Cristina Pozzoli

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.

Strain-dependent effects of the histamine H(4) receptor antagonist JNJ7777120 in a murine model of acute skin inflammation

Abstract:  The effects of the histamine H4 receptor antagonist JNJ7777120 were evaluated in a model of acute skin inflammation induced by local application of croton oil. The influence of strain on the effect of JNJ7777120 was investigated in four different mouse strains (CD‐1, NMRI, BALB/c and C57BL/6J). In CD‐1 mice, JNJ777720 (30–100 mg/kg subcutaneously, s.c.) exerted a dose‐dependent inhibition of croton oil‐induced ear inflammation and polymorphonuclear leucocyte infiltration, as confirmed by histological evaluation of ear tissues. JNJ7777120 (30–100 mg/kg) did not reduce ear oedema in NMRI, BALB/c or C57BL/6J mice. The positive control, dexamethasone (2 mg/kg s.c.) induced significant anti‐inflammatory effects only in CD‐1 and NMRI mice. In these strains, also the histamine H1‐receptor blocker pyrilamine (30 mg/kg s.c.) significantly reduced ear oedema at 2 h after croton oil challenge, being as effective as JNJ7777120 in CD‐1 mice. Taken together, these data demonstrate that the H4 receptor antagonist JNJ7777120 may reduce acute croton oil‐induced skin inflammation as effectively as H1 receptor blockade. However, present experiments evidenced for the first time marked strain‐related differences in the JNJ7777120 pharmacological activity, which have to be carefully considered when using this ligand to characterize histamine H4 receptor functions in murine models and translating preclinical data to clinical human settings.

[3-(1H-imidazol-4-yl)propyl]guanidines containing furoxan moieties: A new class of H3-Antagonists endowed with NO-donor properties

Synthesis and pharmacological characterisation of a series of products obtained by coupling the H(3)-antagonist SKF 91486 through appropriate spacers with the NO-donor 3-phenylfuroxan-4-yloxy and 3-benzenesulfonylfuroxan-4-yloxy moieties, as well as with the corresponding furazan substructures, devoid of NO-donating properties, are reported. All the products were tested for their H(3)-antagonistic and H(2)-agonistic properties on electrically-stimulated guinea-pig ileum segments and guinea-pig papillary muscle, respectively. The whole series of compounds displayed good H(3)-antagonist behaviour and feeble partial H(2)-agonist activity. Among furoxan derivatives, the benzenesulfonyl hybrid 28, a good NO-donor, triggered a dual NO-dependent muscle relaxation and H(3)-antagonistic effect on guinea-pig intestine.

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.

PRESYNAPTIC HISTAMINE H2 RECEPTORS MODULATE THE SYMPATHETIC NERVE TRANSMISSION IN THE ISOLATED RAT VAS DEFERENS ; NO ROLE FOR H3-RECEPTORS

The modulatory activity mediated by histamine receptors on the sympathetic nerve transmission was investigated in the rat vas deferens. Agonists and antagonists acting at the different histamine receptor subtypes (H1, H2 and H3) were tested on electrically-driven preparationsin vitro. Low-frequency stimulation (0.1 Hz) evoked muscle contractions almost completelysustained by ATP release, while at high-frequency stimulation (5–10 Hz) norepinephrine was mainly involved. The H1 receptor agonists, pyridilethylamine and 2-(2 aminoethyl)thiazole, enhanced the electrically evoked twitch responses, but not contractions induced by exogenously-applied norepinephrine and ATP. These effects were prevented by the H1-blocking drugs, mepyramine and phenyramine, but only at high concentrations (10 μmol/l). All these H1-antagonists strongly enhanced muscle response to electrical stimulation. The H2 receptor agonists, dimaprit, amthamine and impromidine, reduced the contractions evoked by field stimulation, but not by exogenously applied norepinephrine and ATP, the effect being antagonised by H2-blocking drugs, ranitidine and famotidine. The H3 receptor agonist,R(α)-methylhistamine, reduced the electrically evoked muscle contractions, the effect being not modified by the selective H3-blocking drug, thioperamide, but prevented by famotidine. These data suggest that rat vas deferens contains presynaptic histamine H2 receptors, able to mediate inhibitory effects on the sympathetic transmission, while histamine H3 receptors are apparently not involved. On the contrary, the role of H1 is still unclear, since both agonists and antagonists may have the same effects.

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.

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

Abstract  The inhibitory neurotransmission of the stomach was investigated in isolated guinea‐pig gastric fundus. In preparations treated with guanethidine (1 μmol L−1) and p‐fluoro‐hexahydro‐sila‐difenidol (1 μmol L−1), electrical stimulation evoked neurogenic inhibitory responses not modified by hexamethonium (100 μmol L−1), suggesting that inhibitory postganglionic non‐adrenergic non‐cholinergic (NANC) nerve fibres are involved. The nitric oxide (NO)‐synthase inhibitor Nω‐nitro‐l‐argininine‐methyl‐ester hydrochloride (1–100 μmol L−1) and the soluble guanylyl cyclase inhibitor ODQ (0.1–3 μmol 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 α2‐adrenoceptor agonist, UK 14 304 (10 nmol L−1–10 μmol 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 μ, δ and κ, muscarinic M1 and M2, histamine H2 and H3 and cannabinoid receptors. The NANC response was insensitive to the P/Q‐type Ca2+‐channel blocker ω‐agatoxin TK (1 nmol L−1–0.1 μ mol L−1), but partially inhibited by the N‐type Ca2+‐channel blocker ω‐conotoxin GVIA (0.1 nmol L−1−0.1 μmol L−1), and by the L‐type Ca2+‐channel blockers nifedipine and calcicludine (0.1 nmol L−1−0.1 μmol 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.

Histamine H3 receptors do not modulate reflex-evoked peristaltic motility in the isolated guinea-pig ileum

We investigated the role played by histamine H3 receptors in the control of intestinal peristalsis, using two different in vitro preparations of guinea-pig ileum. (a) Ileal segments were perfused from the oral end, inducing peristaltic movements (emptying waves), due to the activation of intramural reflexes. Such peristaltic motility was measured as changes in the perfusion pressure during the emptying phase and the threshold pressure for triggering the emptying wave was determined. (b) Ileal segments were mounted horizontally and circular muscle contraction evoked by the ascending peristaltic reflex was triggered by caudal distension of the intestinal wall. In perfused ileal segments, specific agonists acting at histamine H3 receptors, ((R)-alpha-methylhistamine and immepip, 1 nmol-10 micromol/l), did not cause any change in the threshold pressure for triggering the peristaltic wave, or in the rise of the perfusion pressure during the emptying phase. Similarly, circular muscle contractions evoked by caudal distension of the wall were not affected by these histamine H3 receptor agonists up to 10 micromol/l. In the same conditions, a complete inhibition of peristaltic movements was elicited by agonists acting at alpha2-adrenoceptors or adenosine A1 receptors (compound UK 14,304 and N6-cyclopentyladenosine, respectively), their effects being prevented by the respective receptor antagonists, idazoxan and 8-cyclopentyl-1,3-dimethyl-xanthine. These data demonstrate that, contrary to alpha2-adrenoceptors and adenosine A1 receptors, histamine H3 receptors are not primarily involved in the modulation of intramural reflexes that modulate the peristaltic motility of the isolated guinea-pig ileum.

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.

Water-Soluble Nitric-Oxide-Releasing Acetylsalicylic Acid (ASA) Prodrugs

A series of water‐soluble (benzoyloxy)methyl esters of acetylsalicylic acid (ASA), commonly known as aspirin, are described. The new derivatives each have alkyl chains containing a nitric oxide (NO)‐releasing nitrooxy group and a solubilizing moiety bonded to the benzoyl ring. The compounds were synthesized and evaluated as ASA prodrugs. After conversion to the appropriate salt, most of the derivatives are solid at room temperature and all possess good water solubility. They are quite stable in acid solution (pH 1) and less stable at physiological pH. In human serum, these compounds are immediately metabolized by esterases, producing a mixture of ASA, salicylic acid (SA), and of the related NO‐donor benzoic acids, along with other minor products. Due to ASA release, the prodrugs are capable of inhibiting collagen‐induced platelet aggregation of human platelet‐rich plasma. Simple NO‐donor benzoic acids 3‐hydroxy‐4‐(3‐nitrooxypropoxy)benzoic acid (28) and 3‐(morpholin‐4‐ylmethyl)‐4‐[3‐(nitrooxy)propoxy]benzoic acid (48) were also studied as representative models of the whole class of benzoic acids formed following metabolism of the prodrugs in serum. These simplified derivatives did not trigger antiaggregatory activity when tested at 300 μM. Only 28 displays quite potent NO‐dependent vasodilatatory action. Further in vivo evaluation of two selected prodrugs, {[2‐(acetyloxy)benzoyl]oxy}methyl‐3‐[(3‐[aminopropanoyl)oxy]‐4‐[3‐(nitrooxy)propoxy]benzoate⋅HCl (38) and {[2‐(acetyloxy)benzoyl]oxy}methyl 3‐(morpholin‐4‐ylmethyl)‐4‐[3‐(nitrooxy)propoxy]benzoate oxalate (49), revealed that their anti‐inflammatory activities are similar to that of ASA when tested in the carrageenan‐induced paw edema assay in rats. The gastrotoxicity of the two prodrugs was also determined to be lower than that of ASA in a lesion model in rats. Taken together, these results indicated that these NO‐donor ASA prodrugs warrant further investigation for clinical application.