Products & Services

A plant histaminase modulates cardiac anaphylactic response in guinea pig

A plant histaminase modulates cardiac anaphylactic response in guinea pig
of 7
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
  A plant histaminase modulates cardiac anaphylacticresponse in guinea pig Emanuela Masini, a Alfredo Vannacci, a Cosimo Marzocca, a Pier Francesco Mannaioni, a Olivia Befani, b Rodolfo Federico, c Alessandro Toma, c and Bruno Mondov  ıı b,* a Department of Preclinical and Clinical Pharmacology, ‘‘A. Rossi Fanelli’’ and CNR Centre of Molecular Biology,Rome University ‘‘La Sapienza,’’ Piazzale A. Moro 5, 00185 Rome, Italy b Department of Biochemical Sciences, ‘‘A. Rossi Fanelli’’ and CNR Centre of Molecular Biology,Rome University ‘‘La Sapienza,’’ Piazzale A. Moro 5, 00185 Rome, Italy c Department of Biology, University of Rome, Rome, Italy Received 24 July 2002 Abstract The effect of a copper amine oxidase (histaminase) purified from the pea seedling as free or immobilized enzyme on the response tospecific antigen was studied in isolated hearts from actively sensitized guinea pigs. In vitro challenge with the specific antigen of heartsfrom actively sensitized animals evokes a positive inotropic and chronotropic effect, a coronary constriction, followed by dilation andan increase in the amount of histamine and nitrites, the oxidation product of nitric oxide, in the perfusates. In the presence of bothforms of histaminases, the positive inotropic and chronotropic responses as well as the coronary constriction and the release of histamine were fully blocked. The amount of nitrites, appearing in the perfusates when anaphylaxis is elicited in the presence of bothformsofhistaminases,issignificantlyincreased,aswellasnitricoxidesynthaseactivityandcyclicGMPcontentincardiactissue,whilecardiac calcium overload was significantly prevented. These observations demonstrate that the decrease in the anaphylactic release of histamine and the subsequent abatement of the cardiac response to antigen can be accounted for by the inactivation by histaminase of thereleased histamineandbyastimulation ofendogenousnitricoxideproduction.   2002Elsevier Science(USA).All rightsreserved. Keywords:  Pea seedling histaminase; Cardiac anaphylaxis; Histamine; Mast cells; Nitric oxide; Cyclic GMP; Intracellular calcium The release of histamine from tissue mast cells elicitedby the cross-linking of antigen with IgE bound to specificreceptor of mast cells membrane is still considered anevent of paramount relevance in type I allergic reaction.Cardiac anaphylaxis is widely recognized as an exampleof type I hypersensitivity in which the release of hista-mine participates in myocardial damage and arrhyth-mias [1]. The model described by Cesaris Demel [2], inwhich the challenge in vitro with a specific antigen of isolated heart preparations from actively sensitized gui-nea pigs results in an increase in the rate and strength of contraction, arrhythmias, and sudden changes in coro-nary outflow, provides a reproducible tool to study thecardiac anaphylactic reaction. Amine oxidases (AOs) areenzymes widely distributed among living organisms:animals, plants, and bacteria [3]. These enzymes areinvolved in the metabolism of biogenic amines andtherefore play a pathophysiological role of primaryimportance. Histamine released during anaphylactic re-action undergoes oxidative deamination by amine ox-idases, enzymes divided into two main groups-based onthe chemical nature of their attached cofactors. The firstone (E.C. contains flavin adenine dinucleotide(FAD) as cofactor and includes mitochondrial types Aand B monoamine oxidases and cytosolic polyamineoxidases. The second one (E.C. contains copper(CuAO) and in most cases 2, 4, 5 trihydroxyphenylala-nine quinone (TPQ), a cofactor derived from the post-translational oxidation of a tyrosine residue [4].CuAOs catalyze the oxidative deamination of pri-mary amine groups of several biogenic amines in thepresence of molecular oxygen by accepting two electrons Biochemical and Biophysical Research Communications 296 (2002) 840– BBRC * Corresponding author. Fax: +39-06-4440062. E-mail address: (B. Mondov  ıı).0006-291X/02/$ - see front matter    2002 Elsevier Science (USA). All rights reserved.PII: S0006-291X(02)00938-5  from the substrate and transferring them to oxygen ac-cording to the following equationR  –  CH 2 NH 2 þ O 2 þ H 2 O ! R  –  CHO þ NH 3 þ H 2 O 2  ð 1 Þ CuAOs are frequently referred as semicarbazide-sensitive amine oxidases (SSAO) due to their charac-teristic sensitivity of inhibition by this compound.CuAOs include the subclass of diamine oxidases(DAO), which oxidize preferentially putrescine, cada-verine, and histamine. It should be pointed out thatDAO and histaminase are identical enzymes [3]. It iswell known that histamine is a crucial mediator in boththe early and late phase reaction of allergic response [5].Histamine is released by tissue mast cells and circulatingbasophils, and mimics many of the clinical symptomsdetermined by allergen exposure.In this context, the elimination of histamine per se bya specific enzyme could greatly help the therapy of al-lergic diseases. Moreover, a protective effect of bovineserum CuAO (BSAO) on heart damage was demon-strated, probably imputable to an antioxidant propertyof this enzyme against oxygen free radical [6]. Theseresults were obtained with BSAO, which shows a verylow histaminase activity. Taking into account the factthat histamine is a good substrate of the pea seedlingenzyme, the aim of this study was to evaluate the effectof a copper diamine oxidase (histaminase) purified fromthe pea seedlings as a free or immobilized enzyme on amodel of IgE-mediated allergic reaction, that is, cardiacanaphylaxis elicited in vitro, in isolated guinea pig heartsfrom actively sensitized animals. Materials and methods Purification, immobilization, and activity of the enzyme.  Pea seedlinghistaminase was purified according to McGuirl et al. [7], with a specificactivity of at least 38IU ( l moles of substrate oxidized/min) per mgprotein. The enzyme was immobilized on CNBr–Sepharose 6 MB(Pharmacia) according to [8]. Briefly, 250mg activated support wasswollen in 0.001M HCl, washed rapidly with distilled water, andequilibrated with 0.01M phosphate buffer pH 7.2. Plant histaminase(2mg) was added to 0.7ml activated gel and the mixture was incubatedfor 2h at room temperature and then for 15h at 4  C. The enzymaticactivity was assayed with putrescine 10  4 M as substrate, using thefluorimetric method of Matsumoto et al. [9], in which homovanillicacid is converted into a highly fluorescent compound by the releasedH 2 O 2  in the presence of peroxidase. The standard curve was preparedwith serial concentrations of H 2 O 2  from 0 to 20mM and the fluores-cence was read at  k ex  324nm and at  k em  426nm in a Perkin–Elmer LS50B spectrofluorimeter. The protein content was measured by theBiuret method [10], using a Perkin–Elmer  k  9 spectrophotometer. Animals.  Forty-eight male adult albino guinea pigs, Dunhin– Hartley strain, were used. They were purchased from a commercialdealer (Rodentia, Bergamo, Italy) and quarantined for 7 days at 22– 24  C an a 12-h light, 12-h dark cycle before use. Standard laboratorychow (Rodentia), fresh vegetables, and water were available ad libi-tum. The experimental protocol was designed in compliance with therecommendations of the European Economic Community (86/609/CEE) for the care and use of laboratory animals and was approved bythe Animal Care Committee of the University of Florence (Florence,Italy). At the end of the treatments, the animals weighed 350–400g. Induction of cardiac anaphylaxis.  The hearts were isolated fromguinea pigs of either sex (200–250g), sensitized by two intraperitonealinjections of crystallized ovalbumin (1ml of 1% solution) given onconsecutive days [11]. The hearts were taken 15–30 days after sensiti-zation and quickly mounted in a modified Langendorff apparatus andperfused with Tyrode’s solution at 37  C, at a constant pressure of 40cm water and gassed with a mixture of 97% O 2  3% CO 2  giving afinal pH of 7.45. The composition of the perfusion fluid was as follows(mM): Na þ 149.3, K þ 2.7, Ca 2 þ 1.8, M 2 þ 1.05, Cl  145.4, HCO  3  11.9,H 2 PO  4  0.4, and (+)-glucose 5.6 [12]. The hearts were allowed toequilibrate for 1h before further treatments. The heart rate and con-traction were recorded by means of a pressure transducer connected toa clip on the apex of the heart and recorded on a thermic writing os-cillograph (MARB, Florence, Italy). The onset and type of arrhyth-mias were monitored by means of a bipolar surface electrocardiograph.Coronary perfusates were collected over intervals of 5min in gradu-ated tubes to determine coronary flow rates and kept frozen at  ) 20  Cuntil needed. Cardiac anaphylaxis was elicited by injection into theaortic cannula of 0.1ml of 1% solution of ovalbumin in Tyrode’s so-lution 60min after the beginning of the perfusion. Coronary perfusateswere collected every 5min for 30min before and after the antigenchallenge. Free or immobilized histaminase was added to the perfusionfluid at a concentration of 100ng/ml, corresponding to 4IU/ml. At theend of the experiments, samples of cardiac tissue were collected todetermine the content of histamine, calcium, and cyclic GMP, as wellas to evaluate the mast cell degranulation and nitric oxide synthaseactivity. Determination of histamine content in the perfusates and in the heart. The histamine content in the perfusates and in the heart was measuredby a fluorimetric method using an excitation wavelength of 365nm andan emission wavelength of 455nm, according to the method of Shoreet al. [13] as modified by Lorenz et al. [14]. The authenticity of theextracted histamine was checked through the fluorescence spectra.Histaminase was added to the perfusion fluid 5min before the challengewith the antigen and continued until the end of the experiments. At theconcentrations present in the perfusates, either free or immobilizedhistaminase did not interfere with the fluorimetric assay of histamine.The values of histamine release were expressed as the percentage of total ‘‘initial’’ histamine, i.e., the ratio between histamine appearingin the perfusates and that remaining in the heart [15]. Because of fluctuations in the liberation of cardiac histamine [16], the effect of histaminase on the release of histamine was studied by comparinghistamine release in the presence of the drug with a matched control. Computer-assisted densitometry of cardiac mast cells.  Tissue sam-ples from the hearts were fixed by immersion in isotonic formaldehyde-acetic acid (IFAA), dehydrated in graded ethanol, and embedded inparaffin wax. Sections 5- l m thick were cut and stained with Astra blue,which selectively binds heparin contained in mast cell granules. Lighttransmittance across mast cells, which is inversely related to theircontent in secretory granules, was evaluated by a computer-assistedmethod, as described previously [17]. The mast cells were viewed by aCCTV television camera (Sony, Tokyo, Japan) applied to a Reichert– Jung Microstar IV light microscope (Cambridge Instruments, Buffalo,NY) with a 100 oil immersion objective and interfaced with a personalcomputer through a Matrox Marvel G400-TVcard (Matrox Graphics,Dorval, Canada). The card allows for the light transmitted across themicroscopic slide to be determined within a range of 256 gray levels,which are comprised between 0 (black level) and 255 (white level). Thecard also allows for a digitized image of mast cells to be reproduced onthe basis of the values estimated. Measurements of transmittance werecarried out using the Scion Image Beta 4.02 image analysis programScion, Frederick, MD, USA). The transmittance of 100 different mastcells, 10 from each animal of the different groups, was analyzed and themean transmittance value(  SE) was then calculated. E. Masini et al. / Biochemical and Biophysical Research Communications 296 (2002) 840–846   841  Nitrite analysis.  Release of NO  2  , stable end-products of NO me-tabolism, in heart perfusates was measured spectrophotometrically bythe Griess reaction. Half of each perfusate (about 15–20ml) was ly-ophilized separately and resuspended in 4ml water. The samples werecentrifuged and each supernatant was allowed to react with the Griessreagent (1% sulanilamide and 0.1%  N  -[naphthyl]ethylenediamine in 5%phosphoric acid) to form a stable chromophore, which was absorbedat 546nm wavelength. Nitrite concentration was calculated by com-parison with standard concentration of NaNO 2  dissolved in Tyrode’ssolution. Results are expressed as nmoles of NO  2  /ml [18]. Determination of nitric oxide synthase (NOS) activity.  Fragmentsof cardiac tissues were homogenized at  ) 4  C in buffer containing0.32M sucrose, 20mM HEPES (pH 7.2), 0.5M EDTA, and 1MMdithiothreitol. NO synthase activity was carried out by the ½ 3 H  arginineconversion assay, according to [19], with minor modifications. Part of the tissue was used for the determination of total NOS activity. Thesamples (340 l l) were added with 60 l l medium composed: NADPH þ 13.2mM, CaCl 2  3mM, calmodulin (free base, Sigma) 10 l g = ml, LL -arginine 200mM, and  ½ 3 H] L -arginine 32 l Ci = ml. After 60min of incubation at 37  C, the mixture was loaded on to 1ml DowexAG50WX-8/Na þ form column (Sigma) and eluted by 1ml HEPESbuffer, followed by 5ml distilled water. The  ½ 3 H  citrulline obtainedfrom the enzyme activity was measured with a  b -counter (Packard,Zurich, Switzerland) and the ratio between  ½ 3 H  citrulline (nmol) andmg of proteins, with the latter evaluated by the Bradford reagent, wastaken as NOS activity. Another part of the cell homogenate was usedto determine the activity of Ca 2 þ /calmodulin-independent NOS. In thelatter case, the samples were treated as above, except for the use of aCa 2 þ /calmodulin-free medium added with EGTA (6.6mM) and thecalmodulin inhibitor trifluperazine (660mM, Sigma). The activity of Ca 2 þ /calmodulin-dependent NOS was determined from the differencebetween the values of total NOS activity and those of Ca 2 þ /calmod-ulin-independent NOS activity. Calcium content.  Total calcium content was determined in hearttissue fragments by atomic absorption spectrometry, as previouslydescribed [20]. Briefly after washing the heart three times for 5min witha calcium-free buffered solution, 30mg tissue was cut from eachfragment, dried, and digested overnight at 80  C with 65% HNO 3 . Thesamples were dried at 45  C under nitrogen, resuspended in 50 l l of 32% HCl, and added with 5ml lanthanum of an aqueous solution of lanthanum chloride (LaCl 3  7 H 2 O). Lanthanium was used to removethe Ca 2 þ -binding ions, which could interfere with the measurementsupon centrifugation to remove particulate residues. The amounts of calcium in the supernatants were read in a atomic absorption spec-trophotometer (Perkin–Elmer, 303  € UUberlingen, Germany) at 422nmwavelength. The experimental values were determined by comparisonwith a standard curve of CaCl 2  and expressed as ng calcium per mgtissue (dried weight). Evaluation of cGMP.  Because NOS is known to upregulate cGMPlevels through its product NO [21], we looked for possible changes inthe cGMP content in hearts from the different experimental groups.Cardiac tissue samples were homogenized in the presence of 3-isobutyl-1-methylxanthine (IBMX 50 l M) to inhibit the phosphodiesterase ac-tivity. The levels of cGMP were measured in the aqueous phase of 5%TCA extracts of the homogenates, as described previously [22]. Thevalues are expressed as fmol of cGMP per mg protein. The proteinconcentrations were determined by the Bradford reagent [23].Materials. Putrescine, homovanillic acid, horseradish peroxidase,HEPES, and EDTA were obtained from Sigma (Milan, Italy) andCNBr–Sepharose was obtained from Pharmacia (Pharmacia, 100Route 206 North Peapack, New Jersey, USA 07977). Bovine serumalbumin was purcheased from Boehringer (Germany) and  ½ 125 I   cyclicGMP-RIA was from (Amersham, Bucks, UK). Statistical analysis.  Data are expressed as means  SEM. StatisticalanalysiswasperformedbyKruskal–WallisTest.Calculationwascarriedout using a Graphpad Prism 2.0 statistical program (GraphPad Soft-ware, San Diego, CA),  p   <  0 : 05 was considered statistically significant. Results Mechanical activity and coronary flow Antigen challenge of untreated control hearts fromsensitized guinea pigs resulted in a typical anaphylacticcrisis, characterized by sinusal tachycardia, severe ar-rhythmias, and increase in the strength of contraction(Figs. 1A and B). These mechanical and electrical ab-normalities were accompanied by a fast, short-lastingdecrease in the coronary flow, followed a sustainedcoronary dilatation (Fig. 2). When antigen challenge isperformed in the presence of free or immobilized hista-minase (4IU/ml), the positive inotropic and chrono-tropic responses to antigen were fully blocked (Figs. 1Aand B). Moreover, no short-lasting decrease in coronaryflow took place and the subsequent flow increase was Fig. 1. Heart rate (A) and strength of contraction (B) evaluated beforeand after antigen challenge of hearts from sensitized guinea pigs (10 l govalbumin). Free ( j ) and immobilized ( ) histaminase (4IU/ml)prevent the chronotropic and inotropic effects of antigen administra-tion observed in the untreated control hearts ( M ). Measurements weretaken at the peak of the anaphylactic response (5min after antigenchallenge). Data are expressed as means  SEM of eight experiments.*  p   <  0 : 001.842  E. Masini et al. / Biochemical and Biophysical Research Communications 296 (2002) 840–846   significantly higher than that in the untreated controlhearts (Fig. 2). Histamine release and mast cell degranulation Under basal conditions in the control hearts, the levelof histamine released was very low and not affected bythe perfusion with histaminase. The overall histaminerelease was evaluated within 30min after antigen chal-lenge. In control experiments, about 58–59% of the en-dogenous histamine was released, results similar to thoseof previous experiments [24]. The amount of histamineappearing in the perfusates showed a peak (348 : 9  27ng ml  1 ) within the first 5min after antigen challenge,followed by a significant decline (150 : 21  11 : 3ng ml  1 )in the following 5min and further decrease (88 : 4  9 : 2ng ml  1 ) at 30min (Fig. 3). In hearts from sensitizedanimals treated with free or immobilized histaminase,the amount of histamine appearing in the perfusates wassignificantly lower than control values especially in thefirst 5min (from 351 : 3  39 : 6 to 134 : 4  8 : 6ng ml  1 inthe presence of free histaminase, and to 99 : 6  8 : 4ng ml  1 in the presence of immobilized histaminase, n ¼ 8 ;  p   <  0 : 001) (Fig. 3).To establish the mechanisms involved in the protec-tion of histaminase, we determined the amounts of his-tamine retained in the heart and mast cell degranulation.Consistently, the amount of histamine retained in theheart after antigen challenge was not modified by his-taminase treatment (Fig. 4A). The concept that the twohistaminases did not produce any chromoglycate-likeeffect on the anaphylactic degranulation of mast cells isstrengthened by the fact that the light transmittanceacross mast cells, which is related to their content insecretory granules, was markedly increased in the heartchallenged with antigen, in comparison to controls,showing mast cell degranulation. Neither free nor theimmobilized histaminase modifies the increase in lighttransmittance through cardiac mast cells in response toantigen (Fig. 4B), meaning that the enzyme failed toexert any influence on the anaphylactic degranulation of mast cells.  NO   2  release and NOS activity Isolated and perfused guinea pigs hearts constantlyrelease NO  2  , an oxidation product of NO, into thecoronary effluent. Ovalbumin challenge of untreatedsensitized hearts released an amount of NO  2  higherthan those from unsensitized animals, confirming ourprevious results [25]. The amount of NO  2  in the perfu-sates showed a peak (1204 : 12  242pmol ml  1 ) withinthe first 10min after antigen challenge, followed by asignificant decrease (532 : 41  97pmol ml  1 ) at 30min.The release of NO  2  in the perfusates was significantly Fig. 2. Coronary flow in hearts from sensitized guinea pigs challengedwith the antigen (10 l g ovalbumin) compared with the untreatedcontrol hearts (white columns). Free (black columns) or immobilized(gray columns) histaminase (4IU/ml) prevents the short-lasting de-crease in coronary flow, evoked by antigen administration, and pro-motes a subsequent sustained flow increase. Data are expressed asmeans  SEM of eight experiments. *  p   <  0 : 01, **  p   <  0 : 05 vs control.Fig. 3. Histamine release in the perfusates from untreated controlhearts (white columns) and hearts treated with free (black columns) orimmobilized (gray columns) histaminase (4IU/ml). Data are expressedas means  SEM of eight experiments. *  p   <  0 : 001, **  p   <  0 : 01.Fig. 4. Histamine retained in the heart (A) and light transmittanceacross Astra blue stained mast cells (B) of control hearts from un-sensitized animals (a), after antigen challenge in untreated controlhearts from sensitized animals (b) and in hearts treated with free (c) orimmobilized (d) histaminase (4IU/ml). Data are expressed as means  SEM of eight experiments. *  p   <  0 : 001 vs unsensitized animals. E. Masini et al. / Biochemical and Biophysical Research Communications 296 (2002) 840–846   843  increased in the hearts treated with both forms of his-taminases particularly in the late phase of anaphylacticreaction, 20–30min after antigen challenge (Fig. 5A).Moreover, NOS activity measured as  ½ 3 H  arginineconversion assay showed that, as compared with theuntreated controls, the hearts treated with free or im-mobilized histaminase underwent a significant increasein both Ca 2 þ -dependent and, Ca 2 þ , and calmodulin-in-dependent, NOS activity (Fig. 5B). Intracellular cGMP and calcium levels It is well known that NOS activity regulates cyclicGMP levels through its product NO [21] and cyclicnucleotides and tissue calcium levels play a key role assecond messengers in type I anaphylactic reactions [26].In our experiments, antigen challenge produced a smalldiminution of cardiac cyclic GMP levels. In the heartstreated with both histaminases, a significant increase incGMP levels took place as compared with the untreatedcontrol hearts (Fig. 6a). Cyclic GMP inhibits intracel-lular Ca 2 þ fluxes in cardiac preparation both in vitro [27]and in vivo [28]. In our experiments, treatment of thehearts with free or immobilized histaminases before andduring antigen challenge caused a significant reductionof calcium content in myocardial tissue as comparedwith the untreated control hearts (Fig. 6B). Discussion The present study indicates that ex vivo perfusion of hearts isolated from ovalbumin-sensitized guinea pigswith histaminase isolated and purified from pea- seed-ling affords a marked protection against the alterationsinduced by challenge with the specific antigen. Theanaphylactic reaction of isolated sensitized guinea pighearts is characterized by typical changes in the cardiacfunction, accompanied by a release of mediators of anaphylaxis such as histamine, platelet activating fac-tor (PAF), and arachidonate metabolites [16,29–31].The characteristic profile of coronary constriction afterantigen challenge in isolated guinea pig heart can easilybe differentiated into an early, more severe, phase anda late, less pronounced phase. The contribution of endogenous cardiac histamine to the dysfunction as-sociated with immediate hypersensitivity has been ex-tensively investigated in isolated guinea pig hearts[24,32]. Histamine, which is released from cardiac mastcells, is responsible for the inotropic, chronotropic, andidioventricular arrhythmias and for the change incoronary flow that characterizes cardiac anaphylaxis[16,24,25,33,34]. In fact, cardiac anaphylaxis is bluntedby H 2 -receptor against such as 4-methyl-histamine [35],dimaprit, and impromidine [24], which inhibit the Fig. 5. Nitrite release (A) in the perfusates of hearts from sensitizedguinea pigs, challenged with the antigen (10 l g ovalbumin) comparedwith the untreated control hearts (white columns). Free (black col-umns) or immobilized (gray columns) histaminase (4IU/ml) causes asustained increase in nitrite amounts. Data are expressed as means  SEM of 8 experiments. *  p   <  0 : 001, **  p   <  0 : 01. Activity of NOS (B)by hearts from sensitized guinea pigs challenged with the antigen (10 l govalbumin), ½ 3 H]arginine conversion assay showing an increase in bothcalcium-dependent and calcium- and calmodulin-independent NOSactivities in the hearts treated with free (black column) or immobilized(gray column) histaminase (4IU/ml), compared with the untreatedcontrol hearts (white column). Data are expressed as means  SEM of eight experiments. *  p   <  0 : 001 vs unsensitized animals.Fig. 6. Tissue levels of cGMP (A) and tissue calcium (B) in controlhearts (a), after antigen challenge (b) and after antigen challenge inhearts treated with free (c) or immobilized (d) histaminase (4IU/ml).Data are expressed as means  SEM of eight experiments. *  p   <  0 : 001vs control hearts.844  E. Masini et al. / Biochemical and Biophysical Research Communications 296 (2002) 840–846 
Similar documents
View more...
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks