Expression of histamine HI receptors in autoimmune myocarditis mice

NORA GOREN, CLAUDIA PEREZ LBIROS, LEONOW STERIN-BBRDA, AND ENRI BOHBDA~ Centre de Estudios F ~ T ~ Q c Q ~ ~ ~ ~ c Q s y Bothnicos (CEFBIBO), Consejo 1Yacionak de Investdgacidn

CientGcas y TTe'cnic~s (CO~VICETJ '), Serrano 665, H 414 Blaenos Aires, Argentina

Received July 28, 1992

G ~ R E N , N., PEREZ LBIROS, C., STERIN-BORDA, L., and B ~ R D A , E. 1993. Expression sf histamine H, receptors in auto- immune mywarditis mice. Can. J . Physiol. Pharmacol. 'PI: 639 -644.

Two populations of histaminergic H, receptors with distinct high and low affinity binding sites were characterized by the specific H I receptor antagonist [%]mepyrarnine in autoimmune myocardium. No saturable binding of the radiolabelled H, antagonist was observed in normal myocardium. Reaction of autoimmune myocardium with specific H, agonist (2-thiazolyl- ethylarnine (ThEA)) triggered positive inotropy and negative chronotropy, which were inhibited by rnegyramine. Inhibitors s f phospholipase C and protein kinase C attenuated both the inotropic and chronotropic effe~ts of ThEA, suggesting the partic- ipation of phosphoinositide hydrolysis in this phenomenon. The latter was verified by measurement of polyphosphoinositide hydrolysis in autoimmune myocardium following the reaction of ThEA with histarninergic M I receptors. We conclude that functionat HI histaminergic receptors could involve a distinctive mechanism operating in autoimmune myscardiurn as a result of cardiac antigen immunization.

Key words: histamine, H, receptors, myocarditis. autoimmunity

GORBN, N., PEREZ LEHROS, C., STERIN-BORDA, L., et BQRDA, E. 1993. Expression of histamine HI receptors in auto- immune myocarditis mice. Cam. J . Physiol. Pharmacol. '71 : 639-644.

k u x ppulations de rkcepteurs histaminergiques MI ayant des sites de fajlble et de haute affinitC distincts ont 6t6 caractkrisks par 19antagonists spksifique des rkcepteurs HI ["]mdpyrarnine dans le mywarde autoimmun. Il n'y a pas eu de fixation saturable de I'antagoniste H, radiomarque5 dans le myocarde normal. La rkaction du myocarde autoimmun avec I'agoniste H, s@cifique (ThEA) a dkclenchC une inotropie positive et Pane chronotropie nkgative, qui ont kt6 inhibkes par la rnkpyra- mine. Les inhibitems de phospholipase 6 et de protCine kinase G owt attinu6 les effets instropes et chronotropes de ThEA, suggkrant la participation de l'hydrolyse des phosphoinositides, laquelle a CtC vCrifiCe en mesuraaad I'hydrolyse des phosphai- nositides dans le myscarde autoimmun aprbs la rkaction de ThEA avec les rkcepteurs histaminergiques H,. Nous concIuons que les rkcepteurs histarninergiques HI faanctionnels pourraient faire intervenir un mkanisme distinct agissant dans le myo- carde autoirnmun i cause de l'immunisation antigdnique.

Mots cl&s : histamine, rkcepteurs H,, myocarde, autoimmunit6. [Traduit par Ia r6dactionl

Introduction It is well h o w n that histamine, which is a normal consti-

tuent of the mammalian heart, can produce an increase in the force of cardiac contraction in vivs and in vitro (Levi et d. 1982). The positive inotropic effect of histamine is mainly due to activation of H2 receptors in mammalian heart (including human), and is associated with the stimulation of adenylate cyclase activity (Gus et d. 1984; Hattori et d. 1983). However, in guinea pig left atria (Hattori and Kanno 1985) and rabbit papillary muscle (Hattori et al. B988), histamine produces a positive inotropic effect mediated by HI receptors that is not associated with an increase in cyclic AMP (CAMP) levels (Verma and McNeill 1977).

By contrast, histamine HI receptor stimulation has been shown to stimulate the hydrolysis of phosphoinositides (PIS) in guinea pig atria (Hattori et sal. 1989). The hydrolysis of PI results in the formation of two putative intraceUular second messengers, inositol trisphosphate (IP3), and diacylg~ycerol (DAG) (Begridge and Irvine 1984). IP3 and DAG act through different pathways of intracellular signal transduction and cause important physio- logical responses in a variety of tissues (Nishizuka 1984). However, the role of the hydrolysis of PI in the regulation of the haanction of cardiac muscle is still far from clear.

We have previously shown that in normal spontaneously beating mouse atria, histamine in a concentration higher than

receptor effect of histamine is related to the cANIB production (Goren et al. 1991). No effect of histamine on HI receptors was detected.

It is well h o w n that histmine is a mediator of the immediate hypersensitivity reaction of the heart and plays an important role as a modulator sf immune response (Melmsn and Khan 1987). Moreover, the contribution of histamine to the func- tional crisis that characterizes the anaphylactic reaction of the heart has been documented (Eevi and Burke 1980). However, the actions that histamine has ow autoimmune cardiac disease remain open to question.

Immunologic mechanisms can release endogenous histamine from the heart (Eevi and Burke 1988). Thus, the heart may be exposed during the immune response to rather low concentra- tions of histamine that could be enough to modify automaticity and contractility.

In this study we investigated the occurrence sf histarninergic receptors, and the consequences of their stimulation by low concentrations of the arnine on the contractility of the myo- cardium from autoimmune myocarditis mice. We demonstrate that myocardium from experimental autoimmune myocarditis expresses HI receptors not present in normal heart. The stimu- lation of histamine H, receptors by the agonist triggers posi- tive inotropic and negative chronotropic effects, which are related to the activation of PI hydrolysis.

1 pM exerts positive chronotropic accompanied by- negative inotropic effects, both of which are mediated by Hi2 receptors. Materials and methods

Furthermore, we have found that the positive chronotropic H2 of CF% mice, 45 days old, were used in this study. They were sepa-

'Author for csrrespndence. rated into three groups of the same age: (1) immunized with isogenic Printed in Canah / Imprime au Canada

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648 CAN. 5 . PHYSIOL. PH.4RMACOL. VOL. 41, 1993

myocardium and adjuvant; (2) immunized with adjuvant only; and (3) nonimmunized (normal). Groups 2 and 3 were rased as controls.

Immunization procedures were carried out as previously reported (Cossio et al. 19W), according to the animal care guidelines of Argentina institutions. Animals were sacrificed 40 days postimmuni- zation.

Immunization was assessed as previously reported (Cossio et al. 1984; Perez Leiros et al. 1988, 1990), and circulating reactive anti- bodies in cardiac membrane were detected by ELISA (titers were always higher than 300)-

Atria! mechalaicwl activity Mice were decapitated, and auricles were quickly removed and placed

in a glass chamber containing a modified Krebs - Ringer bicarbonate ) solution, gassed with 5 % CO, in oxygen, maintained at pH 7.4

and 30°C. The ionic composition of KRB was (in mM) NaCl, 120.50; KCl, 4.83; CaCl,, 1.22; KH,PO,, 1.21; MgSO,, 1.34; NaHCO,, 24.50; and glucose, 5.5. One end of the preparation was anchored to a glass holder and the other connected to a force transducer coupled to an ink-writing oscillograph (Borda et al. 1984). A constant resting tension of 500 mg was applied to the atria by means of the micro- metric device, and the activity of the isolated spontaneously beating or driven atria was analyzed in terms of amplitude and frequency of the contractions. The atria were allowed to equilibrate for 60 rnin before the interventions; values at 40 min were taken as control (IQO % ). (The absolute values of tension (milligrams) and frequency (katslmin) are given in Table 2.) Concentration - response data were obtained by cumuiative addition sf specific H, agonist to the tissue bath, and the time interval (5 min) between doses was sufficient for each individual dose to produce its maximal effect. Mepyramine (H, antagonist) and enzymatic inhibitors were added to the tissue baths 30 min before the responses to specific H, agonist were tested. Chronotropic effects were evaluated while the atria were beating spontaneously; inotropic effects were assessed from records of isometric tension development during electrical stimulation at a fixed frequency close to the resting heart rate in mice (200 beatslmin). The stimuli had a duration of 2 ms and their voltage was 10% above threshold.

Binding assay [3H]MepymHl%jlne (pyrilaranine, pyridinyl-5-'H) (New England Nuclear

Co., Du Pont, U.S.A.; specific activity 25 Ci/mmol(l Ci = 37 GBq)) binding to mouse cardiac membranes was performed essentially as previously described (Chang et al. 1978, 1979; Tran et al. 1978). This radioligand has been widely used to characterize H, receptors in various tissues (Hill 1990). Freshly dissected hearts from control and autoimmune mice were freed from connective tissue, fat, large vessels, and blood, and then homogenized at 4'C in HO volumes of 5 mM Tris-HCl (pH 7.4), 1 mM MgCl,, and 250 mM sucrose in an Ultra- Turrax at settings 4, 8, and 10 for 15 s each. The hornogenate was centrikged twice at 4OC and 3000 x g for 10 min. Supernatants were sequentially spun at 10 000 X g for 10 rnin and 40 000 x g for 90 min. The resulting pellets were resuspended in 50 mM Na+ - K+ phosphate buffer (pH 7-44), and the protein concentration was adjusted to about 3.0 rng/mL.

Membrane suspension from normal, adjuvant-treated, or auto- immune hearts (200 - 300 pg protein) and various concentrations (0.2-20 nM) of [3H]mepyrarnine were incubated for 30 min at 25°C in a total volume s f 150 p L of Na+-K' phosphate buffer. Non- specific binding, determined by incubating membranes in the presence of 10 FM mepyramine, did not exceed 40% of the total binding at [3H~mepymmine from 0.2 to 8 nM. Incubations were stopped by addition of ice-cold Na ' - K + phosphate buffer followed by rapid filtration on Whatman GF/c under mild pressure. Filters were washed with 16 mL of the same buffer, placed into vials, and counted in Triton -toluene based scintillation cocktail in a Beckman scintillation counter with 65% efficiency. The equilibrium dissociation constant (&) and the maximal number of binding sites (B,,,) were calculated using Scatchard analysis. Competition assays were run similarly except that five concentrations of various dmgs were included in the same incubation volume with [3H]mepyramine, fixed at 0.5 nM.

Measurememt of to tab labelled inositol phosphate Water-soluble HP was extracted after 120 min incubation following

the method of Berridge (1982) and Gorelik et al. (1992). Atria were quickly washed with KRB and homogenized in 0.3 mL of KRB and 10 mM LiCl and 2 rnL chloroform-methad ( 1 2 , v/v) to stop the reactions. Chloroform (0.62 mL) and water (l mE) were subsequently added. Samples were centrifuged at 2800 X g for 15 min, and the aqueous phase of supernatants (1 -2 mL) was applied to a 0.7-mL column of Bio-Wad AG B x 8 anion-exchange resin (100-200 mesh) suspended in 0. 1 M formic acid, which was previously washed with 10 mM Tris-formic, pH 7.4. The resin was then washed with 20 a d - umes of 5 mM myo-inositol (MI) followed by 6 volumes of water, and PIS were eluted with 1 EQ4 ammonium formate in 8.1 M formic acid. Fractions of I mL were recovered and radioactivity was deter- mined by scintillation counting. Beak areas were determined by triangu- lation. Results, corresponding to the second peaks, were expressed as counts per minute (cpm) per milligram tissue wet weight. To deter- mine the absence of [3H]ME in the eluted peaks of IPS, chromatog- raphy in silica gel 60 H254 sheets (Merck, Darmstadt, Germany) was performed using propan-2-01 - 6 N NH,BH (1415) as developing solvent (Hokin-Neaverson and Sadeghian 1976). Spots were located by spraying with freshly prepared 0.1 % ferric chloride in ethanol, followed, after air drying, with H % sulfosalicyclic acid in ethanol, as described (Hokin-Neaverson and Sadeghian 1976). To assay the radio- activity, a histogram was constructed by cutting up of the sheet gel, placing each sample in Triton-toluene based scintillation fluid, and then counting.

Drugs The following dmgs were used: 2-thiazolylethylamine (ThEA),

kindly provided by Smith Uine Lab. (Philadelphia, Pa.), and mepysa- mine, cimetidine, histamine, diphenhydramine. atropine, psopranolol, phentolamine, 1-(5-isoquinolinylsulfony1)-2-methy1piperaz:ine dihydro- chloride (H-7), and 2-nitro-4-carboxyphenyl-N9N-dipheny1carbamate (NCBC) (Sigma Chemical Co., St. Louis, Mo.). Stock solutions were freshly prepared in distilled water, except for NCDC, which was in dimethylsulfoxide. The davgs were diluted in the bath to achieve the final molar concentration indicated in the text.

statistic^ Student's t-test for unpaired values was used to determine the levels

of significance. Differences between means were considered signifi- cant at p 5 0.05.

Results To identify histamine HI receptors on cardiac membrane,

binding studies were carried out with [%H]mepyramine on normal, adjuvant-treated, and autoinmune myocardium. A hnc- tional correlation between binding data and pharmacological activity was used to demonstrate the specificity of the recep- tor site.

[%H]Mepyrarnine specifically and reversibly bound to cardiac membrane from autoimmune myocarditis mouse in a time-, temperature-, and concentration-dependent manner (Fig. I).

In autsimmune heart preparations [%]rnepyramine bound to high and Isw affinity binding sites demonstrable by Scatchard analysis sf saturation curves. The Kd values were 0.33 f 0.M nM for the high affinity component and 12 f 1.9 amM for the Isw affinity component, The B,,, of low affinity bind- ing sites (82 t 15 fmolirng protein) was significantly greater than that of the high affinity binding sites (19 + 1.2 fmolirng protein) (Fig. 2). The Ki values calculated from competition curves of hishrninergic agcsnists and antagonists showed an order of potency typical of HI histarninergic receptors (Hill 1990) (Table 1).

Saturable binding of [3M]rnepyramine is dso observed in adjuvated-treated membranes bound to high and Isw affinity

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@OWEN ET AL.

TIME (rnin)

FIG. I . [3H]Mepyramine (MEP) binding to cardiac membrane of autoimmune myocarditis mice. (A) Time course of the association reaction (0) and the dissociation reaction (@). For the association reaction 200-360 pg of membrane and 0.6 nM E3PI]MEP were incubated at 25OC for different times and the specific binding was determined in the presence of M mepyramine for the dissocia- tion reaction, at 40 rnin M mepyrarnine was added (indicated by the arrow). and the specific binding was determined at the differ- ent times shown. (B) Effect of temperature (T) on E3H]MEP specific binding. (C) Protein concentration dependence of [3H]MEP binding. Binding data are means f SEM of four experiments done in duplicate.

TABLE 1. Competition of histaminegic agonists and antagonists with [3H]mepyramine on membrane

from autoimmune myocarditis mice

Dmgs K, ((cLM)

Mepyramine Diphedydramine ThE A Histamine Cimeaidine

NOTE: Assays were pcdomed at the [3H]mepyramine concentration of 0.4 nM, and different concentrations of uniabelled drugs were used to obtain IC,, values. The equilibrium dissociation constant (K,) was calculated according to Cheng and Pmssoff (19'73) (K, = KJ(l + LIiEl,), whre IC, is the competing ligand concentration which half-maximally inhibib the specific binding of the radioligand at a concentration &. Propranolol, atropine, and pheratslamine at concentrations greater than 0.1 m M were unable to compete with the radioligand.

binding sites (Kd: 1.40 +_ 8.11 and 6.25 + 1.38 nM; B,,,: 25.6 + 2.2 and 84.4 f 3.8 fmsllmg protein). The Kd value of the high affinity component in adjuvant-treated cardiac membrane was significantly higher thaw in autoimmune myo- carditis heart ( p < 0.001). No saturable binding was detected in cardiac preparations from normal mice. To analyze the bio- logical activity of the HI receptor on heart contractility, we studied the effects of the HI receptor agonist TREA on con-

20 .XI 60

B (hollrng protein)

FIG. 2. (A) Saturability of specific [%H]mepyramine (MEB) bind- ing to cardiac membranes from autoimmune myocarditis mice. Prokin (2 mg6mL) was incubated at 25°C for 30 min with different concen- trations of [%W]MEP. Nonspecific binding was determined in the presence of 10-% mepyramine. (B) Scatchard analysis of the data. This assay is representative of six others performed in duplicate. B, bound; F, free.

A

30- .- z 3 20 0 3 zg u .- z , ,@-

32 Ez k- '5 s to- EE lo V

FIG. 3. CaamuBaeive dose -response curve of ThEA on the tension and frequency of atria isolated from mice with autoimmune myo- carditis in the absence (2) and in the presence of 5 x M NCDC (8) and 10-% H-7 (A). NCDC and H-7 were added to the tissue bath 30 min before the response of ThEA was tested. Values are means k SEM of seven experiments in each group. Frequency was evaluated in spntanmusly beating atria and tension was measured in fixed paced rate atria at 220 beats6min.

tractde tension md beating frequency of atria from autoimmune, adjuvant-treated, and normal mice.

The addition of ThEA to spntaneoudy beating atria caused effats that were dependent on the source (control, autoimmune) of the myocardium. In atria from autoimmune myocarditis mice, ThEA exerted positive inotropic and negative chrono- tropic effects in a concentration-dependent manner; however, it was without any effect on normal or adjuvant-treated heart (Table 2; Fig. 3). The effect of ThEA was specific as mepyra- mine blunted the contractile effect of the agonist (Table 2). Developed tension and beating frequency before the addition sf ThEA were similar in normal, adjuvant-treated, and auto- immune atria (Table 2).

To determine the nature of the mechanisms triggering posi- tive inotropy and negative chronotropy by ThEA in autoimune atria, the modifying actions s f inhibitors s f phosphslipase @ (NCDC) and protein Emnase C (H-7) were explored. As shown in Fig. 3 , both NCDC (5 x 10-%) and H-'7 M) reduced the inotropic and chronotropie effects of ThEA on autoimmune atria. However, neither H-'7 nor NGDC at these respective concentrations modified the basal tension and fre- quency (Table 3).

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CAN. J. PHYSIBE. PHARMACOL. V8L. 71, 1993

TABLE 2. Inf'luence of HI agonist and antagonistic dmgs on the tension and frequency of isolated atria from control and autoimmune myocarditis mice

Normal Adjuvant Autoimmune

Additions T F T F T F

None 348+28 193f20 367f26 204f18 342f25 207f16 ThEA 362&-31 1784f19 372k33 198i-15 455f22* H55&1I** ThEA+MEB 341$3fB 2Wf21 359f38 210f19 348f26 200f14

NOTE: T (tensisn) and F (frequency of contraction) are expressed in milligrams and beats per minute, respectively. The concentration of ThEA (lWR M) was that which caused 100% of the maximam response. MEP (mepyramine, M), was added 30 rnin before the initiation of the dose-response curve of the agonist. Values are mean f SEM of six experiments in each group. Asterisks indicate significant difference from none addition: *p < 0.005 and **p < 0.025.

TABLE 3. Effect s f different dmgs on the basal values of tension and frequency of autoimmune atria

Dmg Tension (mg) Frequency (beatstrnin) n

None 350f23 195 f22 6 NCBC 3602 29 182f 18 7 M-4 342 f 20 198+23 4

Noas: Values are mean f SEM. Initial control values (no addi- tions) were recorded after an equilibration period (H? min) follow- ing set up s f the preparations. Experimental values were recorded 38 rnin after adnzinistration of each drug. r t , number sf prepara- tions tested.

TABLE 4. Stimulation of PI turnover by ThEA on autoimmune anyoeardium

PI formation (epnl . mg - l wet weight)

Additions Autointmune Adjuvant Normal

Basal 247f 14(6) 288f l6(6) 254f 15(7) ThEA 51 1 +22*(7) 380+29(7) 270f 18(7) Mepyramine + ThEA 243 f 18(5) 293 f 3 8(5) 246 + 2 B (5) NCDC + ThEA 215f 2045) 287&21(5) 269k 19(5)

NOTE: Atria from normal, autoimmune, and adjuvant-treated CHI mice were incubated fc>r 643 min with 13Hlmvo-inositol and for an additional 30 min in the absence or presence o f bl&cers: 10-% mepyramine or 5 X M To ThEA actions On phosphoiipase (PLC) induced NCDC. Tissues were then left for a further 30 min in the absence (basal) or

intracellular second messengers in cardiac tissue, mice atria in the Dresence of 5 x 10 -9 M T ~ E A . Results are mean + SEM. Numbers - were incubated with [%]MI and EiCl (10 mM) for inositol in parlntheses are number of preparations tested. phosphate ThEA (5 x 18-9 M) produced an *Significantly different from basal value, p < 0.001.

increase in inositol phosphate formation in myocardium from autoimmune mice Gut had no effect on normal or adjuvant- treated heart (Table 4). This effect was abolished by pretreat- ing atria with M mepyramine, suggesting HI receptor participation. Fufihemore, 5 x 10-% NWCDC, a PLC inhib- itor, also abrogated the effect of ThEA on phosphoinositide turnover, suggesting that PLC-mediated hydrolysis sf phos- phoinositide turnover was involved in the phenomenon.

Discussion

The ma~or finding of the present study is that active hista- mine HI receptors are expressed in myocardium from auto- immune mice, while no binding can be detected in normal

non-Hi receptor binding sites in rat liver plasma membrane, with a Kd value close to the one we obtained for low affinity sites. The low affinity site, which appears to be inactive, may represent ethylenediamine recognition sites (Leurs et al. 1989). The high affinity H1 receptors present in autoimmune heart appeared to be in an active state, since they were capable of reacting with the specific HI agonist, modifying PI turnover, and altering the contractile parameters of the autoimmune heart. By contrast, the receptors present in adjuvant-treated myo- cardium seemed to be in an inactive state, since they were unable to mediate agonist responses.

In accordance with the binding results, contractile measure- heart and no active histamine HI receptors appear to be present ments in autoimmune myocardium indicated that H, histamin- in adjuvant-treated heart. ergic agonist (ThEA) induces positive inotropy and negative

~u~oimmune myocardium displayed two populations of [3H]mepyramine binding sites with low and high affinity for the histamine antagonist, as demonstrated in binding studies. The high affinity component seems to be HI selective, since the order of potency of the agonists and antagonists and the values of Kd calculated are similar in both quality and number to that reported in the literature for guinea gig heart, one of the best models of cardiac anaphylaxis (Levi and Burke 1980; Chang et al . 2 979). By contrast, E3H]mepy ramine binding sites expressed in myocardium from adjuvant-immunized mice displayed lower affinity than sites in autoimmune myocardium.

It is possible that the low affinity component does not indi- cate the presence of true histamine Hi receptors. Recently, Leurs et al. ( 1989) demonstrated that ["Imepyramine labels

chronokopy. It is important to note that the kd calculated for the high affinity component of the Hi receptor population predicted by binding competition experiments correlates with the agonist concentration required for half-maximd contractile effect on autoimmune myocardium. This points to a specific coupling of these receptors to HI-agonist response. These effects were observed ody in atria from autoimmune myocarditis mice, sug- gesting that the fbanctional HI histaminergic receptors could involve a distinctive mechanism operating in autoimmune myocardium as a result of cardiac antigen immunization.

It is known that activation of HI receptors stimulates the hydrolysis of phospholnositides, and that this could provide the initial stimulus for calcium release (Hattori et al. 1988). The phosphoinositide hydrolysis could modify the contractile

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GOREN ET AL. 643

behaviour of the heart by production of IP3 and intracellular calcium mobilization, and by production of DAG with subse- quent activation of protein kinase C (Brown et al. 198%). The fact that NCDC and H-7 at concentrations known to inhibit PEC (Walenga et al. 1986) and protein kinase C (Hidaka et al. 19841, respectively, attenuate both the inotropic and chrsno- tropic effects of ThEA in autoimmune myocarditis mice atria suggests that phosphoinositide hydrolysis and probably protein b a s e C activation are involved in the contractile activity. However, because the inhibition of protein kinase C only par- tially blocked the contractile effect of ThEA, it is not possible to rule out the participation of HP3 as another messenger of this effect.

Sakuma et a%. (1988) have reported a rapid accumulation of IP3 after HI receptor stimulation, but evidence obtained with either PLC inhibitor or phorbol ester in guinea pig Heft atria suggests that this may not be the only mechanism operat- ing in the positive instropic effect of the HI agonist (Hattori et al. 1989).

On the contrary, in autoimmune mice heart, our findings show that the phssphsinositide hydrolysis subserves the HI receptor mediated mechanical effect of ThEA, suggesting that this mechanism could play an important role in this model.

The different profile produced by the HI agonist in auto- immune versus control mice myocardium is not related to changes in basal conditions, since auricles from the former group had similar developed tension and beating frequency as those from the latter group, The tact that this high affinity receptor population appeared prior to disturbances of basal contractile parameters (Perez Leiros et al. 1986, 1988) sug- gests that the receptor emergence could be an early signal in the development of cardiac muscle lesion.

Acknowledgemen&% This work was supported by grant PID-BID CONICET

(0352). The authors thank Mrs. Elvita Vannucchi and Beatriz Gostigliolo for technical assistance and typing the manuscript, respectively.

Berridge, M .J. 1982. Lithium amplifies agonist-dependent phospha- tidylinositol responses in brain and salivary glands. Biochem. J . 286: 587 - 595.

Berridge, M.S., and Imine, W.F. 1984. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature (London), 312: 315-321.

Borda, E., Bracco, M., Finiaz, M., and Sterin-Borda, L. 1984. Lymphocytes sensitize rat isolated atria to the inotropic and chronotropic effect of sodium arachidonate. Br. J. Pharnaacol. 81: 75-83.

Brown, S . M . , Buxton, I.L., and Brunton, L.L. 1985. Alpha-1 adrenergic and muscarinic cholinergic stimulation of phospho- inositide hydrolysis in adult rat cardiomyocytes. Circ. Res. 5'7: 532 -537.

Chang, R.S., Tran, V.T., and Snyder, S.H. 1978. Histamine HI receptors on brain labelled with 3H rnepyramine. Eur. J . Pham~acol. 48: 463 - 464.

Chang, R.S., Tran, V.T., and Snyder, S.H. 1979. Characteristics of histamine HI receptors in peripheral tissues labelled with 'H rnepyramine. J. Pharmacol. Exp. Ther. 209: 437 -442.

Cheng, Y., and Pmsoff, W.H. 1973. Relationship between the inhi- bition constant (KJ and the concentration of imhibitor which causes 50% inhibition (I,,) of an enzymatic reaction. Biochern. Phmacol . 22: 3099 -31688.

Cossio, P., Bustuoabad, O., Paterno, E., Lotti, R., Casanova, M., Podesta, M., Bolomo, N., Arana, R., and Pasqualini, C. 1984. Experimental rnyocarditis induced in Swiss mice by homologous heart immunization resembles chronic Chagas heart disease. Clin. Hmmunol. Irnmunopathol. 33: 165 - 175.

Gorelik, G . , Borda, E., Bacman, S., Cremaschi, G . , and Sterin-Borda, L. 1992. Chagasic IgG stimulates phosphoinositide hydrolysis via neurotransmitter receptor activation: role of cal- cium. J . Lipid Mediators, 5: 249-259.

Goren, N., Perez Leiros, C., and Borda, E. 1991. Myocarditis autoimmune experimental: efecto de la hisPamina sobre el corazon. Medicina, 51: 421 -422.

Gus, Z.G., Eevi, R., Graver, M., Robertson, A., and Gay, W.A., Jr. 1984. lnotropic effect of histamine in human myocardium. ,I. Cardiovasc . Pharmacol. 6: 12 10 - 12 15.

Hattori, Y., and Kanno, M. 1985. Effect of Ni" on the multiphasic positive inotropic responses to histamine mediated by H 1 receptors in left atria of guinea pigs. Naunyn-Schmiedekrg's Arch. Pharmacol . 329: 188 - 194.

Hattori, Y., Kimura, S., Fujii, S., and Kanno, M. 1983. Effects of chemical performance and biochemical and electrical activity in the heart of monkeys. Eur. J. Pharmacol. 91: 1 1 - 19.

Hattori, Y., Sakuma, I., and Kanno, M. 1988. Differential effects of histamine mediated H1 and H2 receptors on contractility, spon- taneous rate and cyclic nucleotides in rabbit. Eur. J. Pharmacol. 1853: 221 -229.

Hattori, Y., Endou, M., Shirota, M., and Kannon, M. 1989. Dis- sociation of phosphoinositide hydrolysis and positive inotropic effect of histamine mediated by H1 receptors in guinea pig left atria. Naunyn-Schmiedeberg's Arch. Pharmacol. 340: 196 - 203.

Hidaka, H., Inagaki. M., Kawamoto, S o , and Sasaki, J. 1984. Isoquinoline sulfonamides, novel and potent inhibitor of cyclic nucleotide dependent protein kinase and protein kinase C. Bio- chemistry, 23: 5036 - 5041.

Hill, S . 8990. Distribution, properties and functional characteristics of three classes of histamine receptors. Bhamacol. Rev. 42: 45 - 83.

Hokin-Neaverson, M., and Sadeghian, K. 1976. Separation of 'H-inositol monophosphates and 'H-inositol on silica gel glass- fiber sheets. J. Chromatogr. 128: 502 - 505.

ERurs, W., Bast, A., and T i m e m n , H. 1989. High affinity, saturable 3H-mepyramine binding sites on rat liver plasma membrane do not represent histamine HI -receptors. Biochem. Pharmacol. 38: 2175-2180.

Levi, R., and Burke, J . A. 1980. Cardiac anaphylaxis; SRS-A poten- tiates and extends the effects of released histamine. Eur. 9. Phar- macol. 62: 41 -49.

Levi, R., Owen, D.A.A., and Trzeciakowski, J. 1982. Actions of histamine on the heart and vasculature. &n Pharmacology of hista- mine receptors. Edited by Ce We Ganellit and M.E. Parsons. Wright and Sons Ltd., Bristol, England. pp. 236-297.

Melmon, K.L., and Kan, M.M. 1987. Histamine and its lymphocyte- selective derivatives as immune modulators. Trends Pharmacol. Sci. 8: 437-441.

Nihizuka, H. 1984. The role of protein kinase @ in cell surface signal transduction and tumor promotion. Nature (London), 308: 693 - 698.

Perez Leiros, C., Sterin-Borda, L., Cossio, P.: and Borda, E.S. 1986. Potential role of n~ononuclear cell infiltration on the auto- immune myocardial dysfunction. Clin. Exp. Imunol . 63: 648-655.

Perez Leiros, C., Sterin-Borda, L., and Borda, E.S. 1988. Eyrnpho- cyte infiltration induces dyshnction in autoianrnune myocarditis: role of SWS-A. 9 . Mol. Cell. Cardiol. 20: 149- 158.

Perez Leiros, C., Sterin-Borda, L., Cossio, P., and Borda, E.S. 1990. Muscarinic cholinergic antibody in experimental autoimmune myocarditis regulates cardiac functions. Proc. Ssc. Biol. Exp. Med. 195: 356-363.

Sakuma, I., Gross. S.S., and Levi, R. 1988. Positive inotropic effect of histamine on guinea pig left atrium: HI receptor induced stimu- lation of phosphoinositide turnover. J. Pharnucol. Exp. Ther. 247: 466-472.

Can

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644 CAN. B. PHYSIOL. PHAWMACOL. VOL. 71, 1993

Tran, V.T., Chang, R.S., and Snyder, S.H. 1978. Histamine HI Walenga, W., Vanderhoek, J.Y., and Feinstein, M.B. 1986. Serine receptors identified in mammalian brain with 3H mepyrmirae. esterase inhibitors block stimulus-induced mobilization or arachi- Proc. Natl. Acad. Sci. U.S.A. 75: 6290-6294. donic acid and phospkatidylinositide specific Pl@ activity in platelets.

Vema, S. , and McNeill, J . 1977. Cardiac histamine receptors: differ- J . Bid. @hem. 255: 6024-602'7. ence between left and right atria and right ventricle. J , Pharrnacol. Exp. Ther. 200: 352-362.

Can

. J. P

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