Furnished a Hint of Qualitative Differences. Perhaps the First Of

314

J. Physiol. (1958) I44, 3I4-336

THE ACTION OF SYMPATHOMIMETIC AMINES IN ANIMALS TREATED WITH RESERPINE BY J. H. BURN AND M. J. RAND From the Department of Pharmacology, University of Oxford (Received 3 June 1958) The impression left in the minds of those who have read the account of the sympathomimetic amines written by Barger & Dale in 1910 has been that these substances in the main were found to differ only quantitatively in activity. From time to time, however, there have been observations which furnished a hint of qualitative differences. Perhaps the first of these was the discovery by Tainter & Chang (1927) that under the influence of cocaine the action of tyramine was abolished, although, as Frohlich & Loewi had shown in 1910, the action of adrenaline was potentiated. In 1931 Burn & Tainter observed that tyramine and ephedrine had no action on the denervated pupil, though to adrenaline it was supersensitive. In 1932, Burn recorded that denervation of the cat's foreleg by removal of the stellate ganglion led to a loss of the vasoconstrictor action of tyramine and of ephedrine, though not of that of adrenaline. Burn also found that in the perfused dog's hind leg tyramine and ephedrine had, compared with adrenaline, only a fraction of the constrictor action they would be expected to have, judging from the pressor effect in the spinal cat. He observed, however, that the constrictor action could be restored if adrenaline was added to the blood perfusing the hind leg, and that this restoration could not be effected by adding pituitary (posterior lobe) extract instead. Fleckenstein & Burn (1953) found that after denervation of the nictitating membrane it became more sensitive only to those amines which were catechol derivatives; that to the derivatives of phenylethanolamine it was less sensitive and to the derivatives of phenylethylamine it was nearly insensitive. Flecken- stein & Bass (1953) and Fleckenstein & St6ckle (1955) showed that the normally innervated nictitating membrane after full doses of cocaine behaved in exactly the same way. These various observations have remained incoordinated and unexplained. Recently, however, Carlsson, Rosengren, Bertler & Nilsson (1957) found that, in cats treated with reserpine, the intravenous injection of tyramine had no SYMPATHOMIMETIC AMINES 315 pressor effect. In explanation they said that 'tyramine belongs to a group of sympathomimetic amines which are fully active only in the presence of an intact adrenergic system'. We have recently observed (Burn & Rand, 1957, 1958b) that when rabbits and dogs were treated with reserpine, the noradrenaline-like substance demonstrated by Schmiterl6w (1948) to be present in the wall of the aorta disappeared. We also observed that the constrictor action of nicotine in the perfused rabbit's ear disappeared, and were led to the conclusion that nicotine exerted this constrictor action by releasing a noradrenaline-like substance from the vessel wall or perhaps from chromaffin cells in its neighbourhood. It therefore seemed to us possible that the failure of tyramine to exert its pressor action in the cat after treatment with reserpine was due to the fact that tyramine normally acted by releasing a noradrenaline-like substance from the vessel walls. We now describe experiments to test the action of a series of sympathomimetic amines from this point of view.

METHODS Observations of four kinds have been made. The effect of sympathomimetic amines has been examined (1) on the blood pressure, the spleen and the nictitating membrane of the spinal cat; (2) on the arterial resistance and venous outflow of the perfused dog hind leg; (3) on the outflow of the perfused rabbit ear; and (4) on spiral strips of rabbit aorta set up in an isolated organ bath. Spinal cats were prepared under ether anaesthesia, dividing the cord at the 2nd cervical verte- bra and destroying the brain. Records of the spleen volume and of the contractions of the nictitating membrane were made as described by Burn (1952). For the hind leg perfusion one dog was bled under ether anaesthesia from the carotid artery into a jar containing heparin, and its lungs were prepared for perfusion by one of a pair of Dale-Schuster (1928) pumps. Perfusion was begun through the lungs, which were ventilated by oxygen with 5 % CO2. A second dog was then anaes- thetized. After dividing the rectum and the inferior mesenteric artery between ligatures, the right external iliac artery was tied, and a cannula was inserted pointing towards the aorta. The body wall was divided in sections between ligatures along the course ofthe left external iliac artery. Poupart's ligament was cut. Branches of the left external iliac from the aorta to about 1 cm below Poupart's ligament were tied, as were also the corresponding veins. The continuation of the aorta below the origin of the external iliac arteries was tied. When the dissection was complete, perfusion was begun by the second pump through the left hind leg at the moment of tying the aorta to arrest the natural circulation. In some experiments the left lumbar sympathetic chain was prepared for stimulation; in these the dog was first eviscerated and the left kidney removed. After the arrest of the natural circulation, the dog was cut in half, the perfusing cannula was tied into the aorta and the blood collected from the vena cava. By this method the ganglia of the sympathetic chain were perfused. In these experiments there was an interval of 7 min between the arrest of the natural circulation and the start of the perfusion. In all perfusions mass ligatures were tied so as to enclose the whole circumference of the body wall above the area perfused. An outflow record was obtained by using Stephenson's recorder (1949). When stimulating the sympathetic chain shielded electrodes, each a silver plate 3 mm wide, were used; stimuli were square-wave pulses of 2-5 mA strength, 1 msec duration and 20-25/sec frequency. The perfusion of the rabbit ear was arranged as previously described (Burn, 1952) using Stephen- son's outflow recorder (1948). 316 J. H. BURN AND M. J. RAND Reserpine was dissolved in 20% ascorbic acid solution making the solution up to 10 mg/ml. as required. Injections were made intraperitoneally. Dogs were given 0-5 mg/kg on two successive days, the dog being killed on the third day. Cats were given 2-5-5-0 mg/kg on two successive days, and rabbits were given 3 mg/kg on two successive days.

Fig. 1. Upper record is that of the nictitating membrane; lower record that of blood pressure in the spinal preparation. (a) Normal cat; injection of 10 i&g noradrenaline produced a small rise of blood pressure; 4 mg tyramine hydrochloride produced a large rise of blood pressure and large contraction of the nictitating membrane. (b) Cat treated with reserpine. Note that the effects were reversed; 4 mg tyramine had a very slight effect on the blood pressure and none on the nictitating membrane; 10 iLg noradrenaline had a large effect on the blood pressure. (c) Same as (b); after the intravenous infusion of 0-12 mg noradrenaline, the effect of 4 mg tyramine on the blood pressure was much greater than in (b).

RESULTS Action of tyramine Fig. 1 shows records taken from experiments in two spinal cats, the one a normal cat, and the second a cat treated with reserpine. In Fig. 1la the injection of 10 pg noradrenaline into the normal cat caused a small rise of blood pressure, whereas the injection of 4 mg tyramine hydrochloride caused a large rise of blood pressure and a large contraction of the nictitating membrane. In Fig. 1lb the same injections were made into a cat treated with reserpine. The effect of 4 mg tyramine hydrochloride was very small and there was no contraction of the nictitating membrane, while the effect of 10 pg noradrenaline was very much larger than in the normal cat. A solution of SYMPATHOMIMETIC AMINES 317 noradrenaline was then prepared in a burette and the burette was connected to the femoral vein. An infusion of noradrenaline at a uniform rate was begun and maintained until 012 mg noradrenaline had entered the cat. The infusion caused a large rise of blood pressure, but although the rate of infusion was kept constant, the blood pressure fell as the infusion continued, though the extent of this fall varied in different experiments. At the end of the infusion the point at which the blood pressure returned to its former level was noted, and an injection of 4 mg tyramine was made again, as shown in Fig. 1 c.

Fig. 2. Upper record is that of the spleen volume; lower record that of blood pressure, in the spinal preparation. (a) Normal cat. (b) Cat, treated with reserpine, in which 2 tg adrenaline had a greater effect on the spleen volume than 10 jg in (a), and in which 1 mg tyramine had no action, though it contracted the spleen in (a). An infusion ofnoradrenaline was then made, after which (c) 1 mg tyramine again caused contraction. The rise of blood pressure was much greater than in Fig. 1 b and there was also a small contraction of the nictitating membrane which was absent in Fig. 1 b. The events shown in Fig. 1 were observed in many experiments. When the pressor action of tyramine had been restored, as in Fig. 1 c, then repeated injections of tyramine had a declining effect, but a further infusion of noradrenaline would restore it a second time. In other experiments the spleen volume was recorded, as shown in Fig. 2. In a normal cat (Fig. 2a), the injection of 10 ,g adrenaline and of 1 mg tyramine produced a contraction of the spleen volume. In a cat treated with reserpine (Fig. 2b), 2 ,ug adrenaline produced a greater contraction ofthe spleen volume than 10 pg in the normal cat, and 1 mg tyramine had no appreciable action on the spleen volume. After the intravenous infusion of noradrenaline, both the pressor action of 1 mg tyramine and its action on the spleen volume were partly restored (Fig. 2c). Observations were also made by perfusing 318 J. H. BURN AND M. J. RAND the hind leg of the dog. In a preparation from a dog previously treated with reserpine the injection of 0-25 mg tyramine had no constrictor effect as Fig. 3a shows. Stimulation of the sympathetic chain after the injection of 01 mg atropine to exclude the cholinergic fibres (Biulbring & Burn, 1935) also had no effect. After the slow addition of 0f41 mg noradrenaline to the reservoir and after sufficient time had elapsed for the effect of this noradrenaline on the arterial resistance to have passed off, stimulation was then effective, as shown in Fig. 3b, and the injection of 025 mg tyramine caused a rise in mean arterial resistance of 56 mm.

Fig. 3. Records from perfusion of hind leg of dog treated with reserpine. Upper record is venous outflow; lower record is pressure in the arterial cannula. In (a) at St., stimulation was applied to the lumbar sympathetic after injecting 0.1 mg atropine to exclude cholinergic fibres; it had no effect. Similarly, injection of 0-25 mg tyramine into the cannula had no effect. (b) The records were taken after the addition of 0-41 mg noradrenaline to the venous reservoir during 20 min. When the pressure returned to its previous level, stimulation at St. caused arterial constriction and a fall in outflow. The injection of 0-25 mg tyramine caused vasoconstriction and a fall in outflow. Since reserpine is known to displace hydroxytryptamine from sites where it is bound, and since Woolley & Shaw (1953) state that hydroxytryptamine is present in arteries, it was possible that the failure of tyramine to exert a pressor effect in the reserpine-treated cat was due to absence of hydroxytryptamine. We therefore carried out experiments to see if an infusion of hydroxytryptamine would restore the pressor effect of tyramine, but we failed to observe any sign of such an action either in the spinal cat or in the perfused SYMPATHOMIMETIC AMINES 319 hind leg of the dog; we were surprised to find that the infusion of amounts from 1 to 2 mg hydroxytryptamine had little or no effect on the blood pressure. Phenylethylamine We obtained results with phenylethylamine very similar to those obtained with tyramine, and an illustration in Fig. 4 shows the changes in the nictitating membrane as well as those in the blood pressure when phenylethylamine was examined. The observations in Fig. 4 were made in a cat treated with reserpine. In Fig. 4a the injection of 4 mg phenylethylamine had little effect on the blood pressure or on the nictitating membrane, while the injection of 10 ,ug noradrenaline had a large effect on both. An infusion of a total of 1 mg noradrenaline was then given, and when its effect on the blood pressure had passed off, the injection of 10 ,g noradrenaline now had a much smaller

Fig. 4. Records as in Fig. I from spinal cat treated with reserpine. (a) Injection of 4 mg phenyl. ethylamine had a small effect; (b) 10 jug noradrenaline had a large effect on blood pressure and also on the nictitating membrane; (c) after the intravenous infusion of noradrenaline during 20 min the injections were repeated. The effect of 10 jug noradrenaline was much smaller on the blood pressure and on the nictitating membrane than in (b), the effect of 4mg phenylethylamine was greater on the blood pressure and much greater on the nictitating membrane than in (a). 320 J. H. BURN AND M. J. RAND effect in both records, while the injection of 4 mg phenylethylamine had a much greater effect, the increased contraction of the nictitating membrane being particularly clear. Observations were also made with phenylethylamine in the perfused hind leg of the dog. An example of these is given in Fig. 5, taken from an experiment on a dog given reserpine beforehand. In Fig. 5a the injection of 1 mg phenylethylamine caused a small increase in arterial resistance attended by a slight rise and fall in the venous outflow. Between Fig. 5a and Fig. 5b, an infusion of 1 mg hydroxytryptamine was made; when the

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Fig. 5. Records of perfusion of hind leg of dog treated with reserpine. Upper record is venous outflow; lower record is pressure in arterial cannula. (a) Injection of 1 mg phenylethylamine produced small constriction and small increase in outflow followed by a decrease; (b) after the infusion of 1 mg hydroxytryptamine the, injection of phenylethylamine had less effect than before; (c) after the infusion of 1 mg noradrenaline, the injection of 1 mg phenylethylamine had a much greater constrictor action. injection of phenylethylamine was repeated, apart from a very slight increase in outflow, there was no effect at all. An infusion of I mg noradrenaline was then given, and when its effect on the vascular tone had passed off, the injection of phenylethylamine caused a large vasoconstriction and decrease in outflow (Fig. 5c.) The catecholamines From the evidence so far given and illustrated in Figs. 1 and 4, the effect of reserpine was to remove the pressor action of tyramine and of phenylethylamine and to increase that of noradrenaline. The increased action of nor- SYMPATHOMIMETIC AMINES 321 adrenaline was, however, depressed by an infusion of noradrenaline. Noradrenaline is a derivative of catechol, and we tested other catecholamines to see whether their pressor action in the reserpine-treated cat was depressed by an infusion of noradrenaline. The results in one experiment are shown in Table 1, in which the substance neosynephrine, which has the same structure as adrenaline except that it lacks the -OH group in the 4 position in the ring, was also tested. All the substances in Table 1 were active pressor agents in the reserpine-treated cat in contrast to tyramine and phenylethylamine, and allwere madeless active by an infusion ofnoradrenaline. Since the constrictor action of tyramine in the perfused hind leg was increased not only by the addition of noradrenaline to the blood but also by the addition of adrenaline as shown by Burn (1932a), we decided to test the effect of infusing dopamine. Fig. 6a shows the effect of injecting 4 mg tyramine in a cat treated with reserpine. The injection raised the blood pressure by 22 mm. TABLE 1. Effect of infusing noradrenaline on pressor action in cat treated with reserpine (spinal preparation) Pressor action (mm Hg) After noradrenaline Substance Dose (pg) Initial infusion Noradrenaline 3 104 26 Adrenaline 20 144 54 Epinine 100 132 58 Dopamine 200 122 34 Neosynephrine 100 150 38 A total of 1 mg dopamine was then infused. When the effect of this infusion on the blood pressure had subsided, the injection of 4 mg tyramine then raised the blood pressure by 46 mm, but did not cause contraction of the nictitating membrane. A further infusion of 4 mg dopamine was given. Afterwards the injection shown in Fig. 6b was made, which caused no greater rise of pressure than in 6a, but caused contraction of the nictitating membrane. Finally, an infusion of 1 mg noradrenaline was given, and after it injection of 4 mg tyramine caused the large effects shown in Fig. 6c. The phenylethanolamines These results established the contrast between substances with an ethylamine side chain which were not catecholamines on the one hand, and substances which were in the main catecholamines on the other. We were fortu- nate in being able to examine phenylethanolamine in the form of its two optical isomers which were prepared by Pratesi & Grassi (1953). The one form, the (-)-form has the same configuration as (-)-adrenaline, while the (+)-form has the same configuration as (+)-adrenaline. We found that while the (+ )-form behaved very much as tyramine or phenylethylamine 21 PHYSIO. CXLIV 322 J. H. BURN AND M. J. RAND the (-)-form resembled the catecholamines much more, though not com- pletely. Fig. 7 shows the action of the two forms in a normal cat and also in a cat treated with reserpine. In Fig. 7 a the pressor action of 4 mg (+ )-phenylethanolamine was similar to that of tyramine, the pressure not rising steeply and the rise being prolonged. The pressor action of 2 mg (- )-phenylethanolamine was slightly greater, but the pressure rose more steeply and fell more rapidly. In Fig. 7 b the pressor action of 4 mg ( + )-phenylethanolamine in the

Fig. 6. Records a-s in Fig. 1 -from spinal preparation of cat treated with reserpine. (a) Injection of 4mg tyramine. (b) Repetition of injection of 4 mg tyramine after the infusion of 5 mg dopa. mine; the injection produced a contraction of the nictitating membrane. (c) Repetition of injection of 4 mg tyramine after the infusion of 1 mg noradrenaline; the injection caused a much greater rise of blood pressure and contraction of the nictitating membrane. reserpine-treated cat had almost disappeared, as also had the action on the nictitating membrane; the pressor action of 2 mg (- )-phenylethanolamine was somewhat smaller than in the normal cat but it was even more adrenaline- like in shape and there was also a contraction of the nictitating membrane. These results suggested that the action of (± )-phenylethanolamine was like that of tyramine or phenylethylamine. This conclusion was supported by observations in which noradrenaline was infused into the spinal cat; after the effect of noradrenaline on the blood pressure had passed off, the pressor effect SYMPATHOMIMETIC AMINES 323 of (+ )-phenylethanolamine was increased. Thus in one experiment (see Fig. 8) 4 mng (+)-phenylethanolamine caused a rise of 24 mm with no contraction of the nictitating membrane, but after the infusion of noradrenaline the same amount caused a rise of 72 mm with a contraction of the nictitating membrane of 15 mm.

Fig. 7. Records as in Fig. 1. (a) From spinal preparation of normal cat; injection of 4 mg ( +)-phenylethanolamine and of 2 mg (- )-phenylethanolamine; the rise in blood pressure caused by the second injection was more 'adrenaline-like' and less 'tyramine-like' than that of the first injection. (b) From cat treated with reserpine; the injection of 4 mg ( +)-phenylethanolamine had almost no effect on the bloodpressureand none on thenictitatingmembrane; the injection of 2 mg ( - )-phenylethanolamine was still more 'adrenaline-like', and there was a small contraction of the nictitating membrane.

TABLE 2. Action of 2 mg (- )-phenylethanolamine in cats treated with reserpine Before infusion of After infusion of noradrenaline noradrenaline Nict. memb. Nict. memb. Pressor contraction Pressor contraction Cat effect (mm) (mm) effect (mm) (mm) 1 70 18 4 12 2 164 26 124 39 3 68 19 100 25 4 31 10 40 7 Mean 83 18 67 21 The action of (-)-phenylethanolamine in the reserpine-treated cat was difficult to define. The results of four experiments are shown in Table 2, which shows that the mean responses were very similar before and after the infusion of noradrenaline, so that this substance did not fall into the class of catechol amines on the one hand, or into the class which included tyramine, phenylethylamine and (+)-phenylethanolamine on the other. That it had some affinity to this class was shown by an experiment in a perfused dog's 21-2 324 J. H. BURN AND M. J. RAND hind leg, in which the injection of 0 5 mg (-)-phenylethanolamine increased the arterial resistance by 17 mm, but after the infusion of noradrenaline the injection increased it by 46 mm.

Fig. 8. Records as in Fig. 1 from spinal preparation of cat treated with reserpine. Injection of 4 mg (+)-phenylethanolamine (a) before and (b) after the intravenous infusion of 1 mg noradrenaline; in (b) the injection caused a larger rise of blood pressure and a contraction of the nictitating membrane. The action of ephedrine Ephedrine is a derivative of phenylethanolamine in which a methyl group is attached to the x carbon (adjoining the amine group) and a methyl group is also attached to the nitrogen, as in adrenaline. Ephedrine has a much longer duration of action than the substances considered hitherto. Its action on the nictitating membrane is specially prolonged. There is general agreement that this greatly increased duration of action is due to the presence of the methyl group on the oa carbon atom, which prevents the destruction of ephedrine by amine oxidase. The form of ephedrine used was the (-)-isomer. Fig. 9 shows the effect of2 mg ephedrine on the blood pressure andnictitating membrane of (a) a normal cat, and (b) a cat treated with reserpine. In each the injection of ephedrine was the first injection after making the spinal preparation. In normal cats successive injections caused decreasing effects, but the decrease was much more rapid in cats treated with reserpine. It was not so easy to restore the pressor action of ephredine in the reserpine- treated cat by an infusion of noradrenaline as to restore the pressor action of the substances already described. Fig. 10, however, shows a preparation in SYMPATHOMIMETIC AMINES 325 which an injection of no more than 10 .g noradrenaline had some restoring action, for 1 mg ephedrine, given after it, had more pressor action than when given before, and also more action on the nictitating membrane. In the record it is seen that noradrenaline caused contraction of the nictitating membrane; this it had failed to do when the same injection was made on three previous occasions in this experiment. We concluded that the contraction was a conse- quence of the injection of ephedrine and was due to inhibition of the amine oxidase in the nictitating membrane.

Fig. 9. Records from spinal preparation as in Fig. 1. (a) Normal cat; injection of 2 mg ephedrine. (b) Cat treated with reserpine; the injection of 2 mg ephedrine had much less effect.

In the perfused hind leg of the dog treated with reserpine, the small constrictor effect of ephedrine was increased by an infusion of noradrenaline, though not greatly. We made repeated attempts to see if the pressor action of ephedrine was restored by an infusion of hydroxytryptamine. All the-se experiments failed, and indeed we found that hydroxytryptamine reduced the pressor action. For example, in an experiment on a normal cat the injection of 2 mg ephedrine at approximately 30 min intervals caused rise of 126, 66, 42,' 42 mm. Then five injections each of 0-2 mg hydroxytryptamine were given, after which the injection of 2 mg ephedrine caused a rise of only 3 mm. 326 J. H. BURN AND M. J. RAND

Fig. 10. Records as in Fig. 1 from spinal preparation of cat treated with reserpine. Injection of 1 mg ephedrine at E. had very little pressor action; injection of 10 slg noradrenaline caused large rise of blood pressure and a contraction of the nictitating membrane, which had not been seen previously when 10 ug noradrenaline was injected. The contraction may have been due to inhibition of the amine oxidase by the ephedrine which had been injected just before. An injection of 1 mg ephedrine immediately after the response to noradrenaline produced a greater pressor action than before and a contraction of the nictitating membrane. Action of amphetamine The action of amphetamine was very like that of ephedrine. In the reserpine-treated cat the rise of blood pressure was small, and there was no contraction of the nictitating membrane. We were able to demonstrate the restoration of the effect on the nictitating membrane by an inffusion of noradrenaline, as illustrated in Fig. 11 in which, before the infusion of noradrenaline, we tested the preparation by injecting tyramine to see that the injection of reserpine had been effective. We also increased the constrictor action in the perfused hind leg of the dog by an infusion of noradrenaline, but not greatly. Effect of sympathetic stimulation We observed in several experiments that in cats treated with reserpine, in which tyramine, phenylethylamine and other amines acting like them had SYMPATHOMIMETIC AMINES 327

Fig. 11. Records as in Fig. 1 from spinal preparation of cat treated with reserpine. (a) Injection of 4 mg tyramine to demonstrate that the animal had been effectively treated with reserpine, in which case the injection of 2 mg amphetamine would have had little effect. (b) Injection of 2 mg amphetamine after intravenous infusion of 1 mg noradrenaline; the amphetamine produced a large effect.

Fig. 12. (a) Contraction of nictitating membrane of normal cat in response to 10 sec maximal stimulation of cervical sympathetic chain. (b) Response to the same stimulation in a cat treated with reserpine in whichizothe injection of4 mg tyramine had no effect on the membrane. 328 J. H. BURN AND M. J. RAND little effect on the blood pressure and no effect on the nictitating membrane, maximal stimulation of the cervical sympathetic chain still caused contraction of the nictitating membrane, as shown in Fig. 12b. The contraction followed the stimulus after a slightly greater delay and both the rate of rise and rate of fall were slower than in the normal cat (Fig. 12a). This persistence of the effect of nerve stimulation was observed by Muscholl & Vogt (1958). Action of various amines in the perfused rabbit ear In a previous paper (Burn & Rand, 1958b) we showed that the vasoconstrictor action of nicotine in the perfused rabbit ear was absent in rabbits treated with reserpine. We therefore investigated the constrictor action of tyramine, ephedrine and amphetamine in the perfused rabbit ear, a record being obtained of the outflow, so that the degree of constriction could be measured on the drum. Observations were made in ears from normal rabbits TABLE 3. Mean constrictor action in perfused rabbit ear Reserpine- Substance Dose (jg) Normal (mm) treated (mm) Tyramine 2 28 (4) 0 (1) 4 53 (8) 15 (4) 8 92 (3) 21 (8) 16 - 41 (1) Ephedrine 4 18 (1) 8 47 (5) 17 (3) 16 49 (2) 22 (6) 32 - 56 (2) Amphetamine 1 45 (6) 3 (3) 2 43 (4) 3 (4) 4 49 (3) 11 (4) The figures in parentheses are the numbers of observations. and also in ears from rabbits previously treated with reserpine, and they are summarized in Table 3. The constrictor effect of all three substances was reduced in the ears of rabbits treated with reserpine. The effect of the amines was not abolished, however, and this was in contrast to the effect of nicotine, which was regularly abolished. The same difference was observed in spiral strips of rabbit aorta by our colleague Dr Damrong Bejrablaya. Strips taken from rabbits treated with reserpine were not stimulated by nicotine, but were stimulated by tyramine and by amphetamine. A further important difference between the action of the amines and that of nicotine was that the action of the amines in the perfused rabbit ear was not affected by hexamethonium (see Fig. 13) whereas the action of nicotine was always abolished. SYMPATHOMIMETIC AMINES 329

Fig. 13. Record of outflow from perfused rabbit ear. (a) Diminution of outflow after injection of 5 j&g nicotine acid tartrate. (b) Diminution of outflow after injection of 8 Zg tyramine hydrochloride; outflow unaffected by 5 pg nicotine following 0.1 mg hexamethonium (H.), but outflow decreased as before by 8 Ftg tyramine following 0.1 mg hexamethonium (H.).

Fig. 14. Record of outflow from perfused rabbit ear. Diminution of outflow caused by (a) 0-004 pg noradrenaline, (b) 2 pg amphetamine, (c) 4 ug tyramine. Between (c) and (d) tolazoline (Priscol) 0-2 mg/ml. was added to the perfusing fluid; (d) shows that 0-5 pg noradrenaline caused an increase of outflow, 2 plg amphetamine caused a decrease of outflow and 4 pg tyramine caused an increase of outflow. 330 J. H. BURN AND M. J. RAND

Reversal of effects of amines by tolazoline Knowing that the effect of noradrenaline is reversed by tolazoline (Priscol) in the perfused ear, becoming dilator (Burn & Hutcheon, 1949), we tested the effect of adding tolazoline to the perfusing fluid. We found, as shown in Fig. 14, that when the effect of noradrenaline was reversed, the effect of tyramine was also reversed, but, except in one experiment, the effect of amphetamine was not reversed. Similarly, we found that the effect of ephedrine was reversed, but not that of phenylethylamine. The results are shown in Table 4.

TAIBLE 4. Perfusion of rabbit ears with tolazoline (20 ug/ml.); five experiments with each drug Constrictor action reversed (no. of experiments) Tyramine 5 Phenylethylamine 0 Amphetamine 1 Ephedrine 5

Fig. 15. Record of outflow from perfused rabbit ears, to show the effect of cocaine. (a) shows fall in outflow due to 4 l&g tyramine, (b) shows fall in outflow due to 0-004 jig noradrenaline, 8 l&g ephedrine, 1 jug amphetamine and 3.5 ,sg nicotine acid tartrate. Between (b) and (c) cocaine 5 x 10-6g/ml. was added to the perfusing fluid; in (c) the effect of tyramine was reversed to dilatation, that of noradrenaline was unchanged, those of ephedrine, amphetamine and nicotine were abolished. Abolition of effects of amines by cocaine Tainter & Chang (1927) showed that the pressor action of tyramine was reduced or abolished by cocaine. We therefore tested the effect of adding cocaine to the fluid perfusing the ears of normal rabbits. We observed that the constrictor action of tyramine, ephedrine, amphetamine and nicotine was aboished at a time when that of noradrenaline was unaffected. Such a result is shown in Fig. 15; in addition, the effect of tyramine was reversed by cocaine to a dilator action. This change in the spinal cat was described previously by Burn & Tainter (1931). SYMPATHOMIMETIC AMINES 331

DISCUSSION The results described in this paper suggest that the sympathomimetic amines may be divided into two classes, one consisting of substances like noradrenaline and adrenaline, which act on vessels in both normal and reserpine-treated animals, and the other consisting of substances like tyramine which act only on vessels of normal animals and have little or no action on those treated with reserpine. We have previously demonstrated that reserpine treatment has three consequences; it causes the store of noradrenaline-like substance which is present in the walls of the rabbit aorta to disappear; it causes the noradrenaline-like substance in the skin of the rabbit ear to disappear; and it causes the chromaffin cells adjoining the vessels in the rabbit ear to disappear (Burn & Rand, 1958b). These observations suggest that substances like tyramine, which lose their pressor and constrictor action in the reserpine-treated animal, act in the normal animal by releasing a noradrenaline-like substance which, when released, exerts the pressor and constrictor action. Our observations do not indicate whether this is a general release into the blood stream or whether it is only a local release. This suggestion receives support from the observation that the constrictor action of tyramine can be restored in the vessels of the reserpine-treated animals by perfusing noradrenaline through the vessels for a short period. Both during the infusion and also after it, when the constrictor action of this noradrenaline has passed off, the injection of tyramine, or of substances which act like it, once more causes constriction. One of us showed (Burn, 1932a) that a similar effect was obtained by perfusing adrenaline through the vessels. The vessels of the reserpine-treated animals differ from those of the normal animal, not only in being insensitive to tyramine and substances acting like it, but also in being highly sensitive to the action of noradrenaline and adrenaline. Thus 01 jg noradrenaline was seen to have as great a constrictor effect in the perfused hind leg of a reserpine-treated dog as 2-4 ,g noradrenaline in that of a normal dog. When noradrenaline was infused to restore the sensitivity to tyramine, the high sensitivity to noradrenaline was reduced to its normal level. These observations indicate that there is a store of material in or near the vessel walls from which tyramine, ephedrine, amphetamine and other similar substances release either noradrenaline or adrenaline or both. The action of these 'adrenaline-releasers' differs from that of nicotine and of acetylcholine since it is not abolished by hexamethonium. It is thus conceivable that while nicotine and acetylcholine act on the chromaffin cells which Adams-Ray & Nordenstam (1956) have observed in human skin and which Mr E. H. Leach found in the skin of the rabbit ear (Burn & Rand, 1958b), liberating adrenaline from these cells, tyramine, ephedrine and similar amines act by 332 J. H. BURN AND M. J. RAND displacing what may be mainly noradrenaline from the vessel wall. The fact of importance, however, is that the store does not appear to be an inert store, for its presence reduces the response of the vessel to an injection of adrenaline or noradrenaline into the blood stream. From this it appears that there may normally be some discharge from the store, the noradrenaline-like substance which is set free occupying some of the receptors and playing a part in maintaining the tone. If this were not so, it is difficult to understand how the sensitivity of the vessel to injected noradrenaline could be diminished by the presence of the store. We have already demonstrated such a discharge from the store in the heart by its effect on the rate of the pace-maker (Burn & Rand, 1958a). Thus we appear to have in this store a new factor in the control of vascular tone. Our observations have thrown some light on the action of cocaine. The vessels of a cat treated with reserpine react in a similar manner to those of a normal cat which has received an injection of cocaine. In both there is supersensitivity to the action of noradrenaline and adrenaline, and in both there is greatly depressed sensitivity to the action of tyramine and ephedrine. This similarity suggests that the action of cocaine may be to arrest the release of the noradrenaline-like substance from the store. In the presence of cocaine tyramine and ephedrine are no longer able to act as 'adrenaline-releasers', and in the absence of a release the vessel at once becomes much more sensitive to the action of injected noradrenaline or adrenaline. We may note that cocaine also prevents the release by nicotine. One of us has expressed the opinion (Burn, 1956) that the potentiation of adrenaline by cocaine is explained by inhibition of amine oxidase; the present evidence indicates that this opinion is wrong, as others (Bacq, 1949) have contended. It may, however, be wise to keep in mind the possibility that, in the action of cocaine, diminished destruction of adrenaline plays a part. There is also a similarity between tissues denervated by degeneration of their sympathetic supply and the tissues of an animal treated with reserpine. 'After denervation the tissues become supersensitive to adrenaline and noradrenaline, and less sensitive to the action of tyramine and ephedrine. It is interesting to note that Fleckenstein & Bass (1953) and Fleckenstein & Stockle (1955) have demonstrated a close similarity in the behaviour of the nictitating membrane after denervation to its behaviour after treatment with cocaine. Towards the catechol amines it shows supersensitivity in both cases; towards the phenylethanolamines it shows diminished sensitivity in both cases; towards the phenylethylamines it shows greatly diminished sensitivity in both cases. We know from the work of von Euler & Purkhold (1951) that after degeneration of sympathetic fibres there is a loss mainly of noradrenaline from the spleen, the kidney, the liver and the salivary glands. We know from the work of Goodall (1951) of a similar los in the heart. Examination of the effect of SYMPATHOMIMETIC AMINES 333 denervation of the vessels, which has not yet been made, may show a similar loss, and if so it may indicate that the changes in the reaction of vessels caused by denervation are to be explained in the same way as those caused by injections of reserpine. The evidence that an infusion of noradrenaline, either intravenously in the spinal cat or into the reservoir of blood perfusing the hind leg of a dog, in- creases the pressor and constrictor action of tyramine and of other amines which act like it, shows that the store which is in or near the vessel wall can be replenished from noradrenaline or adrenaline circulating in the blood. This demonstration appears to indicate that the output of amines from the adrenal medulla may have some importance for maintaining the store in the vessel wall. One of us (Burn, 1932 b) observed that in the perfused hind leg of the dog sympathetic stimulation became more effective after an infusion of adrenaline, and we have confirmed this observation in the present experiments after an infusion of noradrenaline. We intend to study it further. From the standpoint of the relation between structure and action the difference in the responses to the two isomers of phenylethanolamine is of interest. While the vascular responses to (+ )-phenylethanolamine were similar to those of phenylethylamine, those to (-)-phenylethanolamine were not. In the reserpine-treated animal the latter compound retained a good deal of its pressor action and of its action on the nictitating membrane; moreover, these actions were neither greatly augmented nor greatly depressed by an infusion of noradrenaline. We came to the conclusion that this compound stood midway between 'adrenaline-releasers' like tyramine on the one hand, and adrenaline-like substances on the other. The (+)-isomer, however, was only an 'adrenaline-releaser'. In 1950 Blaschko pointed out the similarity in the potency of ( + )-adrenaline and of epinine; he suggested that this similarity was explained by the position of the -OH group in (+)-adrenaline, this position being such that it was unable to exert any effect on the combination between (+ )-adrenaline and the receptors; ( +)-adrenaline therefore behaved like epinine. The same may be true for ( + )-phenylethanolamine, which behaves like phenylethylamine. (-)-Phenylethanolamine, however, has a configuration exactly similar to that of (-)-adrenaline, and therefore approximates to it in behaviour. If this explanation has substance we would expect that (-)-ephedrine would also resemble (-)-phenylethanolamine. This, however, was not found to be so, and ephedrine seemed to be in the main an 'adrenaline- releasing' substance like tyramine. Ephedrine, however, has a -CH3 group on the a-carbon atom, and perhaps the proximity of this group interferes with the action of the -OH group on the a-carbon atom, so that this group cannot combine with the receptors. In conclusion, it should be emphasized that the conception of tyramine and similar amines as substances which release adrenaline is a hypothesis only, 334 J. H. BURN AND M. J. RAND and it must remain so until a direct demonstration is made. In the perfused rabbit ear, the constrictor effect of tyramine and that of ephedrine, like that of adrenaline and noradrenaline, is reversed by adding tolazoline to the perfusing fluid. The constrictor effect of phenylethylamine and that of amphetamine is, however, not reversed in this way. Yet if the constrictor action of all four substances is due to release of adrenaline or noradrenaline, this action should be reversed by tolazoline in all four cases. We should add that we are also concerned to discover where hydroxytryptamine comes in. We have observed that, in the perfused hind leg of a dog with treated reserpine, the constrictor effect of hydroxytryptamine is reduced by the infusion of noradrenaline. Further, it has been found by Balzer & Holtz (1956) that the pressor action of hydroxytryptamine is somewhat reduced by cocaine. While therefore the observations described in this paper shed new light on old problems, they also reveal problems requiring further study.

SUMMARY 1. When an animal is injected with reserpine the store of noradrenaline- like substance which is present in the vessel wall, and the chromaffin cells adjoining the vessel wall, have been shown to disappear. 2. In the reserpine-treated animal tyramine and many other sympathomimetic amines which are not derivatives of catechol, such as phenylethylamine, ephedrine, amphetamine and ( + )-phenylethanolamine, lose theirpressor action in the spinal cat and no longer cause contraction of the nictitating membrane or of the volume of the spleen. They also lose their constrictor action in the perfused dog's hind leg. 3. In the reserpine-treated animal, noradrenaline, adrenaline, dopamine, epinine and neosynephrine have, on the other hand, a much greater action than in the normal animal. 4. The action of tyramine and substances acting like it can be restored in the reserpine-treated animal by an infusion of noradrenaline into the blood stream. The restoration is observed after the infusion has stopped and after the rise of pressure which it caused has subsided. 5. When an infusion of noradrenaline has restored the action of tyramine, the actions of noradrenaline, adrenaline, dopamine and neosynephrine are once more reduced to their normal size. 6. The observations suggest that tyramine and similar substances normally act by releasing noradrenaline or adrenaline from the store in the artery wall. They also suggest that there is some continuous small discharge from the store, which diminishes the sensitivity of the vessel wall to injected noradrenaline. This discharge probably plays a part in maintaining vascular tone. SYMPATHOMIMETIC AMINES 335 7. The amines liberated from the adrenal medulla into the blood presum- ably help to maintain the store in the vessel wall. We wish to thank Miss Roneen Hobbs for making the observations on the perfused rabbit ear, Professor P. Pratesi of the University of Pavia for the two isomeric forms of phenylethanolamine and Dr H. J. Bein of the CIBA Laboratories, Basle, for a supply of reserpine. The work was done by one of us (M.J.R.) during the tenure of a Fellowship awarded by the Life Insurance Medical Research Fund of Anstralia and New Zealand.

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