EVIDENCE FOR A FALSE NEUROCHEMICAL TRANSMITTER AS A MECHANISM FOR THE HYPOTENSIVE EFFECT OF MONOAMINE OXIDASE INHIBITORS BY IRWIN J. KOPIN, JOSEF E. FISCHER, JOSE MUSACCHIO, AND W. DALE HORST

LABORATORY OF CLINICAL SCIENCE, NATIONAL INSTITUTE OF MENTAL HEALTH, NATIONAL INSTITUTES OF HEALTH Communicated by Seymour S. Kety, July 14, 1964 Monoamine oxidase inhibitors have been found useful in the treatment of hypertension, angina, and some psychiatric disorders. The mechanism by which these drugs act, however, is not well understood. There is both clinical and experimental evidence that the accumulation of nor- mally produced amines is related to the diminished sympathetic responsiveness seen after monoamine oxidase inhibition. When monoamine oxidase inhibitors are administered, there is rapid inhibition of the enzyme with increased excretion of endogenously formed amines in the urine,' yet the clinical response to the drug develops gradually over a period of days. Furthermore, there appears to be a relation between the degree of monoamine oxidase inhibition and the hypotensive effects observed.2 These clinical observations are consistent with the view that the hypotensive effects are related to an accumulation of amines in the tissues. Kakimoto and Armstrong3 demonstrated that inhibition of monoamine oxidase in the rabbit results in marked elevation of tissue levels of . While p- , 3-methoxytyramine, and were also present in the brain, normetanephrine was the only other monophenolic amine that could be found in the heart. These workers suggested that the beneficial effects of monoamine oxidase inhibitors in the treatment of anginal pain might result from the accumulation of octopamine in the heart. Davey et al.4 showed that chronic, but not acute, treat- ment with a monoamine oxidase inhibitor resulted in diminished release of norepi- nephrine in response to stimulation of the sympathetic nerves of the perfused cat spleen. Day and Rand5 reported that after a monoamine oxidase inhibitor, re- peated tyramine administration resulted in reduced response to sympathetic nerve stimulation, and that this occurred to a lesser extent when the enzyme was not in- hibited. They suggested that amines which accumulate in the tissue following treatment with a monoamine oxidase inhibitor may result in impaired release of from the sympathetic nerve endings. One mechanism by which release of norepinephrine from the nerve ending can be impaired is by replacement of the by another, less active, molecule. It has been shown that the methyl amino acids, a-methyl dopa and a-methyl metatyrosine, are decarboxylated to form a-methyl amines,6 which are resistant to the action of monoamine oxidase. Several groups of investigators have suggested that these foreign amines, or their ,3-hydroxylated derivatives, may enter norepi- nephrine storage sites and then be released7' 8 by nerve stimulation. Following ad- ministration of a-methyl dopa, Muscholl and Maitre9 demonstrated that a-methyl norepinephrine is released from the heart by stimulation of the sympathetic nerves. The ratio of a-methyl norepinephrine to norepinephrine released was similar to 716 Downloaded by guest on September 25, 2021 VOL. 52, 1964 BIOCHEMISTRY: KOPIN ET AL. 717

their ratio in the myocardium. Similar findings have been recently reported for metaraminol,'0 the 63-hydroxy amine derivative of a-methyl metatyrosine. The a-methyl group protects these foreign amines from the action of monoamine oxidase. When monoamine oxidase is inhibited, there is a marked increase in the excretion, and presumably the blood levels, of tyramine and other amines.' Following intra- venous administration of small doses of labeled tyramine, the amine is taken up by the heart, spleen, and salivary gland and rapidly converted to octopamine." This f-hydroxylated amine is selectively retained in the tissue and remains after all the tyramine is destroyed. If the superior cervical ganglion has been removed and the sympathetic nerves allowed to degenerate for several days before the administration of the labeled amine, only negligible amounts of octopamine can be demonstrated in the salivary gland.11-'3 These results indicate that octopamine formation and storage require an intact sympathetic nerve supply. It is probable that these proc- esses occur in the sympathetic nerves. It is generally recognized that to implicate a molecule as a neurotransmitter it is necessary to establish that the substance occurs in the nerve ending, that it is released by nerve stimulation, and that, at physiological concentrations, it mimics the effects of nerve stimulation. Norepinephrine satisfies all three criteria and is generally considered to be the normal sympathetic neurotransmitter. The term, "false neurochemical transmitter," has been applied8' 11 to other substances, nor- mally not present in significant amounts in the sympathetic nerves, which can be made to accumulate in the nerve endings and which can then be discharged by sympathetic nerve stimulation. We have attempted to determine whether octopamine accumulates in the sympathetic nerves after inhibition of monoamine oxidase, and whether this amine can be released as a false neurochemical transmitter. Procedures and Results.-Effect of monoamine oxidase inhibition on octopamine accumulation in intact and denervated tissues: The right superior cervical ganglion was removed from four cats, and sufficient time (more than 1 week) was allowed to elapse to ensure sympathetic nerve fiber degeneration. Pupillary constriction and relaxation of the nictitating membrane on the ganglionectomized side were apparent. The monoamine oxidase inhibitor, pheniprazine, 10 mg/kg, i.p., was administered daily for 2 days. The cats were anesthetized with nembutal, 30 mg/kg, i.p., and the salivary glands removed from the four cats. The denervated and intact sali- vary glands of two cats were pooled and homogenized separately in 10 vol of cold 0.4 N perchloric acid. A trace amount of octopamine-H3 (10-7 mg) was added to each homogenate. Following centrifugation, the slightly cloudy supernatant fluid was adjusted to pH 6.5, using 5 N potassium hydroxide and 0.5 N potassium carbonate. After allowing it to stand overnight in the refrigerator, the solution was decanted from the crystals of potassium perchlorate and the amines absorbed on Dowex-50 (NH4+) and eluted with 3 N NH40H. The eluate was concentrated in vacuo, and the salts precipitated by the addition of 10 vol of acetone. After centrifugation, the clear supernatant solution was decanted into 15-mi centrifuge tubes and the solvent evaporated in a stream of nitrogen. The dry residue was taken up in 0.2 ml of ethanol, and an aliquot containing 60 per cent of the octop- amine-H3 tracer was chromatographed as described by Kakimoto and Armstrong.3 Downloaded by guest on September 25, 2021 718 BIOCHEMISTRY: KOPIN ET AL. PROC. N. A. S.

The octopamine content of the hearts and spleens of these animals was also deter- mined. Although octopamine was not found in the tissues of control animals, this amine was easily demonstrable in the heart, spleen, and salivary glands of cats chronically treated with pheniprazine. The denervated salivary glands, however, contained only traces of octopamine (Table 1). TABLE 1 OCTOPAMINE CONTENT* OF TISSUES OF THE CAT Untreated Pheniprazine-treated Salivary gland Intact <0.05 (4) 0.3 -0.4 (4) Denervated <0.05 (4) 0.05-0.08 (4) Heart <0.05 (2) 0.8 -1.2 (3) Spleen <0.05 (2) 0.2 -0.3 (3) * Results expressed as pg/gm tissue. Number of cats shown in parentheses. Effect of denervation and monoamine oxidase inhibition on accumulation of octop- amine-H3 formed from administered tyramine-H3: In other experiments, the right superior cervical ganglion was removed from male Sprague-Dawley rats weighing 180-220 gm. One week later, half the animals were treated intraperitoneally with a monoamine oxidase inhibitor, pheniprazine (10 mg/kg). One hour after the monoamine oxidase inhibitor, 20 ,uc of tyramine-H3 (1.56 C/mM) or 8 Mc a-methyl tyramine-H3 (2.5 C/mM) was injected intravenously. Animals not treated with pheniprazine served as controls. One hour after administration of the labeled amines, the animals were killed and the salivary gland analyzed for octopamine-H3 as previously described.'3 The innervated salivary gland always contained much more of this labeled ,3-hydroxylated amine than the chronically denervated gland. The monoamine oxidase inhibitor caused a more than tenfold increase of octopamine- H3 in the innervated salivary gland but resulted in no increase on the denervated TABLE 2 AMINE CONTENT OF INNERVATED AND DENERVATED RAT SALIVARY GLANDS Octopamine-H 2 - > -a-Methyl Octopamine-H' Denervated Intact Denervated Intact Untreated 15.0 ± 1.9 49.9 4 4.0 4.1 ± 0.9 99.1 ± 13.7 Pheniprazine 19.9 4 3.3 581. 0* 4 60.5 4. 0 i 0. 6 93.9 i 10.4 * p <0.001. Rats received 20 pc tyramine-H3 or 8 pc a-methyl tyramine-H3 1 week after removal of the superior cervical ganglia. Results are the means for groups of six rats, expressed as mjsc/gm :1: S.E.M. side (Table 2). Pheniprazine did not influence the accumulation of a-methyl octopamine-H3, which is not a substrate for the enzyme. These results indicate that the monoamine oxidase inhibitor facilitated accumulation of octopamine in the innervated salivary gland and that the action of the drug was mediated through enzyme inhibition. Release of octopamine-H3 by sympathetic nerve stimulation: A third group of experiments was performed in which spleens from untreated cats were isolated and perfused by a modification of the technique described by Brown and Gillespie.'5 Tyramine-H3 (50 MA) was infused into the splenic artery, and one-half hour later the effect of sympathetic nerve stimulation on release of octopamine-H3 was observed. Stimulation of the splenic nerve resulted in a marked increase in the rate of appear- Downloaded by guest on September 25, 2021 VOL. 52, 1964 BIOCHEMISTRY: KOPIN ET AL. 719

ance of octopamine-H3 in the effluent (Fig. 1). The amount of octopamine re- leased appears to be related to the number of stimuli. When norepinephrine was infused, the spleen contracted as it did during the nerve stimulation, but there was no increase in octopamine release. The results, which are typical of six such experi- ments, indicate that octopamine can be released by sympathetic nerve stimulation, and not by contraction of the spleen alone. Discussion.-Following inhibition of monoamine oxidase, Kakimoto and Arm- strong3 demonstrated that octopamine accumulated in the tissues of the rabbit. They showed that this amine was normally present in human urine as well as in the urine of animals. It was pointed out that since p-hydroxymandelic acid, the major metabolic product of octopamine, was not excreted in increased amounts by pa- tients with pheochromocytoma or other tumors of the sympathetic nervous system, it appeared unlikely that the adrenals or sympathetic nervous tissues were a major site of octopamine formation. The results reported here in the cat are consistent

20-,

5-

E

Time 30 40 50 60 min

Frequency 10 10 30/sec NE Duration 30 10 lOsec FIG. 1.-Release of octopamine-H3 from the isolated perfused cat spleen. Each bar represents the octopamine-H3 present in the effluent during a 2-min collection interval. The splenic nerve was stimulated supramaximally at the indicated frequencies and durations. At NE, 1.0 mpLg norepinephrine was injected into the splenic artery. These results are typical of six such experi- ments.

with the observations of Kakimoto and Armstrong3 regarding accumulation of octopamine in the tissues following inhibition of monoamine oxidase. This amine did not, however, markedly accumulate in the chronically denervated salivary glands, indicating that an intact sympathetic nerve supply is required for this occurrence. These findings suggest that octopamine may accumulate in the sympathetic nerve endings. It has recently been demonstrated that intravenously administered tyramine-H3 is taken up into the tissues, presumably in the sympathetic nerves", 13 and rapidly converted to octopamine-H3. Octopamine-IP so formed is selectively retained and its distribution in tissue homogenates after centrifugation in a sucrose gradient parallels that of norepinephrine.'2 These findings suggest that, while complete synthesis of octopamine from may not occur to a significant extent in sympathetic nervous tissue, when tyramine is available, it can be taken up by the nerves and converted to octopamine. It would be expected that pretreatment Downloaded by guest on September 25, 2021 720 BIOCHEMISTRY: KOPIN ET AL. PROC. N. A. S.

with a monoamine oxidase inhibitor would enhance this conversion. The results presented show that in the intact rat salivary gland with inhibition of this enzyme there is a tenfold increase in octopamine derived from injected tyramine. Since tyramine excretion is increased by monoamine oxidase inhibitors,' it is likely that octopamine in the tissues, presumably in the sympathetic nerves of the tissue, is derived, at least in part, from tyramine in the blood. The latter amine is a product of tyrosine decarboxylation and may be formed in tissues other than the sympa- thetic nerves. If monoamine oxidase is not inhibited, then the tyramine is almost immediately destroyed so that little enters the circulation. It would not be ex- pected, therefore, that patients with tumors of the sympathetic nervous system would necessarily excrete excessive amounts of p-hydroxymandelic acid. This would occur only if excessive amounts of tyrosine were decarboxylated, and in the sympathetic nerves this reaction may result in only minimal formation of tyramine, compared to tyramine formation in larger organs, such as the liver. The definitive demonstration that octopamine can be made to accumulate in the sympathetic nerves must await histological studies, but presumptive evidence for this has been obtained in the present studies. The release of octopamine-H8 from the spleen of the cat by splenic nerve stimulation provides further evidence that octopamine is present in the nerve and that this substance can be a false transmitter. Preliminary evidence has been obtained that octopamine accumulated in the spleen after monoamine oxidase inhibition and could also be released by nerve stimulation, in amounts comparable to the norepinephrine ordinarily released. Since there is a diminished release of this catecholamine from the spleens of cats pretreated with a monoamine oxidase inhibitor, it is possible that the octopamine has replaced a portion of the normal transmitter under condition of monoamine oxidase inhibition. Norepinephrine is stored in the dense core vesicles of the sympathetic nerve.16 These vesicles contain -fj-oxidasel7 and probably synthesize, as well as store, the neurotransmitter. Studies of spontaneous potentials at sympathetic nerve endings in smooth muscle'8 have indicated that, as in skeletal neuromuscular transmission,19 the nerve ending response to the impulse probably depends on the synchronous release of the contents of a number of these vesicles. The hypothesis is proposed that following inhibition of monoamine oxidase, endogenously formed amines, such as tyramine, usually destroyed by this enzyme, are taken up by these vesicles and converted to the fl-hydroxylated derivatives. These f3-hydroxylated amines take the place of a portion of the norepinephrine normally contained in the vesicles and are released with the catecholamine. If each impulse releases a limited number of transmitter molecules, less norepinephrine would be released, a portion being replaced by less activel4 molecules, even if tissue levels of norepi- nephrine are elevated. This would result in apparent sympathetic blockade, es- pecially at low rates of nerve stimulation. At high, unphysiological rates of nerve stimulation, a sufficient number of vesicles could be made to release their contents so that the apparent block would be overcome. Similarly, it is possible that some drugs, such as or bretylium, are taken up in sympathetic nerve endings, replace a portion of the norepinephrine stores, and exert their sympathetic blocking action by acting as false transmitters which are not rapidly destroyed. This mechanism of alteration of neurohumoral Downloaded by guest on September 25, 2021 VOL. 52, 1964 BIOCHEMISTRY: WALLACH AND KAMAT 721

activity may be of importance following administration of other drugs as well as monoamine oxidase inhibitors, after development of tachyphylaxis to sympathomi- metic amines, and in disease states. 1Sjoerdsma, A., W. Lovenberg, J. A. Oates, J. R. Crout, and S. Udenfriend, Science, 130, 225 (1959). 2Orvis, H. H., I. G. Tamagna, D. Horwitz, and R. Thomas, Ann. N. Y. Acad. Sci., 107, 958 (1963). 3 Kakimoto, Y., and M. D. Armstrong, J. Biol. Chem., 237, 422 (1962). 4Davey, M. J., J. B. Farmer, and H. Reinert, Brit. J. Pharmacol., 20, 121 (1963). 5Day, M. D., and M. J. Rand, Brit. J. Pharmacol., 21, 84 (1963). 6Weissbach, H., W. Lovenberg, and S. Udenfriend, Biochem. Biophys. Res. Commun., 3, 225 (1960); Lovenberg, W., H. Weissbach, and S. Udenfriend, J. Biol. Chem., 237, 89 (1962). Carlsson, A., and M. Lindquist, Acta Physiol. Scand., 54, 87 (1962). 8Day, M. D., and M. J. Rand, J. Pharm. Pharmacol., 15, 221 (1963). 9Muscholl, E., and L. Maitre, Experientia, 19, 658 (1963). 10 Crout, J. R., and P. A. Shore, Clin. Res., 12, 180 (1964). 11 Carlsson, A., and B. Waldeck, Acta Pharmacol. Toxicol., 20, 371 (1964). 12 Musacchio, J., I. J. Kopin, and S. Snyder, Life Sciences, in press. 13 Fischer, J. E., J. Musacchio, I. J. Kopin, and J. Axelrod, Life Sciences, 3, 413 (1964). 14Lands, A. M., and J. I. Grant, J. Pharm. Exptl. Therap., 106, 341 (1952). 15 Brown, G. L., and J. S. Gillespie, J. Physiol., 138, 81 (1957). '6Wolfe, D., L. T. Potter, K. Richardson, and J. Axelrod, Science, 138, 440 (1962). 17 Potter, L. T., and J. Axelrod, J. Pharm. Exptl. Therap., 142, 209 (1963). 18Burnstock, G., and M. E. Holman, J. Physiol., 160, 446 (1962). 19Fatt, P., and B. Katz, J. Physiol., 117, 109 (1952).

PLASMA AND CYTOPLASMIC MEMBRANE FRAGMENTS FROM EHRLICH ASCITES CARCINOMA* BY DONALD F. HOELZL WALLACHt AND VIRENDRA B. KAMAT DEPARTMENT OF BIOLOGICAL CHEMISTRY, HARVARD MEDICAL SCHOOL Communicated by Herman M. Kalckar, July 9, 1964 A major obstacle to the study of plasma membranes has been the isolation of these structures in a reasonably pure form. For this reason we lack details about their specific composition, metabolism, three-dimensional organization, enzyme content, and antigenic properties. The present communication concerns a new approach to this problem, applied to the isolation and purification of the plasma membranes of Ehrlich ascites carcinoma cells. It is based upon the following: (a) the use of natural cell-surface markers, (b) cell rupture under controlled conditions, and (c) separation of membrane com- ponents by density, and by their ionic and permeability properties. Two surface markers were employed in this study: (1) a sodium- and potassium- dependent, magnesium-activated, ouabain-sensitive ATPase;' (2) the surface anti- gens involved in the agglutination of intact Ehrlich ascites carcinoma cells by heterologous antibody.2 When Ehrlich ascites carcinoma cells are ruptured under controlled, isoosmotic conditions by the intracellular cavitation of nitrogen gas,3 the plasma membrane breaks into small fragments, as evidenced by the fact that the cell surface markers require an average force field of 4.5 - 106 g - min to sediment Downloaded by guest on September 25, 2021