Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine 1964 Catechol-O-methyl transferase and monoamine oxidase levels in rabbit ocular tissues following cervical ganglionectomy Stephen R. Waltman Yale University Follow this and additional works at: http://elischolar.library.yale.edu/ymtdl 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 This Open Access Thesis is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale. For more information, please contact [email protected]. Digitized by the Internet Archive in 2017 with funding from The National Endowment for the Humanities and the Arcadia Fund https://archive.org/details/catecholomethyltOOwalt 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 April, 1964 ii: 0 H'/iJKAOHOM CIKA 3> J /iH-oHAHT ' )H03TA OKI )J . , j IAJU: T1 A>I 41 8J2 7M0T0:'[f4(.)Jj; KAx J./ 3IVH3D B I j " 33\ S3 ’ sri ei- JUl 1964 ) 1 *? O s' a, T\\ «/ 0 /y, 'X G a - • £ - .iC :; 9 ■ IJ. , ■ rr: cqv- qa*, v j o irarjij ~si snic s j j tojc c; < £ 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 <xioil£7iqs <: o: BnoiJB 1 c c ■ -' - . - - - s - " • .a srniT3C,z9 : n£ noUiuO asIxsriO .‘i\V c B I sail p©J S£ ■ : , lO'f ~ /I < '10 vv' o ,. ■ J TABLE OF CONTENTS 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 '.•oijOUbO'iJfl A fsi-cr.r.sje'^ bf: n at-oriieM s i> id -A - - si : 1 i ■ -1 - . - : - '{£asA 9aBbixO s0im.sdfioM . - - ' . ' . :: 3 . 3 e - raBi'I ■'■':• - - )£i DvidoBoibsH .U \ - - - . 1 5'iLrgl ■ - yjboA S3£i0lan£'iT I^riJeM .0 £ 3’ii pi;; : . 60 ib Sb.:bixO si unroraoM noitauosiO .VI .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 ?J°J; v ,.rs-J;: dr r/oc.f rr.ajpvr oi,i; ranc-dbe a eft to eons rltoi srfT - - < I ib< • ■ oi j e iJaav ; s e i to :. a( I re s rfja □ * . ' " ' s: : a i £ to a : - ; - ' '■ G iUaxs alsaxsfifb wofftno sift rusan 0,1 • -: ~ . b 1 OB uni bfi£ sonBJei&ai : fA s3£ ■ ss ' ' - (lj e rmi i i i r ;.r - s3 - : ai p " ’ ' - '' ' : i aldj saocj r^tda o:r narft Jeamlni is ■ ■ - ■ i■; aa i bJ >rioa?Bo ibIuoo adJ to : \ . ' is £>ns amaB asifci/Ja i Pib^clois^ifif - 1 ^"oc:s 9gt:-9iwoa:f xuo isdmd oj rbum snot svnri : 3 , Jiddni . iaU ,ej£ ii b 9’iotod sun Bra Iasi woftjuo bns ‘i ■jfjod to: ri LOS JIM) iii 93SSToni cib biiLroi Y©dT -2- 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. 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' - , ■ j j b II j rii siuaaoiq irteliioofcxirri lo neftteit/ . • i« Ja 989ifj V1'1 i..f 33e^i.-a si }£>rf3 -jlc nelsn : il i}£ . ;• ; r . c * ■ , r ' ' fVB S 3 a9ti£99b JfcB do :lt < . : d srij oilorifiis 1 9fl3 li bevlovni 9is rioirlw -3- 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. 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