PARTIAL CHARACTERIZATION OF MONOAMINE OXIDASE IN THE SALIVARY GLAND OF RATS. Kazuko NEMOTO, Seiichi HASHIMOTO, Takeyuki IKENO. and Hiroshi KUZUYA

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1 PARTIAL CHARACTERIZATION OF MONOAMINE OXIDASE IN THE SALIVARY GLAND OF RATS Kazuko NEMOTO, Seiichi HASHIMOTO, Takeyuki IKENO and Hiroshi KUZUYA Department of Biochemistry, Tohoku Dental University, Tomita-machi, Koriyama 963, Japan Accepted November 12, 1979 Abstract-Changes in the substrate specificity of monoamine oxidase in the rat sub maxillary gland were examined after ligation of the excretory duct and after denervation by postganglionic sympathectomy. Atrophy of the parenchymal cells after ligation of the excretory duct was observed, but such was not so clear after the denervation. The rate of decreases in the enzyme activity after the duct ligation was highest with sero tonin, intermediate with dopamine and tyramine and lowest with ~-phenylethylamine, used as substrates. The proportion of form A monoamine oxidase was examined using clorgyline as an inhibitor specific for form A enzyme; approximately 95 %, 90 % and 85 % of the total enzyme activities were attributed to form A enzyme in the intact glands, in the denervated glands and in the duct ligated glands, respectively. These results indicate that 90 % of the enzyme activity in the parenchymal cells and almost of all the enzyme activity in the sympathetic nerves of the gland may be attributed to form A enzyme and the atrophy of parenchymal cells after the duct ligation results in a decrease in form A enzyme more markedly than form B. Monoamine oxidase (MAO) [EC ] in the rat salivary glands has been found mostly in the parenchymal cells and not in the nerves, in studies on duct ligation of the salivary gland and postganglionic sympathectomy of the superior cervical ganglion (1, 2). In most organs, MAO is localized in the mitochondria, but in the salivary glands the enzyme is found in both mitochondrial and microsomal fractions (2-4). On the other hand, the presence of two forms of MAO, form A and form B, has been recognized in various tissues (5, 6). In this paper, the form of MAO in the submaxillary gland of rats was investigated based on the studies on duct ligation and denervation of the glands and on inhibition with clorgyline in vitro. MATERIALS AND METHODS The reagents used were: tyramine hydrochloride (Merck), 3-hydroxytyramine (dopamine) hydrochloride (Calbiochem), 5-hydroxytryptamine (serotonin) creatinine sulfate complex (Calbiochem), 9-phenylethylamine hydrochloride (Sigma), [1-14C]-tyramine hydro chloride (56.2 mci/mmol, New England Nuclear), 3,4-[ethyl-1-14C]-dopamine hydrobromide (45.29 mci/mmol, New England Nuclear), 5-[2-14C]-serotonin binoxalate (49.3 mci/mmol, New England Nuclear), and (3-[ethyl-1-14C]-phenylethylamine hydrochloride (50.98 mci/ mmol, New England Nuclear). purified further, The other reagents were of analytical grade and were not

2 Adult Wistar rats weighing g were used in these experiments. The main excretory ducts of the submaxillary and sublingual glands were ligated unilaterally at two sites near the hilum. The duct was dissected free from the artery and nerves and was ligated with a surgical silk thread. Postganlionic sympathectomy was performed unilaterally by removal of the superior cervical ganglion. The success of the denervation was assessed initially by the ptosis that developed on the operated site. The operations were performed under ether anaesthesia and the unoperated side was used as a paired control. After the rats were decapitated, the salivary gland was excised and homogenized in 19 volumes of 0.25M sucrose for 20 see, twice, in an ice bath using a Polytron homogenizer (KINEMATICA GmbH, Switzerland). The homogenate was filtered through gauze and the filtrate was used for the determination of MAO activity. MAO activity was measured according to a modification of the method by McCaman et al. (7). Amounts of the reagents and the ph and incubation time of the reaction mixture were modified for the salivary gland. Ascorbic acid was also added to the reaction mixture to give a good reproducibility. The enzyme activity was assayed using four substrates, serotonin, dopamine, tyramine and 13-phenylethylamine. The incubation mixture (total volume 200,ul) contained : 80 Id of 0.1 M potassium phosphate buffer (ph 7.4 for tyramine and dopamine, ph 7.6 for serotonin and ph 8.0 for Q-phenylethylamine), 20 PI of 10 mm ascorbic acid, 50 I1 of the filtrate (enzyme preparation) and 50,ul of the substrates (final concentration, 2.5 mm for tyramine, 2.0 mm for dopamine, 1.0 mm for serotonin and 0.5 mm for (3-phenylethylamine. These compounds were dissolved in 0.01 N HCl and the substrate solutions contained 0.05,uCi of 14C-substrate in 50 /il). The mixture was incubated at 37'C for 15 min for serotonin, tyramine and dopamine and for 10 min for (3-phenyl ethylamine as substrates, respectively. As blank, the enzyme preparation inactivated with 20 ul of 3 N HCl was added to the reaction mixture and the incubation at 37'C was omitted. In the case of experiments with clorgyline, the inhibitor and enzyme solution were pre incubated at 22'C for 15 min before addition of the substrates into the reaction mixture (8). The reaction was stopped by the addition of 20 til of 3 N HCL Then 0.5 ml of ethyl acetate was added and the mixture was shaken for 2 min and centrifuged to separate the phase. Four hundred al of the ethyl acetate layer were transferred to a centrifuge tube containing 300 ul of 0.3 N HCL After the mixture was shaken and centrifuged, 200 /il of the ethyl acetate layer were transferred to a counting vial containing 10 ml of Bray's scintillation solution (9). RESULTS 1. Effects of duct ligation on nionoamine oxidase activities of the rat submnaxillary gland The excretory duct of the submaxillary gland of male Wistar rats was ligated unilaterally and the glands were taken at days 1, 4, 7, 10, 14, 21, 28 and 42 after ligation. Changes in weight and MAO activities per one gland and per unit weight of the gland are shown in Table 1, Figs. 1 and 2, respectively. The weight of the submaxillary gland decreased rapidly by approx. 50% in 10 days,

3 TABLE 1. Changes in body and tissue weights after duct ligation or denervation of the rat salivary gland FIG. 1. Changes in total activities of MAO in the rat submaxillary gland after duct ligation. Values are expressed as percent activity (mean+s.e.m.) against the ac tivity of the unoperated gland. O ---0: serotonin, : tyramine, l~ A : dopamine and x x : R-phenylethyl amine. FIG. 2. Changes in activities of MAO per unit weight in the rat submaxillary gland after duct ligation. Values are expressed as percent activity (mean+s.e.m.) against the activity of the unoperated gland. serotonin, --0: tyramine, A : dopamine and x x : (3 phenylethylamine.

4 then slowly, by approx. 30% at 28 and 42 days after ligation. The total activities of MAO per one gland (Fig. 1) changed in a pattern similar to the change seen in the gland weight. somewhat larger than that in the gland weight. However, the rate of decrease in the total activity was With respect to the rate of decreases in the total activities with the four substrates, the differences among the four substrates were observed at day 4 after ligation and continued until day 42, at about the same rate. decreases in the total activity were highest with serotonin, intermediate with tyramine and dopamine, and lowest with a-phenylethylamine. The The differences in the rate of decreases were predominant when the activities were expressed as activity per unit weight of the gland (Fig. 2). The differences among four substrates increased, time-dependently, after ligation. The activities of the materials towards the four substrates used, serotonin, dopamine, tyramine and Q-phenylethylamine, were 41.7±2.9, , 43.6±1.9 and 19.9±1.7 respectively in the glands 2 weeks after unilateral ligation, and 103.7±5.1, 132.7±10.5, and (nmol/g tissue/min, mean A S.E. M., n=4) in the glands on the unligated side, as the control. 2. Effects of postganglionectomy on monoamine oxidase activities of the rat submaxillary gland The superior cervical ganglion of male Wistar rats was excised unilaterally and MAO activities of the submaxillary glands at days 1, 2, 3, 4, 7, 10 and 14 after denervation were measured. Changes in weight and MAO activities per one gland and per unit weight are shown in Table 1 and Figs. 3 and 4, respectively. The gland weight did not vary significantly after denervation, although there was an approx. 10 % increase at day 3. The total activities of MAO per one gland decreased only slightly by approx. 75 % at FIG. 3. Changes in total activities of MAO in the rat submaxillary gland after dener vation. Values are expressed as percent activity (mean±s.e.m.) against the activi ty of the unoperated gland. O U : serotonin, : tyramine, A A: dopamine and x x : ~-phenylethyla mine. FIG. 4. Changes in the activities of MAO per unit weight in the rat submaxillary gland after denervation. Values are expressed as percent activity (mean±s.e.m.) against the activity of the unoperated gland : serotonin, : tyramine, A-- --A: dopamine and x x : phenylethylamine.

5 day 7 and approx. 80 % at day 14 after denervation (Fig. 3). However, such marked de creases as in the case of ligation were not observed. With respect to the rate of decreases in the total activities, these were no apparent differences among four substrates. Changes in the enzyme activities per unit weight after denervation were also similar to those seen in the case of total activities (Fig. 4). The activities of the materials toward serotonin, tyramine, dopamine and (3-phenyl ethylamine, were 68.9±8.5, 91.0±5.0, 52.4±1.0 and , respectively, in the glands of 1 week after unilateral denervation, and 101.4±11.7, , and (nmol/g tissue/min, mean+s.e.m., n=3) in the glands of unoperated side as the control. 3. Effects of clorgyline on monoamine oxidase activities of the rat submaxillary gland The form of MAO of the rat submaxillary gland was examined, using clorgyline as a form A-specific inhibitor, and serotonin and tyramine as substrates, according to the method of Johnston (8). The results are shown in Fig. 5. 1) Inhibition against MAO activities of the unoperated gland (Fig. 5A) : A sigmoid curve was observed when serotonin was used as substrate. At a 10-7 M concentration of clorgyline, the inhibition reached a plateau at approx. 100 % inhibition of the enzyme activity. The plateau observed with tyramine as substrate was approx. 95 % inhibition of the enzyme activity at a 10-6 M concentration of clorgyline. Therefore, using the terminology of Johnston (8), approx. 95 % of the total MAO activity could be attributed to form A MAO. 2) Inhibition against MAO activities of the duct-ligated gland (Fig. 5B): A sigmoid curve was observed when serotonin was used as substrate. In the same manner as the results from the unoperated gland (Fig. 5A), the inhibition reached a plateau at approx. 100% inhibition of the enzyme activity at a 10-7 M concentration of clorgyline. With tyramine was substrate, a pair of sigmoid curves was observed. At a 10-6 M concen tration of clorgyline, the plateau occurred at approx. 85 % inhibition of the enzyme activity. Therefore, approx. 85 % of the total MAO activity could be attributed to form A MAO. 3) Inhibition against MAO activities of the denervated gland (Fig. 5C) : A sigmoid curve FIG. 5. Inhibition of MAO by clorgyline in the unoperated, duct-ligated and denervated submaxillary gland of the rat. The points are mean values for duplicate deter minations on a single homogenate from three rats on day 14 after ligation of the excretory duct and at days 7 after removal of the superior cervical ganglion. 0 0: serotonin (1.0 mm) and 9 : tyramine (2.5 mm).

6 was observed when serotonin was used as substrate. The inhibition reached a plateau at approx. 100% inhibition of the enzyme activity at a 10-1 M concentration. When tyramine was used as substrate, a pair of sigmoid curves was observed. The plateau was observed at approx. 90 % inhibition of the enzyme activity at a 10-6 M concentration of clorgyline, indicating that approx. 90 % of the total MAO activity could be attributed to form A MAO. DISCUSSION It has been reported that a large part of MAO in the rat salivary gland occurs in the parenchymal cells (2, 10). In our study, four substrates, serotonin, dopamine, tyramine and (3-phenylethylamine, were used as substrates for MAO, and the result with the substrate tyramine was similar to findings in previous observations (2) in that the activity toward tyramine after ligation of the excretory duct of the salivary gland decreased more markedly than after postganglionic sympathectomy; the total activity toward tyramine decreased by approx. 25 % at days 14 after ligation (Fig. 1) and by approx. 75 % at days 7 after denervation (Fig. 3). In the duct-ligated submaxillary gland, however, the rates of decreases in the enzyme activities were not same among the four substrates. The presence of two forms of MAO, form A and form B, has been recognized in various tissues (5, 6). The existence of form A MAO in the rat submaxillary gland was examined according to the method of Johnston (8); clorgyline was used as an inhibitor specific for form A MAO, and serotonin and tyramine as the substrates. The inhibition curves by clorgyline indicate that the activities of form A and B MAO in the intact submaxillary gland (Fig. 5A), in which the sympathetic nerves are distributed, exist in an approximate proportion 95 : 5. In the denervated gland, which should reflect the activity of enzyme in the par enchymal cells, the ratio of the activities of form A and B MAO was 90 : 10 (Fig. 5C). The decrease of the ratio of the activity of form A MAO, despite the presence of a small portion of MAO in the nerves of the gland (Fig. 3), suggests that almost all MAO in the nerves belong to form A MAO. Goridis and Neff concluded that sympathetic nerves contain, almost exclusively, form A MAO, as their results indicated that 90% of MAO in the superior cervical ganglion belonged to form A MAO and, after bilateral superior cervical ganglionectomy, form A MAO was hardly detectable in the rat pineal gland, despite no significant change in form B MAO (11). In the duct-ligated submaxillary gland, where the gland cells atrophy but the sympathetic nerve terminals are essentially intact (12), the total activities of MAO decreased markedly (Fig. 3). An increase of the ratio of form A MAO can be expected when the duct is ligated as, in the submaxillary gland, there is an increase in the ratio of intraneural MAO which belongs almost entirely to form A MAO, but the ratio of form A MAO was decreased by 85 % in the duct ligated gland. (Fig. 5B). This result suggests that form A MAO is affected by atrophy of the gland more markedly than is form B MAO. This coincides with the following results (Figs. 1, 2); the rates of decreases in the enzyme activities with the four substrates after ligation of the excretory duct were highest in the case of serotonin (specific

7 for form A MAO), intermediate toward dopamine and tyramine (affected by both form A and B MAO, equally) and lowest toward 9-phenylethylamine (specific for form B MAO), respectively. Acknowledgements: The authors are indebted to Dr. W. Lovenberg (National Heart and Lung Institute, NIH) for providing clorgyline. We thanks Miss K. Nagamine for valuable technical assistance. REFERENCES 1) MUELLER, R. A., DE CHAMPLAIN. J. AND AXELROD. J.: Increased monoamine oxidase activity in isoproterenol-stimulated submaxillary glands. Biochein. Pharmacol. 17, (1968) 2) ALMGREN, 0., ANDI:N, N.-E., JONASON, J., NORBERG, K.-A. AND OLSON, L.: Cellular locali zation of monoamine oxidase in rat salivary glands. Acta Physiol. Scand. 67, (1966) 3) DE CHAMPLAIN, J., AXELROD, J., LAWRENCE, R., KRAKOFF, R.L. AND MUELLER, R.A.: Micro somal localization of monoamine oxidase (MAO) in the heart and salivary gland. Fedrn. Proc. 27, 399 (1968) 4) HARADA, M., OYA, H., NAKANO, G., KUZUYA, H. AND NAGATSU, T.: Intracellular locali zation of monoamine oxidase in mammalian salivary glands. J. dent. Res. 50, (1971) 5) SQUIRES, R.F.: Additional evidence for the existence of several forms of mitochondrial monoamine oxidase in the mouse. Biochem. Pharmacol. 17, (1968) 6) FULLER, R.W.: Kinetic studies and in vivo effects of a new monoamine oxidase inhibitor, N-(2-[o-chlorophenoxy]-ethyl-)-cyclopropylamine. Biochem. Pharmacol. 17, (1968) 7) MCCAMAN, R.E., MCCAMAN, M.W., HUNT, J.M. AND SMITH, M.S.: Microdetermination of monoamine oxidase and 5-hydroxytryptophan decarboxylase activities in nervous tissues. J. Neurochem. 12, (1965) 8) JOHNSTON, J.P.: Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem. Pharmacol. 17, (1968) 9) BRAY, G.A.: A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Biochem. 1, (1960) 10) MARSDEN, C.A., BROCH, JR. O.J. AND GULDBERG, H.C.: Catechol-O-methyltransferase and monoamine oxidase activities in rat submaxillary gland: Effects of ligation, sympathectomy and some drugs. Europ. J. Pharmacol. 15, (1971) 11) GORIDIS, C. AND NEFF, N.H.: Evidence for a specific monoamine oxidase associated with sympathetic nerves. Neuropharmacol. 10, (1971) 12) AND%N, N.-E., NORBERG, K.-A. AND OLSON, L.: The adrenergic nerves of rat salivary glands after excretory duct ligation. Acta Physiol. Scand. 66, (1966)