Involvement of adenosine in the effect of fluoxetine on isolated guinea-pig atria

Pharmacological Research 53 (2006) 44–48

Involvement of adenosine in the effect of fluoxetine on isolated guinea-pig atria Abbas Pousti ∗ , Tara Deemyad, Golrokh Malihi, Kaveh Brumand Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, PO Box 13145-784, Tehran, Iran Accepted 16 August 2005

Abstract The effect of fluoxetine (FL) a selective serotonin reuptake inhibitor antidepressant was studied on the rate and force of contractions of isolated guinea-pig atria. FL (2–16 ␮g/ml) caused a decrease in the rate (13–45%) and contractile force (41–53%) of isolated guinea-pig atria in a dose-dependent manner. These negative inotropic and chronotropic effect of FL (4 ␮g/ml) were not prevented by atropine (1 ␮g/ml) and 3,7-dimethyl-1-propargylxanthine (DMPX; 1.5 ␮g/ml), an adenosine A2 receptor antagonist, but 1,3-dipropargyl-8-cyclopentylxanthine (DPCPX;12 ␮g/ml), a specific adenosine A1 receptor antagonist significantly blocked these effects (P < 0.001) and theophylline (30 ␮g/ml) a non- selective adenosine A1 /A2A receptor antagonist also prevented the inotropic and chronotropic effects of FL. These results suggest that the negative chronotropic and inotropic effect of FL on isolated guinea-pig atria is probably mediated through an inhibition of the reuptake of adenosine or the A1 receptor mechanism. © 2005 Elsevier Ltd. All rights reserved. Keywords: Fluoxetine; Adenosine receptor; Isolated atria

1. Introduction There is some evidence that adenosine is involved in the action of antidepressants. Antidepressant drugs inhibited the uptake of adenosine in rat brain synaptosomes [1] and potentiated the depressant action of adenosine on cortical neurons [2]. There are four recognized subtypes of adenosine receptors: A1 , A2A , A2B and A3 [3]. The experiments in guinea-pig cardiac preparations present evidence that A1 adenosine receptors agonists cause a negative chronotropic effect by slowing the pacemaker rate in the sinus nodes [4] and in the left atria a direct negative inotropic effect due to an activation of a potassium outward current [5]. These effects were abolished by the A1 adenosine receptor antagonist 1,3-dipropargyl-8-cyclopentylxanthine (DPCPX) [6]. The A1 adenosine receptors appear to act on many effectors as a ‘second messenger system’. These receptors mediate the inhibition of G proteins and adenylyl cyclase [7], activation of several types of K+ channels and inactivation of N-P and ∗

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Q type Ca2+ channels. Adenosine is indicated for the prompt conversion of the paroxysmal supraventricular tachycardia to sinus rhythm, slowing the ventricular rate during atrial fibrillation and as an adjunct to thallium cardiac imaging in the evaluation of coronary artery disease in patients unable to exercise adequately [3]. Previously, it was shown that fluoxetine (FL) caused a negative inotropic and chronotropic effect on isolated rat, rabbit and guinea-pig atria [8,9]. FL was also reported to relax vascular [10–12] and intestinal smooth muscle [13]. These effects of FL are similar to those of adenosine A1 receptors agonists on isolated atria. The present study was therefore designed to assess a possible involvement of adenosine receptors in mediating the effects of FL on isolated guinea-pig atria.

2. Materials and methods 2.1. Animals Guinea-pigs of either sex weighing 450–600 g were anaesthetized by ether and exsanguinated. The heart was rapidly

A. Pousti et al. / Pharmacological Research 53 (2006) 44–48

removed; the auricles were dissected from the heart and suspended in a bath containing 50 ml of oxygenated modified Krebs solution at 36–37 ◦ C and pH 7.4. The composition of solution was as follows (mM): NaCl 118.0, KCl 4.7, CaCl2 2.6, MgCl2 1.2, NaH2 PO4 1.0, NaHCO3 25.0, glucose 11.1, EDTA 0.004 and ascorbic acid 0.11. After mounting, the preparation was allowed to stand for 30 min for equilibration. The rate and force of spontaneous contractions were recorded isometrically with a photosensitive transducer on a Beckman RS Dynograph recorder. The diastolic tension on the atrial preparation was adjusted to 0.5 g. Seven atria were used for each experiment. Solution of drugs were prepared so that a constant volume of 0.5 ml for each dose was added to 50 ml of the bathing fluid [14]. The duration of spontaneous rhythmicity of guinea-pig atrial preparations under our experiments conditions was 25–35 min.

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Fig. 1. The effect of fluoxetine (2, 4, 8 and 16 ␮g/ml) on rate and contractile force of isolated guinea-pig atria.

2.2. Drugs The following drugs were used and prepared in distilled water and pH adjusted to 7.3 ± 0.1: fluoxetine (Dista), atropine (Emerk), 3,7-dimethyl-1-propargylxanthine, DMPX (RBI), 1,3-dipropyl-8-cyclopentylxanthine, DPCPX (RBI) and theophylline (Sigma). 2.3. Experimental plan 2.3.1. Group I Twenty-eight atria were examined after a period of 30 min. FL was added to the bath in doses 2, 4, 8 and 16 ␮g/ml. Only one dose of FL was added to each atrium. 2.3.2. Group II Seven atria were pretreated with 1 ␮g/ml of atropine for 10 min. FL (4 ␮g/ml) was then added to the bath in presence of atropine. 2.3.3. Group III Seven atria were pretreated with 30 ␮g/ml theophylline for 10 min. FL (4 ␮g/ml) was added to the bath in the presence of theophylline. 2.3.4. Group IV Seven atria were pretreated with 1.5 ␮g/ml DMPX (3,7dimethyl-1-propargylxanthine) an A2 adenosine receptor antagonist 10 min before adding FL (4 ␮g/ml). 2.3.5. Group V Seven atria were pretreated with 3, 6 and 12 ␮g/ml DPCPX (1,3-dipropyl-8-cyclopentylxanthine), an A1 adenosine receptor antagonist, 10 min before adding FL (4 ␮g/ml).

Fig. 2. Time-course of negative chronotropic response induced by FL (4 ␮g/ml) and FL (4 ␮g/ml) + DPCPX (3, 6 and 12 ␮g/ml) on isolated guineapig atria.

3. Results Fluoxetine at a concentration of 2–16 ␮g/ml (n = 28) caused a decrease in the rate (13–45%) and contractile force (41–53%) of isolated guinea-pig atria in a dose-dependent manner (P < 0.001, Fig. 1). The baseline of the heart rate and length of contractile force of isolated atria before adding the drugs (as a control) were 245 ± 3 min−1 and 15.2 ± 1.5 mm−1 , respectively. Pretreatment with atropine (1 ␮g/ml) or DMPX (3,7dimethyl-1-propargylxanthine) an A2 adenosine receptor antagonist (1.5 ␮g/ml) 10 min before addition FL, did not prevent the effect of FL on the rate and contractions of the atria (Table 1). However, pretreatment with DPCPX (1,3dipropyl-8-cyclopentylxanthine), an A1 adenosine receptor antagonist (3–12 ␮g/ml), significantly prevented the negative chronotropic and inotropic effect of FL at 10, 15 and 20 min in isolated guinea-pig atria (P < 0.001, Figs. 2–4, respectively). This effect was dose-dependent. Theophylline (phosphodiesterase inhibitor) an A1 /A2A adenosine receptor antagonist (30 ␮g/ml) prevented the effect of FL on isolated guinea-pig atria (Table 1).

2.4. Statistical analysis

4. Discussion

Results were expressed on mean ± S.E. Statistical significance was determined using Student’s t-test for paired data.

In isolated guinea-pig atria fluoxetine produced a significant decrease in the rate and contractile force in a dose-

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Table 1 Effects of atropine, DMPX and theophylline on the negative inotropic and chronotropic response of FL in isolated guinea-pig atria Drug

Dose (␮g/ml)

Decrease in contractile force (%)

Decrease in rate (%)

10 min

20 min

10 min

20 min

Control group FL

4

38.80 ± 2.14 (7)

52.60 ± 2.48 (7)

18.48 ± 1.86 (7)

32.10 ± 2.05 (7)

Atropine +FL

1 4

40.80 ± 3.28 (7)

70.20 ± 4.98 (7)

28.82 ± 3.14 (7)

48.78 ± 5.98 (7)

DMPX +FL

1.5 4

49.81 ± 3.98 (7)

68.78 ± 4.05 (7)

29.11 ± 2.10 (7)

35.66 ± 2.78 (7)

32.66 ± 0.78 (7)

41.66a ± 2.88 (7)

8.28a ± 0.91 (7)

18.25a ± 1.05 (7)

Theophylline +FL

30 4

Values are expressed as mean ± S.E. Numbers in parentheses indicate the number of experiments. a P < 0.05 when compared with the control group.

Fig. 3. Time-course of negative inotropic response induced by FL (4 ␮g/ml) and FL (4 ␮g/ml) + DPCPX (3, 6 and 12 ␮g/ml) on isolated guinea-pig atria.

dependent manner (P < 0.001) [9]. These effects are similar to those of adenosine receptor agonists [5]. The effect of antidepressant drugs amitriptyline and citalopram on rat prefrontal cortex was attenuated by local infusion of the adenosine A1 receptor antagonist 8-cyclopentyltheophylline [15]. It has been reported that adenosine receptors are widely distributed both in the brain and peripheral tissue [16]. The antinociceptive activity of several antidepressants was antagonized by the non-selective blocker of A1 /A2A adenosine receptor aminophylline [17]. There are several similarities between A1 adenosine receptor agonist and FL on atria. (1) Both have similar effects on the rate and contractions of isolated guinea-pig atria, the same effect of a selective A1 adenosine receptor agonist ( )-N-phenylisopropyladenosine (R-PIA) on the rate and contractions of atria were abolished by the A1 adenosine receptor

Fig. 4. The pattern of contractions of isolated guinea-pig atria by the fluoxetine alone and in presence of DPCPX at different concentrations.

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antagonist, DPCPX [18,6]. (2) FL produces a direct cardiac action due to the inhibition of cardiac Na+ and Ca2+ channels in isolated guinea-pig atria [19–22]. The action of an A1 adenosine agonist is accompanied by an inhibition of current through the L-type calcium channel [23,24]. (3) Adenosine interact with A1 adenosine receptors present on the extracellular surface of cardiac cells and activates K+ channels in a fashion similar to that produced by acetylcholine. The increase in K+ conductance shortens the atrial action potential duration, hyperpolarizes the membrane potential and decreases atrial contractility. Similar changes occur in sinus and AV nodes [25,7]. Our previous finding indicated that potassium concentration of the atrial tissue is significantly increased by FL [9]. This action may in part explain the negative inotropic and chronotropic action of FL by hyperpolarization [9,26]. However, in our present experiments, atropine and DMPX, an A2 adenosine receptor antagonist, did not prevent the effects of FL on isolated guinea-pig atria (Table 1), but DPCPX, a specific A1 adenosine receptor antagonist, significantly inhibited the negative chronotropic and inotropic effects of FL on isolated atria dose-dependently (P < 0.001, Figs. 2 and 3). Theophylline, an A1 /A2A adenosine receptors antagonist, also prevented the effects of FL on atria. These results help to explain the action of FL on the rate and contractile force of isolated guinea-pig atria that may be through an inhibition of uptake of adenosine, or by an activation of A1 adenosine receptors in atrial tissue.

Acknowledgement This work was supported by the Medical School of Tehran University of Medical Sciences. We also thank Dr. Abidi Pharmaceutical Co. in Iran for the generous supply of fluoxetine powder.

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