Effects of selective α2 -adrenergic receptor agonists on electrical field-stimulated contractions of isolated bronchi in horses.

We investigated the effects of different selective α2 -adrenergic receptor (AR) agonists (detomidine, medetomidine, xylazine, and brimonidine) on the contractions of horse-isolated bronchi induced by electrical field stimulation (EFS) and by carbachol. No effects were observed on the contraction induced by carbachol, while α2 -AR agonists reduced EFS-evoked contractions in a concentration-related fashion. The rank order of potency (pD2 ) was brimonidine (7.40 ± 0.20) >medetomidine (7.09 ± 0.24) >detomidine (6.13 ± 0.55) >xylazine (4.59 ± 0.16). The maximal effects (Emax ) were -56.3% ± 6.3%, -40.4% ± 6.9%, -48.6% ± 9.9%, and -72.7% ± 12.7% for brimonidine, medetomidine, detomidine, and xylazine, respectively. Adrenergic block by guanethidine enhanced the potency (8.10 ± 0.05, 7.30 ± 0.15, 6.83 ± 0.41, and 5.40 ± 0.22) and the efficacy (-95.2% ± 0.7%, -45.2% ± 11.7%, -58.5% ± 9.8%, and -97.9% ± 0.6%) of brimonidine, medetomidine, detomidine, and xylazine, respectively. Selective α2 -AR antagonist, atipamezole, competitively antagonized the inhibition of EFS-evoked contractions induced by all agonists except xylazine. These results suggest the existence of presynaptic α2 -ARs on cholinergic neurons, negatively regulating the release of acetylcholine in horse bronchial muscle, and that α2 -AR agonists may be beneficial against vagally mediated bronchoconstriction.

Sildenafil improves clinical signs and radiographic features in dogs with congenital idiopathic megaoesophagus: a randomised controlled trial.

We evaluated the efficacy of oral sildenafil citrate in dogs with congenital idiopathic megaoesophagus (CIM). Twenty-one puppies were randomly assigned to two groups (treatment and control). The dogs were given sildenafil oral suspension 1 mg/kg every 12 hours for 14 days or placebo in a masked fashion. Clinical signs (frequency of regurgitation and weight gain) and oesophagrams (relative oesophageal diameter, ROD) were evaluated in order to assess the efficacy of drug treatment, by examiners who were unaware of the study protocol. In addition, a set of in vitro experiments on isolated samples of canine lower oesophageal sphincter (LOS) was performed, and the effects of increasing concentrations of sildenafil on basal tone and electrically-stimulated motility were assessed. Sildenafil administration significantly reduced the number of regurgitation episodes (0.88±1.40 v 2.65±1.56, P<0.0001) and significantly increased weight gain in the treated dogs compared to controls (79.76±28.30 per cent v 53.40±19.30 per cent, P=0.034). ROD values, at the end of the treatment period, were significantly decreased in the sildenafil group, compared to pre-treatment values (0.97±0.19 v 0.24±0.14, P<0.0001), in contrast to control subjects (0.98±0.17 v 1.10±0.25, P=0.480). In accordance with the in vivo findings, sildenafil dose-dependently reduced basal tone and increased electrically-induced relaxation of dog LOS samples. These results suggest that sildenafil citrate helps ameliorate clinical and radiographic signs in dogs with CIM by reducing LOS tone, and could represent a novel therapeutic tool for the treatment of this disease.

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.

Inhibition of motility in isolated horse small intestine is mediated by κ but not µ opioid receptors.

The effects of preferential µ (morphine), selective µ (fentanyl), selective κ (compound U69593) opioid receptor agonists, and nonselective (naloxone) and selective µ (naloxonazine) antagonists on equine small intestinal motility were evaluated in vitro. Samples of circular muscle from equine jejunum were placed in isolated organ baths and drug-induced modifications of both spontaneous and electrically evoked contractile activity were measured. None of the opioid agonists induced a significant change in spontaneous contractions. Fentanyl and U69593 reduced electrically induced contractions, whereas morphine reduced them only slightly. Naloxone competitively antagonised U69593, but both naloxone and naloxonazine were unable to counteract the inhibition of contractions induced by fentanyl. The inhibition of contractions shown by fentanyl is therefore probably not mediated by opioid receptors, but due to an anticholinergic activity of this drug. In summary, these data showed an inhibitory effect exerted by κ receptors on equine small intestinal motility, whereas the role of µ receptors seemed marginal and would need further characterisation.

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.

Effects of oral curcumin on indomethacin-induced small intestinal damage in the rat.

Nonsteroidal anti-inflammatory drug (NSAID)-induced injury on gastrointestinal tract is well documented, and jejunal inflammation caused by indomethacin in rats is a broadly used experimental model of enteritis. We evaluated the effect of oral curcumin, a compound known to possess anti-inflammatory and anti-oxidant properties, on indomethacin-induced enteritis in the rat. Curcumin (50, 100, and 300 mg/kg) was given to rats by oral gavage 48, 24, and 1 h before enteritis was induced by intragastric administration of 20 mg/kg indomethacin. After 24 h, intestinal macroscopic lesions, myeloperoxidase activity and lipid peroxidation levels were assessed. Curcumin at the dose of 50 mg/kg was uneffective, while at the dose of 100 and 300 mg/kg significantly reduced macroscopic damage caused by indomethacin. By contrast, curcumin at all tested doses was unable to modify indomethacin-induced increases of myeloperoxidase and lipid peroxidation. Curcumin (100 and 300 mg/kg) significantly increased lipid peroxidation level in normal intestinal tissues of rats. Present data show that oral curcumin protects against macroscopic injury induced by indomethacin, leaving unaffected neutrophil infiltration and oxidative cell damage, thus suggesting that this beneficial effect is due to mechanisms not involving anti-inflammatory or antioxidant activities.

Long-term study of TNBS-induced colitis in rats: focus on mast cells.

OBJECTIVE AND DESIGN: To investigate the severity and duration of colitis induced by two different doses of 2,4,6-trinitrobenzenesulfonic acid (TNBS) and the changes in mast cell number in acute inflammation and in the recovery process of colitis. METHODS: Colitis was induced in rats by an enema of TNBS (10 or 30 mg) in 25% ethanol. Macroscopic and histologic changes of the colon, colon weight and mast cell counts were examined at various times (7, 30 and 60 days) after colitis induction. RESULTS: TNBS induced a colonic damage which was dose-related for both severity and time necessary to complete recovery. On day 7 after colitis induction 10 mg TNBS induced macroscopic and microscopic alterations of colonic architecture that completely resolved at day 60. By contrast, 30 mg TNBS induced massive necrosis, thickening of the colon, severe histologic changes that were only partially reversed after two months. Mast cell number in the submucosa and muscularis propria decreased significantly in the acute phase of inflammation (7 days) and slowly increased thereafter, reaching a maximum level (up to about 5-fold) at day 60 after both doses of TNBS. CONCLUSIONS: Present data confirm the ability of TNBS to induce in rats damage to the colon that was dose-dependent for severity and duration. Moreover, these data unravel a different role of mast cells in TNBS-induced colitis: an early degranulation in the acute phase of inflammation and a subsequent accumulation of mast cells in the late phase of the disease, associated with tissue repair.

Prejunctional modulation of non-adrenergic non-cholinergic (NANC) inhibitory responses in the isolated guinea-pig gastric fundus.

The inhibitory neurotransmission of the stomach was investigated in isolated guinea-pig gastric fundus. In preparations treated with guanethidine (1 micro mol L-1) and p-fluoro-hexahydro-sila-difenidol (1 micro mol L-1), electrical stimulation evoked neurogenic inhibitory responses not modified by hexamethonium (100 micro mol L-1), suggesting that inhibitory postganglionic non-adrenergic non-cholinergic (NANC) nerve fibres are involved. The nitric oxide (NO)-synthase inhibitor Nomega-nitro-l-argininine-methyl-ester hydrochloride (1-100 micro mol L-1) and the soluble guanylyl cyclase inhibitor ODQ (0.1-3 micro mol L-1) also abolished such relaxant response, suggesting the involvement of NO/Cyclic Guanosine 3',5' monophosphate (cGMP) system as the final mechanism of muscle relaxation. The alpha2-adrenoceptor agonist, UK 14 304 (10 nmol L-1-10 micro mol L-1) did not influence the electrical field stimulation (EFS)-evoked NANC responses. These latter responses were also refractory to a variety of receptor agonists and antagonists, acting at Gamma Aminobutyric Acid (GABA), serotonin 5HT1a, opioid micro , delta and kappa, muscarinic M1 and M2, histamine H2 and H3 and cannabinoid receptors. The NANC response was insensitive to the P/Q-type Ca2+-channel blocker omega-agatoxin TK (1 nmol L-1-0.1 micro mol L-1), but partially inhibited by the N-type Ca2+-channel blocker omega-conotoxin GVIA (0.1 nmol L-1-0.1 micro mol L-1), and by the L-type Ca2+-channel blockers nifedipine and calcicludine (0.1 nmol L-1-0.1 micro mol L-1). These data suggest that the NANC relaxation of the isolated guinea-pig gastric fundus is mediated by NO as the final inhibitory (neuro)transmitter at the longitudinal smooth muscle cells. The mechanism(s) promoting NO production is/are Ca2+-dependent, but apparently insensitive to presynaptic modulation. Both N- and L-type channels seem to occur in nitrergic nerve endings, where they contribute to trigger NO diffusion at the synaptic cleft.

Morphological and functional alterations of the myenteric plexus in rats with TNBS-induced colitis.

The 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced model of experimental colitis was used to investigate the time-course of alterations in enteric neurotransmission and/or smooth muscle function that occur in chronic inflammation. Myenteric plexus morphology (immunocytochemical markers), functional integrity of cholinergic neurons (3H-choline uptake, acetylcholine release and contractile response to electrical field stimulation) and smooth muscle integrity (contractile response to exogenous acetylcholine) were determined 2, 7, 15, and 30 days after TNBS treatment. In TNBS-treated rats extensive ulcerations of the mucosa and/or the submucosa and increase in colonic weights were accompanied by significant reduction in 3H-choline uptake, acetylcholine release and contractile response to stimulation of enteric nerves. These changes were maximal 7 and 15 days after TNBS treatment. Immunocytochemical marker (PGP 9.5, SNAP 25, synaptophysin and S100 protein) expression was absent in necrotic areas of colons removed 7 days post-injury and partially reduced in colons removed 15 days after TNBS treatment. By contrast, the contractile response to exogenous acetylcholine was significantly increased after 7 days in both inflamed and uninflamed regions and returned to control values by day 30. Likewise, an almost complete recovery of neural cholinergic function and of myenteric plexus morphology was observed 30 days after TNBS treatment. These data suggest that TNBS-induced colitis is associated with progressive and selective alterations in myenteric plexus structure and function, with consequent reduction of cholinergic neurotransmission and abnormality in colonic contractility. The reversibility of myenteric plexus disruption is a clear indication of neuronal plasticity within enteric nervous system as an adaptative mechanism against inflammatory challenges.

Role of histamine H(3) receptors in the control of gastrointestinal motility. An overview.

Over the last few years, the biochemical and functional characterization of H(3) receptors has been a matter for extensive investigation, culminating in the cloning of the human, guinea pig and rat receptor protein from brain tissues. This discovery contributed to determine the distribution of receptors in the body and to define the molecular mechanisms which follow activation. The major breakthrough in the histamine H(3) receptor field came with the synthesis of selective and potent agonists and antagonists, which unravelled the function of this receptor subtype in the different tissues. As expected from the ubiquitous location of histamine in the body, histamine H(3) receptors have also been identified in virtually every tissue, although they are quantitatively less abundant than H(1) and H(2) receptors. Concerning the gastrointestinal tract, this new receptor subtype seems to have multiple cellular locations, which include neurons, enteric ganglia, paracrine and immune cells and, in some tissues, also smooth muscle cells. Therefore it might be regarded as a general regulatory system of different digestive functions, including motility. The effects mediated by histamine H(3)-receptors mainly reflect the presynaptic inhibition of the release of either excitatory or inhibitory neurotransmitters from the myenteric plexus. The molecular mechanism of presynaptic inhibition seems to involve a restriction of calcium entry into the nerve endings, but other mechanisms (reduction of cAMP), possibly associated to different H(3) receptor subtypes, may be involved. Despite the widespread distribution and the well defined inhibitory effects evoked in the majority of in vitro models of intestinal motility, no clear cut evidence of its involvement in the control of peristalsis could be provided. In vivo models of gastrointestinal transit, indeed, did not reveal a defined effect of histamine H(3) receptor ligands, even though the possibility of a central inhibition was pointed out in several studies. Therefore, it is not clear at the present what is the physiological meaning of the histamine H(3) receptor in the control of gastrointestinal motility and whether it could represent a potential target for novel therapeutic interventions in deranged motility, taking into account that human gastrointestinal tissues are apparently devoid of this receptor.

Antisecretory and gastroprotective activities of compounds endowed with H2 antagonistic and nitric oxide (NO) donor properties.

In spite of the well recognized gastric antisecretory activity, the gastroprotective potential of histamine H2 receptor antagonists is controversial. Most clinical studies in fact indicate that these drugs do not substantially protect the gastric mucosa from aggressive factors. Nitric oxide (NO) has been recently recognized as a fundamental mediator in gastric defence mechanisms, due to its ability to increase gastric mucosal blood flow and mucus production and to inhibit neutrophils adherence to endothelial cells. The aim of this study was to investigate the gastroprotective and H2 receptor antagonistic activity of a series of lamtidine analogues which contain different NO-releasing moieties (furoxan, nitroxy and nitrosothiol). These compounds were tested, in comparison with related H2 antagonists devoid of NO-donor structures, in different H2 receptor assays and in the conscious rat against 0.6 N HCl-induced gastric lesions. All the compounds tested were able to antagonize histamine-mediated responses at cardiac and gastric H2 receptors; however, furoxan and nitroxy derivatives were 10-fold less potent than the analogues devoid of NO-donor properties. By contrast, NO-donor compounds were more active than reference H2 antagonists as gastroprotective agents against mucosal injury induced by 0.6 N HCl. Among the different NO-donor moieties, the furoxan group conferred to the H2 antagonist molecule the highest gastroprotective potential; this finding closely correlates with the characteristics of NO release. In conclusions, lamtidine-analogue H2 antagonists combined with NO-donor moieties are endowed with gastric antisecretory and protective activity and could be the prototypes of a new class of anti-ulcer drugs. Finally, the furoxan NO donor group seems to be the most favourable among the different moieties tested.

Effects of histamine H2 receptor agonists and antagonists on the isolated guinea pig gallbladder.

Histamine H2 receptor-mediated responses were examined on cholecystokinin-octapeptide (CCK-8)-precontracted guinea pig gallbladder in vitro, testing histamine and a series of specific histamine H2 receptor agonists and antagonists. Among the antagonists tested, zolantidine and compound SKF 92857 were previously shown to antagonize H2 receptor-mediated responses in the heart, but not in the stomach. Histamine, in the presence of the H2 receptor antagonist mepyramine (10 microM), and the H2 receptor agonists dimaprit, impromidine and amthamine, inhibited CCK-8 (3 nM)-induced contractions in a concentration-dependent fashion, with the following rank orders of potency: impromidine > amthamine > histamine > dimaprit (pD2 values were 6.73 +/- 0.04, 5.44 +/- 0.07, 4.64 +/- 0.04 and 3.71 +/- 0.05, respectively). The maximal relaxation produced by dimaprit was significantly lower than that produced by histamine, as well as by impromidine and amthamine, which behaved as full agonists. All the H2 receptor antagonists examined were able to inhibit amthamine-induced relaxation. Famotidine (pA2 = 7.09 +/- 0.26), zolantidine (pA2 = 6.54 +/- 0.11), compound SKF 92857 (pA2 = 6.58 +/- 0.13) and aminopotentidine (pA2 = 6.86 +/- 0.06) competitively antagonised the amthamine-induced effect, while iodoaminopotentidine produced surmountable antagonism only at low concentrations (1 microM, pA2 = 6.83 +/- 0.21). Finally, the slowly-dissociable antagonist loxtidine caused a non-parallel displacement to the right of the concentration--response curve to amthamine (pKB = 7.55 +/- 0.24), with a significant depression of the maximal response to the agonist, even at the lowest effective concentration (0.3 microM). In conclusion, histamine H2 receptors in guinea pig gallbladder resemble those of the heart, as regards their sensitivity to zolantidine and compound SKF 92857, which, by contrast, were unable to antagonize histamine effects at gastric H2 receptors in different experimental models.

Synthesis of 1,2-benzisothiazole derivatives and investigation of their putative histaminergic activity.

Some new 2-(1,2-benzisothiazol-3-yl)ethylamine derivatives were synthesised and their putative histaminergic activity was investigated in in vitro gastrointestinal and cardiac preparations. In the isolated guinea pig duodenum, all the compounds induced a tetrodotoxin- and atropine-sensitive contractile activity, which was minimally affected by mepyramine in the case of the compound 2-(1,2-benzisothiazol-3-yl)ethylamine. In the same tissue, all the compounds were devoid of any H3 receptor agonistic or antagonistic activity, but caused a nicotinic and/or 5-HT3 receptor activation. None of these compounds induced any histamine H2 agonistic or antagonistic activity in the isolated guinea pig gastric mucosa or in the isolated papillary muscle. On this latter substrate, the compound N,N,N-trimethyl-2-(1,2-benzisothiazol-3-yl)ethylammonium iodide induced a positive inotropic activity, apparently due to a release of catecholamines. These results demonstrate the substantial inability of 1,2-benzisothiazole derivatives to interact with histamine receptors in functional tests. These compounds, however, possess gangliomimetic properties, related to the activation of 5HT3 and/or nicotinic receptors.

Histamine receptor-independent muscle relaxation elicited by a series of histamine H2-receptor agonists on the isolated guinea pig duodenum: a study into the mechanism of action.

1. The histamine H2 receptor agonists, dimaprit, impromidine, amthamine, and several dimaprit- and impromidine-analogues were investigated for their spasmolytic activity on the guinea pig duodenum, precontracted with acetylcholine or KCl. 2. Almost all the H2 receptor agonists exerted a histamine H2 receptor-independent muscle relaxation, which was more evident on acetylcholine- than on KCl-precontracted preparations. 3. The relaxing activity of these compounds was independent of inhibitory receptors, like beta-adrenergic, GABA-ergic, serotoninergic, etc. Similarly, modifications of cyclic nucleotide metabolism and nitric oxide production did not appear to be involved. 4. The behavior of histamine H2-receptor agonists was shared only by the Na+-blocker procaine, the intracellular Ca2+-antagonist ruthenium red and, at least in terms of efficacy, by the protein kinase C inhibitor, chelerithrine. 5. This spasmolytic effect is probably due to an impairment of receptor-mediated depolarization at the plasma membrane level and/or an inhibitory activity on the protein kinase C-dependent activation of the contractile machinery. 6. Finally, our findings suggest that the histamine H2 receptor-independent muscle relaxation is a general feature shown by H2 receptor agonists endowed with different chemical structure and the putative spasmolytic "receptor" prefers a (substituted) thiazole over a (substituted) imidazole.

SAR studies on H2 antagonists containing alkylamino substituted 1,2, 5-thiadiazole 1-oxide moieties.

A number of ranitidine analogues in which the diamino-1,2,5-thiadiazole 1-oxide substructure bearing alkyl chains of different length is present as the urea equivalent group, were synthesised and studied for their lipophilic and H2 antagonist properties. Derivatives which displayed a logP < or = 3 behaved as competitive antagonists of histamine at H2 receptors present on guinea pig right atrium. The remaining more lipophilic members of the series showed an insurmountable antagonism not completely reversible after prolonged washing. A binding study suggested that an increase in the length of alkyl chain gave rise to hydrophobic interactions with the receptor which were responsible for the apparent irreversible H2 antagonism shown by the higher homologues of the series.