Adrenaline and Noradrenaline

Adrenaline and Noradrenaline J . H. GADDUM

AND

MARGARETHE HOLZBAUER

Department of Pharmacology. University of Edi.nburgh, Scotland

Page I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 I 1 . Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 1. Phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 2. Tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 3 . Dopa (~3,4-Dihydroxyphenylalanine). ......................... 4 . Dopamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 5. Noradrenaline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 6 . Dihydroxyphenylserine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 7. p-Hydroxyphenylserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 8. Tyramine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 9. Formation in Depleted Adrenals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 IT1. Fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 1 . Excretion of Free Catecholamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 2. Excretion of Conjugated Catecholamines . . . . . . . . . . . . . . . . . . . . 160 3 . Destruction of Catecholamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 TV . Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Invertebrates . . . . . . . . . . . . . . . . . . . . . 2 . Metabolic Effects . . . . . . . . . . . . . . . . . . . . . . . 3 . Circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Smooth Muscle . .......................................... 168 5. Skeletal Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 6. Peripheral Nerves . . . . . . . . ........................ . . . . 170 7. Autonomic Ganglia . . . . . . . .................................... 170 171 8. Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 9. Anterior Pituitary Gland and Adrenals., . . . . . . . . . . . . . . . . . . . . . . . . . . V . Estimation . . . . . . ...................... . . . . . . . . . . . . . . . 172 173 1. Preliminary Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Bioassays . . . . . . . . . ................. .................................... 175 b . Rat’s Uterus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 c . Blood Vessels of the Rabbit’s Ear . . . . . . . . . . . . . . . . . . . . d . Intestinal Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Colorimetric Methods . .......................... 177 4. Fluorimetric Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................... 178 .................................... 178 VI . Distribution . ................................................ 179 1. Lower Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 2. Adrenal Medulla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 3 . .4ccessory Chromaffin Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 4 . Chromaffin-Cell Tumors (Pheochromocytomata) . . . . . . . . . . . . . . . . . . . . 181 151

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Paye

5. Peripheral Nerves ............................... 6. Central Nervous System.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Other Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Body Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V11. Release from the Adrenal Glands.. . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Direct Actions on the Adrenals.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Reflex Control of the Adrenals.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Resting Secretion with Splanchnic Nerves Intact.. . . . . . . . . . . . . . . . b. Reflexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Carotid Sinus.. .. .................................... d. Blood Sugar.. . . . ................................... e. Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f. Other Factors .... ............. 4. Independent Release of Adrenaline and Noradrenaline. . . . . . . . . . . . . . . VIII. Release from Adrenergic Nerves.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IY.Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . ......................................

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I. INTRODUCTION

The presence in the adrenal glands of a substance which gives a red color on oxidation was discovered by Vulpian (1856). This substance was eventually shown to be mainly adrenaline and noradrenaline. Oliver and Schafer (1894) and Szymonowicz (1896) working with Cybulski, independently discovered the pressor action of adrenal extracts, and adrenaline was isolated (Abel and Crawford, 1897; Takamine, 1901) and synthesized (Stolz, 1904; Dakin, 1905) a few years later. No general account will be given of early work on this substance, which has been reviewed many times (Trendelenburg, 1929; Hartung, 1931; Rogoff, 1935; Grollman, 1936; Cori and Welch, 1941; Hartman and Brownell, 1949). I n America, the official name is epinephrine and in Britain, it is adrenaline; Adrenalin is a trade name. Noradrenaline (arterenol) is now recognized to be the main substance liberated by postganglionic sympathetic nerves. Much work has been done on this substance in recent years and this work will be discussed in some detail. It has been reviewed by von Euler (1951a, 1956). Lewandowsky (1899) suggested, and Elliott (1904) proved, that the effects of stimulation of postganglionic sympathetic nerves corresponded closely with the effects of adrenaline. Elliott then made the brilliant suggestion that these nerves produced their effects by liberating adrenaline. Loewi (1921) proved that the sympathetic nerves in a frog’s heart actually did liberate a substance resembling adrenaline, and a t about the same time Cannon and Rapport (1922) found that stimulation of sympathetic nerves liberated a substance which was carried by the blood stream and

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produced effects in other parts of the body. Subsequent work in Cannon’s laboratory showed that two adrenaline-like substances appeared in the blood when adrenergic nerves were stimulated (Cannon and Rosenblueth, 1933). Barger and Dale (1910) had called substances acting like adrenaline “sympathomimetic amines,” and they studied the properties of a long series of such substances more or less closely allied to adrenaline in their chemical structure and pharmacological actions. Noradrenaline mas one of these substances, and Barger and Dale came to the inspired conclusion that its action corresponded more closely with that of sympathetic nerves than did that of adrenaline. A t that time the only hint of chemical transmission was Elliott’s speculation that sympathetic nerves acted by liberating adrenaline. Barger and Dale rejected the theory that noradrenaline acted by liberating adrenaline but did not suggest that it might itself be the substance liberated by the nerves. There was no evidence for the natural occurrence of noradrenaline, which had only become available by synthesis, and the natural occurrence of druglike substances in the body was not so clearly recognized then as it is today; auto-pharmacology (Dale, 1933a) was still unborn. The discussion of the mode of action of sympathetic nerves became complicated when it was found that sympathetic preganglionic nerves (Feldberg and Gaddum, 1934) and some sympathetic postganglionic nerves (von Euler and Gaddum, 1931) liberated acetylcholine, so that the pharmacological classification of nerves did not correspond exactly with the anatomical classification. Dale (193313) therefore introduced the terms “adrenergic” and “cholinergic” to facilitate discussion, and in view of the uncertainty of the exact nature of the substances liberated, he defined adrenergic nerves as nerves which acted by liberating a substance like adrenaline. Unfortunately, the term “adrenergic” is sometimes applied to drugs as if it meant the same thing as sympathomimetic. The results of Cannon and his colleagues drew general attention to the problem of the nature of the adrenergic transmitter, and it became clear that several of the sympathomimetic amines studied by Barger and Dale had marly of the required properties. Bacq (1934) was the first to publish the theory that adrenergic nerves liberated a mixture of adrenaline and noradrenaline. The physiological importance of noradrenaline was established by other methods. In a paper submitted for publication in 1944, but published three years later Holtz et al. (1947) presented evidence that urine and adrenal extracts both contained a substance resembling noradrenaline rather than adrenaline in its pharmacological properties. Von Euler (1946a,b) found that extracts of spleen and splenic nerves also contained

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a substance resembling noradrenaline and later (von Euler, 1948a) obtained good evidence that it actually was L-noradrenaline. These results confirmed the theory th at the main substance liberated by adrenergic nerves was noradrenaline itself and not some other closely allied substance. Convincing direct evidence for this conclusion was obtained by Peart (1949) who found that comparatively small amounts of adrenaline were also liberated. Small amounts of noradrenaline were found mixed with the adrenaline in extracts of adrenal glands by independent work in various laboratories (Holtz et al., 1947; Holtz and Schumann, 1949; von Euler and Hamberg, 1949; Coldenberg el al., 1949) and larger amounts in adrenal medullary tumors by Holton (1949). This pharmacological evidence was confirmed when Tullar (1949) isolated L-noradrenaline from adrenal extracts and identified i t by its chemical properties. Evidence for the presence in the body of a third highly active allied substance (isopropyl noradrenaline) has recently been obtained by 1,ockett (1954, 1956).

Ir. FORMATION

T he main metabolic pathway leading to adrenaline is probably that shown by continuous arrows in Fig. 1. The conversion of phenylalanine to adrenaline involves five changes in the molecule; theoretically there are 120 different orders in which these changes might occur and 30 possible intermediates, but only a few of these need be considered. The various possible precursors will be discussed separately. 1. Phenylalanine

Gurin and Delluva (1947) labeled phenylalanine either with H3in the ring or with C14 in the side chain and gave them by mouth or intraperitoneally t o rats. Tracer-free adrenaline was then added t o extracts of adrenals and radioactive adrenaline was isolated. Udenfriend and Wyngaarden (1956) obtained a similar result after several daily intraperitoneal injections of phenylalanine labeled with CL4.These results show that phenylalanine is a precursor of adrenaline. There was some evidence that the side chain remained attached to the benzene ring. L-Phenylalanine is converted to tyrosine by a specific enzyme systrni, found in rat's liver, which is active in the presmcr of DPN and oxygen (ITdenfriend arid Cooper, 1952; Lerner, 1953). 2 . Tyrosine

There is evidence that tyrosine is converted to dopa both by enzymes and by nonenzymatic processes (Raper, 1926, 1932; Lerner, 1953). Udenfriend and Wyngaarden (1956) injected ~ ~ - t y r o s i n e - a - C in' ~rats and ob-

ADRENALINE AND NORADRENALINE

a-c Amines

Amino acids

*&&?-CH--N 5

1

Phenylalanine

-

HO

H

Phenylethylamine H O - ~ -CH~-CH,-N

HO-~-CH,-CH-NH,

I

H~-cH?--N

Ht LOOH

6

155

I

H

COOH Tyrosine

Tyramine

1

HO I

I

Dopa

Dopamine (Hydroxytyramine)

( IAh ydroxyphenylalanine) I

HO I

1

3,1Dihydroxyphenylserine

HO

HO

I

H O - o -CH-cH-N

I

I

1

Noradrenaline (Arterenol)

HcHB H O - - d - C H - c HI ? - N

HcHs

OH

OH COOH

FIG.1 .

Adrenaline (Epinephrine)

tained direct evidence that tyrosine is a precursor of adrenaline by iso1at)inglabeled adrenaline from the adrenals. 3 . Dopa (L-3,4-Dihydrox yphen ylalanine)

Most workers believe that this substance occupies a key position in the synthesis of adrenaline. This view was originally based on studies of the active and specific enzyme known as dopadecarboxylase (Blaschko, 1939), but there is now other evidence. For example, a spot attributed to dopa has been found in paper chromatogranis of extracts of the adrenals of thyroidectomized sheep (Goodall, 1951), and dopa has also been found in a pheochromocytoma (Weil-Malherbe, 1956). Van Arman (1951) de-

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J. H. GADDUM AND MARGARETHE HOLZBAUER

pleted the adrenals of rats with insulin and studied the effects of various possible precursors on the reappearance of adrenaline in the glands during the next 6 hours. Dopa was effective, but phenylalanine, tyrosine, tyramine, phenylserine, and even noradrenaline had no effect. Demis et al. (1956)and Hagen and Welch (1956)incubated dopa-a-C14 with adrenal homogenates and recovered most of it as radioactive dopamine and a small amount as radioactive noradrenaline. Udenfriend and Wyngaarden (1 956) injected dopa-&C14 intraperitoneally for several days in rats and recovered large amounts of radioactive adrenaline from the gland. Dopadecarboxylase was discovered by Holtz et al. (1939),who incubated extracts of guinea pig’s kidneys with dopa, and its action in z+o was demonstrated by Holtz and Credner (1942),who gave dopa by the mouth to man and animals. Dopadecarboxylase acts specifically on the lev0 isomer of dopa to f o m dopamine (Blaschko, 1939). It, or some similar enzyme, also acts slowly on dihydroxyphenylserine (Blaschko, 1950; Holtz and Westermann, 1956), but it does not act at all on N-methyl derivatives of these substances. The fact that dopa is decarboxylated rapidly by these enzymes, while allied substances are decarboxylated slowly or not at all, is evidence that it is an important metabolite. Extracts of the livers of rats fed on pyridoxine-deficient diets had less than the normal dopadecarboxylase activity and this was restored by the addition of pyridoxal-5-phosphate in uitro. This substance thus appears to act as coenzyme (Blaschko el al., 1948; Blaschko, 1950). Pyridoxine deficiency did not affect the adrenaline content of the adrenals of resting rats but diminished the amounts present after depletion with insulin (Blaschko el al., 1951). Dopadecarboxylase was found in the adrenal medulla by Langemann (1950,1951) and Sourkes et al. (1952).Like histidine decarboxylase, i t is in the supernatant fluid after high speed centrifugation (Blaschko et al., 1955). If pyridoxal phosphate is added, the enzyme can be detected in extracts of adrenergic nerves and in the spinal cord, medulla, brain stem, hypothalamus, thalamus, and caudate nucleus. There is little or none in cholinergic nerves (Iloltz and Westermann, 1956). The distribution of this enzyme is thus consistent with the theory that it is essential for the formation of noradrenaline. The effects of competitive inhibitors on dopadecarboxylase have been studied by Hartman et al. (1955a).The most potent known inhibitor is 5-(3,4-dihydroxycinnamoyl)-salicylicacid, which was effective when present in 10-3 times the concentration of the substrate. Clark et al. (1956) found that the intravenous injection of dopa caused a rise of blood pressure in pithed cats and obtained evidence that this was due to the forma-

ADRENALINE AND NORADRENALINE

157

tion of dopamine. This rise of blood pressure was antagonized by the drugs which inhibited the enzyme.

4. Dopamine This substance has been found in urine and in extracts of adreiials and heart (Holtz and Credner, 1942; Goodall, 1951; Shepherd and West, 1953). Adrenergic nerves. and sympathetic ganglia may contain about equal amounts of dopamine and noradrenaline (Schumann, 1956). This finding suggests that dopamine is converted to noradrenaline by hydroxylation in the axons of nerves. Holtz and Kroneberg (1949) incubated adrenal slices with dopamine and observed an increase in the pressor action of the suspension fluid. This change may have been due to the formation of noradrenaline by the introduction of an OH group in the side chain. It did not occur when allied substances which already contained the OH group in the side chain, but not the two OH groups in the ring, were used. Hagen and Welch (1956) demonstrated the transformation of labeled dopamine to labeled noradrenaline by centrifuged particle-free adrenal homogenates. Leeper and Udenfriend (1956) injected d0~amine-a-C'~ in rats and recovered radioactive adrenaline from the adrenals in even larger proportions than those recovered from dopa. 6. Noradrenaline The fact that N-methyl compounds are not decarboxylated suggests that methylation occurs after decarboxylation, and this view is confirmed by the fact that the methylation of noradrenaline to form adrenaline has been demonstrated in perfused adrenals (Biilbring and Burn, 194913) and in minced adrenal tissues (Biilbring, 1949). 6. Dihydroxyphenylserine

This substance resembles dopa but contains the hydroxyl group in the side chain; it can be regarded as the carboxylic acid of noradrenaline. It is slowly decarboxylated by extracts of guinea pig kidney to form noradrenaline (Blaschko et al., 1950). When given by injection to rabbits it caused an increase in the amount of noradrenaline in the urine (Schmiterlow, 1951). When it was incubated with homogenates or extracts of nervous tissues or liver in vitro it caused a small increase in the COzproduction and the formation of noradrenaline (Hartman et d., 1955b; Werle and Jiintgen-Sell, 1955; Holtz and Westermann, 1956). These results suggest a second metabolic pathway in which the hydroxyl group is inserted in the side chain before decarboxylation occurs. The decarboxylation of dihydroxyphenylserine is, however, a much slower process than the de-

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J. H. GADDUM AND MARGARETHE HOLZBAUER

carboxylation of dopa and is probably of minor importance. There is evidence that these two amino acids are decarboxylated by different enzymes (Werle and Juntgen-Sell, 1955). 7. p-Hydroxyphenylserine

This substance is slowly decarboxylated by extracts of various mammalian organs (Werle and Peschel, 1949). The p-hydroxyphenylethanolamine formed in this way is also known as octopamine because it has been found in the salivary gland of an octopus (Erspamer, 1952). This suggests another possible metabolic path, but probably not an important one. 8. Tyramine

Tyramine was isolated from urine and from various tissues many years ago, but it was not realized a t that time that tyrosine is rapidly decarboxylated by bacteria (Gale, 1946), and no precautions were taken to avoid the post-mortem formation of tyramine. There is some evidence that tyrosine is decarboxylated by minced kidney tissues (Holtz, 1937)) but apart from this fact there is no reason to believe that tyramine is normally present in the body. Blaschko (1939) did not detect this change. Some of the work on this question was shown to be based on an unspecific test for tyramine (Verney and Vogt, 1938). Schuler and M’iedemann (1935) tested the hypothesis th a t tyramine might be converted in the body to dopamine by incubating tyramine with adrenal slices. They observed an increase in the pressor activity and in the estimate of adrenaline by Folin’s method, but did not succeed i n establishing this as an important metabolic pathway. T h e observed effects may have been due to the inhibittion of amine oxidase. Udenfriend and Wyngaarden (1956) injected phenylethylan~ine-P-C’4and tyramine-cu-Cl4 in rats and failed to recover radioactive adrenaline from the adrenals. 9. Formation in Depleted Adrfnafs

The total adrenaline content of dogs’ adrenals is about 200 pg./kg. of dog, which is the amount liberated during more than 24 hours at the resting rate of secretion, or more than 30 minutes a t the maximum rate (SatakB, 1955, see below). These figures illustrat,e the well-known fact that the adrenal medulla contains a large store of its active principles. This store can be exhausted by appropriate stimulation, but this stimulation must be prolonged. When the glands have been depleted of adrenaline and noradrenaline by various procedures, the reaccumulation of these substances takes several days (Satow, 1938; Arman, 1951; Udenfriend et al., 1953).

ADRENALINE: A N D NORADRENALINE

159

The adrenal glands of rabbits normally contain adrenaliiie, but. Iitt,Ie or no noradrenaline. Hokfelt (1951) found that after large doses of insulin (6 units/kg.) considerable amounts of noradrenaline appeared in t,he adrenals in a few hours. Outschoorn (1952a) observed a similar absolute increase in the noradrenaline in cats’ adrenals 2 hours after administration of insulin, when there was no appreciable depletion of adrenaline. It is possible that both these results were due to the release of adrenaline and partial resynthesis, the final stage of which was incomplete. In some of Hokfelt’s experiments the splanchnic nerve was cut on one side, and it was found that after 6 days the normal gland had recovered almost completely, while the denervated gland still contained significant amounts of noradrenaline and less than the normal amount of adrenaline. Resynthesis appears to depend in some way on the splanchnic nerves. Butterworth and Mann (1956) found that after depletion tiy acetylcholine there was little increase in the amounts of catecholamines in cats’ adrenals in 2-3 days, but after a week the noradrenaline content had risen to many times the depleted level, while that of adrenaline had risen much less. It is clear that both the amounts of amines and their composition, in the adrenals, are likely to depend on the experiences of the animal during the previous week. 111.

FATE

When adrenaline is injected, it rapidly disappears from the plasma, being taken up by various organs. This uptake may occur so quickly that SO-SO% of a dose of adrenaline is removed from the blood during a single passage through the liver or the lower limbs (Dawes, 1946; Celander and Mellander, 1955). It is probable that adrenaline passes through cell membranes before it is metabolized, and the rate of its disappearance from the blood may depend on the rate of this process rather than on its destruction. However, although these considerations may be important in experiments where single doses are injected, it is improbable that any organ could continue to remove adrenaline from blood for long without destroying it. Significant amounts of catecholamines may be removed by the receptors on which they act (Brown and Gillespie, 1956). Bain and associates (1937) found that when adrenaline was added to blood in vitro nearly half of it was taken up by the cells in 2 hours and could be recovered by hemolyzing the blood. When adrenaline labeled in the alpha or beta position with CI4is injected into rats, practically all the radioactive carbon can be recovered from the urine (Schayer, 1951a; Schayer and Smiley, 1953). It should, therefore, he possible to find all the final products in the urine.

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J. H. GADDUM A N D MARGARETHE HOLZBAUEH

I . Excretion of Free Catecholamines

Cybulski (1895) injected adrenal extracts and detected increased pressor activity in the urine. Holtz et al. (1947) showed that certain extracts of human urine caused a rise of blood pressure in rabbits and inhibition of rabbit’s gut. They gave the name “urosympathin” t o the active substance and came to the conclusion that it consisted of a mixture of adrenaline, noradrenaline, and dopamine. Later work (Kroneberg and Schumann, 1950; von Euler and Hellner, 1951; von Euler et al., 1951; G. P. Burn, 1953; Crawford and Law, 1957) has confirmed the normal presence of free adrenaline and noradrenaline in the urine of man and rats and shown that dopamine is present in even larger quantities but contributes little to the biological activity. According to von Euler and Hellner (1951), normal human urine collected during 24 hours contains 11.5 k 6 (S.D.) pg. of adrenaline and 29 k 12.3 pg. of noradrenaline. During sleep the rate of excretion was about one-fourth of this. When adrenaline was injected subcutaneously in rats, 5-10% of the dose appeared as free adrenaline in the urine (Schayer, 1951b; Crawford and Law, 1957). When noradrenaline was infused intravenously in man (16.4-28 pg. per minute), 1.5-3.3% of the dose appeared as free noradrenaline in the urine; there was no increase in the amount of adrenaline in the urine in these experiments after the injection of noradrenaline (von Euler and Luft, 1951).

2. Excretion of Conjugated Catecholamines Acid hydrolysis of the urine of healthy humans or normal animals usually leads to a small increase in the amount of active adrenaline or noradrenaline (e. g. Kroneberg and Schumann, 1950). This suggests t.hat under physiological conditions conjugation plays a minor part in the inactivation of catecholamines. If large amounts of these substances are given by mouth, large amounts of conjugates appear in the urine. This phenomenon was first described by Richter (1940), who swallowed 15-55 mg. of adrenaline and recovered 30-70% from the urine as a conjugate which liberated adrenaline on acid hydrolysis. Richter suggested that this substance might be a sulfate ester, but there is no evidence for this theory and definite evidence against it. The main facts were confirmed by Richter and MacIntosh (1941), Beyer and Shapiro (1945), and Schayer (1951b), but the conditions for quantitative recovery of the adrenaline are not known. If the boiling is continued until hydrolysis is complete, some of the adrenaline is inactivated. There is good evidence that adrenaline is excreted in conjugation with glucuronic acid. After giving adrenaline by mouth to rabbits, Clark et al.

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ADRENALINE AND NORADRENALINE

(1951) and Clark and Drell (1954) isolated nearly pure adrenaline monoglucuronide from the urine. The elementary analysis agreed with expectations and there can be no real doubt that this substance was correctly identified. Only 70% of the theoretical amount of adrenaline was recovered after acid hydrolysis, but this was to be expected. There was some evidence that this substance was hydrolyzed by a preparation of glucuronidase but not by a preparation of sulfatase. There is no evidence that the percentage of conjugation is increased when large amounts of catecholamines are liberated by an adrenal medullary tumor.

3. Destruction of Catecholamines Adrenaline is very stable at pH 4,but is rapidly inactivated by oxygen in neutral or alkaline solutions. This change is inhibited by serum and

CHe

Catechol oxidase, etc.

I

CHI Adrenochrome

AH3 Adrenoluh e

NH

Adrenaline

3,4-Dihydroxymandelaldehyde

3,4-Dihydroxymandelic acid

FIQ.2.

simple extracts of various tissues (Wiltshire, 1931). Reducing agents are grams/ml.) is commonly particularly effective and ascorbic acid ( used to preserve dilute solutions of adrenaline for experimental work. Some of the oxidation products of adrenaline are shown in Fig. 2. It is easily oxidized in vitro t o form the pink substance adrenochrome, and in the presence of alkali this may be converted to the fluorescent substance adrenolutine and various other substances (Guggenheim, 1951). Noradrenaline undergoes similar changes.

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The actions of phenolases and similar enzymes (catechol oxidase, cytochrome oxidase, etc.) favor the formation of adrenochromes. Bacq (1949) lays great emphasis on this mechanism for the inactivation of adrenaline, which may be especially important because some of the oxidation products of catecholamines have physiological actions which may be the cause of some of the prolonged effectsof adrenaline on metabolism, on capillaries, and on the central nervous system. However, it has not been proved that adrenochrome is formed in the body, which is k n o w to contain substances which prevent this form of oxidation. Schayer and Smiley (1953) failed to confirm this theory by experiments with radioactive substances. They gave labeled adrenochrome to rats and prepared paper chromatograms from extracts of the urine. The distribution of radioactive substances in these chromatograms was quite different from that seen in similar chromatograms prepared after the administration of labeled adrenaline. If adrenochrome were an important metabolite of adrenaline in the body, its chromatographic pattern should appear in the urine after the administration of adrenaline. Schayer and Smiley do, however, discuss the possibility that adrenochrome is formed and metabolized without being released into the general circulation. The oxidation of adrenaline by this route is inhibited by cyanide, but Blaschko et al. (1937) found that, some tissues contain another enzyme which destroys adrenaline and is cyanide-resistant. This enzyme is now known as amine oxidase and its properties have been reviewed by Blaschko (1952). It is widely distributed in the animal kingdom and is bound to cytoplasmic particles. It reacts with many monoanlines when they are in the ionized state, with the formation of an imino compound and H202. The over-all reaction in the presence of catalase has been as written as follows:

+

R-CH2NHR’R”

+ +Oz

= R-CHO

+

+ NHzR’R”

With adrenaline or noradrenaline it forms 3,4-dihydroxymandelaldehyde (see above). It has a similar but quicker action on dopaniine and 5-hydroxytryptamine and various other simple amines; it a& on long(!hain diamines, but not on short-chain diamines. When a methyl group is present in the alpha position, the forniation of an aldehyde is impossible and certain compounds containing the group R C H ( C H I ) . N Hresist ~ oxidation but appear to combine with the enzyme and act as competitive inhibitors. The first compound of this type to be studied was ephedrine, but amphetamine and a number of other such compounds have a similar action. The inhibitory effects of iproniazide (l-isonicotinyl-2-isopropylhydrazine,marsilid ; Zeller et al., 1952) or of

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163

choline-p-tolyl ether (Brown and Hey, 1956) are particularly great, and these substances have been used to inhibit the enzyme in metabolic studies. Low concentrations of ephedrine increase the effects of adrenaline or of the stimulation of adrenergic nerves (e.g. on the rabbit’s ear, the cat’s nictitating membrane, and the frog’s heart). Gaddum and Kwiatkowski (1939) suggested that this effect was due to the inhibition of amine oxidase in the region of the adrenaline receptors, and they showed that ephedrine increased the amount of adrenaline which could be detected in the perfusate after stimulation of the sympathetic nerves to the rabbit’s ear. This theory received support from evidence of various kinds obtained in Oxford (J. H. Burn, 1952). It led directly to the discovery that choline-ptolyl ether is a powerful inhibitor of aniine oxidase (Brown and Hey, 1956). It was predicted that amine oxidase would be found in the arteries of a rabbit’s ear and this prediction was confirmed by Thompson and ‘I’ickner (1951). The main objections t o this theory are that: (1) Ephedrine sensitizes tissues not only to adrenaline but also to corbasil (cobefrin, a-methylnoradrenaline) , and corbasil is not destroyed by amine oxidase (Jang, 1940; Gaddum, 1950a). (2) The histochemical studies of Koelle and Valk (1954) lend no support t o the view that amine oxidase is concentrated near adrenergic nerve endings. (3) Although inhibitors of amine oxidase often cause potentiation, other drugs may have a similar effect (Kamijo et al., 1956). Some of the motor effects which appear to be produced by ephedrine it.self may he due to the inhibition of amine oxidase, but some of them are not (Gaddum and Kwiatkowski, 1938). Concentrations of ephedrine higher than those causing potentiation antagonize the actions of adrenaline-possibly by acting as competitive inhibitors a t the motor receptors. Jang (1940) has drawn attention to the fact that some of the best-known adrenaline antagonists (ergotamine, yohimbine, piperidylmethylbenzodioxane, etc.) resemble ephedrine in that low concentrations increase the effects of adrenaline. It has been suggested that these substances produce this effect by preventing the uptake of catecholamines by the cells, so that they do not come in contact with the oxidizing enzymes associated with intracellular particles (Blaschko, 1954). The t,heory that amine oxidase plays an important part in the metabolism of adrenaline is supported by the work of Schayer and his colleagues, using adrenaline isotopically labeled with C14 in different positions. Racemic adrenaline was used in the early experiments, but the results were confirmed with small quantities of the natural isomer L-adrenaline.

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J. H. GADDUM AND MARGARETHE HOLZBAUER

After the subcutaneous injection of j3-Cl4-~-adrenalinein rats (0.05 mg./kg.) approximately 100% of the radioactivity was recovered from the urine within 17 hours (Schayer et al., 1952). Paper chromatograms of such urines showed at least five distinct radioactive peaks which presumable represent five different metabolites of adrenaline, all containing j3-C14 (Schayer, 1951a). I n similar experiments with N-methyl-CI4 adrenaline, an average of 61% of the radioactivity was recovered from the urine. The difference between these two recoveries indicates that the urine must contain considerable quantities of a compound from which the N-methyl group, but not the j3-carbon group, has been removed. Significant amounts of carbon derived from the methyl group could be detected as radioactive C o nin the expired air, but it is probable that t,he urine also contained metabolites derived from the methyl carbon. The difference between the recoveries with adrenaline labeled in different positions (39%) therefore represents a minimum estimate of the percentage of the adrenaline undergoing fission between the j3-carbon and the methyl carbon. This metabolic change might be due to a transmethylase or a demethylase, but there is evidence for the alternative view that it is due to amine oxidase. This enzyme is inhibited by iproniazide, and when iproniazide was administered to rats shortly before the administration of methyl-C14-adrenaline, almost all the radioactivity was recovered from the urine (Schayer and Smiley, 1953). Amine oxidase would be expected to lead t o the formation of methylamine and the ether-soluble compounds, dihydroxymandelaldehyde and dihydroxymandelic acid. About 20% of the total urinary radioactivity after the administration of a- or p-Cl4-adrenaline was recovered from ether-soluble fractions and was probably due to these two substances (Schayer, 1951b). This view is consistent with the fact that the administration of methyl-C14-adrenaline did not lead to the appearance of ethersoluble radioactive substances in the urine; ether-soluble substances appear to be formed only after the methyl group has been removed. It is clear, however, that these results do not explain the whole of the original discrepancy between the results obtained with adrenaline labeled in different positions. There must be some other metabolite formed which does not contain the methyl carbon and which is not soluble in ether. Paper chromatograms of urine after the administaration of methylC14-adrena1ineshow three radioactive peaks, one of which is presumably due t o the adrenaline. The other two might have been due to products formed from methylamine, but a comparison with chromatograms prepared after the injection of radioactive methylamine suggested that this was unlikely and that they were due to two unknown substances retaining the methyl group (Schayer et al., 1952).

ADRENALINE AND NORADRENALINE

1ti5

In summary, the tentative conclusions of these workers are that about 50% of the adrenaline is inactivated by amine oxidase to form methylamine, which is partly oxidized to COz and partly excreted in the urine as metabolites. The remainder of the molecule after demethylamination is converted into a t least three metabolic products-dihydroxymandelaldehyde, dihydroxymandelic acid, and a third compound, which is not soluble in ether and has not been identified. Apart from adrenaline itself, the urine also contains two other unidentified substances retaining the methyl group.

IV. ACTIONS Barger and Dale (1910) concluded that the essential difference between the actions of adrenaline and noradrenaline was that noradrenaline failed to reproduce the inhibitor actions of adrenaline. These inhibitory actions also differed from excitor actions in that it was difficult or impossible to inhibit them with ergotoxine. Later work has shown that practically all the inhibitor actions of adrenaline are also given by noradrenaline, though many of them require larger doses, so that the difference between these two drugs is probably quantitative rather than qualitative. When they produce opposite effects on the blood pressure, the result can be explained on the theory that both drugs tend to raise the blood pressure by one mechanism and to lower the blood pressure by another mechanism, so that the resultant effect is the balance of the differentactions of the two drugs on these two mechanisms. In classifying the effects of these two drugs, it is convenient to calculate the ratio of the dose of noradrenaline to the dose of adrenaline having the same action. This ratio should be high for inhibitor actions and low for excitor actions, and this is often found to be the case. The dose ratio is generally high for metabolic actions which thus resemble inhibitor actions on smooth muscle. Barger and Dale’s classification does not seem appropriate for metabolic effects, and for this and other reasons it is sometimes convenient to speak of alpha effects and beta effects (Ahlquist, 1948; Gaddum, 1950a). The alpha effects include the excitor effects of Barger and Dale and the inhibitor effects on some intestines. They are readily produced by noradrenaline and inhibited by ergotoxine and other “adrenolytic” drugs (Nickerson, 1949). The beta effects include inhibition of the blood vessels in voluntary muscles and inhibition of certain kinds of uterus and the metabolic effects. They are more readily produced by adrenaline than by noradrenaline and are not easily inhibited hy ergotoxine. The natural isomers L-adrenaline and L-noradrenaline are both much more active than the D-isomers.

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J. H. GADDUM A N D MARGARETHE HOLZBAUER

I . Invertebrates

The actions of adrenaline on invertebrates have been reviewed by Bacq (1947). The hearts of molluscs are generally stimulated by adrenaline or noradrenaline in fairly high concentrations, but the heart of Cardium edule is inhibited (Gaddum and Paasonen, 1955). The muscles of the intestinal tract of invertebrates, unlike those of vertebrates, are stimulated. 2. Metabolic E f e c t s

The metabolic effects of adrenaline and noradrenaline have beer1 reviewed by Trendelenburg (1929) and Lundholm (1949). Von Euler (1956) has collected much evidence about the differences between the actions of adrenaline and noradrenaline. Adrenaline causes glycosuria (Blum, 1901), hyperglycemia (Zuelzer, 1901), and a fall in the glycogen content of the liver (Doyon and Kareff, 1904). These facts suggest that it causes liver glycogen to be liberated as blood glucose, and there is much evidence that this is so. The hepatic glucose output, determined in man by catheterization of the hepatic vein, increases considerably (Bearn et al., 1951). The effect can be studied in tissue slices and, according to Sutherland and Cori (1951), it is due t o the activation of a phosphorylase which converts glycogen to glucose-l-phosphate which then becomes glucose-6-phosphate and eventually glucose. The hyperglycemia is also partly due to a decrease in the uptake of glucose by voluntary muscles (Cori, 1931; Cori et al., 1935; Somogyi, 1950, 1951). This is accompanied by an increased formation of lactic acid from muscle glycogen, which is thus decreased by two different mechanisms. The loss of glycogen can be followed in vitro with isolated muscle in which there is an accumulation of glucose-6-phosphate (Hegnauer and Cori, 1934; Walaas and Walaas, 1950). It has been suggested that this effect is due t o adrenochrome but this does not appear t o be the case (Walaas and Walaas, 1956). Most of these effects on carbohydrate metabolism are also produced by noradrenaline in doses 15-20 times (Schumann, 1949) as great as those of adrenaline (see von Euler, 1956). Adrenaline causes amino acids to disappear from the blood. Noradrenaline had no definite effect in 10 times the dose (Crismon et al., 1940; Brunish and Luck, 1952). An intravenous injection of adrenaline causes a rise in the potassium content of arterial serum lasting 5-7 minutes. Noradrenaline has the same effect but is less active. The rise is followed by a more prolonged fall in the potassium level (D’Silva, 1949).

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167

Subcutaneous injection of adrenaline (10 pg./kg.), or intravenous infusion (0.05 pg./kg./min.), increases the oxygen consumption of man and animals by 20% or more for an hour or two. This phenomenon was discovered by Belawenez (1903). According to Lundholm (1949, 1951), it is due to the oxidation of increased amounts of lactic acid. Noradrenaline has much less effect (see Griffith, 1951). 3. Circulation

Much has been written about the effects of adrenaline on the circulation (McDowall, 1938). The action was discovered by Oliver and Schafer (1895) who found that large doses of adrenal extracts caused a large rise of blood pressure in dogs. When the vagi were cut, the force and rate of the heart increased, but when they were intact the heart was slowed reflexly. The rise of blood pressure was largely due to peripheral vasoconstriction, since it occurred even when the heart was reflexly inhibited and since it. was accompanied by a fall in the volume of limbs and internal organs. Both adrenaline and noradrenaline produce these effects when large doses are injected intravenously. When smaller doses are used, the effects of these two drugs differ in the following ways: (1) The reflex inhibition of the heart is more marked with noradrenaline than with adrenaline. (2) Noradrenaline still increases the peripheral resistance, but adrenaline may decrease it. The result is that adrenaline increases the cardiac output in the whole organism and noradrenaline diminishes it (von Euler and Liljestrand, 1927; Starr et al., 1937; Goldenberg el al., 1948). With certain anesthetics, when the blood pressure is high, small doses of adrenaline cause a fall of blood pressure (Moore and Purinton, 1900). After the injection of ergot alkaloids (Dale, 1905) or other antiadrenalines, all doses of adrenaline cause a fall of blood pressure. It is probable that the mechanisms of the two depressor actions are the same; noradrenaline does not normally cause either effect. Dale and Richards (1918) showed that the depressor effect of small doses of adrenaline was largely due to dilatation of capillaries and the smallest arterioles in voluntary muscles but not in skin. This paper helped to establish the importance of active changes in small vessels, and its conclusions have been confirmed by much later work. Adrenaline dilates the blood vessels in voluntary muscles in conditions where the vascular tone is high, but its effect on most other blood vessels is mainly constrictor. It increases the circulation in the heart and voluntary muscles and decreases the circulation in the skin. Noradrenaline is a more general vasoconstrictor and may increase the

168

J. H. GADDUM A N D MARGARETHE HOLZBAUER

tone of blood vessels which are then dilated by adrenaline (Meier and Bein, 1948). Both drugs dilate the coronary vessels and increase the coronary flow (WBgria, 1951; Schofield and Walker, 1953). Their direct actions on the skin vessels, on the other hand, are almost purely vasoconstrictor, although Gowdey (1948) found that after 2-benzylimidazoline (Priscol) both caused vasodilatation in the perfused ear of a rabbit. I n this preparation adrenaline is a slightly more active vasoconstrictor thaii noradrenaline (Burn and Hutcheon, 1949), but in other tissues, where mixed effects occur, the net vasoconstrictor effect of noradrenaline is the greater. The pressor action of noradrenaline in the whole cat appears greater than that of adrenaline when the meant blood pressure is recorded; noradrenaline raises both systolic and diastolic pressure in man, while adrenaline may raise the systolic pressure and lower the diastolic pressure (Goldenberg et al., 1948). A detailed account of the effects of these substances on the human blood vessels is given by Barcroft and Swan (1953), who conclude that adrenaline increases the blood flow in human voluntary muscle, liver, and brain and decreases them in the skin and kidney, while noradrenaline decreases blood flow in the skin, kidney, and braiti and has little or no effect on blood flow in voluntary muscle or liver. Much work has been done on the direct actions of these drugs on isolated hearts and hearts protected from reflex inhibition by atropine or by section of the vagi. The force and rate of the beat are increased and conduction is improved by both drugs and they are about equally active except in the frog, where noradrenaline may be much less active than adrenaline (West, 1947). The actions of isoprenaline on isolated auricles are much greater than those of either of these drugs (Garb et al., 1956). Nathanson and Miller (1950) injected adrenaline and noradrenaline intravenously in a human case of complete heart block and obtained evidence that adrenaline quickened the heart rate-presumably by acting on the ventricle-and that noradrenaline had little or no such action. The differences in the effects of these drugs on the electrocardiogram have been reviewed by von Euler (1956).

4.

Smooth Muscle

Adrenaline causes contraction of some arteries, the splenic capsule, the ileocolic sphincter, the nictitating membrane, the dilator pupillae, the retractor penis, and the pilomotor muscles which cause erection of the hair and gooseflesh. Adrenaline also causes inhibition of the smooth muscle in the stomach and intestine, the bladder, the gall bladder, and the bronchi. It causes contraction of the uterus in the rabbit and the pregnant cat and inhibition of the movements of the uterus in the guinea pig, rat, and virgin cat. Ergot alkaloids and other antiadrenalines may

ADRENALINE AND NORADRENALINE

169

reverse an excitor action on isolated smooth muscle so that adrenaline causes inhibition (Dale, 1913). There is some evidence (Mohme-Lundholm, 1953) that the inhibitory effects are due to the formation of lactic a.cid in the muscles. The action of noradrerialine is generally but not always (Greeff and IIoltz, 1951) qualitatively similar to that of adrenaline. The ratio of equivalent doses (noradrenaline :adrenaline) varies from 0.1 to 300 or more. According to Barger and Dale (1910), this ratio should be low for excitor actions and high for inhibitor actions, but there are many exceptions to this rule (West, 1947; von Euler, 1956). For example, the ratio is high (6-10) for excitor actions on the pilomotor muscles and the dilator pupillae and low for the inhibitor actions on the intestine. The ratio for any given tissue varies widely and may depend on the circumstances. For example, West (1947) found that when isolated rabbit’s ileum was kept in the cold for 5 days, the ratio fell from 2 to 0.2. Many motor and inhibitor effects of both adrenaline and noradrenaline are increased by degenerative action of sympathetic nerves (Cannon and Rosenblueth, 1949). The nictitating membrane and the dilator pupillae of a cat are normally less sensitive t o noradrenaline than to adrenaline, but after degenerative section of the sympathetic nerves, the reverse is true (Bulbring and Burn, 1949a; Burn and Hutcheon, 1949). This effect of section of the nerves has been attributed to the disappearance of amine oxidase, which destroys noradrenaline more rapidly than adrenaline (Burn, 1952). 6. Skeletal Muscle Oliver and Schafer (1895) observed prolongation of the response of voluntary muscles to nervous stimulation after the injection of adrenal extracts. Later work has shown that adrenaline increases contractions in voluntary muscles when fatigue develops after stimulation of motor nerves (Gruber, 1914). This phenomenon became famous through the work of Orbeli (1923), who concluded that stimulation of sympathetic nerves improved synaptic transmission in voluntary muscles and elsewhere. Dale and Gaddum (1930) studied the effects of adrenaline on mammalian voluntary muscle which had been sensitized to acetylcholine by degenerative section of the motor nerves. Such muscles contract when acetylcholine is injected or when it is liberated in the muscle by the stimulation of vasodilator nerves. Some previous workers had observed depression of these responses after adrenaline, while others had observed increased responses. Dale and Gaddum observed both eff ects-a brief depression followed by a more prolonged increase of tjhe response. They

170

J. H . GADDUM A N D MARGARETHE HOLZBAUER

believed that the initial depression was due to vascular changes which prevented the acetylcholine from reaching the appropriate receptors arid that the increase in the response was due t o an effect of adrenaline on the muscle. In confirmation of this theory, they found that depression was also produced by another vasoconstrictor substance (vasopressin) and that adrenaline increased the effect of acetylcholine on an isolated strip of sensitized muscle from a kitten’s diaphragm. This work was confirmed and extended in a series of papers which have been reviewed by Burn (1945). Simultaneous stimulation of the sympathetic nerves generally increases the response of normal muscle to stimulation of the motor nerve roots (the Orbeli phenomenon), but as in the experiments with adrenaline on denervated muscle, it sometimes has the opposite effect. It has been suggested that the increase of the response may be partly due to improved conduction in the nerve fibers or to improved transmission a t the synapse. Brown et al. (1958) however, came t,o the conclusion that it was mainly due to an effect on the muscle fibers, possibly associated with the mobilization of potassium. Adrenaline and noradrenaline both have these effects and adrenaline is the more active of the two (West and Zaimis, 1949). It has been suggested that these effects may be similar t o the therapeutic effect of ephedrine in myasthenia gravis. 6. Peripheral Nerves

Bulbring and Whitteridge (1941) found that adrenaline increased the electric response of the sciatic nerve of cats to submaximal electric stiniulation, presumably by lowering the threshold; after fatigue this effect was increased. Y. Autonomic Ganglia Experiments with adrenaline on autonomic ganglia, like those 011 voluntary muscle, have given discrepant results; sometimes it improves transmission and sometimes it has the opposite effect. Marrazzi (1939) recorded impulses in postganglionic nerves and found that adrenaline blocked transmission. This effect is due partly to the inhibition of the release of acetylcholine and partly to inhibition of its effects (Paton and Thompson, 1953). Bulbring and Burn (1942) and Bulbring (1944) recorded the physiological effects of postganglionic nerves and found that small amounts of adrenaline improved transmission and that large amounts depressed it. Konzett (1950) found that noradrenaline also improved transmission but was less active than adrenaline. Marrazzi and Marrazzi (1947) found that all doses caused initial depression but that sometimes this was followed by improved transmission. Lundberg (1952), using a technique like Marrazzi’s, confirmed Marrazzi’s results arid

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171

Trendelenburg (1956), using a technique like that of Biilbring and Burn, confirmed their results. When nerve impulses are recorded, the initial depression, which may perhaps be allied to that observed in voluntary muscle, seems important. When muscular responses are recorded, the main effect of small doses is improved transmission. On the other hand, adrenaline and noradrenaline block peristaltic reflexes and specifically antagonize nicotine in isolated guinea pig’s ileum; this effect, which appears t o be due to ganglionic block, is antagonized by low concentrations of sympatholytic drugs (McDougall and West, 1954). 8. Central Nervous System

Biilbring and Burn (1941) perfused the spinal cord of dogs with blood and recorded muscular contractions. They found that the presence of adrenaline in the blood increased the effects of acetylcholine and reflex stimulation. Skoglund (1952) on the other hand found that adrenaline (or noradrenaline) injected into the aorta facilitated extensor reflexes and inhibited flexor reflexes and that acetylcholine had the opposite effects. The intravenous injection of adrenaline causes arousal and anxiety in man; noradrenaline has less effect (Barcroft and Swan, 1953). Adrenaline also causes increased electrical activity in the posterior hypothalamus (Porter, 1952) in anesthetized animals. Bonvallet et al., (1954) recorded the electric responses of different parts of the brains of unanesthetized cats and dogs and found that the injection of adrenaline caused signs of arousal, apparently due to stimulation of the reticular formation. This action increased spinal reflexes, but this increase was opposed by reflexes arising from the pressor receptors which depressed spinal reflexes (Dell el al., 1954). The injection of adrenaline into the cerebral ventricles, on the other hand, causes signs of depression and drowsiness. Noradrenaline has a similar effect (Bass, 1914; Feldberg and Sherwood, 1954; Feldberg, 1956). It, is not known whether this effect is due to excitation or inhibition of centers near the ventricles; i t might possibly be secondary to vascular effects. The first effect on respiration in animals is a temporary apnea (Kahn, 1903) due to improved circulation in the carotid body (Heymans and Bouckaert, 1930). This is followed by increased respiration, which is the main effect in man and is produced by both adrenaline and noradrenaline (Whelan and Young, 1953). It has been suggested that it may be due to the rise of metabolism which is known to occur. Very large doses of adrenaline may cause apnea, even when effects on the carotid body are excluded (Hoff et al., 1950).

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9. Anterior Pituitary Gland and Adrenals

Adrenaline causes the release of corticotropin (ACTH), which then stimulates the formation of hormones in the adrenal cortex. This phenomenon was first detected by the bioassay of hormones in the adrenal blood (Vogt, 1944), but has also been detected by various other means, including the bioassay of corticotropin in the blood of rats (Farrell and h'lcCann, 1952). This action may be a direct one on the pituitary since it, can be demonstrated in transplanted pituitaries, but it may also be secondary to an action of adrenaline on the hypothalamus. It is antagonized by adrenolytic agents such as dihydroergotamine, dibenamine, and dihenzyline, and the effect soon becomes less when the dose of adrenaline is repeated (cf. Vogt, 1954b). Isopropylnoradrenaline is a t least equally effective, but noradrenaline is less so (Jarrett, 1951). The stimulation of the adrenal cortex which follows stress may be secondary to the release of adrenaline from the medulla, but this is not the only mechanism involved, since some kinds of stress cause similar changes in demedullated rats and since suitable doses of an adrenolytic drug may suppress the response to adrenaline but not to stress (Guillemin, 1955). There is also some evidence that adrenaline may have a direct action on the adrenal cortex in the absence of the pituitary (Vogt, 1954b). The relations between the adrenal cortex and medulla are complicated by the fact that, in the absence of cortical hormones, the medullary hormones lose their effects. For example, adrenalectomy diminishes some of the effects of adrenaline and noradrenaline on blood vessels and on glycogen, and cortisone restores these effects. This is what Ingle (1954) calls a permissive action of cortisone (reviewed by Vogt, 195413; von Euler, 1956). There is evidence that adrenaline may also play some part in the release of luteinizing hormone. The direct application of adrenaline to the pituitary has been found to cause ovulation. The adrenolytic substance dibenamine antagonizes naturally occurring ovulation in rats or rabbits (Sawyer et al., 1947, 1949). Some doubt has been cast on t,hese conclusions by Donovan and Harris (1956). V. ESTIMATION Recent advances in our knowledge of the physiology of catecholamines have depended largely upon bioassays. Colorimetric and fluorimetric methods give more results in a given time but are generally less specific than the methods which depend on the pharmacological actions of these

173

ADRENALINE A N D NORADRENALINE

TABLE I Amount (ng.1) required for each test” Methods of assay Biological Cat’s blood pressure Rat’s blood pressure (C,) Rat’s blood pressure (pithed) Rat’s uterus (2-ml. bath) Rabbit’s ear (perfused) Rabbit’s ear (Armin and Grant, 1953) Rabbit’s gut (10-ml. bath) Fowl rectal cecum (2-ml. bath) Chemical Formation of adrenochrome Reduction of arsenomolybdate Formation of adrenolutine Coupling with ethylenediamine a

rldrenaline ‘LOO 50 7 0.1

0.5 0.002

Noradrenaline 100

3

5 15

1

40 2

40 50

10,000 50 20 6

10,000 800 20 6

The amounts required for an accurate bioassay w ould be 5-10 times the amounts given in the table.

substances on smooth muscle. It is probable, however, that improved chemical methods will eventually replace these pharmacological methods. The approximate sensitiveness of various methods are given in Table I . I . Preliminary PuriJication The specificity and sensitivity of both biological and chemical methods can be increased by preliminary treatment which removes interfering substances from extracts and concentrates the catecholamines. The appropriate treatment depends on the method to be used in the final test. The adsorption of the active substances on aluminum hydroxide or alumina, or on various cationic exchangers has been much used. I n the method of Shaw (1938), freshly precipitated A1(OH)3 is shaken with the sollitmioncontaining adrenaline a t p H 4 and then separated by centrifugat ion. This removes some interfering substances. The supernatant is adjusted to pH 8.5 and then shaken again with Al(0H)S which adsorbs adrenaline a t this pH but leaves other substances in solution; the adrenaline is recovered by dissolving the aluminum hydroxide in acid. Yon Euler (194813) modified this procedure by precipitating the aluminum hydroxide in the presence of adrenaline and then precipitating the salts from the acid solution with acetone and alcohol, so that he eventually obtained a solution suitable for bioassay. In most recent methods alumina is used as adsorbent. Lecomte and Fischer (1949) pass their extracts through a column of 1 ng. = 1 nanogram = 10-6mg.

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J. H. GADDUM AND MARGARETHE HOLZBAUEH

‘(Decalso” which adsorbs both adrenaline and noradrenaline. An iodine solution of pH 4 is then used to convert adrenaline to adrenochrome and to elute i t ; noradrenochrome is then eluted a t p H 7. The ion-exchange resin I R C 50 has been successfully used by Rergstrom and Hansson (1951) and by Crawford and Law (1957). Paper chromatography mas first applied to this problem by James (1048) who used phenol as the mobile phase t o separate adrenaline and noradrenaline. The catecholamines can be detected by exposing the paper t o potassium ferricyanide (James, 1948), potassium iodate (Shepherd and West, 1951), P-naphthoquinone-4-sulfonate (Glazko and Dill, 1951), or in other ways. When large amounts (>2 pg.) are present, rough quantitative estimates may be made by spraying the paper with potassium ferricyanide and ferric sulfate and comparing the sizes of the spots (Goldenberg et al., 1949). If suitable precautions are taken to avoid loss by oxidation i t is possible to extract very small amounts of catecholamines from tissues wit.h acid ethanol and t o apply the extract to paper. After chromatographic separation, the amines can be eluted from the appropriate part of the paper and estimated by bioassay (Crawford and Outschoorn, 1951; Vogt, 1954a). With this system the Rf of noradrenaline is about 0.2 and that, of adrenaline is about 0.5; dopamine and histamine both lie between them. The R, of isoprenaline is about 0.7, that of pituitary hormones and of substance P, about 0.9; the actual values are determined on control strips of paper in each experiment. Schumann (1956) separated dopamine from noradrenaline in extracts by two-dimensional paper chromatography using phenol and butanol containing hydrochloric acid; in the latter solvent mixture, dopamine runs faster than either adrenaline or noradrenaline. The dopamine w a b identified by the position of a spot on the paper and also by biological tests after elution. Its biological activity is very low, but in a dose of 3-4 pg. it causes a rise of blood pressure in rats and a fall of blood pressure in guinea pigs. The material eluted from the paper also had these two opposite actions. When blood is collected without special precautions, other pharmacologically active substances, such as 5-hydroxytryptamine, are quickly liberated into the plasma. This introduces an error in bioassays which can generally be eliminated by using heparin and siliconed vessels and by either testing the blood immediately or by rapidly cooling it and separating the plasma in a centrifuge (Gaddum el al., 1949). Complications due to the pharmacological effects of potassium can sometimes be avoided by taking up dried extracts in anhydrous ethanol saturated with sodium chloride (Barsoum and Gaddum, 1935).

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2. Bioassays

a. Blood Pressure. Ever since Oliver and Schafer (1895) discovered the pressor effect of adrenal extracts, observations on the blood pressure have played a n important part in work in this field. The pressor effect on a spinal cat has been much used for the control of commercial solutions of adrenaline (Burn et al., 1950a). Records of the blood pressure of cats have also been used, in conjunction with other records, to detect catecholarnines when these have been liberated into the blood stream (Cannon and Rosenblueth, 1937). The rat’s blood pressure (Landgrebe et al., 1946; Crawford and Outschoorn, 1951) provides a particularly sensitive test for noradrenaline. If hexamethonium (20 mg./kg.) is injected, the preparation becomes much more sensitive to noradrenaline than it is to adrenaline, but it soon loses this sensitivity (Vogt, 1952). If the spinal cord is destroyed (Shipley and Tilden, 19-17), hexamethonium is unnecessary and the preparation gives about equal responses to adrenaline and noradrenaline for as long as 10 hours (Holzbauer and Vogt, 1956). 6 . Rat’s I’lerus. It has long been known that the rat’s uterus is inhibited by adrenaline, but accurate assays were not possible before the work of de Jalon et al. (1945). They eliminated the spontaneous activity of the uterus by modifying the salt solution in which it was bathed arid found the smallest dose of adrenaline that would suppress the effect of acetylcholine. Gaddum ei al. (1949) compared a modification of this method with many other methods and found that it was particularly sensitive t o adrenaline and much less sensitive to noradrenaline (cf. West, 1947). A simple mechanism for applying the choline ester a t regular intervals was described by Gaddum and Lembeck (1949). The ratio of equivalent doses is generally about 150, so that noradrenaline is unlikely to interfere with the use of this preparation for the assay of adrenaline unless it is present in at least ten times the Concentration. In favorable ronditions, less than 0.1 ng.’ of adrenaline can be detected in a 2-nil. bath, and the sensitivity of insensitive uteri can be increased t o surh values with, for example, dibenzyline (Holzbauer and Vogt, 1955). 5-Hydroxyt ryptamine (10-9-10-8 grams/ml.) causes contraction of rat’s uterus and may thus lessen the effect of adrenaline, but its effects can be diminished if necessary with dibenamine (Gaddum ef al., 1955). The result may be complirated if large amounts of oxytocin or aretylcholiiie are present i n the solution t o be tested. c. Blood Vessels of the Rabbit’s Ear. Schlossmann (1927) came to the coriclusion that vasoconstriction in the perfused ear of a rabbit provided the most sensitive and specific test for adrenaline available a t the time.

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The solution of Page and Green (1948) gives particularly sensitive preparations but their apparatus is unnecessarily elaborate; this tissue is very sensitive to noradrenaline (Savini, 1956). 5-Hydroxytryptamine (1-10 ng.) also causes vasoconstriction in the perfused ear, but its effects can be eliminated with lysergic acid diethylamide (Gaddum and Hameed, 1954). Arniin and Grant (1953) have described a method which will detect, adrenaline in a concentration of lo-” grams per milliliter or even less. Drugs are injected centrally into the artery of a rabbit’s ear in situ, sensitized by degenerative section of the nerves. The effect is observed by measuring the diameter of the artery at intervals with a microscope. Armin and Grant (1955) use a special apparatus to transfer samples of blood rapidly to this artery and can thus measure the blood adrenaline a t frequent intervals. d. Intestinal Muscle. Intestinal muscle is generally inhibited by catecholamines. Rabbit’s intestine was used in much of the early work on adrenaline (O’Connor, 1912). It responds well to adrenaline and is also sensitive to noradrenaline (Burn et al., 1950a). Rat’s colon is particularly sensitive to noradrenaline (Gaddum et d., 1949), but it is also sensitive to 5-hydroxytryptamine. grams/ml., The rectal cecum of a fowl is inhibited by adrenaline Barsoum and Gaddum (1935)] and is much less sensitive to noradrenaline (von Euler, 1948a). 3 . Colorimetric Methods

Adrenaline is easily oxidized under suitable conditions to form adrenochrome (Section 111, 3) and other pink indole compounds. A number of different tests using a dozen different oxidizing agents depend on this change, which was first observed by Vulpian (1856) in adrenal ext,racts. These tests are not very sensitive, but they are likely to be fairly specific. (Barker et al., 1932; von Euler and Hamberg, 1950; Suzuki and Ozaki, 19.51; von Euler, 1956). Other methods, in which adrenaline acts as a reducing agent, arc’ more sensitive, but generally less specific, than these. Other reducing substances must therefore be removed before the test is applied. 111 the method of Folin et al. (1913), phosphotungstic acid was used. In the methods of Whitehorn (1935) and Shaw (1938), adrenaline reduces arsenomolybdic acid to a blue compound. The color produced by adretialine may be increased 3-5 times by preliminary treatment with alkali ; no such increase has been observed with allied substances such as noradrenaline. This method of assay is quite sensitive, and a test with alkali

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may be used to coiifirm the identity of the substance measured. Verly (1948) failed to confirm this, but his failure has been attributed to the use of impure noradrenaline (Bischoff and Bakhtiar, 1956).

4. Fluorimetric Methods Paget (1930) observed that adrenaline became fluorescent in the presence of a mixture of various reagents. Gaddum and Schild (1934) found that a solution containing nothing but adrenaline, alkali, and oxygen developed a powerful transient fluorescence, and they based a method of assay on this fact. The fluorescence due to noradrenaline was only 2% of that due to adrenaline, and other allied substances were also comparatively inactive. The fluorescent substance formed from adrenaline has been shown to be adrenolutine (Section 111, 3) (EhrlBn, 1948; Fischer, 1949). Lund (1949a,b,c) developed a method of assay in which adrenaline is oxidized to adrenochrome with manganese dioxide and then converted to adrenolutine in the presence of alkali and ascorbic acid, which prevents further oxidation. The fluorescence is then fairly stable and can be estimated in a fluorimeter. Von Euler and Floding (1955) have described a similar method using potassium ferricyanide as oxidant and have claimed that it is possible in this way to estimate adrenaline in a concentration of 10-9 grams per milliliter. Weil-Malherbe and Bone (1952a) described a different type of test in which a fluorescent substance is formed by condensing adrenochrome with ethylenediamine. This test is sensitive and can estimate adrenaline in a concentration of grams per milliliter, but it is unspecific since catechol itself and various catechol derivatives also form fluorescent compounds. Methods of removing these other substances have been described, but it is doubtful whether these methods have been completely successful. Using this method Weil-Malherbe and Bone (1952b, 1954) obtained evidence that insulin caused a fall in the concentration of adrenaline in the peripheral blood plasma. Holzbauer and Vogt (1954a), using biological methods of assay, failed to confirm this surprising result. Estimates, obtained by pharmacological methods, of the normal concentrations of adrenaline and noradrenaline in the plasma are less than one-fifth of those given by Weil-Malherbe and Bone, and the injection of insulin caused a rise in the pharmacological estimates. Various other workers have used modifications of this fluorimetric method and lower figures have been obtained’for the normal concentrations (Aronow et al., 1956; Richardson ol., 1956; Woods st at., 1956; Montagu, 1956). f7f

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5. Tests of Identitg

The methods of assay discussed above are generally applied to solutions containing very small amounts of catecholamines and much impurity, so that ordinary chemical methods of identification cannot be used. Most of the evidence available a t present depends on parallel assays by different methods. This method was used by Cannon and Rosenblueth (1933) to show that adrenaline was not the only substance liberated by sympathetic nerves. If different tests give widely different estimates of the concentration of adrenaline in a solution (different “ adrenalineequivalents 1 1 ) there must be some other active substance present. If several tests give the same adrenaline-equivalent, there is some reason to believe that this concentration of adrenaline actually is present, but the value of such parallel assays is not great unless it can be shown that the methods used do differentiate between adrenaline and allied substances. Gaddum et al. (1949) compared 11 sympathomiometic amines by various different biological methods and showed that they could all be distinguished from one another by tests on rat’s uterus and colon and rabbit’s ear (Gaddum, 1950b). When biological estimates agree with colorimetric or fluorimetric estimates the results are particularly convincing. Von Euler (1948a) used this fact to show that adrenergic nerves contained the levo isomer of noradrenaline, which gives the same color as racemic noradrenaline but has about twice as much biological activity. Other tests of identity include the test with alkali in Shaw’s colorimetric method, the spectrum of the fluorescent light in Weil-Malherbe’s method, and paper chromatography (see above). 6. Assays of Mixtures

I n certain conditions when Lund’s method of estimation is used, the fluorescence due to adrenaline is equal to the fluorescence due to an equivalent amount of noradrenaline, so that the sum of the concentratmion of these two substances can be estimated in a mixture (Crawford, unpublished data). In most other tests these two substances have unequal effects, and the result of a direct test on a mixture has no precise meaning. If one of these substances is present in a very low concentration, or if the method of assay is very insensitive to it, it may be possible to neglect its effects and estimate the other substance, but often it is necessary to estimate them both. This may be achieved in two ways. (1) Standard solutions of adrenaline and noradrenaline are compared with the unknown mixture by two different methods, one of which is especially sensitive to adrenaline and the other of which is especially

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sensitive to noradrenaline. The concentrations of adrenaline and noradrenaline in the mixture are then calculated by suitable mathematical methods. This method of calculation has been applied to bioassays (von Euler (1948a), cat’s blood pressure and chicken’s rectal cecum; Bulbring (1949), rat’s uterus and rat’s colon; Burn et al. (1950b), cat’s blood pressure and nictitating membrane). The results are subject to large errors (Gaddum and Lembeck, 1949). Similar methods of calculation may be applied to colorimetric (von Euler and Hamberg, 1949) and fluorimetric methods (Lund, 1949c; von Euler and Floding, 1955). In acid conditions adrenaline is more quickly oxidized than noradrenaline and the two results can therefore be obtained by oxidizing the mixture for appropriate times at two different pH values. In Weil-Malherbe and Bone’s (1953) method the two results are obtained by filtering the fluorescent light with two different filters. With a yellow filter the intensity of the fluorescent light from adrenaline is 4.5 times that from noradrenaline; with a blue-green filter, the intensities are equal. (2) When accurate results are needed, or when the proportions in which the substanoes are present invalidate these tests, or when isoprenaline (Lockett, 1954) may be present, the substances should be separated by paper chromatography before estimation (see above). I n this way it is possible to estimate as little as 0.5 ng. of adrenaline by its effect on the rat’s uterus and 15 ng. of noradrenaline by its effect on t,he rat’s blood pressure. VI. DISTRIBUTION 1 . Lower Animals

The ganglionic chain of earthworms contains both adrenaline and noradrenaline. Both of these substances and dopamine have been found in insects of various kinds. The housefly contains particularly large amounts (1 pg. noradrenaline and 0.3 pg. adrenaline per fly). None was found in various species of protozoa, coelenterata, echinodermata, crustacea, or tunicata (Gaskell, 1919; Ostlund, 1954). The livers, spleens, hearts, and interrenal organs of frogs and various kinds of fish contain both adrenaline and noradrenaline. Although large amounts of both catecholamines are present in the heart of the hagfish they do not appear to have any action on this organ (btlund, 1954). 2. Adrenal Medulla Tables summarizing numerous estimates of adrenaline and noradrenaline in adrenal glands are given by von Euler (1956). The total amount

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of catecholamine in the whole gland varies in different species in the range 0.12 to 14 mg. per gram. Human adrenals have been found to contain 0.27-1 mg./gram (n = 13). This figure must depend on the relative amounts of cortex and medulla. Fear and other forms of stress are known to deplete the adrenal medulla (see below), and some of the observed differences between species must be due to differences in the manner of death. According to Hokfelt (1951), the adrenals in female rats contain less catecholamines than those in male rats when the results are calculated per gram of gland and more when they are calculated per kilogram of body weight. The relative amounts of adrenaline and noradrenaline also vary widely. This is sometimes expressed as the percentage of adrenaline and sometimes as the percentage of noradrenaline. In the present review it will be given as the “percentage methylated” which is equal to the amount of adrenaline as a percentage of the sum of the amounts of adrenaline and noradrenaline. Typicbal figures for the percentage methylated are 17 (whale), 30 (fowl), 60 (cat), 80 (horse, cow), 85 (man, rat), 97 (rabbit). The percentage methylated increases with maturity and may be zero in the fetus. In those species where the cortex is relatively large, the percentage methylated is generally high (West, 1955). Other factors which may affect the percentage methylated are discussed below. Most of the pressor activity in adrenal homogenates is contained in granules which have the same sedimentation rate as mitochondria (Blaschko and Welch, 1953; Hillarp et al., 1954). Catecholamines may represent 11-17% of the dry weight of these granules and large amounts of ATP are also present. Amine oxidase and other oxidizing enzymes are in the same granules and dopa decarboxylase is in the supernatant fluid (Blaschko et al., 1955, 1956; Hillarp et al., 1955; Eade, 1956). Hillarp and Hokfelt (1954) and Eriinko (1954) have obtained histological evidence that some cells in the adrenal medulla contain mainly adrenaline while other cells contain mainly noradrenaline. The presence of noradrenaline in some cells is presumably due to incomplete resynthesis after the release of adrenaline (Section 11, 9), but the histological evidence suggests that there are also special islets of tissue which secrete noradrenaline. The fact that certain forms of stimulus appear to release adrenaline or noradrenaline preferentially makes this interpretation of the histological data attractive. 3. Accessory Chromafin Tissue

The application of chromates to sections of adrenal glands causes the appearance of a brown color known as the chromaffin reaction (Henle,

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1865). The brown color is due to oxidized catecholamines (Bennett, 1941). Islands of chromaffin tissue (“paraganglia”), homologous with the adrenal medulla, are found round the large blood vessels behind the peritoneum. The largest accumulations of these are known as the organs of Zuckerkandl: similar cells are found in sympathetic ganglia, in the adrenal cortex, and in various other tissues (Biedl and Wiesel, 1902; Coupland, 1952; von Euler, 1956). Elliott (1913) found that in the newborn child the organs of Zuckerkandl contained 24 times as much adrenaline activity as did the two adrenal glands together, but they atrophy early in life and appear to be of little importance in the adult. The percentage methylated in the organs of Zuckerkandl is similar to that in the adrenals at the same age; it starts practically at zero and rises in humans during the first two years of life (West, 1955).

4. Chromafin-Cell Tumors (Pheochromocytomata) These tumors may arise in man either from the adrenal medulla or in accessory chromaffin tissue. They usually contain large amounts of noradrenaline (Holton, 1949) and comparatively small amounts of adrenaline. These substances are excreted in the urine (Engel and von Euler, 1950), and their estimation in the urine provides the best method of diagnosing the disease. They are usually not present in increased amounts in other forms of hypertension (von Euler et al., 1954b). In some cases the output of noradrenaline per 24 hours has been estimated as more than 3000 pg. instead of the normal 20-50 pg. Large amounts of dopamine have also been found in the urine (von Euler, 1951b). The percentage methylated in the urine has been found to be about the same as the percentage methylated in the tumor. Von Euler (1956) has expressed the view that when this is fairly high (10-50%), the tumor probably arises in the adrenal medulla, and when it is low (0-2%), the tumor probably arises in accessory tissue, and this is confirmed by observations on four cases by T. B. B. Crawford (unpublished data). 6. Peripheral Nerves

Gaddum and Khayyal (Gaddum, 1936) demonstrated the release of an adrenaline-like substance from adrenergic nerve trunks during strong electric stimulation which may have damaged the nerve. Lissbk (1939) proved that extracts of adrenergic nerve trunks contain an adrenalinelike substance. Von Euler (1948a) established the presence of L-noradrenaline in these extracts and obtained evidence that a small amount of adrenaline was also present (von Euler, 1949). Schumann (1956)

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showed that dopamine may also be present in large amounts and failed to detect adrenaline. Splenic nerves from cows and mesenteric nerves from cats and dogs contain particularly high concentrations of noradrenaline (8.5-20 p g . / gram). The concentration seems to depend on the proportion of adrenergic fibers in the nerve, and is low ( < 1 pg./gram) in the cervical sympathetic, which is mainly preganglionic. Sympathetic ganglia contain large amounts of noradrenaline and small amounts of adrenaline (see von Euler, 1956). Prolonged stimulation does not alter the noradrenaline content of nerves (Luco and Gofii, 1948) or ganglia (Vogt, 1954a). I t is decreased by reserpine (Muscholl and Vogt, 1957). When splenic nerves are homogenized and centrifuged, 15-19 ;c of the noradrenaline is found to be attached to small particles (von Euler and Hillarp, 1956). 6. Central Nervous System

The preseiice of adrenaline and noradrenaline in the central nervous system was detected by von Euler (1946b) and Holtz (1950) and attributed to vasomotor nerves. Vogt (1954a) made a detailed study of their distribution in the brains of cats and dogs, using improyed methods. The percentage methylated was about 10. The highest concentration x i as in the hypothalamus (1-1.4 pg./gram) and the midbrain and particularly in areas connected with the sympathetic system; there was also a. high concentration in the area postrema. Other areas generally contained less than one-tenth of these quantities. This specific distribution strongly suggests that these amines play a physiological role in the brain inclependently of their connection with vasomotor activity. This conclusion is confirmed by the facts that the concentrations fell after the inject ioii of drugs causing central stimulation of the sympathetic system and were not affected by extirpation of the superior cervical ganglia. 7 . Other Organs

Most organs contain small amounts of noradrenaline and smaller amounts of adrenaline, presumably located in adrenergic nerves, although the concentrations in some nerve terminals would have to be surprisingly high (von Euler, 1956). No such sympathomimetic substances have been found in nerve-free tissues such as placenta or bone marrow, and only small amounts in lung and voluntary muscle (Racq and Fischer, 1947; Schmiterlow, 1948; Hokfelt, 1951). The heart has also been shown to contain dopamine (Goodall, 1951). Degenerative section of sympathetic nerves causes a fall in the adrenaline-equivalent of organ extracts, which is reversed when the

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nerves regenerate (Cannon and Lisshk, 1939). This fall is mainly due to the disappearance of noradrenaline from the tissue. There is some evidence that adrenaline, which normally represents only 5-10 % of the total, is little if a t all affected by section of the nerves, and it has been suggested that this adrenaline is not in the nerves but in chromaffin tissue (Goodall, 1951; von Euler and Purkhold, 1952). On the other hand it is known that adrenaline is present in extracts of nerve trunks and is liberated when nerves are stimulated. Stimulation of sympathetic nerves does not appear to alter the noradrenaline content of the organs (von Euler and Hellner-Bjorkman, 1955). 8. Body Fluids

In conditions of complete rest, t,he most sensitive and specific methods generally fail t o detect adrenaline or noradrenaline in peripheral blood. In the experiments of Holebauer and Vogt (1954a), the concentrations of' adrenaline and noradrenaline in resting human plasma were certainly grams per milliliter respectively. Armin and less than lO-'O and Grant (1955), found less than lo-" grams per milliliter adrenaline in resting rabbit blood. Factors which cause the release of adrenaline or noradrenaline may increase the concentrations of these substances not oiily in the local venous blood but also in the general circulation (see below). Estimates of adrenaline and noradrenaline in urine give an indication of adrenal and sympathetic activity in the whole body (see below).

VII. RELEASEFROM

THE A D R E N 4 L

GL4NDS

The factors controlling the release of adrenaline from the adrenals have been discussed by Cannon (1928, 1929), McDowall (1938), and Satak6 (1955). Cybulski (1895) was the first to show that the adrenal glands secrete their active principles into the blood. Dreyer (1898) showed that the rate of secretion is increased by stimulation of the splanchnir nerves. Since then much work has been devoted to the effects of various factors on the rate of secretion. IJntJ fairly recently it was assumed that adreiialine was the only substance released. Meier and Bein (1918) found that an infusion of noradrenaline restored normal vascular responses to adrenaline in adrenalectomized dogs, and they suggested that the resting gland was liberating noradrenaline. When it was shown that some noradrenaline actually is released (Riilbring and Burn, 1919a; Gaddum and Lembeck, 19-29), attention was turned to the possihilitv that the two catecholamines might he released independently.

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I. Methods In much of the early work, the adrenal veins were intact and the medullary secretion was carried by the normal circulation and detected by its effects on distant organs such as the heart or pupil (the auto-assay method). It is of course, important that these detector organs should be denervated, or they may show changes mediated by their nerves. Sometimes they are sensitized by cutting the nerves several days before the main experiment. Experiments on the effect of stimulation of splanchnic nerves on the adrenals may be complicated by the liberation of substances from other tissues (see below) unless precautions are 'taken against this source of error. An anastomosis may be made so that the adrenal venous blood drains directly into the circulation of a second animal which is used to deteet the adrenaline released (Tournade and Chabrol, 1921). This technique ensures that the results really are due to substances in the blood from the adrenal. It is easier to interpret the results when samples of adrenal venous blood are first collected and then tested, and this is the only method providing a reliable estimate of the amounts of catecholamines liberated per minute. I n an anesthetized animal, blood can be collected from the adrenal vein through a cannula in the renal vein or a lumbar vein, after all other veins have been tied. Alternatively, blood may be collected in a pocket made by tying all branches of the inferior vena cava except the adrenal veins and then either removed at intervals for testing or released into the general circulation so that its effects can be observed (Rogoff, 1935). Such techniques involve anesthesia and trauma. These may be avoided by passing, under preliminary anesthesia, a tube through a suitable vein so that its end lies in the vena cava near the renal veins (Cannon and de la Paz, 1911; Armin and Grant, 1955), but this method cannot give quantitative results. A good method of collecting adrenal blood quantitatively without stress is that described by SatakC et nl. (1927), in which the sensory roots in the lumbar region of dogs are first cut and allowed to degenerate. A month later, the first lumbar vein is exposed and a cannula inserted for. the collection of blood from one adrenal while the dog is conscious and collaborative (SatakC, 1955). The adrenaline equivalent of the blood collected in this way has usually been estimated by its effects on rabbit's intestine and on the denervated pupil of a cat. These two tests generally agree quantitatively with one another, but not, always, and it is possible that some of the disrrepancie8

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are due t o noradrenaline. Another similar technique for collecting adrenal blood has been described by Hume and Nelson (1954). The complications of operation and anesthesia are also avoided in experiments which depend on the fact that certain forms of intense stimulation applied for several hours cause the disappearance of the hormones from the gland (Elliott, 1912). The adrenaline-equivalents of the two glands in the same animal are generally about equal, but there is some variation between those taken from different animals; sulky cats have lower adrenaline-equivalents than placid cats. When the effect is due to impulses in the splanchnic nerves, it is therefore best to cut one nerve in a preliminary aseptic operation and then to apply the stimulus and compare extracts of the two glands with one another; nerve section by itself has no effect. When the effect is due to a direct action on the gland, the control adrenal is removed at the start of the experiment and the denervated adrenal is then subjected to the stimulus (Elliott, 1912). With either type of action, the average result with one group of animals may be compared with the average result with another group, and the results treated statistically. The amounts of adrenaline and noradrenaline secreted in a given time can also be followed with a minimum of stress by estimating the amounts in the urine. The validity of the conclusions drawn from the results depends on the assumption that there is a constant relation between the amount liberated in the body and the amount excreted in the urine, but if it is borne in mind that any observed effects may be due to changes in the function of the kidney, it is unlikely that this factor will lead t o serious errors. Adrenalectomy causes a fall in the urinary adrenaline without much effect on the urinary noradrenaline in man (von Euler et al., 1954a) and in rats (Crawford and Law, 1957). The urinary adrenaline is therefore taken as an index of the activity of the adrenal gland, and the urinary noradrenaline presumably comes from adrenergic nerves. Conclusions based on this type of experiment are likely to be unreliable when applied to animals whose adrenals release significant amounts of noradrenaline. In such experiments it is important to exclude the influence of the psychological factors discussed below; even the injection of saline may produce effects. 2. Direct Actions on the Adrenals

Factors which act directly on the adrenal glands are most conveniently studied after section of the splanchnic nerves. In these circumstances, the concentrations of adrenaline and noradrenaline in the

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adrenal blood are low and the noradrenaline can be satisfactorily estimated only after chromatographic separation. In the experiments of Vogt (1952), the average sum of the concentrations of these two substances in the adrenal plasma of cats under chloralose was 41 ng. per milliliter. The concentration in the arterial blood of cats is much lower than this and was neglected in calculations which indicated that the glands were secreting on the average 21 ng. of adrenaline and 38 ng. of noradrenaline per cat per minute (38% methylated). Dun& (1953) obtained similar values-a total of 26 ng. per kilogram per minute from a single gland (12 % methylated). The injection of nicotine, choline, or acetylcholine causes the release of adrenal medullary secretion and stimulation of sympathetic ganglia throughout the body (Cannon et al., 1912; Dale and Laidlaw, 1913; Dale, 1914). There is good evidence that acetylcholine is the chemical transmitter of impulses from the splanchnic nerves to the secreting cells in the adrenal medulla. This effect is mainly a nicotine action. The pressor effect of splarichiiic stimulation in cats or of the close arterial injection of acetylcholine can be almost completely abolished by large doses of nicotine. The remaining pressor effect is enhanced by eserine and abolished by atropine. The effect is thus mainly a nicotine action, but there is also n. small muscarine action. Muscarine itself also releases the secretion, and its effect is abolished by atropine (Feldberg et al., 1934). The ganglionblocker tetraethylammonium inhibits the release of medullary hormones after splanchnic stimulation (hlorrison and Farrar, 1949). 5-Hydroxytryptamine (1 pg. by close arterial injection) releases t8he hormone in cats (Reid, 1952). Catecholamines are released by the direct action of potassium on the gland (Houssay and hlolinelli, 1925). Vogt (1952) found that the percentage methylated in the adrenal blood after potassium or after splanchnic stimulation mas about the same as the percentage methylated i n extracts of glands from the same cat after death. Many estimates have been made of the adrenaline-equivalent of adrenal blood daring splanchnic stimulation and the following figures ( p g . per minute) are taken from Trendelenburg (1929): rabbit, 1-2; cat, 8-20; dog, 50-75. These large rates of secretion are accompanied by a two- to threefold increase of the oxygen consumption of the gland and R 507, increase of the blood flow (Broening, 1924). llnlike most other factors which release the medullary hormones, electrical stimulation of the splanchnic nerves causes only a small fall in the stores of hormone left in the gland (Elliott, 1912; Hokfelt and McLean, 1950).

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According t o Bulbring et aE. (1948), the amount of adrenaline released hy splanchnic stimulation depends on the concentration of adrenaline in the arterial blood; when much is present, much is released. The wide variations in the percentages methylated found i n extracts of the adrenals of different species have been mentioned. Similar variations are likely to occur in the composition of the mixture of hormones released. Most of the work on this subject has been done \vith cats, hut the results have nevertheless been variable. This may he partly because the amount of active substance in the gland depends on the recent history of the animal. Bulbring arid Burn (1949s) found on t,he average that the vatecholamities were 59T0 methylated in the adrenal blood of cats whet1 the splanchnic nerve was first stimulated, and the figure fell steadily to 23C;;, 011 the fifth stimulation. In West’s (1950a) experiments 1% ith rabbits, the percentage methylated was 100 at first, but fell and then rose again to 100 at 15 minutes. Other figures for cats are 58% (Outschoorn, 1952a) and 25% (von Euler and Folkow, 1953). Holtz el nI. (1952), who stimulated the splanchnic nerve without cutting it, got the high figure of 95%. Nicotine has been found to increase the percentage methylated in the adrenal blood of dogs from the control figure of 66 to 90 (Houssay and Rapela, 1953). On the other hand, Butterworth and Mann (1956) found that the final effect of acetylcholine was a similar percentage loss of both amities, M hich was quantitatively accounted for by estimates of the amounts secreted into the blood. 3. Reflex Control of the AdrenaIs

The effects of various factors on the adrenal secretion when it is under the control of the central newous system have been studied by various techniques. The trauma of a surgical operation causes reflex secretion, arid this is inhibited hy deep anesthesia but may be increased by light anesthesia. Elliott (1912) found that surgical trauma under ether depleted an innervated adrenal more effectively than stimulation of the cut splanchnic nerve on tLheother side. a. Resting Secretion with Splnnchnic Nerves In f a d . The adrenaliiicequivalent of the secretion of both adrenals i n resting dogs has heen estimated as about 50 ng. per kilogram per minute (Satak6, 1955). Four publications have given estimates of the mean total catecholamines secreted by the left adrenal of cats anesthetized with chloralose; the range is 190 to 277 ng. per kilogram per minute (19-28% methylated). Under Nembutal the rate was 76 ng. per kilogram per minute. The mean percentage methylated in extracts of the glands was consistently reported to be greater than this (55-62%) in five publications (see von Euler, 195G).

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This indicates that noradrenaline is preferentially secreted under these conditions. b. Rejlexes. Elliott (1912) found that innervated adrenals were depleted when sensory nerves were stimulated, and he showed, by dividing the central nervous system a t different levels, that this effect depended on centers situated between the corpora quadrigemina and the upper part of the spinal cold. The glycosuria and hyperglycemia which may be caused by lesioris i n the floor of the fourth ventricle (piqQrediabbtique) were shown by various workers t o be partly due t o increased secretion of the adrenal medulla (see Trendelenburg, 1929). Ranson and Magoun (1939) mapped the parts of the brain in which ail electric stimulus causes a pressor response due to discharge in sympathetic, nerves and found them to be mainly concentrated in the hypothalamus midbrain and medulla. Vogt (1954a) found that these same parts of the brain have the highest concentrations of noradrenaline. Several groups of workers have obtained evidence that electric stimulation of different parts of the hypothalamus may liberate preferentially either adrenaline or noradrenaline (Briicke et al., 1952; Redgate and Gellhorn, 1953; Folkow and von Euler, 1954). The reflexes controlled by these centers are stimulated by a fall of pressure in the carotid sinus, a fall of blood sugar, the stimulation of sensory nerves, muscular work, emotional excitement, asphyxia, cooling, heating, and by various drugs acting through the central nervous system. Some of these stimuli increase the secretion by 100 times or more (Cannon, 1928; Trendelenburg, 1929; Grollman, 1936; McDowall, 1938; Satak6, 1955). c. Carotid Sinus. According to Tournade and Chabrol (1926) and Heymans (1929), the rate of secretion of the adrenal medulla is normally influenced by reflexes arising in the large arteries. When the pressure in the carotid sinus falls, the rate of secretion rises and vice versa. This reflex can be studied by clamping the carotid arteries so as t o cause a fall of pressure in the sinus. Holtz and Schumann (1949) studied the effects of carotid occlusion 011 various organs in the body and concluded that these effects were due to the release of noradrenaline. They thought that this noradrenaline was liberated from the adrenals but did not obtain convincing evidence of this (Driver and Vogt, 1950; Brauner et al., 1950). Kaindl and voii E:ulrr (1951) found that carotid occlusion increased the total secretion 1.4-7 times without any change in the percentage methylated. This is confirmed by other estimates-29% (Holtz et al., 1952) and 28% (von Euler and

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Folkow, 1953). Both amines are liberated by the carotid sinus reflex, but noradrenaline appears to be preferentially liberated, as it is in anesthetized cats without carotid occlusion. d. Blood Sugar. The rate of secretion also depends on the blood sugar. When this is lowered by the injection of insulin, adrenaline is released from the adrenals provided that the splanchnic nerves are intact. That this effectis due to the fall of blood sugar is indicated by the fact that it is prevented by the infusion of glucose (Cannon et al., 1924; Abe, 1924). The adrenaline is released in sufficient quantities to raise the blood sugar in another dog which receives the adrenal blood by cross-circulation (Houssay et al., 1955), and this mechanism must play a part in keeping the blood sugar constant. According to Dun& (1953), it depends on the blood sugar level in the hypothalamus since he found that the injection of glucose in this region decreased the adrenal secretion. Von Euler and Luft (1952) found that the injection of insulin increased the excretion of adrenaline in human urine and had little effect on the excretion of noradrenaline. Dun& (1953, 1954) estimated adrenaline and noradrenaline in cats' adrenal blood by parallel assays and mathematical formulas and found that the infusion of glucose depressed the secretmionof adrenaline and that the injection of insulin had the opposite effect. Neither procedure had any effect on the secretion of noradrenaline. These experiments were done under anesthesia and the effect of a large dose of insulin was comparatively small. This important work should be confirmed on unanesthetized animals with chromatographic separation of the catecholamines. The effect of insulin on the adrenaline concentration in the peripheral blood has already been discussed in the section on methods. According to Weil-Malherbe and Bone (1952b, 1954) it falls, but according to Holzbauer and Vogt (1954a) it rises, as was to be expected. Insulin is one of the most effective agents for depleting the adrenal medulla of adrenaline, and there is evidence that it has less effect on the amount of noradrenaline in the gland (Burn et al., 1950b; Hokfelt, 1951; Outschoorn, 1952a). There is thus evidence of various kinds that the secretion of adrenaline, but not that of noradrenaline, is increased by hypoglycemia. This is sabisfactory for teleologists because adrenaline has much more hyperglycemic action than noradrenaline. e. Drugs. Elliott (1912) cut one splanchnic nerve in cats and then injected various drugs and eventually compared the adrenaline content of the two adrenals. The drugs which caused depletion in the innervated gland in these experiments were 0-tetrahydronaphthylamine,morphine,

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ether, chloroform, urethane, histamine, and diphtheria toxin. Strychnine, pilocarpine, eserine, posterior pituitary extracts, and adrenaline itself had no effect. This effect may either be due to a direct action on the central nervous system or t o various reflexes. Vogt (195.la) also observed depletion after 0-tetrahydronapht hylamine, morphine, or apomorphine, but not after leptazol or caffeine. These drugs also caused the disappearance of noradrenaline from the hypothalamus, and experiments 011 cats with one splanchnic nerve cut showed clear correlation between this effect and the effect on the innervated adrenal, which must have been due to impulses in the splanchriic nerve. Reserpine has a similar action (Holzbauer and Vogt, 1950). The effects of morphine were antagonized by nalorphine but not by chlorpromazine (Holzbauer and Vogt, 1954b). Other work is in agreement with much of this. The increased catecholamines in the adrenal blood have been detected after 0-tetrahydronaphthylamine (Sugawara, 1927), morphine (Stevart and Rogoff, 1922), ether (Bhatia and Burn, 1933), histamine (Dale, 1920, Roth and Kvale, 1954), strychnine (Stewart and Rogoff, 1919), and picrotoxin (Tatum, 1922). f. Other Facfors. Von Euler and Folkow (1953) found that when stimula;ion of a sensory nerve increased the secretion into the adrenal blood, it also increased the percentage methylated, while asphyxia, clamping the carotid arteries, and even splanchnic stimulation did not increase the percent age methylated. Muscular work may increase the secretion (Houssay and Molinelli, 1925; Cannon and Rritton, 1927), but according t o Wada et at. (1935), the effect i n dogs is small and occurs only when the animal is exhausted. Vori Euler and Hellner (1952) and von Euler and Lundberg (1954) found that during strenuous muscular work the excretion of both adrenaline and noradrenaline i n human urine was increased. I t is not always easy to separate the effects of muscular exercise from those of emotion, the effects of which were beautifully demonstrated by Cannon and de la Paz (1911). They collected blood from the region of the adrenal veins in a conscious cat through a tube passed up from the iliac veins and detected the hormone released when a dog appeared. Elliott (1012) found that emotional cats had small amounts of adrenaline in their adrenals. Diethelm et al. (1950) and Funkenstein et al. (1952) found an increased excretion of noradrenaline in human urine during emotion. Crawford and Law (1957) found that when rats were placed in metabolism cages, the excretion of catecholamines was high at first and then fell during the first few days as the rats became accustomed to their new environment. The subcutaneous injection of saline in these trained rats sometimes caused a small increase in the adrenaline-equiir-

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alerit of the urine collected during 24 hours, and if drugs are given it is necessary to compare their effects with those of control injections of saline.

4. Independent Release of Adrenaline and Noradrenaline If i t is true that there are two types of cell or two types of granule iri the adrenal medulla, one of which releases adrenaline and the other noradrenaline, it might be expected that these two substances would be released independently; the evidence on this question has been discussed and will now be summarized. It has been estimated in experiments on resting anesthetized cats that adrenaline represents about 25% of the total secretion. This proportion is lower than that found in extracts of the gland, so that noradrenaline is liberated preferentially during rest, but not exclusively. Carotid occlusion increases the amounts of both amines in the same proportion. The evidence for these conclusions is convincingly consistent, but all the estimates showing a large excess of noradrenaline in adrenal blood are based on the application of mathematical formulas to low concentrations of the catecholamines in unextracted plasma. These low estimates of the percentage methylated might be due to the presence of some substance which stimulated the fowl’s rectal cecum and might thus diminish the apparent concentration of adrenaline. This possibility was discussed by Dun&- (1953), who excluded histamine and acetylcholine as interfering substances, but not 5-hydroxytryptamine or other unknown substances. Most other forms of stimulation release larger amounts of the catecholamines, and the estimates of the percentage methylated are indistinguishable from those in extracts of the adrenals. There is evidence, however, that hypoglycemia causes a preferential liberation of adrenaline.

VIII. RELEA4SEFROM ADRENERGIC NERVES The early history of the work which led to the recognition of noradrenaline as the main chemical transmitter at adrenergic nerve endings was told a t the beginning of this article. The subject has been reviewed by von Euler (1951a). Elliott (1904) suggested the theory th a t something like adrenaline was released, and Loewi (1921) proved it by experiments on frog’s heart. This work was confirmed and extended by other work on tissues isolated from the body in salt solutions (reviewed by Gaddum and Kwiatkowski, 1939). It was shown that the substance liberated in the frog’s heart was unstable and that it caused not only stimulation of the heart but also vasoconstriction and inhibition of the gut; it,s actions lvere inhibited by ergotamine and it was present, in extracts of the heart.

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Loewi (1936) used the test for adrenaline depending on fluorescence (Gaddum and Schild, 1934) to get evidence which seemed to show that the substance liberated in a frog’s heart actually was adrenaline. Finkleman (1930) showed that stimulation of the nerves to an isolated piece of rabbit’s intestine liberated a substance which resembled adrenaline in its inhibitory effect on another piece of intestine. Gaddum et al. (1939; Gaddum and Kwiatkowski, 1939) showed that the sympathetic nerves in the perfused ear of a rabbit liberated a substance which caused vasoconstriction in the rabbit’s ear, stimulated a frog’s heart, inhibited t8hefowl’s rectal cecum, and gave Shaw’s specific test for adrenaline. The quantities were so small that the experiments were difficult, but the results indicated the release of adrenaline by the nerves. Outschoorn (1952b), using improved methods, confirmed this conclusion but showed that noradrenaline was also released. The first evidence that adrenaline was not the only substance liberated by adrenergic nerves came from work in Cannon’s laboratory (Cannon and Rosenblueth, 1937). Cannon and Rapport (1922) observed a small increase in the rate of the denervated heart on splanchnic stimulation even after adrenalectomy. This was shown to be due to the release of a11 adrenaline-like substance by adrenergic nerves. Cannon and Bacq (1931) suggested the word “sympathin” to denote this substance, and Cannon and Rosenblueth (1933) found that its properties were not quite the same as those of adrenaline. For example, adrenaline caused contraction of the nictitating membrane and inhibition of the nonpregnant uterus of a cat, and stimulation of the hepatic nerves in the same experiment caused a larger contraction of the nictitating membrane but had no action on the uterus. Cannon and Rosenblueth developed the theory that adrenaline was liberated by adrenergic nerves and then combined with receptors in the tissues, which either caused excitation and converted the adrenaline to sympathin E or caused inhibition and converted the adrenaline to sympathin I. These sympathins were then carried by the blood stream to cause excitation or inhibition in other parts of the body. It was found that many different sympathetic nerves liberated substances which affected various different tissues in other parts of the body, and all the results were attributed to mixtures of sympathin E and I. Later work has led to the conclusion that substances with purely motor or purely inhibitor effects do not exist and that the terms sympathin E and sympathin I should no longer be used. Cannon and Rosenblueth did not suggest that their results might be due to the liberation of a mixture of adrenaline and noradrenaline, but Bacq (1934) made this suggestion and various other workers (Greer et al., 1!138; Gaddurn and Goodwin, 1947) using similar techniques ohtainml

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results compatible with this theory. This kind of evidence cannot, however, be conclusive since the effects on different organs are affected differently by the rate of injection and the rate of liberation by the nerves is unknown. The work which proved that L-noradrenaline is present in adrenergic nerve fibers is discussed above (Section VI, 5). It left no real doubt that the main substance liberated was noradrenaline, and other independent work soon provided direct evidence of this. Gaddum et al. (1949) developed a method of distinguishing adrenaline and noradrenaline from closely allied substances in low concentrations and Peart (1949) applied this method to show that when the splenic nerves of a cat were stimulated electrically they liberated noradrenaline mixed with a small amount of adrenaline. This conclusion was confirmed by Mann and West (1950), and also by Mirkin and Bonnycastle (1954), who used chromatographic separation and bioassay. By similar methods, West (1950b) and Mann and West (1950, 1951) showed the release of noradrenaline with small amounts of adrenaline from various other tissues in cats on stimulation of sympathetic nerves. According to their estimates the percentages methylated in experiments with intestine and uterus were 23 and 12 respectively. In experiments with spleen and liver the percentages were smaller than this or negligible. Outschoorn and Vogt (1952) showed that noradrenaline was liberated in the dog’s heart when the sympathetic nerves were stimulated. No adrenaline was detected although chromatographic methods were used which would have detected it if the amount had been 3% of the amount of noradrenaline. Folkow (1952) has pointed out that the normal discharge rate in adrenergic nerves is 1-6 impulses per second and that it is possible that in these conditions the noradrenaline is mainly destroyed at the site of release. The experimenters who have detected it in the blood have used higher frequencies. This conclusion is confirmed by work by Brow1 arid Gillespie (1956), who studied the effect of frequency of stimulation on the release of noradrenaline from the cat’s spleen. The maximum recovery after 200 stimuli was obtained when the frequency was 30 per second. They concluded that the loss with lower frequencies was due to the combination of noradrenaline with the receptors, since the yield was increased when these were blocked with dibenamine. The reflexes which control the release of substances from adrenergic nerves have been studied in cats after adrenalectomy by recording the effects of various stimuli on the denervated heart or on the nictitating membrane sensitized by degenerative section of the cervical sympathetic (see Cannon and Rosenblueth, 1937). There is evidence, based on such

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experiments, that sympathin is released by emotion, by the stimulation of sensory nerves, by chilling, and by a fall of blood sugar.

IX. DISCUSSION Speculations about the functions of adrenaline and noradrenaline must depend on the oversimplification of complex and uncertain facts, but they are nevertheless worth making. Noradrenaline is a general vasoconstrictor and its main function is to control the blood pressure by regulating the tone of the arterioles. When the pressure in the carotid sinus falls, noradrenaline is released in the neighborhood of each arteriole, and the evidence suggests that it is also preferentially released by the adrenals. The peripheral resistance to the flow of blood is thus increased and the blood pressure rises. Adrenaline would be unsuitable for this purpose since it decreases the peripheral resistance. Because it is the main transmitter liberated by adrenergic nerves, noradrenaline also plays a part i t 1 all the local activities of the sympathetic system. The relation of adrenaline to blood sugar is like the relation of noradrenaline to blood pressure. Adrenaline raises the blood sugar more effectively than does noradrenaline, and when the blood sugar falls, adrenaline appears to be preferentially released from the adrenal medulla. A sudden burst of violent muscular activity increases the glucose metabolism and tends to lower the blood sugar. The quick reaction which prevents the blood sugar from falling too far depends on the release of adrenaline, which is especially well adapted for this purpose since it not only releases sugar from the liver but also prevents the uptake of sugar by the muscles. The maintenance of the supply of sugar to the brain is perhaps the primary purpose of adrenaline, while the other effects are of secondary importance. The pupil and the bronchi dilate, the pulse yuickens, the muscles become less easily fatigued, the circulation i n t tic brain and muscles increases, and the anterior pituitary releases corticotropin, which Causes the stores of sugar to be replenished. Adrenaline makes all these changes and prepares the animal for a(*tion; noradrenaline makes some of them much less effectively than adrenaline and the others not a t all. Adrenaline is released automatically when the blood sugar falls, but it may also be released by the complex reactions in the brain which lead to emotion. These reactions prepare the animal for an emergency before the emergency actually arises and may lead to the unnecessary release of adrenaline not followed by violent activity. This picture of the functions of adrenaline and noradrenaline is in-

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complete. For example, these substances undoubtedly play a part in the response of body to cold, but little is known about the relative amounts liberated in this response. Each substance helps in its own way to keep the internal &ate of the body constant, but neither of them is indispensable. The body has other mechanisms for controlling the blood sugar and the blood pressure. Cannon et al. (1929) removed all the sympathetic, ganglia from cats, so that all the sympathetic nerves must have degenerated, and the adrenal medulla was cut off from nervous connection with the central nervous system. In one cat, they also removed the hypophysis and one adrenal and the medulla of the other adrenal. These cats lived normally in the laboratory for many months and raised families. There Tvas no change in their natural dispositions; savage and friendly cats, restless and indolent cats, all remained true to themselves after sympathectomy. In the sheltered conditions of the laboratory they lived normally, but they had lost some of their power of responding to eniergencies. When restrained they showed emotion by struggling, but the blood sugar did not rise as it does in normal cats. They had thus lost one safeguard against hypoglycemia, but so long as they kept quiet their blood sugar was maintained by other means. Immediately after sympathectomy, the blood vessels and the pupils dilate and the nictitating membrane relaxes, but after a few days some compensation occurs and these efyects tend t o disappear. These facts illustrate the general rule that, the body does not rely entirely on one mechanism for producing any given result. REFERENCES Ahe, Y. 1924. Naunyn-Schmiedeberg’s Arch. exptl. Pathol. Pharmakol. 103, 73-83. Ahel, J. J., and Crawford A. C. 1897. Bull. Johns Hopkins Hosp. 8, 151-157. Ahlquist, R. P. 1948. Am. J . Physiol. 163, 586-600. Arman, C. G. van. 1951. Am. J . Physiol. 164, 476-479. Armin, J., and Grant, R. T. 1953. J . Physiol. (London) 121, 593-602. Armin, J., and Grant, R. T. 1955. J . Physiol. (London) 128, 511-540. Aronow, L., Howard, F. A., and Wolff, D. 1956. J . Phurmucol. Ezptl. Therap. 116, 1-2. Bacq, Z.M. 1934. Ann. physiot. physicochim. biol. 10, 467-528. Bncq, Z. M. 1947. Biol. Revs. 22, 73-91. Bacq, Z.M. 1949. Pharmacol. Reils. 1, 1-26. Bacq, Z.M., and Fischer, P. 1947. Arch intern. physiol. 66, 73-91. Bain, W. A., Gaunt, W. E., and Suffolk, S. T. 1937. J . Physiol. (London) 91,233-259. Barcroft, H., and Swan, H. J. C. 1953. “Sympathetic Control of Human Blood Vessels,” pp. 1-165. Edward Arnold, London. Barger, G., and Dale, H. H. 1910. J . Physiol. (London) 41, 19-59. Barker, J. H., Eastland, C. J., and Evers, N. 1932. Biochem. J . 26, 2129-2143. Barsoum, G. S., and Gaddum, J. H. 1935. J . Physiol. (London) 86, 1-14. Bass, A. 1914. 2.ges. Neurol. Psychiat. 26, 600-601. Bearn, A. G., Billing, B., and Sherlock, S. 1951. J . Physiol. (London) 116, 430-441.

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