The non-euphoric phytocannabinoid cannabidivarin counteracts intestinal inflammation in mice and cytokine expression in biopsies from UC pediatric patients.

Patients with ulcerative colitis (UC) using marijuana have been reported to experience symptomatic benefit. Cannabidivarin (CBDV) is a safe non-psychoactive phytocannabinoid able to activate and desensitize TRPA1, a member of the TRP channels superfamily, which plays a pivotal role in intestinal inflammation. Here, we have investigated the potential intestinal anti-inflammatory effect of CBDV in mice and in biopsies from pediatric patients with active UC. Colonic inflammation was induced in mice by dinitrobenzenesulfonic acid (DNBS). The effect of orally administered CBDV on macroscopic and microscopic damage, inflammatory parameters (i.e. myeloperoxidase activity, intestinal permeability and cytokine production) and faecal microbiota composition, was evaluated 3 days after DNBS administration. TRPA1 expression was studied by RT-PCR in inflamed colons of mice as well as in mucosal colonic biopsies of children with active UC, whose response to incubation with CBDV was also investigated. CBDV attenuates, in a TRPA1-antagonist sensitive manner, DNBS-induced signs of inflammation including neutrophil infiltration, intestinal permeability, and cytokine (i.e. IL-1ß, IL-6 and the chemokine MCP-1) production. CBDV also alters the dysregulation of gut microbiota associated to colitis. Finally, CBDV lessens cytokine expression in colonic biopsies from pediatric patients with ulcerative colitis, a condition in which TRPA1 was up-regulated. Our preclinical study shows that CBDV exerts intestinal anti-inflammatory effects in mice via TRPA1, and in children with active UC. Since CBDV has a favorable safety profile in humans, it may be considered for possible clinical trials in patients with UC.

Evidence for an alpha/beta T cell-independent mechanism of resistance to mycobacteria. Bacillus-Calmette-Guerin causes progressive infection in severe combined immunodeficient mice, but not in nude mice or in mice depleted of CD4+ and CD8+ T cells.

Depleting thymectomized mice of CD4+ T cells, or CD4+ plus CD8+ T cells, rendered them incapable of resolving Bacillus-Calmette-Guerin (BCG) infection in their lives, spleens, kidneys, and lungs. However, it did not render them incapable of stabilizing infection in the latter three organs after an initial period of BCG growth. Athymic nude mice showed a similar capacity to control BCG growth in these organs after a certain stage of infection. In contrast, congenitally severe combined immunodeficient (SCID) mice appeared to offer no resistance to BCG infection, in that the organism grew progressively in all organs of these mice and was lethal for them beginning on day 55 of infection. The results suggest that, although CD4+ T cells are important for resolving BCG infection, an alpha/beta T cell-independent mechanism of resistance can be acquired at 2-3 wk of infection that is capable of inhibiting further BCG growth in all organs except the lungs. Because this mechanism is absent from SCID mice, it is likely that it depends on the functions of gamma/delta T cells, B cells, or both types of cells. In keeping with this possibility is the additional finding that SCID mice engrafted with lymph node cells depleted of CD4+ or CD8+ T cells were capable of expressing an appreciable level of resistance against BCG infection.

Mycobacterial virulence. Virulent strains of Mycobacteria tuberculosis have faster in vivo doubling times and are better equipped to resist growth-inhibiting functions of macrophages in the presence and absence of specific immunity.

The kinetics of growth of two virulent strains of mycobacteria (M. tuberculosis Erdman and M. tuberculosis H37Rv) and two attenuated strains (M. tuberculosis H37Ra and M. bovis Bacillus Calmette-Guerin [BCG]) were studied in the lungs, livers, spleens, and kidneys of severe combined immunodeficient (SCID) mice and of their coisogenic CB-17 immunocompetent counterparts. It was found, in keeping with the findings of earlier investigators (Pierce, C. H., R. J. Dubos, and W. B. Schaefer. 1953. J. Exp. Med. 97:189.), that in immunocompetent mice, virulent organisms grew progressively only in the lungs, whereas the growth of attenuated organisms was controlled in all organs. In SCID mice, in contrast, virulent mycobacteria grew rapidly and progressively in all organs, as did BCG, although at a slower rate. However, H37Ra failed to grow progressively in any organs of SCID mice, unless the mice were treated with hydrocortisone. In fact, hydrocortisone treatment enabled virulent, as well as attenuated, organisms to grow strikingly more rapidly in all organs of SCID mice and in all organs of CB-17 mice. A histological study showed that in SCID mice, multiplication of mycobacteria in the liver occurs in the cytoplasm of macrophages in granulomas and presumably in macrophages in other organs. It is suggested, therefore, that the macrophages of SCID mice possess a glucocorticoid-sensitive mycobacterial mechanism that prevents virulent and avirulent mycobacteria from expressing their true minimal doubling times. In the absence of this mechanism in the lungs of hydrocortisone-treated SCID mice, the doubling times of Erdman, H37Rv, BCG, and H37Ra were 17.7, 17.4, 44.6, and 98.6 h, respectively. The possible importance of a rapid multiplication rate for mycobacterial virulence is discussed.

Emerging role of cannabinoids in gastrointestinal and liver diseases: basic and clinical aspects.

A multitude of physiological effects and putative pathophysiological roles have been proposed for the endogenous cannabinoid system in the gastrointestinal tract, liver and pancreas. These range from effects on epithelial growth and regeneration, immune function, motor function, appetite control, fibrogenesis and secretion. Cannabinoids have the potential for therapeutic application in gut and liver diseases. Two exciting therapeutic applications in the area of reversing hepatic fibrosis as well as antineoplastic effects may have a significant impact in these diseases. This review critically appraises the experimental and clinical evidence supporting the clinical application of cannabinoid receptor-based drugs in gastrointestinal, liver and pancreatic diseases. Application of modern pharmacological principles will most probably expand the selective modulation of the cannabinoid system peripherally in humans. We anticipate that, in addition to the approval in several countries of the CB(1) antagonist, rimonabant, for the treatment of obesity and associated metabolic dysfunctions, other cannabinoid modulators are likely to have an impact on human disease in the future, including hepatic fibrosis and neoplasia.

Cannabidiol, extracted from Cannabis sativa, selectively inhibits inflammatory hypermotility in mice.

BACKGROUND AND PURPOSE: Cannabidiol is a Cannabis-derived non-psychotropic compound that exerts a plethora of pharmacological actions, including anti-inflammatory, neuroprotective and antitumour effects, with potential therapeutic interest. However, the actions of cannabidiol in the digestive tract are largely unexplored. In the present study, we investigated the effect of cannabidiol on intestinal motility in normal (control) mice and in mice with intestinal inflammation. EXPERIMENTAL APPROACH: Motility in vivo was measured by evaluating the distribution of an orally administered fluorescent marker along the small intestine; intestinal inflammation was induced by the irritant croton oil; contractility in vitro was evaluated by stimulating the isolated ileum, in an organ bath, with ACh. KEY RESULTS: In vivo, cannabidiol did not affect motility in control mice, but normalized croton oil-induced hypermotility. The inhibitory effect of cannabidiol was counteracted by the cannabinoid CB1 receptor antagonist rimonabant, but not by the cannabinoid CB2 receptor antagonist SR144528 (N-[-1S-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide), by the opioid receptor antagonist naloxone or by the alpha2-adrenergic antagonist yohimbine. Cannabidiol did not reduce motility in animals treated with the fatty acid amide hydrolase (FAAH) inhibitor N-arachidonoyl-5-hydroxytryptamine, whereas loperamide was still effective. In vitro, cannabidiol inhibited ACh-induced contractions in the isolated ileum from both control and croton oil-treated mice. CONCLUSIONS AND IMPLICATIONS: Cannabidiol selectively reduces croton oil-induced hypermotility in mice in vivo and this effect involves cannabinoid CB1 receptors and FAAH. In view of its low toxicity in humans, cannabidiol may represent a good candidate to normalize motility in patients with inflammatory bowel disease.

The role of endocannabinoids in the regulation of gastric emptying: alterations in mice fed a high-fat diet.

BACKGROUND AND PURPOSE: Endocannabinoids (via cannabinoid CB(1) receptor activation) are physiological regulators of intestinal motility and food intake. However, their role in the regulation of gastric emptying is largely unexplored. The purpose of the present study was to investigate the involvement of the endocannabinoid system in the regulation of gastric emptying in mice fed either a standard diet (STD) or a high-fat diet (HFD) for 14 weeks. EXPERIMENTAL APPROACH: Gastric emptying was evaluated by measuring the amount of phenol red recovered in the stomach after oral challenge; CB(1) expression was analysed by quantitative reverse transcription-PCR; endocannabinoid (anandamide and 2-arachidonoyl glycerol) levels were measured by liquid chromatography-mass spectrometry. KEY RESULTS: Gastric emptying was reduced by anandamide, an effect counteracted by the CB(1) receptor antagonist rimonabant, but not by the CB(2) receptor antagonist SR144528 or by the transient receptor potential vanilloid type 1 (TRPV1) antagonist 5'-iodoresiniferatoxin. The fatty acid amide hydrolase (FAAH) inhibitor N-arachidonoyl-5-hydroxytryptamine (but not the anandamide uptake inhibitor OMDM-2) reduced gastric emptying in a way partly reduced by rimonabant. Compared to STD mice, HFD mice exhibited significantly higher body weight and fasting glucose levels, delayed gastric emptying and lower anandamide and CB(1) mRNA levels. N-arachidonoylserotonin (but not rimonabant) affected gastric emptying more efficaciously in HFD than STD mice. CONCLUSIONS AND IMPLICATIONS: Gastric emptying is physiologically regulated by the endocannabinoid system, which is downregulated following a HFD leading to overweight.

The hallucinogenic herb Salvia divinorum and its active ingredient salvinorin A reduce inflammation-induced hypermotility in mice.

The hallucinogenic plant Salvia divinorum has been used for medical treatments of gastrointestinal disorders. Here, we evaluated the effect of a standardized extract from the leaves of Salvia divinorum (SDE) and of its active ingredient salvinorin A on motility in vivo, both in physiological states and during croton oil-induced intestinal inflammation. SDE (1-100 mg kg(-1)) significantly inhibited motility only in inflamed, but not in control, mice. In control mice, salvinorin A (0.01-10 mg kg(-1)) significantly inhibited motility only at the highest doses tested (3 and 10 mg kg(-1)) and this effect was not counteracted by naloxone or by the kappa-opioid receptor (KOR) antagonist nor-binaltorphimine. Inflammation significantly increased the potency of salvinorin A (but not of the KOR agonist U-50488) in reducing motility. The inhibitory effects of both salvinorin A and U-50488 in inflamed mice were counteracted by naloxone or by nor-binaltorphimine. We conclude that salvinorin A may reduce motility through activation of different targets. In physiological states, salvinorin A, at high doses, inhibited motility through a non-KOR mediated mechanism. Gut inflammation increased the potency of salvinorin A; this effect was mediated by KOR, but it was not shared by U-50488, thus suggesting that salvinorin A may have target(s) other than KOR in the inflamed gut.

Cannabinoids and gastrointestinal motility: animal and human studies.

The plant Cannabis has been known for centuries to be beneficial in a variety of gastrointestinal diseases, including emesis, diarrhea, inflammatory bowel disease and intestinal pain. delta9-tetrahydrocannabinol, the main psychotropic component of Cannabis, acts via at least two types of cannabinoid receptors, named CB1 and CB2 receptors. CB1 receptors are located primarily on central and peripheral neurons (including the enteric nervous system) where they modulate neurotransmitter release, whereas CB2 receptors are concerned with immune function, inflammation and pain. The discovery of endogenous ligands [i.e. anandamide and 2-arachidonoyl glycerol (2-AG)] for these receptors indicates the presence of a functional endogenous cannabinoid system in the gastrointestinal tract. Anatomical and functional evidence suggests the presence of CB1 receptors in the myenteric plexus, which are associated with cholinergic neurons in a variety of species, including in humans. Activation of prejunctional CB1 receptors reduces excitatory enteric transmission (mainly cholinergic transmission) in different regions of the gastrointestinal tract. Consistently, in vivo studies have shown that cannabinoids reduce gastrointestinal transit in rodents through activation of CB1, but not CB2, receptors. However, in pathophysiological states, both CB1 and CB2 receptors could reduce the increase of intestinal motility induced by inflammatory stimuli. Cannabinoids also reduce gastrointestinal motility in randomized clinical trials. Overall, modulation of the gut endogenous cannabinoid system may provide a useful therapeutic target for disorders of gastrointestinal motility.

Role and regulation of acylethanolamides in energy balance: focus on adipocytes and beta-cells.

The endocannabinoid, arachidonoylethanolamide (AEA), and the peroxisome proliferator-activated receptor (PPAR)-alpha ligand, oleylethanolamide (OEA) produce opposite effects on lipogenesis. The regulation of OEA and its anti-inflammatory congener, palmitoylethanolamide (PEA), in adipocytes and pancreatic beta-cells has not been investigated. We report here the results of studies on acylethanolamide regulation in these cells during obesity and hyperglycaemia, and provide an overview of acylethanolamide role in metabolic control. We analysed by liquid chromatography-mass spectrometry OEA and PEA levels in: 1) mouse 3T3F442A adipocytes during insulin-induced differentiation, 2) rat insulinoma RIN m5F beta-cells kept in 'low' or 'high' glucose, 3) adipose tissue and pancreas of mice with high fat diet-induced obesity (DIO), and 4) in visceral fat or blood of obese or type 2 diabetes (T2D) patients. In adipocytes, OEA levels remain unchanged during differentiation, whereas those of PEA decrease significantly, and are under the negative control of both leptin and PPAR-gamma. PEA is significantly downregulated in subcutaneous adipose tissue of DIO mice. In RIN m5F insulinoma beta-cells, OEA and PEA levels are inhibited by 'very high' glucose, this effect being enhanced by insulin, whereas in cells kept for 24 h in 'high' glucose, they are stimulated by both glucose and insulin. Elevated OEA and PEA levels are found in the blood of T2D patients. Reduced PEA levels in hypertrophic adipocytes might play a role in obesity-related pro-inflammatory states. In beta-cells and human blood, OEA and PEA are down- or up-regulated under conditions of transient or chronic hyperglycaemia, respectively.

The cannabinoid CB(2) receptor: a good friend in the gut.

Mammalian tissues express the cannabinoid 1 (CB(1)) receptor and the cannabinoid 2 (CB(2)) receptor, the latter being involved in inflammation and pain. In somatic nerve pathways, the analgesic effects of CB(2) agonism are well documented. Two papers published in the Journal have provided evidence that CB(2) receptor activation inhibits visceral afferent nerve activity in rodents. These exciting findings are discussed in the context of recent data highlighting the emerging role of CB(2) receptor as a critical target able to counteract hypermotility in pathophysiological states, gut inflammation and possibly colon cancer.

Endocannabinoid overactivity and intestinal inflammation.

Cannabinoid receptors of type 1 and 2 (CB(1) and CB(2)), endogenous ligands that activate them (endocannabinoids), and mechanisms for endocannabinoid biosynthesis and inactivation have been identified in the gastrointestinal system. Activation of CB(1 )receptors by endocannabinoids produces relaxation of the lower oesophageal sphincter and inhibition of gastric acid secretion, intestinal motility, and fluid stimulated secretion. However, stimulation of cannabinoid receptors impacts on gastrointestinal functions in several other ways. Recent data indicate that the endocannabinoid system in the small intestine and colon becomes over stimulated during inflammation in both animal models and human inflammatory disorders. The pathological significance of this "endocannabinoid overactivity" and its possible exploitation for therapeutic purposes are discussed here.

Targeting fatty acid amide hydrolase and transient receptor potential vanilloid-1 simultaneously to modulate colonic motility and visceral sensation in the mouse: A pharmacological intervention with N-arachidonoyl-serotonin (AA-5-HT).

BACKGROUND: Endocannabinoid anandamide (AEA) inhibits intestinal motility and visceral pain, but it may also be proalgesic through transient receptor potential vanilloid-1 (TRPV1). AEA is degraded by fatty acid amide hydrolase (FAAH). This study explored whether dual inhibition of FAAH and TRPV1 reduces diarrhea and abdominal pain. METHODS: Immunostaining was performed on myenteric plexus of the mouse colon. The effects of the dual FAAH/TRPV1 inhibitor AA-5-HT on electrically induced contractility, excitatory junction potential (EJP) and fast (f) and slow (s) inhibitory junction potentials (IJP) in the mouse colon, colonic propulsion and visceromotor response (VMR) to rectal distension were studied. The colonic levels of endocannabinoids and fatty acid amides were measured. KEY RESULTS: CB1-positive neurons exhibited TRPV1; only some TRPV1 positive neurons did not express CB1. CB1 and FAAH did not colocalize. AA-5-HT (100 nM-10 µM) decreased colonic contractility by ~60%; this effect was abolished by TRPV1 antagonist 5'-IRTX, but not by CB1 antagonist, SR141716. AA-5-HT (1 µM-10 µM) inhibited EJP by ~30% and IJPs by ~50%. The effects of AA-5-HT on junction potentials were reversed by SR141716 and 5`-IRTX. AA-5-HT (20 mg/kg; i.p.) inhibited colonic propulsion by ~30%; SR141716 but not 5`-IRTX reversed this effect. AA-5-HT decreased VMR by ~50%-60%; these effects were not blocked by SR141716 or 5`-IRTX. AA-5-HT increased AEA in the colon. CONCLUSIONS AND INFERENCES: The effects of AA-5-HT on visceral sensation and colonic motility are differentially mediated by CB1, TRPV1 and non-CB1/TRPV1 mechanisms, possibly reflecting the distinct neuromodulatory roles of endocannabinoid and endovanilloid FAAH substrates in the mouse intestine.

The hallucinogenic herb Salvia divinorum and its active ingredient salvinorin A inhibit enteric cholinergic transmission in the guinea-pig ileum.

Salvia divinorum is a widespread hallucinogenic herb traditionally employed for divination, as well as a medicament for several disorders including disturbances of gastrointestinal motility. In the present study we evaluated the effect of a standardized extract from the leaves of S. divinorum (SDE) on enteric cholinergic transmission in the guinea-pig ileum. SDE reduced electrically evoked contractions without modifying the contractions elicited by exogenous acetylcholine, thus suggesting a prejunctional site of action. The inhibitory effect of SDE on twitch response was abolished by the opioid receptor antagonist naloxone and by the kappa-opioid antagonist nor-binaltorphimine, but not by naltrindole (a delta-opioid receptor antagonist), CTOP (a mu-opioid receptor antagonist), thioperamide (a H(3) receptor antagonist), yohimbine (an alpha(2)-receptor antagonist), methysergide (a 5-hydroxytryptamine receptor antagonist), N(G)-nitro-L-arginine methyl ester (an inhibitor of NO synthase) or apamin (a blocker of Ca(2+)-activated K(+) channels). Salvinorin A, the main active ingredient of S. divinorum, inhibited in a nor-binaltorphimine- and naloxone-sensitive manner electrically induced contractions. It is concluded that SDE depressed enteric cholinergic transmission likely through activation of kappa-opioid receptors and this may provide the pharmacological basis underlying its traditional antidiarrhoeal use. Salvinorin A might be the chemical ingredient responsible for this activity.

St John's wort: Prozac from the plant kingdom.

Conventional antidepressants are associated with a range of adverse drug reactions. The herb Hypericum perforatum (St John's wort) might offer another approach to the treatment of depression. Biochemical and animal studies suggest that the phloroglucinol derivative hyperforin is the main active ingredient of St John's wort, and inhibits the synaptosomal uptake of 5-HT, noradrenaline, dopamine, glutamate and GABA. St John's wort has been shown to alleviate symptoms of mild to moderate depression, and seems to offer significant advantages over conventional antidepressants because it is associated with fewer adverse reactions. However, important herb--drug interactions have been described. In view of its efficacy and safety records, St John's wort should be considered for the first-line treatment of mild to moderate depression.

Recent findings on the mode of action of laxatives: the role of platelet activating factor and nitric oxide.

Platelet activating factor (PAF) is a phospholipid mediator of inflammation and stimulates anion secretion in animals and in isolated preparations of human colon. Nitric oxide (NO), synthesized from the amino acid L-arginine, is an important enteric inhibitory neurotransmitter. In addition, NO-donating compounds stimulate anion secretion in rat and guinea-pig colon. In this article, Angelo A. Izzo and colleagues review the key pharmacological features of the involvement of NO and PAF in the action of laxatives and propose that the classification of laxatives should take into account the important implications of these endogenous mediators.

The gastrointestinal pharmacology of cannabinoids.

The digestive tract contains endogenous cannabinoids (anandamide and 2-arachidonylglycerol) and cannabinoid CB1 receptors can be found on myenteric and submucosal nerves. Activation of CB1 receptors inhibits gastrointestinal motility, intestinal secretion and gastric acid secretion. The enteric location of CB1 receptors could provide new strategies for the management of gut disorders.

Cannabinoids and the digestive tract.

In the digestive tract there is evidence for the presence of high levels of endocannabinoids (anandamide and 2-arachidonoylglycerol) and enzymes involved in the synthesis and metabolism of endocannabinoids. Immunohistochemical studies have shown the presence of CB1 receptors on myenteric and submucosal nerve plexuses along the alimentary tract. Pharmacological studies have shown that activation of CB1 receptors produces relaxation of the lower oesophageal sphincter, inhibition of gastric motility and acid secretion, as well as intestinal motility and secretion. In general, CB1-induced inhibition of intestinal motility and secretion is due to reduced acetylcholine release from enteric nerves. Conversely, endocannabinoids stimulate intestinal primary sensory neurons via the vanilloid VR1 receptor, resulting in enteritis and enhanced motility. The endogenous cannabinoid system has been found to be involved in the physiological control of colonic motility and in some pathophysiological states, including paralytic ileus, intestinal inflammation and cholera toxin-induced diarrhoea. Cannabinoids also possess antiemetic effects mediated by activation of central and peripheral CB1 receptors. Pharmacological modulation of the endogenous cannabinoid system could provide a new therapeutic target for the treatment of a number of gastrointestinal diseases, including nausea and vomiting, gastric ulcers, secretory diarrhoea, paralytic ileus, inflammatory bowel disease, colon cancer and gastro-oesophageal reflux conditions.

Liver and plasma concentrations in paf-acether and its precursors after partial hepatectomy.

Liver and plasma concentrations in paf-acether (paf) and related phosphocholines, i.e., lysopaf and the ether lipid 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (AAGPC) were studied in rats following two-third hepatectomy. We report a rapid increase in hepatic content of the 3 phospholipids at early steps of the regeneration process, when hepatocytes are switching from G0 to G1 (time 2-6 h). Later on, throughout G1 and at the G1-S transition, these concentrations decreased progressively. They were back to sham-operated or intact control levels at 50 h. In the plasma of hepatectomized animals, no comparable changes were detected. However, an increase in both circulating paf and lipoprotein-bound paf concentrations was measured during the regenerating response. This report is, to our knowledge, the first one on paf level variations following 2/3 hepatectomy. In rats, partial resection of the liver was shown to initiate rapid and complex cascades of biochemical changes involving growth factors, neurotransmitters and interleukins among others. Our data are in good agreement with reported increases in both total phospholipid content and synthesis of phosphatidylcholine, a paf precursor, in the regenerating liver. At present, the possible functional significance of high paf concentrations measured over the 'priming' stage of the induced proliferative wave is suggested as a working hypothesis. However, on the one hand, the observed paf response is noteworthy in view of its cytokine-related action, i.e., stimulation of IL-6 production by different cell types (endothelial, macrophagic). On the other hand, it could represent an in vivo confirmation of previously reported in vitro paf effects inducing c-fos and c-jun expression, two members of the so-called 'cellular immediate-early gene' family.

A matrix metalloproteinase inhibitor promotes granuloma formation during the early phase of Mycobacterium tuberculosis pulmonary infection.

OBJECTIVE: The host response to pulmonary Mycobacterium tuberculosis (Mtb) infection results in granuloma formation in an effort to limit infection, but the host immune cells also provide an environment in which Mtb persists. Granuloma formation requires immune cell infiltration and concurrent extensive remodeling of pulmonary tissue which we hypothesize to be the result of increased matrix metalloproteinases (MMP) activity. DESIGN: C57BL/6 mice infected with virulent Mtb (H37Rv) via intratracheal inoculation were treated with a synthetic inhibitor of MMP activity (BB-94). Mice were assessed for colony forming units, granuloma morphology, leukocyte recruitment and cytokine levels over 90 days of infection. RESULTS: BB-94 treated mice had significantly decreased numbers of pulmonary and blood-borne Mtb early during disease, increased collagen deposition within early granulomas and significantly decreased pulmonary leukocyte recruitment when compared to vehicle-treated, Mtb-infected mice. Cytokine expression did not differ significantly between groups. CONCLUSION: Events of early granuloma formation can be modified by inhibiting MMP activity, by decreasing leukocyte recruitment, a major source of MMPs during infection, enhancing the establishment of granulomas and decreasing blood-borne dissemination of Mtb.

Ascending neural pathways in the isolated guinea-pig ileum: effect of muscarinic M1, M2 and M3 cholinergic antagonists.

The effect of muscarinic cholinoceptor antagonists was investigated on the ascending neural pathways activated by electrical stimulation in the guinea-pig ileum. For comparison, prejunctional and postjunctional effects of muscarinic cholinoceptor antagonists were also studied on circular smooth muscle. A two-compartment (oral and anal compartments) bath was used to study the ascending neural pathways. These were activated by electrical field stimulation in the anal compartment and the resulting contraction of the intestinal circular muscle in the oral compartment was recorded isotonically. Pirenzepine (10-300 nM), a muscarinic M1 cholinoceptor antagonist, reduced the ascending neural contractions in a concentration-dependent fashion when applied either to the oral or anal compartments (11-52% and 13-55% inhibition, respectively, P < 0.05). Pirenzepine inhibited (31+/-7%, P < 0.05) the acetylcholine (100 nM)-induced contractions at a higher non-selective concentration (300 nM), while its effect on the electrically-induced contractions was biphasic (10 and 30nM: 8-15% increase, P<0.05; 100 and 300 nM: 16-28% inhibition, P<0.05). The muscarinic M2 cholinoceptor antagonist methoctramine (3-100 nM) did not modify the contractions produced by 100 nM acetylcholine, electrically-induced contractions and the ascending neural contractions (when applied to either compartment). Parafluorohexahydrosiladifenidol (3-100 nM), a muscarinic M3 cholinoceptor antagonist, inhibited the contractions produced by 100 nM acetylcholine (19-81% and 15-69%), electrically-induced contractions (11-71% and 12-72%) and the ascending neural contractions (13-76% and 866%) when applied to the oral compartment, but it was without effect when applied to the anal compartment. These studies suggest that in the enteric ascending neural pathway, muscarinic M1 receptors are involved in neuroneuronal transmission, muscle contraction is mediated by muscarinic M3 cholinergic receptors, whereas muscarinic M2 receptors do not seem to participate.