release of endogenous catecholamine ins the ischemic myocardium...

689

Release of Endogenous Catecholamines in theIschemic Myocardium of the Rat

Part A: Locally Mediated Release

Albert Schomig, Anthony M. Dart, Rainer Dietz, Eckart Mayer, and Wolfgang KiiblerFrom the Department of Cardiology, University of Heidelberg, Heidelberg, Germany

SUMMARY. The accumulation of endogenous catecholamines within the extracellular space ofthe ischemic myocardium has been studied in the isolated perfused (Langendorff) heart of the ratsubjected to various periods of complete ischemia, with subsequent collection of the reperfusate.Catecholamines and deaminated metabolites were measured by radioenzymatic methods, or highpressure liquid chromatography. Ischemic periods of less than 10 minutes are not associated withan increased overflow of catecholamines or metabolites. Longer periods of ischemia are accom-panied by the overflow of noradrenaline and its deaminated metabolite 3,4-dihydroxyphenylgly-col. This overflow increases with lengthening of the preceding ischemic period (10 minutes: 2.5± 0.6, 20 minutes: 209.8 ± 17.2, 60 minutes: 1270.5 ± 148.1 pmol noradrenaline/g heart).Noradrenaline concentration is highest during the first minute of reperfusion, suggesting that thenoradrenaline detected during reperfusion is released into the extracellular space of the myocar-dium during ischemia and is subsequently eluted. Experiments with variation of extracellularcalcium concentration and with neuronal uptake (uptakei) blocking agents suggest that differentmechanisms of catecholamine release are acting during the course of ischemia. A calcium-independent carrier-mediated efflux of noradrenaline from the nerve terminals is of majorimportance, using the same carrier as is normally responsible for transporting noradrenaline fromthe synaptic clefts into the neuronal varicosities. Thus, various uptake]-blocking agents diminishthe noradrenaline overflow following ischemic periods of between 10 and 40 minutes. Thenoradrenaline overflow following longer periods of ischemia is unaffected by uptake]-blockingagents, and additional noradrenaline release at this time is probably consequent upon dissolutionof cell membranes. Overflow of adrenaline and dopamine occurs to a minor degree (less than 5%of the corresponding noradrenaline overflow), and only after ischemic periods of more than 15minutes. (Circ Res 55: 689-701 1984)

THE importance of the sympathetic nervous systemin modulating the course of myocardial ischemia hasbeen demonstrated both in clinical (Multicentre in-ternational study, 1975; Peter et al., 1978; Yusuf etal., 1980; Norwegian multicenter study group, 1981;Hjalmarson et al., 1981; Beta-blocker heart attacktrial research group, 1982) and experimental studies(Maling et al., 1959; Schaal et al., 1969). This mod-ulation has been shown both with regard to infarctsize (Reimer et al., 1976) and the occurrence ofarrhythmias (Ebert et al., 1970; Menken et al., 1979;Schwartz et al., 1980). However, a quantitative as-sessment of the extracellular accumulation of nor-adrenaline within the myocardium during ischemiaof varying durations is not available. Similarly, therelative contributions of locally and centrally me-diated release of noradrenaline to the total accu-mulation are not known. Therefore, in the presentstudies, these aspects of the interrelationship be-tween the sympathetic nervous system and myocar-dial ischemia were investigated, using the isolatedperfused heart of the rat.

Methods

Rats (Wistar strain) weighing 150-200 g were anesthe-tized with thiobutabarbital (50 mg/kg, ip). The peritonealcavity was opened and 0.1 ml heparin (500 U) was injectedinto the inferior vena cava. The thorax was opened andthe hearts were rapidly removed, weighed, and the aortascannulated. All hearts underwent an initial perfusion witha modified Krebs-Henseleit solution (KHS) at 5 ml/g heartper min for 30 minutes. The composition of KHS in mM;Na+, 144.14; K+, 4.02; Ca++, 1.85; Mg++, 1.05; Cl", 139.8;PO4

B, 0.44; HCO3-, 11.9; pyruvate, 1.82; glucose, 11.1;EDTA, 0.027. The perfusate was gassed with oxygen andthe pH adjusted to 7.4 with carbon dioxide. The temper-ature of the perfusate was adjusted to 37.5°C at the pointof entry into the aorta and the hearts suspended in hum-idified warmed (37.5°C) chambers. The efficacy of myo-cardial temperature regulation during ischemia was dem-onstrated by measurement of the intramyocardial temper-ature, with a fine temperature-sensitive probe. The heartswere subsequently subjected to various periods of globaland total ischemia and then reperfused at 5 ml/g heartper min. Samples for catecholamine estimation were takenfrom the coronary venous effluent before ischemia and

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during the reperfusion period. The concentrations of cat-echolamines in the perfusate were measured either bymeans of electrochemical detection following high pres-sure liquid chromatography (HPLC) separation in series1, or by a radioenzymatic method (Da Prada and Ziircher,1976) in all other series of experiments. The pharmacolog-ical agents used in this study [cocaine (Merck), corticoster-one (Fluka), desipramine (Ciba-Geigy), lidocaine (Astra),nisoxetine (Eli Lilly), (+)-oxaprotiline (Ciba-Geigy), yo-himbine (Serva)] were dissolved in ethanol, with finalconcentrations of ethanol being less than 0.05%, which isfar below the ethanol concentration influencing the spon-taneous or stimulation-induced release of noradrenaline(Gothert and Thielecke, 1976). The drugs (or the solventonly, in the control experiments) were added to the per-fusate 20 minutes prior to ischemia and continued to theend of the experiment.

Experimental Series

Series 1

Figure 1: noradrenaline (NA); Figure 2: adrenaline (A)and 3,4-dihydroxyphenylglycol (DOPEG).

Ischemia: 5, 15, 30 and 60 minutes (each group n = 10).Effluent collections: last minute before ischemia, and 1st,

2nd, 3rd, 4th, 5th, 10th, and 15th minute of reperfusion.Perfusate: Krebs-Henseleit solution (KHS) without drug.HPLC analysis.

Series 2

Figure 3: noradrenaline.Ischemia: 2, 10, 20, and 60 minutes (each time, n = 12).Effluent collection: cumulative 5 minutes before and 5

minutes after.Perfusate: KHS without (n = 24) and with cocaine 30

tiM (n = 24).Radioenzymatic analysis.

Series 3Table 1: noradrenaline, adrenaline, and dopamine (DA).Ischemia: 5, 10, 15, 20, 30, 40, and 60 minutes.Effluent collection: cumulative 5 minutes before and 5

minutes after.Perfusate: KHS without and with desipramine (DMI)

100 nM.Radioenzymatic analysis.

Series 4

Figure 4: noradrenaline.Ischemia: 20 minutes.Effluent collection: cumulative 5 minutes before and 5

minutes after.Perfusate: KHS with varying concentrations of desipra-

mine (0 M, 100 pM, 1 nM, 10 nM, 100 nM, and 1 fiht) (eachgroup «=7).

Radioenzymatic analysis.

Series 5Figure 5: noradrenaline.Ischemia: 20 minutes.Effluent collection: cumulative 5 minutes before and 5

minutes after.Perfusate: KHS without drug (n = 7), with nisoxetine,

100 nM (n = 7), with oxaprotttine, 300 nM (n = 7), with

Circulation Research/Vo/. 55, No. 5, November 1984

yohimbine 1 /*M (n = 7), with lidocaine, 10 /IM (n = 7).Radioenzymatic analysis.

Series 6


minutes after.Perfusate: KHS without (« = 7) and with corticosterone

30 AIM (n = 7), KHS + desipramine, 100 nM, without (« =7) and with corticosterone, 30 /IM (n = 7).


Series 7


minutes after.Perfusate: normal KHS (Ca++, 1.85 HIM) (n = 7), Ca++-

free KHS (5 minute preperfusion) (n = 7), Ca++-free KHS(20 minute preperfusion) (« = 7), Ca++-free KHS + EGTA1 irtM (20 minute preperfusion) (« = 7).


Series 8

Table 2: noradrenaline, adrenaline, and dopamine.Ischemia: 10, 20, 40, and 60 min (each time, n = 14).Effluent collection: cumulative 5 minutes before and 5

minutes after.Perfusate: Ca++-free KHS (20 minute preperfusion)

without (n = 28) and with desipramine, 100 nM (« = 28).Radioenzymatic analysis.

Series 9

Figure 8: heart rate, developed tension.Ischemia: 30 minutes.Measurement of heart rate and tension.Perfusate: KHS without drug (n = 6), KHS with desi-

pramine, 100 nM (n = 6), Ca++-free KHS {n = 6).Measurement of Tension. In series 9, the tension devel-

oped by the heart and the rate of contraction was recordedaccording to the method of Nayler (1979): A force-dis-placement transducer (Grass FTO3C) was attached to theventricular apex via a thread and an atraumatic needle.After amplification of the signal by a carrier amplifier(San-ei 6M41, carrier frequency 5000 Hz), it was displayedon a recorder (San-ei Recti-Horiz-8K).

At the start of the experiment, pretension was adjustedto 500 dyn in all hearts. Developed tension was calculatedas the difference between systolic peak tension (at phasiccontraction) or continuous tension (at contracture) and thepreset tension.

Biochemical Methods

Radioenzymatic AssayThe samples were put on ice, immediately stabilized by

addition of perchloric acid, and stored at —80°C. Radioen-zymatic determinations of concentrations of noradrena-line, adrenaline, and dopamine were performed accordingto the method of Da Prada and Zurcher (1976). Theintraassay coefficient of variation was less than 5%, theinterassay coefficient of variation, less than 10%. Cocaineat 30 IIM, corticosterone at 30 (OA, desipramine at concen-trations from 100 pM to 1 /IM, lidocaine at 10 IIM, nisoxetine


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at 100 riM, oxaprotiline at 300 nM, and yohimbine at 1 /*Mwere without effect on blanks or on the estimation ofstandard concentrations of catecholamines.

HPLC Separation and Electrochemical Detection

The samples were collected into tubes cooled to -60°Cby means of a metal block placed in dry ice. Samples werestored at —80°C until assayed. Before thawing an internalstandard, dihydroxybenzylamine (DBA), and 0.1 ml of anantioxidant mixture containing EDTA (100 HIM) and di-thiothreitol (2 HIM) were added to each 3-ml sample. ThepH was adjusted to 8.4 by addition of 100 /il of 1 M Trisbuffer (pH 8.5). Catecholamines and deaminated metab-olites were adsorbed on aluminum oxide column contain-ing 18 mg acid washed A12O3 (Eriksson and Persson,1982). After two washings with distilled water, the cate-cholamines were eluted with 300 n\ of 0.2 M phosphoricacid. One hundred microliters of the eluate were injectedinto the HPLC system. Separation was performed with aCis 5-^m reversed phase column (Latek), the solvent being0.2 M phosphate buffer, pH 2.7, containing EDTA (40 ^M)and octylsulfate (100 HM). Quantitative analysis was per-formed by electrochemical detection (BAS LC 4B). Theretention times of the catecholamines and their metabo-lites were: noradrenaline 5.0 minutes; adrenaline, 9.1 min-utes; dopamine, 24.2 minutes; DOPEG, 4.1 minutes; 3,4-dihydroxymandelic (DOMA), 3.0 minutes; DBA (internalstandard), 11.2 minutes. Calculations of the concentra-tions included correction for recovery by relation to theinternal standard (Davis, 1981). The limit of detection was0.4 pmol/ml for noradrenaline, adrenaline, and DOPEG,0.8 pmol/ml for DOMA and 2 pmol/ml for dopamine.The coefficients of variation were 8.4% for noradrenaline,8.5% for adrenaline, 11.8% for dopamine, 8.7% for DO-PEG, and 13.3% for DOMA.

Statistical MethodsStatistical evaluation was by analysis of variance or by

paired f-test. Results are expressed as means ± SEM.

Results

Dependence of Catecholamine Overflow on theDuration of the Preceding Ischemia (Table 1,Fig. 3)

The overflow of the catecholamines noradrena-line, adrenaline, and dopamine (measured radioen-zymatically) after various periods of total myocardialischemia (series 3) is given in the top section of Table1. The values represent the cumulative overflowfrom the heart per gram heart weight during a 5-minute period of reperfusion following stop flow.Ischemic periods of 10 minutes result in no increasein noradrenaline overflow in comparison to the con-trol (preischemic) values. Ischemic periods of 15minutes and longer result in a progressive increaseof noradrenaline overflow, more than 1 nmol nor-adrenaline per g heart after 60 minutes of totalischemia.

The overflow of adrenaline and dopamineamounts to less than 5% of the corresponding nor-adrenaline overflow. Adrenaline is detected in thecoronary venous effluent after ischemic periods of15 minutes and longer. Dopamine is not found afterischemic periods of less than 20 minutes.

Figure 3 shows, on a logarithmic scale, the cu-mulative overflow of noradrenaline (radioenzymat-ically measured) during a 5-minute period of reper-fusion after 2, 10, 20, and 60 minutes of myocardialischemia (series 2). The open bars represent thevalues obtained with Krebs Henseleit solution with-out addition of cocaine. The values of noradrenalineoverflow are in good agreement with those givenabove in Table 1. Again, a progressive increase ofnoradrenaline overflow is observed when ischemia

TABLE 1Ischemia-Induced Overflow of Catecholamines from Isolated Perfused Rat Hearts

PerfusateCatechol- Control

amine period

Ischemia

5 min 10 min 15 min 20 min 30 min 40 min 60 min

Krebs- NAHenseleit pmol/gsolution A ND

pmol/gDA NDpmol/g

solution+ DMI,100 nM

3.7 ± 0.6 2.9 ± 0.5 2.5 ± 0.6 72.1 ± 8.0 209.8 ± 17.2 481.2 ± 56.2 817.4 ± 110.5 1270.5 ± 148.1

ND ND 3.4 ±0.6 8.7 ± 1.3 16.0 ± 1.9 23.8 ± 5.0 51.9 ±9.9

ND ND ND 3.1 ±0.8 5.4 ± 1.1 8.3 ± 2.7 18.3 ± 2.3

n = 56 n = 7 n = 14 n = 7 n = 7 n = 7 n = 7 n = 7

Krebs- NA 4.0 ± 0.6 6.0 ± 0.8* 5.1 ± 0.9* 24.1 ± 4.1* 31.4 ± 4.6* 90.7 ± 12.3* 330.1 ± 38.6* 1122.0 ±92.3Henseleit pmol/g

Apmol/gDApmol/g

ND ND ND 0.5 ±0.2* 2.5 + 0.5* 4.1 ± 1.4* 8.1 ± 1.5* 48.8 + 3.6

ND ND ND ND 1.6 ± 1.1 1.2 + 0.6* 8.3 ± 1.3 15.5 ± 1.8

n = 56 n = 7 n = 14 n=7 n = 7 n = 7 n = 7 n = 7Overflow of noradrenaline (NA), adrenaline (A), and dopamine (DA) from isolated perfused rat hearts (n = 112) during a 5-minute

control period before ischemia and during the first 5 minutes of reperfusion after total ischemia of varying duration (5, 10, 15, 20, 30,40, 60 minutes) without (top section) and with (bottom section) blockade of neuronal uptake with 100 nM desipramine (DMI). ND = notdetectable by radioenzymatic assay; mean values ± SEM.

* P < 0.05 (perfusion with DMI vs. without DMI).


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is prolonged for more than 10 minute. The valuesare 3.5 ± 0.4 pmol/g for the control period, 3.7 ±0.5 for 2, 6.6 ± 1.3 for 10, 250.4 ± 63.0 for 20, and1319.8 ± 174.5 pmol/g for 60 minutes of totalischemia. In contrast to series 3, the noradrenalineoverflow after 10 minutes ischemia is slightly, butsignificantly (p < 0.05), higher than control values.

Time Course of Catecholamine Overflow duringReperfusion (Figs. 1 and 2)

The results of experiments in which the overflowof catecholamines and metabolites were determinedby HPLC separation and electrochemical detectionare shown in Figures 1 and 2. The time courses ofthe noradrenaline overflow after ischemic periodsof 5, 15, 30, and 60 minutes are illustrated (Fig. 1).No overflow of endogenous noradrenaline can bedetected during the preischemic perfusion period.There is also no noradrenaline overflow detectableby this method after an ischemic period of 5 min-utes. Following a stop flow ischemia of 15 minutes,23.3 ± 3.9 pmol/g noradrenaline are detected duringthe first minute of reperfusion. This value increasesto 278.6 ± 28.6 pmol/g after a 30-minute period ofischemia, and to 871.0 ± 152.3 pmol/g after a 60-minute period of ischemia. After each period of

overflow ofnoradrenaline

1000.

pmolmln x g

300.

100.

30,

10.

1 j

60min

30min

15min

ischemia

ISmin0 1 2 3 4 5 X)

reperfusion after ischemia

FIGURE 1. Time course of cardiac noradrenaline overflow during thefirst 15 minutes of the reperfusion period following various periodsof total ischemia. After 15, 30, and 60 minutes of ischemia, the mainportion of noradrenaline is washed out during the very early phaseof reperfusion. Mean values ± SEM; each group n = 30.

Circulation Research/VoZ. 55, No. 5, November 1984

ischemia30-

overflow ofadrenaline

10.

pmol

minxg

2J

200.

60min

0 1 2 3 4 5 10 ISmin

100.

overflow ofDOPEG

20.

10.

pmol

minxg

2J

60min

30min

15min

0 1 2 3 4 5 10reperfusion after ischemia

15min

FIGURE 2. Time course of cardiac adrenaline (upper panel) andDOPEC (lower panel) overflow during the first 15 minutes of thereperfusion period following various periods of total ischemia. By theHPLC method, adrenaline is detectable only after 60 minutes ofischemia. The characteristics of the washout is similar to that ofnoradrenaline. DOPEG, the main deaminated metabolite of noradren-aline and adrenaline, is found after 15, 30, and 60 minutes of ischemia.Mean values ± SEM; each group n = 10.

ischemia, there is a continuous decay in the amountof noradrenaline released with the bulk beingwashed out during the first 5 minutes of reperfusion.

No detectable amounts of adrenaline are foundwith the HPLC method after 5, 15, or 30 minutes ofischemia (Fig. 2). Adrenaline is detectable in signif-icant amounts only after 60 minutes of ischemia(26.6 ±4 .1 pmoi during the first minute of reper-fusion). Dopamine cannot be detected by the HPLCmethod during the reperfusion phase after ischemicperiods of 5, 15, 30, or 60 minutes.

DOPEG, the main deaminated metabolite of nor-adrenaline and adrenaline, is not detectable duringnormal perfusion nor during the washout periodafter 5 minutes of ischemia. Prolongation of theischemic period to 15, 30, and 60 minutes results ina progressive overflow of this metabolite from theischemic hearts. During the first minute of reperfu-sion, 7.9 ± 2.6 pmol/g are detected after a 15-minuteperiod of ischemia, 15.8 ± 4.1 pmol/g after a 30-minute period of ischemia, and 125.9 ± 23.7 pmol/g after a 60-minute period of ischemia. DOMA


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overflow ofnoradrenalinepmol/g

2000—

2 0 0 -

2 0 -

¥:*:W

controlperiod

10ischemia

20 60min

FIGURE 3. Cumulative overflow of endogenous noradrenaline fromisolated rat hearts during the first 5 minutes of reperfusion followingvarious periods of total ischemia. Open bars represent the releasewith normal perfusate; hatched bars represent the release with acocaine (30 iiM)-containing perfusate. Ischemic periods of longer than10 minutes lead to a progressive release of noradrenaline. This releaseis increased by blockade of neuronal reuptake with cocaine at ischemicperiods of 2 and 10 minutes, decreased at 20 minutes, and notinfluenced at 60 minutes of ischemia. Mean values ± SEM; each groupn = 6.

cannot be found during normal perfusion nor aftervarious intervals of stop flow ischemia.

Effects of Neuronal Uptake Blockade onCatecholamine Overflow (Table 1; Figs. 3 and 5)

The lower panel in Table 1 represents values fornoradrenaline, adrenaline, and dopamine releaseafter various periods of ischemia in the presence ofdesipramine (100 nM). Uptake blockade with desi-pramine has no significant effect on spontaneousnoradrenaline overflow during the control period,but leads to a 2-fold increase in noradrenaline ov-erflow after ischemic periods of 5 and 10 minutes.In contrast to the enhancement following short is-chemic periods, uptake] blockade causes a reductionof noradrenaline overflow after ischemic periods of15, 20, 30, and 40 minutes. At 20 and 30 minutes,values amount to less than 20% of those obtainedwithout desipramine. Adrenaline and dopamine ov-erflow are both reduced in a similar fashion atischemic periods of 20 and 30 minutes. Followingmyocardial ischemia of 60 minutes, however, cate-

cholamine release is not affected by the presence ofdesipramine.

When uptake] blockade is performed with cocaine(30 MM), similar effects are found (series 2). Additionof cocaine to the perfusate results in an enhancedoverflow of noradrenaline after ischemic periods ofup to 10 minutes. The cumulative overflow (over a5-minute period) of endogenous noradrenaline dur-ing control conditions increases from 3.5 ± 0.4 to5.6 ± 0.8 pmol/g heart (P < 0.05), from 3.7 ± 0.5to 6.5 ± 0.8 pmol/g (P < 0.05) after 2 minutes ofischemia and from 6.6 ± 1.3 to 17.4 ± 2.5 pmol/g(P < 0.01) after 10 minutes of ischemia. However,after 20 minutes of ischemia, noradrenaline over-flow is decreased from 250.4 ± 63.0 to 36.3 ± 3.8pmol/g (P < 0.01). After 60 minutes of ischemia,there is no difference (1319.9 ± 174.5 vs. 1388.3 ±91.4 pmol/g) in the cumulative noradrenaline ov-erflow between hearts with and without cocaine.

The effects of two other agents blocking neuronaluptake on the ischemia-induced noradrenalineoverflow are demonstrated in Figure 5. The concen-trations used (nisoxetine, 100 nM, and (+)-oxaproti-line, 300 nM) are equivalent to 100 nM desipraminerelative to their receptor-binding or uptakej-block-ing properties (Wong and Bymaster, 1976; Raismanet al., 1982; Waldmeier et al., 1982). Both agents areas effective in reducing the ischemia-induced nor-adrenaline release as desipramine and cocaine. Thenoradrenaline overflow from the heart after 20 min-utes of ischemia amounts to 36.8 ± 4.9 pmol/g (P< 0.01) with nisoxetine and to 35.9 ± 2.4 pmol/g(P < 0.01) with (+)-oxaprotiline, compared to 201.8± 40.4 pmol/g without uptake] blockade.

Concentration-Related Effect of Desipramine onIschemia-Induced Noradrenaline Overflow(Fig. 4)

Figure 4 shows the effects of various concentra-tions of desipramine on the noradrenaline releasefollowing a 20-minute period of total ischemia.Whereas 100 pM desipramine has no significanteffect on noradrenaline release, a dose-dependentreduction is observed when the concentration ofdesipramine in the perfusate is increased from 1 to100 nM. Further increase of desipramine concentra-tion of 1 /XM does not result in a further decrease ofnoradrenaline overflow. A 50% inhibition of theischemia-induced noradrenaline release is achievedat a concentration of 2.3 ± 1.1 nM desipramine.

Effects of Local Anesthetic and of a2-ReceptorBlockade on Ischemia-Induced NoradrenalineOverflow (Fig. 5)

The effects of lidocaine (10 /IM) and yohimbine (1HM) on noradrenaline overflow after 20 minutes ofmyocardial ischemia are shown in Figure 5. Neitheragent exerts any significant effect on noradrenalineoverflow. With lidocaine, the noradrenaline over-flow amounts to 205.2 ± 42.6 pmol/g, with yohim-


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overflow of 250,noradrenalinepmol/g

150.

100.

Oj10-10 10-9desipramine

10-8 10-7 10-6 M

FIGURE 4. Cumulative overflow of endogenous noradrenalinefrom isolated rat hearts during reperfusion following 20 min-utes of total ischemia. The open bar represents the overflowwith normal perfusate, the hatched bars the overflow withperfusate containing various concentrations of desipramine.Blockade of neuronal uptake by desipramine leads to a dose-dependent reduction of ischemia-induced noradrenaline re-lease with an lCso of 2.3 nM. Mean values ± SEM; each groupn=7.

bine to 215.5 ± 28.2 pmol/g, compared to 201.8 ±40.4 pmol/g without pharmacological intervention.

Effects of Blockade of Extraneuronal Uptake(Uptake^ on Noradrenaline Overflow (Fig. 6)

Figure 6 shows the effect of blockade of uptake2by corticosterone (30 fiu) on the noradrenaline ov-erflow from the heart during 5 minutes of reperfu-sion after 20 minutes of ischemia. The increase ofnoradrenaline overflow after uptake2 blockade isstatistically significant (P < 0.05) after additionalblockade of uptakej by desipramine (68.8 ± 4.9 vs.46.0 ± 4.3 pmol/g), but not significant withoutuptake, blockade (267.3 ± 28.9 vs. 234.0 ± 25.5pmol/g).

Effects of Calcium-Free Perfusion onCatecholamine Overflow (Fig. 7; Table 2)

Perfusion with calcium-free perfusate has no ef-fect on the cumulative overflow of noradrenalinefrom the rat heart after 20 minutes of ischemia. The

overflow is independent of the duration of calcium-free preperfusion (5 minutes: 242.8 ± 34.6 pmol/g;20 minutes: 252 ± 23.1 pmol/g). Addition of 1 HIMEGTA to the calcium-free perfusate does not influ-ence the overflow of noradrenaline (225.7 ± 17.9pmol/g) as compared to control perfusion (202;0 ±15.6 pmol/g) (Fig. 7). The dependence of catechol-amine overflow on the duration of the precedingischemia remains unaffected when calcium-free-perfused hearts (Table 2) are compared to heartsperfused with calcium containing medium (Table 1).Addition of 100 nM desipramine to the calcium-freeperfusate results in a reduced overflow of catechol-amines after periods of ischemia longer than 10minutes (Table 2).

Heart Rate and Developed Tension before andduring Ischemia (Fig. 8)

Heart rate and developed tension remain stablethroughout an initial perfusion period of 30 minuteswith KHS. After cessation of flow, both heart rate

overflow of 250.,noradrenalinepmol/g

150.

5 0 .

O j

control yohimbine lidocaine

nisoxetine oxaprotiline

•KP 3x10-7 10-6

FIGURE 5. The effect of various agents on the cumulativeoverflow of endogenous noradrenaline from isolated rat heartsduring 5 minutes of reperfusion following 20 minutes of totalischemia. The uptake ̂ blocking agents, nisoxetine (100 nM)and (+)-oxaprotiline (300 nM), decrease the ischemia-inducednoradrenaline release to less than 20% compared to the ov-erflow without blockade. The local anesthetic lidocaine (10IIM) and the ai-blocking agent yohimbine (1 IIM) do not haveany effect on the ischemia-induced noradrenaline overflow.Mean values ± SEM; each group n = 7.


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overflowof noradrenaline

3 0 0 ^

pmol/g

200.

100.

desipramine100 nM

0 J

mmma corticosteroneFIGURE 6. Effect of blockade of the extraneuronal uptake with corti-costerone (30 HM) on the noradrenaline overflow during 5 minutes ofreperfusion following 20 minutes of total ischemia. The increase ofnoradrenaline overflow after blockade of the extraneuronal uptake issignificant after additional blockade of the neuronal uptake withdesipramine (100 nM), but not without blockade of the neuronaluptake. Mean values ± SEM; each group n = 7.

and developed tension decline rapidly, and no spon-taneous beats are detectable 6-10 minutes after thestart of ischemia. During the following period ofischemia, tension rises gradually because of devel-oping contracture. Half-maximal contracture isachieved after 18.8 ± 0.9 min of ischemia.

When 100 nM desipramine is added to the perfus-ate after an initial perfusion with normal KHS, nei-ther the time course of heart rate nor developedtension is altered before or during ischemia. Halfmaximal contracture is achieved after 17.9 ± 0.8min.

When calcium-free perfusion is started after 10minutes of preperfusion with KHS, developed ten-sion ceased immediately. During ischemia, the de-velopment of contracture is retarded significantly (P< 0.05). Half-maximal contracture is delayed to 22.4± 0.6 min.

Discussion

Early Release of NoradrenalineThe experiments demonstrate that periods of total

ischemia of up to 10 minutes duration are not as-sociated with overflow of catecholamines during thereperfusion phase, and such periods are thereforeprobably not associated with the accumulation ofcatecholamines within the extracellular spaces of themyocardium. Ischemic episodes of 10 minutes du-ration are associated with a small and nonconstantnoradrenaline overflow, and this noradrenaline-washout becomes progressively greater with longerperiods of ischemia (Table 1; Figs. 1 and 3).

The absence of noradrenaline overflow after shortperiods of ischemia is in contrast to an earlier studyin which short periods of total ischemia (3-4 min-utes) in isolated rabbit hearts were found to causethe overflow of large quantities (2700 pmol/g heart)of noradrenaline-like fluorescence (Wollenbergerand Shabab, 1965). Similarly, in in vivo experiments

overflow ofnoradrenaline

300-,

pmol/g

200-

100-

0 -

control Ca free

FIGURE 7. Cumulative overflow of endogenous noradrenaline fromisolated rat hearts during 5 minutes of reperfusion following 20minutes of total ischemia. Perfusion with KHS containing 1.85 mMcalcium (control), calcium-free perfusion starting 5 minutes beforeischemia (group A), starting 20 minute before ischemia (group B), andcalcium-free perfusion + 1 mM EGTA starting 20 minutes beforeischemia (group O. No significant difference was found between thegroups. Mean values ± SEM; each group n = 7.


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TABLE 2

Ischemia-Induced Overflow of Catecholamines from Isolated Perfused Rat Hearts (Calcium-Free Perfusion)

Perfusate

Krebs-Henseleit solution,Ca++-free

Krebs-Henseleit solution Ca++-free + DMI

Catechol-amine

NApmol/gApmol/gDApmol/g

NApmol/gApmol/gDApmol/g

Controlperiod

2.2 ± 0.4

ND

ND

n = 28

4.2 ± 0.9*

ND

ND

n = 28

10 min

5.6 + 1.3

ND

ND

n = 7

7 A ± 2.6

ND

ND

n = 7

Ischemia

20 min

211.7 ± 18.0

9.4 ± 1.0

6.5 ± 1.4

n = 7

27.6 ± 3.3*

0.7 ± 0.2*

1.1 ±0.2*

n = 7

40 min

800.1 ± 20.4

34.5 ± 3.0

12.3 + 1.3

n = 7

148.0 ± 12.8*

5.5 ± 0.6*

5.3 ± 0.5*

n = 7

60 min

1490 ± 99

84.7 ± 11.9

14.0 ± 2.1

n =7

1001 ± 89*

43.5 ± 7.4*

16.2 ±4.0

n = 7Overflow of noradrenaline (NA), adrenaline (A), and dopamine (DA) from isolated perfused rat hearts (n = 56) during a 5-minute

control period before ischemia and during the first 5 minutes of reperfusion after total ischemia of varying duration (10, 20, 40, 60minutes) without (top section) and with (bottom section) blockade of neuronal uptake with 100 nM desipramine (DMI), the perfusatebeing calcium-free Krebs-Henseleit solution. ND = not detectable by radioenzymatic assay; mean values ± SEM.

* P < 0.05 (perfusion with DMI vs. without DMI).

in dogs, coronary artery occlusions of 2.5 minutesresulted in a net overflow of noradrenaline (73pmol/g heart) detected in the coronary venous ef-fluent (Shabab and Wollenberger, 1969). Hirche etal. (1980) found an inconsistent noradrenaline ov-erflow following 3 minutes of ischemia in the an-esthetized pig. On the other hand, in a study in theisolated perfused rat heart, Abrahamsson et al.(1981) were unable to find evidence of an earlymyocardial depletion of noradrenaline. However, inall these studies, noradrenaline was assayed fluo-romerrically, a method subsequently shown to beunreliable because of insufficient specificity and sen-sitivity in comparison to the radioenzymatic andHPLC methods.

perfusate 1 | perfusate 2 | global ischemia

3000 _,

tensiondyn

2000.

1000.

0 -250 _,

heartrate,,min"1 2 0 0 .

150-

100.

50 .0-

To overcome these methodological problems, twoindependent methods were used in the current ex-periments to estimate the overflow of noradrenaline,adrenaline, and dopamine from the ischemic ratmyocardium. The advantage of using two differentmethods was to combine the higher sensitivity ofthe radioenzymatic method with the higher specific-ity of the HPLC method. Neither of these methodsprovided evidence of a substantial overflow of nor-adrenaline within the first 10 minutes of total andglobal ischemia (Table 1; Figs. 1 and 3).

In other studies in which a radioenzymatic assayhad been used to determine the noradrenalineoverflow into the coronary venous effluent, 5 min-utes of coronary ligation in dogs did not result in a

FIGURE 8. Developed tension and heart rate inisolated perfused rat hearts before and during30 minutes of total ischemia. The effect of threedifferent perfusates was investigated after 10minutes of initial perfusion with Krebs-Hense-leit solution (KHS) (perfusate V: (V control per-fusion (perfusate 2 = KHS) (n = 6), (2) perfusate2 = KHS containing 100 n\t desipramine (n =6), (3) perfusate 2 = calcium-free KHS (n = 6).After treatment with desipramine, mechanicalaction of the hearts was not different fromcontrol hearts. Perfusion with calcium-free per-fusate resulted in an immediate loss of tension,and the development of ischemia-induced con-tracture was retarded significantly. Mean val-ues ± SEM

contracture

10 20 30 40 50 60min

control -•perfusate2 with desipramine 100nM •— perfusate 2 Ca* * free


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net overflow of noradrenaline (Me Grath et al.,1981). Similarly, 11 minutes of myocardial ischemiain dogs did not lead to higher noradrenaline concen-trations in the ischemic than in the nonischemicvenous effluent, even in combination with left stel-late ganglion stimulation (Forfar et al., 1983). Theseresults are in close agreement with our present re-sults concerning the early noradrenaline release.

In vivo models, however, although closer to theclinical situation, have certain disadvantages: thedegree of flow reduction in such models is notprecisely defined, and flow is not uniform due tothe presence of a collateral circulation. Furthermore,collection of venous overflow from cardiac veinsincludes, to a variable extent, overflow of bloodfrom both ischemic and nonischemic areas, thusmaking interpretation difficult. On the other hand,the model described in this paper cannot answerquestions regarding the release of noradrenaline inresponse to sympathetic nerve activity, a questionwhich is addressed by Dart et al. (1984).

A major advantage of the model chosen is the factthat it is well defined. The results obtained on heartrate and tension during ischemia correspond wellwith the extensively described time course of cardiacdynamics in a comparable model given by Hearse(1977). Thus, our data on transmitter release can berelated to metabolic events occurring in this modelwhich have been described elsewhere (Hearse et al.,1977; Garlick et al., 1979). Direct conclusions fromthe mechanical action of the heart muscle cell to theischemia tolerance of the sympathetic nerve cell arenot possible because of profound quantitative dif-ferences in the energy-consuming processes in thesedifferent types of cells. Therefore, interventions im-proving the tolerance to ischemia of the myocardialmuscle cell by sparing energy due to decreasedmechanical activity do not necessarily have a similarbeneficial effect on sympathetic nerve cells withinthe myocardium.

Release of Noradrenaline Induced by IschemicPeriods of Longer Than 10 Minutes

The depletion of catecholamines from ischemic aswell as nonischemic areas of the canine heart wasassessed in studies conducted by Matthes andGudbjarnason (1971). After 24-hour periods of cor-onary occlusion, a decrease in cardiac noradrenalinecontent to less than 10% of the initial value wasobserved in the infarcted area, and no noradrenalinecould be detected within the ischemic myocardiumafter 4 days. Holmgren et al. (1981) reported agradual decline in cardiac noradrenaline fluores-cence within hours of a coronary artery occlusion inrats. However, these studies do not give informationabout the time course of catecholamine loss duringischemia, the metabolic fate of the catecholamines,or the concentration of the transmitter in the extra-cellular compartment of the myocardium potentiallyavailable for receptor activation. Similarly, studiesin which isolated rat hearts are loaded with radio-

labeled noradrenaline and the overflow of radioac-tivity from the ischemic myocardium subsequentlymeasured (Rochette et al., 1980; Abrahamsson et al.,1983) cannot give any quantitative informationabout the release of endogenous catecholaminesduring the course of ischemia because of an un-known relation between radiolabeled and endoge-nous noradrenaline, an unknown rate of metabolismof the radiolabeled transmitter, and an unknowndistribution of the radiolabeled compound in intra-cellular compartments.

A quantitative assessment can be made by meas-uring the overflow of endogenous noradrenalineduring washout. Assuming the prior uniform distri-bution of noradrenaline within the extracellularspaces of the heart, and assuming an extracellularspace of 20%, then 20-30 minutes of total ischemiacould be expected to produce an extracellular nor-adrenaline concentration in excess of 1 HM under theexperimental conditions described. This concentra-tion is well within the range known to be capableof producing myocardial necrosis, even in the nor-mally perfused heart (Waldenstrom et al., 1978).

The overflow of catecholamines during the reper-fusion period in these experiments could be dueeither to washout from the extracellular space ofnoradrenaline previously released from the sympa-thetic nerves or to a reperfusion-activated release.The very high concentrations of noradrenaline im-mediately after the onset of reperfusion, togetherwith the subsequent fast decay (Fig. 1), stronglysuggest that the detected noradrenaline represents,in large part, noradrenaline previously accumulatedwithin the extracellular space which is then washedout during reperfusion.

Adrenaline and dopamine are found during re-perfusion after ischemic periods longer than 15 min-utes (A) and 20 minutes (DA), respectively (Table1). Their quantities amount to less than 5% of thenoradrenaline washed out during the same reper-fusion period. The ratio between noradrenaline andadrenaline released during ischemia is in the samerange as that found in the rat heart (Saavedra et al.,1981) or in the human heart (Petch and Nayler,1979). It is only possible to speculate about thesource of adrenaline. The presence of phenyletha-nolamine-N-methyl transferase, the enzyme rele-vant for adrenaline synthesis in the myocardium(Axelrod, 1962), makes it conceivable that, besidesadrenaline taken up from the systemic circulation,this transmitter can be synthetized in the heart, atleast in small amounts.

Possible Mechanisms of ExtracellularCatecholamine Accumulation in the IschemicMyocardium

An accumulation of noradrenaline within the ex-tracellular space of the myocardium represents thenet outcome of two opposing processes: (1) therelease of noradrenaline from sympathetic nerveterminals, and (2) its removal.


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Since, in the experimental model used, there is noongoing flow during the course of ischemia, removalof liberated noradrenaline must represent an intra-myocardial clearance process such as the neuronaluptake (uptakd) or extraneuronal uptake (uptake2)of noradrenaline (Iversen, 1971), and a decreasedactivity of clearance mechanisms should result in anenhanced extracellular accumulation of catechol-amines.

An increased release of noradrenaline from neu-rons could be the result of either sympathetic stim-ulation or of an increased extracellular potassiumconcentration of more than 15 HIM (Blaustein et al.,1972; Carpenter et al., 1976). The underlying mech-anism for both possibilities is a Ca++-dependentexocytosis, (Smith and Winkler, 1972; Blaustein,1979) which is modulated by presynaptic receptorssuch as a2-receptors (Starke and Montel, 1974; Lan-ger, 1977; Dart et al., 1984).

Another mechanism which might contribute tothe noradrenaline release induced by ischemia isincreased leakage from intracellular to extracellularcompartments due to passive diffusion. However,due to the poor lipid solubility of the predominantlyionized catecholamines (Mack and Bonisch, 1979),this mechanism can be considered to be importantonly when the structural integrity of cell membranesis lost because of ischemic damage.

A third mechanism might be a carrier-mediatedefflux of catecholamines from the cytoplasm intothe extracellular space. This mechanism was postu-lated by Paton (1973) who, under special experi-mental conditions (blockade of monoaminoxidase,catechol-O-methyltransferase, and vesicular uptake;subsequent loading with radiolabeled noradrena-line; inhibition of the Na+,K+-ATPase; or reductionof the extracellular sodium concentration) found anefflux of [3H]noradrenaline from the rabbit atriawhich could be blocked by cocaine or desipramine.This finding was confirmed by several groups inother experimental models for noradrenaline (Rossand Kelder, 1976; Graefe and Fuchs, 1979; Ross andKelder, 1979; Bonisch et al., 1983) and for dopamine(Raiteri et al., 1977; Raiteri et al., 1979; Liang andRutledge, 1982). Besides the fact that it can beinhibited by various uptake] blockers, this releasemechanism is characterized by its independencefrom extracellular calcium (Ross and Kelder, 1976,1979; Bonisch et al., 1983). No biological signifi-cance has so far been attributed to this mechanism,however.

The relative contribution of these different mech-anisms to the catecholamine accumulation withinthe extracellular space of the myocardium may varyduring the course of ischemia. From the results ofthese studies, three subsequent phases of noradren-aline release from the ischemic myocardium can becharacterized:

Phase 1 (Up to 10 Minutes of Total Ischemia)

During this phase, no increase of spontaneousnoradrenaline overflow is found compared to a non-

Circulation Research/Vol. 55, No. 5, November 1984

ischemic control period. The transmitter overflow isincreased after blockade of neuronal reuptake (Table1; Fig. 3).

Sympathetic stimulation during ischemia resultsin a slightly increased overflow of noradrenalineduring reperfusion. This stimulation-induced over-flow is enhanced by uptake blockade and/or block-ade of presynaptic a2-receptors, but does not reachthe levels obtained by stimulation during nonis-chemic conditions (Dart et al., 1984). The release ofcatecholamines shows the same characteristic duringphase 1 of ischemia as during a nonischemic period.A significant accumulation of catecholamines in theextracellular space of the ischemic myocardium isprevented by a functioning neuronal uptake. (Amore detailed discussion of phase 1 is found in Dartet al., 1984).

Phase 2 (15-40 Minutes of Total Ischemia)

During this second phase, a progressive increaseof the overflow of catecholamines (Table 1), espe-cially of noradrenaline (Fig. 3), is observed. Thisrelease is independent from extracellular calcium(Fig. 7; Table 2). It is neither modulated by localanesthetics, by «2-antagonists (Fig. 5), nor electricalstimulation (Dart et al., 1984).

On the other hand, agents blocking neuronalreuptake are capable of reducing the ischemia-in-duced catecholamine overflow by more than 80%(Table 1; Figs. 3 and 5). This effect is caused byuptake]-blocking agents, which are structurally un-related and which are not transported by the carrier,such as cocaine, desipramine, nisoxetine, and ox-aprotiline. In our preparation, the effect of desipra-mine on the catecholamine release is dose dependentwith an IC50 of 2.3 nM (Fig. 4). This is in the samedose range as both the inhibition constant of desi-pramine for noradrenaline uptake into synapto-somes of rat brain (Koe, 1976) and the bindingconstant of desipramine at membranes of rat heartand cortex cells (Raisman et al., 1982).

Unspecific effects such as inhibition of calmodulinrequire doses 10,000-fold higher than needed foruptakei blockade (Roufogalis, 1983). Modulation ofmetabolic events of ischemia by desipramine can beexcluded from the unchanged time course of me-chanical activity of the heart during ischemia (Fig.8).

Blockade of the extraneuronal uptake with corti-costerone (Iversen and Salt, 1970) increases the is-chemia-induced noradrenaline overflow from theheart by approximately 20 pmol/g (Fig. 6).

These facts exclude most of the above-mentionedmechanisms as the cause of the catecholamine re-lease during phase 2: The exocytotic release of cat-echolamines is absolutely dependent on the pres-ence of extracellular calcium (Rubin, 1970; Bakerand Knight, 1978). However, the present experi-ments demonstrate the complete independence ofischemia-induced catecholamine overflow from ex-tracellular calcium. Moreover, whereas exocytotic


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noradrenaline release is modulated by a2-antago-nists such as yohimbine (Starke et al., 1975; Langer,1977; Dart et al., 1984), this is not the case for theischemia-induced noradrenaline overflow . In con-trast to the increasing effect on noradrenaline over-flow of uptake] blockers when release is exocytotic,the blockade of uptake results in a reduction ofcatecholamine overflow during ischemia.

Overflow of noradrenaline from the heart inducedby passive diffusion of the transmitter out of theneuron would not have been diminished by uptakeiblockade. If a reduction of neuronal inactivation hadcaused the increase in noradrenaline overflow, thena further enhancement would have been expectedafter blockade of neuronal uptake, which is the mainelimination mechanism of extracellular noradrena-line (Iversen, 1971). However, the opposite effecthas been observed during phase 2.

An explanation for the enhanced noradrenalinerelease during phase 2 is a carrier-mediated effluxof catecholamines from the sympathetic varicosities.Since active cation-dependent membrane transportsystems potentially operate in both directions(Crane, 1977), it is conceivable that the neuronaluptake carrier is identical with the efflux carrier.Under nonischemic conditions, this carrier providesthe inward transport of released noradrenaline, en-ergetically dependent on an intact transmembraneNa+ gradient with high extracellular and low intra-cellular concentrations (White, 1976; Sammet andGraefe, 1979). Such carrier-mediated efflux mecha-nism has been described, not only for catechol-amines, but also for the aminoacids, glycine andalanine (Schultz and Curran, 1970; Crane, 1977),for sugars (Crane, 1977), and for 6-hydroxytrypt-amine (Nelson and Rudnick, 1979).

The characteristics of the local metabolic cate-cholamine release during periods of ischemia be-tween 10 and 40 minutes are in accordance with thecharacteristics described for a carrier-mediated ef-flux.

In our model, the outward transport of catechol-amines cannot be inhibited by local anesthetics (Pa-ton, 1973; Ross and Kelder, 1976, 1979) (Fig. 5). Itis independent from extracellular calcium (Wakadeand Kirpekar, 1974; Ross and Kelder, 1976, 1979,Bonisch et al., 1983) (Fig. 7; Table 2). It can beinhibited effectively by uptake] blockade (Paton,1973; Ross and Kelder, 1976, 1979; Graefe andFuchs, 1979; Bonisch et al., 1983). The part of releaseinhibited by uptake] blockade is 67% at 15, 85% at20, 81% at 30, and 60% at 40 minutes of ischemia.

The main prerequisite for a carrier-mediated ef-flux across the axoplasmatic membrane is an in-creased cytoplasmatic concentration of catechol-mines (Paton, 1976). Under normoxic conditions,this cytoplasmatic transmitter concentration is lowbecause of the activity of two mechanisms, both ofwhich are compromised during ischemia: (1) theoxidative deamination catalyzed by monoaminoxi-dase depends on oxygen (Tipton, 1975), and (2) thetransport of catecholamines into the storage vesicles

699

depends on an intact proton gradient across thevesicle membrane (Philips and Apps, 1980; Knothet al., 1981) which is disturbed by the lack of highenergy phosphates (Casey et al., 1977) and thecytoplasmatic acidosis during ischemia (Garlick etal., 1979).

The efflux may be facilitated by additional factors(Paton, 1976), such as decreased transmembranegradient of sodium, an increased extracellular potas-sium concentration, and a changed membrane po-tential which will develop during ischemia (Kleber,1983). However, the quantitative influences of thesefactors, the metabolic prerequisites, and the mech-anisms of catecholamine efflux from storage vesiclesare not elucidated by the present experiments, andremain an object for further studies.

Other mechanisms, such as exocytosis induced byincreased extracellular potassium concentration(Hirsche et al., 1980) and leakage (especially atlonger ischemic periods), may contribute, to a minorextent, to the observed catecholamine overflow dur-ing this phase.

Phase 3 (Periods of Total Ischemia Longer than 40Minutes)

This last phase is characterized by a further in-crease in the spontaneous overflow of catechol-amines which cannot be modulated by blockade ofthe uptake] carrier. Studies of permeability and mor-phology reveal a progressive injury of membranestructures after ischemic periods of 60 minutes orlonger (Ganote et al., 1976) which may cause theobserved leakage of catecholamines out of the sym-pathetic neurons.

Time Course of Catecholamine Release

The time course of the three phases of catechol-amine release depends on the conditions of myocar-dial ischemia. Although total ischemia in vitro isassumed to be a good model of severe ischemia invivo (Jennings et al., 1981), the time course of cate-cholamine release from isolated hearts cannot beextrapolated directly to the in vivo conditions. Insituations of variable and incomplete myocardialischemia (e.g., in vivo experimental myocardial is-chemia and human myocardial infarction), it is con-ceivable that a similar, but temporally dispersed,release would occur. The mechanisms postulated toaccount for this release would be expected to applyalso to incomplete ischemia, but with a more pro-tracted time course. Thus, in situations of heteroge-nous reduction in myocardial blood flow, differentareas of ischemic myocardium (with different resid-ual flows) could be simultaneously at differentphases of noradrenaline release. Such heterogeneity,however, would be difficult to investigate in "invivo" models of myocardial ischemia, since areas ofmyocardium at different phases of catecholaminerelease may be topographically close and drain intothe same veins. Furthermore, uptake] blockade willhave opposite effects on noradrenaline release indifferent parts of the ischemic zone.


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In conclusion, the data demonstrate a catechol-amine accumulation in the extracellular compart-ment during myocardial ischemia. The increase inlocal noradrenaline concentration within the stillviable myocardium (Jennings et al., 1981) may beresponsible for a further deterioration of myocardialfunction during the ischemic process, i.e., accelera-tion of cell damage and induction of arrhythmias.Different mechanisms of extracellular noradrenalineaccumulation have been demonstrated to operateduring the course of ischemia. Among them, thecarrier-mediated efflux of noradrenaline appears tobe of major significance. It is the first time that thismechanism of catecholamine release has been dem-onstrated to play a key role under pathophysiologi-cal conditions such as myocardial ischemia.

Technical assistance was provided by Michaela Brautigam, An-nette Bruckner, Karin Keil, and Peter Stephan.

This work was supported by the Deutsche Forschungsgemeinschaft(SFB 90-Cardiovascular System).

Dr. A.M. Dart was in receipt of a Royal Society European ScienceExchange Fellowship. Current address: Cardiovascular Research Unit,Hugh Robson Building, George Square, Edinburgh, United Kingdom.

Address for reprints: Dr. A. Schomig, Department of Cardiology,lm Neuenheimer Feld 326, 69 Heidelberg, Germany.

Received March 5, 1984; accepted for publication August 14, 1984.

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INDEX TERMS: Catecholamine release • Calcium-independentnoradrenaline release • Carrier-mediated efflux • Myocardialischemia * Isolated rat heart


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A Schömig, A M Dart, R Dietz, E Mayer and W KüblerLocally mediated release.

Release of endogenous catecholamines in the ischemic myocardium of the rat. Part A:

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