REIJI IMAIZUMI*, KIYOSHI OMORI, ATSUSHI UNOKI, KAORU SANO, YASUO WATARI, JIN MAMBA AND KUNIO INUI

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1 PHYSIOLOGICAL SIGNIFICANCE OF MONOAMINE OXIDASE REIJI IMAIZUMI*, KIYOSHI OMORI, ATSUSHI UNOKI, KAORU SANO, YASUO WATARI, JIN MAMBA AND KUNIO INUI Department of Pharmacology, Osaka University Medical School, Osaka Department of Pharmacology, Osaka University Denial School, Osaka Received for publication May 9, 1958 INTRODUCTION Since Hare (1) found monoamine oxidase (abbreviated as MO) in 1923, many reports have been made that MO is the only one enzyme that inactivates adrenaline (abbreviated as AD), and the metabolism of AD has been discussed chiefly in this direction, while the potentiation of AD by ephedrine and cocaine has been explained by Blaschko, Richter, Schlossmann (2), Gaddum (3) and others as it is due to their inhibiting action on MO. On the other hand, Griesemer (4), Kuelle (5) and Euler (6), from their respective experiment, expressed their opinions that AD is hardly attacked by MO, and Udenfriend (7) stated "it is possible that the only physiologic action of MO is on serotonin." It was also asserted by Bacq (8) that MO has no significance in AD metabolism the serum con centration of which is between 10-'M and 10- M, while the Michaelis constant of MO for AD is 1.5 x 10-2M. And it was made clear by Schayer (9) that when radioactive AD is administered to rats, the increased excretion of the labelled AD in urine occurs together with the administration of marsilid and p-tolylether bromide but not with other inhibitors such as ephedrine and cocaine. From these data written above, we have come to doubt if the potentiation ephedrine and cocaine can be well explained by their inhibiting action on MO. of AD by Thus many problems about the physiological significance of MO, esp. about its role in AD metabolism are left unsolved. To investigate the influence of some MO inhibitors upon the action of AD, tyramine, noradrenaline (abbreviated as NA) and adrenalone (abbreviated as AL), and moreover to scrutinize the physiological significance of MO, the following experiments out. I. The Influence of Marsilid upon Vasoconstricting Action by Amines have been carried Method The change in carotid blood pressure of rabbits caused by AD, tyramine and AL injec tions, were recorded on a kymograph before and 1 hour after the intravenous injection of marsilid (20 mg, kg) which is known to inhibit MO in vrvo as well. * Professor

2 Results As shown in Fig. 1, both the blood pressure increase by tyramine and its decrease by AL were strengthened after the administration of marsilid, but in the case of AD, its action was rather weakened than strengthened. FIG. 1. Effect of marsilid on blood pressure response to adrenaline, tyramine and adrenalone. 1, 5 : Adrenaline 4 r 2, 6 : Tyramine 2 mg 3, 7 : Adrenalone 5 mg 4, 8 : Adrenalone 0.4 mg This result together with the fact that MO is the unique enzyme inhibited by marsilid led us to conclude that MO can not be the limiting factor of AD action in i4vo, though it may play some role in cases of tyramine and AL. Methods 11. The Influence of Various MO Inhibitors upon Vasoconstricting Action by Amines Each inhibition rate of cocaine, marsilid and methylene blue was measured and corn pared. Preincubation with these amines was omitted. Soluble MO of guinea pig liver was prepared following the Schayer's method (10) as follows : The 33 per cent homogenate in 0.25 M sucrose was centrifuged at 600 x g and the mito chondria was separated after the re-centrifugation of its supernatant at x g for 30 minutes. After being washed in 0.25 M sucrose, the mitochondria was suspended in 3 times larger than the original liver amount of 0.25 M sucrose, and oscillated for 1 hour at 2-3 C. MO activity was measured by the 0, uptake and the ammonia liberation, the former, by the standard manometric method and the latter, by Conway's method (11). The influence of these three MO inhibitors upon vasoconstricting action was investi gated following the Krawkow-Pissemski's Results method. It became clear from the above experiments that methylene blue showed the strongest inhibition, marsilid less strong and cocaine the weakest inhibition (Fig. 2, 3). In the experiment as to the vasoconstricting effect, concaine in dosis submedicamentosa sensitized AD, but not NA and tyramine (Fig. 4). Marsilid which showed stronger inhibi tion to MO than cocaine increased the vasoconstricting effect by NA and tyramine, but did not potentiate AD. Methylene blue having the strongest inhibition to MO potentiated

3 FIG. 3. Determination of liberated am monia by monoamine oxidase in the presence of some inhibitors. FIG. 2. Inhibition degree of cocaine, marsilid and methylene blue to the monoamine oxidase activity. FIG. 4. Influence of monoamine oxidase inhi bitors on the ear vessel. Adrenaline x c.c. Noradrenaline x 5 x 10, 0.5 c.c. Tyramine : x c.c. Cocaine : x c.c. Marsilid : x 1(3 0.5 c.c. Methyleneblue : x c.c. tyramine, but neither AD nor NA. It was clarified that the inhibition rate of MO inhibitors does not parallel the potentiation degree of AD. The stronger inhibition they have, the weaker potentia tion of AD they show. We assume that the mechanism of AD potentiation can not be attributed to the MO inhibition. On the other hand, Thompson and Tickner (12) have proved MO in blood vessel walls and Burn (13) has reported the decrease of its activity in sympathecto mized vessel walls and in consequence, the stronger appearance of AD action. We followed their experiments in order to investigate the presence of MO in blood vessel walls both chemically and histologically. Method III. Investigation of MO in Blood Vessel Walls 1) Chemical investigation Five hundred mg of rabbit liver and intima of aorta was ground with quartz sand and its extract in 10 c.c. of 1/20 M phosphate buffer was used as enzyme material.

4 Using AD. tvramine and AL as substrates, the oxygen consumption was manometri cally measured and at the same time. ammonia liberated from tvramine. and methylamine from AD and' AL. was measured by Conway's method (11). Thus, the activities of MO in liver and aorta were compared. The Oa consumption by MO was, for convenience' sake, regarded as the inhibited O_ uptake by adding marsilid in the final concentration of M, as enzyme materials used here were not purified at all. 2) Histochemical int estigation The histochemical investigation was carried out by following Francis' method (14): A small piece of aorta was inserted into the rabbit liver tissues, and its frozen section with the breadth of 20 p was prepared by means of Shimizu's cryostat (15), and was transfer red to the object glass and incubated for 15 hours at 37 C in the following solution. After the incubation, it was washed in aqua dest. fixed in 10 formalin for 1 hour and washed in aqua dest. again. The formation of diformazan granules which is assumed to be the reduced form of neotetrazolium was examined. Results 1) Chemical inrestigation In the manometric measurement of O., uptake. the oxidation of AD, tyramine and AL by MO in aorta was very weak compared with that in liver, esp. in the cases of AD and AL as substrates, their oxidation was almost negligible (Fig. 5). The quantitative determination of ammonia ran parallel with the manometric results (Fig. 6). 2) Histochentieal ittt'eslit;ation Endogenous activity, that is to say, the reduction of the tetrazolium salt to its formazan in the absence of added substrate was found to be destroyed by freezing and thus to be negative. The formation of diformazan granules in liver parenchyma, when tyramine was present as substrate, was recognized. but it was quite free in the aorta wall (Fig. 7, A, B). This colored precipitate ' as apparently decreased with the addition of marsilid (Fig. 7, C). That led us to assume it corresponding to the formation of diformazan as a result

5 FIG. 5. Oxidation of adrenaline and tyramine by liver and aorta monoamine oxidase. FIG. 6. Determination of ammonia liberated from adrenaline,, adre nalone and tyramine by liver and aorta monoamine oxidase. of the reduction of neotetrazolium which occurred in the process of oxidation of tyramine by MO. While the color formation was not seen in the aorta wall even by adding tyramine. Thus we failed in detecting MO in aorta walls by this histochemical experiment. Admitting that the slight oxygen uptake and ammonia production were recognized, it must be con cluded that the activity of MO in blood vessel walls is far from being significant even if we must be careful in denying its existence.

6 Judging from these data together with those in chapter II. we, in spite of the Burn's report (13) quoted in chapter II. assumed that the mechanism of potentiation of AD action can hardly be explained b\ the inhibition of MO in blood vessel walls. I1 Affinity o f %l() to Its Suhstrate3 It is well known that there exist many at-nines in living organisms, and these biogenic amines are destroyed through oxidative deamination in eitro. But it is naturally supposed that their affinity to MO is different respectively, and in the coexistence of multiple sub strates as in living organisms, substrates having stronger affinity are more easily destroyed and others having less affinity remains less attacked. Of these monoamines. the affinities of serotonin, AD and AL to MO were examined. Methods MO was prepared from guinea pig liver following the Schayer's method (9) as described in chapter II. The oxygen uptake was measured by using the standard Warburg technique. In the systems involving two amines, namely AD and serotonin, and AD and AL, their total oxygen uptake was first measured, and to clarify how much of the total consumption was attributed to each of the two amines, serotonin which remained unoxidized was determined colorimetrically (16) and AD fluorimetrically (17). Oxygen consumption attributed to AL was calculated by substracting that attributed to AD from the total oxygen consumption. Results As shown in Fig. 8, the oxygen uptakes of serotonin, AD and AL are large in the order listed, namely serotonin>ad>al. However, it does not necessarily indicate the order of affinity to M10. FIG. 8. The effect of amine concentration on the oxidation rate of monoamine oxidase. Time 10 min.

7 TABLE 1. Michaelis constants of serotonin, adrenaline and adrenalone to monoamine oxidase. So, the Michaelis constants of these three amines were approximately estimated from Vmax/2 in Fig. 8, and it was known that the affinity to MO of these amines is strong in the order of serotonin, AL and AD, namely serotonin>al>ad (Table 1). However, the Michaelis constant does not strictly indicate the substrate affinity to the enzyme. So, the two-substrates-systems were investigated. TABLE 2. Oxidation of equimolar mixtures of adrenaline and another amine by monoamine oxidase. The results in the systems involving two amines are presented in Table 2. In both cases, AD oxidation was strikingly inhibited, while scrotonin and AL oxidations were little influenced. These experimental findings were in accord with those obtained from the rough cal culation of the Michaelis constants. From the above data, it was ascertained that AD has little affinity to MO. Since Hare's experiment DISCUSSION (1) in 1923, MO has always been conceived as the most im portant AD-destroying enzyme. Along the tendency to stress the physiological significance of MO, Burn (13) has reported the presence of MO in blood vessel walls and the cor relation after denervation between the change of MO activity and that of AD action, Blaschko, Richter, Schlossmann (2), Gaddum (3) and others have attributed the potentiation of AD by ephedrine and cocaine in their inhibition of MO. However, as quoted in the introduction, Bacq (8), Euler (6) and others have come to reflect on such a conception as attaches too much importance to MO. In our experiments, we also could not help doubting its significance. Cocaine potentiates AD, but has less in hibition to MO than methylerte blue or marsilid which does not potentiate AD. This fact indicates us that MO is not the limiting factor in AD potentiation. Moreover, the activity of MO in blood vessel walls was found too weak to be examined histochemically, and also the affinity of AD to MO was proved to be strikingly small. Thus, it is not right to attribute the potentiation of AD action by cocaine or ephedrine upon peripheral vasoconstriction to the change of MO activity in vessel wall. Nevertheless, it is obviously a mistake to make much of MO in AD action, and this together with the fact that serotonin, having far stronger affinity to MO than AD, exists

8 in living organisms in the concentration 10' times higher than that of AD, naturally led us to the necessity of scrutinizing the role of MO in AD metabolism. On the other hand, the result from the experiments using radioactive AD has been reported by Schayer (18) and Resnick (19) that MO was responsible for at least 50 per cent in AD metabolism. But it does not always follow that AD was directly oxidized by MO. Years ago, Prof. Imaizumi (20-22) of our Department made it clear that AD is, by AD dehydrogenase, oxidized to AL which has a stronger affinity to MO. It can possibly be supposed that Schayer and others might have observed the oxidation of AL by MO after AD had been oxidized to AL. The further studies in the significance of AD dehydrogenase both in AD metabolism and in AD potentiation is an interesting problem now confronting us. SUMMARY 1. Marsilid strengthens the action of tyramine and AL upon blood pressure but does not strengthen that of AD. 2. Methylene blue shows the strongest inhibition to MO, marsilid less strong, and cocaine the weakest inhibition. Methylene blue and marsilid increase the vasoconstricting effect by tyramine but do not increase that by AD, while cocaine increases that by AD, but does not increase that by NA nor that by tyramine. It is clear that the inhibition rate of MO inhibitors is not parallel with their potentiation degree of AD action upon vasoconstriction. 3. From chemical and histochemical investigations, it is assumed that the MO activity in blood vessel walls is extremely weak. 4. Among various substrates of MO viz., serotonin, AL and AD, their affinity to MO is strong in the order of serotonin, AL and AD, namely serotonin>al>ad. It was assumed and discussed that the physiological significance of MO in AD action is doubtful and the potentiation of AD by cocaine and ephedrine can not be well explained by the role of MO. 1) HARE, M.L.C.: Biochem. J. 22, 918 (1928) REFERENCES 2) BLASCHKO, H., RICHTER, D. AND SCHLOSSMANN, H. : Ibid. 31, 2187 (1937) 3) GADDUM, J.H. AND KWIATOWSKI, H. : J. Physiol. 96, 385 (1939) 4) GRIESEMER, E.C., BARSKY, J., DRAGSTEDT, C.A., WELLS, J.A. AND ZELLER, E.A. : Proc. Soc. exp. biol., N.Y. 84, 699 (1953) 5) KUELLE, G.B. AND VALK, A. DU T. JR.: J. Physiol. 126, 434 (1954) 6) EULER, U.S. VON AND ZETTERSTROUR, B. : Acta physiol. scand. 33, Suppl. 118, 26 (1955) 7) SJOERDSMA, A., SMITH, T.E., STEVENSON, T.D. AND UDENFRIEND, S.: Proc. Soc. exp. Biol., N.Y, 89, 36 (1955) 8) BACQ, Z.M.: J. Pharmacol. 95, 1 (1949)

9 9) SCHAYER, R.W.: Proc. Soc. exp. Biol., N.Y. 84, 60 (1953) 10) SCHAYER, R.W., SMILEY, R.L. AND KAPLAN, E.H. : J. biol. Chem. 198, 545 (1952) 11) CONWAY, E.J. AND BYRUE, A.: Biachem. J. 27, 419 (1933) 12) THOMPSON, R.H.S. AND TICHENER, A. : J. Physiol. 115, 34 (1951) 13) BURN, J.H. : Brit. J. Pharmacol. 7, 304 (1952) 14) FRANCIS, C.H. : Nature 171, 701 (1953) 15) SHIMIZU, N., KUBO, Z. AND MORIKAWA, N. : Stain Technol. 31, 105 (1956) 16) UDENFRIEND, S., WEISSBACH, H. AND CARROLL, T.C. : J. biol. Chem. 215, 337 (1955) 17) EULER, U.S. VON AND FLODING, I.: Acta physiol. scand. 33, Suppl. 118, 45 (1955) 18) SCHAYER, R.W., WU, K.Y.T., SMILEY, R.L. AND KOBAYASHI, Y.: J. biol. Chem. 210, 259 (1954) 19) RESNICK, O. AND ELMADJIAN, F. :J. clin. Endocrinol. and Metabol. 18, 28 (1958) 20) IMAIZUMI, R. AND KAWAMOTO, K. : Med. J. Osaka Univ. 3, 269 (1952) 21) IMAIZUMI, R., KAWAMOTO, K., KITA, T. AND SATO, H.: Ibid. 3, 279 (1952) 22) IMAIZUMI, R., YOSHIDA, H., WATAYA, K., TAKAOKA, Y., NODA, T. AND WADA, M.: Ibid. 5, 223 (1954)

Interrelationship between Angiotensin Catecholamines. Tatsuo SATO, M.D., Masaru MAEBASHI, M.D., Koji GOTO, M.D., and Kaoru YOSHINAGA, M.D.

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