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 contra- lateral normal muscle, indicating that the higher the endogenous nor- adrenaline 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 dener- vated 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 anaes- thetized 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 cer- vical 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 contra- lateral 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 signi- ficant 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.