Catechol-O-Methyl Transferase and Monoamine Oxidase Levels in Rabbit Ocular Tissues Following Cervical Ganglionectomy Stephen R

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1964 Catechol-O-methyl transferase and monoamine oxidase levels in rabbit ocular tissues following cervical ganglionectomy Stephen R. Waltman Yale University

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Recommended Citation Waltman, Stephen R., "Catechol-O-methyl transferase and monoamine oxidase levels in rabbit ocular tissues following cervical ganglionectomy" (1964). Yale Medicine Thesis Digital Library. 3278. http://elischolar.library.yale.edu/ymtdl/3278

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CATECHOL-O-METHYL TRANSFERASE AND MONOAMINE OXIDASE LEVELS IN RABBIT OCULAR TISSUES FOLLOWING CERVICAL GANGLIONECTOMY

Stephen Reeves Waltman

Presented to the Faculty of Medicine in partial fulfillment of the requirements for the degree of Doctor of Medicine

Yale University

New Haven, Connecticut

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< £ R ACKNOWLEDGEMENTS

The author wishes to express his appreciation to Dr. Marvin L. Sears of the Department of Surgery for his continuing interest, inspiration, and guidance.

The author is indebted to Dr. Julius Axelrod of the National Institutes of Health for his suggestions regarding the experimental procedures and for donating some of the compounds used in these experiments.

The technical assistance of Mr. Charles Cintron and the secretarial assistance of Miss Lois Childs is greatly appreciated.

This investigation was conducted under a Fight for Sight Student Fellowship of the National Council to Combat Blindness, Inc., New York, New York. ■ i , o : c j'lECiS ; d ' • xvi b • ^ .9 ) of . j b

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Page I. Introduction 1

II. Methods and Materials

A0 Preparation of Tissues 7

B0 Catechol-0-Methyl Transferase Assay 9

C. Monoamine Oxidase Assay 18

III. Results

A. Fluorometric Assay for Catechol - 0-Methyl Transferase 20

B0 Radioactive Tracer Assay for Catechol-0-Methyl Transferase 21

Figure 1

C„ Methyl Transferase Activity 25

Figure 2

D. Radioactive Tracer Assay for Monoamine Oxidase 28

Figure 3

IV. Discussion 32

V. Summary 38

VI. Bibliography 39 rraaTOCQ ho hjsat

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.IV INTRODUCTION

The influence of the adrenergic system upon the steady state maintenance of intraocular pressure in normal and diseased eyes has become the subject of recent investigation,, In particular, studies of the increase in resistance to outflow of aqueous humor in sympathetically denervated, catecholamine depleted rabbit eyes suggest that under normal circumstances the liberation of catecholamines from structures near the outflow channels exerts a continuous effect upon outflow

OQ resistance and intraocular pressure. A puzzling finding, however, is the fact that in normally innervated eyes catecholamines cannot be detected in significant amounts in aqueous humor. It becomes of interest then to study possible modes of inactivation and destruction of the ocular catecholamines since the significant levels present in ocular tissues may exert effects upon intraocular pressure. 2q 7 Physiological studies by Sears and Barany and Barany have done much to further our knowledge about the role of adrenergic mechanisms in the regulation of intraocular pressure in experimental animals. Using rabbits, these investigators studied intraocular pressure and outflow resistance before and after cervical ganglionectomy and the effects of various adrenergic blocking agents on these parameters.

They found an increase in outflow facility 24 hours after ganglionectomy. HOITDUGOHTMJ

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Later the resistance became supra normal and still later returned to control levels. The ’ganglionectomy effect' was prevented by pre- treatment with systemic reserpine or anterior chamber injection of alpha adrenergic blocking agents suggesting that the phenomenon was a manifestation of release of adrenergic material into the aqueous.

Using the same preparation, Sears and Sherk^ found that aqueous outflow resistance was supersensitive to small doses of adrenergic compounds one week after unilateral cervical ganglionectomy. Further studies by these authors indicate that the denervation supersensitivity is much more marked after bilateral ganglionectomy. The mechanism of this super sensitivity has not yet been clarified in these studies.

In the human, Ballintine and Garner^ and others using topical epinephrine therapy for glaucoma simplex, have indicated that the delayed pressure lowering effect of these compounds is related primarily to an increase in the facility of aqueous outflow. The possibility that adrenergic mechanisms may play a role in the normal regulation of intraocular pressure in man as well as in the rabbit is suggested by these studies.

Metabolic correlates for these physiological observations have not been demonstrated for the rabbit eye.

During the past decades two enzymes have been described which are involved in the metabolic inactivation of circulating - -

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catecholamineso Catechol-O-Methyl Transferase (COMT) is an enzyme

that catalyzes the O-methylation of catecholamines using S-adenosyl

methionine as a methyl donor, ^ O-methylation has been shown to be the principal pathway of metabolism of administered epinephrine and 1,22 2 norepinephrine and studies by Axelrod suggest that COMT may be a major enzyme involved in the inactivation of endogenous catecholamines.

Monoamine Oxidase (MAO) is an enzyme that oxidatively deaminates amines. The role of this enzyme in the inactivation of catecholamines 3 is not clear, but Axelrod has suggested that it is mainly concerned with deamination of O-methylated metabolites rather than with catecholamines themselves. Other investigators^1 do not completely accept the subordinate role of MAO and suggest that while O-methylation in the rabbit brain does occur, deamination may be an important pathway for the initial inactivation of norepinephrine. Both enzymes are present in the rabbit brain and it is difficult to decide which plays the more important role in the initial metabolism of catecholamines.

The role of COMT and MAO in inactivation of circulating- catecholamines is fairly well accepted, but what part these enzymes play in local inactivation of catecholamines is not clearly delineated.

According to Axelrod, ^ the observation that the tissues contain more

3 3 H-metanephrine than ' H-epinephrine within two minutes after

O intravenous injection of labeled °H-epinephrine taken together with fcte -O-Jorfi ■ •

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The distribution and relative activity of these enzymes.

Monoamine Oxidase and Catechol-O-Methyl Transferase have been 4 ?i studied in many mammalian species. Krishna et al have studied the distribution of MAO in various pooled ocular tissues of the rabbit and cat using a manometric technique. Further studies on this enzyme and on COMT distribution in the eye are lacking at present. - a -

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Denervated tissues are known to have an increased sensitivity to the neurohumoral transmitters normally released by their effector nerves. The exact mechanism of super sensitivity of structures deprived of their sympathetic innervation is still not 18 known. Considerable work has been done trying to relate supersensitivity to a decreased ability of the denervated tissue to inactivate the chemical mediators. Studies by Burn and Robinson^ showed that

9-12 days after removal of the superior cervical ganglion the MAO content of the nictitating membrane of the cat fell to 60% of the control level, but over the next 30 days rose to control levels. Concomitant removal of the stellate ganglion prevented the delayed return to normal levels. These investigators felt that super sensitivity of the cat nictitating membrane to sympathetic amines was well correlated with the decreased MAO content of this tissue. Further studies by Burn

o et al however, failed to show any change in MAO activity of the cat iris following homolateral superior cervical ganglionectomy, a procedure which renders this tissue supersensitive to norepinephrine. Recently,

Crout and Cooper^ studied myocardial COMT activity after regional neural ablation of the dog heart. Following denervation the catecholamine content of the tissue disappeared and the preparation showed a marked super sensitivity to norepinephrine. The 42% decrease in myocardial

COMT suggested that supersensitivity of the denervated dog heart could not be attributed to a marked decrease in COMT activity. This - - -

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E-'T ~ j-9 ;ir, i £ 03 f °3UC'.'*l33r 9f: 3t>Xi ' fl )0 -6- lack of correlation between enzyme changes and denervation supersensitivity may be due to species variation, but suggests that this theory does not give a satisfactory explanation of all the observed facts.

In view of incomplete knowledge about the enzymatic inactivation of catecholamines in general as well as in the eye and because of the influence of these amines on intraocular pressure this project was undertaken to investigate the distribution of COMT and

MAO in the ocular tissues of rabbits to discover possible sites of inactivation of catecholamines. These same enzymes were then studied in rabbits that had undergone bilateral cervical ganglionectomy to decide whether changes in the concentration of these enzymes might play any role in the denervation supersensitivity phenomena demonstrated in this preparation. i&visns (£ arf a .rrr noi3£i©i:fioo Id jfosl ixl3 l£ri: i < i tnoi3£i*£i s&iosqa 03 ©ub ad [&m .

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. =r irfd i baiEi: METHODS AND MATERIALS

A. Preparation of Tissues

Albino rabbits, mainly males, weighing between L 6 and 3. 2 leg. were used. The animals were killed by the intravenous injection of gallamine triethiodide (Davis and Geek) 5 mg. /kg., followed by

5-10 cc. of air. A few animals convulsed, but only briefly.

Immediately after death the eyes were enucleated and put into chopped ice. The skull was opened and the optic nerves removed, up to, but not including the chiasm. A specimen of liver was also removed and iced. Eyes were dissected and the various tissues from both eyes of each animal pooled; lenses were removed with their capsules intact. Excess blood and vascular tissues were dissected away from the optic nerves. After blotting to remove excess water the tissues were weighed and then homogenized with five volumes of ice cold

1.15% KC1 in an all glass homogenizer and samples were then taken for assay. The average weights of the tissues from each eye were:

Iris-ciliary body = 54 mg., Retina - choroid = 72 mg., Optic nerve =

22 mg., Extraocular muscle = 99 mg., and lens = 312 mg.

Tissues used for COMT determinations were centrifuged at

9, 000 xg for 12 minutes at a temperature of 0-4° C. Those used for

MAO studies were not centrifuged but were kept in ice prior to assay.

Abbreviations E.O.M. = Extraocular muscle S.E.M. = Standard error of the mean. 2, i ( V < l '

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Rabbits were subjected to bilateral cervical ganglionectomy under thiopental sodium (Abbott) anesthesia„ Five to seven days later they were sacrificed and the tissues treated in the same manner as the non-operated animals. -8-

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B. Catechol-O-Methyl Transferase Assay

Several assay methods were used for the determination of COMT.

The fluorometric assay procedure used is a modification of that described by Axelrod and Tomchick^. An aliquot of the soluble supernatant fraction was taken for enzyme assay and the COMT activity determined by measuring the amount of metanephrine formed from epinephrine. The incubation mixture, in a 50 cc, polypropylene centrifuge tube, contained 50imoles phosphate buffer pH = 7,8,

10 /Umoles MgC^, 100 rmutmoles S-adenosyl methionine, 330 m/*.moles of Epinephrine bitartrate and an appropriate amount of enzyme

(mouse liver = 20 mg,, rabbit liver = 33 mg.) in a final volume of

1,0 ml. Blanks were obtained using boiled tissue extract in place of the enzyme. The mixture was incubated at 37° C for 1 hour and then

0. 5 ml. of 0. 5M borate buffer, pH=10. 0, and 30 ml. of washed

Ethylene dichloride was added and the tubes shaken by hand for 5-7 minutes. The aqueous phase was then aspirated and 1.5 cc. of 0. IN

HCL was added to the Ethylene dichloride, and the metanephrine back extracted into the aqueous layer. After centrifugation, this layer was aspirated and the amount of metanephrine present was determined 15 using a modification of the Trihydroxyindole method of Haggendal

(see details below). The values were corrected for the partition of

metanephrine in the above solvent buffer systems (The combined V

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ulev srfl . -10- extraction procedures had a recovery of 40-45%).

The method used for fluorometric determination of metanephrine is as follows:

1) The pH of the acid extract was adjusted to 7. 0 (indicator paper) using IN I^CO^. Indicator paper was used as the Brom

Thymol Blue originally used was found to interfere with the readings.

The volume of the extract was adjusted to 3, 2-3. 3 ml.

2) Immediately after the pH adjustment, a 1.0 ml. sample was added to 0.5 ml. of buffer (equal parts, 0. 6 M citrate and 1 M phosphate, pH=7. 2) and an appropriate amount of H2O.

3) 0.10 ml. of 0.02 M iodine was added and the oxidation allowed to proceed.

4) Three minutes later 0.5 ml. of 5 per cent sodium sulfite containing 2 per cent EDTA was added.

5) 30 seconds later 0.5 ml. of 5 N NaOH was added.

6) After 5 minutes, 0.5 ml. of 10 N acetic acid was added.

7) The final volume was 4.1 ml.

8) Between 2-10 minutes after the acetic acid was added the samples were read in a Turner Fluorometer #110 using an activating wavelength of 436 mu, (Filters #47B and 2A) and a fluorescence wavelength of 525 irym (Filter #58). - -

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9) A reagent blank containing all reagents was used to zero the instrument o

10) The tissue blank used was the same as the sample except that no iodine was added and therefore no oxidation took place.

11) 0.1 ml. of 0o5 M KC1 was added to the metanephrine standard and reagent blank, prior to oxidation, to correct for the added salt in the sample.

12) The internal standard was prepared by adding an appropriate amount of metanephrine to 1.0 ml. of the sample prior to oxidation by the iodine. (See Table l)

Several important modifications were necessary to adopt the

15 original method of Haggendal ' to the available equipment and to make it sensitive enough to measure the small yields of metanephrine expected from the ocular tissues. These modifications were as follows:

1) Haggendal used an excitation wavelength of 440 rr^ and read fluorescence at 510 nyx or 520 myx to measure the metanephrine formed. After considerable trial, filters giving wavelengths of

436 rryiA and 525 mju. were chosen because they were very close to the original wavelengths and because the results obtained were found to be very reproducible. Distilled water blanks gave a consistently reproducible low value when compared to a black plastic background. ■ - • E ifiiJ33riO : E ' ttfSgBOl

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These wavelengths were also very sensitive. Other wavelengths available with the Turner Fluorometer appeared more sensitive, but consistency and specificity were of a lower order of confidence.

2) The final volume of the reaction mixture was 4.1 ml. instead of 3.3 ml. This was necessary to fill the cuvettes to the desired level.

3) Using the present buffer, a pH of 7. 2 gave a much more sensitive method then when a pH of 6. 5 was used. Sensitivity with the Turner Fluorometer was increased approximately 50 fold using the above filters and a pH 7, 2 buffer. Despite the increased sensitivity the reproducibility of the results was excellent.

4) Because we were interested in measuring only metanephrine the fluorescence was measured within 10 minutes after addition of the

HAco The stability of the metanephrine fluorophor was found to be very similar to the values reported by Haggendal and because of this stability and slow linear decay values were not corrected to zero time.

5) As reported by Haggendal citrate-phosphate buffer minimizes the effects of salt on the assay procedure, but to minimize these further an amount of KC1 equivalent to the amount formed by neutalization of the sample was added to the blanks.

With these modifications the assay proved to be a reliable one for the measurement of small amounts of metanephrine in solution. r-

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To check accuracy and linearity of the method multiple determinations were done with known amounts of metanephrine ranging from 0. 0045/tg to 1.125 /xg. Approximately 10 readings were made at each point,,

To obtain reproducible values at low concentrations it was necessary to set the fluorometer at maximum sensitivity. When this was done the higher concentrations used could not be read on the same scale and the sensitivity of the fluorometer had to be decreased. The sensitivity of the instrument can be increased stepwise, each step giving approximately 3 times the sensitivity of the previous one.

To facilitate the performance and analysis of these and future experiments three separate ranges of concentrations were run at three different sensitivity settings. Curves were constructed using values:

1. 0,0045, 0.01125, and 0. 0225 yugram

2. 0.045, 0.1125, and 0. 225 yUgram

3. 0.225, 0.5625, and 1.125 yUgrams of metanephrine

The average of 10 readings at each of these points was determined and three curves plotted. Straight line curves were obtained in each instance. Using these curves the corresponding amounts of metanephrine were then calculated for each point.

The values are summarized in table 2. -': f -

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The radioactive tracer assay procedure used for the determination of COMT activity is a modification of that described by 2 Axelrod et al . An aliquot of the soluble supernatant was taken for enzyme assay and the COMT activity was determined by measuring the amount of ^H-metanephrine formed from -epinephrine. The incubation mixture, in a 10 cc. polypropylene centrifuge tube, contained

12. 5>t moles of phosphate buffer pH=7. 8, (T 675 mmoles MgC^,

25yUmoles S-adenosyl methionine, 4.12 nyumoles (250 rrpucurie) 3 2 H-epinephrine (Epinephrine - 7 - H-hydrochloride from New

England Nuclear Corp. was mixed with nonradioactive epinephrine to obtain the desire specific activity) and 50 yuliters of enzyme preparation in a final volume of 135yu liters. Blanks were obtained by using boiled liver extract in place of enzyme after experiments showed no differences among boiled liver, lens, or iris. Liver was used because of limited quantities of the other enzyme preparations available. The mixture was incubated at 37° C for 1 hour and then

0. 5 ml. of 0. 5M borate buffer, pH=10„0, and 6 ml. of a mixture of toluene: isoamyl alcohol (3:2) were added and the tubes shaken by hand for 7-10 minutes. After centrifugation, a 4 ml. aliquot of the organic phase was added to 1 ml. of ethanol and 10 ml. of 0.4%

2,5 Diphenyloxazole and 0.01% 1, 4-bis-2- (5-Phenyloxazolyl-)

Benzene in toluene and counted in a liquid scintillation counter. ~ r* ~

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Another assay procedure, which is perhaps not as specific, was also used. The incubation mixture contained 50yumoles of phosphate buffer pH=7, 8, 1 fJjcnoles MgCh?, 83 m/rmoles epinephrine, enzyme preparation, and 3,7 m^Umoles of C-S-adenosyl methionine, with the

label on the methyl group, in a final volume of 210 ^liters.

After incubation for 60 minutes at 37° C, borate buffer and toluene: isoamyl alcohol were added and the material extracted into the organic solvent was counted in a system identical to the previously described one. The specificity of this assay procedure will be discussed. 6 IIA .

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C. Monoamine Oxidase Assay

The assay procedure used for the determination of MAO activity 27 is essentially that of Wurtman and Axelrod, Duplicate aliquots of uncentrifuged homogenate were used and the MAO activity determined by measuring the amount of 1_lcC-tryptamine converted to -indole

14 acetaldehyde and C-indole acetic acid. The incubation mixture in a final volume of 300 //.liters contained 100 /Amoles of phosphate buffer pH=7,4, enzyme preparation, and 13,77 myumoles (17,9 rn/xcuries) of ^C-tryptamine bisuccinate (2.22 mg. of Tryptamine-2- -bisuccinate from the New England Nuclear Corp. was dissolved in 14,5 ml. of ice cold 100% ethanol and 25 JjlI. of this preparation used per assay.) The solution was stored at -10° C. Approximately 4-8 mg, of tissue were

27 used per assay. This was more tissue than Wurtman and Axelrod used, but because of the lower specific activity of the tissues assayed the assay procedure was shown to be linear over the range of tissue concentrations used. The mixture was incubated in a 10 cc polypropylene centrifuge tube for 20 minutes at 37° C and then 0. 2 ml. of 2N HC1 was added which lowered the pH to less than 0.5. Six ml. of toluene was then added and the tubes shaken by hand for 5-7 minutes. After centrifugation, 4 ml. of the toluene was added to 10 ml. of phosphor and the samples counted in a liquid scintillation counter. -81-

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Blanks were obtained by using boiled liver extract in place of enzyme preparations after experiments failed to show a difference among boiled liver,, lens, iris, retina* extraocular muscle and optic nerve. The values obtained were corrected for the distribution of the products in the solvent buffer system used. Wurtman and Axelrod^ have shown that this assay is specific for MAO since the products formed have been identified as indole acetaldehyde and indole acetic acid and the reaction is inhibited in vivo and in vitro by tranylcypromine, a compound which inhibits monoamine but not diamine oxidase.

The S-adenosyl methionine and the C-S-adenosyl methionine used were the generous gift of Dr. Axelrod and had been synthesized by him according to the method of Cantoni. ^ These compounds were stored in and diluted with 0„ 01M acetate buffer pH=5. 5 and were stored at -10° C. Preliminary results with commercially available S-adenosyl methionine iodide were disappointing and inconsistent. The COMT activities of several tissues were determined using S-adenosyl methionine iodide. When these were compared with the activities calculated using Dr, Axelrod's

S-adenosyl methionine it was found that the ratios of the activities using these two compounds varied considerably from tissue to tissue. Because of this the S-adenosyl methionine iodide was not used. -

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. 6 RESULTS

A, Fluorometric Assay for Catechol-O-Methyl Transferase

This assay procedure was used in only two tissues, rabbit liver and mouse liver. The results obtained are shown in Table 3,

Table 3

COMT Activity of Liver

Tissue Metanephrine formed/gram/hour {jXmoles)

Rabbit liver 0,10

Mouse liver 1,50

Both results were the average of two assays each done in duplicate. They are in the same range as the values reported by

Axelrod and Tomchick:^ rabbit liver, 0,1 /Amole/gm,/hr,, and mouse liver, 2, 3 u moles/gm, /hr. The difference in values for mouse liver may be due in part to a strain difference. - -

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B° Radioactive Tracer Assay for Catechol-O-Methyl Transferase

Tables 4a and 4b and Figure 1 show the COMT values obtained

O using H-epinephrine in the assay procedure„

Using the Student ’T’ test none of the differences noted in table

4b are significant at a probability level of 0.05,

Without S-adenosyl methionine in the incubation mixture and with liver as the source of enzyme, the yield was 3.5% of the control.

An attempt was made to inhibit the enzyme, in vitro, with pyrogallol (Mallinckrodt Chem. Co„) and the following results were obtained, using liver as a source of enzyme. Epinephrine concentration

= 2.60 x 10'5 M.

Pyrogallol concentration % Yield %Inhibition

0 100 0

1.5 x 10~6M 71 29

1.5 x 10~4M 11 89

1,5 x 10~3M 0 100

Iridial COMT was also inhibited in vitro by pyrogallol. Over

60% inhibition occurred with pyrogallol at a concentration of

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~~Table 4a~~

~~COMT Activity of Rabbit Ocular Tissues~~

~~0 ( H-metanephrine formed per gram in one hour (mju»moles)~~

~~Normal~~

~~Expt, Retina - Iris- Optic EOM Liver Choroid Ciliary body nerve~~

~~1 10 10 - - 81~~

~~2 18 7 - 4 143~~

~~3 12 12 9 5 91~~

~~4 24 24 12 7 117~~

~~5 9 8 15 3 67~~

~~6 11 9 14 3 50~~

~~7 9 12 11 3 -~~

~~8 12 11 10 3 -~~

~~9 11 13 13 5 36~~

~~10 14 17 15 3 68~~

~~11 13 11 9 2 35~~

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~~Table 4a (continued)~~

~~Ganglionectomized~~

~~Expt. Retina Iris- Optic EOM Liver Choroid Ciliary body nerve~~

~~3. 19 16 18 - 114~~

~~2 21 10 11 4 84~~

~~3 12 18 - 4 52~~

~~4 17 12 14 5 88~~

~~5 15 13 18 5 50~~

~~6 12 14 12 4 37~~

~~7 14 10 14 3 54~~

~~8 12 11 22 3 83~~

~~- indicates that no assay was performed for various technical reasons~~

~~-24~~

~~Table 4b~~

~~COMT Activity of Various Ocular Tissues of the Rabbit 3 COMT Activity (m/Amoles of ' H-metanephrine formed per gram in one hour S. E. M.)~~

~~1 issue Control Ganglionectomized~~

~~Retina-Choroid 13.1 T 1.1 (12) 15.3 + 1.2 (8)~~

~~Iris-ciliary body 12.0 t 1.3 (12) 13.0 t E0 (8)~~

~~Optic nerve 12.3 t 0.8 (10) 15. 6 t 1.5 (7)~~

~~Extraocular muscle 3„ 8 1 0.5 (11) 4.0t 0.3 (7)~~

~~Lens 0 0~~

~~Liver 73.0 I 11 (10) 69.4 ‘ 9.3 (8) lo asionri^m) ^JiviiaA TMOD . • . . Big iaq a 10't~~

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~~C. Methyl Transferase Activity~~

~~Tables 5a and 5b and Figure 2 show the results obtained using~~

-labeled S-adenosyl methionine. Results are reported in m/Umoles of CHg transferred to substrate and extracted from pH=10, 0 buffer/gram of tissue/hour. Using the Student 5 t' test none of the differences between the control and ganglionectomized animals are significant at a probability level of 0,05.

~~To determine the specificity of the above method for COMT, epinephrine was omitted from the incubation mixture and the enzymatic activity as a per cent of the control for each tissue was~~

~~measured. The results are reported in table 6.~~

~~Pyrogallol was tried as an in vitro inhibitor of this system.~~

~~Results with liver were: i ii ><: Epinephrine concentration I—* o~~

~~Pyrogallol concentration %Yield %Inhibit~~

~~0 100 0~~

~~1,2x10 6M 92 8~~

~~1,2x10"4M 31 69~~

~~1,2x10"3M 14 86~~

~~The enzymatic activity of lens was inhibited 74% using a -3 concentration of pyrogallol of 1.2x10 M, iLviJ BfilSfl BT . '~~

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~~Table 5a~~

~~Methyl Transferase Activity of Rabbit Ocular Tissues 14 ( C-CH3 transferred per gram of tissue in one hours (myumoles)~~

~~Normal~~

~~Expt. Retina - Iris- Optic EOM Lens Liver Choroid Ciliary body nerve~~

~~1 138 99 71 24 53 496~~

~~2 88 104 57 21 46 435~~

~~3 86 65 53 14 45 543~~

~~4 160 102 78 34 45 600~~

~~5 149 70 47 28 - 532~~

~~6 161 70 58 29 54 666~~

~~7 163 84 77 30 53 554~~

~~Ganglionectomized~~

~~Expt. Retina- Iris- Optic EOM Lens Liver Choroid Ciliary body nerve~~

~~1 44 44 56 - 43 596~~

~~2 97 72 15 30 41 527~~

~~3 97 97 58 28 56 430~~

~~4 114 97 38 23 51 490~~

~~5 170 90 84 - - -~~

~~6 146 104 130 33 46 425~~

~~7 153 20 93 38 51 570~~

~~8 166 110 54 37 - 490 - -~~

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~~\ -27- Table 5b~~

~~Methyl Transferase Activity of Rabbit Ocular Tissues 14 (m/Amoles of C-CH^ transferred to substrate per gram of tissue in one hour 1 S. E. M.)~~

~~Tissue Control Ganglionectomized~~

~~Retina-choroid 135 t 13. 4 (7) 123 15 (8)~~

~~Iris-ciliary body 85 3 7.5 (7) 79 3 11.3 (8)~~

~~Optic nerve 66 14„ 3 (7) 66 3 12 (8)~~

~~Extraocular muscle 26 t 2.5 (7) 32 3 2.3 (6)~~

~~Lens 49 t 1.8 (6) 48 + 2.3 (6)~~

~~Liver 547 ± 28 (7) 518 ! 28 (7)~~

~~Table 6~~

~~Methyl Transferase Activity Without Added Epinephrine~~

~~(as % of values with Epinephrine)~~

~~Tissue %Yield~~

~~Retina-choroid < 5~~

~~Iris-ciliary body < 5~~

~~Optic nerve <5~~

~~Extraocular muscle <15~~

~~Lens 88~~

~~Liver 30 -\T-~~

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~~Table 7a~~

~~MAO Activity of Rabbit Ocular Tissues 1 4 ( C-tryptamine oxidized/gram/20 min, (iii/a moles)~~

~~Normal~~

~~Expto Retina - Iris- Optic EOM Liver Choroid Ciliary body nerve~~

~~1 196 1230 277 172 1060~~

~~2 100 373 190 103 773~~

~~3 103 445 190 102 1132~~

~~4 212 947 305 110 1187~~

~~5 105 340 190 82 600~~

~~6 101 304 186 107 1030~~

~~7 125 345 204 118 1478~~

~~8 130 318 222 89 1015~~

~~9 94 271 172 89 1004~~

~~10 130 753 278 132 820~~

~~11 77 212 114 88 747~~

~~12 77 342 156 88 675~~

~~13 82 233 148 77 960~~

~~14 145 1026 268 129 818 -~~

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~~\ -30-~~

~~Table 7a (Continued)~~

~~Ganglionectomized~~

~~Expt. Retina- Iris Optic EOM Liver Choroid Ciliary body nerve~~

~~1 101 292 175 79 1180~~

~~2 88 206 129 79 1087~~

~~3 87 264 160 102 803~~

~~4 108 280 193 94 987~~

~~5 127 342 180 86 837~~

~~6 93 258 153 96 985 - Sft. j: . : '{tod •fiBili-~~

~~8Q -31-~~

~~Table 7b~~

~~MAO Activity of Rabbit Ocular Tissues~~

~~1 A MAO Activity (m^umoles of C-tryptamine oxidized per gram of tissue in 20 minutes _ S. E. M.)~~

~~Tissue Control Ganglionectomized~~

~~Retina-choroid 120 1" 11 (14) 101 t 6.2 (6) N.S.~~

~~Iris-ciliary body 510% 88 (14) 274 + 18.2 (6) . 01< p <3 02~~

~~Optic nerve 207 + 15 (14) 165 1 9 (6) . 02<^p 05~~

~~Extraocular muscle 106 + 7.6 (14) 89 t 3. 9 (6) . 02~~

~~Lens 0 0~~

~~Liver 950 t 62 (14) 956 t 55 (6) N.S.~~

~~Table 8~~

~~Monoamine Oxidase Inhibition with Tranylcypromine~~

~~Tissue Inhibition (as % of control values)~~

~~Retina-choroid 96.5%~~

~~Iris-ciliary body 96,1%~~

~~Optic nerve 83.5%~~

~~Extraocular muscle 96, 8%~~

~~Liver 93. 3% - ['€-~~

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~~DISCUSSION~~

~~The results reported show an apparent disparity between~~

~~the relative COMT and MAO activities of the various ocular tissues.~~

~~The COMT levels of the different tissues vary, but over a limited range. Iris, retina, and optic nerve all have specific activities within~~

10% of each other while the lens has no activity and EOM about 30% of the retinal content. The relative MAO activities of the various tissues exhibit greater variation as can be seen in Table 9. These values are 20 in the same range as those of Krishna et al who used pooled tissues from 6-12 rabbit eyes and a manometric technique for measuring MAO,

~~Table 9~~

Relative MAO Activity of Rabbit Ocular Tissues*

~~Tissue Present Study Krishna^~~

~~Retina-Choroid 1.0 1.0~~

~~Iris-Ciliary Body 4.25 4.67~~

~~Optic Nerve 1.72 1.67~~

~~EOM 0.88 0. 80~~

~~Lens 0 0~~

*Retina has been arbitrarily assigned a value of 1,

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To study the possible role of COMT in the immediate inactivation of catecholamines in the eye an attempt was made to inhibit the enzyme using an intravitreous injection of 10 ' moles of pyrogallol in a volume of 10 /x.liters. This was the largest amount of inhibitor that could be placed in the eye in 10 ^liters and the initial concentration of pyrogallol in the vitreous was about 1 M„ Iris and retina were taken for COMT assay 3 hours and 6 hours after injections, but no inhibition of COMT could be discerned.

Lack of inhibition with pyrogallol could be related to the dilution that the inhibitor undergoes in preparation of the extract, diffusion away from its site of action within three hours, or failure to reach its intimate site of action within the time allowed for the experiment. That the failure to demonstrate in vivo inhibition was not due to an inactive or inappropriate inhibitor was confirmed by in vitro experiments which demonstrated that the pyrogallol used is a potent inhibitor of COMT„ The unqiue properties of the ocular system studied make it difficult to apply the results of previous experiments using pyrogallol to this system.

~~The differences in the levels of COMT and MAO in the various ocular tissues is also noted after denervation, (Tables 4b and 7b),~~

~~The insignificant effect of bilateral cervical ganglionectomy on~~

~~COMT activity stands in contrast to the significant 46% decrease in~~

~~MAO content of iris and 16% decrease in MAO content of optic nerve and EOM. There are several possible explanations for these results. '~~

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~~The enzymes (MAO and COMT) present in the ocular~~

tissues are either associated with the vessels and nerves supplying them or are inherent in the cellular elements of these tissues. The differences in relative activities of the two enzymes suggest that they are either localized in different structures within each tissue or that they are localized in the same structures, in varying amounts, and are differentially affected by denervation. The changes following denervation could be due to a relative decrease in enzyme activity in either of these possible tissue stores. Koelle and Valk^ have shown that MAO is found in the blood vessels of the nicitating membrane of the rabbit as well as in the ganglion cells of adrenergic fibers supplying this tissue suggesting that this same type of distribution may occur within the ocular tissues. Whether or not MAO is also present in the cellular elements of the ocular tissues is unfortunately not known, but Haggendal and Malmfors have found adrenergic

~~neurons in the inner nuclear layer in the retina of rabbits suggesting that MAO may also be present. No report could be found about the~~

~~histochemical localization of C0MTo Considering all the evidence it~~

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It seems logical to conclude from these experiments that while the role of COMT in the immediate inactivation of endogenous catecholamines is still a matter of debate the super sensitivity of the iris and the aqueous outflow facility following cervical sympathectomy is probably not due to a change in the COMT activity of the ocular tissues. The role of MAO is not clear, but in view of the findings of previous investigators that COMT is probably more intimately 4 involved in the immediate inactivation of the amines and that the cat iris, following denervation, is supersensitive to epinephrine without g a change in MAO activity it seems reasonable to conclude that the moderate decrease in MAO activity noted after denervation probably has little to do with the observed supersensitivity phenomena.

Unfortunately no data on the degree of supersensitivity of the cat iris g following denervation is presented in the work of Burn et al. Data about the time course of the supersensitivity and change in MAO content of cat nicitating membrane following denervation is available. At

8-12 days following denervation there was a correlation between these parameters. There was no correlation, however, at 19-33 days. Quantitative histochemical localization of these two enzymes is necessary to ascertain in which elements of these tissues the enzymes are localized and to be certain that intracellular changes in the distribution of these enzymes are not responsible for the observed denervation phenomena. icrto eiasruj j©ib . .. - u x od ' pJ a c sa

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There appears to be a great deal of species variation in the effect of denervation on the MAO and COMT content of tissues. 14 Grout and Cooper found a 42% decrease in COMT activity of the dog 9 heart following regional neural ablation and Burn and Robinson reported a 40% drop in MAO activity of the cat nicitating membrane

~~o after cervical ganglionectomy. In contrast to this Burn et al reported no change in MAO activity of the cat iris following denervation.~~

These reports as well as those of Koelle and Valk^ and Krishna et al~^ have shown that there is considerable species variation in the relative tissue concentrations of MAO and make it clear that it is impossible to anticipate the effect of denervation on the enzyme content of a given tissue of a given species„ The results reported here on the effect of denervation on the enzyme content of the rabbit ocular tissues represent another variation to be considered in future experiments.

~~The significance of the results obtained using~~

^C-S-adenosyl methionine in the COMT assay is still unclear. The enzyme measured in iris, retina, optic nerve and EOM would appear to be COMT because depletion of epinephrine from the incubation mixture causes an almost complete reduction in the amount of extractable compound formed. The fact that the system was inhibited, in vitro, by pyrogallol lends further support to the argument. When no ocijBV'isnob io 3 si'is sri3

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o observation that the liver enzyme was inhibited 85% by 10 M pyrogallol suggests that the above findings can be explained by variations in the tissue stores of catecholamines. They could also be explained by the presence of an epinephrine activated enzyme that is also inhibited by pyrogallol directly or because the inhibitor interferes with the catalyst role of epinephrine. Treatment with reserpine to deplete the tissue stores of catecholamines would help resolve the question.

~~The enzyme activity measured in the lens is more complex to analyze. Exclusion of epinephrine from the incubation mixture lowers activity only 5-20% and yet pyrogallol inhibits this enzyme~~

_Q 74% at a concentration of 10 ' M. There are many possible explanations for this finding and further studies are needed to determine the compounds formed by the lens with the present incubation system. For now we must conclude that the enzyme measured is a methyl transferase, the specificity of which has yet to be determined. -

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~~SUMMARY~~

~~lo The levels of COMT in several ocular tissues of the rabbit have been measured,~~

~~2, The levels of this enzyme are not affected by bilateral cervical ganglionectomy,~~

~~3, The levels of MAO in several ocular tissues of the rabbit have been measured,~~

4, Bilateral cervical ganglionectomy caused a significant decrease in the MAO content of the optic nerve, extraocular muscle and iris, although the decrease demonstrated for retina was not significant,

5, An incompletely characterized methyltransferase has been measured in the rabbit ocular tissues and its values are unchanged by denervation. There is a significant amount of this enzyme present in the lens,

~~6, These studies indicate that the enzymes MAO and COMT may be localized in different substructures within the ocular tissues,~~

~~7, Denervation super sensitivity phenomena present in the rabbit eye after cervical ganglionectomy are probably unrelated to changes in the tissue concentrations of the enzymes MAO and COMT.~~

~~8, A method is described for the measurement of small~~

~~quantities (4-20 myograms) of metanephrine in solution. or!: ju O' f-iii "iGif.~~

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~~1. Axelrod, ]. et al: O-Methylation - Principal Pathway for the Metabolism of Epinephrine and Norepinephrine. Biochem. andBiophys. Acta, 27:210 (1958).~~

~~2. Axelrod, J. et al: Distribution of COMT in the Nervous System and other Tissues. J. Neurochem. 5:68 (1959).~~

~~3. Axelrod, J.: Metabolism of Sympathomimetic Amines. Physiological Reviews 39:751 (1959).~~

~~4. Axelrod, ].: The Fate of Adrenalin and Noradrenalin, in Ciba Symposium on Adrenergic Mechanisms. Churchill, London 1960 p. 28.~~

~~5. Axelrod, ]. and Tomchick, R.: Enzymatic O-Methylation of Epinephrine and Other Catechols. ]. Biol. Chem. 233:702 (1958).~~

~~6. Ballintine, E., and Garner, L.: Improvement of the Coefficient of Outflow in Glaucomatous Eyes - Prolonged Local Treatment with Epinephrine. A.M. A. Arch. Ophth. 66:314 (1961).~~

~~7. Barany, E. H.: Transient Increase in Outflow Facility After Superior Cervical Ganglionectomy in Rabbits. A.M. A. Arch. Ophth. 67:303 (1962).~~

~~8. Burn, ]. H. et al: Effect of Denervation on Enzymes in Iris and Bloodvessels. Brit. ]. Pharm. 9:423 (1954).~~

~~9. Burn, J. H., and Robinson, ].: Effect of Denervation on Amine Oxidase in Structures Innervated by the Sympathetic. Brit. J. Pharm. 7:304 (1952).~~

~~10. Burn, J.H., and Trendelenberg, U.: The Hypersensitivity of Denervated Nicitating Membrane to Various Substances. Brit. ]. Pharm. 9:202 (1954).~~

~~11. Cantoni, G.L., in Colowick and Kaplan: Methods in Enzymology. Vol. 3. Academic Press, Inc. New York 1957 Pg„ 600.~~

~~12. Crout, J.R. : Inhibition of COMT by Pyrogallol in Rat. Biochem. Pharm. 6:47 (1961). - ' ‘ '.cJ.r:.:A .1~~

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~~13. Crout, JoR. : Effect of Inhibiting both COMT and MAO on Cardio¬ vascular Response to Norepinephrine. Proc. Soc. Exp. Biol, and Med. 108:482;, (1961).~~

~~14. Crout, J.R., and Cooper, T.: Myocardial COMT Activity After Chronic Cardiac Denervation. Nature 194:387 (1962).~~

~~15. Haggendal, J.: Fluorimetric Determination of 3-0-Methylated Derivatives of Adrenaline and Noradrenaline in Tissues and Body Fluids. Acta. Physiol. Scand. 56:758 (1962).~~

~~16. Haggendal, ]. : The Presence of Normetanephrine in Normal Brain Tissues. Acta Physiol. Scand. 59:261 (1963).~~

~~17. Haggendal, J., and Malmfors, T.: Evidence of Dopamine Containing Neurons in the Retina of Rabbits. Acta Physiol. Scand. 59:295 (1963).~~

~~18. Hillarp, Na : Peripheral Autonomic Mechanisms, in Handbook of Physiol. Section 1: Neurophysiology. American Physiological Society, Washington D. C. 1960 p. 979.~~

~~19. Koelle, G.B., and Valk, Jr., A: Physiological Implications of the Histochemical Localization of MAO. J. Physiol. 126:434 (1954).~~

~~20. Krishna, N. et al: Manometric Determination of MAO in Ocular Tissues. A.M. A. Arch. Ophth. 65:338 (1961).~~

~~21. Krishna, N0 et al: Manometric Determination of MAO in Cat Ocular Tissues. A.M.A. Arch. Ophth. 67:488 (1962).~~

~~22. Labrosse, E.A., Axelrod, J. and Kety, S.S.: O-Methylation- Principal Route of Epinephrine Metabolism in Man. Science 128:593 (1958).~~

~~23. Sears, M.L., and Barany, E.H.: Outflow Resistance and Adrenergic Mechanisms. A.M. A. Arch. Ophth. 64:839 (1960).~~

~~24. Sears, M., Sherk, T., Cintron, C. and Mizuno, K.: Investigative Ophthalmology (In Press).~~

~~25. Sears, M.L., and Sherk, T.: Supersensitivity of Aqueous Outflow Resistance in Rabbits After Sympathetic Denervation Nature 197:387 (1963). on sft 3 : .fl -I ,JuoiD .£ I . i iriqs 03 < - ' . £ r~~

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~~26. Shore, P. A. and Cohn, Jr., V.H.: Comparative Effects of Monoamine Oxidase Inhibitors on Monoamine Oxidase and Diamine Oxidase. Biochem. Pharm. 5:91 (I960).~~

~~27. Wurtman, R. and Axelrod, J.: A Sensitive and Specific Assay for the Estimation of Monoamine Oxidase. Biochem. Pharm. 12:1439 (1963). -1 b-~~

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