Scott D. Smid

Cannabinoid 1 (CB1) receptors coupled to cholinergic motorneurones inhibit neurogenic circular muscle contractility in the human colon.

1 The effects of cannabinoid subtype 1 (CB1) receptor activation were determined on smooth muscle, inhibitory and excitatory motorneuronal function in strips of human colonic longitudinal muscle (LM) and circular muscle (CM) in vitro. 2 Electrical field stimulation (EFS; 0.5–20 Hz, 50 V) evoked a relaxation in LM and CM precontracted with a neurokinin‐2 (NK‐2) selective receptor agonist (β‐ala8‐neurokinin A; 10−6 M) in the presence of atropine (10−6 M); this was unaltered following pretreatment with the CB1‐receptor selective agonist arachidonyl‐2‐chloroethylamide (ACEA; 10−6 M). 3 In the presence of nitric oxide synthase blockade with N‐nitro‐L‐arginine (10−4 M), EFS evoked a frequency‐dependent ‘on‐contraction’ during stimulation and an ‘off‐contraction’ following stimulus cessation. On‐contractions were significantly inhibited in CM strips by pretreatment with ACEA (10−6 M). These inhibitory effects were reversed in the presence of the CB1 receptor‐selective antagonist N‐(piperidine‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide (10−7 M). 4 ACEA did not alter LM or CM contractile responses to acetylcholine or NK‐2 receptor‐evoked contraction. 5 Immunohistochemical studies revealed a colocalisation of CB1 receptors to cholinergic neurones in the human colon based on colabelling with choline acetyltransferase, in addition to CB1 receptor labelling in unidentified structures in the CM. 6 In conclusion, activation of CB1 receptors coupled to cholinergic motorneurones selectively and reversibly inhibits excitatory nerve transmission in colonic human colonic CM. These results provide evidence of a direct role for cannabinoids in the modulation of motor activity in the human colon by coupling to cholinergic motorneurones.

GABAB receptor‐mediated effects on vagal pathways to the lower oesophageal sphincter and heart

GABAB receptors influencing vagal pathways to the lower oesophageal sphincter and heart were investigated. In urethane‐anaesthetized ferrets, the GABAB agonist baclofen (7 μmol kg−1 i.v.) increased basal lower oesophageal sphincter (LOS) pressure. This was reversed by antagonism with CGP35348 (100 μmol kg−1 i.v.). Baclofens 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 μmol kg−1) reduced bradycardia and enhanced LOS excitation, which was also seen in animals pretreated with atropine (400 μg 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 μmol kg−1 i.v.) on vagal stimulation‐induced LOS and cardiac responses were unchanged by the GABAB antagonists CGP35348 or CGP36742 (up to 112 μmol kg−1 i.v.), but were reversed by CGP62349 (ED50 37 nmol kg−1 i.v.) or CGP54626 (ED50 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 (200 μ M). We conclude that baclofen reduces vagal output at two peripheral sites: one presynaptically on pre‐ganglionic neurones (CGP35348‐insensitive), and another (CGP35348‐sensitive) that could not be identified. This demonstrates heterogeneity of GABAB receptors through differential sensitivity to antagonists.

Dietary polyphenol-derived protection against neurotoxic β-amyloid protein: from molecular to clinical.

Polyphenolic compounds derived mainly from plant products have demonstrated neuroprotective properties in a number of experimental settings. Such protective effects have often been ascribed to antioxidant capacity, but specific augmentation of other cellular defences and direct interactions with neurotoxic proteins have also been demonstrated. With an emphasis on neurodegenerative conditions, such as Alzheimers disease, we highlight recent findings on the neuroprotection ascribed to bioactive polyphenols capable of directly interfering with the Alzheimers disease hallmark toxic β-amyloid protein (Aβ), thereby inhibiting fibril and aggregate formation. This includes compounds such as the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) and the phytoalexin resveratrol. Targeted studies on the biomolecular interactions between dietary polyphenolics and Aβ have not only improved our understanding of the pathogenic role of β-amyloid, but also offer fundamentally novel treatment options for Alzheimers disease and potentially other amyloidoses.

Vagal neurotransmission to the ferret lower oesophageal sphincter: inhibition via GABAB receptors

GABAB 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 (3×10−4 M) and (±)baclofen (2×10−4 M) inhibited vagally‐stimulated LOS relaxation. The potent GABAB receptor‐selective agonist 3‐APPA dose‐dependently inhibited vagally‐stimulated LOS relaxation, with an EC50 value of 0.7 μM Decreased responses following vagal stimulation in the presence of (±)baclofen or 3‐APPA were reversed with the potent GABAB receptor antagonist CGP 62349. Neither CGP 62349 nor muscimol (GABAA receptor agonist) alone affected LOS responses following vagal stimulation. Agonists of other G protein‐coupled receptors (clonidine (α2‐adrenoceptor) (5×10−6 M), U50488 (κ 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 GABAB receptors on vagal preganglionic fibres serving inhibitory motorneurones in the ferret LOS.

Impaired capsaicin and neurokinin-evoked colonic motility in inflammatory bowel disease

Background:  Inflammatory bowel disease (IBD) is associated with altered sensory and motor function in the human colon. The aim of the present study was to compare neuromuscular function in normal and IBD‐affected colon in vitro, with emphasis on inhibitory enteric nerves, sensory neuropeptides and stimulation of axon collaterals.

Cannabinoid effects on β amyloid fibril and aggregate formation, neuronal and microglial-activated neurotoxicity in vitro.

Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands against β amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter β amyloid (Aβ) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aβ1–42 directly or microglial (BV-2 cells) conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ9-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55 ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aβ1–42. Aβ1–42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aβ-evoked SH-SY5Y cell viability, whereas JWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aβ fibrils and aggregates, there was no clear correlation between effects on Aβ morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.

Açaí (Euterpe oleraceae Mart.) berry extract exerts neuroprotective effects against β-amyloid exposure in vitro

The native South American palm açaí berry (Euterpe oleraceae Mart.) has high polyphenolic and antioxidant levels. This study examined whether açaí berry extract afforded protection against β-amyloid (Aβ)-mediated loss of cell viability and oxidative stress associated with anti-fibrillar effects. PC12 cells were exposed to either Aβ1-42, Aβ25-35 or tert butyl hydroperoxide (t-BHP), alone or in the presence of açaí extract (0.5-50μg/ml). Thioflavin T (ThT) binding assay and transmission electron microscopy were used to determine effects of açaí extract on Aβ1-42 fibril morphology and compared to açaí phenolics gallic acid, cyanidin rutinoside and cyanidin glucoside. Exposure to Aβ1-42, Aβ25-35 or t-BHP decreased PC12 cell viability. Pretreatment with açaí extract significantly improved cell viability following Aβ1-42 exposure, however Aβ25-35 or t-BHP-mediated viability loss was unaltered. Açaí extract inhibited ThT fluorescence and disrupted Aβ1-42 fibril and aggregate morphology. In comparison with other phenolics, açaí was most effective at inhibiting Aβ1-42 aggregation. Inhibition of β-amyloid aggregation may underlie a neuroprotective effect of açaí.

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-1β 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-1β 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.

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 (All) can modulate neuroeffector responses in the vasculature of spontaneously hypertensive rats (SHR). Included in this modulation is an action of All in facilitating release of neurotransmitter from sympathetic nerves by a mechanism involving prejunctional angiotensin receptors. In addition, AH may be a substrate for the carrier processes that operate within sympathetic nerves. Therefore, we examined the influence of All on the responses to sympathetic nerve stimulation in the isolated perfused mesenteric vascular bed preparation and determined whether All was incorporated into neuronal tissue in blood vessels. AII (10-8) shifted the frequency-response curves to the left, and this shift was reversed with addition of the All receptor type 1 (AT1) antagonist losartan (10-6 M). All 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 All 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 AH 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 All 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.

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

BACKGROUND Columnar lined (Barretts) oesophagus is often considered a sequel to chronic severe reflux disease. Aberrant lower oesophageal sphincter (LOS) motility associated with Barretts 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 (Barretts) 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 Barretts 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 Barretts group. After tetrodotoxin pretreatment, responses to KCl and DMPP were significantly reduced in the SCC but not in Barretts LOS. Maximal contraction in response to bethanechol was significantly decreased in Barretts 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 Barretts 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 Barretts 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.