European Journal of Pharmacology 587 (2008) 243–247

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European Journal of Pharmacology

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Glucagon increases contractility in ventricle but not in atrium of the rat heart

Carmen Gonzalez-Muñoz a, Susana Nieto-Cerón b, Juan Cabezas-Herrera b, Jesús Hernández-Cascales a,⁎a Department of Pharmacology, Medical School, University of Murcia, Spainb Research Unit, Clinical Analysis Service, University Hospital Virgen de la Arrixaca, Murcia, Spain

⁎ Corresponding author. Departamento de FarmacoCampus de Espinardo, 30071 Murcia, Spain. Tel.: +34 96

E-mail address: jehernca@um.es (J. Hernández-Casca

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.04.001

A B S T R A C T

A R T I C L E I N F O

Article history:

This study evaluates the inot Received 12 February 2008Accepted 1 April 2008Available online 8 April 2008

Keywords:GlucagonRat heartCyclic nucleotide phosphodiesteraseIsoprenalineCardiac contractilityPertussis toxin

ropic responses to glucagon in electrically driven isolated left and right atria as wellas in right ventricular strips of rat heart. For comparison, the contractile effects resulting from stimulating β-adrenoceptors with isoprenaline in atrial and ventricular tissues were also obtained.Glucagon (0.01–1 μM) produces a concentration-dependent positive inotropic effect in ventricular but not inatrial myocardium. Isoprenaline, however, increases contractility both in atrial and ventricular tissues. Thenonselective phosphodiesterase (PDE) inhibitor 3-isobutylmethylxantine (IBMX,10 μM) enhances the contractileeffect of glucagon on ventricular myocardium. However, glucagon still failed to increase contractility in atrialmyocardium in the presence of 10 μM, IBMX. Also, in left atria of rats pretreated with pertussis toxin, glucagondid not produce any positive inotropic effect, either alone or in the presence of 10 μM, IBMX. Western blottinganalysis indicates that glucagon receptors expression is 5 times higher in ventricular than in atrial myocardium.Taken together, these results indicate that the lack of inotropic effect of glucagon in atrium is not due to Giprotein or PDEs activity but seems to be a consequence of a lower glucagon receptor density in this tissue.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Glucagon is a polypeptide hormone produced and secreted by thealpha cells of the pancreatic islets of Langerhanswhich is considered toincrease cardiac contractility (White,1999). Consequently, it is used forthe treatment of poisoning caused by cardiodepressant drugs such asβ-adrenoceptors blockers or calcium channel blockers (DeWitt andWaksmann, 2004). The mechanism responsible for the contractileeffects of glucagon on heart is stimulation of glucagon receptors as-sociated with Gs protein, which causes adenylyl cyclase activation andthe consequent increase of 3′,5′-cyclic adenosine monophosphate(cAMP) production in the myocardium (White, 1999). The effects ofcAMP-dependent positive inotropic agents are regulated by theactivity of the cyclic nucleotide phosphodiesterase (PDE) enzymeswhich break down cAMP into its chemically inactive product 5′AMP(Bender and Beavo, 2006). On the basis of structure, kinetic properties,substrate specificity and ability to regulate, PDE can be grouped intodifferent families and at least four of these families (PDE 1–4) are pre-sent in the heart of a variety of animal species, including man (Benderand Beavo, 2006; Nicholson et al., 1991). PDE3 and PDE4, hydrolyzescAMP enhancement induced by glucagon in the myocardium andlimits its contractile effect since it is increased by selective inhibitors ofeach of these two isoenzymes (Juan-Fita et al., 2005; Rochais et al.,2006). In addition to Gs protein, glucagon can also stimulate Gi protein

logía, Facultad de Medicina,8367198; fax: +34 968364150.les).

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and this may blunt the positive inotropic effect resulting from Gsactivation (Kilts et al., 2000). The positive inotropic effect of glucagonhas been clearly established in ventricular myocardium (MacLeod etal., 1981; Juan-Fita et al., 2004; Juan-Fita et al., 2005). However,glucagon doesn't seem to produce a similar contractile response in theatrium since either, no effect (Antonaccio and Cavaliere, 1974) or onlyminor inotropic response (Furukawa et al., 1986) has been reported.The reason behind the differences observed between the atrial andventricular effects of glucagon is unknown but it may result from adifferent expression of glucagon receptor in atrial and ventricularmyocardium. Indeed, regional differences in distribution and densityof other receptors, such as adrenoceptors ormuscarinic receptors, havebeen detected in the heart (Myslivecek et al., 2006; Horinouchi et al.,2006). The purpose of the present work was to study the contractileresponses of atrial and ventricular tissues from the rat heart toglucagon. For comparison, we have also studied, in these two tissues,the effect of the activation of β-adrenoceptors by isoprenaline, whichalso produce a cAMP dependant positive inotropic effect by activatingGs/adenylyl cyclase pathway (Brodde and Michel, 1999). We alsoevaluate glucagon receptors distribution as well as the possible role ofPDEs and Gi protein in regulating the effect of glucagon in rat myo-cardium. To our knowledge, this is the first study showing a differentregional distribution of glucagon receptors in the heart which aremainly located at ventricular level.

2. Material and methods

The study was performed in accordance with the EuropeanCommunities Council Directive of 24 November 1986 (86/609/

244 C. Gonzalez-Muñoz et al. / European Journal of Pharmacology 587 (2008) 243–247

EEC) and approved by the Ethical Committee of the University ofMurcia.

2.1. Contractile responses induced in electrically driven rat atrial andventricular myocardium

41 Sprague–Dawley rats of either sex (250–300 g) were stunnedand exsanguinated. The chest was opened, the heart rapidlyremoved and placed in Tyrode solution saturated with 95% O2–5%CO2 and the left atria as well as the right atria and the free wall ofthe right ventricle were excised. All procedures were performed inthe presence of Tyrode solution of the following composition (mM):NaCl 136.9, KCl 5.0, CaCl2 1.8, MgCl2 1.5, NaH2PO4 0.4, NaHCO3 11.9and dextrose 5.0. The left atria, right atria and a strip of the rightventricle (1.5 mm wide, 10 mm long and 1 mm thick) of rat heartwere mounted between two platinum electrodes in Tyrode solutionat 37 °C, pH 7.4 (measured every 15 min with an electrodeMinitrode-Hamilton, Bonaduz, Switzerland) and gassed with 95%O2–5% CO2. The preparations were electrically stimulated (Grass SD-9 stimulator) at a frequency of 1 Hz and 1 ms of duration andsupramaximal (threshold+25%) voltage. A length-force curve wasobtained, and the tissues left at the length associated withmaximum developed force (Juan-Fita et al., 2004). Contractionswere measured using a force-displacement transducer (Grass FT-03)and displayed on a computer screen using a Stemtech amplifier(Stemtech Inc., Houston, Texas) and ACODAS software (DataqInstruments, Inc., Akron, Ohio). Tissues were allowed to equilibratefor 45–60 min before drug challenge.

Cumulative concentration–response curves to glucagon andisoprenaline in left atria and right ventricular strips wereperformed. For comparison with right ventricular tissue, concentra-tion–response curves for glucagon in right atria (devoid of sinusnode to prevent interferences with electrical pacing) were alsocarried out. To ascertain the role of PDEs in regulating glucagonresponses, we also determined concentration–response curves forglucagon in the presence of the nonselective inhibitor of PDEs, 3-isobutyl-1-methylxanthine (IBMX, Bender and Beavo, 2006). Theconcentration of IBMX used was 10 μM which effectively inhibitsPDEs activity in the rat heart (Bian et al., 2000). IBMX was left incontact with the tissue for 15 min before construction of theconcentration–response curves for glucagon. Only one concentra-tion–response curve for glucagon alone or in the presence of IBMXwas performed in the same preparation. Drugs were added to a30 ml organ bath in volumes less than or equal to 0.1 ml.Experiments were concluded by raising the Ca2+ concentration to9 mM which produce a maximal inotropic response and resultsexpressed as percentages of this effect.

To examine the involvement of Gi proteins in the responses toglucagon, rats were treated with pertussis toxin 30 μg/kg byintraperitoneal injection. 3 days later the rats were sacrificed andcumulative concentration–response curves for glucagon wereobtained as described above. To assess the effectiveness of thetreatment with pertussis toxin, tissues were incubated with 20 μmolcarbachol for 5 min shortly after mounting, followed by washing andequilibrating for 90 min. It is known that the negative inotropic effectof carbachol is mediated by Gi protein-coupled M2 receptors and pre-treatment with pertussis toxin inactivates Gi protein and abolishcarbachol induced cardiodepression (Sandirasegarane and Diamond,2004).

2.2. Tissue preparation for Western blotting analyses

After removing the heart, the left atria and the right ventriclewere dissected, fresh-frozen, and stored immediately at −80 °C untiluse. Tissues were homogenized (2.5% w/v) in a lysis buffer composedof 1% triton X-100, 50 mM Tris–ClH (pH 7.5), 1 M NaCl, 50 mM

MgCl2. After centrifugation at 10,000 g for 30 min at 4 °C, thesupernatant was saved. The tissue extracts (50 μg) were transferred,mixed, and boiled in sample buffer [10 mM Tris–ClH, pH 6.8, 2%sodium dodecylsulfate (SDS), 10% glycerol, 1% 2-mercaptoethanol,Merck, Germany]. The lysates were resolved by reductive SDS-polyacrylamide gel electrophoresis (Laemmli, 1970), carried out in12% polyacrilamide-gel slabs. Proteins were electro-transferred tonitrocellulose membranes, blocked with 5% non-fat dried milkdissolved in tris buffer saline tween (TBS-T: 10 mM Tris–ClH, pH7.5, 140 mM NaCl, 0.1% tween-20) and incubated with the blockingsolution containing anti-glucagon receptor antisera (I-16 from SantaCruz Biotechnology, Calif., USA). The membranes were washed threetimes in TBS (5 min each), and then incubated with a horseradishperoxidase conjugated anti-goat IgG antibodies (Sigma-Aldrich,Spain) in the blocking solution. After washing, immunoreactiviywas detected with an enhanced chemiluminescence Western blotdetection system (ELC, Amersham-Pharmacia-Biotechnology,Madrid, Spain) and visualised by Amersham Hyperfilm-ECL. Anti-bodies were stripped from the blots by incubation with strippingbuffer (glycine 25 mM and SDS 1%, pH 2), for 1 h at 37 °C. Blots weresubsequently reblocked and probed with 1:8000 anti-actin (mouseclone 5C5, Sigma-Aldrich, Spain) for detecting actin as a loadingcontrol. The size of the glucagon receptor was estimated in referenceto molecular weight markers (Full Range Rainbow™ MolecularWeight Markers, Amersham Biosciences, U.K.) and the intensity ofthe protein bands was quantified with the GelPro Analyzer Software(version 3.1; Media Cybernetic Inc., Bethesta, MD. USA).

2.3. Drug

Glucagon was generously supplied by Novo Nordisk Pharma S.A.(Madrid, Spain). Isoprenaline, IBMX and pertussis toxinwere obtainedfrom Sigma Chemicals Co. (Madrid, Spain) and dimethyl sulphoxide(DMSO) from Probus (Barcelona, Spain).

Glucagon and IBMX were dissolved in DMSO and Tyrodesolution (20% DMSO in Tyrode) and isoprenaline was dissolved inTyrode solution; this stock solution was diluted into pre-warmedand pre-aerated bathing solution to achieve the final concentrationdesired. The drug was added to the organ bath at an appropriateconcentration so that the concentration of DMSO in the testsolution was less than 0.3%, which produced no effect on thesepreparations.

2.4. Statistical analysis

Results are expressed as mean values±S.E.M. Student's t test or oneway analysis of variance followed by Scheffe' method for multiplecomparisons was used. The criterion for significance was that P valuesshould be less than 0.05.

3. Results

3.1. Effects of glucagon and isoprenaline

Typical responses of atrial and ventricular rat myocardium toglucagon (0.01–1 μmol) are illustrated in Fig. 1A–C. As can be seenthe ventricular contractile force gradually increases with eachglucagon concentration during 2 min and tended to fade thereafter.The Emax of glucagon was 32.2±6.1% (n=6), of the response to 9 mMCa2+. On the contrary, glucagon was virtually devoid of inotropiceffect in atrial myocardium. The glucagon solvent DMSO, at the sameconcentrations, was devoid of effect in this preparation (data notshown). Further experiments were carried out and the results aresummarized in Fig. 1D.

Isoprenaline (0.1–1000 nM) produced a concentration-dependentpositive inotropic effect, both in atrial and ventricular rat myocardium

Fig. 1. Effect of glucagon on the basal force of contraction in rat myocardium.Representative traces showing that glucagon is devoid of contractile effect in (A) leftand (B) right atria but produces a concentration-dependent positive inotropic effectin (C) right ventricle strips. (D) Cumulative concentration–response curves for theeffect of glucagon in left atria (■), right atria (□) and right ventricle (●) of rat heart.Inotropic responses are expressed as a percentage of the effect caused by 9 mM Ca2+.Each point represents the mean value±S.E.M. (vertical bars) of 6 experiments.

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(Fig. 2). However, although its maximal efficacy (Emax) is similar atatrial (Emax=87.5±2.3, n=4) and at ventricular level (Emax=86.5+2.8,n=5) PN0.05, its potency in the left atria (−log EC50=8.6±0.07, n=4) is

Fig. 2. Cumulative concentration–response curves for the inotropic effect of isoprena-line in left atria (■) and right ventricle (●) of rat heart. Each point represents the meanvalue±S.E.M. (vertical bars) of 4 experiments. Further details as in legend to Fig. 1.

higher than in the right ventricle (−log EC50=7.32±0.06, n=5)(Pb0.05).

3.2. Effects of glucagon in combination with IBMX or in rats pretreatedwith pertussis toxin

Fig. 3 shows the effects of IBMX on rat atrial and ventricularmyocardium responses to glucagon. IBMX (10 μmol) is devoid ofinotropic effect, on its own, in ventricular myocardium but increasesatrial contractility by 23.5±9.7% (n=10). In left atria glucagon fails toproduce a positive inotropic response in the absence and in thepresence of IBMX (Fig. 3A). However, in ventricular myocardium (Fig.3B) IBMX produces a shift to the left of the concentration–responsecurve to glucagon and changed its −log EC50 from 7.3±0.07 (n=6),alone to 8.3+0.08 (n=5), in the presence of IBMX (Pb0.05). Also, itincreases the Emax value of glucagon (32.2±4.8, n=6, in the absenceand 89.3±6.9, n=5, in the presence of IBMX, Pb0.05), in ventricularmyocardium.

In order to investigate whether Gi protein is responsible for thelack of glucagon response in atrial myocardium, 10 rats werepretreated with pertussis toxin. In these preparations, carbacholdoes not reduce contractility in contrast to non pretreated ratswhere carbachol decreases inotropy by 85±5% (n=7). Glucagon,however, still fails to increase contractility either alone or in the

Fig. 3. Effects of IBMX (10 μM) and pre-treatment with pertussis toxin (30 μg/kgintraperitoneal injection) on the contractile responses of rat myocardium toglucagon. IBMX on its own produces a positive inotropic effect in left atria (□)but not in right ventricle (○). Concentration–response curves to glucagon in leftatria (A), in the absence (■) and in the presence (□) of IBMX of non treated (solidlines) and pertussis toxin treated (dotted lines) rats and in right ventricular strips(B), in the absence (●) and in the presence (○) of IBMX. Each point represents themean value±S.E.M. (vertical bars) of 5–7 experiments. Further details as in legend toFig. 1.

Fig. 4. Expression of glucagon receptors in atrium and ventricular myocardium of ratheart. Protein levels were determined with Western blot analysis using β-actin as aninternal control. Band intensity was determined with densitometry and the valuespresented are the mean of 5 independent experiments; bars±S.E. M.(*Pb0.05).

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presence of IBMX (10 μmol) in left atria of rats pretreated withpertussis toxin (Fig. 3A).

3.3. Expression of glucagon receptors in rat atrial and ventricularmyocardium

Next, we examined glucagon receptor expression in atrial andventricular rat myocardium in which we measured contractility. Ascan be seen in Fig. 4, glucagon receptor level is about 5 times higher inventricle when compared to atrium.

4. Discussion

Our results demonstrate that glucagons receptors in the rat heartare mainly localized at ventricular level and glucagon only increasescontractility in this tissue.

Glucagon is considered to produce a positive inotropic effect whichis consequence of the activation of the glucagon receptor/Gs/adenylylcyclase/cAMP pathway (White, 1999). This is consistent with aglucagon induced increase in ventricular contractility reported by usin the present and in previous work (Juan-Fita et al., 2004; Juan-Fita etal., 2005). On the contrary, no positive inotropic effect of glucagonwasobtained in the left atria of the rat in our results, which also agreeswithprevious finding indicating that glucagon only produces a marginal(Furukawa et al., 1986), or no effect at all (Antonaccio and Cavaliere,1974) on contractility in atrial tissue. The reason behind this differentinotropic effect of glucagon on atrial and ventricular contractility wasnot known and it was the purpose of the present work.

The effects of cAMP-dependent positive inotropic agents areregulated by the activity of PDEs which break down cAMP into itschemically inactive product 5′AMP (Bender and Beavo, 2006). On thebasis of structure, kinetic properties or substrate specificity, PDEs canbe grouped into different families and, at least four of these families(PDE 1–4) are present in the heart of a variety of animal species,including man (Bender and Beavo, 2006; Nicholson et al., 1991). PDE3and PDE4 are the main isoenzymes which limits the effects of cAMP-dependent positive inotropic agents, including glucagon (Juan-Fita etal., 2005; Rochais et al., 2006) and selective PDE3 and PDE4 inhibitorsenhance the Emax and reduced the fade of the contractile effect ofglucagon in rat ventricular myocardium (Juan-Fita et al., 2004; Juan-

Fita et al., 2005). To assess whether the inotropic effect of glucagonwas reduced by PDEs we used IBMX which inhibits multiple PDEs,including PDE3-4 (Bender and Beavo, 2006). IBMX, on its own, wasdevoid of effect on ventricular myocardium but increases contractilityon the left atria of the rat, probably due to the different distribution ofPDEs in cardiac tissue (Bode et al., 1991). As expected, IBMX clearlypotentiates the effect of glucagon in the right ventricle of the rat heart.However, in left rat atria, IBMX failed to reveal any positive inotropiceffect of glucagon, thus, indicating that the absence of glucagon effecton atrial contractility is not consequence of PDEs activity.

In addition to Gs, glucagon can also stimulate Gi protein and thismay blunt the positive inotropic effect of Gs activation (Kilts et al.,2000). For example, β2-adrenoceptors which are also coupled to Gsand Gi signalling can only elicit a contractile effect in cardiac myocytesafter Gi signalling is inhibited by pre-treatment with pertussis toxin(Xiao et al., 1999). However, Gi protein doesn't seem to be responsiblefor the lack of effect of glucagon in atrial myocardium since it stillpersist after pre-treatment with pertussis toxin. Furthermore, even acombined inhibition of Gi protein with pertussis toxin and PDEs withIBMX is unable to reveal a positive inotropic effect of glucagon in ourresults, thus indicating that the absence of effect of glucagon on atrialcontractility is not due to either Gi proteins or PDEs activity.

The existence of a different regional distribution of receptors in theheart is known (Brodde and Michel, 1999). For example, muscarinicreceptors are predominant in atrial tissue (Myslivecek et al., 2006) butα-adrenoceptors are mainly located in ventricular myocardium(Brodde and Michel, 1999; Horinouchi et al., 2006). β-adrenoceptorsare highly expressed through all the heart but more abundant in atrialmyocardium (Myslivecek et al., 2006; Horinouchi et al., 2006). This isconsistent with our results showing that β-adrenoceptors activationby isoprenaline increases contractility in both atrial and ventricularmyocardium, being more potent at atrial level. The regional distribu-tion of glucagon receptor in the heart was not known but results of thepresent work show that it is about 5 times higher in ventricular thanin atrial myocardium. Although this lower concentration of glucagonreceptor in atrial myocardium may play a role in modulatingmetabolic pathways (Anousis et al., 2004), it doesn't seem to behigh enough to increase contractility in this tissue.

Despite its positive inotropic effect, glucagon is not considered auseful drug for disorders characterized by a reduced cardiac outputdue to its lack of therapeutic effect (Stevenson, 2003; London andSena, 2006). Indeed, patients with heart failure or circulatory shockwho received no beneficial effect with glucagon subsequentlyresponded well to digitalis (Nord et al., 1970). In beta blockers orcalcium channel blockers overdose, clinical improvement has beenassociated with glucagon administration in multiple case reports butits clinical efficacy has not been assessed in any controlled clinicaltrial and some of the beneficial effect reported could have been dueto other concomitant therapies received by these patients (DeWittand Waksmann, 2004). Indeed, glucagon does not consistentlyimprove survival in these patients and failure to respond toglucagon, particularly in subjects with propranolol toxicity, hasbeen reported (Shepherd, 2006). Atrial contractility contribute up to30% of the cardiac output (Matsuda et al., 1983) and the lack ofinotropic effect of glucagon on atrium as well as its relatively weakcontractile effect on ventricular myocardium (less than half of thatof isoprenaline) reported in the present paper may, at least partially,explain its low therapeutic efficacy as an inotropic agent.

Acknowledgements

This studywas supported by grant 05/2338 from the “Ministerio deSanidad y Consumo” (Spain). Juan Cabezas-Herrera belongs to theResearch Programme of “Sistema Nacional de Salud” (Ref 01/3025)and Susana Nieto-Ceron holds a fellowship from “Instituto de SaludCarlos III”.

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