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 octopamine. While p- tyramine, 3-methoxytyramine, and normetanephrine 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 norepinephrine from the sympathetic nerve endings. One mechanism by which release of norepinephrine from the nerve ending can be impaired is by replacement of the catecholamine 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.