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The effects of neuronal uptake blockade on the cardiac responses to sympathetic nerve stimulation and norepinephrine infusion in anesthetized dogs
Journal of the Autonomic Nervous System, 10 (1984) 1-17
1
Elsevier JAN 00322 Research Papers
The effects of neuronal uptake blockade on the cardiac responses to sympathetic nerve stimulation and norepinephrine infusion in anesthetized dogs * Y u k i t a k a M a s u d a a n d M a t t h e w N. L e v y Division of lnvestigative Medicine, The Mt. Sinai Medical Center, Cleveland, OH 44106 and Case Western Reserve University, Cleveland, OH 44106 (U.S.A.)
(Received June 16th, 1983) (Revised version receivedNovember 1st, 1983) (Accepted November 7th, 1983)
K e y words." cardiac sympathetic nerves - heart rate - myocardial contractile force -
neuronal uptake blockade - norepinephrine release
Summary In anesthetized dogs, cocaine (COC) was administered intravenously in order to block the neuronal uptake of norepinephrine (NE) by the heart. COC had relatively little effect on the magnitudes of the inotropic a n d chronotropic responses to sympathetic stimulation, but it did prolong the decay times, especially those of the chronotropic responses. The marked prolongation of the decay times of the chronotropic responses indicates that neuronal uptake must be the main mechanism for dissipating the adrenergic transmitter in the sinus node. However, this mechanism appears to be less important in the ventricular myocardium. At all dosage levels, COC increased the overflow of N E into the coronary sinus blood during cardiac sympathetic stimulation. The extraction of exogenously infused norepinephrine by heart tissue varied inversely with the dose of COC, indicating that the extent of neuronal uptake blockade increased with the dose. Nevertheless, N E overflow into the coronary sinus blood after a relatively large dose (5.7 m g / k g ) of COC was less than that observed after a much smaller dose (0.5 m g / k g ) , suggesting that relatively * A preliminary report of this paper was presented at the FASEB meetings in New Orleans in April, 1982. Correspondence: M.N. Levy, Division of InvestigativeMedicine, The Mt. Sinai Medical Center, Cleveland, OH 44106, U.S.A. 0165-1838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
large doses of COC also tend to inhibit the neuronal release of NE. These results indicate that the effects of COC on the cardiac responses to sympathetic stimulation depend on the balance between its influences on the release and dissipation of neurotransmitter in the neuroeffector gap.
Introduction
The cardiac responses to sympathetic neural activity depend in part on the concentration of norepinephrine (NE) in the 'biophase', which is the pool of interstitial fluid in contact with the cardiac effector cells. The NE concentration in the biophase at any time reflects the balance between the rate of neuronal release of NE into the biophase and the rate of its disappearance from the biophase. The principal mechanisms involved in the disappearance of NE from the biophase are the neuronal and extraneuronal uptake processes and diffusion into the bloodstream
[2]. The neuronal uptake process can be blocked by a number of drugs, the prototype of which is cocaine (COC). The inotropic and chronotropic responses of the heart to neurally released NE are not appreciably augmented by neuronal uptake blockade in whole-animal preparations whereas, in such preparations [14,18,21,24], the cardiac responses to exogenous NE are substantially enhanced [14,18,21,23]. In isolated heart preparations, on the other hand, COC and other neuronal uptake blocking agents augment the cardiac responses substantially, both to neurally released and to exogenous NE [4,5,7,10,26,31,32]. Regardless of the type of preparation, however, the decays of the cardiac responses to endogenous or exogenous NE are consistently prolonged by COC and other uptake blocking agents; the decay of the chronotropic response is especially prolonged [6,8,14,21,23]. The dosage of COC that had been employed in most of the in vivo studies cited above was about 5 mg/kg. This dosage level not only blocks the neuronal uptake mechanism, but it also has an appreciable local anesthetic action, which tends to diminish the rate of neurotransmitter release [9,11,12,33]. In the experiments to be described, we compared the effects of much smaller doses of COC with those of the more standard dose of about 5 mg/kg, in an attempt to explain why the cardiac responses to sympathetic nerve stimulation were not appreciably enhanced by the more standard doses of COC in intact animals. We performed two series of experiments, one to determine the effects of COC on the cardiac responses to sympathetic nerve stimulation, and the other to determine the effects of COC on the cardiac responses to exogenous NE. In the first series, the overflow of NE into the coronary sinus blood after neuronal uptake blockade was used as an index of the rate of release of NE from the sympathetic nerve terminals in the heart. In the second series, the extraction of NE from the coronary circulation by the cardiac tissues was used as an index of the inhibition of the neuronal uptake mechanism.
Materials and Methods Experiments were conducted on 24 mongrel dogs with a mean body weight of 20.6 + 3.9 (S.D.) kg. The dogs were anesthetized with sodium pentobarbital, 30 m g / k g i.v. A tracheal cannula was inserted through a midline cervical incision, and intermittent positive-pressure ventilation was begun. The chest was opened through a transverse incision in the fourth intercostal space. Both cervical vagi and the upper poles of both stellate ganglia were crushed by tight ligatures, in order to interrupt almost all of the tonic autonomic neural activity to the heart [22]. Arterial blood pressure was measured from a femoral artery by means of a Statham transducer (P23AA). A Walton-Brodie strain-gauge arch was used to measure the myocardial contractile force. It was attached to the right ventricle, parallel and about 1 cm lateral to the anterior descending coronary artery, at a site about halfway between the apex and base of the heart. Cardiac cycle length was derived from the strain gauge arch output. After heparin, 500 u n i t s / k g i.v., was administered to prevent blood coagulation, a wide-bore cannula was introduced into the azygos vein and threaded into the coronary sinus. The tip of the cannula was fixed in position by a suture placed in the posterior wall of the right atrium around the coronary sinus, and within 5 m m of its ostium. The venous outflow from the coronary sinus was led through the extracorporeal probe of an electromagnetic flowmeter (Biotronex, Model BL615), and it was returned to the venous system through a cannula in the right external jugular vein. Coronary sinus blood samples were obtained from a T-tube in this external line. The arterial blood pressure, right ventricular contractile force, cardiac cycle length, and coronary sinus blood flow were recorded on a direct-writing oscillograph (Brush, Mark 260). At selected times in each experiment, arterial and coronary sinus blood samples were withdrawn simultaneously for N E analysis. The blood concentrations of N E were assayed spectrophotometrically by a modification of the methods of Anton and Sayre [1] and Laverty and Taylor [20]. Series L Responses to sympathetic nerve stimulation Each animal was assigned either to an experimental group or to a control group. Animals in the former group received COC at appropriate times in the experiment, whereas those in the latter group were not given COC. A randomization scheme was used that assured an equal number (n = 6) of animals in each group. Each experiment was subdivided into 4 observation periods. During the hrst period (P1), regardless of the group, the decentralized right ansa subclavia was stimulated at frequencies of 1 and 4 Hz in the absence of COC. The order of applying these stimulation frequencies was randomized in this and in the subsequent periods. Each stimulation consisted of a 90-s train of square wave pulses (Grass stimulator, model $9); each pulse was 2 ms in duration, and of supramaximal voltage (usually 15 V). These stimulus characteristics are similar to those that have been used in previous studies in our laboratory [21,24,25]. The changes in contractile force, cardiac cycle length, coronary sinus blood flow, and arterial blood pressure
evoked by sympathetic stimulation were determined. Coronary sinus blood samples were withdrawn for NE analysis when the steady-state response to sympathetic nerve stimulation had been attained, usually between 60 and 90 s after the beginning of stimulation. ' N E overflow' from the cardiac tissues was calculated as the product of coronary sinus blood flow and the NE concentration in the coronary sinus blood. After the completion of the first observation period (P1), the animals in the experimental group received an intravenous infusion of cocaine hydrochloride, 10 /xg.kg - 1 . m i n -1, for 75 min. The cardiac responses to ansal stimulation were determined at the same two stimulation frequencies, beginning 15 min after the start of the COC infusion. At this time the cumulative dose of COC was 0.15 mg/kg. Coronary sinus blood samples were withdrawn for NE analysis near the end of each train of stimulation. This constituted the second observation period (P2). The third period (P3) was begun 50 min after the beginning of the COC infusion. At the beginning of P3, the cumulative dose of COC was 0.5 mg/kg. The cardiac responses and NE overflows evoked by ansal stimulation were again determined, as described above. After completion of P3, the COC infusion rate was increased to 330/xg. kg-1. rain-1 for a 15-min period, and then was reduced to one-tenth of that rate for the remainder of the experiment. After this slower rate had been started, the cardiac responses to ansal stimulation were again determined at the two stimulation frequencies, and coronary sinus blood samples were withdrawn. This constituted the fourth observation period (P4). The cumulative dose of COC was 5.7 mg/kg at the beginning of P4. In the control group, COC was not infused, but the responses to ansal stimulation were determined and coronary sinus blood samples were withdrawn during 4 observation periods that were equivalent in timing to the 4 periods in the experimental group. The data were analyzed by means of a 4-way, mixed-model analysis of variance [30]. The 4 factors were the groups (G), observation periods (P), sympathetic stimulation frequencies (S), and individual animals (A). The factors G, P, and S were considered to evoke fixed treatment effects, whereas factor A was considered to induce a random effect. A single degree of freedom test was used for a priori comparisons [30]. Seheffr's test was used for a posteriori comparisons and for any other comparison for which the single degree of freedom test was not applicable [29].
Series 11. Responses to norepinephrine infusions Each animal was assigned randomly either to an experimental group (that received COC) or to a control group (that did not receive COC); there were 6 animals in each group. Each experiment was subdivided into 4 observation periods. Instead of cardiac sympathetic nerve stimulation, l-norepinephrine bitartrate (Winthrop Labs) was infused intravenously at a rate of 0.1/~g. kg-1. min-1 (of the base) during each observation period for precisely 3 rain. The cardiac responses to the infusions reached steady-state within this time interval. The changes in contractile force, cardiac cycle length, and arterial blood pressure evoked by the NE infusions were determined. Coronary sinus and arterial blood samples were
withdrawn for N E analysis near the end of each N E infusion. The N E extraction ratio was calculated as the ratio of the arteriovenous N E concentration difference to the arterial N E concentration. During period P1 in both groups of animals, the cardiac responses evoked by the N E infusion and the N E extraction ratio were determined in the absence of COC. After the completion of this first observation period, COC was infused in the experimental group during periods P2 to P4, according to the same dosage regimen that had been used in the first series. The standard N E infusion was administered intravenously during each of these periods, and the cardiac responses and extraction ratios were again determined. In the control group, COC was not infused. The cardiac responses to the standard N E infusions and the N E extraction ratios were determined during 4 observation periods that were equivalent in timing to the four periods in the experimental group. The data were analyzed by means of a three-way, mixed-model analysis of variance [30]. The 3 factors were the groups (G), observation periods (P), and individual animals (A). The factors G and P were considered to evoke fixed treatment effects, whereas factor A was considered to induce a random effect.
Results
Series I. Responses to sympathetic nerve stimulation Representative experiment Fig. 1 shows the changes in coronary blood flow, cardiac cycle length, and right ventricular contractile force elicited by stimulation of the right ansa subclavia in a
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representative experiment. The record was obtained during the first observation period, in the absence of an infusion of COC. Ansal stimulation (1 Hz, between the arrows) evoked a 34% increase in coronary sinus blood flow, a 132 ms reduction in cardiac cycle length, and a 79% increase in contractile force. After cessation of stimulation, these variables returned toward their control levels. The '50% recovery times' for contractile force and cycle length were 27 s and 16 s, respectively. These values are the times required for the responses to return halfway to their steady-state recovery levels, and they reflect the 'decay times' of the responses after cessation of stimulation.
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(a) Inotropic responses In both the experimental and the control groups, ansal stimulation increased the ventricular contractile force, and this was usually associated with a 10-15% increase in the mean arterial blood pressure. The magnitude of the change in contractile force
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Fig. 2. The magnitudes and decay times of the.inotroplc responses to cardiac sympathetic stimulation in an experimental group (open circles) of dogs, which received cocaine, and in a control group (closed squares), which did not receive cocaine. The magnitude of the inotropic response is expressed as a percent of the prestimulation contractile force. The decay times are measured as the 507o recovery times; i.e. the time required for the inotropic response to retura haffwa~to the steatdy-state recovery level after cessation of neural stimulation. In the experimental g l a i r , t ~ ~ d 0 0 ~ Of Cocaine were 0.15, 0.5 and 5.7 m g / k g , administered during observation periods P2. P3 and P4, respectively. In any given panel, each point represents the mean value averaged over all unspecified factors. For example, panels A and C represent the interactions bctweea groups ((3) and stimulation frequency (F). Hence, each point in panels A and C reprgsents the mean value averaged over all observation periods (P) and over all animals. Similarly, in panels B and D, each point represents the mean value averaged over the two stimulation frequencies (F) and over all animals.
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evoked by ansal stimulation varied directly with the frequency of stimulation (Fig. 2A). The main effect of stimulation frequency was significant (P = 0.01, Table I). The inotropic responses in the control group happened to be greater than those in the experimental group (Fig. 2B). However, such differences could not have been ascribable to the effects of COC. Note that in the experimental group, the inotropic responses elicited at any of the dosage levels of COC (P2 to P4) were not appreciably different from the responses obtained before COC had been given (P1). In both groups of animals, the decay times of the inotropic responses, as assessed by the 50% recovery time, increased with the frequency of ansal stimulation (Fig. 2C). In the control group, the decay times of the inotropic responses remained virtually constant from period to period (Fig. 2D). In the experimental group, the decay times of the inotropic responses at all of the dosage levels of COC (P2 to P4) were significantly greater (P = 0.01) than they were before COC had been given (P1). The decay times of the responses during P2, P3 and P4 in the experimental group were not significantly different from each other, however. The main effects of groups, observation periods, and stimulation frequencies were all significant (P _< 0.05, Table I), but the interactions were not significant.
(b) Chronotropic responses Ansal stimulation diminished the cardiac cycle length (Fig. 3A), and the magnitude of this chronotropic response varied with the stimulation frequency (P = 0.01, Table I). The responses to ansal stimulation in the control group did not change appreciably over the course of the 4 observation periods (Fig. 3B). In the experimental group, the magnitudes of the chronotropic responses to ansal stimulation were not significantly affected by the smaller doses of COC (P2 and P3). However. after the largest dose of COC (P4), the reduction in cardiac cycle length was significantly greater (P = 0.05) than it was during any of the preceding periods. In the control group, the decay times of the chronotropic responses were not appreciably different at the two stimulation frequencies (Fig. 3C), and they remained virtually constant from period to period (Fig. 3D). In the experimental group, the decay times of the responses to ansal stimulation were greater at 4 Hz than at 1 Hz (Fig. 3C), and they were much greater (P < 0.001) in the animals that received COC than in those that did not (Fig. 3D). After the smallest dose of COC (P2), the decay time of the chronotropic response was significantly longer (P = 0.05) than it was before COC had been given (P1; Fig. 3D). Furthermore, the decay times of the response became progressively greater (P < 0.025) as the cumulative dose of COC was increased (P2 to P4). The mare effects of groups, observation periods, stimulation frequencies, and all the interactions were highly significant (P _< 0.01, Table I).
(c) Coronary blood flow The increase in coronary blood flow evoked by ansal stimulation (Fig. 4A) were approximately equal in the control and experimental groups, and the responses were frequency dependent ( P = 0.01, Table I). In the experimental group (Fig. 4B), the increments in coronary blood flow evoked by ansal stimulation were greater ( P 0.05) during the infusion of COC (P2 to P4) than before COC had been adminis-
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Fig. 3. The magnitudes and decay times of the chronotropic responses to cardiac sympathetic stimulation in the experimental (open circles) and control (closed squares) groups. The chronotropic responses are expressed as the mean changes in cycle length from the prestimulation (basal) value. The mean basal values (+ S.D.) of the cardiac cycle lengths in the control and experimental groups were 501 + 79 and 511 + 61 ms, respectively. Fig. 4. The changes in coronary sinus blood flow and in norepinephrine overflow into the coronary sinus blood evoked by cardiac sympathetic stimulation in the experimental (open circles) and control (closed squares) groups. The mean basal values ( _ S.D.) of the coronary sinus blood flow and the norepinephrine overflow in the control groups were 30.2 + 11.9 ml/min and 3.76 + 2.60 ng/min, respectively, whereas those in the experimental group were 27.6 +_8.5 ml/min and 4.22 + 3.44 ng/min, respectively. tered (P1). The coronary blood flow did fluctuate from period to period in the control group, but the changes were neither systematic nor statistically significant.
(d) Norepinephrine overflow The rate of overflow of N E into the coronary sinus blood increased ( P = 0.01) with the frequency of ansal stimulation in both groups (Fig. 4C). In the control group, the N E overflow evoked by ansal stimulation tended to decrease slightly, but not significantly, throughout the 4 observation periods (Fig. 4D). In the experimental group, on the other hand, the N E overflow was significantly greater ( P = 0.05) at each of the dosage levels of C O C (P2 to P4) than it was before C O C had been infused (P1). However, after the largest dose of C O C (P4), the rate of N E overflow was less ( P = 0.05) than it had been during the preceding observation period (P3).
Series 11. Responses to norepinephrine infusion (a) Inotropic responses In the control group the magnitudes of the inotropic responses to the N E infusions did not change significantly from period to period (Fig. 5A). In the
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experimental group, the inotropic responses were not significantly affected by, the two lower doses of COC (P2 and P3). However, there was a pronounced enhancement of the inotropic response at the highest dosage level of COC (P4). The main effects of groups and periods and the interaction between them were all significant (Table II). The decay times of the inotropic responses did not vary appreciably from period to period in the control group (Fig. 5B). The two smaller doses of COC (P2 and P3) did not significantly prolong the decay times, but the largest dose of COC did evoke a significant prolongation (P < 0.05). The first-order interaction was also significant (P = 0.001, Table II).
(b) Chronotropic responses The reductions in cardiac cycle length evoked by the infusions of NE were fairly constant from period to period in the control group (Fig. 6A). Conversely, the reductions in cycle length in the experimental group were significantly greater (P < 0.05) than in the control group at all dosage levels of COC (P2 to P4). The mare effects of experimental groups and observation periods and the interaction between them were all significant (P < 0.05, Table II). The decay times of the chronotropic responses were fairly constant over the 4 periods in the control group (Fig. 6B). Conversely, in the experimental group the decay times of the chronotropic responses were significantly prolonged at atl dosage levels of COC. However, the decay times were especially prolonged after the largest dose of COC (P4). The main effects of groups and periods and the interaction between them were all significant (P < 0.01, Table II). (c) Norepinephrine extraction ratio In the control group, the mean NE extraction ratio over the 4 periods was
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0.55 ± 0.05 (mean ± S.E.), and it did not change appreciably from period to period (Fig. 7). In the experimental group, on the other hand, the NE extraction ratio decreased as the dose of COC was increased. At the lowest dosage level of COC (P2), this ratio decreased to 0.40 ± 0.11 from a control value (P1) of 0.69+ 0.08 (P = 0.05). At the intermediate (P3) and hi~est (P4) dosage levels, the extraction ratios were further reduced to 0.28 ± 0.07 and 0.24 ± 0,07, respectively. These ratios were also significantly less than the control ratio obtained during P1. The main
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13 effect of the observation periods and the interaction between groups and periods were significant (Table II).
Discussion
Effects of COC dosage on NE release and reuptake During sympathetic neural activity, the concentration of NE in the biophase (interstitial fluid in contact with the cardiac effector cells) is determined by the following factors: (a) the rate of release of N E from the sympathetic nerve endings; (b) the rate of neuronal uptake; (c) the rate of extraneuronal uptake; and (d) the rate of diffusion (and possibly other physical transport mechanisms) of neurotransmitter away from the synaptic clefts and into the coronary circulation. The extraneuronal uptake mechanism is relatively unimportant as a determinant of the cardiac responses to sympathetic activity [11,15,19,23,24]. Furthermore, COC has no appreciable effect on extraneuronal uptake; its effect on the uptake of NE is virtually restricted to the neuronal mechanism [11,12]. Hence, in our considerations of the effects of COC on the processes that add NE to and remove it from the biophase, the rate of extraneuronal uptake (factor (c) above) can be ignored. Under steady-state conditions, the rate of addition of N E to the biophase (factor (a)) equals the rate of its removal (factors (b) and (d)). Hence, under steady-state conditions, the rate of diffusion of N E out of the biophase and into the coronary circulation reflects essentially the difference between the rates of release and reuptake of neurotransmitter. In our experiments, even the smallest dose of COC (0.15 mg/kg) had a significant depressant effect on the neuronal uptake of NE. This is supported by the observations that this dosage level evoked a substantial reduction in the N E extraction ratio (Fig. 7; P2) and appreciable prolongations of the decay times of the chronotropic responses to sympathetic nerve stimulation (Fig. 3D; P2) and to N E infusion (Fig. 6; P2). In the experimental group, the N E overflow rates (Fig. 4D) during ansal stimulation were greater after COC (P2 to P4), regardless of the dose, than they were before COC had been infused (P1). For a substance, such as COC, that depresses both the release and the uptake of NE, the increase in the overflow of N E induced by COC signifies that this blocking agent must exert a greater depressant effect on the rate of neuronal uptake of neurotransmitter than on the rate of its neuronal release. However, after the largest dose of COC (P4), the N E overflow was significantly less than it was after the intermediate dose (P3). The data in Fig. 7 reveal that the inhibition of NE uptake was certainly just as intense, if not more intense, after the largest dose of COC than after the intermediate dose. Other factors being equal, the more complete the inhibition of neuronal uptake, the greater would be the NE overflow rate. Hence, the data in Fig. 7 indicate that raising the dose of COC from the intermediate (P3) to the highest (P4) level must have had a greater incremental effect on the neuronal release mechanism than on the neuronal uptake mechanism.
14 Analogous observations have been made in vitro. Low concentrations of COC (less than 5/~g/ml) were found to increase the amount of N E released by electrical field stimulation in the rabbit portal vein and vas deferens [9]. Conversely, higher concentrations of COC diminished the rate of NE release. Similar results were obtained in isolated rabbit hearts [33]. Relatively low concentrations of COC augmented the overflow of NE evoked by sympathetic stimulation, whereas higher concentrations of COC diminished the NE overflow.
Effects of COC dosage on the magnitudes of the inotropic and chronotropic responses In the present study, the inotropic responses to sympathetic stimulation were not augmented after the neuronal uptake mechanism was blocked by COC (Fig. 2B). The chronotropic responses were somewhat enhanced after the largest dose of COC, but not after the two smaller doses (Fig. 3B). If there were no change in the responsiveness of the cardiac effector cells, the magnitude of a given cardiac response should reflect the concentration of neurotransmitter in the biophase. Other factors remaining constant, neuronal uptake blockade would be expected to increase the concentration of N E in the biophase. Hence, it might be anticipated that such blockade would augment the cardiac responses, unless COC also: (a) reduced significantly the rate of NE release from the nerve endings; (b) facilitated the diffusion away from the synaptic clefts; or (c) reduced the responsiveness of the effector cells. An indication of effector cell responsiveness was provided by the second series of experiments, in which the neurotransmitter was infused intravenously. The inotropic and chronotropic responses to the N E infusions were greater over the range of COC doses that were used in this study (Figs. 5 and 6). Therefore, if COC did depress the responsiveness of the effector cells, its depressant effect must have been sufficiently weak that it did not mask the tendency for its neuronal uptake blocking action to augment the inotropic and chronotropic responses to exogenous NE. The studies of Kalsner and Nickerson [17] indicate that COC tends to augment, rather than to depress, the responsiveness of certain effector cells, at least in strips of rabbit aorta. The neuronal release of NE during ansal stimulation was probably not impaired substantially by the lowest and intermediate dosage levels of COC in the present study. In the experimental group, the NE overflow rates during P2 and P3 were greater than that during the control period (Fig. 4D). The increases in NE overflow during P2 and P3 were accompanied by increases in coronary blood flow (Fig. 4B). Such a change in coronary blood flow would tend to facilitate the diffusion of N E out of the biophase [28,36]. Hence, the concomitant increase in coronary blood flow might account, at least in part, for the absence of any detectable potentiation of the cardiac responses during P2 and P3 (Figs. 2B and 3B). Effects of COC dosage on the decay times of the inotropic and chronotropic responses After cessation of sympathetic stimulation, the concentration (C) of N E in the biophase tends to decrease with time. The rate of change of the N E concentration, d C / d t , in the biophase would be determined principally by the rates of neuronal uptake and diffusion of neurotransmitter. After cessation of ansal stimulation,
15 d C / d t is reflected by the 'decay time' of the cardiac response, at least when C is within the linear portion of the dose-response curve [34]. Suppression of the neuronal uptake mechanism retards the removaI of neurotransmitter from the biophase. Therefore, it tends to prolong the decay times of the cardiac responses. The decay time of the chronotropic response to sympathetic neural stimulation is a very sensitive index of the suppression of neuronal uptake in the heart [3,18,21,24,25]. In the present experiments (Fig. 3D), we found that after the smallest dose of COC (P2), the decay time of the chronotropic response to ansal stimulation was much more prolonged than it was in the absence of COC (P1). At the intermediate (P3) and largest doses (P4), the prolongations were significantly greater. These results indicate that the neuronal uptake mechanism was inhibited substantially even after the lowest dose of COC, and the extent of the inhibition increased with the dose. If the increase in coronary blood flow engendered by sympathetic stimulation in the animals that received COC (experimental group, periods P2 to P4, Fig. 4B) was as great in the S-A nodal region as in the cardiac tissues as a whole, the tendency for NE to diffuse out of the biophase in the S-A nodal region and into the coronary circulation would be enhanced. The great prolongation of the decay time of the chronotropic response elicited by COC (Fig. 3D) indicates that the inhibition of neuronal uptake must have been disproportionately greater than any concomitant facilitation of diffusion. The experiments of James and Nadeau [13] suggest that the vascular response to increased sympathetic activity in the S-A nodal region may be just the opposite of the response in the myocardial tissues, i.e. the vessels of the S-A node may actually constrict in response to increased sympathetic activity. This would constitute an impediment to diffusion, which would account at least partially for the marked prolongation of the decay time of the chronotropic response. The relative importance of the dissipation mechanisms in the ventricular muscle seems to differ from that in the sinus node. The decay times of the inotropic responses (Fig. 2D) to sympathetic stimulation were much less prolonged by COC than were the decay times of the chronotropic responses (Fig. 3D). It seems likely that the suppression of the neuronal uptake induced by a given dose of COC would be similar in the nerve endings in the various regions of the heart. Hence, the observed disparities in the decay times in different cardiac tissues after COC suggest differences in the balance among the various factors involved in the dissipation of neurotransmitter. Diffusion of N E (or other physical transfer processes) may play a more important role in the removal of neurotransmitter from the biophase in the ventricular myocardium than it does from the biophase in the S-A node. The results of recent unpublished studies from our laboratory suggest that the cardiac contraction itself, possibly by a 'massaging action', facilitates the transport of neurotransmitter from the biophase to the coronary blood stream. This process would be expected to be much more effective in the ventricular myocardium than in the S-A node. Conversely, the neuronal uptake of N E may be more effective in the S-A nodal region than in the ventricular myocardium. Neuronal uptake processes are known to be more effective in the termination of neurotransmitter action in tissues that have
lt,
relatively narrow neuroeffector gaps [16,17]. The width of the neuroeffector gap in papillary muscle is about 2000 A [27], whereas that in the S-A node is only 100 A [35]. Hence, neuronal uptake blockade would be expected to prolong the decay time of the chronotropic response more than it would that of the inotropic response. The relative importance of these various dissipative mechanisms in the different cardiac structures remains to be established.
Acknowledgements The authors are indebted to Mr. Herrick Finkelstein for his skillful technical assistance. This work was supported by the U,S. Public Health Ser,Ace Grant H L 15758.
References 1 Anton, A.H. and Sayre, D.F., A study of the factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of catccholamines, J. Pharmacol. exp. Ther., 138 (1962) 360-375. 2 Axelrod, J. and Wei~[ilb0um, K., Caiecholamines, N. En$1. J. Med., 287 (1972) 237-242. 3 Bell, C. and Kus]~.~nsky,,~., .Involvement of uptake 1 and uptake 2 in terminating the cardiovascular activity of noradrenaline in normotensive and genetically hypertensive rats, J. Physiol. (Lond.), 283 (1978) 41-51. 4 Chang, C.C. and Lee, M.C., Studies of the effect of phenoxybenzamine on the uptake of noradrenaline and on the response to sympathetic stimulation of rat hearts, Arch. int. Pharmacodyn. Ther., 201 (1973) 400-414. 5 Furchgott, R.F., Kirpekar, S.M., Rieker, M. and Schwab, M.. Actions and interactions of norepinephfine, tyramine and cocaine on aortic stops of rabbit and left atria of guinea pig and cat, J. Pharmacol. exp. Ther.. 142 (1963) 39-58. 6 Furnival, C.M., Linden. R.J. and Snow. H.M.. The inotropic and chronotropic effects of catecholamines on the dog heart, J. Physiol. (Lond.), 214 (1971) 15-28. 7 Gillis, C.N. and Schneider. F.H.. Frequency-dependent potentiation by various drugs of chronotropic response of isolated cat atria to sympathetic nerve stimulation, Brit. J. Pharmacol. Chemother., 30 (1967) 541-553. 8 Hardman, J.G., Mayer, N.F. and Clark, B., Cocaine potentiation of the cardiac inotropic and phosphorylase responses to catecholamines as related to the uptake of [3H]-catecholamines, J. Pharmacol. exp. Ther.. 150 (1965) 341-348. 9 Hughes. J.. Evaluation of mechanisms controlling the release and inactivation of the adrenergi¢ transmitter in the rabbit portal vein and vas defcrcns, Brit. J. Pharmacol., 4,4 (1972) 472~491. 10 Hukovic. S. and MusehoU, E., Die Noradrenalin-Abgabe aus dem isolierten Kanincl~nherzen bei sympathischer Nervenreizung und ihre pharmacologische Beeinflussung, Naunyn-Schmiedberg's Arch. Pharmacol.. 244 (1962) 81-96. 11 Iverscn. L.L., Catecholamine uptake process, Brit. Med. Bull., 29 (1973) 130-135. 12 Iversen. L.L.. Uptake processes for biogenic attunes. In L.L. Iverscn, S.D. Iversen and S.H. Snyder (Eds.), Handbook of Psychopharmacology, Plenum Press, New York, 1975. pp. 381-442. 13 James. T.N. and Nadeau, R.A., Relation of retrograde pressure in the sinus node artery to sinus tachycardia from stellate stimulation, J. Lab. Clin. Med., 62 (1963) 777-786. 14 Johnson, G . L and Kahn, J.B., Cocaine and antihistaminic compounds: comparison of effects of some cardiovascular actions of norepinephrine, tyramine and bretylium. J. Pharmacol. exp. Ther., 152 (1966) 458-468.
17 15 Junstad, M., Stj~irne, L. and Wennmalm, ,~., On the relative importance of extraneuronal uptake of noradrenaline released by nerve stimulation in the rabbit heart, Acta physiol, scand., 88 (1973) 67-70. 16 Kalsner, S., Termination of effector responses to agonists: an analysis of agonist disposition mechanisms, Canad. J. Physiol. Pharmacol., 55 (1977) 315-331. 17 Kalsner, S. and Nickerson, M., Mechanism of cocaine potentiation of responses to amine, Brit. J. Pharmacol., 35 (1969) 428-439. 18 Koerker, R.L. and Moran, N.C., An evaluation of the inability of cocaine to potentiate the responses to cardiac sympathetic nerve stimulation in the dog, J. Pharmacol. exp. Ther., 178 (1971) 482-496. 19 Langer, S.Z. and Rubio, M.C., Effects of the noradrenaline metabolites on the adrenergic receptors, Naunyn-Schmiedberg's Arch. Pharmacol., 276 (1973) 71-78. 20 Laverty, R. and Taylor, K.M., The fluorometric assay of catecholamines and related compounds: improvements and extensions to hydroxyindole technique, Analyt. Biochem., 22 (19681 269-279. 21 Levy, M.N. and Blattberg, B., The influence of cocaine and desipramine on the cardiac responses to exogenous and endogenous norepinephrine, Europ. J. Pharmacol., 48 (1978) 37-49. 22 Levy, M.N. and Zieske, H., Functional distribution of peripheral cardiac sympathetic pathways, Circular. Res., 19 (1966) 650-661. 23 Masuda, Y., Matsuda, Y. and Levy, M.N., The effects of cocaine and metanephrine on the cardiac responses to norepinephrine infusions, J. Pharmacol. exp. Ther., 215 (1980) 20-27. 24 Matsuda, Y., Masuda, Y. and Levy, M.N., The effects of cocaine and metanephrine on the cardiac responses to sympathetic nerve stimulation in dogs, Circulat. Res., 45 (1979) 180-187. 25 Matsuda, Y., Masuda, Y. and Levy, M.N., The effects of cocaine, chlorpheniramine and tripelennamine on the cardiac responses to sympathetic nerve stimulation, Europ. J. Pharmacol., 63 (1980) 25-33. 26 Matsuo, S. and Toda, N., The influence of desmethylimipramine on the chronotropic response to endogenous and exogenous noradrenaline in the isolated atria, Brit. J. Pharmacol. Chemother., 32 (1968) 473-482. 27 Novi, A., An electron microscopic study of the innervation of the papillary muscle of the rat, Anat. Rec., 160 (1968) 123-142. 28 Schafer, D.E. and Johnson, J.A., Permeability of mammalian heart capillaries to sucrose and inulin, Amer. J. Physiol., 206 (1964) 985-991. 29 Scheff& H., A method for judging all contrasts in the analysis of variance, Biometrika, 40 (1953) 87 104~ 30 Sokal, R.R. and Rohlf, F.J., Biometry, W.H. Freeman, San Francisco, 1969, pp. 343. 31 Stafford, A., Potentiation of some catecholamines by phenoxybenzamine, guanethidine and cocaine Brit. J. Pharmacol, 21 (1963) 361-367. 32 Starke, K. and Schiimann, H.J., Interactions of angiotensin, phenoxybenzamine and propranolol on noradrenaline release during sympathetic nerve stimulation, Europ. J. Pharmacol., 18 (1972) 27-30. 33 Starke, K., Wagner, J. and Schiimann, H.J., Adrenergic neuron blockade by clonidine: comparison with guanethidine and local anesthetics, Arch. Int. Pharmacodyn., 195 (1972) 291-308. 34 Trendelenburg, U. and Henseling, M., Factors determining the rate of relaxation of rabbit aortic strips after an exposure to noradrenaline, Naunyn-Schmiedberg's Arch. Pharmacol., 293 (1976) 235-244. 35 Yamauchi, A., Ultrastructure of innervation of the mammalian heart. In C.E. Challice and S. Viragh (Eds.), Ultrastructure of the Mammalian Heart, Academic Press, New York, 1973, pp. 127-129. 36 Young, D.A.B., Factors controlling the washout of the interstitial space on the isolated, perfused rat heart, J. Phsyiol. (Lond.), 196 (1968) 747-759.
1
Elsevier JAN 00322 Research Papers
The effects of neuronal uptake blockade on the cardiac responses to sympathetic nerve stimulation and norepinephrine infusion in anesthetized dogs * Y u k i t a k a M a s u d a a n d M a t t h e w N. L e v y Division of lnvestigative Medicine, The Mt. Sinai Medical Center, Cleveland, OH 44106 and Case Western Reserve University, Cleveland, OH 44106 (U.S.A.)
(Received June 16th, 1983) (Revised version receivedNovember 1st, 1983) (Accepted November 7th, 1983)
K e y words." cardiac sympathetic nerves - heart rate - myocardial contractile force -
neuronal uptake blockade - norepinephrine release
Summary In anesthetized dogs, cocaine (COC) was administered intravenously in order to block the neuronal uptake of norepinephrine (NE) by the heart. COC had relatively little effect on the magnitudes of the inotropic a n d chronotropic responses to sympathetic stimulation, but it did prolong the decay times, especially those of the chronotropic responses. The marked prolongation of the decay times of the chronotropic responses indicates that neuronal uptake must be the main mechanism for dissipating the adrenergic transmitter in the sinus node. However, this mechanism appears to be less important in the ventricular myocardium. At all dosage levels, COC increased the overflow of N E into the coronary sinus blood during cardiac sympathetic stimulation. The extraction of exogenously infused norepinephrine by heart tissue varied inversely with the dose of COC, indicating that the extent of neuronal uptake blockade increased with the dose. Nevertheless, N E overflow into the coronary sinus blood after a relatively large dose (5.7 m g / k g ) of COC was less than that observed after a much smaller dose (0.5 m g / k g ) , suggesting that relatively * A preliminary report of this paper was presented at the FASEB meetings in New Orleans in April, 1982. Correspondence: M.N. Levy, Division of InvestigativeMedicine, The Mt. Sinai Medical Center, Cleveland, OH 44106, U.S.A. 0165-1838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
large doses of COC also tend to inhibit the neuronal release of NE. These results indicate that the effects of COC on the cardiac responses to sympathetic stimulation depend on the balance between its influences on the release and dissipation of neurotransmitter in the neuroeffector gap.
Introduction
The cardiac responses to sympathetic neural activity depend in part on the concentration of norepinephrine (NE) in the 'biophase', which is the pool of interstitial fluid in contact with the cardiac effector cells. The NE concentration in the biophase at any time reflects the balance between the rate of neuronal release of NE into the biophase and the rate of its disappearance from the biophase. The principal mechanisms involved in the disappearance of NE from the biophase are the neuronal and extraneuronal uptake processes and diffusion into the bloodstream
[2]. The neuronal uptake process can be blocked by a number of drugs, the prototype of which is cocaine (COC). The inotropic and chronotropic responses of the heart to neurally released NE are not appreciably augmented by neuronal uptake blockade in whole-animal preparations whereas, in such preparations [14,18,21,24], the cardiac responses to exogenous NE are substantially enhanced [14,18,21,23]. In isolated heart preparations, on the other hand, COC and other neuronal uptake blocking agents augment the cardiac responses substantially, both to neurally released and to exogenous NE [4,5,7,10,26,31,32]. Regardless of the type of preparation, however, the decays of the cardiac responses to endogenous or exogenous NE are consistently prolonged by COC and other uptake blocking agents; the decay of the chronotropic response is especially prolonged [6,8,14,21,23]. The dosage of COC that had been employed in most of the in vivo studies cited above was about 5 mg/kg. This dosage level not only blocks the neuronal uptake mechanism, but it also has an appreciable local anesthetic action, which tends to diminish the rate of neurotransmitter release [9,11,12,33]. In the experiments to be described, we compared the effects of much smaller doses of COC with those of the more standard dose of about 5 mg/kg, in an attempt to explain why the cardiac responses to sympathetic nerve stimulation were not appreciably enhanced by the more standard doses of COC in intact animals. We performed two series of experiments, one to determine the effects of COC on the cardiac responses to sympathetic nerve stimulation, and the other to determine the effects of COC on the cardiac responses to exogenous NE. In the first series, the overflow of NE into the coronary sinus blood after neuronal uptake blockade was used as an index of the rate of release of NE from the sympathetic nerve terminals in the heart. In the second series, the extraction of NE from the coronary circulation by the cardiac tissues was used as an index of the inhibition of the neuronal uptake mechanism.
Materials and Methods Experiments were conducted on 24 mongrel dogs with a mean body weight of 20.6 + 3.9 (S.D.) kg. The dogs were anesthetized with sodium pentobarbital, 30 m g / k g i.v. A tracheal cannula was inserted through a midline cervical incision, and intermittent positive-pressure ventilation was begun. The chest was opened through a transverse incision in the fourth intercostal space. Both cervical vagi and the upper poles of both stellate ganglia were crushed by tight ligatures, in order to interrupt almost all of the tonic autonomic neural activity to the heart [22]. Arterial blood pressure was measured from a femoral artery by means of a Statham transducer (P23AA). A Walton-Brodie strain-gauge arch was used to measure the myocardial contractile force. It was attached to the right ventricle, parallel and about 1 cm lateral to the anterior descending coronary artery, at a site about halfway between the apex and base of the heart. Cardiac cycle length was derived from the strain gauge arch output. After heparin, 500 u n i t s / k g i.v., was administered to prevent blood coagulation, a wide-bore cannula was introduced into the azygos vein and threaded into the coronary sinus. The tip of the cannula was fixed in position by a suture placed in the posterior wall of the right atrium around the coronary sinus, and within 5 m m of its ostium. The venous outflow from the coronary sinus was led through the extracorporeal probe of an electromagnetic flowmeter (Biotronex, Model BL615), and it was returned to the venous system through a cannula in the right external jugular vein. Coronary sinus blood samples were obtained from a T-tube in this external line. The arterial blood pressure, right ventricular contractile force, cardiac cycle length, and coronary sinus blood flow were recorded on a direct-writing oscillograph (Brush, Mark 260). At selected times in each experiment, arterial and coronary sinus blood samples were withdrawn simultaneously for N E analysis. The blood concentrations of N E were assayed spectrophotometrically by a modification of the methods of Anton and Sayre [1] and Laverty and Taylor [20]. Series L Responses to sympathetic nerve stimulation Each animal was assigned either to an experimental group or to a control group. Animals in the former group received COC at appropriate times in the experiment, whereas those in the latter group were not given COC. A randomization scheme was used that assured an equal number (n = 6) of animals in each group. Each experiment was subdivided into 4 observation periods. During the hrst period (P1), regardless of the group, the decentralized right ansa subclavia was stimulated at frequencies of 1 and 4 Hz in the absence of COC. The order of applying these stimulation frequencies was randomized in this and in the subsequent periods. Each stimulation consisted of a 90-s train of square wave pulses (Grass stimulator, model $9); each pulse was 2 ms in duration, and of supramaximal voltage (usually 15 V). These stimulus characteristics are similar to those that have been used in previous studies in our laboratory [21,24,25]. The changes in contractile force, cardiac cycle length, coronary sinus blood flow, and arterial blood pressure
evoked by sympathetic stimulation were determined. Coronary sinus blood samples were withdrawn for NE analysis when the steady-state response to sympathetic nerve stimulation had been attained, usually between 60 and 90 s after the beginning of stimulation. ' N E overflow' from the cardiac tissues was calculated as the product of coronary sinus blood flow and the NE concentration in the coronary sinus blood. After the completion of the first observation period (P1), the animals in the experimental group received an intravenous infusion of cocaine hydrochloride, 10 /xg.kg - 1 . m i n -1, for 75 min. The cardiac responses to ansal stimulation were determined at the same two stimulation frequencies, beginning 15 min after the start of the COC infusion. At this time the cumulative dose of COC was 0.15 mg/kg. Coronary sinus blood samples were withdrawn for NE analysis near the end of each train of stimulation. This constituted the second observation period (P2). The third period (P3) was begun 50 min after the beginning of the COC infusion. At the beginning of P3, the cumulative dose of COC was 0.5 mg/kg. The cardiac responses and NE overflows evoked by ansal stimulation were again determined, as described above. After completion of P3, the COC infusion rate was increased to 330/xg. kg-1. rain-1 for a 15-min period, and then was reduced to one-tenth of that rate for the remainder of the experiment. After this slower rate had been started, the cardiac responses to ansal stimulation were again determined at the two stimulation frequencies, and coronary sinus blood samples were withdrawn. This constituted the fourth observation period (P4). The cumulative dose of COC was 5.7 mg/kg at the beginning of P4. In the control group, COC was not infused, but the responses to ansal stimulation were determined and coronary sinus blood samples were withdrawn during 4 observation periods that were equivalent in timing to the 4 periods in the experimental group. The data were analyzed by means of a 4-way, mixed-model analysis of variance [30]. The 4 factors were the groups (G), observation periods (P), sympathetic stimulation frequencies (S), and individual animals (A). The factors G, P, and S were considered to evoke fixed treatment effects, whereas factor A was considered to induce a random effect. A single degree of freedom test was used for a priori comparisons [30]. Seheffr's test was used for a posteriori comparisons and for any other comparison for which the single degree of freedom test was not applicable [29].
Series 11. Responses to norepinephrine infusions Each animal was assigned randomly either to an experimental group (that received COC) or to a control group (that did not receive COC); there were 6 animals in each group. Each experiment was subdivided into 4 observation periods. Instead of cardiac sympathetic nerve stimulation, l-norepinephrine bitartrate (Winthrop Labs) was infused intravenously at a rate of 0.1/~g. kg-1. min-1 (of the base) during each observation period for precisely 3 rain. The cardiac responses to the infusions reached steady-state within this time interval. The changes in contractile force, cardiac cycle length, and arterial blood pressure evoked by the NE infusions were determined. Coronary sinus and arterial blood samples were
withdrawn for N E analysis near the end of each N E infusion. The N E extraction ratio was calculated as the ratio of the arteriovenous N E concentration difference to the arterial N E concentration. During period P1 in both groups of animals, the cardiac responses evoked by the N E infusion and the N E extraction ratio were determined in the absence of COC. After the completion of this first observation period, COC was infused in the experimental group during periods P2 to P4, according to the same dosage regimen that had been used in the first series. The standard N E infusion was administered intravenously during each of these periods, and the cardiac responses and extraction ratios were again determined. In the control group, COC was not infused. The cardiac responses to the standard N E infusions and the N E extraction ratios were determined during 4 observation periods that were equivalent in timing to the four periods in the experimental group. The data were analyzed by means of a three-way, mixed-model analysis of variance [30]. The 3 factors were the groups (G), observation periods (P), and individual animals (A). The factors G and P were considered to evoke fixed treatment effects, whereas factor A was considered to induce a random effect.
Results
Series I. Responses to sympathetic nerve stimulation Representative experiment Fig. 1 shows the changes in coronary blood flow, cardiac cycle length, and right ventricular contractile force elicited by stimulation of the right ansa subclavia in a
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representative experiment. The record was obtained during the first observation period, in the absence of an infusion of COC. Ansal stimulation (1 Hz, between the arrows) evoked a 34% increase in coronary sinus blood flow, a 132 ms reduction in cardiac cycle length, and a 79% increase in contractile force. After cessation of stimulation, these variables returned toward their control levels. The '50% recovery times' for contractile force and cycle length were 27 s and 16 s, respectively. These values are the times required for the responses to return halfway to their steady-state recovery levels, and they reflect the 'decay times' of the responses after cessation of stimulation.
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evoked by ansal stimulation varied directly with the frequency of stimulation (Fig. 2A). The main effect of stimulation frequency was significant (P = 0.01, Table I). The inotropic responses in the control group happened to be greater than those in the experimental group (Fig. 2B). However, such differences could not have been ascribable to the effects of COC. Note that in the experimental group, the inotropic responses elicited at any of the dosage levels of COC (P2 to P4) were not appreciably different from the responses obtained before COC had been given (P1). In both groups of animals, the decay times of the inotropic responses, as assessed by the 50% recovery time, increased with the frequency of ansal stimulation (Fig. 2C). In the control group, the decay times of the inotropic responses remained virtually constant from period to period (Fig. 2D). In the experimental group, the decay times of the inotropic responses at all of the dosage levels of COC (P2 to P4) were significantly greater (P = 0.01) than they were before COC had been given (P1). The decay times of the responses during P2, P3 and P4 in the experimental group were not significantly different from each other, however. The main effects of groups, observation periods, and stimulation frequencies were all significant (P _< 0.05, Table I), but the interactions were not significant.
(b) Chronotropic responses Ansal stimulation diminished the cardiac cycle length (Fig. 3A), and the magnitude of this chronotropic response varied with the stimulation frequency (P = 0.01, Table I). The responses to ansal stimulation in the control group did not change appreciably over the course of the 4 observation periods (Fig. 3B). In the experimental group, the magnitudes of the chronotropic responses to ansal stimulation were not significantly affected by the smaller doses of COC (P2 and P3). However. after the largest dose of COC (P4), the reduction in cardiac cycle length was significantly greater (P = 0.05) than it was during any of the preceding periods. In the control group, the decay times of the chronotropic responses were not appreciably different at the two stimulation frequencies (Fig. 3C), and they remained virtually constant from period to period (Fig. 3D). In the experimental group, the decay times of the responses to ansal stimulation were greater at 4 Hz than at 1 Hz (Fig. 3C), and they were much greater (P < 0.001) in the animals that received COC than in those that did not (Fig. 3D). After the smallest dose of COC (P2), the decay time of the chronotropic response was significantly longer (P = 0.05) than it was before COC had been given (P1; Fig. 3D). Furthermore, the decay times of the response became progressively greater (P < 0.025) as the cumulative dose of COC was increased (P2 to P4). The mare effects of groups, observation periods, stimulation frequencies, and all the interactions were highly significant (P _< 0.01, Table I).
(c) Coronary blood flow The increase in coronary blood flow evoked by ansal stimulation (Fig. 4A) were approximately equal in the control and experimental groups, and the responses were frequency dependent ( P = 0.01, Table I). In the experimental group (Fig. 4B), the increments in coronary blood flow evoked by ansal stimulation were greater ( P 0.05) during the infusion of COC (P2 to P4) than before COC had been adminis-
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Fig. 3. The magnitudes and decay times of the chronotropic responses to cardiac sympathetic stimulation in the experimental (open circles) and control (closed squares) groups. The chronotropic responses are expressed as the mean changes in cycle length from the prestimulation (basal) value. The mean basal values (+ S.D.) of the cardiac cycle lengths in the control and experimental groups were 501 + 79 and 511 + 61 ms, respectively. Fig. 4. The changes in coronary sinus blood flow and in norepinephrine overflow into the coronary sinus blood evoked by cardiac sympathetic stimulation in the experimental (open circles) and control (closed squares) groups. The mean basal values ( _ S.D.) of the coronary sinus blood flow and the norepinephrine overflow in the control groups were 30.2 + 11.9 ml/min and 3.76 + 2.60 ng/min, respectively, whereas those in the experimental group were 27.6 +_8.5 ml/min and 4.22 + 3.44 ng/min, respectively. tered (P1). The coronary blood flow did fluctuate from period to period in the control group, but the changes were neither systematic nor statistically significant.
(d) Norepinephrine overflow The rate of overflow of N E into the coronary sinus blood increased ( P = 0.01) with the frequency of ansal stimulation in both groups (Fig. 4C). In the control group, the N E overflow evoked by ansal stimulation tended to decrease slightly, but not significantly, throughout the 4 observation periods (Fig. 4D). In the experimental group, on the other hand, the N E overflow was significantly greater ( P = 0.05) at each of the dosage levels of C O C (P2 to P4) than it was before C O C had been infused (P1). However, after the largest dose of C O C (P4), the rate of N E overflow was less ( P = 0.05) than it had been during the preceding observation period (P3).
Series 11. Responses to norepinephrine infusion (a) Inotropic responses In the control group the magnitudes of the inotropic responses to the N E infusions did not change significantly from period to period (Fig. 5A). In the
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experimental group, the inotropic responses were not significantly affected by, the two lower doses of COC (P2 and P3). However, there was a pronounced enhancement of the inotropic response at the highest dosage level of COC (P4). The main effects of groups and periods and the interaction between them were all significant (Table II). The decay times of the inotropic responses did not vary appreciably from period to period in the control group (Fig. 5B). The two smaller doses of COC (P2 and P3) did not significantly prolong the decay times, but the largest dose of COC did evoke a significant prolongation (P < 0.05). The first-order interaction was also significant (P = 0.001, Table II).
(b) Chronotropic responses The reductions in cardiac cycle length evoked by the infusions of NE were fairly constant from period to period in the control group (Fig. 6A). Conversely, the reductions in cycle length in the experimental group were significantly greater (P < 0.05) than in the control group at all dosage levels of COC (P2 to P4). The mare effects of experimental groups and observation periods and the interaction between them were all significant (P < 0.05, Table II). The decay times of the chronotropic responses were fairly constant over the 4 periods in the control group (Fig. 6B). Conversely, in the experimental group the decay times of the chronotropic responses were significantly prolonged at atl dosage levels of COC. However, the decay times were especially prolonged after the largest dose of COC (P4). The main effects of groups and periods and the interaction between them were all significant (P < 0.01, Table II). (c) Norepinephrine extraction ratio In the control group, the mean NE extraction ratio over the 4 periods was
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0.55 ± 0.05 (mean ± S.E.), and it did not change appreciably from period to period (Fig. 7). In the experimental group, on the other hand, the NE extraction ratio decreased as the dose of COC was increased. At the lowest dosage level of COC (P2), this ratio decreased to 0.40 ± 0.11 from a control value (P1) of 0.69+ 0.08 (P = 0.05). At the intermediate (P3) and hi~est (P4) dosage levels, the extraction ratios were further reduced to 0.28 ± 0.07 and 0.24 ± 0,07, respectively. These ratios were also significantly less than the control ratio obtained during P1. The main
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13 effect of the observation periods and the interaction between groups and periods were significant (Table II).
Discussion
Effects of COC dosage on NE release and reuptake During sympathetic neural activity, the concentration of NE in the biophase (interstitial fluid in contact with the cardiac effector cells) is determined by the following factors: (a) the rate of release of N E from the sympathetic nerve endings; (b) the rate of neuronal uptake; (c) the rate of extraneuronal uptake; and (d) the rate of diffusion (and possibly other physical transport mechanisms) of neurotransmitter away from the synaptic clefts and into the coronary circulation. The extraneuronal uptake mechanism is relatively unimportant as a determinant of the cardiac responses to sympathetic activity [11,15,19,23,24]. Furthermore, COC has no appreciable effect on extraneuronal uptake; its effect on the uptake of NE is virtually restricted to the neuronal mechanism [11,12]. Hence, in our considerations of the effects of COC on the processes that add NE to and remove it from the biophase, the rate of extraneuronal uptake (factor (c) above) can be ignored. Under steady-state conditions, the rate of addition of N E to the biophase (factor (a)) equals the rate of its removal (factors (b) and (d)). Hence, under steady-state conditions, the rate of diffusion of N E out of the biophase and into the coronary circulation reflects essentially the difference between the rates of release and reuptake of neurotransmitter. In our experiments, even the smallest dose of COC (0.15 mg/kg) had a significant depressant effect on the neuronal uptake of NE. This is supported by the observations that this dosage level evoked a substantial reduction in the N E extraction ratio (Fig. 7; P2) and appreciable prolongations of the decay times of the chronotropic responses to sympathetic nerve stimulation (Fig. 3D; P2) and to N E infusion (Fig. 6; P2). In the experimental group, the N E overflow rates (Fig. 4D) during ansal stimulation were greater after COC (P2 to P4), regardless of the dose, than they were before COC had been infused (P1). For a substance, such as COC, that depresses both the release and the uptake of NE, the increase in the overflow of N E induced by COC signifies that this blocking agent must exert a greater depressant effect on the rate of neuronal uptake of neurotransmitter than on the rate of its neuronal release. However, after the largest dose of COC (P4), the N E overflow was significantly less than it was after the intermediate dose (P3). The data in Fig. 7 reveal that the inhibition of NE uptake was certainly just as intense, if not more intense, after the largest dose of COC than after the intermediate dose. Other factors being equal, the more complete the inhibition of neuronal uptake, the greater would be the NE overflow rate. Hence, the data in Fig. 7 indicate that raising the dose of COC from the intermediate (P3) to the highest (P4) level must have had a greater incremental effect on the neuronal release mechanism than on the neuronal uptake mechanism.
14 Analogous observations have been made in vitro. Low concentrations of COC (less than 5/~g/ml) were found to increase the amount of N E released by electrical field stimulation in the rabbit portal vein and vas deferens [9]. Conversely, higher concentrations of COC diminished the rate of NE release. Similar results were obtained in isolated rabbit hearts [33]. Relatively low concentrations of COC augmented the overflow of NE evoked by sympathetic stimulation, whereas higher concentrations of COC diminished the NE overflow.
Effects of COC dosage on the magnitudes of the inotropic and chronotropic responses In the present study, the inotropic responses to sympathetic stimulation were not augmented after the neuronal uptake mechanism was blocked by COC (Fig. 2B). The chronotropic responses were somewhat enhanced after the largest dose of COC, but not after the two smaller doses (Fig. 3B). If there were no change in the responsiveness of the cardiac effector cells, the magnitude of a given cardiac response should reflect the concentration of neurotransmitter in the biophase. Other factors remaining constant, neuronal uptake blockade would be expected to increase the concentration of N E in the biophase. Hence, it might be anticipated that such blockade would augment the cardiac responses, unless COC also: (a) reduced significantly the rate of NE release from the nerve endings; (b) facilitated the diffusion away from the synaptic clefts; or (c) reduced the responsiveness of the effector cells. An indication of effector cell responsiveness was provided by the second series of experiments, in which the neurotransmitter was infused intravenously. The inotropic and chronotropic responses to the N E infusions were greater over the range of COC doses that were used in this study (Figs. 5 and 6). Therefore, if COC did depress the responsiveness of the effector cells, its depressant effect must have been sufficiently weak that it did not mask the tendency for its neuronal uptake blocking action to augment the inotropic and chronotropic responses to exogenous NE. The studies of Kalsner and Nickerson [17] indicate that COC tends to augment, rather than to depress, the responsiveness of certain effector cells, at least in strips of rabbit aorta. The neuronal release of NE during ansal stimulation was probably not impaired substantially by the lowest and intermediate dosage levels of COC in the present study. In the experimental group, the NE overflow rates during P2 and P3 were greater than that during the control period (Fig. 4D). The increases in NE overflow during P2 and P3 were accompanied by increases in coronary blood flow (Fig. 4B). Such a change in coronary blood flow would tend to facilitate the diffusion of N E out of the biophase [28,36]. Hence, the concomitant increase in coronary blood flow might account, at least in part, for the absence of any detectable potentiation of the cardiac responses during P2 and P3 (Figs. 2B and 3B). Effects of COC dosage on the decay times of the inotropic and chronotropic responses After cessation of sympathetic stimulation, the concentration (C) of N E in the biophase tends to decrease with time. The rate of change of the N E concentration, d C / d t , in the biophase would be determined principally by the rates of neuronal uptake and diffusion of neurotransmitter. After cessation of ansal stimulation,
15 d C / d t is reflected by the 'decay time' of the cardiac response, at least when C is within the linear portion of the dose-response curve [34]. Suppression of the neuronal uptake mechanism retards the removaI of neurotransmitter from the biophase. Therefore, it tends to prolong the decay times of the cardiac responses. The decay time of the chronotropic response to sympathetic neural stimulation is a very sensitive index of the suppression of neuronal uptake in the heart [3,18,21,24,25]. In the present experiments (Fig. 3D), we found that after the smallest dose of COC (P2), the decay time of the chronotropic response to ansal stimulation was much more prolonged than it was in the absence of COC (P1). At the intermediate (P3) and largest doses (P4), the prolongations were significantly greater. These results indicate that the neuronal uptake mechanism was inhibited substantially even after the lowest dose of COC, and the extent of the inhibition increased with the dose. If the increase in coronary blood flow engendered by sympathetic stimulation in the animals that received COC (experimental group, periods P2 to P4, Fig. 4B) was as great in the S-A nodal region as in the cardiac tissues as a whole, the tendency for NE to diffuse out of the biophase in the S-A nodal region and into the coronary circulation would be enhanced. The great prolongation of the decay time of the chronotropic response elicited by COC (Fig. 3D) indicates that the inhibition of neuronal uptake must have been disproportionately greater than any concomitant facilitation of diffusion. The experiments of James and Nadeau [13] suggest that the vascular response to increased sympathetic activity in the S-A nodal region may be just the opposite of the response in the myocardial tissues, i.e. the vessels of the S-A node may actually constrict in response to increased sympathetic activity. This would constitute an impediment to diffusion, which would account at least partially for the marked prolongation of the decay time of the chronotropic response. The relative importance of the dissipation mechanisms in the ventricular muscle seems to differ from that in the sinus node. The decay times of the inotropic responses (Fig. 2D) to sympathetic stimulation were much less prolonged by COC than were the decay times of the chronotropic responses (Fig. 3D). It seems likely that the suppression of the neuronal uptake induced by a given dose of COC would be similar in the nerve endings in the various regions of the heart. Hence, the observed disparities in the decay times in different cardiac tissues after COC suggest differences in the balance among the various factors involved in the dissipation of neurotransmitter. Diffusion of N E (or other physical transfer processes) may play a more important role in the removal of neurotransmitter from the biophase in the ventricular myocardium than it does from the biophase in the S-A node. The results of recent unpublished studies from our laboratory suggest that the cardiac contraction itself, possibly by a 'massaging action', facilitates the transport of neurotransmitter from the biophase to the coronary blood stream. This process would be expected to be much more effective in the ventricular myocardium than in the S-A node. Conversely, the neuronal uptake of N E may be more effective in the S-A nodal region than in the ventricular myocardium. Neuronal uptake processes are known to be more effective in the termination of neurotransmitter action in tissues that have
lt,
relatively narrow neuroeffector gaps [16,17]. The width of the neuroeffector gap in papillary muscle is about 2000 A [27], whereas that in the S-A node is only 100 A [35]. Hence, neuronal uptake blockade would be expected to prolong the decay time of the chronotropic response more than it would that of the inotropic response. The relative importance of these various dissipative mechanisms in the different cardiac structures remains to be established.
Acknowledgements The authors are indebted to Mr. Herrick Finkelstein for his skillful technical assistance. This work was supported by the U,S. Public Health Ser,Ace Grant H L 15758.
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