Effects of pro-inflammatory cytokines, lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability.

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1ß and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE2, PGF2α) or their corresponding ethanolamides (PGE2-EA or PGF2α-EA) over 48h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1ß and bacterial LPS incubations in an explant setting over 20h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10(-5)M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10(-4)M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE2 transiently increased Caco-2 monolayer permeability at 24h, while LPS (10µg/ml) increased permeability over 24-48h. These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE2 transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.

Interleukin 17A evoked mucosal damage is attenuated by cannabidiol and anandamide in a human colonic explant model.

Interleukin 17A (IL-17A) is a cytokine linked to inflammatory bowel disease. We investigated IL-17A expression in human colonic mucosa, whether IL-17A can elicit colonic mucosal damage in a human explant model and modulate gastrointestinal epithelial permeability in cell culture. We also tested if select cannabinoid ligands, shown to be protective in colitis models could attenuate damage caused by IL-17A. In addition, the ability of pro-inflammatory cytokines TNF-α and IL-1ß to modulate levels of IL-17A in the explant colitis model was also explored. IL-17A incubation caused significant mucosal epithelial and crypt damage which were attenuated following hydrocortisone treatment, and also reduced following anandamide or cannabidiol incubation. IL-17A-evoked mucosal damage was also associated with an increase in matrix metalloprotease activity. However, IL-17A did not induce any significant changes in epithelial permeability in confluent Caco-2 cell monolayers over a 48h incubation period. IL-17A was located predominantly in human mucosal epithelium together with IL-17C, but both IL-17A and IL-17C were also expressed in the lamina propria and submucosa. Incubation of human colonic mucosal tissue or Caco-2 cells with pro-inflammatory cytokines TNF-α and IL-1ß however did not alter IL-17A expression. These results indicate IL-17A has a widespread distribution in the human colon and the capacity to elicit mucosal damage which can be attenuated by cannabinoid ligands.

Cannabinoid CB2 receptor activation attenuates cytokine-evoked mucosal damage in a human colonic explant model without changing epithelial permeability.

Cannabinoid receptor activation is protective in animal colitis models. We sought to investigate if cannabinoids attenuated colitis-like tissue damage in human colonic specimens, with the hypothesis that cannabinoids would be protective in a cytokine-driven model of human colonic mucosal damage. Healthy human colonic mucosa was incubated with pro-inflammatory cytokines TNF-α and IL-1ß to elicit colitis-like tissue damage. The cytokine-driven increase in scored crypt and mucosal damage and lymphocyte density was attenuated with concomitant hydrocortisone pretreatment. The cannabinoid receptor 2 (CB2) receptor-selective agonist JWH-015 significantly reduced colitis scores following cytokine incubation, as evidenced by a reduction in mucosal crypt and luminal epithelial damage and lymphocyte density in the lamina propria. The effect of JWH-015 was reversed in the presence of the CB2 receptor inverse agonist JTE-907. Anandamide was also protective in the cytokine-incubated explant colitis model in a manner reversible with JTE-907, while CB1 receptor agonism with ACEA was without effect. TNF-α and IL-1ß together evoked an increase in paracellular epithelial permeability in Caco-2 cell monolayers over 48h of incubation. However, neither CB2 nor CB1 receptor activation altered the cytokine-evoked increase in permeability. These findings support a discrete role for CB2 receptors in the attenuation of detrimental pro-inflammatory cytokine-mediated mucosal damage in the human colon without directly affecting mucosal epithelial barrier function.

GABA(B)R expressed on vagal afferent neurones inhibit gastric mechanosensitivity in ferret proximal stomach.

GABA(B)-receptor (GABA(B)R) agonists reduce transient lower esophageal sphincter relaxation (TLESR) and reflux episodes through an action on vagal pathways. In this study, we determined whether GABA(B)R are expressed on vagal afferent neurones and whether they modulate distension-evoked discharge of vagal afferents in the isolated stomach. Vagal mehanoreceptor activity was recorded following distensions of the isolated ferret proximal stomach before and after perfusion with the GABA(B)R-selective agonists baclofen and 3-aminopropylphosphinic acid (3-APPiA). Retrograde labeling and immunohistochemistry were used to identify GABA(B)R located on vagal afferent neurones in the nodose ganglia. Vagal afferent fibers responded to isovolumetric gastric distension with an increase in discharge. The GABA(B)-receptor agonists baclofen (5 x 10(-5) M) and 3-APPiA (10(-6) to 10(-5) M) but not muscimol (GABA(A)-selective agonist: 1.3 x 10(-5) M) significantly decreased afferent distension-response curves. The effect of baclofen (5 x 10(-5) M) was reversed by the GABA(B)-receptor antagonist CGP 62349 (10(-5) M). Over 93% of retrogradely labeled gastric vagal afferents in the nodose ganglia expressed immunoreactivity for the GABA(B)R. GABA(B)R expressed on vagal afferent fibers directly inhibit gastric mechanosensory activity. This is likely a contributing mechanism to the efficacy of GABA(B)-receptor agonists in reducing TLESR and reflux episodes in vivo.

Vagal neurotransmission to the ferret lower oesophageal sphincter: inhibition via GABA(B) receptors.

GABA(B) receptors modulate the function of the lower oesophageal sphincter (LOS) in vivo by inhibiting neurotransmitter release in the vagal pathway controlling LOS relaxation. We aimed to determine whether this effect was mediated peripherally on vagal motor outflow to the ferret LOS in vitro. The LOS, with intact vagal innervation, was prepared from adult ferrets and LOS tension measured. Vagal stimulation (0.5 - 10 Hz, 30 V) evoked a tetrodotoxin-sensitive, frequency-dependent relaxation. Both GABA (3x10(-4) M) and (+/-)baclofen (2x10(-4) M) inhibited vagally-stimulated LOS relaxation. The potent GABA(B) receptor-selective agonist 3-APPA dose-dependently inhibited vagally-stimulated LOS relaxation, with an EC(50) value of 0.7 microM Decreased responses following vagal stimulation in the presence of (+/-)baclofen or 3-APPA were reversed with the potent GABA(B) receptor antagonist CGP 62349. Neither CGP 62349 nor muscimol (GABA(A) receptor agonist) alone affected LOS responses following vagal stimulation. Agonists of other G protein-coupled receptors (clonidine (alpha(2)-adrenoceptor) (5x10(-6) M), U50488 (kappa opioid) (10(-5) M), neuropeptide Y (10(-6) M)) did not affect vagally-mediated LOS relaxation. The present study supports a discrete presynaptic inhibitory role for GABA(B) receptors on vagal preganglionic fibres serving inhibitory motorneurones in the ferret LOS.

Triggering of transient LES relaxations in ferrets: role of sympathetic pathways and effects of baclofen.

Activation of gastric vagal mechanoreceptors by distention is thought to be the trigger for transient lower esophageal sphincter relaxations (TLESR), which lead to gastroesophageal reflux. The contribution of higher-threshold gastric splanchnic mechanoreceptors is uninvestigated. GABA(B) receptor agonists, including baclofen, potently reduce triggering of TLESR by low-level gastric distention. We aimed to determine first whether this effect of baclofen is maintained at high-level distention and second the role of splanchnic pathways in triggering TLESR. Micromanometric/pH studies in conscious ferrets showed that intragastric glucose infusion (25 ml) increased triggering of TLESR and reflux. Both were significantly reduced by baclofen (7 micromol/kg ip) (P < 0.05). When 40 ml of air was added to the glucose infusion, more TLESR occurred than with glucose alone (P < 0.01). These were also reduced by baclofen (P < 0.001). TLESR after glucose/air infusion were assessed before and after splanchnectomy (2-4, 9-11, and 23-25 days), which revealed no change. Baclofen inhibits TLESR after both low- and high-level gastric distention. Splanchnic pathways do not contribute to increased triggering of TLESR by high-level gastric distention.

GABA(B) receptor-mediated effects on vagal pathways to the lower oesophageal sphincter and heart.

GABA(B) receptors influencing vagal pathways to the lower oesophageal sphincter and heart were investigated. In urethane-anaesthetized ferrets, the GABA(B) agonist baclofen (7 micromol kg(-1) i.v.) increased basal lower oesophageal sphincter (LOS) pressure. This was reversed by antagonism with CGP35348 (100 micromol kg(-1) i.v.). Baclofen's effect was abolished by vagotomy, suggesting a central action, yet it was ineffective when given centrally (3 - 6 nmol i.c.v.). Peripheral vagal stimulation (10 Hz, 5 s duration) caused LOS inhibition, followed by excitation, then prolonged inhibition. Bradycardia was also evoked during stimulation. Bradycardia and LOS responses were abolished after chronic supranodose vagotomy, indicating that they were due to stimulation of vagal pre-ganglionic neurones, not antidromic stimulation of afferents. Baclofen (1 - 10 micromol kg(-1)) reduced bradycardia and enhanced LOS excitation, which was also seen in animals pretreated with atropine (400 microgram kg(-1) i.v.) and guanethidine (5 mg kg(-1) i.v.), but not in those pretreated with L-NAME (100 mg kg(-1) i.v.). Effects of baclofen (7 micromol kg(-1) i.v.) on vagal stimulation-induced LOS and cardiac responses were unchanged by the GABA(B) antagonists CGP35348 or CGP36742 (up to 112 micromol kg(-1) i.v.), but were reversed by CGP62349 (ED(50) 37 nmol kg(-1) i.v.) or CGP54626 (ED(50) 100 nmol kg(-1) i.v.). Responses of isolated LOS strips to electrical stimulation, capsaicin, NK-1, NK-2 and nicotinic receptor agonists were all unaffected by baclofen (

Neuromuscular function of the human lower oesophageal sphincter in reflux disease and Barrett's oesophagus.

BACKGROUND: Columnar lined (Barrett's) oesophagus is often considered a sequel to chronic severe reflux disease. Aberrant lower oesophageal sphincter (LOS) motility associated with Barrett's oesophagus includes reduced basal LOS pressures. The aim of this study was to characterise neuromuscular function of the LOS in normal (squamous cell carcinoma (SCC) with uninvolved LOS) and reflux affected (Barrett's) oesophagus in vitro. METHODS: Strips of LOS muscle were prepared at biopsy following oesophagectomy from 16 patients with SCC and seven patients with oesophageal adenocarcinoma and Barrett's oesophagus associated with a history of reflux disease. LOS smooth muscle responses were recorded in response to electrical field stimulation (EFS), potassium chloride (KCl), DMPP, isoprenaline, capsaicin, bethanechol, and tachykinins. RESULTS: Basal LOS tone and LOS relaxations in response to isoprenaline, EFS, and DMPP were not significantly altered in the Barrett's group. After tetrodotoxin pretreatment, responses to KCl and DMPP were significantly reduced in the SCC but not in Barrett's LOS. Maximal contraction in response to bethanechol was significantly decreased in Barrett's LOS while substance P and NK-2 receptor mediated contraction was unaltered. Capsaicin, NK-1, and NK-3 receptor agonists exerted negligible effects on LOS tone. CONCLUSIONS: LOS muscle strips from patients with reflux associated Barrett's oesophagus exhibit a reduction in cholinergic muscle contraction while retaining similar features of basal tone, responses to tachykinins, and inhibitory muscle and neural function. Enteric inhibitory neurones in LOS muscle strips from patients with reflux associated Barrett's oesophagus display resistance to axonal sodium channel blockade. No evidence for functional NK-1 or NK-3 receptors or capsaicin sensitive axon collateral reflexes was observed in the human LOS.

Vagal ganglionic and nonadrenergic noncholinergic neurotransmission to the ferret lower oesophageal sphincter.

In the present study we aimed to discretely characterise ganglionic and neuroeffector transmission to the ferret lower oesophageal sphincter (LOS) using a novel preparation of LOS muscle with intact vagal innervation in conjunction with isolated LOS muscle strips. In this way we could compare vagally mediated LOS relaxation with that of enteric inhibitory motorneurones which were directly stimulated. Preparations of LOS muscle, with or without attached vagus nerves, were dissected from adult ferrets and maintained under preload in organ baths, where LOS muscle developed spontaneous tone. LOS relaxations in response to vagal stimulation (0.5-5 Hz, 30 V) were recorded, alone and following pretreatment with tetrodotoxin (TTX), hexamethonium (Hex), Hex and atropine and NG-nitro-L-arginine (L-NNA). Direct activation of enteric inhibitory motorneurones was performed via electrical field stimulation (EFS). Vagal stimulation elicited frequency-dependent relaxations of the LOS that were abolished by tetrodotoxin (1 microM) and markedly reduced following L-NNA pretreatment (100 microM), but unaltered following pretreatment with the selective VIP or PACAP receptor antagonists VIP (10-28) or PACAP (6-38), respectively (each at 5 microM). The potent NOS inhibitor S-methyl-L-thiocitrulline (100 microM) inhibited LOS relaxation to the same degree at 5 Hz. Hex alone (500 microM) reduced maximal relaxation by 50%; in combination with atropine (2 microM), relaxation was almost abolished. In isolated LOS muscle strips, neither VIP (10-28) nor PACAP (6-38) altered EFS-induced relaxation. Taken together, these results suggest ganglionic neurotransmission to the ferret LOS occurs mainly through a combination of nicotinic and muscarinic receptors and utilises nitroxidergic enteric inhibitory motorneurones to relax the LOS. Moreover, LOS relaxation due to direct activation of inhibitory motorneurones also utilises primarily nitric oxide and other as yet undefined neurotransmitters. Neither VIP nor PACAP are involved in vagally mediated or direct enteric neuronally stimulated LOS relaxation in the ferret.

Oesophagitis-induced changes in capsaicin-sensitive tachykininergic pathways in the ferret lower oesophageal sphincter.

Prolonged oesophageal acidification may impair lower oesophageal sphincter (LOS) function in reflux disease. The aim of this study was to investigate aspects of altered LOS innervation in a model of oesophagitis. Oesophagitis was induced by acid (HCl, 0.15 M) and pepsin (0.1% w/v) infusions in anaesthetized ferrets. LOS muscle strip responses to the following stimuli were measured in vitro from control and acid/pepsin-treated ferrets: electrical field stimulation (EFS; 1-50 Hz), potassium chloride KCl; 20 mM), substance P, [beta-Ala8]-neurokinin A 4-10, [Sar9, Met (O2)11]-substance P (all 10(-10) to 10(-6) M) and capsaicin (10(-8) to 10(-6) M). LOS relaxation occurred in response to all stimuli except [beta-Ala8]-neurokinin A 4-10, which evoked contraction. In muscle strips from acid/pepsin-treated animals there were no differences in amplitude or sensitivity of relaxation following EFS, KCl or substance P vs controls. However, the inhibitory response to capsaicin was increased four-fold (10(-8) M; P < 0.05) and an increased sensitivity of the inhibitory response to [Sar9, Met (O2)11]-substance P occurred (pD2 = 8.64 +/- 0.12 acid/pepsin-treated vs 7.94 +/- 0.24 control, P < 0.05). We conclude that in acute oesophagitis, increased sensitivity of capsaicin-activated inhibitory pathways occurs in which activation of NK-1 receptors plays an integral role in the ferret LOS.

Activation of non-adrenergic non-cholinergic inhibitory pathways by endogenous and exogenous tachykinins in the ferret lower oesophageal sphincter.

Repeated oesophageal acidification causes lower oesophageal sphincter (LOS) relaxation in the anaesthetized ferret which is mediated by a peripheral neurokinin (NK-1) receptor mechanism. Our aim in this study was to characterize neural pathways in the LOS activated by capsaicin and tachykinin receptor agonists in vitro. Circular muscle strips of LOS (two per animal) from a total of 24 ferrets were maintained in organ baths. Electrical field stimulation (EFS, 50 V, 5-50 Hz) caused frequency-dependent LOS relaxation which was abolished by tetrodotoxin (TTX; 10(-6) M: P < 0.001) and reduced by N(G)-nitro-L-arginine (L-NNA; 10(-4) M: P < 0.01). Substance P and [Sar9, Met (O2)11]-substance P (selective NK-1 agonist) caused dose-dependent relaxation, while the NK-2 receptor agonist [beta-Ala8]-NKA 4-10 evoked excitation. Capsaicin (10(-6) M) caused relaxation and desensitization that was overcome by long recovery periods and substance P dosing (10(-8) M). After pretreatment with the NK-1 receptor antagonist CP 99994 (10(-7) M), substance P (10(-8) M; P < 0.001) and capsaicin (10(-6) M: P < 0.01)-induced relaxations were reduced. In the presence of TTX (10(-6) M), excitation resulted in response to substance P (10(-8) M; P < 0.05) and [Sar9, Met (O2)11]-substance P (10(-8) M; P < 0.001), while the response to [beta-Ala8]-NKA 4-10 (10(-7) M) was unaffected. In the presence of L-NNA (10(-4) M), substance P and [Sar9, Met (O2)11]-substance P-induced relaxations were reduced (10(-8) M; P < 0.01), while the response to [beta-Ala8]-NKA 4-10 (10(-7) M) was unaffected. These results show that functional coupling between capsaicin-sensitive sensory neurones and NANC inhibitory neural pathways occurs via NK-1 receptors in the ferret LOS. NK-2 (and some NK-1) receptors activate non-neural excitatory mechanisms. Substance P and NK-1 receptors coupling sensory and NANC inhibitory neurones may be important in the reflex control of LOS motility.

Functional tolerance to alpha-adrenergic receptor blockade in the spontaneously hypertensive rat highlights the multifunctional role of vascular angiotensin II in the development of hypertension.

Treatment of spontaneously hypertensive rats (SHR) with alpha-adrenoceptor antagonists failed to alter the development of hypertension in this animal model. However, agents such as captopril (CAP) and losartan (LOS) which interfere with the renin-angiotensin system effectively prevented the development of hypertension. When tolerance occurred in the presence of doxazosin (DOX) or phenoxybenzamine, there was an enhanced sensitivity to the blood pressure lowering influence of LOS. In the presence of CAP, at a dose that did not retard the development of blood pressure in the SHR, DOX treatment significantly offset the development of hypertension in this strain. These results suggest that a functional tolerance to agents that interfere with the sympathetic nervous system is mediated by the renin-angiotensin system. Angiotensin-converting enzyme inhibition was associated with a normalization of the enhanced contraction of the mesenteric vascular bed seen in preparations from the SHR and a suppression in the development of the vascular amplifier. The results suggest that the sympathetic nervous system is unable to maintain an elevated blood pressure in the SHR during interference with the functioning of the renin-angiotensin system. Conversely, under conditions of alpha-adrenoceptor blockade, angiotensin II can maintain an elevated blood pressure in the SHR.

Angiotensin II-mediated facilitation of sympathetic neurotransmission in the spontaneously hypertensive rat is not associated with neuronal uptake of the peptide.

Evidence suggests that angiotensin II (AII) can modulate neuroeffector responses in the vasculature of spontaneously hypertensive rats (SHR). Included in this modulation is an action of AII in facilitating release of neurotransmitter from sympathetic nerves by a mechanism involving prejunctional angiotensin receptors. In addition, AII may be a substrate for the carrier processes that operate within sympathetic nerves. Therefore, we examined the influence of AII on the responses to sympathetic nerve stimulation in the isolated perfused mesenteric vascular bed preparation and determined whether AII was incorporated into neuronal tissue in blood vessels. AII (10(-8) M) shifted the frequency-response curves to the left, and this shift was reversed with addition of the AII receptor type 1 (AT1) antagonist losartan (10(-6) M). AII uptake into mesenteric artery, thoracic aorta, kidney, and skeletal muscle was determined in tissues taken from SHR and normotensive Wistar-Kyoto rats (WKY). Additional tissues were taken from animals that had been subjected to chemical sympathectomy with 6-hydroxydopamine (6-OHDA). Although AII accumulation was evident in all tissues examined, in no case was this accumulation diminished by 6-OHDA treatment. Subsequent studies using segments of kidney and skeletal muscle demonstrated that a large proportion of AII accumulation was temperature sensitive and was also sensitive to the metabolic inhibitor 2-4-dinitrophenol (DNP 10(-3) M). The results confirm the role of AII in potentiating the responses to sympathetic nerve stimulation through a process involving AT1 receptors, but this process is not associated with neuronal accumulation of the peptide.