Reprinted from THE JOURNAL OF PHARMACOLOOY AND EXPERIMENTAL THERAPEUTICS Vol. 130, No. 4 Copyright @ 1960 by The Williams & Wilkins Co. December, 1960 Printed in U.S.A. 8430 INCREASED RATE OF METABOLISM OF EPINEPHRTNE AND NOREPINEPHRINE BY SYMPATHOMIMETIC AMINES JULIUS AXELROD AND ROBERT TOMCHICK Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland Received for publication March 26, 1960 It has been previously shown that many types of drugs alter the rate of metabolism of the catecholamine hormones (Axelrod and Laroche, 1959; Axelrod and Tomchick, 1959; Axelrod, 1960). The psychotropic drugs reserpine, chlor- promazine and imipramine speed the metabolism of epinephrine and norepinephrine, while the catechol-O-methyl transferase inhibitors, py- rogdlol and quercetin, block the metabolism of these compounds. Although iproniazid, a mono- amine oxidase inhibitor, does not affect the rate of disappearance of catecholamines themselves, it blocks the deamination of their 0-methylated metabolites, metanephrine and normetanephrine (Axelrod et al., 1958). In this communication we wish to report that a number of sympathomi- metic amines increase the rate of disappearance of epinephrine and norepinephrine in the intact mouse. MATERIALS AND METHODS. Adult male mice (NIH stock) weighing 25 to 30 g were used. H3-Epinephrine bitartrate (267 rc/mg) and Ha-7-norepinephrine bitartrate (1720 &mg) were prepared by the New England Nuclear Corp., Boston, Mass. These compounds were chromato- graphically pure and when dissolved in 2% sodium metabisulfite and kept at -10oC did not decom- pose or lose tritium after several months' storage. Estimation of Ha-epinephrine and Hz-norepi- nephrine in whole mouse. Mice were killed by a blow on the head and the whole animal was homog- enized with 100 ml of 0.1 N HCl in a Waring blen- dor. An aliquot of the homogenate was centrifuged at 10,090 X g and Ha-epinephrine was determined as described previously (Axelrod el al., 1959b). Ha-Norepinephrine was determined by the same procedure, except that the catecholamine was eluted from the alumina column with 0.1 N HCl. Specificity of the method. Eluates from whole mouse extracts obtained by the procedure de- scribed above were applied to Whatman $1 paper and developed in butanol-acetic acid-water, 8:2:2, or in phenol saturated with water and SOS. Depending on the catecholamine administered, single peaks of radioactivity having the same R&s as authentic epinephrine or norepinephrine were found. Catechol-O-methyl transferase activity was determined, using Ha-epinephrine as a substrate, by a procedure described previously (Axelrod et al., 1959a). RESULTS. Disappearance of epinephrine and norepinephrine in th.e whole mouse. Mice were given 3 rg H3-epinephrine (free base) or 1 pg norepinephrine (free base) in the tail vein. Ten minutes later the mice were killed and assayed for epinephrine or norepinephrine remaining. Zero time values were obtained by homogenizing untreated mice and then adding 3 pg epinephrine or 1 pg norepinephrine to the whole homogenate. After 10 minutes about 33% of the administered epinephrine was found in the mouse (table 1). The rate of metabolism of the hormone in the mouse was the same after the administration of 1 to 10 pg epinephrine. The disappearance of norepinephrine was slower than that of epi- nephrine, about 56% remaining after 10 minutes. E$ect of sympathomimetic amines on the me- tabolism of cakcholamines in v&o. The rate of metabolism of epinephrine and norepinephrine in the whole mouse after the administration of various sympathomimetic amines was examined. Mice were first given the sympathomimetic amine and then the catecholamine was adminis- tered intravenously. Details of drug dosage are described in table 1. The animals were killed 10 minutes after the injection of the catecholamine and assayed for the amount of hormone remain- ing. The sympathomimetic amines phenylethyl- amine, tyramine, phenylaminobutane, d-am- phetamine and ephedrine markedly increased the rate of metabolism of catecholamines in the whole mouse; Paredrine (p-hydroxyamphet- amine) had a moderate effect, and Synephrine 367 368 AXELROD AND TOMCHICK Vol. 130 TABLE 1 dugnaentation of catecholamine metabolism by sympathomimetic amines Amount of Drug Time Drugs Were Catecholamine Remaining in Whole Mouse Drug Gi~;u~:ch Given before Catecholamine Epinephrine Norepinephrine vlkg min % % None 38 f 4.5 i 56 f 5.1 Phenylethylamine 100 10 18 f 3.4 15 f 3.1 Ephedrine 50 10 20 f 3.1 22 f 2.2 d-Amphetamine 20 10 18 f 3.5 19 zt 2.6 Tyramine 80 30, 10 ; 22 f 3.3 35 zt 4.0 Phenylaminobutane 40 10 23 f 4.5 37 xk 2.5 Paredrine 40 10 27 f 2.2 34 zt 5.3 Synephrine 40 10 35 f 8.8 54 f 3.5 * All sympathomimetic amines were given intraperitoneally. Mice received 3 pg H3-epinephrine or 1 pg norepinephrine in the tail vein and were killed 10 minutes later. Results are expressed as per cent of administered catecholamine remaining in the whole animal (rt standard deviation of the mean) in 10 minutes. Six to 10 mice were used in each group. (p-hydroxy(methylaminomethy1) benzyl alcohol) had a negligible effect. Lack of effect of sympathomim.etic amines on catechol-O-methyl transjerase. almost all of the epinephrine and norepinephrinc that disappeared in the whole mouse could be accounted for as free and conjugated 0-methylated metabolites metanephrine (Axelrod and Laroche, 1959) and normetanephrine (Whitby et al., 1960). Since the catecholamines were metabolized primarily by 0-methylation, the sympathomimetic amines might elicit their response by enhancing catechol- O-methyl transferase activity. Consequently, the effect of sympathomimetic amines on catechol-o- methyl transferase activity was examined. The enzyme was prepared by homogenizing the mouse liver in 5 volumes of ice-cold isotonic potassium chloride in a Waring blendor. After centrifuging the homogenate at 4oC at 80,000 X g for 30 minutes, an aliquot of the clear soluble super- natant fraction was assayed for catechol-o- methyl transferase activity (Axelrod et al., 1959a) (at a concentration of 5 X 10e4 M and 5 x 1W >I). Sympathomimetic amines (d- amphetamine, ephedrine, tyramine, phenylethyl- amine) had no effect on catechol-O-methyl transferase activity. DISCUSSION. We have shown that epinephrine (Axelrod et aE., 1959b) and norepinephrine (Whitby et al., 1960) disappear from the whole animal in two phases. In the first 5 minutes, part of the catecholamines are rapidly metabolized, primarily by 0-methylation. Following this, the catecholamines disappear slowly over a period of many hours. The slower second phase indicates that part of the catecholamines are bound to tissue constituents and then slowly released and metabolized. Since a larger fraction of norepi- nephrine is found in the whole mouse as compared to epinephrine, it appears likely that a larger amount of norepinephrine is bound. Reserpine, a compound that has been shown to release cate- cholamines from tissue (Holzbauer and Vogt, 1956), also speeds the metabolism of epinephrine (Axelrod and Tomchick, 1959) and norepineph- rine (Axelrod, 1960). This compound is pre- sumably acting by preventing the binding of the administered catecholamines, thus exposing them to enzymatic attack and more rapid destruction. The findings reported here, that sympathomi- metic amines increase the rate of metabolism of epinephrine and norepinephrine, suggest that they might also prevent the protective binding by tissue constituents. Many hypotheses have been proposed ex- plaining the mode of action of sympathomimetic amines. Until recently, it was generally believed that these compounds act directly on the adren- ergic receptors or by inhibiting monoamine oxidase. Carlsson et al. (1957) and Burn and Rand (1958) have shown that sympathomimetic amines will not elicit their responses in the absence of tissue catecholamines. As a result of these observations, Burn and Rand (1958) postulated that sympathomimetic amines act by liberating catecholamines. Our findings tend to 1960 CATECHOLAMINE METABOLISM 369 support such a mechanism. We have shown that sympathomimetic amines speed the metabolism of administered catecholamines, presumably by preventing the protective binding of these hormones. By affecting the biding sites, the sympathomimetic amines might also cause the release of bound endogenous catecholamines. Burn and Rand (1958) have found that tyramine, phenylethylamine, ephedrine and d-amphetamine will not produce a pressor action when the catecholamine stores are depleted. Likewise, we have noted that these compounds also increase the rate of metabolism of catecholamines. On the other hand, it has also been observed by Burn and Rand (1958) that Neosynephrine (phenylephrine) increased pressor response in animals depleted of catecholamines. Synephrine, a congener of Neosynephrine, had little effect on the speed of metabolism of catecholamines. SUMMARY Epinephrine disappears from the whole mouse more rapidly tha.n norepinephrine. The sympathomimetic amines tyramine, phenylethylamine, ephedrine, d-amphetamine, phenylaminobutane and Paredrine (p-hydroxy- amphetamine) markedly increase the rate of disappearance of epinephrine and norepinephrine in the whole mouse. REFERENCES AXELROD, J.: in Ciba Foundation Symposium on Adrenergic Mechanisms, Churchill, London, in press, 1960. AXELROD. J.. ALBERS. R. W. AND CLEMENTE. C. D.: .i. Nkurochem: 6: 68. 1959a. A&LROD, J., INSCOE, J. l?., SENOH, S. AND WITKOP, B.: Biochim. biophys. Acta 27: 210, 1958. AXELROD, J. AND LAROCHE, M.-J.: Science 130: FQO, 1959. AXELROD, J. AND TOMCHICK, R.: Nat,ure, Lond. 184: 2027, 1959. AXELROD, J., WEIL-MALHERBE, H. AND TOMCHICK, R.: THIS JOURNAL 127: 251, 1959b. BURN, J. H. AND RAND, M. J.: J. Physiol. 144: 314, 1958. CARLSSON, A., ROSENGREN, E., BERTLER, A. AND NILSSON, J.: in Psychotropic Drugs, ed. by S. Garattini and V. Ghetti, p. 363, Elsevier Publishing Company, Amsterdam, 1957. HOLZBAUER, V. AND VOGT, M.: J. Neurochem. 1: 8, 1956. WHITBY, L. G., WEIL-MALHERBE, H. AND AXELROD, J.: Fed. Proc. 19: 296, 1960.