ISOLATED GUINEA-PIG ATRIA

Transcription

1 THE JOURNAL OF PHARMAOLOGY AND EXPERIMEN ral THERAPEUTIS opyright 1969 by The Williams & Wilkins o. vol. 168, No. 2 Printed in U.S.A. THE NEGATIVE HRONOTROPI EFFET OF TYRAMINE ON ISOLATED GUINEA-PIG ATRIA T. H. TSAI, R. L. WENDT AND W. R. MGRATH8 Department of Pharmacology, The Wm. S. Merrell ompany, Division of Richardson-Merrell Inc., incinnati, Ohio Accepted for publication April 23, 1969 ABSTRAT TSAI, T. H., R. L. WENDT AND W. R. MGRATH: The negative chronotropic effect of tyramine on isolated guinea-pig atria. J. Pharmacol. Exp. Ther. 168: , The influence of various agents on the negative chronotropic effect of tyramine was studied on atria isolated from guinea pigs pretreated with reserpine (5 mg/kg i.p. 24 hours prior to the experiment). In this atrial preparation, tyramine at concentrations of 1 M or higher produced a negative chronotropic effect. This effect of tyramine was not antagonized by atropine (1#{176} to 1 M), scopolamine (1 to 1 M) or phentolamine (3 X 1 M). However, in the presence of propranolol (1 to 1 M) or physostigmine (1 M), the negative chronotropic effect of tyramine was enhanced. Physostigmine, even in the presence of propranolol, further enhanced the effect of tyramine. The enhancement of the effect of tyramine by physostigmine was blocked by atropine but not by hexamethonium (1O M). Atropine did not block the enhancement of the effect of tyramine by propranolol. The results suggest that the negative chronotropic effect of tyramine is not mediated through alpha or beta adrenergic receptors or muscarinic receptors. Although tyramine appears to be able to release acetyicholine or an acetylcholine-like substance, this activity does not contribute to the observed effect of tyramine except in the presence of physostigmine. Thus, the present results support the view that the negative chronotropic effect of tyramine is due to a direct depressant effect on the atrial pacemaker. The effect of tyramine on spontaneously beating atria isolated from guinea pigs is characterized by a bell-shaped dose-response curve (Muskus, 1962; Trendelenburg et cii., 1963; Smith, 1966). It has first an ascending limb (accelerating action) followed by a descending limb (decelerating action). Trendelenburg et cii. (1963) showed that treatment of guinea pigs with a large dose of reserpine 24 hours prior to the experiment antagonized the positive chronotropic effect of tyramine but not the negative chronotropic effect. These authors suggested that the negative chronotropic effect of tyramine was a direct action on the atrial pacemaker. However, more recently handra et cii. (1965) observed in atria isolated from reserpine-treated Received for publication January 2, Presented at Ohio Valley Section of the Society for Experimental Biology and Medicine, Present address: Department of Pharmacology, Wyeth Laboratories, Philadelphia, Pa Present address: Enzomedic Laboratories, Inc., Seattle, Wash Send reprint requests to: T. H. Tsai, Ph.D., Department of Pharmacology, The Wm. S. Merrell o.. 11 Amity Rd., incinnati, Ohio rabbits that tyra.mine produced a weak and transient positive inotropic effect, but that in the presence of physostigmine the atria produced a marked and prolonged negative motropic effect in these preparations. Since this negative inotropic effect was blocked by atropine, they concluded that this effect is due to the release of acetylcholine. The present experiments were done in an attempt to clarify further the mechanism of the negative chronotropic effect of tyramine on isolated guinea-pig atria. METHODS. Atria were dissected from hearts of guinea pigs of 3 to 5 g b.wt. that had received 5 mg/kg of reserpine i.p. 24 hours before the experiment. The atria were suspended in a 15-mi organ bath containing Locke s solution (9 g of NaI,.42 g of K1, 24 g of al2,.5 g of NaHO8 and 2 g of dextrose per liter) saturated with oxygen. Before any drugs were added, the preparations were allowed to equilibrate in the bath and were washed every 15 minutes until the spontaneous rate did not change more than 5 beats/mm over a 1-minute period. This equilibration period was usually about 1 hour. The spontaneous rate of the preparations was recorded with 29

2 1969 NEGATIVE HRONOThOPI EFFETS OF TYRAMINE 291 a Grass force-displacement transducer and a Grass et al. (1963). At 1 M, the effects of tyramine model 7 polygraph. were variable; it produced a weak positive umulative dose-response curves for tyramine chronotropic effect in four of seven preparations, were determined by the sequential addition of a weak negative chronotropic effect in one drugs to the bath in amounts that increased the preparation and no effect in two preparations. total concentration in steps of approximately #{189} log unit. Each drug addition was made as soon as omplete arrest of the spontaneously beating the effect of the previous addition had reached a atria could be obtained by increasing the concentration of tyramine to 1 M or higher, and, steady level. Only one dose-response curve was obtained with each preparation. Whenever a doseresponse curve of tyramine was determined in the tyramine added did not exceed 3 x 1 M. for this reason, the maximal concentration of presence of an additional drug, the addition of Influence of atro pine and scopolamine. After tynamine was begun only after the response to the treatment of the atrial preparations with either drug had reached a steady level. atropine or scopolamine, 1 M, for 15 minutes, The following substances were used: atropine cumulative dose-response curves of tyramine sulfate, hexamethonium chloride, phentolamine were determined in the presence of atropine or H1, physostigmine sulfate, propranolol H1, scopolamine H1 and reserpine (5 mg/ml dissolved scopolamine. Neither atropine nor scopolamine in 2% ascorbic acid solution). Drug concentration altered the effects of tyramine (fig. 1, A and B). refers to the final concentration in the bath. At 1 M, both atropine and scopolamine had Statistical significance of differences among no effect on the atrial preparations. At 1-i M, various results was evaluated by Student s t test. atropine depressed the rate of spontaneously RESULTS. Response to tyramine. In atria from guinea pigs treated with 5 mg/kg (i.p.) of reserpine 24 hours prior to the experiment, cumulative dose-response curves of tyramine were determined. Tyramine, in concentrations of 3 x 1 M or higher, produced a negative chronotropic effect (figs. 1 and 2, solid lines). The magnitude of the decelerating action of tyramine agrees with that reported by Trendelenburg beating atria (-33. ± 2. beats/mm) but did not inhibit the effect of tyramine. Influence of phentolamine. umulative doseresponse curves of tyra.mine were determined on atria that had been exposed for 3 minutes to phentolamine (3 x 1-f M). As shown in figure 1, phentolamine failed to alter the effect of tyramine on atnia. This concentration of phentolamine itself did not change the atrial rate and I 8).c 8. io- Tyrornine io (M) Fia. 1. The influence of atropine, scopolamine, phentolamine and propranolol on dose-response curves of tyramine. Isolated guinea-pig atnia were obtained from animals pretreated with 5 mg/kg (i.p.) of reserpine 24 hours prior to the experiment. hange in rats is given in beats per minute; initial rats =. Shown are dose-response curves of tyramine. -, controls;, dose-response curves determined in the presence of atropine (A), scopolamine (B), phentolamine () and propranolol (D). Given are the mean responses and their standard errors (vertical bars). Numbers in parentheses at the end of each curve indicate the number of experiments.

3 292 TSAI ET AL. Vol. 168.c E. 8) 8. 2 #{176} -loot Tyromine (M) Fie. 2. The influence of physostigmine on dose-response curves of tynamine. Isolated guinea-pig atnia were obtained from animals pretreated with 5 ms/kg (i.p.) of reserpine 24 hours prior to the experiment. hange in nate is given in beats per minute; initial rate =. Shown are dose-response curves of tyramine. -, controls;, dose-response curves determined in the presence of physostigmine and/or various agents as shown. Given are the mean responses and their standard errors (vertical bars). Numbers in parentheses at the end of each curve indicate the number of experiments. was found to antagonize the effect of l-norepinephrine on the isolated nictitating membrane of the cat (Arthur and Fleming, 1968) and on rabbit aortic strips (Hudgins and Fleming, 1966). Influence of propranolol. After exposure to 1 M propranolol for 3 minutes, the effects of 3 x 1 M and 1 M tyramine on atria were enhanced (P <.5; fig. 1D). A higher concentration of propranolol (16 M) did not further enhance the effect of tyramine. These concentrations of propranolol were shown to antagonize the effect of l-isoproterenol in isolated guinea-pig atria (Blinks, 1967; Tsai et cii., 1967). Influence of physostigmine. umulative doseresponse curves of tyramine were determined after exposure of atrial preparations to 1 M physostigmine for 15 minutes. This concentration of physostigmine did not itself affect the rate of spontaneously beating atria but significantly (P <.5) enhanced the negative chronotropic effect of all but the highest dose of tyramine (fig. 2A, open circles). When atropine, i M, was present in the bath, the enhancement of the effects of tyramine by physostigmine was not observed (fig. 2A, triangles). In contrast, exposure of atnia to 1 M hexamethonium for 3 minutes did not block the effect of physostigmine (fig. 2B, triangles). This concentration of hexamethonium has been shown to block the response of isolated guineapig atria to nicotine (Trendelenburg, 196). Since propranolol augmented the negative chronotropic effect of tyramine (fig. 1D), the influence of a combination of propranolol and physostigmine on the effect of tyramine was evaluated. As shown in figure 2, the combination of propranolol and physostigmine caused a greater enhancement of the effects of tyramine than did propranolol alone. In the presence of atropine, however, the effect of the combination was reduced to the effect of propranolol alone (fig. 2D). DISUSSION. The present results confirm the observation made by Trendelenburg et cii. (1963) that tyramine produces a negative chronotropic effect on atnia from reserpinetreated guinea pigs. The weak positive chronotropic effect observed in some preparations at 1 M tyramine may reflect incomplete depletion of norepinephrine by reserpine. rout et al. (1962) have shown that the schedule of reserpine treatment used leaves about 1% of the normal quantity of norepinephrine in guinea-pig atria. The release of this residual norepinephrine may account for the weak positive chronotropic effect of tyramine at doses lower than those causing a negative chronotropic effect. The enhancement of the negative chronotropic effect of tyramine by propranolol further supports this view and suggests that the effect of tyramine, at any con-

4 1969 NEGATIVE HRONOTROPI EFFETS OF TYRAMINE 293 centration, represents the net effect of a weak acceleration and a strong deceleration. The failure of phentolamine, propnanolol, atropine or scopolamine to antagonize the negative chronotropic effect of tyramine suggests that the effect is not mediated through alpha or beta adrenergic receptors or muscaninic receptors. However, the enhancement of the effect of tyramine by physostigmine and the blockade of this enhancement by atropine suggest that acetylcholine (Ah) or an Ah-like substance may be released by tyramine, but in a quantity that makes no significant contribution to the effect of tyramine. This may be because the rate of release of Ah or Ah-like substance by tyramine is not greater than the rate of hydrolysis by cholinesterase. It should be pointed out that Koppanyi and MacFarlane (1964) observed that atropine, in high concentrations, did not block the effect of Ah (1.tgJml) on atnia isolated from rosenpine-treated rabbits. They postulated that reserpine pretreatment either altered the binding sites for atropine or unmasked an atropine-resistant muscarinic receptor. According to this postulation, the failure of atropine or scopolamine to antagonize the negative chronotropic effects of tyramine in our experiments would not rule out Ah as a mediator. However, the dose of acetylcholine used by Koppanyi and I\ IacFarlane (1964) was in the range of supramaximal doses and may have been too high to be significantly blocked by atropine. Furthermore, the results of our experiments in which the enhanced effect of tyramine in the presence of physostigmine was blocked by atropine suggest that atropine was effective in blocking Ah under the present experimental conditions. Therefore, it appears that the suggestion made by handra et cii. (1965) that Ah mediates the depressant effect of tyramine can only apply to the observed enhancement of the tyramine effect in the presence of physostigmine. Although the negative chronotropic effect of tyramine was also enhanced by propranolol, the failure of atropine to antagonize this enhanced effect (fig. 2D; Thompson and West, 1968) suggests that, even when any possible positive chronotropic effect of tyramine is blocked, the release of Ah or an Ah-like substance makes no significant contribution to the overt effect of tyramine. The failure of hexamethonium to antagonize the enhanced effect of tyramine in the presence of physostigmine indicates that ganglionic effects are not involved and that, whatever the quantity of Ah released by tyramine, the release is probably not through ganglionic stimulation. It is possible that tynamine released Ah from the storage sites in cardiac muscle, which accounts for most of the releasable Ah (Lewartowski and Bielecki, 1963). The present results, thus, support the suggestion of Trendelenburg et at. (1963) that the negative chronotropic effect of tyramine on atnial preparations from reserpine-treated guinea pigs is a direct depressant action on the atrial pacemaker. Although tyramine appears to be able to release Ah, this activity does not contribute to the effect of tyramine observed. ONLUSIONS. The negative chronotropic effect of tyramine on isolated guinea-pig atria is not mediated through alpha or beta adrenergic receptors or muscarinic receptors. Although tyramine appears to be able to release acetylcholine or an acetyicholine-like substance, this activity does not contribute to the observed effect of tyramine except in the presence of physostigmine. The results support the suggestion made by Trendelenbung et cii. (1963) that the negative chronotropic effect of tyramine is due to the direct depressant action on the atnial pacemaker. REFERENES ARTHUR, M. S. AND FLEMING. W. W.: The effects of tyramine on the isolated nictitating membrane of the cat. J. Pharmacol. Exp. Then. 162: , BLINKS, J. R.: Evaluation of the cardiac effects of several beta adnenengic blocking agents. Ann. N.Y. Acad. Sci. 139: , HANDRA,., DHAWAN K. N. An GuI TA, G. P.: Release of acetylcholine by tynamine. Arch. Int. Pharmacodyn. Then. 157: , RoTJT, J. R., MUSKUS, A. J. AND TRENDELENBURG, U.: Effect of tyramine on isolated uinea pig atnia in relation to their nonadrenahne stones. Brit. J. Phanmacol. hemothen. 18: 6-Oil, HUDOINs, P. M. AND FLEMING, W. W.: A relatively nonspecific supersensitivity in aortic strips nesuiting from pretreatment with nesenpine. J. Phanmacol. Exp. Then. 153: 7-8, KOPPANYI, T. AND MAFARi..&es, M. D.: The effect of atropine on responses of normal and rosenpinized rabbit atria to acetylcholine. Life Sci. 3: , LEwAlrrowsxI, B. AND Bimzcsi, K.: The influence of hemicholinium no. 3 and vagal stimulation on acetyicholine content of rabbit atria. J. Pharmacol. Exp. Ther. 142: 24-3, MUSKUS, A. J.: Effect of pretreatment with reser-

5 294 TSAI ET AL. Vol. 168 pine and reserpine analogs on the response of isolated guinea pig atnia to tyramine. J. Pharmacol. Exp. Ther. 138: 296-3,1962. SwrH,. B.: The role of monoamine oxidase in the intraneuronal metabolism of norepinephrine released by indirectly acting sympathomimetic amines or by adrenergic nerve stimulation. J. Pharmacol. Exp. Ther. 151: 27-22, THOMPSON, E. B. ax WEST, T..: Antagonism by propranolol of the cardiac chronotropic response to norepinephrine or to transmural electrical stimulation. J. Pharmacol. Exp. Then. 161: , TRENDELENBURG, U.: The action of histamine and 5-hydroxytryptamine on isolated mammalian atnia. J. Pharmacol. Exp. Then. 13: 45-46, 196. TRENDELENBURG, U., GOMEZ, AIoNzo DE LA SIERRA, B. awn MUSKUS, A.: Modification by neserpine of the response of the atnial pacemaker to sympathomimetic amines. J. Pharmacol. Exp. Then. 141: 31-39, TaM, T. H., Lawosa, S. Z. AND TRENDELENBURG, U.: Effects of dopamine and c-methyl-dopamine on smooth muscle and on the cardiac pacemaker. J. Pharmacol. Exp. Then. 156: , 1967.