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Effects of thromboxane agonists on cardiac adrenergic neurotransmission
European Journal of Pharmacology.', 213 ( 1992) 79- 85
79
~:'~ 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.110
EJP 52344
Effects of thromboxane agonists on cardiac adrenergic neurotransmission Laura Mantclli, Sandra Amcrini, Annalisa Rubino and Fabrizio Ledda Department of Pharmacoh>gy, Unicersity of Florence, Viah"G.B. Morgagni 65, 50134 Florence, Italy Received 16 May 1991, revised MS received 3 December 1991, accepted 24 December 1991
The effects of thromboxanc B 2 (TxB 2) and of two thromboxane mimetics, dl-(9,11), (11,12)-dimethano-TxA 2 (ONO 11006) and 9,11-dideoxy-ll,9-epoxymcthano prostaglandin F2,, (U46619) on the cardiac response to adrenergic nerve stimulation in isolated guinea-pig atria were evaluated. All the agonists dose dependently reduced the positive inotropic effect induced by field stimulation, U46619 being the most active. The inhibitory effect of U46619 was reduced by the thromboxane receptor antagonists, sulotroban and AH 23848B. U46619 did not significantly reduce the positive inotropic effect induced by exogenous noradrenaline. However U46619 was unable to modify the tritium overflow induced by field stimulation in preparations preloaded with [3H]noradrenaline. In addition to this influencc on adrencrgic neurotransmission, U46619 also had a direct positive inotropic effect on cardiac contractility, which was antagonized by AH 23848B. These results indicate that U46619 reduces the cardiac response to sympathetic nerve stimulation and that is also has a direct stimulatory effect on cardiac muscle. Heart; Adrenergic neurotransmission: U46619; ONO 11006; Thromboxane B,
1. Introduction
Prostaglandins have been shown to inhibit sympathetic neurotransmission in several peripheral preparations (for reviews see Hedqvist, 1977; Malik, 1978), including rabbit (Hedqvist et al., 1970; Wennmalm, 1979) and rat (Khan and Malik, 1982) whole heart. Recently we have confirmed that prostaglandins of the E and I series reduce the response to adrenergic nerve stimulation in isolated guinea-pig atria (Mantelli et al., 1990; Mantelli et al., 19911. Like prostaglandins, thromboxane A 2 (TxA 2) is a cyclooxygcnase metabolite that has been reported to have effects opposite to those of prostaglandins in vascular muscle and platelets ( H a m b e r g et al., 1975; Moncada and Vane, 1979; McGiff, 1981). However, the data concerning the effect of TxA 2 on neurotransmission arc controversial. Analogues of TxA 2, such as U46619 and carbocyclic thromboxane A 2 ( c T x A 2 ) , have been shown to potentiate noradrenaline release in several peripheral preparations from rabbits, i.e. the mescnteric artery (Trachtc and Stein, 1989), the vas deferens (Trachte, 1986) and the portal vein (Trachte and Stein, 1988; Stein and Trachte, 1989). On the other hand, cTxA 2 did not
Correspondence to: L. Mantelli, Department of Pharmacology, University of Florence, Viale G.B. Morgagni 65, 50134 Florence, Italy. Tel. 39.55.437 7272, fax 39.55.436 1613.
modify field stimulation-induced noradrenaline release in dog mesenteric artery (Nakajima and Toda, 1986). Although U46619 induced an increase in vascular tone and enhancement of the pressor response to nerve stimulation in rat isolated kidney, it did not affect the stimulation-induced release of noradrenaline from the same preparation ( R u m p and Schollmeyer, 1989). It has also been reported that cTxA z reduced the amplitude of the excitatory action potentials and of the contractions elicited by nerve stimulation in the guinea-pig mesentcric artery, while the effect of exogenous noradrenaline was enhanced by the agonist (Makita, 1983). We now studied the effects of natural TxB 2 and of two synthetic analogues, O N O 11006 and U46619, on cardiac adrenergic neurotransmission in isolated guinea pig atria, by evaluating both the functional response to adrenergic nerve stimulation and the stimulation-induced [3H]noradrenaline overflow.
2. Materials and methods
2.1. Preparations Isolated atria were obtained from male guinea-pigs weighing 200-300 g. The preparations were mounted vertically in a 15-ml glass chamber containing Tyrode solution of the following composition (mM): NaCl 115;
80 KCI 4.7; CaCI 2 1.8; MgSO 4 1.2; K H 2 P O 4 1.2; NaHCO 3 25; glucose 10. The solution was aerated with a gas mixture of 95% 0 2 and 5% CO 2 and had a pH of 7.4. The temperature was kept at 30°C in order to reduce the metabolic demand of the preparations. Atria were stretched to yield a basal force of 700 mg. The preparations were stimulated at a constant rate (4 Hz) slightly higher than the spontaneous rate, by square wave pulses of 0.5 ms duration and twice the threshold voltage intensity through two platinum punctate electrodes connected with a Tektronix pulse generator. Isometric contraction was recorded by an isometric transducer on a pen recorded (Battaglia Rangoni KV 135) and on a dual beam oscilloscope (Tektronix D 13).
2.2. Neurotransmission studies Sympathetic nerve terminals were stimulated by field stimulation during the cardiac refractory period, as previously described (Ledda and Mantelli, 1984). Trains of field pulses (50-75 mA, 1 ms) were applied at 3-rain intervals through two platinum plates parallel to the preparations, connected with a second pulse generator (MARB 8 2 / 2 / 2 0 0 ) . A control circuit allowed timing of the field pulses to begin 10-20 ms after each driving pulse, during the absolute refractory period. Field pulses were delivered one per consecutive contraction. Stimulus-inotropic-response curves were obtained by increasing the number of field pulses, with the following sequence: 2, 4, 6, 8, 10, 12. Subsequent stimulusresponse curves were separated by 30 min. The responses induced by field stimulation were expressed as % changes from the steady state contraction. All the experiments were carried out in the presence of 1 p.M atropine in order to eliminate the parasympathetic component of the response to field stimulation. The effect of the drugs on the stimulus-response curve was studied according to the following protocol: two control stimulus-response curves were obtained with an interval of 30 min. Thus, the agonist was added after 20 min and the stimulus-response curve was repeated after a contact period of 10 min. The ICso values were calculated by evaluating the response induced by a train of intermediate degree (8 pulses). In the experiments on the effects of thromboxane receptor agonists and antagonists, trains of field pulses of an intermediate value (eight pulses) were applied at 15-min intervals. Cumulative concentrations of the agonist were added 10 min before field stimulation. The experiments with the antagonists were done with different preparations, and the antagonists were added to the bathing solution before starting field stimulation. Concentration-response curves for drugs affecting basal contractility (noradrenaline, isoprenaline, TxB 2 and U46619) were made by adding cumulative concentrations of the agonists. The concentration-response curves for U46619 in the absence and presence of the
antagonist were obtained in different preparations since it was not possible to obtain a reproducible concentration-effect curve for U46619, probably as a consequence of desensitization produced by the agonist, as previously described for other peripheral preparations (Liel et al., 1988).
2.3. [~H]Noradrenaline overflow detection Guinea-pig atria were incubated with 5 izCi of [3H]noradrenaline (specific activity 13.7 C i / m m o l ) for 40 min in 2 ml Tyrode solution oxygenated and maintained at 30°C. After this period the preparations were placed in 50 ml Tyrode solution containing 300 lzM ascorbic acid, 1 tzM atropine and 1 IzM phenoxybenzamine; the radioactivity was washed out for 30 min by replacing the solution 4 times. The atria were then mounted vertically in an organ bath similar to that used for functional studies but containing 2.5 ml Tyrode solution. As previously described the preparations were stimulated at a constant rate (4 Hz); train of 100 field pulses were applied during the absolute refractory period, in order to obtain detectable [3H]noradrenaline release, at 15-min intervals. The incubation medium was collected every 5 min. After two collections of bathing solution (for measurement of basal tritium overflow) the first train was applied at the 13th min (SI), and Tyrode solution was again sampled at the 15th min. Five consecutive trains of field pulses were repeated. When the effect of U46619 was tested the drug was added to Tyrode solution 5 min after $2 in order to evaluate the effect on basal and stimulus-induced overflow. Insta-gel, 15 ml, was added to each sample, and the radioactivity was measured on a Packard 1500 Tricarb liquid scintillation analyzer. The curves were compared using the analysis of variance followed by Tukey's test. Student's paired t-test was used for comparing two groups of data. The drugs used were: TxB 2 (Sigma); ONO 11006, kindly supplied by Ono Pharmaceutical (Osaka, Japan); U46619, kindly supplied by Upjohn (Kalamazoo, USA); AH23848B, a gift of Dr. B.M. Bain of Glaxo Group Research (Greenford, England). Sulotroban was kindly supplied by Boehringer Mannheim; noradrenaline hydrochioride and isoprenaline hydrochloride (Sigma); atropine sulphate (BDH); phenoxybenzamine hydrochloride was kindly supplied by SKF; [3H]noradrenaline (Amersham).
3. Results
3.1. Effect of T x B 2 and thromboxane analogues on the cardiac response to field stimulation Guinea-pig atria, electrically driven at 4 Hz, developed a basal tension of 389 _+ 16 mg (n = 20). Trains of
81
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FIELD PULSE NUMBER
4 6 5 I;3 12 F,ELD PULSe" NUMBEq
Fig. 1. Effect of TxB 2 on the stimulus-inotropic-response curve obtained with isolated guinea-pig atria stimulated electrically at 4 Hz with increasing numbers of field pulses. Mean values_+S.E.M. of six experiments. TxB2, 1 and 10/zM, significantly (P < 0.05) reduced the response to field stimulation.
Fig. 2. Effect of ONO 11006 on the stimulus-inotropic response curve obtained in isolated guinea-pig atria stimulated electrically at 4 Ilz by increasing numbers of field pulses. Mean values+S.E.M, of four experiments. All the points of the curves in the presence of ONO 11006 were significantly different from those of the control curve (at least P < 0.05).
i n c r e a s i n g n u m b e r s o f field refractory period induced a effect: the t e n s i o n i n c r e a s e d 618 4-32 mg a f t e r trains o f respectively (n = 20).
U46619 was the most active d r u g tested, since it was fully active at c o n c e n t r a t i o n s r a n g i n g from 0.1 to 10 nM and p r o d u c e d a m a x i m u m d e g r e e o f inhibition g r e a t e r than that i n d u c e d by the o t h e r two agonists (fig. 3). T h e IC50 valuc for U46619 was 1.35 + 0.13 nM. In o r d e r to e v a l u a t e w h e t h e r the a n t a g o n i s m by U46619 was d u e to i n t e r f e r e n c e with p o s t j u n c t i o n a l /3-adrcnoceptors, the effect o f the t h r o m b o x a n e a n a l o g u e on the c o n c e n t r a t i o n - i n o t r o p i c r e s p o n s e curves for n o r a d r e n a l i n e a n d i s o p r e n a l i n e was tested. U46619, 10 nM, slightly but insignificantly shifted to the right the conc e n t r a t i o n - r e s p o n s e curve for n o r a d r e n a l i n e (fig. 4) a n d that for i s o p r e n a l i n e (n = 4, not shown). In a d d i tion to the inhibitory effect on a d r e n e r g i c n e u r o t r a n s mission, U46619 s h o w e d a direct positive i n o t r o p i c effect on atrial p r e p a r a t i o n s (table I). T h e m a x i m u m d r u g effect c o n s i s t e d o f an i n c r e a s e o f a b o u t 35% in c o n t r a c t i l e force with 100 nM U46619. T h e positive i n o t r o p i c effect i n d u c e d by U46619 was not a f f e c t e d by
pulses a p p l i e d d u r i n g the g r a d u a l positive i n o t r o p i c to 458 + 18, 555 + 27 a n d 4, 8 a n d 12 field pulses,
TxB 2 ( 0 . 1 - 1 0 /zM) c o n c e n t r a t i o n d e p e n d e n t l y red u c e d the c a r d i a c r e s p o n s e to a d r e n e r g i c nerve stimulation (fig. 1). T h e e x t r a p o l a t e d IC50 v a l u e for TxB 2 was 20.7 + 4 . 3 / z M . T h e basal c o n t r a c t i l e force o f atrial p r e p a r a t i o n s was slightly i n c r e a s e d by TxB 2 ( t a b l e 1); at the highest c o n c e n t r a t i o n t e s t e d (10 / , M ) the cont r a c t i o n a m p l i t u d e was i n c r e a s e d by a b o u t 16%. O N O 11006, a synthetic stable a n a l o g u e o f T x A z , was m u c h m o r e effective t h a n TxB 2 to r e d u c e the c a r d i a c r e s p o n s e to s y m p a t h e t i c nerve s t i m u l a t i o n . In fact O N O 11006 significantly r e d u c e d the positive ino t r o p i c effect o f field s t i m u l a t i o n at a c o n c e n t r a t i o n as low as 1 n M (fig. 2). H i g h e r c o n c e n t r a t i o n s o f O N O 11006 ( 1 0 - 1 0 0 n M ) f u r t h e r r e d u c e d this c a r d i a c response; the IC5o values for the agonist was 20.0 + 4.0 nM. O N O 11006 was devoid o f any d i r e c t effect on c a r d i a c contractility.
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Control
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TABLE 1 Change in contractile tension (mg) induced by TxB 2 and by U46619 alone and in the presence of the thromboxane receptor antagonist, AH23848B.
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Numbers indicate mean values _+S.E.M. of at least four experiments. TxB 2
/*M concentration 0 0.1 Contractile tension 434+55 450+65
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nM concentration 0 1 10 100 1000 Contractile tension 377_+45 377_+45 380_+47 386_+49 390+51
Fig. 3. Effect of U46619 on the stimulus-inotropic-responsc curve obtained in isolated guinea-pig atria stimulated electrically at 4 Hz by increasing numbers of field pulses. Mean values of six experiments. S.E.s are included in symbols. All the points of the curves in the presence of U46619 were significantly different from those of the control curve (at least P < 11.05).
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Fig. 4. Increase in contractile tension (expressed in mg) induced by noradrenaline alone and in the presence of U46619 in isolated guinea-pig atria stimulated electrically at 4 Hz. The values are means _+S.E.M. of eight experiments. The points of the curve in the presence of U46619 were not significantly different from those of the c o n t r o l curve.
pretreatment of the preparations with 0.1 /.tM CGP 20721A, a selective/3j-receptor antagonist, with 10/zM ranitidine, an H 2 receptor antagonist and also with 1 /zM capsaicin (data not shown). The positive inotropic response was however completely antagonized by the thromboxanc receptor antagonist, AH23848B, at a concentration of 10 nM (table 1).
3.2. Effect of U46619 in the presence of antagonists In order to characterize the effect of thromboxane agonists on cardiac adrenergic neurotransmission further experiments were performed using the most active drug, U46619. A single train of eight pulses was applied to atrial preparations in the absence and pres-
100
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ence of increasing concentrations of the agonist, and the inhibitory effcct of U46619 was studied in the presence of two thromboxane receptor antagonists, sulotroban and AH 23848B. Sulotroban (0.1-1 /zM) shiftcd to the right in a concentration-dependent and significant manner the concentration-effect curve for U46619 (fig. 5A). The inhibitory effect of the thromboxane analogue was also strongly antagonized by a more potent thromboxane receptor antagonist, AH23848B, at concentrations ranging from 0.1 to 10 nM (fig. 5B). Neither AH 23848B nor sulotroban, at the highest concentrations tested, were able to modify the basal contractility of isolated atria or the cardiac response to adrenergic nerve stimulation.
0 ! ~uM ~00
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Fig. 6. Basal and stimulation-ew)ked tritium overflow in isolated guinea-pig atria preloaded with [311]noradrenaline and incubated in Tyrode .solution containing 1 /zM phenoxybenzamine, in the absence and presence of U46619. Trains of 1(10 field pulses were applied to produce noradrenaline release. Thc valucs are means_+ S.E.M. of four experiments.
A U46619
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Fig. 5. Antagonism by increasing concentrations of sulotroban (A) and AH2384B (B) of the inhibitory effect of U46619 on the cardiac response to a train of eight field pulses. The values are means ± S.E.M. of at least five experiments per group. The antagonists shifted the concentration-effect curve for U46619 significantly to the right (at least P < 0.05).
83
3.3. Effect of U46619 on [~H]noradrenaline ot,erflow In order to test whether the inhibitory effect of U46619 on adrenergic neurotransmission was duc to a reduction in noradrenaline release, experiments were performed in which the effect of U46619 on field stimulation-induced [3H]noradrenaline overflow was measured. In the presence of 1 tzM phenoxybenzamine a train of 100 ficld pulses produced a significant increase in tritium overflow (fig. 6). U46619, at a concentration (10 nM) which reduccd by 7 6 . 0 + 4 . 6 % thc cardiac response to a train of eight pulses in preparations untreated with phenoxybenzaminc (see fig. 5), did not modify either the basal or thc stimulation-induced overflow of [3H]noradrenaline (fig. 6). However, undcr these conditions, the agonist was still able to reduce by 35.2 + 2.9% the functional response to field stimulation.
4. Discussion
There are contradictory reports on the effects of thromboxane analogues on adrenergic neurotransmission. A reduction in the amplitude of the contractions evoked by perivascular nerve stimulation by both cTxA 2 and prostaglandin Iz has been observed in the guineapig mesenteric artery; the response of the same preparation to exogenous noradrenaline was potentiated by cTxA 2. The results of this study, in which no attempt was made to evaluate the possible changes in neurotransmitter release, suggested that the thromboxanc analogue exerted a prejunctional inhibitory role on adrenergic neurotransmission (Makita, 1983). In contrast, potentiation of noradrenaline release by thromboxane mimetics has been demonstrated repeatedly in several peripheral preparations of the rabbit, such as vas deferens (Trachte, 1986), portal vein (Trachte and Stein, 1988) and mesenteric artery (Trachte and Stein, 1989). A potentiating effect of U46619 on the response to field stimulation has been shown in the rat anococcygeous muscle; however, this effect seemed to be due to a postjunctional action, since the thromboxane agonist also enhanced the response to exogenous noradrenaline (Timini et al., 1978). Results qualitatively similar to those reported above were obtained by R u m p and Schollemeyer (1989) with the rat kidney. Enhancement of the response to nerve stimulation by cTxA 2 has also been demonstrated in the dog mesenteric artery: the observation that the agonist increased the response to exogenous noradrenaline, leaving the stimulation-induced [3H]noradrenaline overflow unaffected, suggested that the cTxA 2 effect was attributable to a postjunctional action (Nakajima and Toda, 1986). However, in the same preparation TxB2,
besides potentiating thc response to exogenous noradrenalinc, also increased [3H]noradrenaline overflow, suggesting an excitatory effect both at prejunctional and postjunctional sites (Okamura et al., 1988). Moreover, indirect evidence obtained in vivo with thromboxanc synthetase inhibitors, suggcstcd that thromboxane may have a potentiating role on sympathetic neurotransmission in the mesentcric and renal vascular beds of the rat (Jackson et al., 1984; Jackson, 1985; Robinette and Conger, 1990). Thus, on the whole, available reports indicate that thromboxanc mimetics can inhibit or enhance adrencrgic neurotransmission through either prejunctional or postjunctional mechanisms, depending on thc animal species and the kind of peripheral preparation employed. The results of the present study, showing an inhibitory effect of thromboxane receptor agonists on the functional response to adrenergic nerve stimulation in guinea-pig atrial preparations, are in agreement with those obtained by Makita (1983) for mesenteric artery of the same animals species. All the agonists tested showed a qualitatively similar inhibitory cffcct with the following potency order: TxB 2 < O N O 11006 < U46619. The synthetic stable analogues of TxA 2, O N O 11006 and U46619, wcre active at concentrations 100 and 1000 times lower than those of TxB 2, and the maximum inhibitory effect, as well as the IC5o of the two analogues, were greater than those displayed by TxB 2. The obscrved inhibition of the cardiac response to field stimulation by thromboxane mimetics was probably not attributable to interference with the postsynaptic/3-adrenoceptor response. In fact U46619, at a concentration (10 nM) which was able to reducc the response to field stimulation by about 75%, affected slightly but not significantly the positive inotropic response to exogenous noradrenaline and isoprcnaline. Moreover, our results indicate that the inhibitory effect of U46619 was due to stimulation of specific thromboxane receptors. The inhibitory effect of the thromboxane analogue on adrenergic neurotransmission was greatly reduced by two different thromboxane receptor antagonists, A H 23848B and sulotroban. The results of the experiments in which the effect of U46619 on the [3H]noradrenaline overflow was tested failed to confirm the hypothesis that the inhibition of the cardiac adrenergic neurotransmission induced by thromboxane analogues was mediated through a prejunctional inhibitory effect, since the agonist did not show any inhibitory effect on transmitter overflow. However, our experimental conditions for the overflow studies were not identical to those used in the experiments in which the functional effects of the thromboxane mimetics were studied . The number of field pulses used in order to obtain a detectable amount of released [3H]noradrenaline was much higher than in
84
functional studies; moreover, the non-competitive areceptor blocking agent, phenoxybenzamine, was added to the perfusion fluid at a concentration (! /.tM) able to inhibit, not only the prejunctional a-adrenoceptors, but also the neuronal (Sanchez-Garcia, 1977) and the extraneuronal (lversen and Langer, 1969) uptake to noradrenaline. Thus, unlike the experiments in which the effect of thromboxane receptor agonists on the functional response to adrenergic stimulation was tested, the overflow experiments were performed under conditions leading to a pronounced enhancement of adrenergic neurotransmission. Therefore, the hypothesis may bc advanced that the failure of U46619 to affect [3H]noradrenaline overflow may be due to the fact that the thromboxane receptor-mediated inhibitory effect only operates at a low degree of neuronal activity. A similar pattern of frequency-dependent effect on cardiac adrenergic neurotransmission was seen with other agonists such as opioid peptidcs, whose prejunctional inhibitory effect was manifest at a low, but not at a high stimulation frequency (Lcdda et al., 1984). Obviously other hypotheses can be advanced to account for thc discrepancy between the results we now obtained for the functional and the overflow experiments. For instance, U46619 might interfere with noradrenaline metabolism a n d / o r redistribution in the adrenergic terminals. In this case, measurement of the tritium overflow induced by field stimulation could not indicate the exact amount of active noradrenalinc in the synaptic cleft, since a significant fraction of the transmitter overflow is collected as metabolitcs (Langer, 1970) and metabolism of [3H]noradrenaline can be prevented by the neuronal uptake inhibitor, phenoxybenzamine (Langer, 1974). Moreover, the hypothesis has been proposed that in many tissues the prclabelled radioactive transmitter may not be stored and released in a manner analogous to that of the endogenous substance (Moura et al., 1990). In any case, it is possible to conclude from our results that the inhibitory effect of U46619 was not attributable to a postsynaptic mechanism, since the agonist was unable either to induce a negative inotropic response or to reduce the response to exogenous noradrenaline. However, the techniques applied in the present study failed to identify the exact site at which thromboxane analogues could act to reduce the cardiac response to sympathetic nerve stimulation. The finding that cardiac contractility was increased by U46619, and that this effect was not affected by fl-adrenoceptor or H 2 receptor antagonists but was antagonized by A H 23848B, represents an interesting observation since it indicates that postjunctional thromboxane receptors are probably present in cardiac muscular tissue. This conclusion is consistent with recent observations showing that U46619 induced a positive inotropic effect in the guinea-pig left atrium that
was not affected by adrenoceptor and histamine receptor antagonists, but was antagonized by a thromboxane receptor antagonist, and was associated with an increase in cellular levels of inositol phosphates (Sakuma et al., 1989). In conclusion, thromboxane mimetics induce both a direct positive inotropic effect and inhibition of the functional response to adrencrgic nerve stimulation in guinea-pig cardiac preparations; these effects involve the activation of specific receptors. However, the site at which the inhibitory effect on adrenergic ncurotransmission is exerted, as well as the mechanism by which it is produced, still remain unclear and require further study.
Acknowledgement This study was supported by a grant from the Ministry of Education and of Scientific and Technological Research.
References Hamberg, M., J. Svensson and B. Samuelsson, 1975, Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides, Proc. Natl. Acad. Sci. 72, 2994. Hedqvist, P., 1977, Basic mechanisms of prostaglandin action on autonomic neurotransmission, Ann. Rev. Pharmacol. Toxicol. 17, 259. Hedqvist, P., L. Stj~irnc and A. Wennmalm, 1970, Inhibition by prostaglandin E 2 of sympathetic neurotransmission in the rabbit heart, Acta Physiol. Scand. 79, 139. Iversen, L.L. and S.Z. Langer, 1969, Effects of phenoxybenzaminc on the uptake and metabolism of noradrenaline in the rat heart and vas deferens, Br. J. Pharmacol. 37, 627. Jackson, E.K., 1985, Effects of thromboxane synthase inhibition on vascular responsiveness in the in vivo rat mesentery, J. Clin. lnvest. 76, 2286. Jackson, E.K., H.D. Uderman, W.A. Herzer and R.A. Branch, 1984, Attenuation of noradrenergic neurotransmission by the thromboxane synthetase inhibitor, UK 38,485, Life Sci. 35, 221. Khan, M.T. and K.U. Malik, 1982, Modulation by prostaglandins of the release of ~H noradrenaline evoked by potassium and nerve stimulation in the isolated rat heart, Eur. J. Pharmacol. 78, 213. Langer, S.Z., 1970, The metabolism of [~H]noradrenaline released by electrical stimulation from the isolated nictitating membrane of the cat and from the vas deferens of the rat, J. Physiol. 208, 515. Langer, S.Z., 1974, Presynaptic regulation of catecholaminc release, Biochem. Pharmacol. 23, 1973. Ledda, F. and L. Mantefli, 1984, Differences between the prejunctional effects of phenylephrine and clonidine in guinea-pig isolated atria, Br. J. Pharmacol. 81,491. Ledda, F., L. Mantelli, V. Corti and R. Fantozzi, 1984, Inhibition of the cardiac response to sympathetic nerve stimulation by opioid peptides and its potentiation by morphine and methadone, Eur. J. Pharmacol. 102, 443. Liel, N., D.E. Mais and P.V. Halushka, 1988, Desensitization of platelet thromboxanc A2/prostaglandin H 2 receptors by the mimetic U46619, J. Pharmacol. Exp. Ther. 247, 1133.
85 Makita, Y., 1983, Effects of prostaglandin 12 and carbocyclic thromboxane A 2 on smooth muscle cells and neuromuscular transmission in the guinea-pig mesenteric artery, Br. J. Pharmacol. 78, 517. Malik, K.U., 1978, Prostaglandins - modulation of adrenergic nervous system, Fed. Proc. 37, 203. Mantelli, L., S. Amerini and F. Ledda, 1990, Different effects of prostaglandins on adrenergic neurotransmission in atrial and ventricular preparations, Br. J. Pharmacol. 99, 717. Mantelli, L., S. Amerini, A. Rubino and F. Ledda, 1991, Prejunctional prostanoid receptors on cardiac adrenergic terminals beking to the EP 3 subtype, Br. J. Pharmacol. 102, 573. McGiff. J., 1981, Prostaglandins, prostacyclin, and thromboxanes, Ann. Rev. Pharmacol. Toxicol. 21,479. Moncada, S. and J.R. Vane, 1979, Pharmacology and endogenous role of prostaglandin endoperoxides, thromboxane A 2 and prostacyclin, Pharmacol. Rev. 30, 293. Moura, D., I. Azevedo and S. Guimaraes, 1990, Differential distribution in, and release from, sympathetic nerve endings of endogenous noradrenaline and recently incorporated catecholamines, Naunyn-Schmiedeb. Arch. Pharmacol. 342, 153. Nakajima, M. and N. Toda, 1986, Prejunctional and postjunctional actions of prostaglandins F2,' and 12 and carbocyclic thromboxane A 2 in isolated dog mesenteric arteries, Eur. J. Pharmacol. 120, 309. Okamura, T., M. Nakajima and N. Toda, 1988, Neuroeffector actions of thrombo×ane B 2 in dog isolated mesenteric arteries, Br. J. Pharmacol. 93, 367. Robinette, J.B. and J.D. Conger, 1990, Angiotensin and thromboxane in the enhanced renal adrenergic nerve sensitivity of acute renal failure, J. Clin. Invest. 86, 1532.
Rump, L.C. and P. Schollmeyer, 1989, Effects of endogenous and synthetic prostaglandins, the thromboxane A 2 receptor agonist U-46619 and arachidonic acid on ~H-noradrenaline release and vascular tone in rat isolated kidney, Br. J. Pharmacol. 97, 819. Sakuma. I., S.S. Gross and R. Levi, 1989, Positive inotropic effect of the thromboxane analog U-46619 on guinea pig left atrium: mediation by specific receptors and asu~ciation with increased phosphoinositide turnover, Can. J. Physiol. Pharmacol. 67, 943. Sanchez-Garcia, P., A.G. Garcia, R. Martinez-Sierra and A. Velasco-Martin, 1977, Inhibition of norepinephrine uptake by phenoxybenzamine and desmethylimipramine in the isolated guineapig atrium, J. Pharmacol. Exp. Ther. 201, 192. Stein, E.A. and G.J. Trachte, 1989, Thromboxane mimetics enhance adrenergic neurotransmission in the rabbit-isolated portal vein, J. Cardiovasc. Pharmacol. 14, 469. Timini, K.S., J.R. Bedwani and A.W.B. Stanton, 1978, Effects of prostaglandin E 2 and prostaglandin endoperoxide analogue on neuroeffector transmission in the rat anococcygeous muscle, Br. J. Pharmacol. 63, 167. Trachte, G.J., 1986, Thromboxane agonist (U46619) potentiates norepinephrine efflux from adrenergic nerves, J. Pharmacol. Exp. Ther. 237, 473. Trachte, G.J. and E.A. Stein, 1988, Platelet-generated thromboxane A 2 enhances norepinephrine release from adrenergic nerves, J. Pharmacol. Exp. Ther. 247, 1139. Trachte, G.J. and E.A. Stein, 1989, Thromboxane receptor agonists enhance adrenergic neurotransmission in rabbit isolated mesenteric arteries, J. Pharmacol. Exp. Ther. 249, 216. Wennmalm, A., 1979, Prostaglandin-mediated inhibition of noradrenaline release, Acta Physiol. Scand. 105, 254.
79
~:'~ 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.110
EJP 52344
Effects of thromboxane agonists on cardiac adrenergic neurotransmission Laura Mantclli, Sandra Amcrini, Annalisa Rubino and Fabrizio Ledda Department of Pharmacoh>gy, Unicersity of Florence, Viah"G.B. Morgagni 65, 50134 Florence, Italy Received 16 May 1991, revised MS received 3 December 1991, accepted 24 December 1991
The effects of thromboxanc B 2 (TxB 2) and of two thromboxane mimetics, dl-(9,11), (11,12)-dimethano-TxA 2 (ONO 11006) and 9,11-dideoxy-ll,9-epoxymcthano prostaglandin F2,, (U46619) on the cardiac response to adrenergic nerve stimulation in isolated guinea-pig atria were evaluated. All the agonists dose dependently reduced the positive inotropic effect induced by field stimulation, U46619 being the most active. The inhibitory effect of U46619 was reduced by the thromboxane receptor antagonists, sulotroban and AH 23848B. U46619 did not significantly reduce the positive inotropic effect induced by exogenous noradrenaline. However U46619 was unable to modify the tritium overflow induced by field stimulation in preparations preloaded with [3H]noradrenaline. In addition to this influencc on adrencrgic neurotransmission, U46619 also had a direct positive inotropic effect on cardiac contractility, which was antagonized by AH 23848B. These results indicate that U46619 reduces the cardiac response to sympathetic nerve stimulation and that is also has a direct stimulatory effect on cardiac muscle. Heart; Adrenergic neurotransmission: U46619; ONO 11006; Thromboxane B,
1. Introduction
Prostaglandins have been shown to inhibit sympathetic neurotransmission in several peripheral preparations (for reviews see Hedqvist, 1977; Malik, 1978), including rabbit (Hedqvist et al., 1970; Wennmalm, 1979) and rat (Khan and Malik, 1982) whole heart. Recently we have confirmed that prostaglandins of the E and I series reduce the response to adrenergic nerve stimulation in isolated guinea-pig atria (Mantelli et al., 1990; Mantelli et al., 19911. Like prostaglandins, thromboxane A 2 (TxA 2) is a cyclooxygcnase metabolite that has been reported to have effects opposite to those of prostaglandins in vascular muscle and platelets ( H a m b e r g et al., 1975; Moncada and Vane, 1979; McGiff, 1981). However, the data concerning the effect of TxA 2 on neurotransmission arc controversial. Analogues of TxA 2, such as U46619 and carbocyclic thromboxane A 2 ( c T x A 2 ) , have been shown to potentiate noradrenaline release in several peripheral preparations from rabbits, i.e. the mescnteric artery (Trachtc and Stein, 1989), the vas deferens (Trachte, 1986) and the portal vein (Trachte and Stein, 1988; Stein and Trachte, 1989). On the other hand, cTxA 2 did not
Correspondence to: L. Mantelli, Department of Pharmacology, University of Florence, Viale G.B. Morgagni 65, 50134 Florence, Italy. Tel. 39.55.437 7272, fax 39.55.436 1613.
modify field stimulation-induced noradrenaline release in dog mesenteric artery (Nakajima and Toda, 1986). Although U46619 induced an increase in vascular tone and enhancement of the pressor response to nerve stimulation in rat isolated kidney, it did not affect the stimulation-induced release of noradrenaline from the same preparation ( R u m p and Schollmeyer, 1989). It has also been reported that cTxA z reduced the amplitude of the excitatory action potentials and of the contractions elicited by nerve stimulation in the guinea-pig mesentcric artery, while the effect of exogenous noradrenaline was enhanced by the agonist (Makita, 1983). We now studied the effects of natural TxB 2 and of two synthetic analogues, O N O 11006 and U46619, on cardiac adrenergic neurotransmission in isolated guinea pig atria, by evaluating both the functional response to adrenergic nerve stimulation and the stimulation-induced [3H]noradrenaline overflow.
2. Materials and methods
2.1. Preparations Isolated atria were obtained from male guinea-pigs weighing 200-300 g. The preparations were mounted vertically in a 15-ml glass chamber containing Tyrode solution of the following composition (mM): NaCl 115;
80 KCI 4.7; CaCI 2 1.8; MgSO 4 1.2; K H 2 P O 4 1.2; NaHCO 3 25; glucose 10. The solution was aerated with a gas mixture of 95% 0 2 and 5% CO 2 and had a pH of 7.4. The temperature was kept at 30°C in order to reduce the metabolic demand of the preparations. Atria were stretched to yield a basal force of 700 mg. The preparations were stimulated at a constant rate (4 Hz) slightly higher than the spontaneous rate, by square wave pulses of 0.5 ms duration and twice the threshold voltage intensity through two platinum punctate electrodes connected with a Tektronix pulse generator. Isometric contraction was recorded by an isometric transducer on a pen recorded (Battaglia Rangoni KV 135) and on a dual beam oscilloscope (Tektronix D 13).
2.2. Neurotransmission studies Sympathetic nerve terminals were stimulated by field stimulation during the cardiac refractory period, as previously described (Ledda and Mantelli, 1984). Trains of field pulses (50-75 mA, 1 ms) were applied at 3-rain intervals through two platinum plates parallel to the preparations, connected with a second pulse generator (MARB 8 2 / 2 / 2 0 0 ) . A control circuit allowed timing of the field pulses to begin 10-20 ms after each driving pulse, during the absolute refractory period. Field pulses were delivered one per consecutive contraction. Stimulus-inotropic-response curves were obtained by increasing the number of field pulses, with the following sequence: 2, 4, 6, 8, 10, 12. Subsequent stimulusresponse curves were separated by 30 min. The responses induced by field stimulation were expressed as % changes from the steady state contraction. All the experiments were carried out in the presence of 1 p.M atropine in order to eliminate the parasympathetic component of the response to field stimulation. The effect of the drugs on the stimulus-response curve was studied according to the following protocol: two control stimulus-response curves were obtained with an interval of 30 min. Thus, the agonist was added after 20 min and the stimulus-response curve was repeated after a contact period of 10 min. The ICso values were calculated by evaluating the response induced by a train of intermediate degree (8 pulses). In the experiments on the effects of thromboxane receptor agonists and antagonists, trains of field pulses of an intermediate value (eight pulses) were applied at 15-min intervals. Cumulative concentrations of the agonist were added 10 min before field stimulation. The experiments with the antagonists were done with different preparations, and the antagonists were added to the bathing solution before starting field stimulation. Concentration-response curves for drugs affecting basal contractility (noradrenaline, isoprenaline, TxB 2 and U46619) were made by adding cumulative concentrations of the agonists. The concentration-response curves for U46619 in the absence and presence of the
antagonist were obtained in different preparations since it was not possible to obtain a reproducible concentration-effect curve for U46619, probably as a consequence of desensitization produced by the agonist, as previously described for other peripheral preparations (Liel et al., 1988).
2.3. [~H]Noradrenaline overflow detection Guinea-pig atria were incubated with 5 izCi of [3H]noradrenaline (specific activity 13.7 C i / m m o l ) for 40 min in 2 ml Tyrode solution oxygenated and maintained at 30°C. After this period the preparations were placed in 50 ml Tyrode solution containing 300 lzM ascorbic acid, 1 tzM atropine and 1 IzM phenoxybenzamine; the radioactivity was washed out for 30 min by replacing the solution 4 times. The atria were then mounted vertically in an organ bath similar to that used for functional studies but containing 2.5 ml Tyrode solution. As previously described the preparations were stimulated at a constant rate (4 Hz); train of 100 field pulses were applied during the absolute refractory period, in order to obtain detectable [3H]noradrenaline release, at 15-min intervals. The incubation medium was collected every 5 min. After two collections of bathing solution (for measurement of basal tritium overflow) the first train was applied at the 13th min (SI), and Tyrode solution was again sampled at the 15th min. Five consecutive trains of field pulses were repeated. When the effect of U46619 was tested the drug was added to Tyrode solution 5 min after $2 in order to evaluate the effect on basal and stimulus-induced overflow. Insta-gel, 15 ml, was added to each sample, and the radioactivity was measured on a Packard 1500 Tricarb liquid scintillation analyzer. The curves were compared using the analysis of variance followed by Tukey's test. Student's paired t-test was used for comparing two groups of data. The drugs used were: TxB 2 (Sigma); ONO 11006, kindly supplied by Ono Pharmaceutical (Osaka, Japan); U46619, kindly supplied by Upjohn (Kalamazoo, USA); AH23848B, a gift of Dr. B.M. Bain of Glaxo Group Research (Greenford, England). Sulotroban was kindly supplied by Boehringer Mannheim; noradrenaline hydrochioride and isoprenaline hydrochloride (Sigma); atropine sulphate (BDH); phenoxybenzamine hydrochloride was kindly supplied by SKF; [3H]noradrenaline (Amersham).
3. Results
3.1. Effect of T x B 2 and thromboxane analogues on the cardiac response to field stimulation Guinea-pig atria, electrically driven at 4 Hz, developed a basal tension of 389 _+ 16 mg (n = 20). Trains of
81
O'~ 0 n
100
0-0
Control
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+
Tx8 2 0.1 ~M
ZI-A
+
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+ Txg, 10 pM /
0-0
Control
~--~ ÷ ONO11006 t nMo/C
1 pM a
L~J ¢n Z
~
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÷
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*
0N0 11006 tOO nM
n~
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~ 5o
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<
<
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/'e~
iJ/i~
"
.A~ ,.-'"
-
C
0
2
FIELD PULSE NUMBER
4 6 5 I;3 12 F,ELD PULSe" NUMBEq
Fig. 1. Effect of TxB 2 on the stimulus-inotropic-response curve obtained with isolated guinea-pig atria stimulated electrically at 4 Hz with increasing numbers of field pulses. Mean values_+S.E.M. of six experiments. TxB2, 1 and 10/zM, significantly (P < 0.05) reduced the response to field stimulation.
Fig. 2. Effect of ONO 11006 on the stimulus-inotropic response curve obtained in isolated guinea-pig atria stimulated electrically at 4 Ilz by increasing numbers of field pulses. Mean values+S.E.M, of four experiments. All the points of the curves in the presence of ONO 11006 were significantly different from those of the control curve (at least P < 0.05).
i n c r e a s i n g n u m b e r s o f field refractory period induced a effect: the t e n s i o n i n c r e a s e d 618 4-32 mg a f t e r trains o f respectively (n = 20).
U46619 was the most active d r u g tested, since it was fully active at c o n c e n t r a t i o n s r a n g i n g from 0.1 to 10 nM and p r o d u c e d a m a x i m u m d e g r e e o f inhibition g r e a t e r than that i n d u c e d by the o t h e r two agonists (fig. 3). T h e IC50 valuc for U46619 was 1.35 + 0.13 nM. In o r d e r to e v a l u a t e w h e t h e r the a n t a g o n i s m by U46619 was d u e to i n t e r f e r e n c e with p o s t j u n c t i o n a l /3-adrcnoceptors, the effect o f the t h r o m b o x a n e a n a l o g u e on the c o n c e n t r a t i o n - i n o t r o p i c r e s p o n s e curves for n o r a d r e n a l i n e a n d i s o p r e n a l i n e was tested. U46619, 10 nM, slightly but insignificantly shifted to the right the conc e n t r a t i o n - r e s p o n s e curve for n o r a d r e n a l i n e (fig. 4) a n d that for i s o p r e n a l i n e (n = 4, not shown). In a d d i tion to the inhibitory effect on a d r e n e r g i c n e u r o t r a n s mission, U46619 s h o w e d a direct positive i n o t r o p i c effect on atrial p r e p a r a t i o n s (table I). T h e m a x i m u m d r u g effect c o n s i s t e d o f an i n c r e a s e o f a b o u t 35% in c o n t r a c t i l e force with 100 nM U46619. T h e positive i n o t r o p i c effect i n d u c e d by U46619 was not a f f e c t e d by
pulses a p p l i e d d u r i n g the g r a d u a l positive i n o t r o p i c to 458 + 18, 555 + 27 a n d 4, 8 a n d 12 field pulses,
TxB 2 ( 0 . 1 - 1 0 /zM) c o n c e n t r a t i o n d e p e n d e n t l y red u c e d the c a r d i a c r e s p o n s e to a d r e n e r g i c nerve stimulation (fig. 1). T h e e x t r a p o l a t e d IC50 v a l u e for TxB 2 was 20.7 + 4 . 3 / z M . T h e basal c o n t r a c t i l e force o f atrial p r e p a r a t i o n s was slightly i n c r e a s e d by TxB 2 ( t a b l e 1); at the highest c o n c e n t r a t i o n t e s t e d (10 / , M ) the cont r a c t i o n a m p l i t u d e was i n c r e a s e d by a b o u t 16%. O N O 11006, a synthetic stable a n a l o g u e o f T x A z , was m u c h m o r e effective t h a n TxB 2 to r e d u c e the c a r d i a c r e s p o n s e to s y m p a t h e t i c nerve s t i m u l a t i o n . In fact O N O 11006 significantly r e d u c e d the positive ino t r o p i c effect o f field s t i m u l a t i o n at a c o n c e n t r a t i o n as low as 1 n M (fig. 2). H i g h e r c o n c e n t r a t i o n s o f O N O 11006 ( 1 0 - 1 0 0 n M ) f u r t h e r r e d u c e d this c a r d i a c response; the IC5o values for the agonist was 20.0 + 4.0 nM. O N O 11006 was devoid o f any d i r e c t effect on c a r d i a c contractility.
~0
1O0
O-O
Control
e-e
+
U46619
tx-a
+
U46619
1 nM
A--A
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0.1 nM
0
TABLE 1 Change in contractile tension (mg) induced by TxB 2 and by U46619 alone and in the presence of the thromboxane receptor antagonist, AH23848B.
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o
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Numbers indicate mean values _+S.E.M. of at least four experiments. TxB 2
/*M concentration 0 0.1 Contractile tension 434+55 450+65
1 470+70
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nM concentration 0 0.1 1 10 Contractile tension 341 _+35 352_+35 378_+37 435 + 43
0
,
,
.
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4
.
.
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.
8
.
10
12
FIELD PULSE NUMBER
100 463-+53
U46619 + 10 nM AH23848B
nM concentration 0 1 10 100 1000 Contractile tension 377_+45 377_+45 380_+47 386_+49 390+51
Fig. 3. Effect of U46619 on the stimulus-inotropic-responsc curve obtained in isolated guinea-pig atria stimulated electrically at 4 Hz by increasing numbers of field pulses. Mean values of six experiments. S.E.s are included in symbols. All the points of the curves in the presence of U46619 were significantly different from those of the control curve (at least P < 11.05).
82 0 - 0
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+
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10
L
nM
U46619
150
F 400 -
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Fig. 4. Increase in contractile tension (expressed in mg) induced by noradrenaline alone and in the presence of U46619 in isolated guinea-pig atria stimulated electrically at 4 Hz. The values are means _+S.E.M. of eight experiments. The points of the curve in the presence of U46619 were not significantly different from those of the c o n t r o l curve.
pretreatment of the preparations with 0.1 /.tM CGP 20721A, a selective/3j-receptor antagonist, with 10/zM ranitidine, an H 2 receptor antagonist and also with 1 /zM capsaicin (data not shown). The positive inotropic response was however completely antagonized by the thromboxanc receptor antagonist, AH23848B, at a concentration of 10 nM (table 1).
3.2. Effect of U46619 in the presence of antagonists In order to characterize the effect of thromboxane agonists on cardiac adrenergic neurotransmission further experiments were performed using the most active drug, U46619. A single train of eight pulses was applied to atrial preparations in the absence and pres-
100
0--0
+
Sulotrobon
A--&
+
Sulo[robon
I
30
:
45
( rain
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I
1
60
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ence of increasing concentrations of the agonist, and the inhibitory effcct of U46619 was studied in the presence of two thromboxane receptor antagonists, sulotroban and AH 23848B. Sulotroban (0.1-1 /zM) shiftcd to the right in a concentration-dependent and significant manner the concentration-effect curve for U46619 (fig. 5A). The inhibitory effect of the thromboxane analogue was also strongly antagonized by a more potent thromboxane receptor antagonist, AH23848B, at concentrations ranging from 0.1 to 10 nM (fig. 5B). Neither AH 23848B nor sulotroban, at the highest concentrations tested, were able to modify the basal contractility of isolated atria or the cardiac response to adrenergic nerve stimulation.
0 ! ~uM ~00
Z O
I
I5
$4
Fig. 6. Basal and stimulation-ew)ked tritium overflow in isolated guinea-pig atria preloaded with [311]noradrenaline and incubated in Tyrode .solution containing 1 /zM phenoxybenzamine, in the absence and presence of U46619. Trains of 1(10 field pulses were applied to produce noradrenaline release. Thc valucs are means_+ S.E.M. of four experiments.
A U46619
S3
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Time
CONC[-NTRATION (pM)
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./A
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Fig. 5. Antagonism by increasing concentrations of sulotroban (A) and AH2384B (B) of the inhibitory effect of U46619 on the cardiac response to a train of eight field pulses. The values are means ± S.E.M. of at least five experiments per group. The antagonists shifted the concentration-effect curve for U46619 significantly to the right (at least P < 0.05).
83
3.3. Effect of U46619 on [~H]noradrenaline ot,erflow In order to test whether the inhibitory effect of U46619 on adrenergic neurotransmission was duc to a reduction in noradrenaline release, experiments were performed in which the effect of U46619 on field stimulation-induced [3H]noradrenaline overflow was measured. In the presence of 1 tzM phenoxybenzamine a train of 100 ficld pulses produced a significant increase in tritium overflow (fig. 6). U46619, at a concentration (10 nM) which reduccd by 7 6 . 0 + 4 . 6 % thc cardiac response to a train of eight pulses in preparations untreated with phenoxybenzaminc (see fig. 5), did not modify either the basal or thc stimulation-induced overflow of [3H]noradrenaline (fig. 6). However, undcr these conditions, the agonist was still able to reduce by 35.2 + 2.9% the functional response to field stimulation.
4. Discussion
There are contradictory reports on the effects of thromboxane analogues on adrenergic neurotransmission. A reduction in the amplitude of the contractions evoked by perivascular nerve stimulation by both cTxA 2 and prostaglandin Iz has been observed in the guineapig mesenteric artery; the response of the same preparation to exogenous noradrenaline was potentiated by cTxA 2. The results of this study, in which no attempt was made to evaluate the possible changes in neurotransmitter release, suggested that the thromboxanc analogue exerted a prejunctional inhibitory role on adrenergic neurotransmission (Makita, 1983). In contrast, potentiation of noradrenaline release by thromboxane mimetics has been demonstrated repeatedly in several peripheral preparations of the rabbit, such as vas deferens (Trachte, 1986), portal vein (Trachte and Stein, 1988) and mesenteric artery (Trachte and Stein, 1989). A potentiating effect of U46619 on the response to field stimulation has been shown in the rat anococcygeous muscle; however, this effect seemed to be due to a postjunctional action, since the thromboxane agonist also enhanced the response to exogenous noradrenaline (Timini et al., 1978). Results qualitatively similar to those reported above were obtained by R u m p and Schollemeyer (1989) with the rat kidney. Enhancement of the response to nerve stimulation by cTxA 2 has also been demonstrated in the dog mesenteric artery: the observation that the agonist increased the response to exogenous noradrenaline, leaving the stimulation-induced [3H]noradrenaline overflow unaffected, suggested that the cTxA 2 effect was attributable to a postjunctional action (Nakajima and Toda, 1986). However, in the same preparation TxB2,
besides potentiating thc response to exogenous noradrenalinc, also increased [3H]noradrenaline overflow, suggesting an excitatory effect both at prejunctional and postjunctional sites (Okamura et al., 1988). Moreover, indirect evidence obtained in vivo with thromboxanc synthetase inhibitors, suggcstcd that thromboxane may have a potentiating role on sympathetic neurotransmission in the mesentcric and renal vascular beds of the rat (Jackson et al., 1984; Jackson, 1985; Robinette and Conger, 1990). Thus, on the whole, available reports indicate that thromboxanc mimetics can inhibit or enhance adrencrgic neurotransmission through either prejunctional or postjunctional mechanisms, depending on thc animal species and the kind of peripheral preparation employed. The results of the present study, showing an inhibitory effect of thromboxane receptor agonists on the functional response to adrenergic nerve stimulation in guinea-pig atrial preparations, are in agreement with those obtained by Makita (1983) for mesenteric artery of the same animals species. All the agonists tested showed a qualitatively similar inhibitory cffcct with the following potency order: TxB 2 < O N O 11006 < U46619. The synthetic stable analogues of TxA 2, O N O 11006 and U46619, wcre active at concentrations 100 and 1000 times lower than those of TxB 2, and the maximum inhibitory effect, as well as the IC5o of the two analogues, were greater than those displayed by TxB 2. The obscrved inhibition of the cardiac response to field stimulation by thromboxane mimetics was probably not attributable to interference with the postsynaptic/3-adrenoceptor response. In fact U46619, at a concentration (10 nM) which was able to reducc the response to field stimulation by about 75%, affected slightly but not significantly the positive inotropic response to exogenous noradrenaline and isoprcnaline. Moreover, our results indicate that the inhibitory effect of U46619 was due to stimulation of specific thromboxane receptors. The inhibitory effect of the thromboxane analogue on adrenergic neurotransmission was greatly reduced by two different thromboxane receptor antagonists, A H 23848B and sulotroban. The results of the experiments in which the effect of U46619 on the [3H]noradrenaline overflow was tested failed to confirm the hypothesis that the inhibition of the cardiac adrenergic neurotransmission induced by thromboxane analogues was mediated through a prejunctional inhibitory effect, since the agonist did not show any inhibitory effect on transmitter overflow. However, our experimental conditions for the overflow studies were not identical to those used in the experiments in which the functional effects of the thromboxane mimetics were studied . The number of field pulses used in order to obtain a detectable amount of released [3H]noradrenaline was much higher than in
84
functional studies; moreover, the non-competitive areceptor blocking agent, phenoxybenzamine, was added to the perfusion fluid at a concentration (! /.tM) able to inhibit, not only the prejunctional a-adrenoceptors, but also the neuronal (Sanchez-Garcia, 1977) and the extraneuronal (lversen and Langer, 1969) uptake to noradrenaline. Thus, unlike the experiments in which the effect of thromboxane receptor agonists on the functional response to adrenergic stimulation was tested, the overflow experiments were performed under conditions leading to a pronounced enhancement of adrenergic neurotransmission. Therefore, the hypothesis may bc advanced that the failure of U46619 to affect [3H]noradrenaline overflow may be due to the fact that the thromboxane receptor-mediated inhibitory effect only operates at a low degree of neuronal activity. A similar pattern of frequency-dependent effect on cardiac adrenergic neurotransmission was seen with other agonists such as opioid peptidcs, whose prejunctional inhibitory effect was manifest at a low, but not at a high stimulation frequency (Lcdda et al., 1984). Obviously other hypotheses can be advanced to account for thc discrepancy between the results we now obtained for the functional and the overflow experiments. For instance, U46619 might interfere with noradrenaline metabolism a n d / o r redistribution in the adrenergic terminals. In this case, measurement of the tritium overflow induced by field stimulation could not indicate the exact amount of active noradrenalinc in the synaptic cleft, since a significant fraction of the transmitter overflow is collected as metabolitcs (Langer, 1970) and metabolism of [3H]noradrenaline can be prevented by the neuronal uptake inhibitor, phenoxybenzamine (Langer, 1974). Moreover, the hypothesis has been proposed that in many tissues the prclabelled radioactive transmitter may not be stored and released in a manner analogous to that of the endogenous substance (Moura et al., 1990). In any case, it is possible to conclude from our results that the inhibitory effect of U46619 was not attributable to a postsynaptic mechanism, since the agonist was unable either to induce a negative inotropic response or to reduce the response to exogenous noradrenaline. However, the techniques applied in the present study failed to identify the exact site at which thromboxane analogues could act to reduce the cardiac response to sympathetic nerve stimulation. The finding that cardiac contractility was increased by U46619, and that this effect was not affected by fl-adrenoceptor or H 2 receptor antagonists but was antagonized by A H 23848B, represents an interesting observation since it indicates that postjunctional thromboxane receptors are probably present in cardiac muscular tissue. This conclusion is consistent with recent observations showing that U46619 induced a positive inotropic effect in the guinea-pig left atrium that
was not affected by adrenoceptor and histamine receptor antagonists, but was antagonized by a thromboxane receptor antagonist, and was associated with an increase in cellular levels of inositol phosphates (Sakuma et al., 1989). In conclusion, thromboxane mimetics induce both a direct positive inotropic effect and inhibition of the functional response to adrencrgic nerve stimulation in guinea-pig cardiac preparations; these effects involve the activation of specific receptors. However, the site at which the inhibitory effect on adrenergic ncurotransmission is exerted, as well as the mechanism by which it is produced, still remain unclear and require further study.
Acknowledgement This study was supported by a grant from the Ministry of Education and of Scientific and Technological Research.
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