Selective release of newly synthesized cardiac norepinephrine induced by angiotensin II

EUROPEAN JOURNAL OF PHARMACOLOGY 15 (1971) 8-14. NORTH-HOt LAND PUBLISHING COMPANY

S E L E C T I V E R E L E A S E O F NEWLY S Y N T H E S I Z E D C A R D I A C N O R E P I N E P H R I N E I N D U C E D BY A N G I O T E N S I N II Claude CHEVILLARD, Nicole DUCH~NE and Jean-Michel ALEXANDRE Centre de Recherches sur l'Hypertension Art~rielle and Ddpartement de Pharmacologie de la Facultd de M~decine Broussais-H6tel Dieu, H6pital Broussais, 96, rue Didot, Paris XIVe. France

Accepted 1 February 1971

Received 7 October 1970

C. CHEVILLARD, N. DUCHENE and J.-M. ALEXANDRE, Selective release of newly synthesized cardiac norepinephrine induced by angiotensin 11, European J. Pharmacol. 15 (1971) 8-14. The releasing action of angiotensin II (Angll) and of amphetamine on endogenous norepinephrine (NE), 3 H-NElabeled pools, and 3H-NE newly synthesized from 3H-dopamine (3H-DA) was investigated in rat cardiac slices.

In contrast to amphetamine, AnglI did not affect endogenousNE or 3H-NE stored for a long period. Both drugs induced release of aH-NE newly synthesizedfrom a H-DA. Consequent to this utilization, Angll enhanced aH-NE synthesis from aH-DA,while amphetaminedecreased it. Increase in synthesis was related to increase in tissue aH-DA accumulation, and, decrease by amphetamine, to its reduction. Norepinephrine

Angiotenfin II

1. INTRODUCTION In the last few years, interactions between angiotensin II (AnglI) and the sympathetic system have been reported: stimulation of vasomotor centres (Bickerton and Buckley, 1961; Schmitt and Schmitt, 1968a; Scroop and Lowe, 1968) and of ganglionic transmission (Lewis and Reit, 1965; Panisset, Biron and Beaulnes, 1966), potentiation of endogenous norepinephrine (NE) released by indirectly acting sympathomimetic amines (McCubbin and Page, 1963; Schmitt and Schmitt, 1967; Day and Owen, 1969) or by nervous sympathetic stimulation (Zimmermann and Gomez, 1965; Benelli, Della BeUa and Gandini, 1967; Panisset and Bourdois, 1968; Starke, Werner and Schiimann, 1969), and increase in synthesis of cardiac NE from tyrosine (Boadle, Hugues and Roth, 1969). Angll has also been claimed to inhibit axonal uptake of NE (Panisset and Bourdois, 1968; Peach,

Amnhetamine

Heart

Bumpus and Khairallah, 1969), but more recent workers could not confirm these results (Pals, Fulton and Masucci, 1968; CheviUard and Alexandre, 1970; Schiimann et al., 1970). Moreover, AnglI releases vascular (Disfler, Liebau and Wolff, 1965; Kiran and Khairallah, 1969) and adrenal catecholamines (CA) (Cession and CessionFossion, 1963; Feldberg and Lewis, 1964), but not cardiac NE (Westfall and Peach, 1965; Peach and Ford, 1968; Starke, Werner and Schiimann, 1969), in experiments performed on isolated hearts or in situ. However, increased release of NE by AnglI from a small pool could be a possible explanation for the stimulation of cardiac NE synthesis. It was, therefore, of interest to reinvestigate the effects of AnglI on the different cardiac NE pools. For this purpose, actions ofAnglI on endogenous NE, aH-NE stored for a long period and aH-NE newly synthesized from tritiated dopamine (3H-DA), have been investigated and compared with those of amphetamine.

C.Chevillardet al., Angiotensin H action on cardiacArE 2. METHODS 2.1. Action of AnglI and amphetamine on endogenous NE and CA pool labelled with aH.NE Mate Wistar rats, weighing 200-250 g were used. 25 #Ci of aH-dl-norepinephrine (27 Ci/mmole, C.E.N. Belgium) were administered intravenously. The rats were killed 60 min later. Hearts were quickly excised, dissected on ice. Left ventricles were weighed and cut into slices (300 mg wet weight) which were incubated at 37°C, in 10 ml of Krebs solution containing EDTA (0.5 mg/ml), ascorbic acid (0.2 mg/ml) and pargyline (1.25 × 10-~ M) for 45 or 90 min. The solution was bubbled with a mixture of 95% O2 and 5% CO2. Incubations were performed, as above (controls) or with AnglI (5× lO-l°M and 5× 10-4M) or dl-amphetamine (1 × 10-~ M). After the incubation periods, the slices were washed four times with cold saline and homogenized (Ultra-Turrax) in a mixture of ethanol-water (74:26, v/v). 3H.NE and endogenous NE were separated from their metabolites by ion exchange chromatography on Amberlite CG50 and adsorption on alumina (Thierry et al., 1968). The 3H-amine was determined by liquid scintillation counting. Non-labelled NE was determined by the spectrophotofluorimetric method of Anton and Sayre (1962). 3H-NE and NE values were corrected for respective average recoveries of 80% and 70%. 2.2. Action of AnglI and amphetamine on newly synthesized SH-NE Slices (2500 rag; wet weight) of rat left ventricles were first incubated in 20 rnl of the previously described incubation medium with 25/~Ci of aH-DA (dopamine [ring-T ((3)] hydrochloride, 200 mCi/ mmole, Radiochemical Center, Amersham), for 90 rain, to allow synthesis of 3H-NA. Slices containing aH.NE, newly synthesized from aH-DA, were washed four times with Krebs solution, and then reincubated as previously described, for 30, 90 or 150 min with AngII or dl-amphetamine, to test the releasing action of these substances. After the incubation period, cardiac slices were removed from the incubation medium and homogenized with 0.4 N perchloric acid; concentrated perchloric acid was added to the incubation mixtures until a concentration of 0.4 N was

9

reached. Thereafter, aH-NE was separated from a H-DA by adsorption on alumina and ion exchange chromatography on Dowex (AG-50 WX4 Na*) (Glowinski, Iversen and Axelrod, 1966). The radioactivity of the different fractions was measured by 'iquid scintillation counting. Average recoveries were 75 and 65% for aH-NE and aH-DA, respectively. Contamination of aH-NE by aH-DA never exceeded 5%. All data were corrected for recovery, and for a contamination of 5%. In some experiments, identification of aH-NE and aH-DA present in Dowex column eluates was performed by paper chromatography after acetylation of the amines and organic extraction of the acetylated derivatives (Laverty and Sharman, 1963). 2.3. Action of AnglI and amphetamine on axonal SH-DA uptake Slices (200 mg, wet weight) of rat left ventricles were incubated as previously, for 5 min, in 10 ml of Krebs solution containing 25 nCi of SH-DA, alone or with the drugs to be tested. SH-DA was than separated, as previously described. 2.4. Statistical analysis Student's t test (1909) was used. However, distributions with significatively different variances as demonstrated by the Snedecor's test (1956) were compared using the Darmois' test (1954). This test provides a single significance level of 10%.

3. RESULTS 3.1. Action of angiotensin and amphetamine on endogenous ArE and aH-NE labelled pools Table 1 indicates that AngII, in contrast to dlamphetamine, did not deplete cardiac endogenous NE or aH-NE stored for a long period. 3.2. Action of angiotensin and amphetamine on aliNE newly synthesized from aH-DA and SH.DA, Table 2 shows that AngII and amphetamine reduced the tissue levels of aH-NE and increased the amount of aH-NE in the incubation mixture. Therefore, both drugs released aH-NE newly synthesized from aH-DA. The higher dose of AngII significantly reduced the

C Chevillard et aL. Angiotensin H action on cardiac N E

10

Table 1 Influence o f AnglI and a m p h e t a m i n e on the release o f endogenous and tritiated norepinephrine. AnglI Determinations

Controls

Amphetamine lX 10-6M

5 × 10 - l ° M

5 X 10-6 M

45 rain incubation 3H-NE (nCi/g) E n d o g e n o u s NE (nmole/g) n

738 +- 36 4.73 ± 0.33 8

540 -+ 36 ** 2.71 ± 0.35 *** 7

746 -+ 53 4.28 ± 0.29 7

763 ± 53 4.88 ± 0.31 7

443 -+ 35 3.04 -+ 0.24 9

211 ± 23 *** 0.91 ± 0.03 * 7

375 ± 38 2.84 ± 0.18 10

483 ± 31 2.84 +- 0.27 7

90 min incubation 3H-NE ( n O / g ) E n d o g e n o u s N E (nmole/g) n

Rats received 25 ~Ci 3H-NE intravenously, and were killed 60 m i n later. Cardiac slices (300 mg, wet weight) were incubated for 45 or 90 m i n in 10 ml o f Krebs solution, either alone (controls) or, with AnglI or dl-amphetamine. Slices were t h e n analyzed for e n d o g e n o u s NE and 3H-NE. Data are reported as m e a n ± S.E.M, * p < 0.10 (Darmois' test); ** p < 0.01; *** p < 0.001.

Table 2 Influence of Angll and a m p h e t a m i n e on the cardiac norepinephrine newly synthesized f r o m dopamine. a H-NE (nCi/g) AnglI Controls

Amphetamine lX 10-6M

5×10 -l°M

5 X 10- 6 M

30 min incubation Incubation m e d i u m Slices Incubation m e d i u m + slices n

51.5 ± 4.3 382.5 ± 20 433.0 ± 21 11

85 ± 16 ** 328 ± 25 413 ± 27.5 9

98,5 ± 18.1 ** 319,5 ± 31.6 418 ± 26 12

126.3 ± 21.1 ** 299.2 ± 10.8 * 425.5 ± 33 9

97 423 520

166 ± 15.5 ** 305 ± 2 0 " * 471 ± 29.3 8

164.4 ± 9.5 ** 321.6 ± 11.3 * 486.0 ± 16.3 12

229 470 699

90 min incubation Incubation m e d i u m Slices Incubation m e d i u m + slices n

± 6.6 ± 17.3 ± 20 17

± 11.5 ** ± 26.8 ± 34.1 ** 9

Cardiac aH-NE was synthesized in a preliminary incubation for 90 m i n o f 2500 m g (wet weight) o f rat h e a r t slices in 20 ml of Krebs solution containing 25 #Ci aH-DA. Cardiac slices (300 rag, w e t weight) were t h e n incubated in 10 ml o f Krebs solution, alone (controls) or with angiotensin II (AngII: 5 X 10 -10 M and 5 × 10 -6 M), or dl-amphetamine, for 30 or 90 rain. NE release is d e m o n s t r a t e d by an increase of aH-NE in incubation m e d i u m ; NE synthesis is d e m o n s t r a t e d by an increase in the s u m of concentrations o f SH-NE in slices and incubation m e d i u m . Data is reported as m e a n ± S.E.M. * p < 0 . 1 0 (Darmois' test); ** p < 0.001.

C Chevillard et al., Angiotensin H action on cardiac NE

cardiac levels at the 30th min, while after the smaller dose the change was only significant at the 90th min. However, at the higher dose, the tissue concentration o f newly synthesized aH-NE had returned to control values b y the 90th minute although release into the incubation mixture was still increased. Therefore, the significant reduction of cardiac aH-NE levels had increased 3H-NE synthesis, to compensate for tissue losses. Accelerated synthesis, induced by the higher dose o f AnglI, was demonstrated by addition o f cardiac aH-NE levels to the amount o f aH-NE released into the incubation mixture. Synthesis occurred from the a H-DA, which was taken up b y cardiac sympathetic nerve endings and had not undergone conversion into 3 H-NE, during the first incubation, and which accumulated or was taken up again after release, during the second incubation period. As shown in table 2, amphetamine (10 -4 M) had almost the same releasing action as the lower dose o f AnglI, and, likewise, did not promote an enhancement o f NE synthesis. To further investigate the action o f amphetamine on synthesis, incubation was prolonged for 150 min. Table 3 indicates that, in spite o f a large release, amphetamine antagonized synthesis. Table 4 shows the modifications o f a H-DA distribution induced b y AnglI and amphetamine. Amphetamine significantly increased the amount o f a H-DA in the incubation medium at 30 min without changing the cardiac a H-DA level. Moreover, at the

11

Table 3 Influence of amphetamine on the cardiac 3H-NE newly synthesized from dopamine and on the distribution of 3H-DA after a long-lasting incubation of 150 rain.

Medium analyzed

Amphetamine 10-6 M

Controls (n = 8)

(n = 10) 3H-NE (nO/g)

Incubation medium Slices Incubation medium + slices

141.3 ± 7.8 370 ± 23.5

171.1 ± 11 * 248.3 ± 14.8 ***

511.3 ± 31

419.4 ± 22.6 *

263.6 ± 12.8 115 -+ 8

411 ± 5.3 t" 60.7± 3 t

378.6 ± 16.1

471.7 ± 18 **

3H-DA (nCi/g/

Incubation medium Slices Incubation medium + slices

For further information concerning the experiment, see the legend of table 2. * p < O.O5; ** p < O.Ol, *** p < O.O01;

t p < 0.10 (Darmois' test). 90th min, amphetamine depleted cardiac a H-DA. The higher dose o f AnglI had th e opposite effect: at first, AnglI only diminished the amount o f s H-DA in the incubation medium and second, AnglI also caused a greater cardiac z H-DA accumulation. The lower dose o f AnglI did not affect a H-DA distribution.

Table 4 aH-DA distribution in slices and incubation medium after amphetamine and angiotensln treatment. a H-DA (nCi) Medium analyzed

Controls

Amphetamine 1 x 10-6M

AngII 5× 10- l ° M

5X 10-6M

30 min incubation

Incubation medium Slices

446 ± 18 865 + 21.3

536 ± 32 * 855 ± 32

406 ± 35 836 ± 31

322 ± 32 *** 876 ± 33

473 ± 20 651 ± 40

605 ± 41 ** 436 ± 15 t

440 ± 28 630 ± 45

251 ± 26 *** 758 ± 26 *

90 min incubation

Incubation medium Slices

For further information concerning the experiment, see the legend of table 2. * p < 0.05; ** p < 0.01 ; *** p < 0.001 ; ~" p < 0 . 1 0 ( D a r m o i s ' test).

12

C.Chevillard et al., Angiotensin H action on cardiac NE

Table 5 Influence of Angll and amphetamine on cardiac axonal 3H-DA uptake.

Controls (n = 10) 3H-DA (nCi/g)

3.03 ± 0.37

Amphetamine 10-6 M

AnglI

(n -- 9)

5 X 10-l° M (n = 10)

5 X 10-6 M (n = 10)

3.25 ± 0.21

5.13 ± 0.51 *

5.86 ± 0.63 **

Slices (200 mg, wet weight) of left ventricles were incubated for 5 min in 10ml of Krebs solution containing 25 nCi of 3H-DA, alone (controls), or with amphetamine, or AnglI. Sliceswere, then, analyzed for 3H-DA. Data is reported as mean ± S.E.M. * p < 0.01; ** p < 0.10 (Darmois' test).

3 . 3 . A c t i o n o / d r u g s on the axonal aH-DA uptake

From table 5, it is shown that AnglI favoured a H-DA uptake, while amphetamine had no consistent action.

4. DISCUSSION In agreement with previous reports (Westfall and Peach, 1965; Peach and Ford, 1968; Starke, Wemer and Schiimann, 1969), AnglI, in contrast with amphetamine (Axelrod, Hertting and Potter, 1962; Chidsey, Harrison and Braunwald, 1962) did not release endogenous NE or a H-NE-labelled pods. However, like amphetamine, AnglI released a H-NE newly synthesized from 3 H-DA. Since some cardiac haemodynamic effects of AnglI are thought to depend upon sympathetic tone (Nishith, Davis and Youmans, 1962; Beaulnes, 1963; Krasney et al., 1966; Farr and Grupp, 1967; Hugues, 1968), it is suggested that this pool of newly synthesized NE represents a functional compartment. This pool is probably too small to be investigated by determination of endogenous NE or 3 H-NE stored for a long period. Likewise, preferential utilization of newly synthesized CA by the CNS has been demonstrated by Glowinski et al., after administration of d-amphetamine or desipramine (Besson, Cheramy and Glowinski, 1969) and during mild stress (Thierry, Blanc and Glowinski, 1970). In addition, stimulation of sympathetic neurones is known to selectively release newly synthesized NE (Kopin et al., 1968). It thus appears that AnglI, amphetamine and sympathetic nervous stimulation act on the same pool of newly synthe-

sized NE. This could explain, potentation of amphetamine cardiovascular action by AnglI (Schmitt and Schmitt, 1967), potentation of angiotensin pressor action by amphetamine (Schmitt and Schmitt, 1968b; Commarato and Lum, 1970), and activation by AnglI of NE release during cardiac sympathetic stimulation (Starke, Werner and SchiJmann, 1969). AnglI, by causing utilization of newly synthesized NE, could abolish the negative feed-back on tyrosine hydroxylation and thereby stimulate NE synthesis from tyrosine, as demonstrated by Boadle, Hugues and Roth (1969). In addition, AnglI stimulated cardiac a H-NE synthesis from a H-DA (table 2), by increasing DA O-hydroxylation. It therefore appears that CA synthesis may be regulated at the DA/3-hydroxylation stage, as reported by Goldstein and Nakajima (1967), and indicated by recent results on the CNS (Glowinski, personal communication). Stimulation of DA fl-hydroxylation demonstrated in this paper could result, either from a direct activation of DA/3-hydroxylase by AnglI, or, indirectly from an increase of DA accumulation by adrenergic granules where the hydroxylating enzyme is located. This latter suggestion is substantiated by the fact that increased cardiac 3H-DA accumulation occurred together with enhanced a H-NE synthesis at the higher dose of AnglI, and, that neither increase of accumulation, nor stimulation of synthesis was observed at the lower dose of AnglI. Therefore both phenomena, synthesis and tissue DA accumulation, appeared to be related. According to this correlation, amphetamine which releases newly synthesized a H-NE, but, also reduces cardiac a H-DA level should not be expected to

C.Chevillard et al., Angiotensin H action on cardiac N E

stimulate aH-NE synthesis from aH-DA. In fact, it was found that amphetamine did not increase NE synthesis; moreover, amphetamine decreased this synthesis during a prolonged releasing period. From these results, itis clear, that facilitation o f cardiac NE synthesis from DA requires, not only a previous release of newly synthesized NE, but also an increase in tissue DA accumulation. After incubation for 5 rain, amphetamine (10 "~ M), in contrast to its action on NE uptake (unpublished observations) did not inhibit axonal 3H-DA uptake. Therefore, it appears that amphetamine induced tissue 3H-DA depletion through release of the amine. It was also-obvious, that both doses of AngII strongly increased 3H-DA uptake. There is apparently a discrepancy between these results and those obtained during release-experiments where increased accumulation of 3H-DA cardiac only occurred at the higher dose of AngII and only by the 90th rain. However, in this latter situation, it must be remembered that 3H-DA had first to be released before AngII could act upon reuptake. Moreover reuptake could be indirectly dependent on the synthesis rate since enhancement o f 3 H-DA penetration into the adrenergic granules leads to the maintenance o f an appropriate gradient of DA concentration across the two sides of the axonal membrane. Thus, in this paper, evidence is given that AngII induces a selective release of newly synthesized cardiac 3H-NE, and thereafter, an enhancement of 3 H-NE synthesis from an increased accumulation of tissue 3H-DA. Amphetamine, despite its NE releasing action, antagonizes synthesis by reducing the tissue concentration of precursor available for conversion.

ACKNOWLEDGEMENTS This investigation was supported by grants of the Institut National de la Sante" et de la Recherche M&ticale, and Association Claude-Bernard.

REFERENCES Anton, A.H. and D.F. Sayre, 1962, A study of the factors affecting the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines, J. Pharmacol. Exptl. Therap., 138, 360.

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Axelrod, J., G. Hertting and T.L. Potter, 1962, Effect of drugs on the uptake and release of 3H-norepinephrine in the rat heart, Nature 194, 297. Beaulnes, A., 1963, Effets de l'angiotenslne sur le coeur, Biochem. Pharmacol. 12, suppl.~ 181. Benelli, B., D. Della Bella and A. Gandini, 1964, Angiotensin and peripheral sympathetic nerve activity, Brit. J. Pharmacol. 22, 211. Besson, M.J., A. Cheramy and J. Glowinski, 1969, Effects of amphetamine and desmethylimipramine on amine synthesis and release in central catecholamine-containing neurons, European J. Pharmacol. 7, 111. Bickerton, R.K. and J.P. Buckley, 1961, Evidence for a central mechanism in angiotensin induced hypertension, Proc. Soc. Exptl. Biol. Med. 106,834. Boadle, M.C., J. Hugues and R.H. Roth, 1969, Angiotensin accelerates catecholamines biosynthesis in sympathetically innervated tissues, Nature 222, 987. Cession,G. and A. Cession-Fossion, 1963, Action excitosurr~nalienne de 1' angiotensine chez le rat, Comp. Rend. Soc. Biol. 157, 1830. C~evillard, C. and J.M. Alexandre, 1970, Action, in vivo et in vitro, de l'angiotensine II sur le captage cardiaque de la noradrenaline, Experientia 26, 1334. Chidsey, C.A., D.C. Harrison and E. Braunwald, 1962, Release of norepinephrine from the heart by vasoactive amines, Proc. Soc. Exptl. Biol. Med. 109, 488. Commarato, M.A. and B.K.B. Lure, 1970, Potentiation of the pressor response to angiotensin by amphetamine and ephedrine, European J. Pharmacol. 10, 25. Darmois, G., 1954, Comparaison des moyennes de deux populations normales d'~carts types inconnus et diff,rents, Revue de Statistique Appliqu6e 2, 37. Day, M.D. and D.A.A. Owen, 1969, Potentiation by angiotensln of responses to endogenously released noradrenaline in the pithed rat, Arch. Intern. Pharmacodyn. 179, 469. Distier, A., H. Liebau and H.P. Wolff, 1965, Action of angiotensln on sympathetic nerve ending in isolated blood vessels, Nature 207,764. Farr, W.C. and P. Grupp, 1967, Sympathetically mediated effects of angiotensin on the dog heart in sltu, J. Pharmacol. Expti. Therap. 156,528. Feldberg, W. and G.P. Lewis, 1964, The action of peptides on the adrenal medulla. Release of adrenaline by bradykinin and angiotensin, J. Physiol. (London) 171, 98. Glowinski, J., L.L. Iversen and J. Axelrod, 1966, Storage and synthesis of norepinephrine in the reserpine-treated rat brain, J. Pharmacol. Exptl. Therap. 151,385. Goldstein, M. and K. Nakajima, 1967, The effect of disulfiram on catecholamine levels in the brain, J. Pharmacol. Exptl. Therap. 157, 96. Hugues, I.E., 1968, Cltronotropic and pressor response to angiotensln in rats anaesthetized with urethane, European J. Pharmacol. 3,189. Kiran, B.K. and P.A. Khairallah, 1969, Angiotensin and norepinephrine efflux, European J. Pharmacol. 6, 102. Kopin, I.J., G.R. Breese, K.R. Krauss and V.K. Weise, 1968,

14

C. Chevillard et al., Angiotensin H action on cardiac N E

Selective release of newly synthesized norepinephrine from the cat spleen during sympathetic nerve stimulation, J. Pharmacol. Exptl. Therap. 161,271. Krasney, J.A., F.T. Paudler, P.M. Hogan, R.F. Lowe and W.B. Youmans, 1966, Peripheral adrenergic basis for cardiovascular action of angiotensin, Am. J. Physiol. 211, 1447. Laverty, R. and D.F. Sharman, 1963, The estimation of small quantities of dihydroxyphenylethylamine in tissues, Brit. J. Pharmacol. 24, 538. Lewis, G.P, and F. Reit, 1965, The action of angiotensin and bradykinin on the superior cervical ganglion of the cat, J. Physiol. (London) 179, 538. McCubbin, J.W. and I.H. Page, 1963, Renal pressor system and neurogenie control of arterial pressure, Circulation Res. 12, 553. Nishith, S.D., L.D. Davis and W.B. Youmans, 1962, Cardioaccelerator action of angiotensin, Am. J. Physiol. 202, 237. Pals, D.T., R.W. Fulton and F.D. Masucci, 1968, Angiotensin, cocaine and desipramine: comparison of effects on blood pressure responses to norepinephrine, tyramine and phenylephrine in the pithed rat, J. Pharmacol. Exptl. Therap. 162, 85. Panisset, ].C., P. Biron and A. Beaulnes, 1966, Effects of angiotensin on the superior cervical ganglion of the cat, Experientia 22, 394. Panisset, J.C. and R. Bourdois, 1968, Effects of angiotensin on the response to noradrenaline and sympathetic nerve stimulation, and, on a H-noradrenaline uptake in cat ruesenteric blood vessels, Can. J. Physiol. Pharmacol. 46, 125. Peach, M.J. and G.P. Ford, 1968, The actions of angiotensin II on canine myocardial and plasma catecholamines, J. Pharmacol. Exptl. Therap. 162, 92. Peach, M.J., F.M. Bumpus and P.A. Khairallah, 1969, Inhibition of norepinephrine uptake in hearts by angiotensin II and analogs, J. Pharmacol. Exptl. Therap. 167, 291. Schmitt, H. and H. Schmitt, 1967, Interr61ations entre catecholamines et angiotensine, Comp. Rend. Soc. Biol. 161,753.

Schmitt, H. and H. Schmitt, 1968a, Increased activity in sympathetic nerves induced by angiotensin, Rev. Can Biol. 27,255. Schmitt, H. arid H. Schmitt, 1968b, Modifications des effets hypertenseurs de l'angiotensine par les agents adr~nergiques et anti-adr~nergiques, Arch. Intern. Pharmacol. 171, 31. Schi/mann, H.J., K~ Starke, U. Werner and R. Hellenfurth, 1970, The influence of angiotensin on the uptake of noradrenaline by the isolated heart of the rabbit, J. Pharm. Pharmacol. 22,441. Scroop, G.C. and R.D. Lowe, 1968, Central pressor effect of angiotensin mediated by the parasympathetic nervous system, Nature 220, 1331. Snedecor, G.W., 1956, Statistical Methods (Iowa State University Press, Ames). Starke, K., U. Werner and H.J. Schlimann, 1969, Wirkung von Angiotensin auf Funktion und Noradrenalinabgabe isolierter Kaninchenherzen in Ruhe und bei Sympathicusreizung, Arch. Exptl. Pathol. Pharmakol. 265,170. "Student", 1908, The probable error of a mean, Biometrika 6,1. Thierry, A.M., F. Javoy, J. Glowinski and S.S. Kety, 1968, Effects of stress on the metabolism of norepinephrine, doparnine, and serotonine in the central nervous system of the rat. 1. Modification of norepinephrine turnover, J. Pharrnacol. Exptl. Therap. 163,163. Thierry, A.M., G. Blanc and J. Glowinski, 1970, Preferential utilization of newly synthesized norepinephrine in the brain stem of stressed rats, European J. Pharmacol. 10, 139. Westfail, T.C. and M.J. Peach, 1965, Action of angiotensin on myocardial and renal catecholamines in the rabbit, Biochem. Pharmacol. 14, 1916. Zimmermann, B.G. and J. Gomez, 1965, Increased response to sympathetic stimulation in the cutaneous vasculature in presence of angiotensin, Intern. J. Neuropharmacol. 4, 185.