Ischemia-induced noradrenaline release in the isolated rat heart: Influence of perfusion substrate and duration of ischemia

Ischemia-induced noradrenaline release in the isolated rat heart: Influence of perfusion substrate and duration of ischemia

J Mol Cell Cardiol 15, Ischemia-Induced Influence 821430 (1983: Noradrenaline Release of Perfusion Substrate and T. Abrahamssonl*, 0. Almgren...

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Ischemia-Induced Influence



Noradrenaline Release of Perfusion Substrate and T. Abrahamssonl*,

0. Almgrenl

in the Isolated Rat Heart: Duration of Ischemia and

L. Carlsson2

1 Department of Pharmacolokgy, AB Hiissle, M&da& and 2 Department of Pharmacology, University of Giiteborg, Sweden (Received 23 December 1981, accepted in revisedform

13 May 1983)


0. ALMGREN AND L. CARLSSON. Ischemia-Induced Noradrenaline Release in the Isolated Influence of Perfusion Substrate and Duration of Ischemia. Journal of Molecular and Cellular Cardiology (1983) 15,82 l-830. To examine some of the characteristics of the local noradrenaline (NA) release in myocardial ischemia a study was made on Langendorff-perfused rat hearts, prelabelled with 3H-NA. The left coronary artery was ligated for 15, 30, 60 or 120 min, followed by 10 min of reperfusion. The coronary effluent was collected and analyzed for radioactivity to indicate release of $H-NA. In some of the experiments the fraction of aH-NA was determined. As substrate in the perfusion medium either glucose (11.1 mM) or sodium lactate (5.0 mM) was used. During the ischemic period there was a slight decrease in the outflow of radioactivity. However, reperfusion was associated with a rapid and marked outflow of radioactivity, including an increased fraction of aH-NA, in the effluent. Compared to glucose as perfusion substrate, lactate caused a significantly higher (P < 0.01) outflow of tritiated substances after 60 and 120 min of regional ischemia. With lactate there was an almost linear relationship between reperfusion efflux of 3H and duration of ischemia. With glucose, the reperfusion outflow increased less rapidly after a duration of ischemia longer than 15 min. It is concluded that the degree of local NA release in myocardial ischemia depends on both the duration of the ischemic period and the substrate used. Glucose attenuates the reperfusion outflow of NA, especially after longer periods of ischemia. The effect may be due to decreased myocardial cell damage with this substrate and/or a direct protection of the adrenergic nerve endings. Rat






Introduction A number of experimental findings suggest an increased adrenergic activity in the acutely ischemic myocardium [I, 10, 19, 29, 32, 331. This increased activity and adrenoceptor stimulation may have a significant role in the process of ischemia: both by contributing to the occurrence of ventricular arrhythmias and by increasing metabolic derangement and myocardial cell damage [9, 18, 2.51. In myocardial ischemia there are several possible mechanisms which could cause increased release of noradrenaline (NA). A release of NA may be mediated by an increased nerve impulse flow to the heart, either as a part of a general reflex sympathetic activation [22, 351 or by a local cardiac reflex [5, 71. NA release may also be induced by a direct effect on the * To











adrenergic neurons. Accumulation of extra.cellular potassium, acidosis, hypoxia and energy deficiency within the neuron itself may all be factors which could participate in the mediation of such a local release of NA, independent of nerve impulses [4, 14, 28, 32., 371. The aim of the present study was tCJ examine some of the characteristics of this nerve impulse-independent, ischemiainduced, NA release. As an experimental. model of myocardial ischemia we used the Langendorff-perfused rat heart with acute left coronary artery ligation. In this model we studied the influence of the time period of ischemia and the perfusion substrate used (glucose or lactate) on the degree of NA release.

be addressed. 0








T. Abrahamsson Materials



Male rats (body weight 300-400g) of the Wistar strain, fed ad libitum, were used. Animal preparation The rats were anesthetized with pentobarbitone (60 mg/kgi.p.) and heparin (1000 IU/kg) was given i.v. An endotracheal tube was inserted and the animals were artificially ventilated with room air. Through the fourth intercostal space a left thoracotomy was performed. The pericardium was dissected free, and, in order to prepare the heart for coronary artery ligation in vitro, a piece of silk (5/O) was introduced under the left coronary artery and vein a few millimetres distal to the aortic root with an atraumatic needle. To detect the coronary artery in the left ventricle a light microscope was used. Perfusion procedure The heart was quickly excised, inserted in ice-cold buffer solution, and mounted on the perfusion apparatus for retrograde Langendorff perfusion. The perfusion apparatus contained two perfusate reservoirs and two bubble-traps connected just above the aortic perfusion cannula. This design permits an easy and rapid exchange between different perfusion media. Hearts were perfused via the aorta at a perfusion pressure of 80 cm H,O. During the experimental period, the effluent was collected for determination of coronary flow and for the biochemical measurements. For the perfusion a Krebs-Henseleit bicarbonate buffer (pH 7.4) was used, equilibrated with O,/CO, (95:5) at 37” containing: NaCl 116 mM; KC1 4.7 mM; CaCl, 2.6 mM; KH,PO, 1.2 mM; MgSO, 1.2 mM; NaHCO, 25 mM and EDTA 0.5 m&l. The perfusion substrate was either glucose (11 .l mM) or lactate (5.0 mM, dl-lactic acid sodium salt), however, in one group of glucose-perfused hearts the perfusion substrate was changed to lactate prior to reperfusion. Normetanephrine (10-s M) was added in order to reduce the extraneuronal uptake of NA [13]. In some of the experiments the heart rate was recorded by a cardiotachometer triggered by a bipolar lead ECG. One electrode was attached to the metal aortic perfusion cannula. while the other was a thin and flexible

et al.

copper wire inserted into the free wall of the left ventricle. The recordings were made on a Grass polygraph. After a 15-min equilibration perfusion, 20 @i of 3H-NA ( 1-7-3H-NA, specific activity 4.3-2.7 Ci/mmol; NEN) was administered to the perfusion medium. A cannula was inserted through the polyethylene tube close to the aorta and the sH-NA solution was injected over a period of 2 min. After a 25-min wash-out perfusion, the perfusion medium was changed to a solution containing desipramine ( 10m6 M). Desipramine was included in order to prevent the neuronal reuptake of noradrenaline released by the adrenergic nerve terminals [ZO]. Ten min later, the left coronary artery was ligated by tightening the piece of silk previously placed under it. The ligature included a polyvinyl tubing which allowed the coronary ligature to be cut atraumatically for the reperfusion [24]. The ligature was kept for periods of 15, 30, 60 or 120 min, followed by 10 min of reperfusion. In some of the experiments (n = 20), after the reperfusion period, potassium 90 mM (1 ml infused during 1 min) was injected to test the ability of the adrenergic neuron to respond to depolarization. The high potassium caused an at least tenfold increase in 3H-outRow (DPM/min) j indicating the presence of a neuronal vesicular NA compartment [36]. After the reperfusion the hearts were removed, dried with filter paper and weighed (1.08 & 0.02 g; 12 = 71). The time sequence for the perfusion and the administration of substances is shown in Fig. 1. Experiments were excluded if severe arrhythmias were observed during the pre-ischemic period, if ligation caused a reduction in coronary flow less than 200/, or if coronary flow during reperfusion was less than SOY/, of preligation value. Biochemical


The outflow of 3H-labelled compounds was measured by collection of coronary effluent during 1-min periods. 10 ml of a scintillation solution (Insta-Gel, Packard) was added to the effluent and the radioactivity was measured in a liquid scintillation counter (Searle). Quenching was monitored by an external standard. In some hearts, where the






3H-NA Id





20 Time

FIGURE effluent medium. reperfusion. substances taneous slope for the efflux from the











1. A 3H efflux curve from one of the lactate-perfused hearts. The amount of 3H collected in th,e is expressed on a log scale and plotted against time after the administration of 3H-NA to the perfusion In this experiment, left coronary artery ligation was carried out for 30 min followed by a IO-mm Desipramine (DMI) was added 10 min prior to ligation. For calculation of the amount of tritiated during reperfusion, we approximated the outflow of radioactivity which occurred above the sponefflux curve. In order to to this, rat hearts without coronary artery ligation were perfused to obtain the the spontaneous efflux of aH (see inset). A line with the calculated slope (see Table 1) was drawn from curve just prior to ligation. During 10 min of reperfusion the total amount of 3H-outflow was calculated area above this line (see Material and Methods).

coronary artery was ligated for 60 min, the fraction of 3H-NA in the effluent was determined [3]. The effluent was collected in tubes containing 0.5 ml 0.5 M perchloric acid. The separation was performed on a strong cation exchange resin, Dowex 50 W, x 4. The values were corrected for recovery, which was determined Ruorimetrically with unlabelled NA and found to be 94.394 & 2.7 (n = 11). In a separate set of experiments, in which the hearts were perfused in a similar way, creatine kinase (CK) activity was measured in the effluent. In these experiments the hearts were not prelabelled with SH-NA, and following release of the ligature CK was analyzed

only during the first 4 min reperfusion. ‘I’he enzyme activity was assayed at 25°C using standard UV test kits (Biochemical Test Gembination, Boehringer-Mannheim GmbH), as previously described [I]. The CK activity was expressed as milliunits (mu) released per minute per gram of tissue (wet weight), Calculation

qf radioactivity mashed out during reperfusion

For calculation of the amount of tritiated substances washed out during reperfusion we approximated the outflow of radioactivity which occurred above the spontaneous efflux

T. Abrahamsson


1. Slopes


for the spontaneous


interval n


of 3H from Glucose (fmol/min)h




-17.1 * (-0.94)




-13.1 (-

et hearts

al. perfused







Lactate (fmol/min)b



-15.5 & 3.2 (-0.89)

5 1.7 0.94)


- 11.4 & 2.3 (-0.89)




-8.8 * (-0.93)



-7.0 * (-0.88)





-5.5 & 0.7 (-0.91)


-3.8 * (-0.82)


The hearts were perfused with either glucose (11.1 mu) or sodium lactate (5.0 ITIM) as substrate. Values mean f S.E.M. n = number of experiments. r-ualue (calculated for each interval) shown within parenthesis. a See materials and methods for further information. b fmol 3H-NA and tritiated metabolites/min.

curve (Fig. 1) . In order to do this, rat hearts without coronary ligation were perfused to obtain the slopes for the efflux of 3H. From the efflux curve a slope was calculated for each time interval of coronary ligation (Table 1). This was performed in hearts both when glucose and lactate were used as substrate. Then, for each experiment with left coronary artery ligation, a line with the calculated slope was drawn from the efflux curve just prior to ligation. During 10 min of reperfusion the total amount of 3H was calculated from the area above this line, as indicated in Fig. 1. Statistical


The results are expressed as mean k S.E.M. The Student’s t test and the non-parametric Mann-Whitney two-tailed test were used for determination of differences between the various experimental groups. The slopes of the slow efflux curves for the various intervals were calculated using regression analysis (Table 1). Results

Coronary JIow (Table


Preligation Ilow was significantly (P < 0.001, Student’s t test) higher when lactate was used as the perfusion substrate. The preligation coronary flow (mean from all experiments) with lactate was 7.6 & 0.2 ml/min/g (n = 28) and with glucose 6.2 & 0.4 ml/min/g (n = 3 1).


Ligation of the left coronary artery reduced the coronary flow by 43.0 f 2.8:& (glucose; n = 31) and 41.4 f 2.3:/, (lactate; n = 28) within the first 5 min. Following reperfusion the preligation Ilow levels were more or less regained. A return of the coronary flow to a value above or similar to that before ligation was found after shorter periods of ischemia. However, reperfusion flow after a longer period of ischemia (60 to 120 min) did not quite reach the level prior to ligation. In lactate-perfused hearts a somewhat higher heart rate was observed (265 & 12 beats/min; n = 13) as compared to those perfused with glucose (244 i 7 beats/min; n = 18). However, this difference was not statistically significant. The heart rate was not significantly altered after coronary ligation (240 * 11 beats/min for glucose hearts and 250 & 11 beats/min for lactate hearts 5 min post ligation). Release of tritiated


Reperfusion of an ischemic myocardium was accompanied by an increased amount of 3H in the effluent (Fig. 1). The outflow was sudden and reached a maximum within 1 to 2 min. The amount of 3H appearing during reperfusion was dependent on both the perfusion substrate and the duration of the ischemic period (Fig. 2). With lactate as substrate a more pronounced efflux of radioactivity during reperfusion was observed,





2. Influence of perfusion substrate and left coronary artery ligation coronary flow during the perfusion experiments Coronary

Duration of ischemia perfusion substrate

5 min post ligation

1 min pre reperfusion


15 min Lactate Glucose

7, 6

7.4 * 0.5 6.0 & 0.6

4.3 * 0.3 3.8 & 0.7

4.1 * 3.8 *

min Lactate Glucose

8 6

7.8 j, 0.4 6.5 & 0.6

4.3 & 0.6 3.6 * 0.7



7.4 * 0.4 7.3 & 0.4

6 5

8.0 + 0.3 6.2 & 0.2

min Lactate Glucose



min Lactate Glucose

The hearts were perfused with mean

& S.E.M.

n =


by reperfusion


2 min post reperfusion

-10 min post reperfusion -

either of experiments.


0.3 0.7

8.2 & 0.7 7.5 & 0.5

7.8 & 0.6 6.7 & 0.7

4.1 & 0.6 3.6 & 0.5

8.2 & 0.5 7.7 * 0.4

7.9 & 0.5 7.9 & 0.4

4.1 + 0.4 4.0 & 0.4

3.5 + 0.3 3.4 & 0.3

5.9 * 6.7 k

0.5 0.4

6.1 + 0.3 6.7 i 0.5

4.8 & 0.5 3.5 & 0.3

3.7 f 0.3 2.4 & 0.3

5.5 & 0.3 4.9 & 0.5

5.5 5 0.4 5.2 k 0.4


especially after longer periods of coronary artery ligation. Lactate-perfused hearts, ligated for 60 and 120 min, showed a consistently higher output of 3H (P < 0.01 and P < 0.001, respectively) than corresponding glucose hearts (Fig. 2). In one group of glucose-perfused hearts, with coronary artery ligation for 60 min, the perfusion substrate was altered to lactate just prior to reperfusion. In this group the total 3H-outRow during 10 min reperfusion was 3.2 + 0.2 pmol/g (n = 5). In hearts perfused with glucose during both the ischemic period and reperfusion the 3H-output was 3.1 * 0.6 pmol/g (n = 6) during the lo-min reperfusion period. A few experiments were also carried out in lactate-perfused hearts with glucose during reperfusion confirming the observation that the magnitude of 3H-outRow during reperfusion depends on the perfusion substrate present during the ischemic period. In some of the experiments with coronary ligation for 60 min, isolation of sH-NA from its tritiated metabolites was carried out in the effluent (Table 3). Before ligation, 3H-NA accounted for 13.1”/, of total 3H efflux both bf.C.C.


flow (ml/minis)






or sodium




as substrate.



1 Time (min)


2. Effects of perfusion



duration of ischemia on the magnitude of reperfusion sH outflow. Results are expressed as total 3H, 3H-NA and tritiated metabolites (in pmol/g heart tissue wet weight) during 10 min of reperfusion following the various time periods of coronary artery ligation (15, 3#0, 60 or 120 min). Hearts were perfused with either glucose (11.1 mM) or sodium lactate (5.0 mn) as substrate. Shown are mean values * S.E.M. Number of hearts indicated in the figure. **P < 0.01. ***P < 0.001 between glucoseand lactate-perfused hearts (Student’s t test). l --- 0, lactate; @---a glucose. 2P


T. Abrahamsson


et al.

3. Fraction noradrenaline (3H-NA) of total 3H in the effluent from perfused hearts: effects of coronary artery ligation and reperfusion *,, 3H-NA of total 3H-outflow

Perfusion substrate Glucose Lactate



20 min post ligation

60 min post ligation

1 min post reperfusion

2 min post reperfusion

8 4

13.1 f 2.0 13.1 * 4.3

15.6 + 2.1 13.4 * 3.7

13.7 * 1.9 11.1 f 2.0

26.7 * 3.7a 23.6 & 4.2

21.8 j, 2.3a 20.0 + 3.55

a P < 0.05 v. 60 min post-ligation The artery

hearts were was ligated




value (Student’s glucose (11.1

t test for paired or sodium


values). lactate (5.0


as substrate.



for 60 min followed by 10 min of reperfusion. ‘l’alues are mea* + s.E.M. n = number of experiments.

when glucose and lactate were used as substrate. This fraction did not change significantly during the 60-min ischemic period. At reperfusion, the fraction of 3H-NA significantly increased to about 259;, in both cases. C’K activity in coronary ejluent Before ligation, CK activity in the effluent was 7 & 0.7 and 6 f 3 (mU/g/min) for the glucose-perfused and lactate-perfused hearts, respectively (n = 4). During coronary ligation for 1 h no detectable increase in CK activity of the effluent was observed. However, reperfusion of the ischemic myocardium was associated with an increased outflow of CK. An analysis was made during the first 4 min of reperfusion and the release of CK during this period was significantly higher (P < 0.05, Mann-Whitney two-tailed test) with lactate (452 & 107 mU/g/min) compared to glucose (148 * 45 mU/g/min). Discus&on

In the rat, ligation of the left coronary artery a few mm distal to the aortic root leads to ischemia and infarction in a large part of the left ventricular wall [15, 341. In the isolated perfused rat heart, left coronary artery occlusion causes an acute flow reduction in the entire left ventricular free wall, and this is most pronounced in the centre of the wall

r211. The degree of cell damage in an ischemic myocardium may be influenced by a number

of factors. Studies on isolated perfused hearts have shown that the nature of the perfusion substrate may influence both enzyme release [S, II] and release of NA [30] during ischemia and reperfusion. Furthermore, the amount of enzyme released during reperfusion depends on the duration of the preceding ischemic period [16]. In the present study we examined the influence of perfusion substrate, either glucose or lactate, and the duration of the ischemic period on the degree of NA release. The lactate concentration used in the experiments was similar to the reported arterial level of lactate obtained during moderate exercise [12]. In agreement with previous findings [6], the outflow of enzymes during reperfusion was more pronounced with lactate as substrate, indicating that lactate perfusion in the present study was associated with more ischemic and reperfusion cell damage. The present results demonstrate a sudden output of aH-NA and sH-metabolites in the effluent following reperfusion of an ischemic myocardium. This enhanced outllow of 3H-NA may be induced by the reperfusion per se or, more likely, NA which is lost from the adrenergic ,neurons during the ligation period may accumulate in the ischemic area and thus together with formed metabolites of NA be washed out during the reperfusion. The latter is partly supported by the rate of 3H efflux, which was highest during the first 1 to 2 min following reperfusion and then declined rapidly. However, to what extent



reperfusion itself causes the release of NA from the nerve endings is unknown and needs further examination. In hearts ligated for 60 min the fraction of 3H-NA was determined in the effluent. Before ligation the 3H-NA fraction was 13qb, which is in agreement with what has been reported for spontaneous outflow of the transmitter in other tissues [23]. During the first minute of reperfusion unmetabolized 3H-NA accounted for 25O$ of the total radioactivity. Considering that the ischemic region comprises only 25 to 35O;, of the total heart tissue [21, 341, the NA fraction of the tritiated substances washed out from the ischemic area may be even higher, since it is diluted by the outflow from non-ischemic tissue. At present, it is not known to what extent the increase in the reperfusion NA fraction reflects increased release or decreased transmitter metabolism in the ischemic area and this needs further evaluation. In the present study, the amount of 3H-NA and tritiated NA-metabolites in the outflow following reperfusion was dependent on both the duration of the ischemic period and the perfusion substrate used. Compared to glucose, lactate caused a more pronounced outflow of 3H during reperfusion. One important difference between the two substrates is the anaerobic ATP formation from glycolysis. With glucose as perfusion substrate, a higher rate of glycolytic ATP formation was observed in the ischemic rat myocardium as compared to pyruvate and acetate [6]. Bricknell and Opie [6] speculated that glycolytic ATP might have a special role in maintaining membrane integrity and function in the ischemic cell and thus limit enzyme release. However, glycolytic ATP does not seem to play any significant role in the maintenance of the overall production of metabolic energy. Even in rather severe ischemic regions, the predominant ATP formation, although markedly reduced, is still aerobic via oxidative phosphorylation [26, 271. Although lactate has been shown to be the preferred substrate for energy production in the healthy heart [12], lactate is probably a poor substrate for ATP formation in myocardial &hernia [27]. In addition, lactate has been suggested to inhibit glycolytic flux in ischemia 131). In non-ischemic tissue




lactate has been shown to be utilized as energy substrate for the NA accumulation in adrenergic nerve terminals [28]. In the ischemic myocardium several factors may be involved in a local release of NA. Hypoxia in the isolated heart has been shown to initiate a sudden release of NA [3.?]. Increased extracellular potassium, which occurs early in ischemia [ 171, may depolarize the adrenergic nerve terminals and cause release of NA [4]. In addition, acidosis may disrupt the NA granular storage vesicles [14]. Several processes in the adrenergic neuron, such as uptake and storage of the transmitter, require high-energy phosphates [28, 371. Energy-deficiency within the neurons may thus cause an increased leakage of NA from the nerve endings. Several possible mechanisms may be considered for the increased reperfusion efflux of 3H-NA and tritiated NA-metabolites observed in the present study after coronary ligation when lactate was the substrate. The effect of lactate may be primarily indirect via a high degree of ischemic myocardial cell injury associated with this substrate. The increase in cell damage could lead to a more pronounced leakage and extracellular accumulation of various cellular compounds, such as potassium, which may produce a local release of NA by acting on the nerve terminals [4]. In this situation at high initial NA release may potentiate the metabolic derangement and ischemic cell damage leading to further NA release, thus causing a vicious circle. A possible alternative or additional mechanism is that the substrate used in the experiments could have a direct effect on the energy metabolism and the transmitter homeostasis in the adrenergic neuron itself. Previous studies have shown that anoxia, in the presence of intact glycolysis, does not affect uptake and retention of NA, while anoxia combined with inhibition of glycolysis markedly reduces these processes [28, 371. These findings and the results obtained in the present study may thus indicate a role for glucose and glycolytic ATP in protecting the adrenergic nerve terminals and thus attenuating the release of NA in the ischemic myocardium. The primary function of this glycolytic ATP may be, at least in our experimental model where the neuronal cell membrane 2P2


T. Abrahamsson

amine uptake was blocked by desipramine, to serve the Mgs+-ATP-dependent granular (vesicular) uptake-storage mechanism. The importance of the granular uptake-storage mechanism for the retention of the transmitter within the nerve terminal is evident from, for instance, the effect of reserpine, which specifically inhibits this function [8]. In the rat heart reserpine has been shown to decrease NA levels by 70:4, within 1 h [Z]. In the present experimental model, the protective effect of glucose was more pronounced for ischemic periods longer than 30 min. More than one mechanism may be

et al. involved in the ischemia-induced release of NA. Possibly, the mechanism for release is different in the early stage (e.g. potassium depolarization) [32] compared to later in the ischemic process (e.g. impaired NA retention). Acknowledgements We are indebted to MS A. Fougberg and MS C. K&mark for the isolation of 3H-NA from its metabolites and Mr L. Svensson for CK determination. MS U. Kaufmann’s expert help in preparing figures and typing the manuscript is gratefully acknowledged.

References 1



12 13 14 15

16 17


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?3 24


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