Summary We evaluated the effect of glucagon on cardiac automaticity as well as the possible role of cyclic nucleotide phosphodiesterases (PDE) in regulating this effect. Concentration response curves for glucagon in the absence and in the presence of the non-selective PDE inhibitor IBMX were performed in the isolated right ventricle of the rat. We found that glucagon produces only a minor increase of ventricular automaticity (11.0±4.1, n=5) when compared to the full agonist of β-adrenoceptor isoproterenol These results indicate that PDE blunts proarrhythmic effects of glucagon in rat myocardium

C Gonzalez-Muñoz

J Hernández

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PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online)                              
© 2011 Institute of Physiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic 
Fax +420 241 062 164, e-mail: physres@biomed.cas.cz, www.biomed.cas.cz/physiolres 
 
Physiol. Res. 60: 189-192, 2011 
 
SHORT COMMUNICATION 
 
Phosphodiesterases Inhibition Enhances the Effect of Glucagon  
on Cardiac Automaticity in the Isolated Right Ventricle of the Rat 
 
 
C. GONZALEZ-MUÑOZ1, J. HERNÁNDEZ1 
 
1Department of Pharmacology, Medical School, University of Murcia, Murcia, Spain 
 
Received April 27, 2010 
Accepted July 9, 2010 
On-line October 15, 2010 
 
 
Summary 
We evaluated the effect of glucagon on cardiac automaticity as 
well as the possible role of cyclic nucleotide phosphodiesterases 
(PDE) in regulating this effect. Concentration response curves for 
glucagon in the absence and in the presence of the non-selective 
PDE inhibitor IBMX were performed in the isolated right ventricle 
of the rat. We found that glucagon produces only a minor 
increase of ventricular automaticity (11.0±4.1, n=5) when 
compared to the full agonist of β-adrenoceptor isoproterenol 
(182.2±25.3, n=7). However, IBMX enhances the maximal 
efficacy of glucagon on cardiac automaticity (11.0±4.1, in the 
absence and 45.3±3.2 in the presence of IBMX, n=5, P<0.05). 
These results indicate that PDE blunts proarrhythmic effects of 
glucagon in rat myocardium. 
 
Key words 
Glucagon • Cyclic nucleotide phosphodiesterases (PDE) • Cardiac 
automaticity • Isoproterenol 
 
Corresponding author 
J. Hernández, Departamento de Farmacología, Facultad de 
Medicina, Campus de Espinardo, 30071 Murcia, Spain. Fax: +34 
868864150. E-mail: jehernca@um.es 
 
 Glucagon is a polypeptide hormone produced 
and secreted by the alpha cells of the pancreatic islets of 
Langerhans which increases heart rate and contractility 
(White 1999). Consequently, it is used for the 
management of poisoning caused by cardiodepressant 
drugs such as β-adrenoceptors (β-AR) blockers or 
calcium channel blockers (DeWitt and Waksmann 2004). 
Cardiac effects of glucagon are considered to be resultant 
from stimulation of glucagon receptors associated with 
Gs protein, which causes adenylyl cyclase (AC) 
activation and the consequent increase of 3’,5’,-cyclic 
adenosine monophosphate (cAMP) production (White 
1999).  
 Cell cAMP levels are regulated by the activity of 
cyclic nucleotide phosphodiesterases (PDE) enzymes 
which break down cAMP into its chemically inactive 
product 5’AMP (Bender and Beavo 2006). In contrast to 
the positive inotropic and chronotropic effects of 
glucagon which are well established, there is controversy 
about its effect on cardiac arrhythmias since no 
proarrhythmic and arrhythmogenic effects have been 
reported for glucagon (Farah 1983). Abnormal 
automaticity is an important mechanism underlying 
cardiac arrhythmias (Shu et al. 2009) and cAMP 
increases the spontaneous firing rate of pacemaker cells 
by inducing a local Ca2+ release resulting from a protein 
kinase A-dependent phosphorylation of sarcolemmal 
ryanodyne receptors (Vinogradova and Lakatta 2009). 
The isolated right ventricle of the rat contains pacemaker 
cells, in the His-Purkinje fibers, which develop 
spontaneous activity resembling an idioventricular 
rhythm (Mangoni and Nargeot 2008) and it is a useful 
experimental model for assessing the effect of drugs on 
ventricular automaticity (Hernandez et al. 1994, 
Hernandez and Ribeiro 1996). The present work was 
aimed to evaluate the effect of glucagon in this 
experimental model as well as the possible role of PDEs 
in regulating this effect. For comparison, we have studied 
the effect of the full β-AR agonist isoproterenol which 
also produces its cardiac effects by activating the 
190   Gonzalez-Muñoz and Hernández  Vol. 60 
 
 
Gs/AC/cAMP pathway (Brodde and Michel 1999). To 
our knowledge, this is the first report showing that PDE 
inhibition increases the effect of glucagon on cardiac 
automaticity in this tissue.  
 This study was performed in accordance with the 
European Communities Council Directive of 
24 November 1986 (86/609/EEC) and approved by the 
Ethical Committee of the University of Murcia. 
17 Sprague-Dawley rats of either sex, 250-300 g were 
stunned and exsanguinated, hearts were removed and 
placed in warm Tyrode solution containing (mM): NaCl 
136.9, KCl 5, MgCl2 1.05, CaCl2 1.8, NaH2PO4 0.4, 
NaHCO3 11.9 and dextrose 5. The right ventricle was 
freed, the atrial end fixed to a metallic support and the 
apical end was attached to a Grass FT 03 force-
displacement transducer. A 30 ml organ bath was used 
and the bathing Tyrode solution was maintained at 37 ºC, 
pH 7.4 and bubbled with a mixture of 95 % O2 and 
5 % CO2. Contractions were displayed on the screen of a 
computer by using a Stemtech Inc., Houston, U.S.A.) and 
the AT-CODAS computer programme (Dataq 
Instruments, Ohio, U.S.A.). The resting tension was set at 
1 g and the preparations were allowed to stabilize for at 
least 30 min. After the stabilization period, drugs were 
applied in volumes less than or equal to 0.1 ml and each 
concentration left 5 min; after, higher concentration of the 
test drug was applied until a cumulative concentration-
response curve had been constructed. To ascertain the 
role of PDEs in regulating glucagon responses, we also 
determined concentration response curves for glucagon in 
the presence of the nonselective PDE inhibitor,  
3-isobutyl-1-methylxanthine (IBMX) (Bender and Beavo 
2006). The concentration of IBMX used was 10 μM 
which effectively inhibits PDEs activity in the rat heart 
(Bian et al. 2000). IBMX was left in contact with the 
tissue for 15 min before construction of the concentration 
response curves for glucagon. Recorded data were then 
processed and the spontaneous ventricular frequency 
(beats min-1) was calculated as the average rate of the 
preparation recorded during the incubation period for 
each drug concentration. The change of frequency, 
compared with the control rate, was considered to be the 
effect of a given drug concentration. 
 Glucagon was generously supplied by Novo 
Nordisk Pharma S.A. (Madrid, Spain). Isoproterenol and 
IBMX were obtained from Sigma Chemicals Co. 
(Madrid, Spain) and dimethyl sulphoxide (DMSO) from 
Probus (Barcelona, Spain). Glucagon and IBMX were 
dissolved in DMSO and Tyrode solution (20 % DMSO in 
Tyrode) and isoproterenol was dissolved in Tyrode 
solution. The drugs were added to the organ bath at an 
appropriate concentration so that the concentration of 
DMSO in the test solution was less than 0.3 %, which 
produced no effect on these preparations. Results are 
expressed as mean values ± S.E.M. Student’s t test or one 
way analysis of variance followed by Scheffe’s method 
for multiple comparisons was used. The criterion for 
significance was that P values should be less than 0.05. 
 
 
 
 
Fig. 1. Recorded data from representative experiments showing 
the effects of 1 µM concentration (applied at the arrow) of 
glucagon in the absence (a) and in the presence (b) of the non 
selective phosphodiesterase inhibitor IBMX (10 µM) and 
isoproterenol (c) on the spontanously beating right ventricle of 
the rat heart. 
 
 
 We performed a total of 17 experiments in 
which a spontaneous ventricular rate was obtained by 
using the technique above described. The mean frequency 
before addition of any drug was 12±5 beats min-1. 
Typical effects of glucagon (Fig. 1a) and isoproterenol 
(Fig. 1c) on ventricular automaticity are shown. As it can 
be seen, glucagon produces a small increase in ventricular 
frequency when compared to isoproterenol. For instance, 
a concentration 1 µM of either glucagon and 
isoproterenol increases ventricular frequency by 10.1+3.5 
(n=5) and 182.2+25.3 (n=7) beats min-1 respectively 
(P<0.05, Figure 2). This agrees with results obtained in 
the dog heart showing that glucagon is devoid of effect 
(Shanks and Zaidi 1972, Wilkerson et al. 1977), or only 
produces a minor increase in ventricular automaticity in 
contrast to the huge enhancement of it induced by 
isoproterenol (Wilkerson et al. 1971). Cardiac effects of 
both, glucagon and isoproterenol are considered to be 
2011 Effects of Glucagon and Phosphodiesterases on Ventricular Automaticity   191  
   
resultant of the stimulation of specific receptors 
(glucagon receptors and β-adrenergic receptors, 
respectively) associated with Gs protein, which causes 
AC activation and the consequent increase of cAMP 
production in myocardium (Brodde and Michel 1999, 
White 1999). However, glucagon seems to be weaker 
than isoproterenol in activating AC in rodents (Ostrom et 
al. 2000) as well as in human hearts (Kilts et al. 2000). 
This agrees with the lower efficacy of glucagon when 
compared to isoproterenol for increasing automaticity 
(present results) as well as contractility in the rat 
ventricular myocardium (Gonzalez-Muñoz et al. 2008).  
 
 
 
 
Fig. 2. Effects of isoproterenol and glucagon on ventricular 
automaticity. Concentration-response curves to isoproterenol (□) 
and glucagon in the absence (●) and in the presence (○) of IBMX 
(10 µM). The frequency (contraction rate per min) was calculated 
as the average rate of the preparation recorded during the 
incubation period for each drug concentration. Each point 
represents the mean value + SEM (vertical bars) of 5-7 
experiments. * P<0.05 versus glucagon alone. 
 
 
 PDE hydrolyzes cAMP produced by glucagon 
and limits its contractile effect in rat myocardium since 
inhibition of PDE enhance both cAMP levels and 
inotropic responses to glucagon in this tissue (Juan-Fita et 
al. 2004, Juan-Fita et al. 2005), but whether or not PDE 
blunts its effect on ventricular automaticity was unknown 
and was investigated in the present work. To this 
purpose, we studied the effects of glucagon in the 
presence of the non selective PDE inhibitor IBMX. 
IBMX (10 μM), on its own, increased ventricular 
frequency by 9.5±4.3 beats min-1, (n=5). This is 
consistent with other results showing enhancement of 
cardiac automaticity after PDE inhibition and suggest a 
basal PDE activity which reduces ventricular 
automaticity by hydrolyzing cAMP (Vinogradova and 
Lakatta 2009). Our results reveal that IBMX increases the 
effect of glucagon on ventricular automaticity (Fig. 1b, 
Fig. 2). Indeed, the maximal efficacy of glucagon 
increased from 11.0±4.1 beats min-1 in the absence to 
45.3±3.2 beats min-1 in the presence of IBMX (n=5, 
p<0.05); thus, indicating that PDE also blunts 
proarrhythmic effects of glucagon in this tissue. 
Consequently, basal PDE activity seems to limit 
ventricular automaticity and protects myocardium against 
arrhythmias resulting from latent pacemaker overactivity 
induced either spontaneously or by cAMP producing 
agents such as glucagon. Therefore, PDE inhibition might 
be deleterious due to a potential proarrhythmic effects 
resulting from enhancement of cardiac automaticity. 
Indeed, a higher incidence of sudden death, attributed to 
cardiac arrhythmias has been reported in patients taken 
PDE inhibitors (Crickshank 1993). 
 In summary, the present study provides the first 
evidence that PDE limits proarrhythmic effects of 
glucagon. This might have some practical interest since 
both glucagon and PDE inhibitors are considered to be 
useful agents for treating toxicity induced by 
cardiodepressant agents such as β-adrenoceptor blockers 
or calcium channels blockers (DeWitt and Waksmann 
2004). Although combining PDE inhibitors and glucagon 
produce a synergist inotropic effect (Gonzalez-Muñoz 
2008), which should be beneficial in this case, it might 
also enhance automaticity thus increasing the risk of 
cardiac arrhythmias. However, the clinical relevance of 
these finding remains to be determined. 
 
Conflict of Interest 
There is no conflict of interest. 
 
Acknowledgements  
We are grateful to Prof. Pilar Martinez-Pelegrin 
(University of Murcia) for her review of the English 
version. 
 
References 
 
BENDER AT, BEAVO JA: Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev 
58: 488-520, 2006. 
192   Gonzalez-Muñoz and Hernández  Vol. 60 
 
 
BIAN JS, ZHANG WM, PEI JM, WONG TM: The role of phosphodiesterase in mediating the effect of protein kinase 
C on cyclic AMP accumulation upon κ-opiod receptor stimulation in the rat heart. J Pharmacol Exp Ther 292: 
1065-1070, 2000. 
BRODDE OE, MICHEL MC: Adrenergic and muscarinic receptors in the human heart. Pharmacol Rev 51: 651-689, 
1999. 
CRUICKSHANK JM: Phosphodiesterase III inhibitors: long-term risks and short-term benefits. Cardiovasc Drugs Ther 
7: 655-660, 1993. 
DEWITT CR, WAKSMANN JC: Pharmacology, pathophysiology and management of calcium channel blocker and β-
blocker toxicity. Toxicol Rev 23: 223-238, 2004. 
FARAH AE: Glucagon and the circulation. Pharmacol Rev 35: 181-217, 1983. 
GONZALEZ-MUÑOZ C, NIETO-CERON S, CABEZAS-HERRERA J, HERNANDEZ-CASCALES J: Glucagon 
increases contractility in ventricle but not in atrium of the rat heart. Eur J Pharmacol 587: 243-247, 2008. 
HERNANDEZ J, PINTO F, RIBEIRO JA: Evidence for a cooperation between adenosine A2 receptors and β1-
adrenoceptors on cardiac automaticity in the isolated right ventricle of the rat. Br J Pharmacol 111: 1316-1320, 
1994. 
HERNÁNDEZ J, RIBEIRO JA: Excitatory actions of adenosine on ventricular automaticity. Trends Pharmacol Sci 17: 
141-144, 1996. 
JUAN-FITA MJ, VARGAS ML, KAUMANN AJ, HERNANDEZ-CASCALES J: Rolipram reduces the inotropic 
tachyfilaxis of glucagon in rat ventricular myocardium. Naunyn-Schmiedeberg’s Arch Pharmacol 370: 324-
329, 2004. 
JUAN-FITA MJ, VARGAS ML, HERNANDEZ J: The phosphodiesterase 3 inhibitor cilostamide enhances inotropic 
responses to glucagon but not to dobutamine in rat ventricular myocardium. Eur J Pharmacol 512: 207-213, 
2005. 
KILTS JD, GERHARDT MA, RICHADSON MD, SREERAM G, MACKENSEN GB, GROCOTT HP, WHITE WD, 
DAVIS D, NEWMAN MF, REVES JG, SCHWINN DA, KWATRA MM: β2-adrenergic and several other G 
protein-coupled receptors in human atrial membranes activate both Gs and Gi. Circ Res 87: 705-709, 2000. 
MANGONI ME, NARGEOT J: Genesis and regulation of heart automaticity. Physiol Rev 88: 919-982, 2009. 
OSTROM RS, VIOLIN JD, COLEMAN S, INSEL PA: Selective enhancement of β-adrenergic receptor signalling by 
overexpression of adenylyl cyclase type 6: colocalization of receptor and adenylyl cyclase in caveolae of 
cardiac myocyte. Mol Pharmacol 57: 1075-1079, 2000. 
SHANKS RG, ZAIDI SA: Comparison of some effects of glucagon and isoprenaline on the cardiovascular system of 
the anaesthetized dog. Br J Anaesth 44: 427-432, 1972. 
SHU J, ZHOU J, PATEL CY, YAN GX: Pharmacotherapy of cardiac arrhythmias-Basic science for clinicians. PACE 
32: 1454-1465, 2009. 
VINOGRADOVA TM, LAKATTA EG: Regulation of basal and reserve cardiac pacemaker function by interactions of 
cAMP-mediated PKA-dependent Ca2+ cycling with surface membrana channels. J Mol Cell Cardiol 47: 456-
474. 2009. 
WHITE CM: A review of potential cardiovascular uses of intravenous glucagon administration. J Clin Pharmacol 39: 
442-447, 1999. 
WILKERSON RD, PRUETT JK, WOODS EF: Glucagon-enhanced ventricular automaticity in dogs. Its concealment 
by positive chronotropism. Circ Res 29: 616-625, 1971. 
WILKERSON RD, PARTLOW DB, PRUETT JK, PATTERSON CW: A possible mechanism of the antiarrhythmic 
action of glucagon. Eur J Pharmacol 44: 57-63, 1977. 
 
 


Phosphodiesterases Inhibition Enhances the Effect of Glucagon on Cardiac Automaticity in the Isolated Right Ventricle of the Rat

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Phosphodiesterases Inhibition Enhances the Effect of Glucagon on Cardiac Automaticity in the Isolated Right Ventricle of the Rat

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