European Journal of Pharmacology, 46 (1977) 371--376
371
© Elsevier/North-Holland Biomedical Press
T R A N S M I T T E R R E L E A S E AND A D R E N E R G I C MECHANICAL RESPONSES IN T H E H E A RT: E F F E C T OF PA PA V E R I N E AND IMIDAZOLE PHAM-HUU-CHANH * and ,~KE WENNMALM Department of Clinical Physiology at Karolinska Instituter, Huddinge Hospital, S-141 86 Huddinge, Sweden
Received 24 August 1977, accepted 26 August 1977
P.-H. CHANH and /~. WENNMALM, Transmitter release and adrenergic mechanical responses in the heart: effect ofpapaverine and imidazole, European J. Pharmacol. 46 (1977) 371--376. Chronotropic and inotropic responses were elicited in isolated rabbit hearts by stimulation of the sympathetic nerves or by infusion of noradrenaline or adrenaline and the effects of papaverine and imidazole (10-7--10 -6 M) on these responses were studied. The outflow of noradrenaline induced by sympathetic nerve stimulation was assayed in the absence and in the presence of papaverine and imidazole (10-7--5 x 10-7 M). Papaverine increased the outflow of transmitter during nerve stimulation by 45% and potentiated both the chronotropic and inotropic responses induced by nerve stimulation and those induced by infusion of catecholamines. Imidazole inhibited the outflow of transmitter during nerve stimulation by 33%. The data indicate that the "second messenger" cyclic AMP is active in more than one step in adrenergic neurotransmission and receptor activation in the heart. Furthermore, tissue cyclic AMP seems to be involved not only in the inotropy induced by circulating catecholamines but also in the more "physiological" inotropy elicited by sympathetic nerve stimulation. Isolated heart Cyclic AMP
Sympathetic nerve stimulation Adrenergic transmitter
1. I n t r o d u c t i o n Increased sympathetic discharge in the mammalian heart leads to fi-adrenoceptor activation, which in t ur n gives rise to positive c h r o n o t r o p i c and inotropic responses in the organ. Cyclic AMP (cAMP) has been suggested as playing a role in several o f these steps o f sympathetic n e u r o - e f f e c t o r transmission. It has been proposed t h a t pre-synaptically, the release o f transmitter from nerve endings was cAMP-dependent (Poisner, 1970; Rasmussen, 1970) and th at postsynaptically, t he cyclic nucleotide could be involved in b o t h the c h r o n o t r o p i c and inotropic responses of the heart. The relation b et w e e n the c h r o n o t r o p i c response and intracellular cAMP has been * Present address: Institut de Physiologie, 2 Rue Francois Magendie, F-31400 Toulouse, France.
Papaverine
Imidazole
studied by applying derivatives o f cAMP to isolated Purkinje fibres (Borasio and Vasalle, 1971; Tsien et al., 1972) or by assaying cAMP in these fibres after application o f adrenaline (A) (Danilo et al., 1974). In addition, there is evidence for a c o m m o n mechanism behind the positive c h r o n o t r o p i c effect o f phosphodiesterase (PDE) inhibitors and o f A (Tsien, 1974). It seems generally agreed t hat the positive inotropic response of the heart t o fi-adrenergic stimulation is mediated via increased intracellular levels of cAMP (Suthefland et al., 1968; Kukovetz and PSch, 1972; Wollenberger and Krause, 1973; Kukovetz et al., 1975). However, adrenergic drugs may, u n d e r certain circumstances, e x e r t a positive inotropic effect w i t h o u t increasing intracellular cAMP (Shanfield et al., 1969; Sobel and Mayer, 1973). On the basis o f these apparently divergent results it has been proposed t h a t only
372 fl-adrenergic intotropy is cAMP-dependent; ~-adrenergic inotropy would, according to this hypothesis, not be mediated by the cyclic nucleotide (Osnes and Oye, 1975). Thus, in the heart, the chain of events between increased sympathetic discharge and augmented mechanical activity may include several cAMP-dependent steps. Nevertheless, studies have n o t appeared hitherto on the overall effect of induced changes in cAMP activity in the heart during increased sympathetic activity. In the current investigation we have therefore compared the chronotropic and inotropic responses induced by sympathetic nerve stimulation on the one hand with those induced by infusion of noradrenaline (NA) or A on the other, with respect to their sensitivity to papaverine and imidazole, drugs which are known to change the activity of the cAMP-inactivating enzyme PDE. Furthermore, we have studied ' h o w these drugs affected the release of NA into the venous effluent of the heart during sympathetic nerve stimulation.
2. Materials and methods Rabbits of mixed strains and sexes were used. The weight of the animals varied from 1.2 to 2.4 kg. After a blow on the head, the animal was exsanguinated by cutting the left carotid artery. The heart was dissected free, in some experiments with its left and right sympathetic nerve supply kept intact (Hukovid and Muscholl, 1962). The heart was subsequently transferred to the perfusion apparatus, where it was perfused according to Langendorff with Tyrode solution of the following composition (in mM): NaC1 137, KC1 2.7, CaC12 1.8, MgC12 1.0; NaHCO3 12, NaH2PO4 0.4, glucose 5.6. The solution was gassed continuously with 5% CO2 in 02. The pH of the gassed solution was 7.4--7.5, its temperature 37°C. A small water-filled latex balloon was inserted into the left ventricle via an incision in the left atrium and connected via a 0.5 mm polyethylene tube to a Statham P3 BC pres-
P.-H. CHANH,/~. WENNMALM sure transducer. Heart rate and contractile force were recorded on a Grass Model 5 D Polygraph. Solutions of NA and A in saline (25 #g/ml) were infused at a rate of 2 ml/min through a cannula immediately above the aorta. The left and right sympathetic nerve trunks to the heart were stimulated simultaneously by separate platinum electrodes connected to a Grass Model $88 stimulator with rectangular pulse trains of 2 msec duration at a rate of 5 Hz. The voltage over the electrodes was adjusted to 25 V. In the experiments in which the sympathetic nerves were stimulated, the efflux of NA from the heart was assayed. The effluent was collected during the period of nerve stimulation and for the following 90 sec. It was subsequently chilled and acidified. NA was adsorbed on alumina and analysed fluorimetrically, using the trihydroxy-indole method. The experiments were started after the hearts had been beating spontaneously in the perfusion apparatus for 15--20 min. NA or A were infused or the nerves were stimulated during two 30 sec periods, with in interval of 15 min. The first infusion or nerve stimulation was performed during perfusion of the the heart with drug-free Tyrode solution. Immediately after this infusion or nerve stimulation, perfusion was switched to a Tyrode solution containing papaverine hydrochloride or imidazole (10 -7 to 10 -6 M). This solution was used for perfusion during the rest of the experiment, the second NA/A infusion or nerve stimulation thus being performed in the presence of papaverine or imidazole in the solution perfusing the heart. The chronotropic and inotropic responses of the heart during catecholamine infusion or nerve stimulation were estimated by cutting out the recordings and weighing the area of increase in heart rate or contractile force. The effect of papaverine or imidazole on the chronotropic and inotropic responses to catecholamine infusion or nerve stimulation are expressed as the ratio between the responses during the second ($2, in the presence of drug) and first ($1, no drug) catecholamine
cAMP-MEDIATED EFFECTS IN THE HEART infusion or nerve stimulation. The effect of papaverine or imidazole on the overflow of NA in response to nerve stimulation is expressed as the ratio between the NA overflow during S: and $1. The data in the text are given as mean + S.E. Figures in parentheses indicate the n u m b e r of observations. Student's t-test was used, for analysis of statistical differences when applicable.
3. Results
3.1. General The spontaneous beating rate of the hearts in the perfusion apparatus was 100--140/min. Stimulation of the sympathetic nerves at 5 Hz for 30 sec elicited a rapid increase of 18 + 1% (50) in heart rate and of 76 + 5% (49) in contractile force. Following cessation of the nerve stimulation, the heart rate and contractile force returned to the prestimulation level within 60--90 sec. The venous effluent content of NA, which was n o t measurable at rest, increased to 88 + 3 ng (36) during the period of sympathetic nerve stimulation. A second nerve stimulation, performed 15 min after the first one, with no drug added to the Tyrode solution, elicited mainly the same chronotropic and inotropic responses. The o u t f l o w of NA decreased, to 92 -+ 3% (4) of the outflow during the first stimulation. When NA (25/~g during 30 sec) was infused, the heart rate was increased by 28 -+ 1% (50) and the contractile force by 101-+ 5% (50). After discontinuation of the infusion, the mechanical activity of the heart returned to the pre-infusion level within 60-90 sec. Infusion of A (25 #g during 30 sec) also elicited marked chronotropic and inotropic responses, amounting to 30-+ 2% (54) and 92-+ 5% (54), respectively. In analogy with the mechanical response to NA, t h e increases in heart rate and contractile f o r c e induced by A had completely vanished 60--90 sec after the end of the infusion of A. A second infusion of NA or A in the absence of
373 drug in the Tyrode solution, performed 15 min after the first one, elicited chronotropic and inotropic responses of the same magnitudes as the first infusion.
3.2. Effect of papaverine The overflow of NA from the heart in response to nerve stimulation was significantly (p < 0.01) and dose-dependently increased by papaverine; at a drug concentration of 5 × 10 -7 M, the overflow increased by 45 + 9% (fig. 1). Papaverine potentiated the chronotropic response to catecholamine infusion and to nerve stimulation in a dose
371
© Elsevier/North-Holland Biomedical Press
T R A N S M I T T E R R E L E A S E AND A D R E N E R G I C MECHANICAL RESPONSES IN T H E H E A RT: E F F E C T OF PA PA V E R I N E AND IMIDAZOLE PHAM-HUU-CHANH * and ,~KE WENNMALM Department of Clinical Physiology at Karolinska Instituter, Huddinge Hospital, S-141 86 Huddinge, Sweden
Received 24 August 1977, accepted 26 August 1977
P.-H. CHANH and /~. WENNMALM, Transmitter release and adrenergic mechanical responses in the heart: effect ofpapaverine and imidazole, European J. Pharmacol. 46 (1977) 371--376. Chronotropic and inotropic responses were elicited in isolated rabbit hearts by stimulation of the sympathetic nerves or by infusion of noradrenaline or adrenaline and the effects of papaverine and imidazole (10-7--10 -6 M) on these responses were studied. The outflow of noradrenaline induced by sympathetic nerve stimulation was assayed in the absence and in the presence of papaverine and imidazole (10-7--5 x 10-7 M). Papaverine increased the outflow of transmitter during nerve stimulation by 45% and potentiated both the chronotropic and inotropic responses induced by nerve stimulation and those induced by infusion of catecholamines. Imidazole inhibited the outflow of transmitter during nerve stimulation by 33%. The data indicate that the "second messenger" cyclic AMP is active in more than one step in adrenergic neurotransmission and receptor activation in the heart. Furthermore, tissue cyclic AMP seems to be involved not only in the inotropy induced by circulating catecholamines but also in the more "physiological" inotropy elicited by sympathetic nerve stimulation. Isolated heart Cyclic AMP
Sympathetic nerve stimulation Adrenergic transmitter
1. I n t r o d u c t i o n Increased sympathetic discharge in the mammalian heart leads to fi-adrenoceptor activation, which in t ur n gives rise to positive c h r o n o t r o p i c and inotropic responses in the organ. Cyclic AMP (cAMP) has been suggested as playing a role in several o f these steps o f sympathetic n e u r o - e f f e c t o r transmission. It has been proposed t h a t pre-synaptically, the release o f transmitter from nerve endings was cAMP-dependent (Poisner, 1970; Rasmussen, 1970) and th at postsynaptically, t he cyclic nucleotide could be involved in b o t h the c h r o n o t r o p i c and inotropic responses of the heart. The relation b et w e e n the c h r o n o t r o p i c response and intracellular cAMP has been * Present address: Institut de Physiologie, 2 Rue Francois Magendie, F-31400 Toulouse, France.
Papaverine
Imidazole
studied by applying derivatives o f cAMP to isolated Purkinje fibres (Borasio and Vasalle, 1971; Tsien et al., 1972) or by assaying cAMP in these fibres after application o f adrenaline (A) (Danilo et al., 1974). In addition, there is evidence for a c o m m o n mechanism behind the positive c h r o n o t r o p i c effect o f phosphodiesterase (PDE) inhibitors and o f A (Tsien, 1974). It seems generally agreed t hat the positive inotropic response of the heart t o fi-adrenergic stimulation is mediated via increased intracellular levels of cAMP (Suthefland et al., 1968; Kukovetz and PSch, 1972; Wollenberger and Krause, 1973; Kukovetz et al., 1975). However, adrenergic drugs may, u n d e r certain circumstances, e x e r t a positive inotropic effect w i t h o u t increasing intracellular cAMP (Shanfield et al., 1969; Sobel and Mayer, 1973). On the basis o f these apparently divergent results it has been proposed t h a t only
372 fl-adrenergic intotropy is cAMP-dependent; ~-adrenergic inotropy would, according to this hypothesis, not be mediated by the cyclic nucleotide (Osnes and Oye, 1975). Thus, in the heart, the chain of events between increased sympathetic discharge and augmented mechanical activity may include several cAMP-dependent steps. Nevertheless, studies have n o t appeared hitherto on the overall effect of induced changes in cAMP activity in the heart during increased sympathetic activity. In the current investigation we have therefore compared the chronotropic and inotropic responses induced by sympathetic nerve stimulation on the one hand with those induced by infusion of noradrenaline (NA) or A on the other, with respect to their sensitivity to papaverine and imidazole, drugs which are known to change the activity of the cAMP-inactivating enzyme PDE. Furthermore, we have studied ' h o w these drugs affected the release of NA into the venous effluent of the heart during sympathetic nerve stimulation.
2. Materials and methods Rabbits of mixed strains and sexes were used. The weight of the animals varied from 1.2 to 2.4 kg. After a blow on the head, the animal was exsanguinated by cutting the left carotid artery. The heart was dissected free, in some experiments with its left and right sympathetic nerve supply kept intact (Hukovid and Muscholl, 1962). The heart was subsequently transferred to the perfusion apparatus, where it was perfused according to Langendorff with Tyrode solution of the following composition (in mM): NaC1 137, KC1 2.7, CaC12 1.8, MgC12 1.0; NaHCO3 12, NaH2PO4 0.4, glucose 5.6. The solution was gassed continuously with 5% CO2 in 02. The pH of the gassed solution was 7.4--7.5, its temperature 37°C. A small water-filled latex balloon was inserted into the left ventricle via an incision in the left atrium and connected via a 0.5 mm polyethylene tube to a Statham P3 BC pres-
P.-H. CHANH,/~. WENNMALM sure transducer. Heart rate and contractile force were recorded on a Grass Model 5 D Polygraph. Solutions of NA and A in saline (25 #g/ml) were infused at a rate of 2 ml/min through a cannula immediately above the aorta. The left and right sympathetic nerve trunks to the heart were stimulated simultaneously by separate platinum electrodes connected to a Grass Model $88 stimulator with rectangular pulse trains of 2 msec duration at a rate of 5 Hz. The voltage over the electrodes was adjusted to 25 V. In the experiments in which the sympathetic nerves were stimulated, the efflux of NA from the heart was assayed. The effluent was collected during the period of nerve stimulation and for the following 90 sec. It was subsequently chilled and acidified. NA was adsorbed on alumina and analysed fluorimetrically, using the trihydroxy-indole method. The experiments were started after the hearts had been beating spontaneously in the perfusion apparatus for 15--20 min. NA or A were infused or the nerves were stimulated during two 30 sec periods, with in interval of 15 min. The first infusion or nerve stimulation was performed during perfusion of the the heart with drug-free Tyrode solution. Immediately after this infusion or nerve stimulation, perfusion was switched to a Tyrode solution containing papaverine hydrochloride or imidazole (10 -7 to 10 -6 M). This solution was used for perfusion during the rest of the experiment, the second NA/A infusion or nerve stimulation thus being performed in the presence of papaverine or imidazole in the solution perfusing the heart. The chronotropic and inotropic responses of the heart during catecholamine infusion or nerve stimulation were estimated by cutting out the recordings and weighing the area of increase in heart rate or contractile force. The effect of papaverine or imidazole on the chronotropic and inotropic responses to catecholamine infusion or nerve stimulation are expressed as the ratio between the responses during the second ($2, in the presence of drug) and first ($1, no drug) catecholamine
cAMP-MEDIATED EFFECTS IN THE HEART infusion or nerve stimulation. The effect of papaverine or imidazole on the overflow of NA in response to nerve stimulation is expressed as the ratio between the NA overflow during S: and $1. The data in the text are given as mean + S.E. Figures in parentheses indicate the n u m b e r of observations. Student's t-test was used, for analysis of statistical differences when applicable.
3. Results
3.1. General The spontaneous beating rate of the hearts in the perfusion apparatus was 100--140/min. Stimulation of the sympathetic nerves at 5 Hz for 30 sec elicited a rapid increase of 18 + 1% (50) in heart rate and of 76 + 5% (49) in contractile force. Following cessation of the nerve stimulation, the heart rate and contractile force returned to the prestimulation level within 60--90 sec. The venous effluent content of NA, which was n o t measurable at rest, increased to 88 + 3 ng (36) during the period of sympathetic nerve stimulation. A second nerve stimulation, performed 15 min after the first one, with no drug added to the Tyrode solution, elicited mainly the same chronotropic and inotropic responses. The o u t f l o w of NA decreased, to 92 -+ 3% (4) of the outflow during the first stimulation. When NA (25/~g during 30 sec) was infused, the heart rate was increased by 28 -+ 1% (50) and the contractile force by 101-+ 5% (50). After discontinuation of the infusion, the mechanical activity of the heart returned to the pre-infusion level within 60-90 sec. Infusion of A (25 #g during 30 sec) also elicited marked chronotropic and inotropic responses, amounting to 30-+ 2% (54) and 92-+ 5% (54), respectively. In analogy with the mechanical response to NA, t h e increases in heart rate and contractile f o r c e induced by A had completely vanished 60--90 sec after the end of the infusion of A. A second infusion of NA or A in the absence of
373 drug in the Tyrode solution, performed 15 min after the first one, elicited chronotropic and inotropic responses of the same magnitudes as the first infusion.
3.2. Effect of papaverine The overflow of NA from the heart in response to nerve stimulation was significantly (p < 0.01) and dose-dependently increased by papaverine; at a drug concentration of 5 × 10 -7 M, the overflow increased by 45 + 9% (fig. 1). Papaverine potentiated the chronotropic response to catecholamine infusion and to nerve stimulation in a dose
