Nitric oxide modulates cardiac and mast cell anaphylaxis

Anaphylaxis in the isolated guinea-pig heart was associated with a sudden release of histamine with a long-lasting release of nitrite (NO ₂ ^– ), an oxidation product of NO.N ^G-monomethyl-l-arginine (MeArg, 300 M) increased the severity of cardiac anaphylaxis, as shown by the decrease in the coronary flow and by a prolonged duration of antigen-induced arrhythmias. Concomitantly, MeArg increased the release of histamine while decreasing the release of nitrite. Sodium nitroprusside (NaNP, 10^–5–10^–4 M) reduced the severity of cardiac anaphylaxis by increasing coronary flow and shortening the duration of antigen-induced arrhythmias. Concomitantly, NaNP decreased the release of histamine while increasing the release of nitrite. In mast cells isolated from actively sensitized guinea-pigs, the release of histamine elicited by specific antigen was increased by MeArg and decreased by NaNP.In conclusion, endogenous and exogenous NO antagonizes the effect of vasoconstrictor mediators released after antigen challenge and plays a protective role in anaphylactic reactions in vitro,     

The effect of nitric oxide generators on ischemia reperfusion injury and histamine release in isolated perfused guinea-pig heart

Experiments were carried out to provide evidence of the effect ofl-arginine (l-Arg), its analogue N^G-monomethyl-l-arginine (MeArg) and of some nitrovasodilators (sodium nitroprusside, NaNP; 3-morpholino-sydnonimine, SIN-1) which spontaneously release nitric oxide (NO) on ischemia-reperfusion injury, histamine release and mast cell degranulation, occurring after multiple ligature and release of the left anterior descending (LAD) coronary artery in isolated perfused guinea-pig hearts. The reopening of the LAD coronary artery leads to a release of histamine related to a decrease in microdensitometry of cardiac mast cells and to calcium overload. The perfusion of the heart with NO-donors significantly reduces either the release of histamine, the loss of mast cell metachromasia and the overload of calcium. These effects were potentiated by SOD. The results suggest that the endogenous formation of NO and molecules able to generate NO have a role in the prevention of post-ischemic tissue injury.     

Defibrotide decreases histamine release in a guinea-pig model of myocardial ischemia and reperfusion “in vitro”

It has been shown that defibrotide (DFT), a single stranded polydeoxyribonucleotide obtained from bovine lungs, has significant anti-thrombotic, profibrinolytic properties and stimulates the synthesis of prostacyclin. The present study was designed to evaluate the effects of DFT in the isolated perfused guinea-pig heart submitted to ischemia and reperfusion.

The release of histamine and lactate dehydrogenase (LDH) after left anterior descending coronary artery ligation and reopening was measured, and the changes in electrocardiographic parameters and the computer-aided analysis of cardiac mast cell metachromasia were evaluated. DFT (8.4×10⁻⁶ M) significantly decreased both histamine and LDH release during reperfusion but not in the ischemic phase and attenuated ventricular arrhythmias induced by ischemia-reperfusion, leaving the heart rate unchanged. DFT also reduced the loss of mast cell granule metachromasia and calcium overload consequent to ischemia-reperfusion. Indomethacin (10⁻⁶ M) reduced the protective effect of DFT.

     

The effects of heavy metal ions (Cd2+, Hg2+, Pb2+, Bi3+) on histamine release from human adenoidal and cutaneous mast cells

The influence of lead (Pb[CH₃COO]₂), mercury (HgCl₂), cadmium (CdSO₄) and bismuth (BiO[ClO₄]) on the spontaneous and stimulated histamine release from human adenoidal and cutaneous mast cells was tested in the concentration range 10⁻⁸–10⁻⁴ M. Lead displayed a bell shaped dose-response relationship in adenoidal mast cells with a maximum at 10⁻⁶ M whereas in cutaneous cells only the spontaneous release was slightly enhanced at 10⁻⁴ M. Mercury induced a presumably toxic histamine release in adenoidal and cutaneous mast cells at 10⁻⁴ M. Cadmium increased the histamine release in adenoidal cells at 10⁻⁴ M but in cutaneous cells only the stimulated release (10⁻⁸–10⁻⁵ M) was affected. Bismuth inhibited the histamine release at 10⁻⁴ M in the adenoidal mast cells only. In conclusion, human adenoidal and cutaneous mast cells are affected differently by metal ions.

     

Modulation of anaphylactic histamine release by calcium channel agonists and antagonists

Calcium antagonists have been reported to exert protective effects in hypersensitivity reactions in man and animals. However, their effect on anaphylactic histamine release is highly variable and controversial. In the present paper we evaluate the effect of calcium entry blockers and BAY K 8644 on the response to specific antigen in isolated hearts taken from actively sensitized guinea-pigs and from isolated rat and guinea-pig mast cells, actively or passively sensitized. Verapamil, diltiazem, nifedipine and prenylamine dose-dependently decreased anaphylactic histamine release in isolated actively sensitized guinea-pig mast cells. BAY K 8644 was found to be ineffective. In isolated, passively sensitized rat mast cells, verapamil showed a highly signficant inhibitory effect, while prenylamine (10^–4 M) was able to evoke a histamine releasing effect. In cardiac anaphylaxis verapamil, diltiazem, prenylamine, but not nifedipine, were active in reducing the release of histamine without modifying the antigen-induced arrhythmias and positive chronotropic and inotropic effects.     

The involvement of nitric oxide synthase in the effect of histamine on guinea-pig airway smooth musclein vitro

The influence of histamine on nitric oxide synthase (NOS) in the development of airway smooth muscle hyperresponsiveness to histamine was investigatedin vitro. In the absence of histamine,N ^G-nitro-l-arginine methyl ester (l-NAME, 100 μM) had no significant effect on the basal tone. However, precontraction of the tissues with histamine (0.3 μM) resulted in a significant contractile response tol-NAME in the preparations with intact epithelium. Removal of the epithelial layer decreased the responses tol-NAME.l-arginine could partially reverse the contraction produced byl-NAME.l-NAME enhanced the maximal response to histamine, but the sensitivity of the tracheal smooth muscle to histamine was not affected. These results suggest that, in the airway, histamine can activate NOS, resulting in the release of nitric oxide. The latter may be regarded as a local negative modulator to maintain the tissue in a physiological homeostasis.

     

Tumor necrosis factor — a novel stimulus for human skin mast cells to secrete histamine and tryptase

Mast cells from infant foreskin obtained during circumcision were dispersed by an enzymatic technique, pooled, washed and purified. Mast cells, with a purity of 70–90% were incubated with 10⁻¹¹ to 10⁻⁷ M rTNFα. Histamine and tryptase levels were assessed in the cell supernatant and a concentration dependent release of histamine was observed. Histamine reached a maximum of 11.5±2.2 nmol/10⁶ cells at 10⁻⁸ M rTNF. Tryptase reached a maximum of 293±105 mU/10⁶ cells (10⁻⁸ M rTNF). rTNFα thus appears to be a direct stimulus for mast cells to degranulate and to release both histamine and tryptase.

     

Synergy between tumour necrosis factor α and granulocyte-macrophage colony-stimulating factor in neutrophil stimulation

We have examined the interaction between the cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor α (TNFα) on polymorphonuclear leukocyte (PMN) function and on PMN responses to further stimulation with formyl Met Leu Phe (fMLP). Incubation of PMN with TNFα (0.3 nM) and, to a lesser extent, with GM-CSF (1 nM) directly stimulated superoxide anion (O ⁻₂ ) generation and increased the response to subsequent stimulation of PMN by fMLP (100 nM). However, the combination of GM-CSF and TNFα did not result in increased O ⁻₂ generation and there was no synergistic effect of the combination of these cytokines on the priming of fMLP-induced O ⁻₂ generation. The combination of TNFα and GM-CSF did result in a striking synergism in the stimulation of PAF generation, and, whereas neither stimulus alone resulted in detectable PAF release, the combination elicited the release of significant levels of PAF. The observation of significant PAF release from PMN exposed to TNFα and GM-CSF indicates that overt neutrophil stimulation with phagocytic or soluble stimuli may not be required for expression of at least some of the PMN pro-inflammatory capacity.

     

Role of positive charges of neuropeptide Y fragments in mast cell activation

The incubation of neuropeptide Y (NPY) or of NPY C-terminal fragments with rat peritoneal mast cells resulted in a dose-dependent histamine secretion (10⁻⁹–10⁻⁵ M). A linear correlation between the number of net positive charges of the peptides and histamine release potencies was obtained. The histamine secretion induced by NPY fragments was inhibited by the treatment of mast cells with benzalkonium chloride and pertussis toxin indicating the involvement of G proteins.

     

The effect of calcium on cardiac anaphylaxis in guinea-pig Langendorff heart preparations

This study was designed to determine the effects of different calcium concentrations on the perfused isolated guinea-pig heart preparation subjected to cardiac anaphylaxis. Following challenge both physiological and biochemical effects were determined on hearts from guinea-pigs previously sensitized to ovalbumin. Perfusion media containing either 1, 2.54 or 5 mM of calcium was used.In comparison to nonsensitized controls challenged to ovalbumin, challenged sensitized hearts (CSH) perfused with 1 mM Ca²⁺ showed an initial increase indF/dt, a prolonged rise in H.R. and depressed coronary flow. Raising the calcium concentration to either 2.54 or 5 mM in CSH preparations resulted in a marked increase in the release of lactate dehydrogenase (LDH) into the coronary effluent and depressed coronary flow. Perfusing CSH preparations with increasingly higher calcium concentrations more often produced severe tachyarrhythmias and fibrillation. The highest level of histamine released into the coronary effluent occurred immediately following challenge and then declined exponentially over the next 20 min. Both challenge and the administration of histamine induced an immediate but transient increase in H.R., a rise indF/dt, and LDH release. The infusion of histamine produced an increase in coronary flow, but on porcine tubular coronary arterial segments only a direct constricting effect was obtained. The prior administration of cimetidine (10^–5 M) attenuated the rise in LDH anddF/dt in CSH and nonsensitized preparations infused with histamine (3 g). However, although cimetidine did not affect the decreased coronary flow in CSH preparations, it initially attenuated the rise in coronary flow in preparations infused with histamine.These results suggest that calcium enhances the liklihood of tachyarrhythmias in cardiac anaphylaxis. The release of LDH in histamine-infused preparations and those CSH preparations perfused with 2.54 and 5 mM calcium-containing medias also suggests the possibility that calcium enhances the damaging effects on the myocardial cell in cardiac anaphylaxis.This study was supported by grants from the G. and L. Pfeiffer Medical Research Foundation, the Oregon Heart Association and NIH HL-25831. The authors wish to acknowledge the excellent technical assistance of Mr Dan Austin, and the use of equipment in the laboratory of Dr J.M. Hanifin.     

Forskolin inhibits the release of histamine from human basophils and mast cells

We found that forskolin (10^–7 to 3×10^–5 M) caused dose-related inhibition of antigen-induced histamine release from human basophil leukocytes. The dose-response inhibition curve was paralleled by a forskolin-induced increase in cyclic AMP (cAMP) levels in human leukocyte preparations. The kinetics of inhibition of histamine release and of the increase in leukocyte cAMP were the same.In a second series of experiments we evaluated the effect of forskolin on antigen-induced histamine release from chopped human lung passively sensitized with serum from an allergic patient. Forskolin (10^–7 to 3×10^–5 M) dosedependently inhibited the release of histamine from human lung mast cells. Thus forskolin appears to modulate the release of mediators of the immediate hypersensitivity reaction, presumably through activation of adenylate cyclase in human basophils and mast cells.Supported by Grants from the C.N.R. (83.00430.04 and 84.01756.04) and M.P.I. (Rome, Italy).     

Potential anti-anaphylactic activity of clenbuterol,a beta-agonist with calcium antagonist properties

Clenbuterol 10^–8 to 10^–6 M inhibited antigen-induced histamine release from passively sensitized human lung tissue. This inhibition was not antagonized by propranolol, whereas the inhibitions observed with isoprenaline and fenoterol were reduced by propranolol. Clenbuterol also inhibited compound 48/80-induced histamine release and immunological histamine secretion from actively sensitized peritoneal rat mast cells. High concentrations of clenbuterol were required (10^–5 to 10^–3 M) and propranolol did not antagonize these inhibitions of histamine release. Isoprenaline and salbutamol did not modify the secretion from rat mast cells. The potential anti-anaphylactic activity of clenbuterol, might be partly related to its calcium antagonist property.     

Role of nitric oxide in the vasodilator but not exudative component of mustard oil-induced inflammation in rat skin

The participation of nitric oxide (NO) in the neurogenic inflammatory reaction of the rat hindpaw skin to topical application of mustard oil was examined by the use ofN ^G-nitro-l-arginine methyl ester (l-NAME, 43 μmol kg⁻¹ i.v.), an inhibitor of NO formation. Control rats received the same dose of the inactive enantiomerd-NAME. Vasodilatation was recorded by contactless infrared emission thermography, and plasma protein exudation was measured by the Evans Blue leakage technique and by measurement of the paw volume in anaesthetized rats.l-NAME reduced the cutaneous hyperaemia caused by topical administration of mustard oil by about 50% but did not change the exudative reaction to mustard oil. These findings indicate that NO plays a mediator role in the vasodilator component of neurogenic inflammation in the rat paw skin, whereas the increase in vascular permeability does not appear to depend on NO.

     

Does TNF-α directly increase endothelial cell monolayer permeability?

The effects of dexamethasone (DEX) and ϖ methyl ester (l-NAME) on the tumour necrosis factor-α (TNF-α)-induced increase in permeability of human umbilical vein endothelial cell (HUVEC) monolayer to [¹²⁵I] labelled bovine serum albumin (BSA) were examined. Preincubation of HUVEC monolayers with DEX (1μM, 2 h) completely abolished the effect of TNF-α (5 ng/ml, 18 h). Administration of DEX 2 h after TNF-α also reduced the effect of TNF-α, whilel-NAME (5 ng/ml, 1 mM, 18 h) had no significant effect.

The observed inhibition of the TNF-α-induced permeability increase on preincubation with DEX would suggest a role for nitric oxide (NO) in mediating the permeability response. However, this is not confirmed by the experiments withl-NAME. The inhibition caused by DEX administered after TNF-α would suggest alternative mechanisms by which DEX may be acting in addition to inhibition of NO synthase induction.

     

Inhibition of histamine release from human FcεRI+ cells by nimesulide

We have previously demonstrated that pharmacological concentrations of non-steroidal anti-inflammatory drugs (NSAID) such as indomethacin, acetylsalicylic acid, and meclofenamic acid enhance IgE-mediated histamine release (HR) from human basophils. We have now examined the effects of nimesulide (NIM), a new NSAID, on mediator release from human basophils. Preincubation (10 min at 37°C) of basophils with pharmacological concentrations (10⁻⁶−10⁻³ M) of NIM resulted in a concentration-dependent decrease of HR induced by anti-IgE, the Ca²⁺-ionophore A23187 and the formylated tripeptide f-Met-Leu-Phe (FMLP). Maximal inhibition of anti-IgE-induced HR was achieved after preincubation with 10⁻⁴ M NIM and ranged between 14.5% and 44.5% (mean±SEM, 29.7±4.5%). The drug had a marked inhibitory effect on HR from basophils induced by A23187 (80.6±5.5%), FMLP (63.5±10.3%), 12-O-tetradecanoyl-phorbol-13-acetate (57.0±8.7%) and bryostatin 1 (65.7±7.6%). NIM also inhibited the IgE-mediated synthesis of peptide leukotriene C₄ from basophils.

     

Selective activation of human mast cells by general anesthetics

We investigated thein vitro effects of increasing concentrations of three general anesthetics commonly used in clinical practice, i.e., ketamine (10⁻⁶–10⁻³ M), thiopentone (10⁻⁵–8×10⁻⁴ M) and propofol (5–70 μg/ml), on histamine release (HR) from human peripheral blood basophils and mast cells isolated from lung parenchyma (HLMC) and skin tissues (HSMC). None of the drugs induced HR from basophils. Ketamine caused HR from HLMC (p<0.001 compared to spontaneous release [SR]) and HSMC (p<0.05 compared to SR). Thiopentone induced limited HR from HLMC (p<0.05 compared to SR). Propofol induced HR from HLMC (p<0.005 compared to SR) and HSMC (p<0.05 compared to SR). Thus, general anesthetics induce HR selectively from human mast cells.

     

Defibrotide decreases histamine release in a guinea-pig model of myocardial ischemia and reperfusion “in vitro”

It has been shown that defibrotide (DFT), a single stranded polydeoxyribonucleotide obtained from bovine lungs, has significant anti-thrombotic, profibrinolytic properties and stimulates the synthesis of prostacyclin. The present study was designed to evaluate the effects of DFT in the isolated perfused guinea-pig heart submitted to ischemia and reperfusion.The release of histamine and lactate dehydrogenase (LDH) after left anterior descending coronary artery ligation and reopening was measured, and the changes in electrocardiographic parameters and the computer-aided analysis of cardiac mast cell metachromasia were evaluated. DFT (8.4×10^–6 M) significantly decreased both histamine and LDH release during reperfusion but not in the ischemic phase and attenuated ventricular arrhythmias induced by ischemia-reperfusion, leaving the heart rate unchanged. DFT also reduced the loss of mast cell granule metachromasia and calcium overload consequent to ischemia-reperfusion. Indomethacin (10^–6 M) reduced the protective effect of DFT.     

Calcium uptake in mast cells,energy metabolism and histamine secretion

The inhibition of energy metabolism of mast cells causes an inhibition of histamine secretion. As the secretion is generally initiated by the influx of calcium into the cell, we have made correlative studies of the effect of blocking the energy metabolism on calcium uptake and histamine secretion.When the influx of calcium is increased by exposing the cells to low concentrations of saponin or ionophore A23187, histamine release occurs, having the character of a secretory response. Brief incubation of the cells with antimycin A, 10^–9 M–10^–7 M, prior to exposure to saponin or the calcium ionophore gave similar dose-response curves for the inhibitory effect of antimycin A on calcium uptake and histamine release.The inhibition of calcium uptake in untreated mast cells by antimycin A, 10^–9 M–10^–7 M, showed good correlation to the inhibition of anaphylactic histamine release and the release induced by compound 48/80. The antigen-induced histamine release is dependent on extracellular calcium and an inhibition of its uptake by antimycin A could by itself inhibit the release. Compound 48/80 on the other hand induces histamine release both in the presence and absence of calcium, and both are similarly inhibited by 10^–9 M–10^–7 M antimycin A. This indicates that antimycin A has other sites of action apart from the inhibition of the influx of extracellular calcium. The inhibitory effect of antimycin A on compound 48/80-induced histamine secretion in the absence of extracellular calcium may be due to an inhibitio of energy requiring steps in the final phase of the secretory process. These steps could be phosphorylation and activation of enzymes involved in the secretion. Since calmodulin has been demonstrated in a high concentration in the cytosol of the mast cell, and histamine release induced by antigen, compound 48/80, saponin and the calcium ionophore, is inhibited by calmodulin-antagonists, it seems likely that calmodulin-dependent protein kinase plays an important role in the release process.It may then be tentatively concluded that the activation of calcium channels in the plasma membrane as well as the activation of the enzymes involved in the final phase of secretion are both are energyrequiring steps, dependent on ATP.     

The effect of Ro 21-7634 on the antigen-induced production of bronchoconstrictive arachidonic acid metabolites in the guinea pig lung

Ro 21-7634 has previously been shown to inhibit histamine and SRS-A release from actively-sensitized guinea pig lung fragments upon antigen challenge. In the studies described herein, it was observed that Ro 21-7634 does not decrease SRS-A release but instead acts to inhibit the synthesis of this mediator. This was confirmed by studying SRS-A synthesisin vitro in rat peritoneal cells after challenge with ionophore A23187. In the peritoneal cell system, Ro 21-7634 exhibited an IC₅₀ of 500 M, in comparison with 5,8,11,14-eicosatetraynoic acid, phenidone and BW755C (IC₅₀'s of 2, 100, and 100 M, respectively). When studied at 10^–4 and 10^–3 M in perfused guinea pig lung, Ro 21-7634 inhibited antigen-induced thromboxane A₂ production by 68 and 96%, respectively. In this system, antigen is believed to induce thromboxane A₂ production through the release of histamine and SRS-A from lung tissue. These mediators then interact at receptor sites in the lung parenchyma to induce thromboxane A₂ synthesis. Ro 21-7634 could thus be inhibiting thromboxane A₂ production by preventing the release of histamine and synthesis of SRS-A in the perfused lung system. Such a mechanism is suggested by the fact that although Ro 21-7634 was effective in inhibiting antigen-induced thromboxane production, it was ineffective in inhibiting thromboxane A₂ production induced in the guinea pig lung system by the direct perfusion of histamine or SRS-A through the lung.     

Heterogeneity of human basophils and mast cells in response to muscle relaxants

We investigated thein vitro effects of increasing concentrations (10⁻⁵–10⁻³ M) of four muscle relaxants (succinylcholine, d-tubocurarine, vecuronium and atracurium) on histamine release (HR) from human peripheral blood basophils and mast cells isolated from lung parenchyma (HLMC) and skin tissues (HSMC). Basophils released less than 5% of their histamine content when incubated with any one of the muscle relaxants. In contrast, mast cells showed a marked heterogeneity in their response. Succinylcholine did not induce HR from any type of mast cell, and only high concentrations of d-tubocurarine (10⁻³ M) caused HR from HSMC and HLMC. Vecuronium concentration-dependently induced HR from HLMC and HSMC. Atracurium concentration-dependently caused marked HR from HLMC and HSMC up to a maximum of 46.2±15.1% and 30.6±6.0%, respectively. From both HLMC and HSMC HR caused by atracurium and vecuronium was extremely rapid (t_1/2<1 min). The releasing activity of atracurium and vecuronium on HLMC and HSMC was reduced, but not abolished, by lowering the temperature of the incubation buffer to 22°C and 4°C. These results confirm that there are functional differences between human basophils and mast cells and among mast cells isolated from different anatomical sites in response to the muscle relaxants tested.