Gen. Pharmac., 1976, Vol. 7, pp. 71 to 73. Pergamon Press• Printed in Great Britain
EFFECTS OF INHIBITORS D U R I N G EYE INDOLEAMINE MATURATION ROBERT E. BUDAAND PETER C. BAKER Department of Biology and Health Sciences, Cleveland State University, Cleveland, OH 44115, U.S.A.
(Received 1 July 1975) Abstract--1. The eye of CFW albino strain mice were measured in vitro for 5-hydroxytryptophan decarboxylase (5-HTPD) and monoamine oxidase (MAO) following intraperitoneal acute exposure to enzymatic inhibitor drugs. 2. The ability to inhibit 5-HTPD in the eye increases as the animal matures being greatest at 8 weeks postpartum. 3. The ability to inhibit MAO in the eye increases as the animal matures being greatest at 2 weeks or 8 weeks depending upon the drug used. INTRODUCTION The presence of indoleamine metabolism in the mammalian eye has been substantiated in a number of species including mouse (Erank6, Niemi & Merenmies, 1961; Erank6 & Merenmies, 1961), rats and frogs (Erank6 et aL, 1961), rabbits (Shanthaveerappa & Bourne, 1964), lower vertebrates (Welsch, 1964) and man (Kojima, Iida, Majimi & Okada, 1961). Maturational studies of the eye's indoleamine metabolism are minimal although Smith (1973) and Smith & Baker (1974) have described the maturation of a number of the pathway components in the mouse eye. The present study has dealt with the drug response of two enzymes, 5-hydroxytryptophan decarboxylase (5-HTPD) and monoamine oxidase (MAO), to inhibition of their action during maturation. It was found that during maturation the indoleamine pathway of the young animal responds in a different manner to administered drugs than does the adult. MATERIALS AND METHODS
tion. The eyes were removed, cleaned of extraneous tissue, and weighed to the nearest mg before homogenization by hand in cold buffer in glass tissue-grinders. 5-HTPD activity was determined by the method of Snyder & Axelrod (1964), as modified by Baker (1966a) and MAO activity was measured by the method of Wurtman & Axelrod (1963), as modified by Baker (1966b). RESULTS No significant inhibition resulted with NSD-1034 in 1 and 5 day, or 2-week-old animals for any dose given. In 8-week-old mice significant inhibition was only achieved with medium and high doses, with no dose response effect observed. Carbidopa given to young animals (1 and 5 day) did not produce the degree of inhibition exhibited in older animals (2 and 8 week). The low dose did not result in significant inhibition in either 1 or 5 day animals (Fig. 1).
oo~°~ o--~ ~Oo o,b u b I10
V
V
V
V
V
V
V
V
90 70
Mice of the Carworth Farms CFW albino strain were 50 used at postpartum stages of 1 and 5 day, and 2 and 8 week. Animals were injected intraperitoneally with drug 3 0 -I • i solutions and littermates, injected with vehicle only, I0 - I were used as controls. Drugs were given in the following concentrations: L-a-hydrazinomethyl-13-(3,4-dihydroxyLMH LMH LMH LMH phenyl)-propionic acid (MK-486; Carbidopa) 50, 25, Id0y .Sdoy 2week 8week 12.Smg/kg of body weight, N-(3-hydroxybenzyl)-NNSD-1034 methyl hydrazine dihydrogen phosphate, NSD-1034, 500, 250, and 125 mg/kg of body weight; (from Smith & Fig. 1. 5-Hydroxytryptophan deearboxylase activity following NSD-1034 administration. Nephew Ltd.) were used to inhibit 5-HTPD. Trans-2phenylcyclopylamine (Tranylcypromine; Parnate) 5, Control value given as 100% with experimental values 2.5, and 1.25 mg/kg and 1-isonicotinyl-2-Coenzylearbox- compared to controls. P represents the probability amidoethyl) hydrazine (Nialamide) 10, 5, and 2"5 mg/kg determined from Student-Fisher t-test. C represents the of body weight were used for MAO inhibition. Both control, L represents the low dose (125 mg/kg), M MAO inhibitors were obtained from the Sigma Chemical represents the medium dose (250 mg/kg), and H represents Co. Thirty minutes after injection, the animals were the high dose (500mg/kg). S.E.M. represented by brackets at top of bar. sacrificed in the third hour of their light cycle by deeapita71
72
R.E. BUDAAND P. C. BAKER
Significant inhibition resulted after the administration of the medium and high doses in all ages except day 1. Only 8-week-old animals showed a dose response effect. --000
ooo 0 O0
ooo 000
000
- V# V = V
0oo
V V V
60¢5
6oo
90-o00
~,0-~d.
~."",~. ~."'~. ~.~.~.
V VV
VV
V
3O
LMH
LMH
LMH
LMH
Iday
5day
2week
8week
Tranylc y_prornine
Fig. 2. Monoamine oxidase activity following tranylcypromine administration. Control value given as 100~o with experimental values compared to controls. P represents the probability determined from Student-Fisher t-test. C represents the control, L represents the low dose (1-25 mg/kg), M represents the medium dose (2-5 mg/kg), and H represents the high dose (5 mg/kg). S. E. M. represented by brackets at top of bar. oo 06 I10
V
.o o V
~ 60 00o
ooo 0 oo
~,&~.
~""
LMH 2 week
LMH 8 week
V V V
V VV
90 70 50
30
I
IO' LMH I doy
LMH 5 day Niolornide
Fig. 3. Monoamine oxidase activity following nialamide administration. Control value given as 100K with experimental values compared to controls. P represents the probability determined from Student-Fisher t-test. C represents the control, L represents the low dose (2.5 mg/kg), M represents the medium dose (5 mg/kg), and H represents the high dose (10 mg/kg). S. E. M. represented by brackets at top of bar. The M A O inhibitors nialamide and tranylcypromine produced less inhibition in young animals (I and 5 day) than in older animals (2 and 8 week). A dose response effect was observed for all ages studies (Figs. 2 and 3). DISCUSSION
Smith (1973) has shown that the enzymes of the indoleamine pathway undergo radical and characteristic changes during the first postnatal week. She found that 5-HTPD activity increased after day 1
reached its peak at day 3, then dropped to mature levels by day 7. M A O activity dropped after day 1 and then increased to reach its peak at day 5, after which it decreased to mature levels at 2 weeks. Smith's data show that there are higher levels of 5-HTPD and M A O during the first postnatal week, than during any subsequent period. Indeed the general pattern of indoleamine maturation in eye is one of decline toward adult status. This generalization is further supported by falling levels of the two enzyme's metabolites, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) during postnatal life (Smith & Baker, 1974). Our inhibitor data show that it is not possible to achieve the same degree of inhibition during the early postnatal period. The variable inhibition noted with the M A O inhibitors suggests the possibility of multiple M A O forms. Although an increasing interest in multiple M A O forms is developing (Sandier & Youdim, 1972), there are few embryonic or maturational studies. Shih & Eiduson (1971) found that chick M A O exists in multiple forms in various tissues and that neonatal tissues possess fewer M A O forms than do corresponding adult tissues. It is possible that the preponderant neonatal forms in the eye are not as subject to drug inhibition as the preponderant forms in older animals, and that changing relationships during maturation result in a shift to forms that are more easily inhibited. Multiple forms of 5-HTPD have not been suggested, although its identity with D O P A decarboxylase is still being argued (Christenson, Dairman & Udenfriend, 1972). Variations in drug action upon 5-HTPD can not, therefore, be ascribed to variations in enzyme form during maturation. The pathway's first enzyme, Tryptophan-5hydroxylase (T5-H) is believed to be the regulatory factor in maturation of 5-HT containing neurons in rat brain (Bennett & Giarman, 1965), however, Smith & Baker (1974) failed to find T5-H in the lateral eye of the mouse and as a result it has been postulated that the eye, unlike the brain (GrahameSmith, 1967), depends upon an extra-ocular source of 5-HTPD substrate. Non-neuronal 5-HTPD activitity is much reduced in the immature animal (Kellogg & Lundborg, 1972), although nonneuronal T5-H is elevated during the same time (Wapnir, Hawkins & Stevenson, 197l). The effect this may have on neuronal metabolism, including the eye, is unclear. Competition between inhibitor and substrate may be playing a role in the effects we find, and the variable relationships of extramural enzymes during maturation could increase the amount of 5-HTPD substrate available in early stages. At the same time high 5-HT levels (Smith & Baker, 1974) may influence the relationship between M A O and inhibitor during early life. Although these inhibitor studies have failed to clarify any of the confusing aspects of the eye's indoleamine maturation they do reinforce the
Inhibitors and maturing eye earlier conclusion that the maturation of this biochemistry in the eye is in some way unique, and dissimilar to brain (Smith & Baker, 1974). Acknowledgements--This research was supported in part by grant number NS0963 from the National Institute of Neurologic Disease and Stroke. Special thanks are extended to Dr. Irving M. Katz for supplying the drug, carbidopa MK-486, Merck Sharp & Dohrne, West Point, Pennsylvania.
REFERENCES
BAKERP. C. (1966a) Development of 5-hydroxytryptophan decarboxylase in the brain, eye and whole embryo of Xenopus laevis. Neuroendocrinology 1, 257-264. BAKERP. C. (1966b) Monoamine oxidase in the eye, brain and whole embryo of developing Xenopus laevis. Devl BioL 14, 267-277. BEN~mTT D. S. & G l ~ N. J. (1965) Schedule of appearance of 5-hydroxytryptamine (serotonin) and associated enzymes in the developing rat brain. J. Neurochem. 12, 9II-918. CrmmT~NSONJ. G., D ~ A N W. & UDEN]~.m~rDS. (1972) On the identity of DOPA-decarboxylase and 5-hydroxytryptophan decarboxylase. Prec. natn Acad. Sci. U.S. 69, 343-347. ~K60. & M,~.ENMmSE. (1961) In rive inhibition of histochemically demonstrabable amino oxidase in the retina of the mouse. Acta ophthaL 39, 335-352. ERAS6 O., N m ~ M. & Mm~t~nr.s E. (1961) Histochemical observations on esterases and oxidative enzymes of the retina. In The Structure of the Eye. (F_xlited by S~AmtSERG. K.), pp. 159-171. Academic Press, New York.
73
GRAHA~-S~nTH D. G. (1967) The biosynthesis of 5hydroxytryptamine in brain. Biochem. Jr. 105, 351-360. KELLOGG C. • LUNDBORO P. (1972) Uptake and utilization of SH-5-hydroxytryptophan by brain tissue during development. NeuropharmacoL 11, 363-372. KOJIMA K., IIDA M., MAJIM[ Y. & OKADA S. (1961) Histochemical studies of the human retina. Jap. J. OphthaL 5, 205-210. SANDLER M. & YOUDIM M. B. H. (1972) Multiple forms of monoamine oxidase: functional significance. Pharmacol. Rev. 24, 331-348. SHANTHAVEERAPPA T. R. & BOURNE G. H. (1964) Monoamine oxidase distribution in the rabbit eye. jr.Histochem. Cytochem. 12, 281-287. SmH J-H. C. & EIDUSONS. (1971) Multiple forms of monoamine oxidase in tissue in developing brain: tissue and substrate specificities. J. Neurochem 18, 1221-1227. SMrrR M. D. (1973) 5-Hydroxytryptophan decarboxylase (5-HTPD) and monoamine oxidase (MAO) in the maturing mouse eye. Comp. gen. P/tarmac. 4, 175-178. SMITH M. D. & BAKERP. C. (1974) The maturation of indoleaminemetabolism in the lateral eye of the mouse. Comp. Biochem. Physiol. 49A 281-286. SNYDSRS. & AXELRODJ. (1964) A sensitive assay for 5hydroxytryptophan decarboxylase. Biochem. Pharmac. 13, 805-806. W ~ N m R. A., I-~wKn~s R. L. & STEVENSON J. H. (1971) Ontogenesis of phenylalanineand tryptophan hydroxylation in rat brain and liver. BioL Neonate 18, 85-93. WELSH J. H. (1964) The quantitative distribution of 5hydroxytryptamine in the nervous system, eyes and other organs of some vertebrates. In Comparative Neurochemistry. (Edited by RaCHT~), pp. 355-366. Pergamon Press, Oxford. WtrgTMAN R. J. & AXEL~ODJ. (1963) A sensitive and specific activity for the estimation of monamine oxidase. Biochem. Pharmac. 12, 1439-1447.
EFFECTS OF INHIBITORS D U R I N G EYE INDOLEAMINE MATURATION ROBERT E. BUDAAND PETER C. BAKER Department of Biology and Health Sciences, Cleveland State University, Cleveland, OH 44115, U.S.A.
(Received 1 July 1975) Abstract--1. The eye of CFW albino strain mice were measured in vitro for 5-hydroxytryptophan decarboxylase (5-HTPD) and monoamine oxidase (MAO) following intraperitoneal acute exposure to enzymatic inhibitor drugs. 2. The ability to inhibit 5-HTPD in the eye increases as the animal matures being greatest at 8 weeks postpartum. 3. The ability to inhibit MAO in the eye increases as the animal matures being greatest at 2 weeks or 8 weeks depending upon the drug used. INTRODUCTION The presence of indoleamine metabolism in the mammalian eye has been substantiated in a number of species including mouse (Erank6, Niemi & Merenmies, 1961; Erank6 & Merenmies, 1961), rats and frogs (Erank6 et aL, 1961), rabbits (Shanthaveerappa & Bourne, 1964), lower vertebrates (Welsch, 1964) and man (Kojima, Iida, Majimi & Okada, 1961). Maturational studies of the eye's indoleamine metabolism are minimal although Smith (1973) and Smith & Baker (1974) have described the maturation of a number of the pathway components in the mouse eye. The present study has dealt with the drug response of two enzymes, 5-hydroxytryptophan decarboxylase (5-HTPD) and monoamine oxidase (MAO), to inhibition of their action during maturation. It was found that during maturation the indoleamine pathway of the young animal responds in a different manner to administered drugs than does the adult. MATERIALS AND METHODS
tion. The eyes were removed, cleaned of extraneous tissue, and weighed to the nearest mg before homogenization by hand in cold buffer in glass tissue-grinders. 5-HTPD activity was determined by the method of Snyder & Axelrod (1964), as modified by Baker (1966a) and MAO activity was measured by the method of Wurtman & Axelrod (1963), as modified by Baker (1966b). RESULTS No significant inhibition resulted with NSD-1034 in 1 and 5 day, or 2-week-old animals for any dose given. In 8-week-old mice significant inhibition was only achieved with medium and high doses, with no dose response effect observed. Carbidopa given to young animals (1 and 5 day) did not produce the degree of inhibition exhibited in older animals (2 and 8 week). The low dose did not result in significant inhibition in either 1 or 5 day animals (Fig. 1).
oo~°~ o--~ ~Oo o,b u b I10
V
V
V
V
V
V
V
V
90 70
Mice of the Carworth Farms CFW albino strain were 50 used at postpartum stages of 1 and 5 day, and 2 and 8 week. Animals were injected intraperitoneally with drug 3 0 -I • i solutions and littermates, injected with vehicle only, I0 - I were used as controls. Drugs were given in the following concentrations: L-a-hydrazinomethyl-13-(3,4-dihydroxyLMH LMH LMH LMH phenyl)-propionic acid (MK-486; Carbidopa) 50, 25, Id0y .Sdoy 2week 8week 12.Smg/kg of body weight, N-(3-hydroxybenzyl)-NNSD-1034 methyl hydrazine dihydrogen phosphate, NSD-1034, 500, 250, and 125 mg/kg of body weight; (from Smith & Fig. 1. 5-Hydroxytryptophan deearboxylase activity following NSD-1034 administration. Nephew Ltd.) were used to inhibit 5-HTPD. Trans-2phenylcyclopylamine (Tranylcypromine; Parnate) 5, Control value given as 100% with experimental values 2.5, and 1.25 mg/kg and 1-isonicotinyl-2-Coenzylearbox- compared to controls. P represents the probability amidoethyl) hydrazine (Nialamide) 10, 5, and 2"5 mg/kg determined from Student-Fisher t-test. C represents the of body weight were used for MAO inhibition. Both control, L represents the low dose (125 mg/kg), M MAO inhibitors were obtained from the Sigma Chemical represents the medium dose (250 mg/kg), and H represents Co. Thirty minutes after injection, the animals were the high dose (500mg/kg). S.E.M. represented by brackets at top of bar. sacrificed in the third hour of their light cycle by deeapita71
72
R.E. BUDAAND P. C. BAKER
Significant inhibition resulted after the administration of the medium and high doses in all ages except day 1. Only 8-week-old animals showed a dose response effect. --000
ooo 0 O0
ooo 000
000
- V# V = V
0oo
V V V
60¢5
6oo
90-o00
~,0-~d.
~."",~. ~."'~. ~.~.~.
V VV
VV
V
3O
LMH
LMH
LMH
LMH
Iday
5day
2week
8week
Tranylc y_prornine
Fig. 2. Monoamine oxidase activity following tranylcypromine administration. Control value given as 100~o with experimental values compared to controls. P represents the probability determined from Student-Fisher t-test. C represents the control, L represents the low dose (1-25 mg/kg), M represents the medium dose (2-5 mg/kg), and H represents the high dose (5 mg/kg). S. E. M. represented by brackets at top of bar. oo 06 I10
V
.o o V
~ 60 00o
ooo 0 oo
~,&~.
~""
LMH 2 week
LMH 8 week
V V V
V VV
90 70 50
30
I
IO' LMH I doy
LMH 5 day Niolornide
Fig. 3. Monoamine oxidase activity following nialamide administration. Control value given as 100K with experimental values compared to controls. P represents the probability determined from Student-Fisher t-test. C represents the control, L represents the low dose (2.5 mg/kg), M represents the medium dose (5 mg/kg), and H represents the high dose (10 mg/kg). S. E. M. represented by brackets at top of bar. The M A O inhibitors nialamide and tranylcypromine produced less inhibition in young animals (I and 5 day) than in older animals (2 and 8 week). A dose response effect was observed for all ages studies (Figs. 2 and 3). DISCUSSION
Smith (1973) has shown that the enzymes of the indoleamine pathway undergo radical and characteristic changes during the first postnatal week. She found that 5-HTPD activity increased after day 1
reached its peak at day 3, then dropped to mature levels by day 7. M A O activity dropped after day 1 and then increased to reach its peak at day 5, after which it decreased to mature levels at 2 weeks. Smith's data show that there are higher levels of 5-HTPD and M A O during the first postnatal week, than during any subsequent period. Indeed the general pattern of indoleamine maturation in eye is one of decline toward adult status. This generalization is further supported by falling levels of the two enzyme's metabolites, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) during postnatal life (Smith & Baker, 1974). Our inhibitor data show that it is not possible to achieve the same degree of inhibition during the early postnatal period. The variable inhibition noted with the M A O inhibitors suggests the possibility of multiple M A O forms. Although an increasing interest in multiple M A O forms is developing (Sandier & Youdim, 1972), there are few embryonic or maturational studies. Shih & Eiduson (1971) found that chick M A O exists in multiple forms in various tissues and that neonatal tissues possess fewer M A O forms than do corresponding adult tissues. It is possible that the preponderant neonatal forms in the eye are not as subject to drug inhibition as the preponderant forms in older animals, and that changing relationships during maturation result in a shift to forms that are more easily inhibited. Multiple forms of 5-HTPD have not been suggested, although its identity with D O P A decarboxylase is still being argued (Christenson, Dairman & Udenfriend, 1972). Variations in drug action upon 5-HTPD can not, therefore, be ascribed to variations in enzyme form during maturation. The pathway's first enzyme, Tryptophan-5hydroxylase (T5-H) is believed to be the regulatory factor in maturation of 5-HT containing neurons in rat brain (Bennett & Giarman, 1965), however, Smith & Baker (1974) failed to find T5-H in the lateral eye of the mouse and as a result it has been postulated that the eye, unlike the brain (GrahameSmith, 1967), depends upon an extra-ocular source of 5-HTPD substrate. Non-neuronal 5-HTPD activitity is much reduced in the immature animal (Kellogg & Lundborg, 1972), although nonneuronal T5-H is elevated during the same time (Wapnir, Hawkins & Stevenson, 197l). The effect this may have on neuronal metabolism, including the eye, is unclear. Competition between inhibitor and substrate may be playing a role in the effects we find, and the variable relationships of extramural enzymes during maturation could increase the amount of 5-HTPD substrate available in early stages. At the same time high 5-HT levels (Smith & Baker, 1974) may influence the relationship between M A O and inhibitor during early life. Although these inhibitor studies have failed to clarify any of the confusing aspects of the eye's indoleamine maturation they do reinforce the
Inhibitors and maturing eye earlier conclusion that the maturation of this biochemistry in the eye is in some way unique, and dissimilar to brain (Smith & Baker, 1974). Acknowledgements--This research was supported in part by grant number NS0963 from the National Institute of Neurologic Disease and Stroke. Special thanks are extended to Dr. Irving M. Katz for supplying the drug, carbidopa MK-486, Merck Sharp & Dohrne, West Point, Pennsylvania.
REFERENCES
BAKERP. C. (1966a) Development of 5-hydroxytryptophan decarboxylase in the brain, eye and whole embryo of Xenopus laevis. Neuroendocrinology 1, 257-264. BAKERP. C. (1966b) Monoamine oxidase in the eye, brain and whole embryo of developing Xenopus laevis. Devl BioL 14, 267-277. BEN~mTT D. S. & G l ~ N. J. (1965) Schedule of appearance of 5-hydroxytryptamine (serotonin) and associated enzymes in the developing rat brain. J. Neurochem. 12, 9II-918. CrmmT~NSONJ. G., D ~ A N W. & UDEN]~.m~rDS. (1972) On the identity of DOPA-decarboxylase and 5-hydroxytryptophan decarboxylase. Prec. natn Acad. Sci. U.S. 69, 343-347. ~K60. & M,~.ENMmSE. (1961) In rive inhibition of histochemically demonstrabable amino oxidase in the retina of the mouse. Acta ophthaL 39, 335-352. ERAS6 O., N m ~ M. & Mm~t~nr.s E. (1961) Histochemical observations on esterases and oxidative enzymes of the retina. In The Structure of the Eye. (F_xlited by S~AmtSERG. K.), pp. 159-171. Academic Press, New York.
73
GRAHA~-S~nTH D. G. (1967) The biosynthesis of 5hydroxytryptamine in brain. Biochem. Jr. 105, 351-360. KELLOGG C. • LUNDBORO P. (1972) Uptake and utilization of SH-5-hydroxytryptophan by brain tissue during development. NeuropharmacoL 11, 363-372. KOJIMA K., IIDA M., MAJIM[ Y. & OKADA S. (1961) Histochemical studies of the human retina. Jap. J. OphthaL 5, 205-210. SANDLER M. & YOUDIM M. B. H. (1972) Multiple forms of monoamine oxidase: functional significance. Pharmacol. Rev. 24, 331-348. SHANTHAVEERAPPA T. R. & BOURNE G. H. (1964) Monoamine oxidase distribution in the rabbit eye. jr.Histochem. Cytochem. 12, 281-287. SmH J-H. C. & EIDUSONS. (1971) Multiple forms of monoamine oxidase in tissue in developing brain: tissue and substrate specificities. J. Neurochem 18, 1221-1227. SMrrR M. D. (1973) 5-Hydroxytryptophan decarboxylase (5-HTPD) and monoamine oxidase (MAO) in the maturing mouse eye. Comp. gen. P/tarmac. 4, 175-178. SMITH M. D. & BAKERP. C. (1974) The maturation of indoleaminemetabolism in the lateral eye of the mouse. Comp. Biochem. Physiol. 49A 281-286. SNYDSRS. & AXELRODJ. (1964) A sensitive assay for 5hydroxytryptophan decarboxylase. Biochem. Pharmac. 13, 805-806. W ~ N m R. A., I-~wKn~s R. L. & STEVENSON J. H. (1971) Ontogenesis of phenylalanineand tryptophan hydroxylation in rat brain and liver. BioL Neonate 18, 85-93. WELSH J. H. (1964) The quantitative distribution of 5hydroxytryptamine in the nervous system, eyes and other organs of some vertebrates. In Comparative Neurochemistry. (Edited by RaCHT~), pp. 355-366. Pergamon Press, Oxford. WtrgTMAN R. J. & AXEL~ODJ. (1963) A sensitive and specific activity for the estimation of monamine oxidase. Biochem. Pharmac. 12, 1439-1447.
