Activation of cannabinoid receptors prevents antigen-induced asthma-like reaction in guinea pigs.

In this study we evaluated the effects of the CB1/CB2 cannabinoid receptor agonist CP55, 940 (CP) on antigen-induced asthma-like reaction in sensitized guinea pigs and we tested the ability of the specific CB2 receptor antagonist SR144528 (SR) and CB1 receptor antagonist AM251 (AM) to interfere with the effects of CP. Ovalbumin-sensitized guinea pigs placed in a respiratory chamber were challenged with the antigen given by aerosol. CP (0.4 mg/kg b.wt.) was given i.p. 3 hrs before ovalbumin challenge. Sixty minutes before CP administration, some animals were treated i.p. with either AM, or SR, or both (0.1 mg/kg b.wt.). Respiratory parameters were recorded and quantified. Lung tissue specimens were then taken for histopathological and morphometric analyses and for eosinophilic major basic protein immunohistochemistry. Moreover, myeloperoxidase activity, 8-hydroxy-2-deoxyguanosine, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) levels, and CB1 and CB2 receptor protein expression by Western blotting were evaluated in lung tissue extracts. In the bronchoalveolar lavage fluid, the levels of prostaglandin D2 and tumour necrosis factor-alpha TNF-alpha were measured. Ovalbumin challenge caused marked abnormalities in the respiratory, morphological and biochemical parameters assayed. Treatment with CP significantly reduced these abnormalities. Pre-treatment with SR, AM or both reverted the protective effects of CP, indicating that both CB1 and CB2 receptors are involved in lung protection. The noted treatments did not change the expression of cannabinoid receptor proteins, as shown by Western blotting. These findings suggest that targeting cannabinoid receptors could be a novel preventative therapeutic strategy in asthmatic patients.

Carbon monoxide: the bad and the good side of the coin, from neuronal death to anti-inflammatory activity.

The double origin of carbon monoxide (CO) as an atmospheric pollutant or as an endogenous gaseous modulator of many pathophysiological processes prompted us to review some aspects of the bad side and of the good side of coin among the pleiotropic effects of CO. On the bad side of the coin, we focus on the interval form in acute CO poisoning, discussing experimental evidence suggesting that the delayed neuropathology after CO poisoning is a free radical-driven event. In this context, we challenge the mandatory place of hyperbaric oxygen therapy (HBO) in CO poisoning as a possible summation of oxy-radicals generated by HBO and the free radical cascade set in motion during the reoxygenation phase of acute CO-poisoning. We also discuss an opposing view, which provides evidence suggesting that HBO therapy actually decreases the load of free radicals in acute CO-poisoning and may be beneficial in preventing delayed neuropsychiatric sequelae.On the good side of the coin, we briefly outline the endogenous generation of CO and the leading role of heme-oxygenases (HO) in relation to the place of CO in biology and medicine. The main focus of this section is on the growing literature on CO and inflammation. Here we report on in-vitro and in-vivo studies on the modulation afforded by exogenously administered/endogenously produced CO in a variety of experimental and clinical settings of inflammation. Our recent studies on experimental models of allergic inflammation are also discussed, and the CO-releasing molecules envisaged as potential anti-inflammatory drugs suitable for clinical use.

Protection from cardiac injury by induction of heme oxygenase-1 and nitric oxide synthase in a focal ischaemia-reperfusion model.

The enzymes heme oxygenase (HO) generate carbon monoxide (CO) in living organisms during heme degradation. Carbon monoxide has recently been shown to dilate blood vessels and to possess anti-inflammatory properties. It is also known that nitric oxide (NO) donors ameliorate cardiac ischaemia-reperfusion injury in experimental models of global or focal ischaemia-reperfusion (FIR). The two gaseous mediators share the same mechanism of action via the stimulation of soluble guanylyl cyclase and the increase in cellular levels of cyclic GMP. We studied the effects of manipulating the HO system and the possible interaction between CO and NO in an experimental in vivo model of FIR in the rat heart. FIR-subjected rats had necrotic area in the left ventricle, ventricular arrhythmias and a shortening of survival time in comparison to sham-operated animals. Resident mast cells underwent a heavy degranulation, malonyldialdehyde was produced by myocardial cell membranes, and tissue calcium levels were increased. High levels of myeloperoxidase were also detected, suggesting a FIR-related inflammatory process. In animals pre-treated with the HO-1 inducer, hemin, all the biochemical and morphometric markers of FIR were minimized or fully abated. Consistently, the biochemical and morphometric markers of FIR were reversed in rats treated with the HO-1 blocker, ZnPP-IX, prior to hemin administration. Pre-treatment with hemin significantly increases the expression and activity of both cardiac HO-1 and iNOS, suggesting that CO and NO cooperate in the cardioprotective effect against FIR-induced damage, and that there is a therapeutic synergism between NO-donors and CO-releasing molecules, via the common stimulation of increase in cGMP levels and decrease in calcium overload.

Histamine dilutions modulate basophil activation.

BACKGROUND: In order to demonstrate that high dilutions of histamine are able to inhibit basophil activation in a reproducible fashion, several techniques were used in different research laboratories. OBJECTIVE: The aim of the study was to investigate the action of histamine dilutions on basophil activation. METHODS: Basophil activation was assessed by alcian blue staining, measurement of histamine release and CD63 expression. Study 1 used a blinded multi-centre approach in 4 centres. Study 2, related to the confirmation of the multi-centre study by flow cytometry, was performed independently in 3 laboratories. Study 3 examined the histamine release (one laboratory) and the activity of H(2) receptor antagonists and structural analogues (two laboratories). RESULTS: High dilutions of histamine (10(-30)-10(-38) M) influence the activation of human basophils measured by alcian blue staining. The degree of inhibition depends on the initial level of anti-IgE induced stimulation, with the greatest inhibitory effects seen at lower levels of stimulation. This multicentre study was confirmed in the three laboratories by using flow cytometry and in one laboratory by histamine release. Inhibition of CD63 expression by histamine high dilutions was reversed by cimetidine (effect observed in two laboratories) and not by ranitidine (one laboratory). Histidine tested in parallel with histamine showed no activity on this model. CONCLUSIONS: In 3 different types of experiment, it has been shown that high dilutions of histamine may indeed exert an effect on basophil activity. This activity observed by staining basophils with alcian blue was confirmed by flow cytometry. Inhibition by histamine was reversed by anti-H2 and was not observed with histidine these results being in favour of the specificity of this effect We are however unable to explain our findings and are reporting them to encourage others to investigate this phenomenon.

Histaminergic receptors modulate the coronary vascular response in isolated guinea pig hearts. Role of nitric oxide.

OBJECTIVE: The effects of histamine and of histamine receptor agonists and antagonists on the coronary outflow and on the generation of nitric oxide (NO) were evaluated on isolated guinea pig hearts. METHODS: Isolated guinea pig hearts were perfused for 50 min in a Langendorff apparatus with histamine (10(-7)- 10(-8) M), in the absence or in the presence of N(G)-monomethyl-L-arginine (L-NMMA, 10(-4) M), a NO synthase inhibitor and of triprolidine (3.10(-8) M) and cimetidine (10(-7) M), H(1) receptor and H(2) receptor antagonists, and with trifluoromethyl-phenylhistamine (TFMPH, 10(-7) M) and dimaprit (10(-7) M), H(1) and H(2) receptor agonists. The effects of (R)-alpha-methylhistamine (10(-7) M), a H(3) receptor agonist and of FUB 181 (10(-7) M), a H(3) receptor antagonist, were studied in the presence of bradykinin (10(-7) M). RESULTS: Histamine increases the coronary outflow and the generation of NO in a concentration-dependent fashion. The effects were completely abolished by blocking NO-synthase (NOS) with L-NMMA (10 (-4 ) M). The effects were also abolished by cimetidine (10 (-7 ) M), H (2 ) receptor antagonist, and only scarcely affected by triprolidine (3.10 (-8 ) M), H (1 ) receptor antagonist. The effects were reproduced by dimaprit (10 (-7 ) M), H (2 ) receptor agonist, and only scarcely by TFMPH (10 (-7 ) M), a selective H (1 ) receptor agonist. Bradykinin (10 (-7 ) M) produces a sustained coronary dilation paralleled by a marked increase in the generation of NO; the effects were significantly reduced by L-NMMA. The stimulation of H (3 ) receptors by (R)-alpha-methylhistamine (10 (-7 ) M) significantly reduced both effects, which reverted to normal with FUB 181 (10 (-7 ) M), an H (3 ) receptor antagonist. CONCLUSION: These results suggest that, in isolated guinea pig hearts, histamine produces coronary dilation through an H (2 )/H (3 )-dependent mechanism involving the generation of nitric oxide.

Heme oxygenase-1 and the ischemia-reperfusion injury in the rat heart.

Carbon monoxide (CO) is a signaling gas produced intracellularly by heme oxygenase (HO) enzymes using heme as a substrate. During heme breakdown, HO-1 and HO-2 release CO, biliverdin, and Fe(2+). In this study, we investigated the effects of manipulation of the HO-1 system in an in vivo model of focal ischemia-reperfusion (FIR) in the rat heart. Male Wistar albino rats, under general anesthesia and artificial ventilation, underwent thoracotomy, the pericardium was opened, and a silk suture was placed around the left descending coronary artery; ischemia was induced by tightening the suture and was monitored for 30 min. Subsequently, the ligature was released to allow reperfusion lasting for 60 min. The first group of rats was sham operated and injected intraperitoneally (i.p.) with saline. The second group underwent FIR. The third group was treated ip 18 hr before FIR with hemin (4 mg/kg). The fourth group was pretreated ip 24 hr before FIR and 6 hr before hemin with zinc protoporphyrin IX (ZnPP-IX, 50 microg/kg). Specimens of the left ventricle were taken for determination of HO expression and activity, infarct size, malonyldialdehyde (MDA) production, and tissue calcium content. FIR led to a significant increase in the generation of MDA and notably raised tissue calcium levels. Induction of HO-1 by hemin significantly decreased infarct size, incidence of reperfusion arrhythmias, MDA generation, and calcium overload induced by FIR. These effects were prevented by the HO-1 inhibitor ZnPP-IX. The present experiments show that the concerted actions of CO, iron, and biliverdin/bilirubin modulate the FIR-induced myocardial injury.

Protective effect of relaxin in cardiac anaphylaxis: involvement of the nitric oxide pathway.

1 Relaxin (RLX) is a multifunctional hormone best known for its role in pregnancy and parturition, that has been also shown to influence coronary perfusion and mast cell activation through the generation of endogenous nitric oxide (NO). In this study we report on the effects of RLX on the biochemical and mechanical changes of ex vivo perfused hearts isolated from ovalbumin-sensitized guinea-pigs induced by challenge with the specific antigen. The possible involvement of NO in the RLX action has been also investigated. 2 A 30-min perfusion with RLX (30 ng ml(-1)) before ovalbumin challenge fully abated the positive chronotropic and inotropic effects evoked by anaphylactic reaction to the antigen. RLX also blunted the short-term coronary constriction following to antigen challenge. Conversely, perfusion with chemically inactivated RLX had no effect. 3 The release of histamine in the perfusate and the accumulation of calcium in heart tissue induced by antigen challenge were significantly decreased by RLX, while the amounts of nitrites in the perfusate were significantly increased, as were NO synthase activity and expression and cGMP levels in heart tissue. 4 These findings indicate that RLX has a protective effect in cardiac anaphylaxis which involves an up-regulation of the NO biosynthetic pathway.

Differential effects of amphotericin B and liposomal amphotericin B on inflammatory cells in vitro.

OBJECTIVE AND DESIGN: To understand whether the pseudo-allergic reactions to amphotericin B (AmB) administration are due to AmB or to the solubilizing vehicles, a study was designed to evaluate the effects of AmB, liposomal AmB, (L-AmB), AmB-deoxycholate micellar complex (AmB-DC) and deoxycholate (DC) on the responses of rat serosal mast cells (RSMC) and of human basophils (HB), in vitro. MATERIALS AND METHODS: Serosal mast cells were obtained by density gradient centrifugations from male Wistar albino rats. Partially purified HB were obtained from healthy donors. The cells were exposed to AmB, L-AmB, AmB-DC and DC. Histamine release was measured fluorometrically, and the release of lactic dehydrogenase (LDH) was measured spectrophotometrically. HB activation was evaluated cytofluorimetrically by CD63 expression. RESULTS: AmB and L-AmB did not evoke histamine or LDH release from either RSMC or HB. CD63 expression was not induced in HB challenged with AmB and L-AMB. On the other hand, AmB-DC and DC produced a cytotoxic histamine release from both RSMC and HB, and a sustained increase of CD63 expression on HB. CONCLUSIONS: Only AmB-DC was able to induce the release of inflammatory mediators from RSMC and HB. Conceivably, the cytotoxic effect is accounted for by the micellar complexes with DC, which has been confirmed as a powerful histamine releaser, and held responsible for the pseudo-allergic reactions after AmB-DC administration. The data lend support to a better safety profile of L-AmB.

Haeme oxygenase-1 and cardiac anaphylaxis.

1. Haeme oxygenase (HO) is an enzyme mainly localized in the smooth endoplasmic reticulum and involved in haeme degradation and in the generation of carbon monoxide (CO). Here we investigate (1) whether the inducible isoform of HO (HO-1) is expressed in the isolated heart of the guinea-pig and (2) the functional significance of HO-1 on the response to antigen in isolated hearts taken from actively sensitized guinea-pigs. 2. Both the HO-1 expression and activity are consistently increased in hearts from guinea-pigs pretreated with hemin, an HO-1 inducer (4 mg kg(-1) i.p., 18 h before antigen challenge). The administration of the HO-1 inhibitor zinc-protoporphyrin IX (ZnPP-IX, 50 micromol kg(-1), i.p., 6 h before hemin) abolished the increase of both the HO-1 expression and activity. 3. In vitro challenge with the specific antigen of hearts from actively sensitized animals evokes a positive inotropic and chronotropic effect, a coronary constriction followed by dilation and an increase in the amount of histamine in the perfusates. In hearts from hemin-pretreated animals, antigen challenge did not modify the heart rate and the force of contraction; the coronary outflow was significantly increased and a diminution of the release of histamine was observed. The patterns of cardiac anaphylaxis were fully restored in hearts from animals treated with ZnPP-IX 6 h before hemin. 4. In isolated hearts perfused with a Tyrode solution gassed with 100% CO for 5 min and successively reoxygenated, the response to antigen was similar to that observed in hearts from hemin-pretreated animals. 5. Pretreatment with hemin or the exposure to exogenous CO were linked to an increase in cardiac cyclic GMP levels and to a decrease of tissue Ca(2+) levels. 6. The study demonstrates that overexpression of HO-1 inhibits cardiac anaphylaxis through the generation of CO which, in turn, decreases the release of histamine through a cyclic GMP- and Ca(2+)-dependent mechanism.

Histamine H3 receptor-mediated impairment of contextual fear conditioning and in-vivo inhibition of cholinergic transmission in the rat basolateral amygdala.

We investigated the effects of agents acting at histamine receptors on both, spontaneous release of ACh from the basolateral amygdala (BLA) of freely moving rats, and fear conditioning. Extensive evidence suggests that the effects of histamine on cognition might be explained by the modulation of cholinergic systems. Using the microdialysis technique in freely moving rats, we demonstrated that perfusion of the BLA with histaminergic compounds modulates the spontaneous release of ACh. The addition of 100 mm KCl to the perfusion medium strongly stimulated ACh release, whereas, 0.5 microm tetrodotoxin (TTX) inhibited spontaneous ACh release by more than 50%. Histaminergic H3 antagonists (ciproxifan, clobenpropit and thioperamide), directly administered to the BLA, decreased ACh spontaneous release, an effect fully antagonized by the simultaneous perfusion of the BLA with cimetidine, an H2 antagonist. Local administration of cimetidine alone increased ACh spontaneous release slightly, but significantly. Conversely, the administration of H1 antagonists failed to alter ACh spontaneous release. Rats receiving intra-BLA, bilateral injections of the H3 antagonists at doses similar to those inhibiting ACh spontaneous release, immediately after contextual fear conditioning, showed memory consolidation impairment of contextual fear conditioning. Post-training, bilateral injections of 50 microg scopolamine also had an adverse effect on memory retention. These observations provide the first evidence that histamine receptors are involved in the modulation of cholinergic tone in the amygdala and in the consolidation of fear conditioning.

Interactions between histaminergic and cholinergic systems in learning and memory.

The aim of this review is to survey biochemical, electrophysiological and behavioral evidence of the interactions between the cholinergic and histaminergic systems and evaluate their possible involvement in cognitive processes. The cholinergic system has long been implicated in cognition, and there is a plethora of data showing that cholinergic deficits parallel cognitive impairments in animal models and those accompanying neurodegenerative diseases or normal aging in humans. Several other neurotransmitters, though, are clearly implicated in cognitive processes and interact with the cholinergic system. The neuromodulatory effect that histamine exerts on acetylcholine release is complex and multifarious. There is clear evidence indicating that histamine controls the release of central acetylcholine (ACh) locally in the cortex and amygdala, and activating cholinergic neurones in the nucleus basalis magnocellularis (NBM) and the medial septal area-diagonal band that project to the cortex and to the hippocampus, respectively. Extensive experimental evidence supports the involvement of histamine in learning and memory and the procognitive effects of H(3) receptor antagonists. However, any attempt to strictly correlate cholinergic/histaminergic interactions with behavioral outcomes without taking into account the contribution of other neurotransmitter systems is illegitimate. Our understanding of the role of histamine in learning and memory is still at its dawn, but progresses are being made to the point of suggesting potential treatment strategies that may produce beneficial effects on neurodegenerative disorders associated with impaired cholinergic function.