Action of angiotensin on uptake, release and metabolism of 14C-noradrenaline by isolated rabbit hearts

EUROPEAN JOURNAL OF PHARMACOLOGY 14 (19 71 ) 112-12 3. NORTH-HOLLANDPUBLISHING COMPANY

ACTION OF ANGIOTENSIN ON UPTAKE, RELEASE AND METABOLISM O F 14 C - N O R A D R E N A L I N E

BY ISOLATED RABBIT HEARTS

Klaus STARKE Institute o f Pharmacology, Klinikum Essen, Ruhr University, 43 Essen, Germany

Received 29 July 1970

Accepted 4 December 1970

K. STARKE, Action o f angiotensin on uptake, release and metabolism o f 14C-noradrenaline by isolated rabbit hearts, European J. Pharmacol. 14 (1971) 112-123. The influence of angiotensin on the uptake, release and metabolism of 14C-dl-noradrenaline,and on the release of endogenous noradrenaline by tyramine, was studied in the isolated perfused rabbit heart. After 12 min perfusion with 10 ng/ml 14C-noradrenaline, 43.4% of the labelled amine was recovered from the perfusate, 52.6% from the heart; the rest was metabolised. There was no measurable exchange with the endogenous transmitter stores. Up to 100 ng/ml angiotensin did not cause any change. 10 t~g/mlinhibited tissue accumulation; noradrenaline deamination was decreased, probably as a consequence of diminished intraneuronal uptake. After labelling of the noradrenaline stores, the efflux of 14C-noradrenaline, deaminated 14C-catechols and O-methylated 14C-metabolites declined in two phases. The second components followed single exponential curves and probably corresponded to release from intraneuronal sites. The linearity, after logarithmic transformation, of the second phases was not abolished by infusions of anglotensin in final concentrations up to 10/~g/ml, but was abolished by 1/zg/ml tyramine. Sympathetic nerve stimulation greatly increased 14C-noradrenaline efflux, whereas the outflow of metabolites was not significantly augmented. Two stimulation periods (S 1, $2) were appfied in each experiment. The output of 14C-noradrenaline caused by S2 was only 75% of that caused by $1 under control conditions, but 105%, if 1 ng/ml angiotensin was present during $2 (P < 0.02). Angiotensin favoured the O-methylation of noradrenaline during nerve stimulation, probably by increasing the extraneuronal noradrenaline concentration. Up to 1/~g/ml angiotensin did not influence the output of endogenous noradrenaline caused by tyramine. It is concluded that angiotensin increases the amount of transmitter released, per nerve impulse, from the sympathetic nerve terminals. The peptide seems to influence some step involved in the liberation of noradrenaline by nerve impulses, but not in spontaneous and tyramine-induced release. Rabbit heart, isolated Postganglionic sympathetic nerves

Angiotensin Tyramine

1. INTRODUCTION The overflow of noradrenaline from isolated perfused rabbit hearts during stimulation of the sympathetic cardiac nerves is increased by low concentrations of angiotensin ( 0 . 1 3 n g / m l ; Starke, Werner and SchiJmann, 1969). If the perfusion pressure is kept constant, the peptide diminishes perfusion volume. Reductions of coronary flow caused by other methods decrease, rather than increase, norad-

Noradrenaline release and uptake

renaline overflow (Starke, Werner, Hellerforth and Sch[imann, 1970). The usual connexion of flow reduction and diminution of transmitter output is overcome by some specific action of angiotensin on the sympathetic nerve terminals. Concentrations of angiotensin up to 130 ng/ml do not interfere with the removal of infused 1-noradrenaline from the perfusion medium by rabbit hearts, nor with the net-uptake from high noradrenaline concentrations (Sch/imann, Starke, Werner and

K.Starke, Angiotensin and sympathetic cardiacnerves Hellerforth, 1970). It seems unlikely that the small inhibition of uptake by excessive concentrations of angiotensin (13/.tg/ml) has anything to do with the potentiation of amine overflow by much smaller doses. Moreover, after inhibition of re-uptake by cocaine, desipramine, protriptyline or pronethalol, the potentiating effect of angiotensin on the output of noradrenaline is left unimpaired, while that of a second uptake inhibitor is abolished (Starke, 1970; Schtimann, Starke and Werner, 1970). We concluded from these results that angiotensin, instead of interfering with the re-uptake of liberated noradrenaline, increases the amount released, per nerve impulse, from the nerve terminals. The present experiments were performed to obtain further information about the mechanism of the potentiating effect of angiotensin.

2. METHODS 2.1. Perfusion of hearts Isolated rabbit hearts, in some of the experiments with their sympathetic nerve supply intact (Hukovi6 and Muscholl, 1962), were perfused by means of a roller pump. The rate of flow was 10 ml/min in all isotope experiments, and 25 ml/min in experiments on the release of endogenous noradrenaline by tyramine. The composition of the physiological salt solution (PSS) was as follows (g/l): NaC18.0; KC10.2; CaC12 0.2; MgC12 0.1; NaHCO3 1.0; NaH2PO4 0.05; glucose 1.0; ascorbic acid 0.01 ; disodium-EDTA 0.01. It was saturated with a mixture of 95% 02 and 5% CO2 and warmed to 34°C. All experiments started after one hour perfusion with PSS. 2.2. Uptake of 14C.noradrenaline ~4C-dl-Noradrenaline (specific activity 42.9-47.3 Ci/mole) was infused into the aortic cannula for 12 rain to give a final concentration of 10 ng/ml. The venous effluent was collected in periods of 3 min and acidified. The heart was then immediately removed, rinsed with PSS, blotted, weighed, and homogenized in 20 ml 0.4 N HC104 containing 0.1% EDTA and ascorbid acid. After centrifugation at 1500g for 5 min, the residue was once more extracted with 20 ml HC104 and centrifuged, and the combined supernatants made up to 50 ml. The amount of

113

noradrenaline infused was determined in each experiment by infusing, after removal of the heart, the amine solution into the Langendorff apparatus and collecting the "perfusate" under equal conditions. To examine the influence of angiotensin, the peptide was infused 10 rain before and during the infusion of 14 C-noradrenaline.

2.3. Efflux of t 4C.noradrenaline and metabolites Immediately after 15 min infusion of 14C-dl-noradrenaline in a final concentration of 10 ng/ml, the perfusate was collected in 3 min samples for 42 rain. Angiotensin and tyramine were infused for 9 rain, starting 21 rain after the end of the infusion of noradrenaline. In some experiments, the intact postganglionic sympathetic nerves were stimulated twice for periods of 2 rain, starting 18 and 36 min after the end of the infusion of noradrenaline. Each 2 min period consisted of eight 15 sec periods of alternative stimulation of the right and left cardiac nerves by rectangular impulses of 1 msec duration, 5 impulses/sec and 8 mA. 2.4. Release of endogenous noradrenaline by tyramine In these experiments, the coronary arteries were perfused at a rate of 25 ml[min. Tyramine was infused twice for 8 min, leaving an interval of 30 min. Perfusate samples were collected 2 - 4 , 4 - 6 and 6 - 8 min after the onset of each infusion. The noradrenaline contents of the three samples were averaged. Infusions of angiotensin started 10 min before the second tyramine infusion. 2.5. Fluorimetric determination of noradrenaline The catechols were adsorbed on A1203 (Aluminiumoxid basisch, Woelm, Eschwege) at pH 8.5 under stirring and eluted with 0.1 N HC1. In the eluates, noradrenaline was determined according to v.Euler and Floding (1955). Appropriate standards were adsorbed to estimate recovery. 2.6. Analysis of radioactivity Unlabelled carrier noradrenaline was added to perfusates collected for determination of the efflux of radioactivity. The catechols wer adsorbed on A1203. After elution with 0.1 N HCI, the deaminated

114

K.Starke, A ngiotensin and sympathetic cardiac nerves

14C-catechols were extracted by adding 0.5 ml 1.0 N HC1 to 1.0 ml of eluate and shaking for 1 min with 6.0 ml of ethyl acetate. A single extraction removes 40% of the deaminated catechols present in the sample (Crout, 1964). 0.05-+ 0.01% (n = 3) of the 14C-noradrenaline present in the sample were recovered from the organic layer. The counting solution contained 4 g 2,5-diphenyloxazole and 50 mg p-bis[2-(5-phenyloxazolyl)]benzene per liter. 10 ml of this solution, 3 (for ethyl acetate 1.5) ml of absolute ethanol and 0.2 ml of aqueous samples or 1.5 ml of ethyl acetate were added to each vial. Counting efficiency was determined by external standardization. The data were used to calculate: a)total radioactivity; b) 14C_noradrenaline; c) deaminated ~4Ccatechols (3,4-dihydroxy-mandelic acid; 3,4-dihydroxy-phenylglycol); and d) O-methylated 14C_ metabolites (normetanephrine; 3-methoxy-4-hydroxy-mandelic acid; 3-methoxy-4-hydroxy-phenylglycol) as: d = a - ( b +c). 2.7. Drugs 1-Noradrenaline base (Hoechst, Frankfurt/M.Hoechst); ~aC-dl-noradrenaline (NEN Chemicals, Dreieichenhain); ValS-angiotensin II-Aspl-/3-amide (Ciba, Basel); tyramine hydrochloride (Merck, Darmstadt). All substances are calculated as free bases. 2.8. Statistics Unless otherwise stated, means + S.E. are given throughout this paper. Means were compared by Student's t test. Regression equations of the efflux of radioactivity were calculated after logarithmic transformation of the original values. Linearity was examined by F-test (Sachs, 1968). n = number of experiments.

3. RESULTS

3.1. Effect of angiotensin on the uptake of 14C_nor. adrenaline Part of the ~4C-dl-noradrenaline infused into the aortic cannula to give a final concentration of 10 ng/ ml was retained during the passage through the coronary vessels (table 1). In control experiments, 43.4% of the labelled amine infused in 12 min was

recovered unchanged from the perfusate, 52.6% from the heart. Only a small portion was metabolised by deamination (3,4-dihydroxy-mandelic acid; 3,4-dihydroxy-phenylglycol) and/or O-methylation (nor° metanephrine; 3-methoxy-4-hydroxy-mandelic acid; 3-methoxy-4-hydroxy-phenylglycol). Some of the 14C.noradrenaline present in the heart at the end of the infusion was located in blood vessel lumina and other extracellular spaces; this part was, however, small in comparison with the amount actually bound by the tissue and was therefore neglected. 1 and 100 ng/ml angiotensin, infused 10 rain before and during the infusion of noradrenaline, did "not

NorOdrenaline removal 80--

'/0-o c ~c

6(-

c

5¢--

4C6("00 oc

5C-

xx

t~

T

:o 4

-

I

I

I

0-3

3-6

6-9

I 9-12 min

Fig. 1. Removal of 14C-noradrenaline from the perfusion medium by isolated rabbit hearts. During 12 rain infusion of

14C-dl-noradrenaline, final concentration 10ng/ml, the venous effluent was collected in 3 min samples. Angiotensin was infused 10 min before and during noradrenaline infusions. Noradrenaline was determined fluorimetrically. Means + S.E. o o controls (n = 5); o Dangiotensin (1 ng/ml, n =4); ~ v angiotensin (100 ng/ml, n =4); ~ ~x angiotensin (10 #g/ml, n = 4); * Significantly different from controls (p ~0.02). ** Significantly different from controls 6o<0.01).

115

K.Starke, Angiotensin and sympathetic cardiac nerves

Table 1 Influence of angiotensin on the percentage recovery of 14C-noradrenaline infused into isolated rabbit hearts from cardiac tissue and venous effluent. Angiotensin (ng/ml) Controls

I

100

10

Heart Noradrenaline

52.6 +-2.9

48.9 + 2.0

54.1 + 1.0

44.0 + 1.6 *

Deaminated catechols

0.3 + 0.1

0.4 --+-0.1

0.5 + 0.1

0.2 + 0.1

O-Methylated metabolites

0.4 + 0.5

0.1 + 0.8

1.1 + 0.5

0.6 + 0.3

53.4 + 2.9

49.4 +-2.2

56.0 +- 1.4

44.9 + 1.6 *

51.7 + 1.3 **

Total Perfusate Noradrenaline

43.4 + 1.6

46.0 + 2.5

39.3 + 2.2

Deaminated catechols

1.3 + 0.1

1.3 + 0.1

1.3 + 0.1

1.0 + 0.1

O-Methylated metabolites

2.3 + 0.3

2.6 -+0.4

2.8 + 0.9

1.7 + 0.2

47.0 -+ 1.6

49.9 + 2.8

43.4 + 2.7

54.5 + 1.2 **

100.4 + 1.9

99.3 + 0.6

99.4 + 1.8

99.4 + 0.6

4

4

4

Total Total recovery Number of experiments

5

The amounts of 14C-noradrenaline and 14C-metabolites recovered, after 12 rain perfusion with 10 ng/ml 14C-dl-noradrenaline, from heart and perfusion medium are expressed as % of the infused radioactivity. Angiotensin was present 10 min before and during the infusion of noradrenaline. Means + S.E. * Significantly different from controls (p < 0.05) ** Significantly different from controls (p < 0.01) change this pattern of distribution. With 10/ag/ml, however, the amount of total radioactivity and 14C-noradrenaline accumulated in the heart was significantly reduced, while that remaining in the perfusate was significantly augmented (table 1). In fig. 1, the removal of noradrenaline is plotted against time. For this figure, fluorimetric determinations of noradrenaline were evaluated instead of radioactivity measurements. However, evaluation of isotope countings gave essentially the same results, since the specific activity of infused ~4C-noradrenaline was not changed during passage through the heart (see below). In each of the four experimental groups, the rate of noradrenaline removal, expressed as ng/ heart/min (upper panel) increased from the first to

the second collection period, then remained approximately constant; on the other hand, the percentage of the infused amine removed (lower panel) was similar in all collection periods. This apparent discrepancy results from the fact that the dead space, consisting of the aortic cannula and the extracellular space, delayed the increase, after the onset of the infusion, of the concentration of noradrenaline in the vicinity of the uptake sites. During the first 3 min, therefore, a percentage similar to later collection periods was removed, but from a lower absolute amount of noradrenaline. The removal of noradrenaline was inhibited by angiotensin only at the very high concentration of 10/ag/ml. The metabolism of infused noradrenaline was not

K.Starke, Angiotensin and sympathetic cardiac nerves

116

greatly changed by angiotensin (table 1). However, from an analysis of single collection periods, a significant alteration occurred (fig. 2). In control experiments, as well as during perfusion with angiotensin, the percentage of total radioactivity present as unchanged noradrenaline declined with time, that o f deaminated ~4C-catechols rose, while that of methylated 14C_metabolite s was little affected. Angiotension significantly increased the percentage of noradrenaline and decreased that of deaminated catechols; lower concentrations were ineffective. The accumulation of labelled noradrenaline in the heart may be a consequence not only of net-uptake from the perfusion fluid, but also of an exchange with the endogenous transmitter stores. An exchange should decrease the specific activity of the radioactive amine remaining in the perfusate. Table 2 contains the ratios of venous to arterial specific activity for control experiments and different concentrations of angiotensin. In no case did this ratio, to any important degree, deviate from unity.

Influence of angiotensin and tyramine on the spontaneous outflow o f ~4C-noradrenaline and metabolites 3.2.

After loading hearts by 15 min perfusion with 10 ng/ml ~4C-dt-noradrenaline, the efflux of labelled noradrenaline and its metabolites declined gradually. For control experiments, this is illustrated in fig. 3. The efflux curves of 14C-noradrenaline and deaminated 14C-catechols can be resolved into two components, rate constants k~ and k2. The initial rapid efflux of 14C-noradrenaline with a half-life of 1.2 min probably corresponds to the washout of the

"1. of total radioactivity 100 -

.~ ~ o

95

? 90--

~ ~= ®,~ E~ ~ E

t 0

~,, C..C

t

B

5E

0 / 6

o [

I

I

0-3

3-6

6-9

I 9-12 rain

Fig. 2. Percentage composition of total radioactivity in the venous effluent from isolated rabbit heart during 12 min infusion of 14C.dl.noradrenaline, final concentration 10 ng/ml. The perfusate was collected in four 3 min samples. Angiotensin was infused 10 min before and during noradrenaline infusions, o o controls (n = 5); ~ a angiotensin (10 pg/ml, n = 4). Means + S.E. * Significantly different from controls (p ~0.05).

Table 2 Venous to arterial specific activity ratio during infusion of 14C-noradrenaline min after onset of noradrenaline infusion

Controls (n = 5)

Angiotensin (ng/ml) 1 100 (n = 4) (n = 4)

10 (n = 4)

0-3 3-6 6-9 9-12

1.01 + 0.01 0.98 + 0.01 0.97 + 0.01 1.01 +0.03

1.00 -+0.01 1.04 -+0.01 0.99 + 0.03 0.97 -+0.02

1.01 + 0.01 1.03 + 0.02 1.01 + 0.02 0.98 +0.02

1.00 + 0.01 1.02 -+0.02 0.97 + 0.01 0.96 +0.03

14C-dl-Noradrenalinewas infused for 12 min, final arterial concentration 10 ng/ml. The venous effluent was collected in four 3 min samples. Angiotensin was infused 10 rain before and during the infusion of noradrenaline. Means + S.E. n, number of experiments.

K.Starke, A ngiotensin and sympathetic cardiac nerves

117

n Ci I 3 rain I~ ~0 I

Total radioactivity

k2= 0,011 min-1

1

0

N°r::seon.°5sinen-I I

: ~

k2=0,022min-1

0,5

0.1 095

Deaminated catechol~

1 ~

kI = 0,50min-I k2=0.018mln"1

0,5 0,1 o.o5 5 3 2

methylated metabolites

Ok2=0 . 0 0 7

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min -t

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I

I

I

I

I

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1

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0 3 6 9 12 15 111 21 26 27 30 38 36 39 42

min

Fig. 3. Efflux of total radioactivity, 14C-noradrenaline and 14C-metabolites from isolated rabbit hearts. After 15 min perfusion with 10ng/ml ]4C-dl-noradrenaline, the venous effluent was collected in 3 min samples. Values are the means +S.E. of 6 experiments. The equations of the second efflux phases were calculated by regression analysis, those of the first phase were determined graphically, k 1, k2, rate constants of first and second efflux components. extracellular space and the dead space of the perfusion system; the slope of the second part of the curve follows a single exponential decline with a half-life of 32 rain, probably corresponding to the spontaneous release from intracellular sites. The regression coefficients of the slow components (k2) of the outflow of noradrenaline and deaminated catechols are not significantly different (table 3); k2 of the efflux of

O-methylated metabolites is, however, significantly smaller (p < 0.05), i.e. the percentage of total radioactivity present as metabolites increases with time. For total radioactivity as well as 14C-noradrenaline and 14C-metabolites, the efflux is, after logarithmic transformation of the original values, linear (see F-test, table 3). Whereas during infusion of 14C-noradrenaline

118

K.Starke, A ngiotensin and sympathetic cardiac nerves

Table 3 Influence of angiotensin and tyramine on rate constants and linearity of the efflux of 14C-noradrenaline and 14C-metabolites from isolated rabbit hearts Total radioactivity

Noradrenaline F

Deaminated catechols

F

O-Methylated metabolites F

F

k2

fl," f2

k2

fl;f2

k2

fl;f2

k2

fl;f2

0.01,1 +0.002

1.230 11 ;65

0.022 +0.005

0.482 9 ;55

0.018 +0.004

0.113 10 ;60

0.007 +0.002

0.385 11 ;65

6

Angiotensin (1 ng/ml)

0.007 + 0.003

2.045 10 ; 36

0.024 + 0.008

0.721 9 ; 22

0.009 + 0.004

1.002 10 ; 24

0.007 - 0.004

0.621 11 ; 26

3

Angiotensin (100 ng/ml)

0.010 + 0.003

1.550 11 ; 39

0.008 + 0.006

0.270 9 ; 33

0.015 + 0.007

0.098 10 ; 36

0.004 + 0.003

1.200 11 ; 39

4

Angiotensin (10 t~g/ml)

0.009 + 0.003

0.946 10 ; 24

0.015 -+0.005

0.594 9 ; 22

0.016 + 0.003

1.604 10 ; 24

0.008 + 0.003

0.407 11 ; 26

3

3.187 I1 ;26

3

Controls

Tyramine (1 ug/ml)

26.921 11 ; 26

121.79 9 ; 22

14.285 10 ; 24

The noradrenaline stores were labelled by 15 min perfusion with 10 ng/ml 14C-dl-noradrenaline. The venous effluent was subsequently collected in fourteen 3 min periods. Angiotensin and tyramine were infused from 21 to 30 min after the end of the infusion of noradrenaline, k2, rate constants + standard deviations of the second, slow efflux phases. F, ratio of variances to test the linearity of regression, f ; , fz, corresponding degrees of freedom, n, number of experiments.

most o f the radioactivity in the outflowing liquid consisted of noradrenaline (table 1, fig. 2), the composition was quite different after infusion. The percentage o f unchanged amine decreased rapidly, parallel with the wash-out o f the extracellular space. 0 - 3 min after the end o f the infusion, 48.6% o f the total radioactivity was noradrenaline. 3 9 - 4 2 rain after the infusion, only 5.6% was noradrenaline, while deaminated catechols accounted for 9.6% and methylated metabolites for 84.8%. Angiotensin was infused during the second, slow efflux phase, from 21 to 30 min after the end o f the noradrenaline infusion, to give final concentrations of 1, 100 and 10,000 ng/ml. In most experiments, outflow of radioactive compounds was not affected. As a statistical test o f significance, the second efflux components were again examined for linearity. As can be seen from the data of table 3, linearity o f the outflow of noradrenaline and its metabolites was not abolished by angiotensin. Moreover, the rate constants were not significantly changed. Angiotensin, thus, does not increase spontaneous outflow or metabolic degradation of 14C-noradrenaline. The action o f tyramine, drug with known nor-

adrenaline-releasing activity, is illustrated in fig. 4. Comparing the perfusate samples collected 1 8 - 2 1 min after the infusion o f noradrenaline, i.e. before, and 2 4 - 2 7 min, i.e. during tyramine infusion, the outflow of 14C-noradrenaline was increased 28 fold, that o f deaminated catechols 5 fold, that o f methylated metabolites 2.5 fold. The efflux is not linear (table 3), at a high level of significance. 3.3. Influence o f angiotensin on the outflow o f

14 C-noradrenaline and metabolites during sympathetic nerve stimulation During the second phase o f the efflux o f radioactivity, the postganglionic sympathetic nerves were stimulated twice ($1, $2). Control hearts were perfused with PSS throughout the experiment (fig. 5a); in experiments with angiotensin (fig. 5b), $2 was applied in the presence of 1 ng/ml o f the peptide. Sympathetic nerve stimulation always potentiated the efflux of 14C-noradrenaline. Though there was, in most experiments, a concomitant increase of the output of metabolites, this was not significant, if compa'red with the spontaneous outflow during the preceding collection period.

K.Starke, A ngiotensin and sympathetic cardiac nerves

3

OLI 0

I I l I l I l I l = l I I 3 6 9 12 15 18

I

21

24

I1

27 30 313 316 319 /,12 rain

I

J T y r a m i n e lpg/mt

Fig. 4. Influence of tyramine on the efflux of 14C-noradrenaline and 14C.metabolites from isolated rabbit hearts. After 15 min perfusion with 10ng/ml 14,C-dl-noradrenaline, the venous effluent was collected in 3 min samples. Means -+ S.E. of 3 experiments. • 14C_Noradrenaline; e deaminated 14C.catechols; o O-methylated 14C_metabolites.

119

"Stimulation-induced overflow" was calculated by subtracting the spontaneous outflow obtained in the preceding sample. Stimulation-induced 14C_noradren_ aline overflow was, in each control experiment, lower during $2 than during $1 (by -1061 +- 255 pCi); it was in contrast higher than during $1 in angiotensin experiments (by +242 + 270 pCi; p < 0.02). Expressed as relative values: noradrenaline overflow induced by $2 was in control experiments: 74.7 + 1.7% of that induced by $1, if however, angiotensin was present during $2, it was 104.9-+6.7% of S~ (p < 0.02). - The stimulation-induced overflow of deaminated 14C-catechols was similar during $1 and $2 and was not influenced by angiotensin. - The stimulation-induced overflow of O-methylated 14C.metabolites was, in each control experiment, less during $2 than during $1 (by -556-+ 108 pCi); it was in contrast higher in each experiment with angiotensin (by +404 + 138 pCi; p < 0.002). There are thus, two effects of angiotensin: a significant augmentation of the stimulation-induced overflow of noradrenaline and O-methylated metabolites. The correlation of the stimulation-induced overflow of noradrenaline and O-methylated metabolites

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5 .c E 4

4 ._c E

L3 c 3

3

2

1

0

I 0

o I

I

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i

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i

3

6

9

12

15

18

21

24

27

30

33

36

39

S~

S2

I

42 min

3

6

9

12

15

18

21

24 27

30

33

36

39

42 min

S~ Angiotensin

I nglml

Fig. 5. Interaction of sympathetic nerve stimulation and angiotensin on the outflow of 14C.noradrenalin e and 14C.metabolite s from isolated rabbit hearts. After 15 min perfusion with 10 ng/ml 14C-dl-noradrenaline, the venous effluent was collected in 3 min samples. Two 2 rain stimulation periods (St, $2) were applied in each experiment. In control experiments (a), the hearts were perfused with PSS only; in angiotensin experiments (b), peptide infusions started 9 min before $2. Means + S.E. of 4 experiments for each of the two groups. • 14C.Noradrenaline; • deaminated 14C.catechols; D methylated l 4C.metabolite s

120

K.Starke, A ngiotensin and sympathetic cardiac nerves nCi +2

O--methytated

noradrenaline during the first perfusion with 1/ag/ml tyramine was 38.0 + 2.3 ng/min. The noradrenaline output induced, in control experiments, by the second infusion of tyramine amounted to 91% (0.3/ag/ml) or 85% (1/~g/ml) o f the first infusion (table 4). It was not significantly augmented by any of the three concentrations of angiotensin.

mql(aboUtes

0 0

+1

0 0 0

0

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I0

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4

5

6

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2

3

4. DISCUSSION

nCi noradrenaUne

Fig. 6. Relation between the efflux from isolated rabbit hearts, induced by sympathetic nerve stimulation, of 14Cnoradrenaline and O-methylated ] 4C-metabolites. The resting outflow was subtracted. o Perfusion with PSS. • Perfusion with 1 ng/ml angiotensin. was examined for all of the 16 stimulation periods of these experiments (fig. 6). The correlation coefficient was r = 0.4465 ( 0 . 0 5 < p < 0 . 1 ) . The values for stimulation periods in the presence of angiotensin are evenly distributed among the other points. 3.4. Influence o f angiotensin on the ou tput o f endogenous noradrenaline caused by tyramine In every experiment tyramine was infused twice for 8 min. The noradrenaline contents of the three venous effluent samples collected during each infusion were similar (cf. Lindmar, Loeffelholz and Muscholl 1967). The three values were averaged. In 10 experiments, the output of noradrenaline during the first perfusion with 0.3Atg/ml tyramine was 14.6 +- 1.4 ng/minkin 21 experiments, the output of

Our present results are in good agreement "with those obtained previously with endogenous or unlabelled l-noradrenaline. Thus, removal of 1-noradrenaline from the perfusion medium by isolated rabbit hearts was impaired by angiotensin only at the high concentration of 13/~g/ml (SchiJmann et al., 1970), comparable to the minimal inhibitory concentration of 10/ag/ml in the present experiments. In contrast, Peach, Bumpus and Khairallah (1969) reported a 50% inhibition, under similar conditions, of the accumulation of 3H-dl-noradrenaline in rabbit hearts by angiotensin at concentrations as low as 0.05 ng/ml. We originally discussed three hypotheses to reconcile our results on the removal of 1-noradrenaline (SchiJmann et al., 1970) with those of Peach et al. on the accumulation o f 3H-dl-noradrenaline. None of them has, in the present investigation, proved valid. Experiments with racemic noradrenaline do not give results essentially different from experiments with the 1-isomer (cf. Drask6czy and Trendelenburg, 1968); angiotensin does not decrease the cardiac accumulation of noradrenaline by accelerating metabolic degradation; and there is no important exchange with the endoge-

Table 4 Influence of angiotensin on the tyramine-induced output of noradrenaline from isolated rabbit hearts. Tyramine (t~g/ml)

Controls

Angiotensin (ng/ml) 1

100

0.3

90.8 + 8.0 (3)

90.8 + 2.6 (3)

94.6 + 5.9 (4)

1.0

84.8 + 3.5 (7)

90.4 + 4.3 (5)

92.7 + 4.7 (6)

1

93.4 + 2.0 (3)

Tyramine was infused twice in each experiment. Angiotensin was added 10 min before and during the second tyramine infusion. Values represent the output of noradrenaline caused by the second tyramine infusion as % of that caused by the first infusion. Means + S.E. Figures in parenthesis: number of experiments.

K.Starke, Angiotensin and sympathetic cardiac nerves

nous transmitter stores which angiotensin might prevent without impairing net-uptake. At present, we cannot explain the difference. An exchange of exogenous labelled noradrenahne with endogenous stores should lead to a decrease of the specific activity during the passage through the heart; this was not observed. At a concentration of 10 ng/ml, 14C-noradrenahne is accumulated in the heart by net-uptake. This is in agreement with the experiments on rat hearts by Iversen (1963) and Lindmar and Muscholl (1964). Our results confirm, moreover, that the rate of degradation is small in comparison with the rate of uptake (Lindmar and MuschoU, 1964), and that the arterio-venous difference is, therefore, very close to the amount taken up. Inhibition of the uptake of noradrenaline has been obtained, in most organs tested, only by very high concentrations of angiotensin (Thoenen, Huerlimann and Haefely, 1965; Palaic and Khairallah, 1967a,b, Panisset and Bourdois, 1968; Palaic and Panisset, 1969). In particular, angiotensin in concentrations up to 1 pg/ml failed to diminish the uptake of labelled noradrenaline by isolated rat hearts (Hughes and Roth, 1969). In vivo, the accumulation of labelled noradrenaline in rat hearts was not reduced by angiotensin (Pals and Masucci, 1968) or even augmented (Fischer, 1969). The spontaneous efflux of 14C-noradrenahne was not significantly influenced by angiotensin in doses up to 10/.tg/ml. This is in accordance with our previous results (Starke et al., 1969) using fluorimetric measurement of the output of endogenous noradrenaline. Tyramine, on the other hand, caused the expected acceleration of amine effiux. Similarly, angiotensin failed to increase the spontaneous output of noradrenaline from the cat spleen (Hertting and Suko, 1966), the dog kidney (Zimmerman and Gisslen, 1968) and the rabbit portal vein (Hughes and Roth, 1969). In contrast, 10 ng/ml-10/~g/ml angiotensin augmented the outflow of noradrenaline from isolated aortae of rats (Liebau, Distler and Wolff, 1969) and rabbits (Kiran and Khairallah, 1969). Whereas the resting output of noradrenaline from the dog kidney, the rabbit portal vein and the rabbit heart is not changed by angiotensin, the output during stimulation of the sympathetic nerves is potentiated (Zimmerman and Gisslen, 1968; Hughes and Roth, 1969; Starke et al., 1969; and the present

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investigation). The isolated rabbit heart seems to be particularly sensitive, 0.13 ng/ml angiotensin, being significantly effective. The degree of potentiation was smaller in the present experiments than in those previously published (Starke et al., 1969). The difference may be explained by two experimental modifications: a coronary flow of 10 instead of 25 ml/min; and counting of exogenous 14C-dl-noradrenaline instead of fluorimetric determination of endogenous noradrenaline. The augmentation of stimulation-induced noradrenaline overflow is probably not brought about by an inhibition of re-uptake (SchUmann et al., 1970; Starke, 1970, Schiimann, Starke and Werner, 1970; and the present investigation). Much higher doses of angiotensin are necessary to produce a small inhibition of uptake than to increase overflow. The present results demonstrate moreover, that angiotensin does not augment the overflow by diminishing metabolic degradation. On the contrary, an increase of the outflow of O-methylated 14C-metabolites coincided with the increase of noradrenaline output. In general, there is some connexion between noradrenaline liberation and the formation of normetanephrine, one of the compounds of the group of "O-methylated metabolites" (Hertting and Axelrod, 1961 ; Kopin and Gordon, 1963). There was indeed a weak correlation of the stimulation-induced overflow of noradrenaline and methylated metabolites. Lacking further evidence of a direct activation of O-methyl-transferase by angiotensin, we think it more probable that the increase of the output of methylated compounds is an indirect consequence of the elevated extraneuronal concentration of noradrenaline, i.e., of elevated substrate concentration. Similarly, the observed decrease of the deamination of infused ~4C-noradrenaline in the presence of 10/ag/ml angiotensin is probably a result of the diminished intraneuronal uptake of noradrenaline, i.e. of a diminished concentration of the substrate of intraneuronal monoamine oxydase. Not only the spontaneous, but also the tyramineinduced output of noradrenaline was angiotensinresistant, though several concentrations of both drugs were tried. Potentiation of the effects of tyramine by angiotensin has been repeatedly described and explained by an increase of the release of noradrenaline (e.g. McCubbin and Page, 1963). Our results confirm, however, the conclusions of Illanes, Perez-Olea,

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K.Starke, A ngiotensin and sympathetic cardiac nerves

Quevedo, Oriz and Lazo (1967) from experiments with isolated rabbit atria, that angiotensin does not favour the release o f noradrenaline by tyramine in this organ. The present, and previous studies, indicate that angiotensin does not enhance the overflow of noradrenaline during sympathetic nerve stimulation by inhibiting its inactivation: neither re-uptake nor metabolic degradation are impaired by doses that maximally potentiate overflow. Per exclusionem, we assume that angiotensin aug~nents the amount o f transmitter liberated, per nerve impulse, from the sympathetic nerve terminals. The mechanism of this neural action of angiotensin, as well as the mechanism of the release of the sympathetic transmitter, is not clear. Any hypothesis has to take into account that angiotensin potentiates only the release of transmitter caused by nerve impulses; the peptide seems to attack some step not involved in spontaneous or tyramineinduced release.

ACKNOWLEDGEMENT I wish to thank Miss B. Rawe for skilful technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft.

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