BY B. JARROTT* and S. Z. Langert from the Department of Pharmacology, University of Cambridge, and the Agricultural Research

J. Physiol. (1971), 212, pp. 549-559 549 With 2 text-figure8 Printed in Great Britain

CHANGES IN MONOAMINE OXIDASE AND CATECHOL-O-METHYL TRANSFERASE ACTIVITIES AFTER DENERVATION OF THE NICTITATING MEMBRANE OF THE CAT BY B. JARROTT* AND S. Z. LANGERt From the Department of Pharmacology, University of Cambridge, and the Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge (Received 10 August 1970)

SUMMARY 1. The question of cellular localizations of monoamine oxidase and catechol-o-methyl transferase, enzymes involved in the metabolism of noradrenaline, has been investigated by following the changes in the enzyme activity of the smooth muscle of the cat nictitating membrane after sympathetic denervation. Any falls in enzyme activity coinciding with the time course for degeneration of the sympathetic nerve endings (2-3 days) can reasonably be ascribed to a former localization of the enzyme within the sympathetic nerve. 2. Monoamine oxidase activity of the inferior and medial smooth muscle was significantly reduced 3 days after sympathectomy and re- mained reduced 14 days after sympathectomy. 3. A correlation was found between the fall in monoamine oxidase activity and the endogenous noradrenaline concentration of the contralateral normal muscle, indicating that the higher the endogenous noradrenaline concentration, the greater the fall in monoamine oxidase activity after denervation. 4. It was concluded that the fall in monoamine oxidase activity after denervation indicated a former localization of the enzyme within the sympathetic nerve endings. 5. The fall in monoamine oxidase activity of the denervated muscle was significantly less measured with benzylamine as a substrate than with tyramine. This suggested that the neuronal monoamine oxidase may have different properties than the extraneuronal enzyme. * Present address: Department of Physiology, Monash University, Clayton, Vic. 3168, Australia. t Present address: Instituto de Investigaciones Farmacologicas, Paraguay 2155, Piso 70, Buenos Aires, Argentina. 550 B. JARROTT AND S. Z. LANGER 6. A small fall in the catechol-o-methyl transferase activity of denervated smooth muscle was found and a correlation between the fall in enzyme activity and endogenous noradrenaline indicated that in this smooth muscle a proportion of the catechol-o-methyl transferase activity may be of presynaptic origin.

INTRODUCTION Monoamine oxidase and catechol-o-methyl transferase are the enzymes responsible for the catabolism of the sympathetic neurotransmitter noradrenaline, but little is known concerning the cellular localization of these enzymes in sympathetically innervated tissues. In an attempt to gain insight into the cellular localization of these enzymes, a study was undertaken of the time course of any changes in monoamine oxidase and catechol-o-methyl transferase activities after post-ganglionic denervation in the cat nictitating membrane, a tissue with a high density ofsympathetic innervation.

METHODS Denervation and dissection of nictitating membranes. Cats of l-8-3 3 kg bodyweight and of either sex received atropine sulphate (0.1 mg/kg, s.c.) and were then anaesthetized with ether. The right superior cervical ganglion was removed under aseptic conditions. At 3 or 14 days post-operatively the cats were anaesthetized with sodium pentobarbitone (40 mg/kg, I.P.) and the trachea cannulated. The medial and inferior smooth muscles of the nictitating membrane were isolated by a modification of the method of Thompson (1958). The eyeball was excised and the nictitating membrane with all the adjoining tissue was removed from the orbit. The tissue was placed in a slightly modified Krebs solution saturated with 95 % oxygen and 5 % carbon dioxide and maintained at room temperature. Each muscle was then dissected free. The whole procedure took about 20 min per eye. The composition of the Krebs solution (for 1 1.) was as follows: NaCl 6-0 g; KCl 0-35 g; MgCl2 (anhydrous) 0411 g; NaH2PO4 (anhydrous) 0-12 g; CaCl2 (anhydrous) 0-28 g; NaHCO3 2 1 g; ascorbic acid 20 mg; EDTA (disodium salt) 1-5 mg; and glucose 2 g. Biochemical estimation. Membranes were homogenized in ten volumes of ice-cold potassium phosphate buffer (0.005 M, pH 7.0) with an all-glass hand homogenizer. In some experiments, an aliquot of the homogenate (10 gl.) was taken immediately for estimation of endogenous noradrenaline. The remainder of the homogenate was frozen and assayed for enzyme activity and protein on the following day. Measurement of noradrenaline. 101. homogenate was used for estimation of endogenous noradrenaline using a modification of the enzymatic method of Saelens, Schoen & Kovacsics (Iversen & Jarrott, 1970). Measurement of monoamine oxidase activity. The method of McCaman, McCaman, Hunt & Smith (1965) as modified by Jarrott (1970a) was used. The reaction mixture contained 1 mm [3H]tyramine (specific activity 2 /uc/#u-mole) or [14C]benzylamine (specific activity 1 gc/cs-mole) as substrate and tissue homogenate (25 ,ul.) in a final volume of 100 Ad. Blank values were obtained by incubating the buffered substrate without homogenate. With this assay procedure 25 ,d. homogenate yielded 6-8 times the number of counts in the reagent blank after a 20 min incubation at 370 C. DENERVATED NICTITATING MEMBRANES 551 Measurement of catechol-o-methyl transferase activity. A modification of the method of McCaman (1965) was used (Jarrott, 1970b). The reaction mixture contained 1 mM-3,4-dihydroxybenzoic acid and 250,uM [3H]S-adenosylmethionine (specific activity 10 gcc/,t-mole) as substrates and tissue homogenate (50 /1d.) in a final volume of 100 jaI. Blank values were obtained by incubating buffered substrate without homogenate. 50 4zl. tissue homogenate produced 4-5 times the number of counts than the blank. Measure of protein. This was measured by the method of Lowry, Rosebrough, Farr & Randall (1951) using 10 uld. tissue homogenate. Calculation of enzyme activities. The enzyme activity was calculated from the known specific activity of the substrate and expressed as either 'mit-mole per mg wet weight per hour' or 'm/s-mole per mg protein per hour'. Materials. [14C]Benzylamine (specific activity 1.6 mc/m-mole, Mallinckrodt Nuclear, Florida, U.S.), [3H]tyramine (specific activity 2 1 c/m-mole, NEN Chemicals, Dreieichenhain, W. Germany) and S-adenosyl-L-methionine_[3H]methyl (specific activity 5-6 c/m-mole, The Radiochemical Centre, Amersham, U.K.) were diluted to the required specific activity with non-radioactive benzylamine (Koch-Light, Colnbrook), tyramine HCl (Sigma, London) and S-adenosyl-L-methionine chloride (Koch-Light) respectively. All other reagents were of analytical grade purity.

RESULTS Monoamine oxidase activity after denervation. A preliminary experiment was carried out with four cats to compare the activity of monoamine oxidase and catechol-o-methyl transferase in the smooth muscle of the right and left eye. No significant difference was found in the enzyme activities between right and left eyes (Table 1) and sothe right superior cervical ganglion was routinely removed to achieve sympathetic denervation. When monoamine oxidase activity was measured at 3 or 14 days after ganglionectomy, there was a significant reduction in the enzyme activity of the denervated muscles (Table 2). As the variation in absolute activity of the normal muscles for a number of cats was large, the fall in activity of the denervated muscle was expressed as a percentage of the contralateral muscle and the results analysed by a t test ofthe difference (Snedecor, 1956). It is well known that the wet weight and protein content of skeletal muscle changes markedly after motor denervation (McCaman, 1966), but no significant changes in either the wet weight or protein content of the nictitating membrane were found after denervation (Table 2). No significant difference was found between the percentage fall in monoamine oxidase activity 3 or 14 days post-operatively. Burn & Robinson (1952, 1953) and Cervoni (1969) have reported similar falls in monoamine oxidase activity after chronic denervation (8-12 days); the present results show that the fall has reached a maximum at 3 days and most likely parallels the time course of degeneration ofthe sympathetic nerves in the nictitating membrane. 552 B. JARROTT AND S. Z. LANGER Endogenous noradrenaline concentration and fall in monoamine oxidase activity. The noradrenaline concentration of the medial smooth muscle was found to be higher than that of the inferior smooth muscle and this con- firms the findings of Trendelenburg, Drask6czy & Pluchino (1969). Wide variation was found with some cats having twice as much noradrenaline in the medial muscle as in the inferior muscle whilst others contained equal

TABLE 1. Monoamine oxidase (tyramine as substrate) and catechol-o-methyl transferase (COMT) activities of the medial and inferior smooth muscles from the right and left side nictitating membranes (n = 4). The difference between right and left muscles was not statistically significant Right Left R/L Right Left R/L inferior inferior % medial medial % MAO activity 89 16-1 83 + 16.0 107 + 2.1 86 +12.9 87 + 11-8 99+ 7-2 m/z-mole/ hr. mg protein COMT 2*15+ 0281 2*44+ 0*156 87 + 7 2 2*46+ 0302 2*50 + 0.314 99+ 66 activity mpu-mole/ hr. mg protein

TABLE 2. Monoamine oxidase activity (tyramine as substrate), wet weight and protein content of normal muscles and muscles sympathetically denervated 3 or 14 days previous. The % fall in MAO activity after denervation was significant for each muscle (P < 0.05) Wet wt. Protein MAO* (mg) (Qg/mg) activity (%) fall 3 days after ganglionectomy (n = 12) Normal medial 29-7 + 3-25 57-4± 3-69 67-9 ± 11*05 Denervated 28-9 + 2 30 59-8 + 4-31 41-5±5+81 37 + 3.3 medial Normal inferior 32-1 + 2-88 49-5 * 4-62 80 3 + 11-24 Denervated 37-0 + 3*44 56-7 + 4 30 45-9 + 8-42 43 + 5.7 inferior 14 days after ganglionectomy (n = 11) Normal medial 28-4 + 2*73 59-1+ 6*86 65-9 + 11*51 Denervated 31-8 + 1-93 71-8 + 5-92 37-9 + 3-70 49+ 77 medial Normalinferior 36-8+3366 57.3+5.77 72*0+ 11-62 Denervated 41-8 ± 4-91 57-5 + 4-21 40 5 + 7-46 38+ 7-6 inferior * mi/-mole/hr. mg protein. amounts. On the average, the noradrenaline content of the medial muscle was 54 + 22-4 0/ higher than that of the inferior muscle. After denervation, all the endogenous noradrenaline disappeared indicating that all the noradrenaline was present within the sympathetic nerves of the muscle. A correlation was found between the noradrenaline concentration in the DENERVATED NICTITATING MEMBRANES 553 normal membrane and the percentage fall in monoamine oxidase activity of the contralateral denervated muscle (r = 0x48, P < 0 05, n = 18) (Fig. 1). In other words, the higher the endogenous noradrenaline concentration, the larger the fall in monoamine oxidase activity of the denervated contralateral muscle. Monoamine oxidase activity measured with different substrates. The decrease in monoamine oxidase activity in nictitating membranes 3 days after denervation was compared with tyramine and benzylamine as substrates. With benzylamine, the fall in activity was significantly less than 7-0 * 0 6-0

' 5-0 CbO . *0 <4-0 z °3.0 o0D A Lu 2.0_ w2-0 _ 1-0

10 20 30 40 50 60 % fall in MAO Fig. 1. Relationship between the endogenous noradrenaline (NA) concentration of the normal smooth muscle of the nictitating membrane and the % fall in monoamine oxidase (MAO) activity (tyramine as substrate) of the contralateral denervated muscle. the fall in activity, measured with tyramine as a substrate (Fig. 2) (t = 2-5, P < 0.05). However, 14 days after ganglionectomy, the difference in percentage fall in activity, although similar to that at 3 days, was not significantly different when either benzylamine or tyramine were used as substrates (t = 0-89, P = 0.4-0.3). Catechol-o-methyl transferase activity after denervation. When the catechol-o-methyl transferase activity of the medial muscle was determined after ganglionectomy, the enzyme activity was reduced by approximately 25 % (Table 3). There was also a slight fall in the catechol-o-methyl transferase activity of the inferior muscle but this was not significant, 554 B. JARROTT AND S. Z. LANGER probably because of the wide variation seen. The fall in enzyme activity of the medial muscle was similar in muscles denervated for 3 or 14 days. A correlation was also obtained between the noradrenaline concentration of the left normal membrane and the percentage fall in catechol-o-methyl transferase activity of the denervated contralateral muscle (r= 069, P < 0-02, n = 18). 100 Tyramine

60_

E240 0. E

d m 80 Benzylamine 0T 60' E 40

0 Medial Inferior Fig. 2. Monoamine oxidase activity in normal (open bars) and 3 days denervated (cross-hatched) smooth muscle of the nictitating membrane determined with tyramine and benzylamine as substrates. TABLE 3. Catechol-o-methyl transferase (COMT) activity in normal and denervated muscles Days after denervation 3 14 Number of cats 4 7 COMT activity in normal inferior muscle 2.471 + 0.4942 1.977 ± 0.1616 COMT activity in denervated inferior 1-444 + 0-1921 1-772 + 0-1896 muscle Fall in inferior muscle () 33 + 16-5 10 + 8-2 COMT activity in normal medial muscle 2-730 + 0-3432 2-615 + 0-3800 COMT activity in denervated medial 2-010 + 0-2602 1-916 + 0-3072 muscle Fall in medial muscle (%) 25* + 8-0 25* + 8-7 * P < 0-05 compared to contralateral muscle by a paired t test. t mgt mole/hr. mg protein. DENERVATED NICTITATING MEMBRANES 555

DISCUSSION Monoamine oxidase (MAO) occurs widely throughout the body and highest activities are found in certain non-neuronal tissues such as liver and kidney (Pletscher, Gey & Burkard, 1965). It has been postulated that the enzyme is also present in post-ganglionic neurones of the sympathetic nervous system where it may be responsible for controlling the steady- state level of endogenous noradrenaline (Kopin, 1964; Malmfors, 1965). This postulate is based on pharmacological (Kopin & Gordon, 1963; Smith, 1966; Furchgott & Sanchez Garcia, 1968), histochemical (Malmfors, 1965) and biochemical evidence (Jarrott & Iversen, 1970). In this latter study, it was found that in surgically sympathectomized rat vas deferens, the MAO activity declined by 50 % at a rate similar to the onset of degeneration of the sympathetic innervation and it was suggested that this indicated a former neuronal localization of the lost enzyme and the remai ing enzyme activity indicating an extraneuronal cellular localization. The reduced monoamine oxidase activity ofthe cat nictitating membrane shown in the present work is similar to that reported by Burn & Robinson (1952) and Cervoni (1969) in cats at 8-12 days after sympathectomy. How- ever, Burn (1952) interpreted the fall as a loss of enzyme from the 'neigh- bourhood of the sympathetic nerve endings'. We believe that the fall in monoamine oxidase activity represents a former localization of the enzyme within the sympathetic nerves for the following reasons: (a) The fall in activity was complete at 3 days after ganglionectomy and it is known from physiological, biochemical and morphological studies that the nerve endings of the nictitating membrane are in an advanced state of degeneration after 2 days (Langer, 1966; Langer, Draskoczy & Trendelen- burg, 1967; Van Orden, Bensch, Langer & Trendelenburg, 1967). Mono- amine oxidase activity did not further decrease between 3 and 14 daysafter ganglionectomy, suggesting that there is no long-term post-synaptic change in activity. (b) A comparison of the spontaneous release of radioactivity from normal and denervated nictitating membranes previously exposed to [3H]noradrenaline revealed a fall in deaminated metabolites from denervated membranes (Langer, 1970). (c) A correlation was obtained between the endogenous noradrenaline concentration of the normal muscle and the percentage fall of MAO activity in the contralateral denervated organ. In other words, the higher the endogenous noradrenaline concentration in a normal muscle, the denser the sympathetic innervation and the larger the percentage fall in MAO activity of the contralateral muscle after ganglionectomy. However, this is only indirect evidence for the presence of MAO in 556 B. JARROTT AND S. Z. LANGER sympathetic nerves and final proof will probably come from procedures such as the electron microscope histochemical technique recently developed by Boadle & Bloom (1969). With regard to the monoamine oxidase activity in denervated muscle, we presume this to be located extraneuronally, probably in smooth muscle cells, since the morphological studies of Van Orden et at. (1967) showed marked degenerative changes in sympathetic nerves of the nictitating membrane 48 hours after ganglionectomy. Again, a histochemical technique for MAO would yield useful information on the localization of the remaining enzyme activity. When monoamine oxidase activity was measured in membranes 3 days after ganglionectomy using equimolar quantities of benzylamine and tyramine as substrates, the fall in monoamine oxidase activity was significantly less with benzylamine than with tyramine. Differences in the fall of activity with a number of substrates have been observed in sympathetically denervated salivary glands (Almgren et al. 1966; Fujiwara, Tanaka, Hirosaka & Okagawa, 1966) and vas deferens (Jarrott, 1970a) and the present results suggest that in the cat nictitating membrane there may also be a similar difference in the properties of neuronal and extraneuronal monoamine oxidase. Catechol-o-methyl transferase has often been assumed to have an extraneuronal cellular localization (Axelrod, 1966). However, the denervated nictitating membrane showed a fall in catechol-o-methyl transferase activity at 3 and 14 days and a correlation was obtained between the fall in enzyme activity and endogenous noradrenaline concentration similar to that observed for monoamine oxidase. This suggests that a small amount of catechol-o-methyl transferase may have a neuronal localization with the remainder in extraneuronal cells. Similar findings have recently been obtained with the sympathetically denervated rat vas deferens (Jarrott & Iversen, 1970). In spite of this fall of enzyme activity after denervation, the formation of o-methylated metabolites of exogenous noradrenaline actually increases when denervated nictitating membranes are incubated in vitro (Langer, 1970). This apparent paradox can be explained by the loss of presynaptic noradrenaline uptake sites in the denervated muscle and the subsequent prolonged exposure of the catecholamine to the remaining extraneuronal catechol-o-methyl transferase. Furthermore, in the normal cat nictitating membrane previously incubated in vitro with [3H]- noradrenaline, phenoxybenzamine prevents formation ofnormetanephrine (a metabolite which requires catechol-o-methyl transferase in its formation) from noradrenaline released by nerve stimulation (Langer, 1970). This effect is related to the ability of phenoxybenzamine to block extraneuronal uptake, which normally allows noradrenaline to gain access to the post-synaptic metabolizing enzymes. At the same time, phenoxy- DENERVATED NICTITATING MEMBRANES 557 benzamine fails to interfere with the metabolism of noradrenaline released spontaneously from nerves. The latter finding is compatible with the view that normetanephrine released spontaneously is formed by presynaptic catechol-o-methyl transferase and thus is not affected by the block of access to extraneuronal catechol-o-methyl transferase produced by phenoxybenzamine. It should be noted that the fall in catechol-o-methyl transferase activity observed 3 and 14 days after denervation of the nictitating membrane does not prove the neuronal presence of the enzyme. Nevertheless, the present results are compatible with the view that a small fraction of the catechol-o-methyl transferase activity of the nictitating membrane is located in sympathetic nerve endings. In this context it should be noted that Alberci, Rodriguez De Arnaiz & De Robertis (1965) found that approximately 50 % of the catechol-o-methyl transferase activity of rat brain homogenates was present in a synaptosomal fraction and this enzyme has also been found in neuroblastomas (La Brosse & Karon, 1962). Whilst the evidence for the presence of presynaptic monoamine oxidase in sympathetic nerves seems now to be well established, additional evidence is required to further elucidate the distribution and physiological role of catechol-o-methyl transferase in the sympathetic synapse. We wish to thank Dr L. L. Iversen for advice and criticism throughout this work. B. J. gratefully acknowledges financial support from Roche Products Ltd. and S. Z. L. was supported by a fellowship from the Wellcome Foundation.

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