Naunyn-Schrniedeberg's

Archivesof Pharmacology

Naunyn-Schmiedeberg~s Arch. Pharmacol. 300, 273-279 (1977)

9 by Springer-Verlag 1977

Monoamine Oxidase in Rat Reticulocytes" Subcellular Localization and Identification of Isoenzymes* ** K. QUIRING and S. HUBERTUS*** Zentrum der Pharmakologie, Klinikum der Universit~it Frankfurt, D-6000 Frankfurt 70, Federal Republic of Germany

Summary. After "chemically induced reticulocytosis" in rats by treatment with acetyl-phenylhydrazide, monoamine oxidase (MAO) activities were determined in erythrocyte preparations of these animals. Studies on subcellular fractions obtained by differential centrifugation showed that the enzyme activity of rat reticulocytes is a classical mitochondrial MAO. The patterns of inhibition produced by clorgyline (A-type MAO), deprenil (B-type MAO) and pargyline or tranylcypromine (both types of MAO) in reticulocytes were determined in vitro using tryptamine as a substrate for both types of MAO and phenylethylamine as a substrate for the B-type. The results indicate that both A-type ( ~ 75 ~) and B-type ( ~ 25 ~) MAO are present in rat reticulocytes; while tryptamine was mainly deaminated by the A-type enzyme, both types of MAO were shown to contribute to the deamination of phenylethylamine, These findings were confirmed in-investigations on the thermostabilities of the tryptamine and phenylethylamine deaminating activities of rat reticulocyte MAO. Key words: Reticulocytes - MAO - Isoenzymes Clorgyline - Deprenil.

INTRODUCTION Reticulocytes of several mammalian and avian species contain an adrenergic E-receptor system (for review, see Kaiser et al., 1977); work done on rats in our laboratory showed that reticulocytes also possess the Send offprint requests to: K. Quiring, Institut fiir Arzneimittel, Bundesgesundbeitsamt, D-1000 Berlin 33 * This work was supported by a grant from the Deutsche Forschungsgemeinschaft ** Preliminary accounts on this work were presented at the 16. Fr/ihjahrstagung der Deutschen Pharmakologischen Gesellschaft (Quiring and Hubertus, 1975) and at the 6th International Congress of Pharmacology (Quiring et al., 1975 a) *** The experimental work presented in this paper includes the work done by S. H, for his doctoral thesis

catecholamine-inactivating enzymes catechol-O-methyltransferase (Quiring et al., 1973, 1975b) and monoamine oxidase (MAO; Quiring et aI., ~973, ~976). It is a prerequisite for determinations of reticulocyte enzyme activities in total red cell preparations that the portion of circulating reticulocytes is high (Quiring et al., 1973). This prerequisite can be experimentally fulfilled in two ways: 1. by using very young and rapidly growing animals which show a "physiological reticulocytosis" due to a growth-induced dilution anaemia (Ganzoni, 1970), or 2, by stimulating the haemopoietic system of older animals ("chemically induced reticulocytosis"); this is achieved by treating them with a haemolytic agent like acetyl-phenylhydrazide. Our previous work on rat reticulocyte MAO (Quiring et al., 1973) had made it probable that this enzyme be a "classical" monoamine oxidase according to the criteria "of Blaschko (1963). One of Blaschko's criteria is mitochondrial location of the enzyme; in recent experiments usin.g the "physiological reticulocytosis model" we obtained indirect evidence for mitochondrial location of the reticulocyte MAO (Quiring et al., 1976). It was considered of interest to confirm these results in the "chemically induced reticulocytosis model". During the last ;~0 years, '~ forms" or "isoenzymes" of the classical mitochondrial monoamine oxidase have been described in many organs of numerous species (for review see Sandler and Youdim, 1972; Blaschko, 1974; Tipton et al., 1976). Since rat reticulocyte MAO proved to be of mitochondrial origin, it seemed of interest also to characterize the enzyme by studying its substrate and inhibitor specificities as either A-type or B-type MAO 0br review, see Neff and Yang, 1974).

METHODS Male Wistar rats (150-200 g body weight) were used in all experiments. In order to induce reactive reticulocytosis, the animals

274 were treated with single daily i.m. doses (50 mg/kg) of the haemolytic agent, acetyl-phenylhydrazide, on 3 consecutive days. The animals were killed by decapitation on the 7th day after beginning of treatment, that is, at the peak of reticulocytosis (about 60 ~ reticulocytes, see Gauger et al., 1973). Blood from about 10 animals was pooled in cooled heparinized pIastic beakers and all subsequent procedures were carried out at 0 - 4 ~ Processing of Blood Samples. Plasma and corpuscular elements other than erythrocytes were removed as described previously (Quiring et al., 1975c). Erythrocyte homogenates were made from the red cell suspensions by gentle hypotonic haemolysis (1:2 v/v dilutions of packed cells with 17 mOsm phosphate buffer pH 7.4) and subsequent homogenization in a glass-teflon homogenizer. Before incubation, the homogenates were subject 5 times to freezing-thawing. In several experiments, membrane preparations were made according to Gauger et al. (1975). Subcellular fractions were prepared from the homogenates by the fractionated centrifugation method of Schneider (1948) modified essentially as described by Quiring and Palm (1970). Determination of Monoamine Oxidase Activity. MAO activity was determined according to Wurtman and Axelrod (1963). In the assay samples (total volume 0.42 ml), the substrates l~C-tryptamine or 14C-phenylethylamine were usually present in concentrations of 10-5 M and 10-4 M, resp.; enzyme concentrations were adjusted to 1 mg protein or equivalent to 4 to 8 x 10s cells (homogenates) per sample. Inhibitors (pargyline, clorgyline, deprenil or tranylcypromine) were preincubated with the enzyme preparations for 15 min. All assays were run in duplicates. Reaction rates were linear with respect to enzyme concentrations and incubation time (10 rain). Cytoehrome oxidase activity was determined according to Cooperstein and Lazarow (1951). Protein concentrations were determined according to the fluorimetric method of Resch et al. (1971). ReticulocTte counts ( ~ of total erythrocytes) were made using blood smears stained with brilliant cresyl blue. Cell counts (erythrocyte concentrations) were determined in a Coulter counter. Evaluation of ExperimentalResults. The values given are means from N experiments _+ S.E.M. Regression lines were calculated by the method of least squares. Student's t-test was used for statistical evaluations. ICs0 values were determined graphically. In the Figures, inhibitor concentrations are given as pI values (negative logarithm of inhibitor concentration). Substances. 1-AcetyI-2-phenyl-hydrazide (Merck-Schuchardt, Miinchen); clorgyline-HC11 (May & Baker, Dagenham/Essex); cytochrome c (Boehringer, Mannheim); deprenil (kindly supplied by Prof. Knoll, Budapest); heparin (Hoffmann-LaRoche, Basel); pargyline-HCl 1 (Abbott, North Chicago); phenylethylamine-HC1 (K & K Laboratories, New York); phenylethylamine-(ethyl-l-14C)HC1 (2-10mCi/mmole; New England Nuclear Corporation, Boston); serum albumin, bovine (Behringwerke, Marburg); tranylcypromine sulfate 1 (SK & F, Welwyn Garden City); tryptamineHC1 (Carl Roth, Karlsruhe); tryp~amine-(2-14C)-bisuccinate (60 mCi/mmole; New England Nuclear Corporation, Boston). All laboratory chemicals used were products of Merck, Darmstadt. 1 We are gratefuI for samples kindly supplied by the respective firms

Naunyn-Schmiedeberg's Arch. Pharmacol. 300 (1977) RESULTS F o r the investigations on the subcellular location o f monoamine oxidase ( M A O ) tryptamine was used as substrate; tryptamine is k n o w n to be equally m e t a b o lized by both A-type and B-type M A O . Figure 1 shows that in crude mitochondrial preparations f r o m red cell suspensions o f acetyl-phenylhydrazide-treated rats M A O activity was 5 - 6 times higher than in the respective h o m o g e n a t e s and cytoplasmic m e m b r a n e preparations; considerably lower enzyme activity was f o u n d in the m i c r o s o m a l fractions. The relatively high M A O activity in m e m b r a n e fractions and also the finding o f tryptamine deaminating activity in the m i c r o s o m a l fractions m a y be attributed to contamination o f both preparations with mitochondrial protein. Essentially the same pattern o f subcellular distribution was f o u n d for the mitochondrial m a r k e r enzyme, c y t o c h r o m e oxidase (Fig. l). However, the limited sensitivity o f the m e t h o d o f Cooperstein and L a z a r o w (1951) did not allow positive determinations of cytoc h r o m e oxidase activity in other than mitochondrial preparations. All together, the results o f the fractionation experiments show that reticulocyte M A O is located in reticulocyte mitochondria. This confirms our assumption based on previous findings (Quiring et al., 1973, 1976) that reticulocyte M A O is a "classical" m o n o a m i n e oxidase according to the criteria of Blaschko (1963). The use o f specific substrates and inhibitors for the biochemical characterization o f M A 0 isoenzymes (types A and B) was introduced by J o h n s t o n (1968) and has since f o u n d widespread application (for review, see Neff and Yang, 1974). In the present experiments, two substrates and three inhibitors were used in order to identify A - t y p e and B-type M A O activities in rat reticulocytes: the c o m m o n substrate, tryptamine, and the B-type substrate, phenylethylamine; the c o m m o n inhibitors pargyline and tranylcypromine, the A-type inhibitor clorgyline and the B-type inhibitor deprenil (see Neff and Yang, 1974). In preliminary experiments the effects o f the inhibitors on tryptamine deaminating enzyme activity were determined in m e m b r a n e preparations (15 000 x g sediments; see G a u g e r et al., 1975); the ICso values f o u n d were 1 0 - 8 M (clorgyline), 2 • 1 0 - 6 M (pargyline) a n d 10 -5 M (deprenil). Since these values and also the slopes o f the concentration-response curves were nearly identical when red cell h o m o g e n a t e s were used instead o f m e m b r a n e preparations, the m o r e 9 easily prepared h o m o g e n a t e s were used in the experiments described below. Figure 2 shows the inhibitory effects of pargyline on the d e a m i n a t i o n rates o f the two substrates. W h e n

K. Quiring and S. Hubertus : Monoamine Oxidase in Rat Reticulocytes

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Fig. 1, .Specific activiti~ of monoamine oxidase and cytochrome oxidase activities in subcellular fractions (prepared by differential cemrifugation) of erythrocyte homogenates from blood of acetylphenylhydrazide-treatedrats. The activities of the two enzymes show very similar patterns of subcellular distribution, both activities being highly concentrated in the mitochondrial fraction

Fig.2. Inhibition in vitro by pargyline of monoamine oxidase activity (substrates: lr 1.5 x 10 s M, and t~C-phenylethylamine, 10 r M) in erythrocyte homogenates from the blood of acetyl-phenylhydrazide-treated rats (N = 3 - 4 ) . Although the slopes of the concentration-responsecurves for pargyline are slighdy different, the ICs0 vatues for pargyline with each of the two substrates are in the same order of magnitude (3 • 10-6 and 10 - s M, resp.)

phenylethylamine was used as substrate, the concentration-response curve was less steep than with tryptamine, and complete inhibition of phenylethylamine deamination was not achieved even if pargyline concentration was 10 3 M. The ICs0 value for pargyline was about t0 .5 M with phenylethylamineas substrate; this value is about 3 times the ICs0 for pargyline with tryptamine as substrate. These findings do not rule out the presence of MAO isoenzymes in rat reticulocytes, but are compatible with a single form of MAO. According to several authors (for review, see Neff and Yang, 1974), pargyline is assumed to inhibit the B-type preferentially to the A-type enzyme; if this were true, the above findings would be in favour of an A-type MAO in rat reticulocytes. This conclusion was not supported by experiments (not shown) with tranylcypromine, an inhibitor common to both type A and type B (Green and Youdim, 1975): the ICs0 values for

tranylcypromine were nearly identical (7• 10-TM with tryptamine, 10 .6 M with phenylethylamine) and Complete inhibition occurred when tranylcypromine concentrations were 10 -r to 10 . 3 M. Figure 3 shows the effects of the A-type inhibitor cIorgyline on MAO activity determined with both substrates. In contrast to the very strong inhibitory effect of clorgyline on tryptamine degradation (ICso about 10 -8 M), phenylethylamine deamination was only slightly inhibited over the concentration range of clorgyline investigated and maximal inhibition was below 50 % even with 10-4 to 10 -3 M clorgyline. From these results it may be assumed that, besides a large portion of A-type MAO, there is also some B-type enzyme present in rat reticulocytes. Phenylethylamine may be assumed to be partially metabolized by the B-type MAO, the activity of which is readily inhibited by pargyline but not by the A-type inhibitor, clot-

276

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Fig. 3. Inhibition in vitro by clorgyline of monoamine oxidase activity (substrates: 14C-tryptamine, 10 5 M, and ~4C-phenylethylamine, 10 .4 M) in erythrocyte homogenates from the blood of acetyl-phenylhydrazide-treated rats (N = 3 - 4 ) . In contrast to its strong inhibitory effect on tryptamine deamination (ICso < i0-8 M), clorgyline is onIy a weak inhibitor of phenylethylamine deamination

gyline. Another part of phenylethylamine deamination is obviously brought about by a clorgyline-sensitive A-type enzyme. Figure 4 shows the effects of the B-type inhibitor, deprenil. With tryptamine as substrate, a typical concentration-response curve for deprenil was obtained, the ICso value being 10 -5 M. The inhibitory effect of increasing concentrations of deprenil on phenylethylamine deamination, however, occurred at two steps. There was slight but significant inhibition by concentrations of as low as 10 .9 M and a plateau of about 25 % inhibition was maintained while deprenil concentrations were raised up to 10 -s M. Only with concentrations higher than 10 .2 M there was a further dosedependent increase of inhibition, reaching about 100 ~o at 10 -2 M deprenil. From this result it is obvious that in homogenates of rat reticulocytes, phenylethylamine is deaminated by 2 different monoamine oxidases, E1 and E; (Fig.4). At the substrate concentration of 10 C M phenylethylamine, the enzyme E~ contributes 25 ~o and the enzyme E2 75 % to the total

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Monoamine oxidase in rat reticulocytes: subcellular localization and identification of isoenzymes.

Naunyn-Schrniedeberg's Archivesof Pharmacology Naunyn-Schmiedeberg~s Arch. Pharmacol. 300, 273-279 (1977) 9 by Springer-Verlag 1977 Monoamine Oxid...
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