Alessandro Menozzi

Effects of Cannabinoid Receptor Agonists on Rat Gastric Acid Secretion: Discrepancy Between In Vitro and In Vivo Data

The effects of the cannabinoid (CB)-receptor agonists WIN55,212-2 and HU-210 and the selective CB1-receptor antagonist SR141716A were tested on in vitro and in vivo acid secretion assays from the rat. In the isolated gastric fundus from immature rats, WIN55,212-2 (0.001–30 μ M), HU-210 (0.001–10 μ M), or SR141716A (0.1–10 μ M) did not change the basal acid output or acid responses to histamine, pentagastrin, or electrical field stimulation. HU-210 (0.3 μ mol/kg, intravenously) inhibited the acid response to pentagastrin in anesthetized adult, young, or immature rats with lumen-perfused stomachs; moreover, HU-210 reduced vagally induced acid secretion in adult animals, its antisecretory effect being reversed by SR141716A (0.65 μ mol/kg, intravenously). In vitro and in vivo data indicate that CB1 receptors are not located on parietal cells but, rather, on vagal pathways (possibly at preganglionic sites) supplying the gastric mucosa. The lack of effect of CB-receptor ligands in vitro cannot be ascribed to the use of immature rats, since HU-210 inhibited stimulated acid secretion in vivo, irrespective of the animal age.

Effects of α2-adrenergic drugs on small intestinal motility in the horse: an in vitro study.

The effects of selective α(2)-agonists (xylazine, detomidine and medetomidine) and antagonists (yohimbine and atipamezole) on in vitro small intestine motility in the horse were evaluated. Samples of equine jejunum were placed in isolated organ baths and drug-induced modifications of motility were measured by means of an isotonic transducer. All tested α(2)-agonists dose-dependently reduced both spontaneous and electrically-evoked phasic contractions. Conversely, α(2)-antagonists were ineffective when tested alone, and showed a heterogeneous and dose-independent ability to inhibit agonist activity. In particular, the antagonism exerted by higher concentrations of both yohimbine and atipamezole against α(2)-agonists was weaker than when lower concentrations were used. The data are indicative of the presence of both pre- and post-synaptic α(2)-adrenoceptors with inhibitory activity on equine jejunum motility, and support a possible therapeutic utility of these drugs in horse intestinal disorders associated with hypermotility.

Effects of nonselective and selective cyclooxygenase inhibitors on small intestinal motility in the horse.

We investigated the effects of nonselective cyclooxygenase (COX) inhibitors (indomethacin and flunixin meglumine) and selective COX-1 (SC-560) or COX-2 (celecoxib, DUP-398 and NS-697) inhibitors on horse small bowel motility in vitro. At this purpose, samples of equine ileum were put in isolated organ baths for the motility experiments. Nonselective COX inhibitors were devoid of major effects on motility, except for an inhibition of tonic contraction shown by flunixin meglumine. SC-560, selective COX-1 inhibitor, was devoid of significant effects on ileal motility. Selective COX-2 inhibitors reduced both tonic contraction and spontaneous phasic contractions, while prostaglandin (PG) receptor antagonists were uneffective. Some of the intestinal samples were submitted to histological investigation or reverse transcription-polymerase chain reaction (RT-PCR), which revealed the presence of an inflammation reaction and the presence of both COX isoforms mRNAs. Present data support the hypothesis that the effects of COX inhibitors on horse small intestinal motility are not linked to PG depletion.

Effect of the Macrolide Antibacterial Drug, Tylosin, on TNBS-Induced Colitis in the Rat

Bacterial antigens, such as intestinal microflora, are known to play a role in the pathogenesis of human inflammatory bowel disease (IBD). Tylosin, a macrolide antimicrobial agent, has proven to be effective in cat and dog chronic colitis, but the reasons underlying this efficacy are still unclear. In the present study we evaluated the effects of tylosin on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in the rat, in comparison with the antibacterial drug metronidazole and the corticosteroid budesonide. Colitis was induced by a single intrarectal administration of 10 mg TNBS under light ether anesthesia. Tylosin (20 mg/kg twice a day), metronidazole (160 mg/kg twice a day) and budesonide (500 µg/kg once a day) were given orally for up to 6 days to separate groups of rats. The animals were sacrificed after 6 days and colonic lesions evaluated (colon weight, macroscopic and histologic damage, myeloperoxidase activity). Tylosin and metronidazole significantly lowered macroscopic lesion score, reduced colon weight, the severity of histologic lesions and myeloperoxidase activity; budesonide did not significantly change the parameters of colonic inflammation. These data indicate a protective effect of tylosin against intestinal inflammation, suggesting a major role for bacteria, anaerobes in particular, in the development of TNBS-induced mucosal damage.

Diazoxide attenuates indomethacin-induced small intestinal damage in the rat.

ATP-sensitive potassium (K(ATP)) channel openers have been shown to protect against cellular damage in neurons, cardiac muscle, and kidney and to effectively reduce nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage in rats. We investigated the effects of K(ATP) channel opener diazoxide on small intestinal injury induced in rats by indomethacin administration. The effect of glibenclamide, a K(ATP) channel blocker, was also evaluated. Diazoxide (15, 45 and 135mg/kg) or glibenclamide (18mg/kg), were given by oral gavage 1h before and 6h after indomethacin treatment (20mg/kg p.o.). After 24h, macroscopic and histologic lesions, myeloperoxidase (MPO) activity and lipid peroxidation levels were evaluated. Diazoxide at 15mg/kg was ineffective, while at doses of 45mg/kg and 135mg/kg was able to significantly improve all damage parameters. Glibenclamide administration enhanced intestinal injury. These results show for the first time a beneficial effect of diazoxide in indomethacin-induced enteritis in the rat. Several mechanisms, such as oxidative phosphorylation uncoupling and hypermotility seem particularly important in NSAID-induced intestinal injury. Such events lead to increased mucosal permeability and to penetration of noxious lumen components, which ignite the inflammatory response. Since K(ATP) channel openers were shown to protect against mitochondrial damage, to reduce intercellular permeability and to relax smooth muscle, we suggest that diazoxide could exert its beneficial effects by one or more of these actions.

Effects of cyclooxygenase-1 and -2 inhibition on gastric acid secretion and cardiovascular functions in rats.

The discovery of a second isoform of cyclooxygenase has led to a re-evaluation of the mechanisms underlying the adverse effects of nonsteroidal anti-inflammatory drugs, focusing in particular on the gastrointestinal system. We investigated the involvement of cyclooxygenase-1 and -2 in the regulation of gastric acid secretion and cardiovascular functions in anesthetized rats, after acute intravenous administration of the selective cyclooxygenase-1 inhibitor SC-560, the selective cyclooxygenase-2 inhibitor celecoxib and the nonselective inhibitor indomethacin. Indomethacin, celecoxib and SC-560 did not significantly modify basal acid secretion. Indomethacin and celecoxib were also ineffective on the acid secretion stimulated by pentagastrin; by contrast, SC-560 significantly enhanced the acid secretion stimulated by pentagastrin, electrical vagal stimulation or histamine. The stimulatory effects of SC-560 were prevented by cervical vagotomy, atropine and famotidine. Indomethacin caused either no change, increasing or decreasing effects on mean arterial pressure and heart rate. By contrast, SC-560 was unable to change cardiovascular parameters at 5 mg/kg, while inducing a marked bradycardia at 10 mg/kg. Celecoxib was ineffective. Our findings indicate that cyclooxygenase-1-derived prostaglandins are involved in the regulation of stimulated acid secretion and of basal heart rate; the role of prostaglandins in the acute control of systemic blood pressure under resting conditions seems to be negligible.

Effects of histamine H4 receptor ligands in a mouse model of gastric ulceration.

Aim: In the present study we examined whether histamine H4 receptors (H4Rs) have a role in gastric ulcerogenesis using a mouse model of gastric damage. Methods: The H4R antagonist JNJ7777120 and the H4R agonists VUF8430 and VUF10460 were investigated in fasted CD-1 mice against the ulcerogenic effect induced by co-administration of indomethacin (IND, 30 mg/kg s.c.) and bethanechol (BET, 5 mg/kg i.p.). Both macroscopic and histologic lesions were examined. Strain-related differences were investigated by testing JNJ7777120 also in NMRI, BALB/c and C57BL/6J mice. Results: Neither JNJ7777120 nor the H4R agonists displayed effects in the normal stomach at any dose tested (10 and 30 mg/kg s.c.). As expected, IND+BET provoked several lesions in the fundic mucosa, which were significantly reduced by JNJ7777120 (10 and 30 mg/kg s.c.). The gastroprotective effect of JNJ7777120 (10 and 30 mg/kg s.c.) was observed in CD-1, NMRI and BALB/c, but not in C57BL/6J, mice. In CD-1 mice, the H4R agonists VUF8430 and VUF10460 (both at 10 and 30 mg/kg s.c.) did not modify the damage induced by IND+BET, however VUF8430 (10 mg/kg s.c.) prevented the gastroprotection induced by JNJ7777120 (10 mg/kg s.c.). Conclusions: Data obtained with selective ligands suggest that the H4R may have a role in mouse gastric ulcerogenesis. If confirmed in humans, these data would emphasize the potential advantage of H4R blockers as gastrosparing anti-inflammatory drugs. The lack of effects of JNJ7777120 in C57BL/6J mice has to be carefully considered in the pharmacological characterization of H4R functions and/or new selective ligands.

Pharmacological characterization of muscarinic receptors in the contractions of isolated bronchi in the horse

We investigated the effects of nonselective muscarinic antagonist (atropine) and of selective muscarinic subtype 1 (M1), 2 (M2), 3 (M3) antagonists (VU0255035, methoctramine, pFHHSiD, respectively) on the contractions evoked by electrical field stimulation (EFS) or by exogenous ACh in isolated horse bronchial muscle. Atropine completely inhibited neurogenic contractions in a concentration-dependent fashion, whereas selective muscarinic antagonists induced relevant modifications only at the highest concentration tested. Experiments with selective muscarinic antagonists in combination showed that only the simultaneous blockade of M1 /M3 or M2 /M3 receptors was able to induce a nearly complete suppression of contractions. The contractions induced by exogenous ACh were competitively antagonized only by atropine (pA2 = 9.01 ± 0.05). M3 selective antagonist, up to 10(-6) m, caused a moderate concentration-dependent rightward shift of ACh curve (pA2 = 7.96 ± 0.10). These data show that M3 muscarinic receptors possess a central role in mediating cholinergic contraction of horse bronchi, while M1 and M2 receptors seem to have a cooperative role. Selective muscarinic antagonists seem unlikely to be useful against bronchoconstriction associated with airway diseases in horses. Conversely, compounds with selectivity for both M1 and M3 receptors could be as effective as traditional anticholinergics and induce fewer cardiac side effects.

Benzisothiazoles and β-adrenoceptors: Synthesis and pharmacological investigation of novel propanolamine and oxypro-panolamine derivatives in isolated rat tissues

In an attempt to examine the ability of benzisothiazole-based drugs to interact with β-adreno-ceptors, a series of 1,2-benzisothiazole derivatives, which were substituted with various propanolamine or oxypropanolamine side chains in the 2 or 3 position, were synthesised and tested. The pharmacological activity of these compounds at the β-adrenoceptors was examined using isolated rat atria and small intestinal segments, which preferentially express the β1-and β3-adrenoceptor-mediated responses, respectively. None of these products showed any β- adrenoceptor agonistic activity. In contrast, the 2- and 3-substituted isopropyl,tert-butyl, benzyl, and piperonyl derivatives2a- d and3a- d elicited surmountable inhibition of the isoprena-line-induced chronotropic effects in the atria, suggesting competitive antagonism at the β1-recognition site. The pA2 values revealedtert-butyl3b and the isopropyl substituted piperonyl derivatives3a to be the most effective. Remarkably, many of the 2-substituted propanola-mines were less active than the corresponding 3-substituted oxypropanolamines. With the exception of compound3b, none of these drugs antagonised the muscle relaxant activity of isoprenaline in the intestine, suggesting no effect on the β3-adrenoceptors. These results confirm the ability of the benzisothiazole ring to interact with the β-adrenoceptors, and demonstrate that 2-substitution with propanolamine or 3-substitution with oxypropanolamine groups yields compounds with preferential antagonistic activity at the cardiac β1-adrenoceptors. The degree of antagonism depends strongly on both the nature of the substituent and its position on the benzisothiazole ring.

Assessment of enteral bacteria.

The disruption of intestinal barrier leads to the penetration of noxious luminal compounds into the gut wall, causing further damage. This unit describes the assessment of enteric bacteria translocation into the intestinal wall of rats, an established method for the evaluation of bowel damage to the mucosal epithelial barrier. The Basic Protocol provided in the present unit describes collection and preparation of small intestine sample, performing of sample serial dilutions for bacterial culture, performing of the culture of aerobic and anaerobic bacteria on petri dishes, incubation of the cultured plates, and counting of bacterial colonies. The Support Protocols describes the procedures for the preparation of petri dishes for the culture, using different employable media for aerobes or anaerobes. The Alternate Protocol describes the use of the “inclusion method,” suitable for the culture of anaerobic bacteria. Curr. Protoc. Toxicol. 44:21.3.1‐21.3.11.