS (8-32 Hz) caused significant, frequency-dependent increases in the outflow of VIP- and PHI-LI from the strips. The EFS-induced release of VIP-LI was approximately 20% of the PHI-LI release. These findings indicate that PHI is involved in EFS-induced NANC relaxation of the rat gastric fundus, the major physiological role of VIP and PHI is the maintenance of smooth muscle relaxation and VIP is co-released in equimolar amounts mainly with PHI.