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Modulation of noradrenaline release from isolated human atrial appendages
Journal of the Autonomic Nervous System 61 Ž1996. 269–276
Modulation of noradrenaline release from isolated human atrial appendages Claire Abadie
a,)
, Sylvain Foucart a , Pierre Page´ b, Reginald Nadeau ´
c
a
Research Centre, Hopital du Sacre-Coeur de Montreal, ˆ ´ ´ 5400 boul. Gouin ouest, Groupe de Recherche sur le Systeme ` NerÕeux Autonome, Department of Physiology, UniÕersite´ de Montreal, Canada, H4J 1C5 ´ Montreal, ´ Quebec, ´ b Research Centre, Hopital du Sacre-Coeur de Montreal, ˆ ´ ´ Groupe de Recherche sur le Systeme ` NerÕeux Autonome, Department of Surgery, UniÕersite´ de Montreal, Canada ´ Montreal, ´ Quebec, ´ c Research Centre, Hopital du Sacre-Coeur de Montreal, ˆ ´ ´ Groupe de Recherche sur le Systeme ` NerÕeux Autonome, Department of Medicine, UniÕersite´ de Montreal, Canada ´ Montreal, ´ Quebec, ´ Received 9 April 1996; revised 4 July 1996; accepted 19 July 1996
Abstract Prejunctional modulation of noradrenaline release has been studied extensively in various experimental preparations. However, the presence and importance of prejunctional noradrenaline release modulation in human cardiac tissue is still unclear. In this study, we have used superfused human right atrial appendages excised from patients undergoing open heart surgery. The tissues were cut into six pieces and incubated with w 3 Hxnoradrenaline Ž4 m Cirml, 0.2 m M. for 30 min at 378C. The tissues were then inserted into a suprafusion system and washed for 75 min with a Krebs–Henseleit solution at a rate of 0.4 mlrmin. The experimental protocol consisted of a 60-min perfusion period during which a field stimulation Ž2 ms pulses, 60 s, 50 mA, 5 Hz. was delivered at 10 and 45 min. The effect of the drugs on the stimulation-induced outflow of radioactivity was determined by adding them 20 min before the second stimulation. Each experiment was carried out with or without desipramine Ž1 m M. to study the influence of the reuptake blockade. Fenoterol Ž1–1000 nM., a b 2-adrenoceptor agonist, and angiotensin II Ž1–1000 nM. significantly increased noradrenaline release in a concentration-dependent manner. The administration of arecaidine propargyl ester Ž0.03–3 m M., a non-specific muscarinic receptor agonist, and propylnorapomorphine Ž0.1 nM–1 m M., a DA 2-dopaminergic agonist, produced a concentration-dependent inhibition of the stimulation-induced outflow of radioactivity. The a 2-adrenoceptor agonist, oxymetazoline Ž1 m M., inhibited noradrenaline release at a stimulation frequency of 2 Hz, but not at 5 and 10 Hz. The a 2-adrenoceptor antagonist, idazoxan Ž1 m M., significantly increased the release of noradrenaline at 2 and 5 Hz but not at 10 Hz. The results obtained in the present study demonstrated the presence of the facilitatory b 2-adrenoceptor and angiotensin II receptor as well as the presence of inhibitory a 2-adrenoceptor, muscarinic and DA 2-dopamine receptors in the human atrial appendage. Keywords: Human atrial appendage; Noradrenaline release; Presynaptic modulation; ß 2 -adrenoceptors; Angiotensin II; Muscarinic receptor; Dopaminergic receptor
1. Introduction In most studies, the role of prejunctional modulation of noradrenaline ŽNA. release has been evaluated using in vitro or in vivo experimental preparations of animal tissues Žfor review see Refs. w17,18,29x.. In humans, the i.v. administration of NA has revealed the presence of an a-adrenoceptor-mediated inhibition of NA release, whereas the i.v. administration of adrenaline showed the presence )
Corresponding author. Tel: q1 514 3382518; fax: q1 514 3382694; e-mail: [email protected].
of a b-adrenoceptor-mediated facilitation w3,12,13x. The renin–angiotensin system is also involved since the decrease in plasma NA induced by the administration of converting enzyme inhibitors is linked with the loss of the facilitatory effect mediated by angiotensin II on NA release w34x. The modulatory actions of b 2-adrenergic agonists and angiotensin II w25x, dopaminergic and a-adrenergic agonists w27x have recently been observed in pieces of human atrial appendage. These data clearly suggest that prejunctional modulation of NA release is occurring in humans. However, more studies are needed to enable a better understanding of these modulatory mechanisms.
0165-1838r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 6 . 0 0 0 9 3 - 8
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The aim of the present study was the identification of potential neuromodulators of NA release from sympathetic nerves in human atrial tissue. Several receptors have been identified for their ability to modulate positively or negatively the release of NA from sympathetic nerve terminals w23,29,35,36x. The results obtained demonstrate the presence of facilitatory mechanisms via the activation of b 2adrenoceptors and angiotensin II receptors, and inhibitory mechanisms via the activation of a 2-adrenoceptors, and muscarinic and DA 2-dopamine receptors in the human atrial appendages.
2. Materials and methods 2.1. Human atrial tissue Pieces of human atrial appendages were obtained from patients undergoing open heart coronary artery or valvular surgery. The excision of the atrial appendage was performed at the time of cannulation for the establishment of extracorporeal circulation. The experimental protocol was conducted with the understanding and the consent of each subject and was approved by the hospital ethics committee. 2.2. Experimental protocol Immediately after excision, specimens of atrial appendage Žweighing around 200 mg. were placed in an ice-cold Krebs–Henseleit solution ŽpH 7.4., previously gassed with a mixture of O 2rCO 2 Ž95%r5%., and quickly transferred to the laboratory, cleared from fat tissue and sliced into 6 pieces of equal dimensions. These pieces were then incubated with w 3 HxNA Ž4 m Cirml, 0.2 m molrl. for 30 min at 378C. Thereafter, the tissues were transferred to 0.3 ml perfusion chambers. The rate of perfusion was 0.4 mlrmin and the temperature of the suprafusion system ŽBrandel SF-6 suprafusion system, Brandel, Gaithersburg, USA. was kept constant at 378C. The tissues were washed for 75 min with Krebs–Henseleit solution, during which a priming stimulation Ž5 Hz frequency, 60 s duration, 50 mA intensity, 2 ms pulses. was given at 40 min to remove the unbound or loosely bound w 3 HxNA. After washing, the effluent was collected, for radioactivity estimation during twelve 5-min sampling periods for 60 min. During this procedure, the tissues were field-stimulated twice Ž5 Hz frequency, 60 s duration, 50 mA intensity, 2 ms pulses. at 10 min ŽS 1 . and 45 min ŽS 2 .. Twenty minutes before S 2 , the drugs were added to the suprafusion system to determine their effects on the stimulation-induced ŽS-I. outflow of radioactivity. The drugs were dissolved in Krebs– Henseleit solution. RŽy.-propylnorapomorphine hydrochloride and pimozide were dissolved in methanol and dimethyl sulfoxide, respectively. Experiments were performed either in the presence or in the absence of 1 m M desipramine to block NA uptake.
2.3. Determination of NA release At the end of the experiment, the atria were dissolved in tissue solubilizer NCS-II ŽAmersham Corp, Mississauga, Ontario, Canada.. The radioactivity present in the bathing solution was determined by liquid scintillation counting ŽBeckman model LS3801, Beckman Corp., Irvine, USA., the solution was then mixed with biodegradable counting scintillant ŽBCS, Amersham Corp., Mississauga, Ontario, Canada.. Organic counting scintillant ŽOCS, Amersham Corp., Mississauga, Ontario, Canada. was used to estimate the radioactivity present in the dissolved atria. Corrections for counting efficiency were made by automatic external standardization. The resting radioactive outflow was taken during the 5-min period before the stimulation and the S-I component of the radioactivity outflow was calculated by subtracting the resting radioactive outflow from the total radioactivity outflow during the 5-min period involving the stimulation. The S-I outflow of radioactivity measured during the second period of stimulation ŽS 2 . was expressed as the percentage of the first period of stimulation ŽS 1 .. The values were standardized for the total tissue radioactivity ŽTTR. measured at the end of the experiments and therefore expressed as the fractional release ŽFR. of w 3 HxNA using the ratio FR 2rFR 1 to assess the effect of the various treatments. 2.4. Determination of tissue leÕels of endogenous catecholamines The catecholamine tissue content was determined by HPLC coupled to electrochemical detection by using the method developed by Boudreau et al. w2x with minor modifications. Briefly, the tissues were homogenized in perchloric acid Ž0.1 M. and centrifuged for 20 min at 48C Ž3500 rpm.. To 500 m l of aqueous phase was added 200 m l of dihydroxybenzylamine Ž40 ngrml., 100 m l of sodium metabisulfite Ž5.26 nM., 100 m l of solution containing Tris Ž2 M. with 2% EDTA and alumina Ž50 mg.. The samples were vigorously shaken and centrifuged for 5 min at 3000 rpm. The aqueous solution was removed and the alumina was washed with 3 ml of HPLC grade water. This washing procedure was repeated 3 times. The catecholamines in the alumina fraction were extracted with 200 m l of hydrochloric acid Ž0.2 M.. The samples were shaken, centrifuged and 150 m l was mixed with 150 m l of the HPLC ŽWaters 460 electrochemical detector combined with a automatic injector Wisp 710-B, Waters Associates, Milford, USA. mobile phase solution and injected directly into the HPLC column ŽC 18 reverse-phase column, CSC-SpherisorbODS2, 3 m m, Chromatography Sciences Company, Montreal, ´ Canada.. The mobile phase solution contained potassium phosphate monobasic Ž0.05 M., sodium acetate Ž0.05 M., octyl sodium sulfate Ž0.7%., EDTA Ž0.5%. and methanol Ž10%.. All reagents used were HPLC grade. Before use, the mobile phase solution was filtered ŽMilli-
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pore type HA, 0.45 m m. and degassed. Typical recoveries ranged from 90 to 98% for NA, adrenaline and dopamine. 2.5. Drugs and Õehicles The Krebs–Henseleit solution for the suprafusion experiment contained ŽmM.: NaCl 118, KCl 4.7, CaCl 2 2.5, MgSO4 0.45, glucose 11.1, EDTA 0.067, KH 2 PO4 1.03, NaHCO 3 25, and ascorbic acid 0.14. Desipramine Ž1 m M. was added to the Krebs–Henseleit solution before the washing procedure to block NA uptake throughout the experiment. Idazoxan, oxymetazoline, desipramine, and atropine were obtained from Sigma Chemical Co ŽSt. Louis, MO, USA.; leÕo-wring-2,5,6-3 Hxnoradrenaline from Du Pont ŽMississauga, Ontario, Canada.; angiotensin II from Boehringer Mannheim ŽLaval, Quebec, Canada.; arecai´ dine propargyl ester, RŽy.-propylnorapomorphine hydrochloride, pirenzepine dihydrochloride, pimozide and ICI 118,551 from Research Biochemicals International ŽRBI, Natick, MA, USA. and fenoterol hydrobromide from Boehringer Ingelheim ŽBurlington, Ontario, Canada.. Losartan was generously supplied by Merk Frosst Canada ŽPointe Claire-Dorval, Quebec, Canada.. ´ 2.6. Statistical methods All the results are expressed as the mean " S.E.M. Statistical differences were assessed by a one-way or a two-way ANOVA followed by a Newman–Keuls test or a Bonferroni test. A P value of less than 0.05 was considered significant. The IC 50 and EC 50 were calculated using the ‘allfit’ program w7x.
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and dopamine were measured by HPLC in 10 atrial appendages from an independent group of patients. The values were 1.31 " 0.12 m g per gram of fresh tissue for NA, 31 " 6 pg for adrenaline and 24 " 3 pg for dopamine. Desipramine Ž1 m M. did not change the outflow of NA evoked by electrical stimulation. The FR 1 values were 0.47 " 0.04 Ž n s 38. without desipramine and 0.50 " 0.04 Ž n s 33. with desipramine. Since the modulatory effect on radioactivity of fenoterol, angiotensin II, oxymetazoline and idazoxan did not differ in the absence or presence of desipramine in various concentrations Ždata not shown., all the results presented were obtained in the presence of 1 m M desipramine. 3.1. Effect of b2-adrenoceptor agonist on S-I outflow of radioactiÕity The b 2-adrenergic receptor agonist fenoterol Ž1–1000 nM. increased the S-I outflow of radioactivity in a concentration-dependent manner ŽFig. 1.. The maximal facilitatory effect was reached at the concentration of 100 nM ŽFR 2rFR 1 s 121.7 " 8.0%, n s 8, versus control 90.2 " 3.9%, n s 33.. The calculated EC 50 was 93.8 nM. The b 2-adrenergic receptor antagonist ICI 118,551 Ž1 m M, n s 10. totally abolished the facilitatory effect of fenoterol 100 nM ŽFig. 1.. By itself, ICI 118,551 had no effect on the S-I outflow of radioactivity Ž90.0 " 9.2, n s 11.. The resting outflow ratio ŽR 2rR 1 . was not changed by any treatment Ždata not shown..
3. Results Pieces of atrial appendage were obtained from 132 patients Ž103 men and 29 women, 35–81 years old, mean age: 62 " 1 years. undergoing open heart surgery for coronary by-pass Ž123 patients., aortic stenosisrinsufficiency Žaortic valve replacement, 8 patients. or both Ž1 patient.. Most of the patients had received calcium antagonists, b-adrenoceptor blocking drugs, angiotensin converting enzyme inhibitors, nitroglycerin and diuretics, alone or in combination. The total tissue radioactivity ŽTTR. measured at the end of each experiment was 3.85 = 10 5 " 0.96 = 10 4 c.p.m. Ž n s 541, total number of experiments.. The fractional resting outflow of radioactivity ŽR 1 ., measured before S 1 and expressed as a percentage of the TTR was 0.39 " 0.01% Ž n s 541. per 5 min sampling period. The S-I outflow of radioactivity during S 1 in the absence of treatment ŽFR 1 ., expressed as a percentage of TTR, was 0.53 " 0.01% Ž n s 541. as measured at the frequency of 5 Hz. Tissue concentrations of endogenous NA, adrenaline
Fig. 1. Effect of fenoterol Ž1–1000 nM., a b 2 -receptor agonist, on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages and its blockade by the b 2-receptor antagonist ICI 118,551 Ž1 m M.. The data is expressed as mean"S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test; Ža. P - 0.05 versus fenoterolqICI 118,551 as determined by a two-way ANOVA followed by a Bonferroni test.
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3.2. Effect of angiotensin II on S-I outflow of radioactiÕity Fig. 2 shows that angiotensin II Ž1–1000 nM. significantly enhanced the S-I concentration-dependent outflow of radioactivity starting at the 10 nM concentration Ž121.1 " 8.7%, n s 6. compared to the control Ž90.2 " 3.9%, n s 33.. The calculated EC 50 was 62.8 nM. This facilitatory effect of angiotensin II Ž100 nM. was totally abolished in the presence of losartan Ž1 m M, n s 9., an AT1 receptor antagonist ŽFig. 2.. Administration of losartan alone did not change the S-I outflow of radioactivity Ž82.9 " 5.3, n s 8.. The resting outflow ratio ŽR 2rR 1 . was not changed by any treatment Ždata not shown.. 3.3. Effects of muscarinic and dopamine receptor agonists on S-I outflow of radioactiÕity
Fig. 2. Effect of angiotensin II Ž1–1000 nM. on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages and its blockade by the AT1 receptor antagonist losartan Ž1 m M.. The data are expressed as mean"S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test; Ža. P - 0.05 versus angiotensin IIqlosartan, as determined by a two-way ANOVA followed by a Bonferroni test.
Fig. 3. Effect of arecaidine propargyl ester ŽAPE, 0.03–3 m M., a non-specific muscarinic receptor agonist, on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages and its blockade by the M 1 muscarinic receptor antagonist pirenzepine Ž1 m M., and the non-specific muscarinic receptor antagonist atropine Ž1 m M.. The data is expressed as mean"S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test; Ža. P - 0.05 versus APEqatropine; Žb. P - 0.05 versus APEq pirenzepine as determined by a two-way ANOVA followed by a Bonferroni test.
Arecaidine propargyl ester Ž30–3000 nM., a nonspecific muscarinic receptor agonist, produced a significant inhibition of the S-I outflow of radioactivity in a concentration-dependent manner ŽFig. 3.. This effect reached significance at the concentration of 300 nM Ž66.7 " 2.3%, n s 9. versus control Ž90.2 " 3.9%, n s 33. and was maximal at a 1 m M concentration Ž44.6 " 5.8%, n s 8.. The calculated IC 50 was 288 nM. The inhibitory effect of arecaidine propargyl ester Ž1 m M. was totally abolished by atropine Ž1 m M, n s 9., a non-specific muscarinic receptor antagonist, and significantly abolished in the presence of pirenzepine Ž1 m M, n s 9., a specific muscarinic M 1
Fig. 4. Effect of propylnorapomorphine ŽNPA, 0.1–1000 nM., a DA 2 -receptor agonist, on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages and its blockade by pimozide Ž10 m M., a DA 2 -receptor antagonist. The data is expressed as mean" S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test; Ža. P - 0.05 versus NPAqpimozide as determined by a two-way ANOVA followed by a Bonferroni test.
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Fig. 4.. Pimozide Ž10 m M. had no effect on the S-I outflow of radioactivity Ž89.2 " 9.0, n s 8.. By themselves, propylnorapomorphine and pimozide did not alter the resting outflow ratio at any of the concentrations tested Ždata not shown.. 3.4. Effects of a 2-adrenoceptor agonist and antagonist on S-I outflow of radioactiÕity
Fig. 5. Effect of idazoxan Ž0.1–10 m M. on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages. The data are expressed as mean"S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test.
receptor antagonist ŽFig. 3.. The two antagonists, atropine Ž83.1 " 5.3, n s 8. and pirenzepine Ž85.9 " 5.3, n s 9., did not alter the S-I outflow of radioactivity. By themselves, arecaidine propargyl ester, atropine and pirenzepine had no effect on the resting outflow ratio Ždata not shown.. Similarly, propylnorapomorphine Ž0.1–1000 nM., a DA 2 receptor agonist, inhibited the radioactivity S-I outflow in a concentration-dependent manner ŽFig. 4.. The maximal facilitatory effect was reached at the concentration of 10 nM ŽFR 2rFR 1 s 46.7 " 7.3%, n s 8, versus control 100.5 " 6.4%, n s 8.. The calculated IC 50 was 0.3 nM. This inhibitory effect of propylnorapomorphine was partially abolished in the presence of pimozide Ž1 m M. a dopaminergic DA 2 receptor antagonist Ž70.7 " 7.3%, n s 8, versus 84.1 " 2.8 %, n s 8, data not shown., but was totally abolished by pimozide at a concentration of 10 m M Ž87.7 " 7.6%, n s 8, versus control 84.1 " 2.8 %, n s 8,
The a 2-adrenergic agonist, oxymetazoline, did not modify the S-I outflow of radioactivity at either 1 m M Ž85.6 " 7.6%, n s 11. or 10 m M Ž83.0 " 4.6%, n s 6.. However, idazoxan, an a 2-adrenergic antagonist, increased the S-I outflow of radioactivity in a concentration-dependent manner ŽFig. 5.. This effect was significant at a concentration of 1 m M Ž119.6 " 5.5%, n s 6. compared to the control Ž90.2 " 3.9%, n s 33.. The resting outflow ratio of radioactivity was not changed by either idazoxan or oxymetazoline at the concentrations tested Ždata not shown.. Since the modulatory action of prejunctional autoreceptors is influenced by the intensity of the stimulation w33x, we tested the effect of both idazoxan and oxymetazoline at the frequencies of 2 and 10 Hz. At the frequency of 2 Hz, the TTR value was 4.68 = 10 5 " 3.00 = 10 4 c.p.m. Ž n s 8., the R 1 value was 0.45 " 0.02% Ž n s 8. and the FR 1 value was 0.21 " 0.04% Ž n s 8.. At the frequency of 10 Hz, the TTR value was 5.71 = 10 5 " 4.36 = 10 4 c.p.m. Ž n s 8., the R 1 value was 0.40 " 0.02% Ž n s 8. and the FR 1 value was 1.18 " 0.15% Ž n s 8.. The results observed show that at 2 Hz, oxymetazoline Ž1 m M. inhibited significantly the S-I outflow of radioactivity ŽFR 2rFR 1 s 59.7 " 8.8%, n s 8, versus control 86.3 " 9.3%, n s 8, Fig. 6.. At 10 Hz, oxymetazoline had no effect. Idazoxan Ž1 m M. enhanced significantly the S-I outflow of radioactivity at 2 Hz Ž138.3 " 11.7%, n s 8, versus control 86.3 " 9.3%, n s 8. but had no modulatory action at 10 Hz.
4. Discussion In the present study, the electrical S-I outflow of radioactivity from human atria was taken as an index of
Fig. 6. Effect of oxymetazoline ŽOXY, 1 m M. or idazoxan ŽIDA, 1 m M. on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages at stimulation frequencies of 2 Hz ŽA., 5 Hz ŽB. and 10 Hz ŽC.. The data are expressed as mean" S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test.
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endogenous NA release from the sympathetic nerve fibres w14x. Rump et al. w25x have previously demonstrated that this experimental model is appropriate for studying exocytotic NA release since the release of radioactivity is abolished by tetrodotoxin and under Ca2q-free conditions. The uptake of w 3 HxNA as measured by the TTR value obtained at the end of the experimental protocol reflects the presence of sympathetic nerve endings in the human atrial appendages. In addition, the tissue levels of catecholamines, as measured by HPLC, were comparable to those obtained by Chidsey et al. w5x and Rump et al. w25x. Moreover, we observed a small degree of variability in the release parameters and on the modulatory responses to the various agents tested despite the relative heterogeneity of the patient population Žage, sex, race, pathology and medication.. The b 2-adrenergic receptor agonist fenoterol increased the S-I outflow of radioactivity in a concentration-dependent manner. The facilitatory effect of fenoterol was totally abolished by the selective b 2-adrenergic receptor antagonist ICI 118,551 Ž1 m M.. Therefore, our data support the presence of facilitatory prejunctional b 2-adrenoceptors. Similar results were previously observed with isoproterenol, a b 2-adrenergic receptor agonist, on isolated human right atrium w25x, and kidney w26x, or in vivo on human forearm w30x. The absence of an ICI 118,551-induced effect on the S-I outflow of radioactivity would seem to indicate that the b 2-facilitation is not tonically active under the experimental conditions used. Angiotensin II receptors have been shown to enhance the release of NA w22,23,35x. In the present study, angiotensin II increased the S-I outflow of radioactivity from the human atrium. However, the facilitatory effect of angiotensin II was totally abolished by losartan, the first non-peptidic AT1 receptor antagonist w8,31x suggesting that the angiotensin II effect was mediated by prejunctional AT1 receptors. Our results are in accordance with the findings of Rump et al. w25x on human atrial appendages. The identification of AT1 receptors in human cardiac tissues has an important clinical relevance, since AT1 receptor antagonists are effective in reducing blood pressure in hypertensive patients Žfor review see Refs. w1,32x.. Increases in angiotensin II receptor density have been demonstrated in steptozotocin-induced diabetic rat hearts w4x, in SHR rats w10x and in rats with coronary artery-ligation-induced infarction w20x. Therefore, the facilitatory effect of angiotensin II on NA release needs to be reassessed in defined populations of patients with heart failure and an associated pathology such as hypertension or diabetes. It has been shown that muscarinic agonists inhibit the stimulation-evoked NA release w15x. In our study, the muscarinic receptor agonist, arecaidine propargyl ester, inhibited the S-I outflow of radioactivity from human atrium in a concentration-dependent manner. This effect was totally abolished by the non-selective muscarinic receptor antagonist, atropine, and by the selective M 1 recep-
tor antagonist, pirenzepine. This suggests that NA release can be inhibited by M 1 receptor activation in human atrial appendages. We cannot rule out the possible involvement of other muscarinic receptor subtypes since M 2 receptor activation was found to inhibit NA release from rat gastric sympathetic nerves w37x or guinea pig heart w11x and M 3 receptor activation was found to inhibit NA release from human papillary muscle w19x. The observation that both atropine and pirenzepine did not increase the S-I outflow of radioactivity from the isolated human atrial appendage indicate that the muscarinic inhibitory receptors are not tonically active under the experimental conditions used. Similar observation was made by Rump et al. w27x. The DA 2-receptor agonist, propylnorapomorphine, inhibited the S-I outflow of radioactivity in a concentrationdependent manner with a very high sensitivity. However, pimozide Ž1 m M., a specific DA 2-receptor antagonist, did not totally abolish this propylnorapomorphine effect. By itself, pimozide had no effect on the S-I outflow of radioactivity even at the high concentration of 10 m M. Our results strongly suggest the presence of inhibitory DA 2-receptors in the human atrial appendage and support the findings obtained by Rump et al. w27x. In their study, they found that quinpirole, a DA 2-receptor agonist, inhibited NA release with an IC 50 of 30 nM which is 100-fold higher than the calculated IC 50 obtained for propylnorapomorphine Ž0.3 nM.. This difference in efficacy for these two agonists may be of relevance for the clinical use of DA 2-dopaminergic agonist for the treatment of heart failure. In the present study, the a 2-adrenoceptor agonist, oxymetazoline, inhibited release at 2 Hz but not at 5 and 10 Hz. Such an observation suggests that the a 2-adrenoceptor-mediated inhibitory mechanism is already fully activated by the endogenous NA release at a frequency of 5 Hz and more. This characteristic is typical of inhibitory presynaptic autoreceptors w13,28,36x. However, the results obtained with the a 2-adrenoceptor antagonist, idazoxan, do not support this possibility since its administration resulted in a significant increase of NA release at low frequencies of stimulation but not at 10 Hz. The reason for the discrepancy between these two results is unknown. Oxymetazoline and idazoxan have been used in the isolated rat atria and were found to be very effective in either inhibiting or enhancing respectively the release of NA w9x. In the current study, the magnitude of the modulatory effect observed with both a 2-adrenoceptor agonist and antagonist was relatively small compared with those found by Rump et al. w27x. Thus, as observed with the DA 2dopaminergic agonists Žsee above., the efficacy of the a 2-adrenoceptor agonist and antagonist may be very different in human tissues compared to animal tissues. In addition, the efficacy of the a 2-adrenoceptor agents may depend on the state of regulation of the receptors themselves. Therefore, an increased sympathetic activity whether acute and induced by the surgical procedures w6x or chronic and
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dependent on the pathology of the patient w16,21,24x, may be responsible for the relatively poor modulatory responses observed with the a 2-adrenoceptor agents. In conclusion, evidence has been obtained that the release of NA in human atrial appendages is subject to presynaptic modulation through a variety of receptors. The activation of b 2-adrenoceptors and AT1 receptors enhance NA release, whereas the activation of a 2-adrenoceptors, M 1-muscarinic and DA 2-dopaminergic receptors resulted in its inhibition.
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Acknowledgements We thank Ms Louise Grondin for her expert technical assistance. This work was supported by a grant from the Medical Research Council of Canada to S.F. and R.N. S.F. is a junior scholar from the Fonds de la Recherche en Sante´ du Quebec. R.N. is an MRC career investigator. ´
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w18x
References w19x w1x Bauer, J.H. and Reams, G.P., The angiotensin II type 1 receptor antagonist. A new class of antihypertensive drugs, Arch. Intern. Med., 155 Ž1995. 1361–1368. w2x Boudreau, G., Peronnet, F., de Champlain, J. and Nadeau, R., ´ Presynaptic effects of epinephrine on norepinephrine release from cardiac sympathetic nerves in dogs, Am. J. Physiol., 265 Ž1993. H205–H211. w3x Brown, M.J., Struthers, A.D., Burrin, J.M., Di Silvio, L. and Brown, D.C., The physiological and pharmacological role of presynaptic aand b-adrenoceptors in man, Br. J. Clin. Pharmacol., 20 Ž1985. 649–658. w4x Brown, L. and Sernia, C., Angiotensin II receptors in diabetes in rats, Abs., Clin. Exp. Pharmacol. Physiol., Suppl. 1 Ž1993. 10. w5x Chidsey, C.A., Braunwald, E., Morrow, A.G. and Mason, T.D., Myocardial norepinephrine concentration in man: effects of reserpine and of congestive heart failure, N. Engl. J. Med., 269 Ž1963. 653–658. w6x Deegan, R., He, H.B., Krivoruk, Y., Wood, A.J.J. and Wood, M., Regulation of norepinephrine release by b 2 -adrenergic receptors during halothane anaesthesia, Anaesthesiology, 82 Ž1995. 1417– 1425. w7x DeLean, A., Munson, P.J. and Rodbard, R., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radio ligand assay, and physiological dose-response curves, Am. J. Physiol., 235 Ž1978. E97–E102. w8x Duncia, J.V., Carini, D.J., Chiu, A.T., Johnson, A.L., Prince, W.A., Wong, P.C., et al., The discovery of DuP 753, a potent, orally non-peptide angiotensin II receptor antagonist, Med. Res. Rev., 12 Ž1992. 149–191. w9x Foucart, S., Wang, R., Moreau, P., Sauve, ´ R., de Champlain, J., Yamaguchi, N., Bai, L. and Lu, X.R., Effects of Buthus martensii Karsch scorpion venom on the release of noradrenaline from in vitro and in vivo preparations, Can. J. Physiol. Pharmacol., 72 Ž1994. 855–861. w10x Gutkind, J.S., Kurihara, M., Castren, E. and Saavedra, J.M., Increased concentration of angiotensin II binding sites in selected brain areas of spontaneous hypertensive rats, J. Hypertension, 6 Ž1988. 79–84. w11x Haunstetter, A., Haass, M., Yi, X., Kruger, C. and Kubler, W., Muscarinic inhibition of cardia norepinephrine and neuropeptide Y
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A.D., Sympathetically mediated vasoconstriction is augmented by angiotensin II in man, J. Hypertension, 6 Ž1988. S542–S543. w35x Westfall, T.C., Local regulation of adrenergic transmission, Physiol. Rev., 57 Ž1977. 659–728. w36x Yamaguchi, N., de Champlain, J. and Nadeau, R., Regulation of norepinephrine release from cardiac sympathetic fibres in the dog by presynaptic a- and b-receptors, Circ. Res., 41 Ž1977. 108–117. w37x Yokotani, K. and Osumi, Y., Cholinergic M 2 muscarinic receptormediated inhibition of endogenous noradrenaline from the isolated vascularly perfused rat stomach, J. Pharmacol. Exp. Ther., 264 Ž1993. 54–60.
Modulation of noradrenaline release from isolated human atrial appendages Claire Abadie
a,)
, Sylvain Foucart a , Pierre Page´ b, Reginald Nadeau ´
c
a
Research Centre, Hopital du Sacre-Coeur de Montreal, ˆ ´ ´ 5400 boul. Gouin ouest, Groupe de Recherche sur le Systeme ` NerÕeux Autonome, Department of Physiology, UniÕersite´ de Montreal, Canada, H4J 1C5 ´ Montreal, ´ Quebec, ´ b Research Centre, Hopital du Sacre-Coeur de Montreal, ˆ ´ ´ Groupe de Recherche sur le Systeme ` NerÕeux Autonome, Department of Surgery, UniÕersite´ de Montreal, Canada ´ Montreal, ´ Quebec, ´ c Research Centre, Hopital du Sacre-Coeur de Montreal, ˆ ´ ´ Groupe de Recherche sur le Systeme ` NerÕeux Autonome, Department of Medicine, UniÕersite´ de Montreal, Canada ´ Montreal, ´ Quebec, ´ Received 9 April 1996; revised 4 July 1996; accepted 19 July 1996
Abstract Prejunctional modulation of noradrenaline release has been studied extensively in various experimental preparations. However, the presence and importance of prejunctional noradrenaline release modulation in human cardiac tissue is still unclear. In this study, we have used superfused human right atrial appendages excised from patients undergoing open heart surgery. The tissues were cut into six pieces and incubated with w 3 Hxnoradrenaline Ž4 m Cirml, 0.2 m M. for 30 min at 378C. The tissues were then inserted into a suprafusion system and washed for 75 min with a Krebs–Henseleit solution at a rate of 0.4 mlrmin. The experimental protocol consisted of a 60-min perfusion period during which a field stimulation Ž2 ms pulses, 60 s, 50 mA, 5 Hz. was delivered at 10 and 45 min. The effect of the drugs on the stimulation-induced outflow of radioactivity was determined by adding them 20 min before the second stimulation. Each experiment was carried out with or without desipramine Ž1 m M. to study the influence of the reuptake blockade. Fenoterol Ž1–1000 nM., a b 2-adrenoceptor agonist, and angiotensin II Ž1–1000 nM. significantly increased noradrenaline release in a concentration-dependent manner. The administration of arecaidine propargyl ester Ž0.03–3 m M., a non-specific muscarinic receptor agonist, and propylnorapomorphine Ž0.1 nM–1 m M., a DA 2-dopaminergic agonist, produced a concentration-dependent inhibition of the stimulation-induced outflow of radioactivity. The a 2-adrenoceptor agonist, oxymetazoline Ž1 m M., inhibited noradrenaline release at a stimulation frequency of 2 Hz, but not at 5 and 10 Hz. The a 2-adrenoceptor antagonist, idazoxan Ž1 m M., significantly increased the release of noradrenaline at 2 and 5 Hz but not at 10 Hz. The results obtained in the present study demonstrated the presence of the facilitatory b 2-adrenoceptor and angiotensin II receptor as well as the presence of inhibitory a 2-adrenoceptor, muscarinic and DA 2-dopamine receptors in the human atrial appendage. Keywords: Human atrial appendage; Noradrenaline release; Presynaptic modulation; ß 2 -adrenoceptors; Angiotensin II; Muscarinic receptor; Dopaminergic receptor
1. Introduction In most studies, the role of prejunctional modulation of noradrenaline ŽNA. release has been evaluated using in vitro or in vivo experimental preparations of animal tissues Žfor review see Refs. w17,18,29x.. In humans, the i.v. administration of NA has revealed the presence of an a-adrenoceptor-mediated inhibition of NA release, whereas the i.v. administration of adrenaline showed the presence )
Corresponding author. Tel: q1 514 3382518; fax: q1 514 3382694; e-mail: [email protected].
of a b-adrenoceptor-mediated facilitation w3,12,13x. The renin–angiotensin system is also involved since the decrease in plasma NA induced by the administration of converting enzyme inhibitors is linked with the loss of the facilitatory effect mediated by angiotensin II on NA release w34x. The modulatory actions of b 2-adrenergic agonists and angiotensin II w25x, dopaminergic and a-adrenergic agonists w27x have recently been observed in pieces of human atrial appendage. These data clearly suggest that prejunctional modulation of NA release is occurring in humans. However, more studies are needed to enable a better understanding of these modulatory mechanisms.
0165-1838r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 6 . 0 0 0 9 3 - 8
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The aim of the present study was the identification of potential neuromodulators of NA release from sympathetic nerves in human atrial tissue. Several receptors have been identified for their ability to modulate positively or negatively the release of NA from sympathetic nerve terminals w23,29,35,36x. The results obtained demonstrate the presence of facilitatory mechanisms via the activation of b 2adrenoceptors and angiotensin II receptors, and inhibitory mechanisms via the activation of a 2-adrenoceptors, and muscarinic and DA 2-dopamine receptors in the human atrial appendages.
2. Materials and methods 2.1. Human atrial tissue Pieces of human atrial appendages were obtained from patients undergoing open heart coronary artery or valvular surgery. The excision of the atrial appendage was performed at the time of cannulation for the establishment of extracorporeal circulation. The experimental protocol was conducted with the understanding and the consent of each subject and was approved by the hospital ethics committee. 2.2. Experimental protocol Immediately after excision, specimens of atrial appendage Žweighing around 200 mg. were placed in an ice-cold Krebs–Henseleit solution ŽpH 7.4., previously gassed with a mixture of O 2rCO 2 Ž95%r5%., and quickly transferred to the laboratory, cleared from fat tissue and sliced into 6 pieces of equal dimensions. These pieces were then incubated with w 3 HxNA Ž4 m Cirml, 0.2 m molrl. for 30 min at 378C. Thereafter, the tissues were transferred to 0.3 ml perfusion chambers. The rate of perfusion was 0.4 mlrmin and the temperature of the suprafusion system ŽBrandel SF-6 suprafusion system, Brandel, Gaithersburg, USA. was kept constant at 378C. The tissues were washed for 75 min with Krebs–Henseleit solution, during which a priming stimulation Ž5 Hz frequency, 60 s duration, 50 mA intensity, 2 ms pulses. was given at 40 min to remove the unbound or loosely bound w 3 HxNA. After washing, the effluent was collected, for radioactivity estimation during twelve 5-min sampling periods for 60 min. During this procedure, the tissues were field-stimulated twice Ž5 Hz frequency, 60 s duration, 50 mA intensity, 2 ms pulses. at 10 min ŽS 1 . and 45 min ŽS 2 .. Twenty minutes before S 2 , the drugs were added to the suprafusion system to determine their effects on the stimulation-induced ŽS-I. outflow of radioactivity. The drugs were dissolved in Krebs– Henseleit solution. RŽy.-propylnorapomorphine hydrochloride and pimozide were dissolved in methanol and dimethyl sulfoxide, respectively. Experiments were performed either in the presence or in the absence of 1 m M desipramine to block NA uptake.
2.3. Determination of NA release At the end of the experiment, the atria were dissolved in tissue solubilizer NCS-II ŽAmersham Corp, Mississauga, Ontario, Canada.. The radioactivity present in the bathing solution was determined by liquid scintillation counting ŽBeckman model LS3801, Beckman Corp., Irvine, USA., the solution was then mixed with biodegradable counting scintillant ŽBCS, Amersham Corp., Mississauga, Ontario, Canada.. Organic counting scintillant ŽOCS, Amersham Corp., Mississauga, Ontario, Canada. was used to estimate the radioactivity present in the dissolved atria. Corrections for counting efficiency were made by automatic external standardization. The resting radioactive outflow was taken during the 5-min period before the stimulation and the S-I component of the radioactivity outflow was calculated by subtracting the resting radioactive outflow from the total radioactivity outflow during the 5-min period involving the stimulation. The S-I outflow of radioactivity measured during the second period of stimulation ŽS 2 . was expressed as the percentage of the first period of stimulation ŽS 1 .. The values were standardized for the total tissue radioactivity ŽTTR. measured at the end of the experiments and therefore expressed as the fractional release ŽFR. of w 3 HxNA using the ratio FR 2rFR 1 to assess the effect of the various treatments. 2.4. Determination of tissue leÕels of endogenous catecholamines The catecholamine tissue content was determined by HPLC coupled to electrochemical detection by using the method developed by Boudreau et al. w2x with minor modifications. Briefly, the tissues were homogenized in perchloric acid Ž0.1 M. and centrifuged for 20 min at 48C Ž3500 rpm.. To 500 m l of aqueous phase was added 200 m l of dihydroxybenzylamine Ž40 ngrml., 100 m l of sodium metabisulfite Ž5.26 nM., 100 m l of solution containing Tris Ž2 M. with 2% EDTA and alumina Ž50 mg.. The samples were vigorously shaken and centrifuged for 5 min at 3000 rpm. The aqueous solution was removed and the alumina was washed with 3 ml of HPLC grade water. This washing procedure was repeated 3 times. The catecholamines in the alumina fraction were extracted with 200 m l of hydrochloric acid Ž0.2 M.. The samples were shaken, centrifuged and 150 m l was mixed with 150 m l of the HPLC ŽWaters 460 electrochemical detector combined with a automatic injector Wisp 710-B, Waters Associates, Milford, USA. mobile phase solution and injected directly into the HPLC column ŽC 18 reverse-phase column, CSC-SpherisorbODS2, 3 m m, Chromatography Sciences Company, Montreal, ´ Canada.. The mobile phase solution contained potassium phosphate monobasic Ž0.05 M., sodium acetate Ž0.05 M., octyl sodium sulfate Ž0.7%., EDTA Ž0.5%. and methanol Ž10%.. All reagents used were HPLC grade. Before use, the mobile phase solution was filtered ŽMilli-
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pore type HA, 0.45 m m. and degassed. Typical recoveries ranged from 90 to 98% for NA, adrenaline and dopamine. 2.5. Drugs and Õehicles The Krebs–Henseleit solution for the suprafusion experiment contained ŽmM.: NaCl 118, KCl 4.7, CaCl 2 2.5, MgSO4 0.45, glucose 11.1, EDTA 0.067, KH 2 PO4 1.03, NaHCO 3 25, and ascorbic acid 0.14. Desipramine Ž1 m M. was added to the Krebs–Henseleit solution before the washing procedure to block NA uptake throughout the experiment. Idazoxan, oxymetazoline, desipramine, and atropine were obtained from Sigma Chemical Co ŽSt. Louis, MO, USA.; leÕo-wring-2,5,6-3 Hxnoradrenaline from Du Pont ŽMississauga, Ontario, Canada.; angiotensin II from Boehringer Mannheim ŽLaval, Quebec, Canada.; arecai´ dine propargyl ester, RŽy.-propylnorapomorphine hydrochloride, pirenzepine dihydrochloride, pimozide and ICI 118,551 from Research Biochemicals International ŽRBI, Natick, MA, USA. and fenoterol hydrobromide from Boehringer Ingelheim ŽBurlington, Ontario, Canada.. Losartan was generously supplied by Merk Frosst Canada ŽPointe Claire-Dorval, Quebec, Canada.. ´ 2.6. Statistical methods All the results are expressed as the mean " S.E.M. Statistical differences were assessed by a one-way or a two-way ANOVA followed by a Newman–Keuls test or a Bonferroni test. A P value of less than 0.05 was considered significant. The IC 50 and EC 50 were calculated using the ‘allfit’ program w7x.
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and dopamine were measured by HPLC in 10 atrial appendages from an independent group of patients. The values were 1.31 " 0.12 m g per gram of fresh tissue for NA, 31 " 6 pg for adrenaline and 24 " 3 pg for dopamine. Desipramine Ž1 m M. did not change the outflow of NA evoked by electrical stimulation. The FR 1 values were 0.47 " 0.04 Ž n s 38. without desipramine and 0.50 " 0.04 Ž n s 33. with desipramine. Since the modulatory effect on radioactivity of fenoterol, angiotensin II, oxymetazoline and idazoxan did not differ in the absence or presence of desipramine in various concentrations Ždata not shown., all the results presented were obtained in the presence of 1 m M desipramine. 3.1. Effect of b2-adrenoceptor agonist on S-I outflow of radioactiÕity The b 2-adrenergic receptor agonist fenoterol Ž1–1000 nM. increased the S-I outflow of radioactivity in a concentration-dependent manner ŽFig. 1.. The maximal facilitatory effect was reached at the concentration of 100 nM ŽFR 2rFR 1 s 121.7 " 8.0%, n s 8, versus control 90.2 " 3.9%, n s 33.. The calculated EC 50 was 93.8 nM. The b 2-adrenergic receptor antagonist ICI 118,551 Ž1 m M, n s 10. totally abolished the facilitatory effect of fenoterol 100 nM ŽFig. 1.. By itself, ICI 118,551 had no effect on the S-I outflow of radioactivity Ž90.0 " 9.2, n s 11.. The resting outflow ratio ŽR 2rR 1 . was not changed by any treatment Ždata not shown..
3. Results Pieces of atrial appendage were obtained from 132 patients Ž103 men and 29 women, 35–81 years old, mean age: 62 " 1 years. undergoing open heart surgery for coronary by-pass Ž123 patients., aortic stenosisrinsufficiency Žaortic valve replacement, 8 patients. or both Ž1 patient.. Most of the patients had received calcium antagonists, b-adrenoceptor blocking drugs, angiotensin converting enzyme inhibitors, nitroglycerin and diuretics, alone or in combination. The total tissue radioactivity ŽTTR. measured at the end of each experiment was 3.85 = 10 5 " 0.96 = 10 4 c.p.m. Ž n s 541, total number of experiments.. The fractional resting outflow of radioactivity ŽR 1 ., measured before S 1 and expressed as a percentage of the TTR was 0.39 " 0.01% Ž n s 541. per 5 min sampling period. The S-I outflow of radioactivity during S 1 in the absence of treatment ŽFR 1 ., expressed as a percentage of TTR, was 0.53 " 0.01% Ž n s 541. as measured at the frequency of 5 Hz. Tissue concentrations of endogenous NA, adrenaline
Fig. 1. Effect of fenoterol Ž1–1000 nM., a b 2 -receptor agonist, on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages and its blockade by the b 2-receptor antagonist ICI 118,551 Ž1 m M.. The data is expressed as mean"S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test; Ža. P - 0.05 versus fenoterolqICI 118,551 as determined by a two-way ANOVA followed by a Bonferroni test.
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3.2. Effect of angiotensin II on S-I outflow of radioactiÕity Fig. 2 shows that angiotensin II Ž1–1000 nM. significantly enhanced the S-I concentration-dependent outflow of radioactivity starting at the 10 nM concentration Ž121.1 " 8.7%, n s 6. compared to the control Ž90.2 " 3.9%, n s 33.. The calculated EC 50 was 62.8 nM. This facilitatory effect of angiotensin II Ž100 nM. was totally abolished in the presence of losartan Ž1 m M, n s 9., an AT1 receptor antagonist ŽFig. 2.. Administration of losartan alone did not change the S-I outflow of radioactivity Ž82.9 " 5.3, n s 8.. The resting outflow ratio ŽR 2rR 1 . was not changed by any treatment Ždata not shown.. 3.3. Effects of muscarinic and dopamine receptor agonists on S-I outflow of radioactiÕity
Fig. 2. Effect of angiotensin II Ž1–1000 nM. on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages and its blockade by the AT1 receptor antagonist losartan Ž1 m M.. The data are expressed as mean"S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test; Ža. P - 0.05 versus angiotensin IIqlosartan, as determined by a two-way ANOVA followed by a Bonferroni test.
Fig. 3. Effect of arecaidine propargyl ester ŽAPE, 0.03–3 m M., a non-specific muscarinic receptor agonist, on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages and its blockade by the M 1 muscarinic receptor antagonist pirenzepine Ž1 m M., and the non-specific muscarinic receptor antagonist atropine Ž1 m M.. The data is expressed as mean"S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test; Ža. P - 0.05 versus APEqatropine; Žb. P - 0.05 versus APEq pirenzepine as determined by a two-way ANOVA followed by a Bonferroni test.
Arecaidine propargyl ester Ž30–3000 nM., a nonspecific muscarinic receptor agonist, produced a significant inhibition of the S-I outflow of radioactivity in a concentration-dependent manner ŽFig. 3.. This effect reached significance at the concentration of 300 nM Ž66.7 " 2.3%, n s 9. versus control Ž90.2 " 3.9%, n s 33. and was maximal at a 1 m M concentration Ž44.6 " 5.8%, n s 8.. The calculated IC 50 was 288 nM. The inhibitory effect of arecaidine propargyl ester Ž1 m M. was totally abolished by atropine Ž1 m M, n s 9., a non-specific muscarinic receptor antagonist, and significantly abolished in the presence of pirenzepine Ž1 m M, n s 9., a specific muscarinic M 1
Fig. 4. Effect of propylnorapomorphine ŽNPA, 0.1–1000 nM., a DA 2 -receptor agonist, on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages and its blockade by pimozide Ž10 m M., a DA 2 -receptor antagonist. The data is expressed as mean" S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test; Ža. P - 0.05 versus NPAqpimozide as determined by a two-way ANOVA followed by a Bonferroni test.
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Fig. 4.. Pimozide Ž10 m M. had no effect on the S-I outflow of radioactivity Ž89.2 " 9.0, n s 8.. By themselves, propylnorapomorphine and pimozide did not alter the resting outflow ratio at any of the concentrations tested Ždata not shown.. 3.4. Effects of a 2-adrenoceptor agonist and antagonist on S-I outflow of radioactiÕity
Fig. 5. Effect of idazoxan Ž0.1–10 m M. on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages. The data are expressed as mean"S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test.
receptor antagonist ŽFig. 3.. The two antagonists, atropine Ž83.1 " 5.3, n s 8. and pirenzepine Ž85.9 " 5.3, n s 9., did not alter the S-I outflow of radioactivity. By themselves, arecaidine propargyl ester, atropine and pirenzepine had no effect on the resting outflow ratio Ždata not shown.. Similarly, propylnorapomorphine Ž0.1–1000 nM., a DA 2 receptor agonist, inhibited the radioactivity S-I outflow in a concentration-dependent manner ŽFig. 4.. The maximal facilitatory effect was reached at the concentration of 10 nM ŽFR 2rFR 1 s 46.7 " 7.3%, n s 8, versus control 100.5 " 6.4%, n s 8.. The calculated IC 50 was 0.3 nM. This inhibitory effect of propylnorapomorphine was partially abolished in the presence of pimozide Ž1 m M. a dopaminergic DA 2 receptor antagonist Ž70.7 " 7.3%, n s 8, versus 84.1 " 2.8 %, n s 8, data not shown., but was totally abolished by pimozide at a concentration of 10 m M Ž87.7 " 7.6%, n s 8, versus control 84.1 " 2.8 %, n s 8,
The a 2-adrenergic agonist, oxymetazoline, did not modify the S-I outflow of radioactivity at either 1 m M Ž85.6 " 7.6%, n s 11. or 10 m M Ž83.0 " 4.6%, n s 6.. However, idazoxan, an a 2-adrenergic antagonist, increased the S-I outflow of radioactivity in a concentration-dependent manner ŽFig. 5.. This effect was significant at a concentration of 1 m M Ž119.6 " 5.5%, n s 6. compared to the control Ž90.2 " 3.9%, n s 33.. The resting outflow ratio of radioactivity was not changed by either idazoxan or oxymetazoline at the concentrations tested Ždata not shown.. Since the modulatory action of prejunctional autoreceptors is influenced by the intensity of the stimulation w33x, we tested the effect of both idazoxan and oxymetazoline at the frequencies of 2 and 10 Hz. At the frequency of 2 Hz, the TTR value was 4.68 = 10 5 " 3.00 = 10 4 c.p.m. Ž n s 8., the R 1 value was 0.45 " 0.02% Ž n s 8. and the FR 1 value was 0.21 " 0.04% Ž n s 8.. At the frequency of 10 Hz, the TTR value was 5.71 = 10 5 " 4.36 = 10 4 c.p.m. Ž n s 8., the R 1 value was 0.40 " 0.02% Ž n s 8. and the FR 1 value was 1.18 " 0.15% Ž n s 8.. The results observed show that at 2 Hz, oxymetazoline Ž1 m M. inhibited significantly the S-I outflow of radioactivity ŽFR 2rFR 1 s 59.7 " 8.8%, n s 8, versus control 86.3 " 9.3%, n s 8, Fig. 6.. At 10 Hz, oxymetazoline had no effect. Idazoxan Ž1 m M. enhanced significantly the S-I outflow of radioactivity at 2 Hz Ž138.3 " 11.7%, n s 8, versus control 86.3 " 9.3%, n s 8. but had no modulatory action at 10 Hz.
4. Discussion In the present study, the electrical S-I outflow of radioactivity from human atria was taken as an index of
Fig. 6. Effect of oxymetazoline ŽOXY, 1 m M. or idazoxan ŽIDA, 1 m M. on the fractional stimulation-induced ŽS-I. outflow of radioactivity from human atrial appendages at stimulation frequencies of 2 Hz ŽA., 5 Hz ŽB. and 10 Hz ŽC.. The data are expressed as mean" S.E.M. Ž). P - 0.05 versus control as determined by a one-way ANOVA followed by a Newman–Keuls test.
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endogenous NA release from the sympathetic nerve fibres w14x. Rump et al. w25x have previously demonstrated that this experimental model is appropriate for studying exocytotic NA release since the release of radioactivity is abolished by tetrodotoxin and under Ca2q-free conditions. The uptake of w 3 HxNA as measured by the TTR value obtained at the end of the experimental protocol reflects the presence of sympathetic nerve endings in the human atrial appendages. In addition, the tissue levels of catecholamines, as measured by HPLC, were comparable to those obtained by Chidsey et al. w5x and Rump et al. w25x. Moreover, we observed a small degree of variability in the release parameters and on the modulatory responses to the various agents tested despite the relative heterogeneity of the patient population Žage, sex, race, pathology and medication.. The b 2-adrenergic receptor agonist fenoterol increased the S-I outflow of radioactivity in a concentration-dependent manner. The facilitatory effect of fenoterol was totally abolished by the selective b 2-adrenergic receptor antagonist ICI 118,551 Ž1 m M.. Therefore, our data support the presence of facilitatory prejunctional b 2-adrenoceptors. Similar results were previously observed with isoproterenol, a b 2-adrenergic receptor agonist, on isolated human right atrium w25x, and kidney w26x, or in vivo on human forearm w30x. The absence of an ICI 118,551-induced effect on the S-I outflow of radioactivity would seem to indicate that the b 2-facilitation is not tonically active under the experimental conditions used. Angiotensin II receptors have been shown to enhance the release of NA w22,23,35x. In the present study, angiotensin II increased the S-I outflow of radioactivity from the human atrium. However, the facilitatory effect of angiotensin II was totally abolished by losartan, the first non-peptidic AT1 receptor antagonist w8,31x suggesting that the angiotensin II effect was mediated by prejunctional AT1 receptors. Our results are in accordance with the findings of Rump et al. w25x on human atrial appendages. The identification of AT1 receptors in human cardiac tissues has an important clinical relevance, since AT1 receptor antagonists are effective in reducing blood pressure in hypertensive patients Žfor review see Refs. w1,32x.. Increases in angiotensin II receptor density have been demonstrated in steptozotocin-induced diabetic rat hearts w4x, in SHR rats w10x and in rats with coronary artery-ligation-induced infarction w20x. Therefore, the facilitatory effect of angiotensin II on NA release needs to be reassessed in defined populations of patients with heart failure and an associated pathology such as hypertension or diabetes. It has been shown that muscarinic agonists inhibit the stimulation-evoked NA release w15x. In our study, the muscarinic receptor agonist, arecaidine propargyl ester, inhibited the S-I outflow of radioactivity from human atrium in a concentration-dependent manner. This effect was totally abolished by the non-selective muscarinic receptor antagonist, atropine, and by the selective M 1 recep-
tor antagonist, pirenzepine. This suggests that NA release can be inhibited by M 1 receptor activation in human atrial appendages. We cannot rule out the possible involvement of other muscarinic receptor subtypes since M 2 receptor activation was found to inhibit NA release from rat gastric sympathetic nerves w37x or guinea pig heart w11x and M 3 receptor activation was found to inhibit NA release from human papillary muscle w19x. The observation that both atropine and pirenzepine did not increase the S-I outflow of radioactivity from the isolated human atrial appendage indicate that the muscarinic inhibitory receptors are not tonically active under the experimental conditions used. Similar observation was made by Rump et al. w27x. The DA 2-receptor agonist, propylnorapomorphine, inhibited the S-I outflow of radioactivity in a concentrationdependent manner with a very high sensitivity. However, pimozide Ž1 m M., a specific DA 2-receptor antagonist, did not totally abolish this propylnorapomorphine effect. By itself, pimozide had no effect on the S-I outflow of radioactivity even at the high concentration of 10 m M. Our results strongly suggest the presence of inhibitory DA 2-receptors in the human atrial appendage and support the findings obtained by Rump et al. w27x. In their study, they found that quinpirole, a DA 2-receptor agonist, inhibited NA release with an IC 50 of 30 nM which is 100-fold higher than the calculated IC 50 obtained for propylnorapomorphine Ž0.3 nM.. This difference in efficacy for these two agonists may be of relevance for the clinical use of DA 2-dopaminergic agonist for the treatment of heart failure. In the present study, the a 2-adrenoceptor agonist, oxymetazoline, inhibited release at 2 Hz but not at 5 and 10 Hz. Such an observation suggests that the a 2-adrenoceptor-mediated inhibitory mechanism is already fully activated by the endogenous NA release at a frequency of 5 Hz and more. This characteristic is typical of inhibitory presynaptic autoreceptors w13,28,36x. However, the results obtained with the a 2-adrenoceptor antagonist, idazoxan, do not support this possibility since its administration resulted in a significant increase of NA release at low frequencies of stimulation but not at 10 Hz. The reason for the discrepancy between these two results is unknown. Oxymetazoline and idazoxan have been used in the isolated rat atria and were found to be very effective in either inhibiting or enhancing respectively the release of NA w9x. In the current study, the magnitude of the modulatory effect observed with both a 2-adrenoceptor agonist and antagonist was relatively small compared with those found by Rump et al. w27x. Thus, as observed with the DA 2dopaminergic agonists Žsee above., the efficacy of the a 2-adrenoceptor agonist and antagonist may be very different in human tissues compared to animal tissues. In addition, the efficacy of the a 2-adrenoceptor agents may depend on the state of regulation of the receptors themselves. Therefore, an increased sympathetic activity whether acute and induced by the surgical procedures w6x or chronic and
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dependent on the pathology of the patient w16,21,24x, may be responsible for the relatively poor modulatory responses observed with the a 2-adrenoceptor agents. In conclusion, evidence has been obtained that the release of NA in human atrial appendages is subject to presynaptic modulation through a variety of receptors. The activation of b 2-adrenoceptors and AT1 receptors enhance NA release, whereas the activation of a 2-adrenoceptors, M 1-muscarinic and DA 2-dopaminergic receptors resulted in its inhibition.
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Acknowledgements We thank Ms Louise Grondin for her expert technical assistance. This work was supported by a grant from the Medical Research Council of Canada to S.F. and R.N. S.F. is a junior scholar from the Fonds de la Recherche en Sante´ du Quebec. R.N. is an MRC career investigator. ´
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