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Monoamine oxidase in the eye, brain, and whole embryo of developing Xenopus laevis
DEVELOPhlENTAL
BI0LOT.Y
14, 267-277
(l%ili)
Monoamine Oxidase in the Eye, Brain, and Whole Embryo of Developing Xenopus laevis PETER C. BAKER’
Accepted
March
17, 1966
5-Hydroxytryptamine (5HT) and related indole compounds are at present the subject of intensive investigation in a wide variety of animals. One of the most active areas of interest is the vertebrate central nervous system, where these compounds are being seriously considered as synaptic transmitters and hormones (see reviews of Douglas, 1965; Quay, 1965,a ; and Sharman, 1965). The major part of this research has been concerned with adult animals, and until recently, developing forms and the information they might provide have been generally ignored (Buznikov et al., 1964; Bennett and Giarman, 1965; Bourne, 1965; Oshika and Nakai, 1965). This work is part of a series of measurements designed to obtain some information about the metabolism of these compounds in a developing lower vertebrate, Xcnopu.s Zaecis, the South African clawed toad. Previous studies in this biochemical pathway have measured S-IIT ( Baker, 1965 ), the 5-FIT forming enzyme, 5-hydroxytryptophan decarboxylase ( Baker, 1966 ) , and the melatonin-forming enzyme, hvdroxyindole-0-methyltransferase (Baker et al., 1965). These investigations have indicated a pattern of increasing 5hydroxyand 5-methoxyindole metabolism during development and a concentration of this metabolism in the cycs and brain. _\Ionoaminc oxidase ( MAO ), the enzyme responsible for the destruction of 5HT, is the subject of the mcasurcments presented here. The results indicate that LIAO is similar to both 5-hydroxytryptophan de1Sllppwtvtl by U.S. Public Health Service I’rdoct~d Fellowship, 20,888~02, from tl-w Nationd Institute of Gcnerd hledical Sciences. 267
ELFI-GLLI-
268
PETER
C. BAKER
carboxylase and hydroxyindole-0-methyltransferase in its pattern of increase during development. Concentration of MAO activity in the brain is also demonstrated, although concentration in the eyes is found only during some stages. MATERIALS
AND METHODS
Fertilized eggs of Xenopus Zuevis were obtained by artificial ovulation in November and December of 19658.Embryos were allowed to develop at room temperature and were staged in keeping with the Normal Table of Nieuwkoop and Faber (1956). Measurements were made on whole embryos between stage 12, gastrula, and stage 48, premetamorphic larva. Lateral eyes were measured between stage 38, posthatching, and stage 48. Brains were measured between stage 40 and stage 48. The stage of earliest recovery for each structure was dictated by ease of clean dissection in numbers large enough for use. Before hatching, embryos were removed from surrounding jelly and capsule. Dissection of parts was carried out in cold 0.1 M, pH 7.4 phosphate buffer, and homogenization was by hand in all-glass homogenizers, in ice water. The method of Wurtman and Axelrod (1963) for assay of MAO has been modified for use here. The use of tryptamine as substrate was rejected in favor of 5-hydroxytryptamine, for reasons discussed below. It was necessary then to change the extracting solvent from toluene to diethyl ether. Toluene extracts 5-HT, and diethyl ether does not (Quay, 1963). Samples of three whole embryos, six eye pairs, or six brains were homogenized in 300 PI cold, 0.1 M, pH 7.4 phosphate buffer. An aliquot of 290 ,J of each homogenate was transferred to 5-ml centrifuge tubes in ice water. The tubes then received 10 ~1 carbon-14 S-HT solution (New England Nuclear Corporation 5-hydroxytryptamine-2-l%; 6.25 mpmoles, 1.65 mC/mmole). They were then incubated for 20 minutes at 3’7°C in a shaking water bath. After incubation, 150 ,J 1.0 N HCl and 3 ml diethyl ether were added to each tube. The tubes were then stoppered, shaken, and centrifuged; 2.7 ml of the ether phase of each tube was then transferred to a series of scintillation counting vials. The vials received 10 ml POPOP-PPO phosphor and 1 ml ethanol. They were counted for 10 minutes in a Packard, automatic, dual channel, TriCarb spectrometer. Blank values were determined by means of heated enzyme incubations treated as
hf.40 IN DEVELOPING
Xenopus
269
above, A value of 40 counts per minute was found to represent the amount of carbon-14 5HT being carried over in the extraction. methods were used to determine the Spectrophotofluorometric efficiency of the diethyl ether as extracting solvent. Known amounts of 5-hydroxyindoleacetic acid (5-HIAA) in phosphate buffer were extracted as above. The diethyl ether was transferred and reextracted in a tube containing S ml of heptane and a mixture of 200 ~1 of buffer and 150 ~1 1.0 N HCI. The 5-HIAA in the aqueous, buffer-acid, phase was then read in an Aminco-Bowman spectrophotofluorometer, as described by Quay ( 1963). Tubes containing known amounts of 5HIAA in 200 ~1 buffer, plus 150 pl 1.0 N HCI, plus S ml heptane, received diethyl ether that had been sham extracted over a similar buffer-acid mixture without 5-HIAA. These were read as standards. The resulting data indicated that 95% of the available product, 5HIAA, was extracted into the diethyl ether phase. Possible products of tissue incubated with 5-HT were considered to be 5HIAA, 5-hydroxytryptophol, and N-acetyl serotonin (Quay, 1965a). Ascending, one-dimensional, thin layer chromatography was used to determine the homogeneity of the product at various times during the developmental series. Chromatography plates coated with silica gel G were sprayed with ascorbic acid in methanol (Quay and Bagnera, 1964). Extracts of incubation were run in alternate spots with standard solutions containing all three possible products. Plates were run for 45 minutes in the cold using methyl acetate (Eastman) as the solvent. After drying, each plate was masked to cover the areas containing the incubation extracts and the areas containing standards were sprayed with van Urk’s reagent. Rf values of the standards were then determined, The incubation extract areas were marked off in a grid, cut off with razor blades, and added to a series of scintillation vials, which then received 1 ml ethanol and 10 ml phosphor. When the vials were counted, the location of any radioactive products on the chromatogram could be determined. Dry weights used for the calculation of relative enzyme concentrations were made from a number of egg batches during previous experiments. Continued weighings were halted due to the constancy of values from batch to batch, and a table of standard weights was made up. The dry weights were determined by placing whole embryos, eye pairs, or brains in groups of 3 to 10 on preweighed aluminum pans and drying in a forced draft oven overnight at 100°C. After
270
PETER C. BAKER
drying, pans plus tissues were weighed and dry weights were determined by subtraction. All weighings were made on a Cahn electrobalance to the nearest microgram. RESULTS
Two separate batches of eggs from two separate pairs of parents were used here. These have been called batches A and B, and are TABLE 1 MONOAXUNE OXIDASE ACTIVITY EXFRESSED AS i%CROMICROMOLES OF 5-HIAA FORMED PER HOUR PER STRUCTURE” Batch A stage Whole 12 20 25 30 35 38 40 41 42 46 47 48 Eyes 38 40 41 42 46 47 48 Brain 40 41 42 46 47 48
Batch B
H
N
SE
H
N
SE
AVt?K&
0.14 4.79 11.60 35.85 126.45 255.09 423.09 627.18 840.34 1616.13 1999.41 1958.99
10 10 10 10 10 10 10 10 9 10 10 10
0.44 0.64 0.97 1.69 8.21 15.31 19.07 19.76 38.76 60.91 104.67 56.54
0.17 0.87 7.23 12.88 44.48 99.77 235 72 303.4i 396.71 1186.90 1384 87 1178.62
8 10 10 9 10 10 10 10 10 10 10 10
0.92 0.83 0.58 1.17 2.07 2.84 8.97 14.61 12.82 28.62 66.47 22.39
0.16 2.83 9.42 24.37 85.47 176.93 329.41 465 33 618.53 1401.52 1692.14 1568.81
12.06 18.83 29.86 33.22 71.15 93.5i 91.11
10 10 10 10 10 10 10
0.33 0.65 0.64 0.45 2.00 0.59 3.49
2.43 7.5’2 11.51 22.02 3’2.82 36.63 41.95
10 10 10 10 10 7 10
0.30 0.44 0.45 ‘2 75 1. 0.82 0.69 1.09
7.25 13.18 20.79 27.62 51.99 65.10 61. .53
42.96 60.30 73.75 120.41 184.27 167.81
10 10 8 10 10 10
1.65 2.77 2.20 4 ‘2“1 5.95 .3 56
16.61 29.09
9 10 10 10 8 10
0.96 1 .02 3.25 1.88 3.43 2.79
29.79 44.70 55.57 99.72 143.29 134.92
errors
of the mean
35.39 is.03 102.31 102.03
a hlenns (x), number of determinations (S), (SE) are shown separately for bakhes A and B. b Average of the means of batches A and B.
and st,andard
MAO
IN
DEVELOPING
&3l0j?tlS
371
listed separately in Table 1. After stage 25, batch B is always significantly lower than batch A. In spite of this, the shapes of the curves derived from each batch are essentially the same. This batch difference will be discussed more fully belo\v. Table 1 includes a section which was derived l)y averaging the means of headed “Average” batches A and R. The data in Fig. 1 and Table 2 were computed from these averages and the: drv nrcights. The probabilities in Tables 3 and 4 are shover separatel!~ for each batch. Dry n-eights are givren in Table 5.
FIG.
1.
Monoamine oxidase activity
acetic acid formed
per hour per milligram
expressed as moles of 5-hyclroxyindoledry weight per structure.
In Table 1 the data are presented as micromicromoles of 5-HIAA formed per hour per structure. There is an increasing enzyme activity in all structures from the time of earliest measurement until stage 47, after which there is a leveling off. Whole embryo activity at stage 12 is almost undetectable, but slowly increases to stage 35. The level rises sharply between stages 35 and 38, the period of hatching. It con-
272
PETER
C.
BAKER
tinues increasing to stage 46. After this there is a decline followed by leveling off after stage 47. Eyes and brain show much lower levels, and a rather even rise to stage 47, after which they too level off. Most of the changes seen in Table 1 are statistically significant as noted in Table 3. Figure 1 and Table 2 show changes as micromicromoles 5-HIAA produced per milligram dry weight per structure per hour. Brain shows clearly as a center of high MAO activity through all stages. Eyes too concentrate MAO activity but only from stage 40 through TABLE
2
MONOAMIXE OXIDASE ACTIVITY EXPRESSED AS MICROMICROMOLES OF 5-HIAA FORMED PER HOCR PER MILLIGRAM DRY WEIGHT PER STRUCTCRE~
stage
Whole
12 20 25 30 35
0.36 6 .31) 21.4L x5 89 106.94
3x 40 41 48 46 47 48
407 ,67 76!) 65 1118.5!) 1501 20 3.584.45 4464 75 4183.40
a These values are derived
Eyes
Brain
-
-
725 00 1318.00 2079 00 2iB’L. 00 3!)!X) .23 3255.00 c? 2RO’. 61
from the average v&es
2291 .15 3438.08 4’2i4.62 63%. 50 6513.18 5X66.09
shown on Table 1.
stage 42. The statistical significance of eye and brain concentrations as compared to whole embryo are to be found in Table 4. Thin layer chromatograms made at stages 20, 25, 30, 35, 38, 41, 46, and 48, establish the incubation product as 5-HIAA for all three structures, except whole embryo at stage 20, where the yield was so low it did not rise above background. The RI values determined for 91; 5-hydroxytryptophol, the three possible products were: 5-HIAA, 80; and N-acetylserotonin, 36. DISCUSSION
The developmental pattern of MAO activity is in many respects similar to the pattern of other enzymes acting in the same pathway. 5-Hydroxytryptophandecarboxylase ( 5-HTPDC ) , which effects the
I’ Hl:tllk qxtces represent no measurement, as in stage 3X for brain, or levels I)elow whole ernhryo, 3s in stages 46, 4’7, :x11(14S for eyes. P (prol~:tldily) derived from Stutlelrt-l:isher t. XS = not signifiant. V:dues have IWSL determined sep:rr:ttely for 1xttc11es h :mtl R.
final step in S-EIT synthesis methyltransferase (HIOMT), synthesis from SE-IT, have Xenopus. In whole embryos
from tryptophan, and hydroxyindole-Owhich effects the final step in melatonin already been measured in developing these two enzymes showed low relative
274
PETER
C. BAKER
levels before hatching, sharp rises during hatching, continued increase to stage 47, and leveling off thereafter. This is similar to the changes shown here for MAO. These two enzymes also showed a lowered level of activity at stage 25, around the time of first spontaneous movement. This lowering of activity is not seen here with MAO. 5-HTPDC and HIOMT activity in eyes and brain generally was high, and these two structures seem to be centers of activity for the two enzymes. For all three of these enzymes the extent of increased activity in whole embryo between posthatching, stage 38, and limb bud, stage 47, is seven- to tenfold. The absolute levels of activity are of differDRY
JVEIGHTS
I~XPRESRED AS,)
12 20 2.5 30
HRAIX
AS MILLIGRAFJS FROM STAGE
OF Q'IIOLE 12 TO k&GE
EMBRYO,
F:YE PAIR,
48
0.450 0 .4G 0.440
:v5 38 49
0 4x 0.434 0 ,434 0 4%
41 4’2 46
0.416 0.412 0, 301
0.010 0.010 0.01:3
0 OlB 0.013 0.013 0.016
47 48
0. :3x 0 .355
0.020 0.023
0.0%2 0.033
0 010
0 010
ing orders of magnitude, but the relative increases are not. With minor exceptions all three enzymes show much the same shape of curve. The high concentration of this system in embryonic brain is very much in keeping with high concentrations found in adult brain. In vertebrates the central nervous system is second only to the gastrointestinal tract as a center of 5HT metabolism (Douglas, 1965; Erspamer, 1961; Sharman, 1965). The lateral eyes have received far less consideration in this respect. Welsh (1964) has found 5-HT in the retina and pigment epithelium of four vertebrate classes, and HIOMT in the retina of a variety Quay ( 196513) h as demonstrated of lower vertebrates. The indole compounds being considered here are derived from tryptophan (Udenfriend et al., 1956). Investigations of amino acid
metabolism in developing amphibians have not included tryptophan (Dcuchar, 1856, 1963), although assay by M’allace (1963) indicates that tryptophan is a yolk constituent. In a recent study of yolk utilization in developing Xcnopw, Sclman and Pawsey (1965) reported that SCVNI out of twelve tissues initiated yolk lm~akdown around the time of hatching. Tlic oiisct of increased indolc metal~olism at tlw same time may bc related to tryptophan rcleaw 1)): yolk. In this same c’ontest. tlw obserwd hatch difhwncc in 1IAO activity might lw the result of \wiatioiis in volk constitution from batch to hatch. A sin&u lwtcll diffcrcwcc was.fountl in data for S-IITPDC and IIIO\IT measurcmcwts. although not so pronomwrd. Lccpcr et rrl. ( lHfj8 ) and l~ellman and Roth ( 19655) havr shon-n that \I,40 is not sul,strate spc~cific~for 5-IIT. Oswald and Strittnratter ( 1963) haw iudicatctl that thcw may 1~ distinct J1.40~ for each tissuri. \\‘ith thcwh ~onsidclatiolls in mind tlw method of \\.urtman ancl Axctlrod ( 1963 ) \vas modified to Lw $5-IIT. not trvptarnilw, as the substlatr~.
Quantitative comparison of the ill citro cnpacit)- for making 5IIT ( 5-HTPDC activity) and destroying 5I1T ( 11,40 activity) indicates that the drstructiw wpacit) is about tenfold greater in developing Xcn0pu.s. Sinw 3-HT has lwcn fom~d. the capacity for destruction must somehow 1~ isolated. Other investigations have shown that there is a structural separation of some parts of the pathway. Bodanski et al. (195’7) have fomld that 5-HTPDC is located in the supcrnatant fraction, and Rodriguez dc Lores Arnaiz and De Robertis ( 1962) have shown that 11AO is associated with the mitochondria. Two clear conclusions cali be drawn. There is an incrcasc in MAO activity in dewloping Xenopzls embryos, and a concentration of that activity in the brain. Previous measurements in other parts of the same pathway support these conclusions. SUhlhfARY
Monoamine oxidase ( MAO ) activity was measured in whole embryos, brains, and lateral eyes of developing Xennpzrs km&. ‘iC-Shydroxytryptamine precursor was introduced into an in citro system, and after incubation radioactive S-hydroxyindoleacetic acid product was extracted and measured in a liquid scintillation counter. The MAO activity of whole embryos, measured from gastrula to premetamorphic larva, row from almost undetectable to very high
276
PETER
C. BAKER
levels, which leveled off at limb bud stage, Eyes, measured from immediately after hatching, showed low activity which rose to Iimb bud stage and then leveled off. Brains, measured from somewhat later after hatching, had about twice the activity found in eyes but followed about the same pattern. Brains showed a higher concentration of MAO activity than whole embryos. These results were considered in relation to other enzymes measured in this same pathway and on the same animal. There appears to be an increase in indole enzyme activity during development and a concentration of this activity in the brain. The possible relation between available substrate released from stored yolk and increased enzyme activity was discussed. I would like to thank Dr. W. B. Quay for the time, offered during the cnwse of this investigation.
consideration,
ancl help
REFERENCES BAKER, P. C. (1965). Acta Embr~yol. Morphol. Exptl. in press. BAKER, P. C. (1966). Neuroendocrinology in press. BAKER, P. C., QUAY, W. B., and AXELHOD, J. ( 1965). Development of hydroxyindole-0methyltmnsfernse activity in eye and brain of the nmphibian, Xenopzls laevis. Life Sci. 4, 1981-1987. BENNETT, D. S., and GIAR~~AN, N. J. ( 1965). Schedule of appearance of 5hydroxytryptamine ( serotonin ) and associated enzymes in the developing rat brain. .I. Nezcrochem. 12, 911-918. BODANSKI, D. F., WEISSBACH, H., and WDENFHIEND, S. (1957). The distribution of serotonin, 5-hydroxytryptophnn decnrboxylase and mononmine oxidase in brain. J. Netwochem. 1, 272-278. BOURNS, B. B. ( 1965). Metabolism of amines in the brain of the chick during embryonic development. Life Sci. 4, 583-592. BUZNIKOV, G. A., CHUDAKOVA, I. V., and ZVEZUINA, N. D. (1964). The role of nemohumors in early embryogenesis. I. Serotonin content of developing embryos of SE;I urchin and loach. J. Emhryol. Exptl. Morphol. 12, 563-573. DEUCHAII, E. M. (1956). Amino ncids in developing tissues of Xrnopus laevis. J. Emlqol. Exptl. Morphol. 4, 327-346. DEUCHAH, E. hl. ( 1963). Amino acids and differentintion in animal embryos. Symp. Sot. Exptl. Bid. 17, 58-73. DOUGLAS, W. W. (1965). 5.IIydroxytryptamine and antagonists. In “The Phar(L. Goodman and A. Gilman, ed.), pp. macological Basis of Therapeutics” 644-664. Macmillan, New York. ERSPAMER, V. ( 1961). Recent research in the field of 5-hydroxytryptamine and related inclolealkylamines. Progr. Drug Res. 3, 151-367. inhibition of amine FELL~UAN, J. H., and ROTH, E. S. (1965). Th e competitive oxidase with special reference to the carcinoid syndrome. Cnn. J. Biochem. 43, 909-914.
hlAQ
~EI’EII,
L.
c.,
\~EISSBACH,
IS
H.,
DEVELOPISC,
md
~DEXFKIENI),
277
XClWpZtS
s.
( 19%).
StlldieS
in
the
mctnl)olism of norepinephrine, epincphrine and their O-methyl analogs hy partially purified enzyme lxcpxxtions. Arch. Biochcm. 77, 417-427. NIEUWKOOP, I’. D., and FABEK, J. (1956). “Normal Table of Xenoprrs Zocvi,s ( Dwdin) .” North-IIolland l’nldishing Co., Amsterdam. OSHIKA, H., and N.sKAI, K. ( 1965). Effect of \IAO inhibitor on drvelopentnl mechanism in the amphibian. Jnpn. J. Pharmncol. 15, 82-87. OSWALD, E. O., and STIUTTMATTEH, C. F. ( 1963). Comparative studies in the characterization of monoamine oxidnses. Proc. Sot. Exptl. Hid. Med. 114, 668-673. QUAY, 1:‘. R. ( 1969). Differential extractions for the sp-~trophotofluorometric measun’rmcnt of diverse Fj-hytlrouy and li-methoxy indo!es. And. Riochev1. 5, 51-59. Qu~\Y, 11’. 13. ( 1365a). Intlole derivatives of pined ant1 related neural and retinal tissues. ~klT~Jl~Jcd. Rec. 17, 321-345. QUAY, TV. B. ( 19651)). Retinal ant1 pineal lr\droxyindolc-0-methYltr~lnsf(~r~lse activitv in vertebrates. Life Sci. 4, 989-991. QUAY, G7. R., and BACNAHA, J, T. ( 1964). R c,I,‘1t’IVC potencies of indolic and relatctl co~nlx~unds in the body-lightening reaction of lar\al Xenoprs. Ad. Itrfrrn. Phtrr,,lcrcotl!/n. 150, 137-148. Roo~rrcu~z I)E LCHIES AI
R. A. ( 196:‘). Studies on ampl~il~ian yolk. IL’. AII anal!.~is of thr nt;lillhotly componnnt of Lolk platelets. Niocl~int. Ru)ph!/.~. Adu 74, 5OEi-518. ‘Il’er.s~r, 1. II. ( 1964 ) The quantitative distribution of .5-h~dro\!trvptamine in thca IICYVOLIS system, eyes and other organs of some vertebrate<. 11; “Comprative Neurochemistry” ( D. Richter, cd.), pp. 355-3366. l’crgamon Press, London. \%‘UKThIAN, R. J., ant1 AXEI.IW~, 1. (1983). A sensitiw and specific assay for the estimation of monoamine oxidase. Bioclrem. Phurnlmol. 12, 14398-1441, ~fl’ALLACE,
BI0LOT.Y
14, 267-277
(l%ili)
Monoamine Oxidase in the Eye, Brain, and Whole Embryo of Developing Xenopus laevis PETER C. BAKER’
Accepted
March
17, 1966
5-Hydroxytryptamine (5HT) and related indole compounds are at present the subject of intensive investigation in a wide variety of animals. One of the most active areas of interest is the vertebrate central nervous system, where these compounds are being seriously considered as synaptic transmitters and hormones (see reviews of Douglas, 1965; Quay, 1965,a ; and Sharman, 1965). The major part of this research has been concerned with adult animals, and until recently, developing forms and the information they might provide have been generally ignored (Buznikov et al., 1964; Bennett and Giarman, 1965; Bourne, 1965; Oshika and Nakai, 1965). This work is part of a series of measurements designed to obtain some information about the metabolism of these compounds in a developing lower vertebrate, Xcnopu.s Zaecis, the South African clawed toad. Previous studies in this biochemical pathway have measured S-IIT ( Baker, 1965 ), the 5-FIT forming enzyme, 5-hydroxytryptophan decarboxylase ( Baker, 1966 ) , and the melatonin-forming enzyme, hvdroxyindole-0-methyltransferase (Baker et al., 1965). These investigations have indicated a pattern of increasing 5hydroxyand 5-methoxyindole metabolism during development and a concentration of this metabolism in the cycs and brain. _\Ionoaminc oxidase ( MAO ), the enzyme responsible for the destruction of 5HT, is the subject of the mcasurcments presented here. The results indicate that LIAO is similar to both 5-hydroxytryptophan de1Sllppwtvtl by U.S. Public Health Service I’rdoct~d Fellowship, 20,888~02, from tl-w Nationd Institute of Gcnerd hledical Sciences. 267
ELFI-GLLI-
268
PETER
C. BAKER
carboxylase and hydroxyindole-0-methyltransferase in its pattern of increase during development. Concentration of MAO activity in the brain is also demonstrated, although concentration in the eyes is found only during some stages. MATERIALS
AND METHODS
Fertilized eggs of Xenopus Zuevis were obtained by artificial ovulation in November and December of 19658.Embryos were allowed to develop at room temperature and were staged in keeping with the Normal Table of Nieuwkoop and Faber (1956). Measurements were made on whole embryos between stage 12, gastrula, and stage 48, premetamorphic larva. Lateral eyes were measured between stage 38, posthatching, and stage 48. Brains were measured between stage 40 and stage 48. The stage of earliest recovery for each structure was dictated by ease of clean dissection in numbers large enough for use. Before hatching, embryos were removed from surrounding jelly and capsule. Dissection of parts was carried out in cold 0.1 M, pH 7.4 phosphate buffer, and homogenization was by hand in all-glass homogenizers, in ice water. The method of Wurtman and Axelrod (1963) for assay of MAO has been modified for use here. The use of tryptamine as substrate was rejected in favor of 5-hydroxytryptamine, for reasons discussed below. It was necessary then to change the extracting solvent from toluene to diethyl ether. Toluene extracts 5-HT, and diethyl ether does not (Quay, 1963). Samples of three whole embryos, six eye pairs, or six brains were homogenized in 300 PI cold, 0.1 M, pH 7.4 phosphate buffer. An aliquot of 290 ,J of each homogenate was transferred to 5-ml centrifuge tubes in ice water. The tubes then received 10 ~1 carbon-14 S-HT solution (New England Nuclear Corporation 5-hydroxytryptamine-2-l%; 6.25 mpmoles, 1.65 mC/mmole). They were then incubated for 20 minutes at 3’7°C in a shaking water bath. After incubation, 150 ,J 1.0 N HCl and 3 ml diethyl ether were added to each tube. The tubes were then stoppered, shaken, and centrifuged; 2.7 ml of the ether phase of each tube was then transferred to a series of scintillation counting vials. The vials received 10 ml POPOP-PPO phosphor and 1 ml ethanol. They were counted for 10 minutes in a Packard, automatic, dual channel, TriCarb spectrometer. Blank values were determined by means of heated enzyme incubations treated as
hf.40 IN DEVELOPING
Xenopus
269
above, A value of 40 counts per minute was found to represent the amount of carbon-14 5HT being carried over in the extraction. methods were used to determine the Spectrophotofluorometric efficiency of the diethyl ether as extracting solvent. Known amounts of 5-hydroxyindoleacetic acid (5-HIAA) in phosphate buffer were extracted as above. The diethyl ether was transferred and reextracted in a tube containing S ml of heptane and a mixture of 200 ~1 of buffer and 150 ~1 1.0 N HCI. The 5-HIAA in the aqueous, buffer-acid, phase was then read in an Aminco-Bowman spectrophotofluorometer, as described by Quay ( 1963). Tubes containing known amounts of 5HIAA in 200 ~1 buffer, plus 150 pl 1.0 N HCI, plus S ml heptane, received diethyl ether that had been sham extracted over a similar buffer-acid mixture without 5-HIAA. These were read as standards. The resulting data indicated that 95% of the available product, 5HIAA, was extracted into the diethyl ether phase. Possible products of tissue incubated with 5-HT were considered to be 5HIAA, 5-hydroxytryptophol, and N-acetyl serotonin (Quay, 1965a). Ascending, one-dimensional, thin layer chromatography was used to determine the homogeneity of the product at various times during the developmental series. Chromatography plates coated with silica gel G were sprayed with ascorbic acid in methanol (Quay and Bagnera, 1964). Extracts of incubation were run in alternate spots with standard solutions containing all three possible products. Plates were run for 45 minutes in the cold using methyl acetate (Eastman) as the solvent. After drying, each plate was masked to cover the areas containing the incubation extracts and the areas containing standards were sprayed with van Urk’s reagent. Rf values of the standards were then determined, The incubation extract areas were marked off in a grid, cut off with razor blades, and added to a series of scintillation vials, which then received 1 ml ethanol and 10 ml phosphor. When the vials were counted, the location of any radioactive products on the chromatogram could be determined. Dry weights used for the calculation of relative enzyme concentrations were made from a number of egg batches during previous experiments. Continued weighings were halted due to the constancy of values from batch to batch, and a table of standard weights was made up. The dry weights were determined by placing whole embryos, eye pairs, or brains in groups of 3 to 10 on preweighed aluminum pans and drying in a forced draft oven overnight at 100°C. After
270
PETER C. BAKER
drying, pans plus tissues were weighed and dry weights were determined by subtraction. All weighings were made on a Cahn electrobalance to the nearest microgram. RESULTS
Two separate batches of eggs from two separate pairs of parents were used here. These have been called batches A and B, and are TABLE 1 MONOAXUNE OXIDASE ACTIVITY EXFRESSED AS i%CROMICROMOLES OF 5-HIAA FORMED PER HOUR PER STRUCTURE” Batch A stage Whole 12 20 25 30 35 38 40 41 42 46 47 48 Eyes 38 40 41 42 46 47 48 Brain 40 41 42 46 47 48
Batch B
H
N
SE
H
N
SE
AVt?K&
0.14 4.79 11.60 35.85 126.45 255.09 423.09 627.18 840.34 1616.13 1999.41 1958.99
10 10 10 10 10 10 10 10 9 10 10 10
0.44 0.64 0.97 1.69 8.21 15.31 19.07 19.76 38.76 60.91 104.67 56.54
0.17 0.87 7.23 12.88 44.48 99.77 235 72 303.4i 396.71 1186.90 1384 87 1178.62
8 10 10 9 10 10 10 10 10 10 10 10
0.92 0.83 0.58 1.17 2.07 2.84 8.97 14.61 12.82 28.62 66.47 22.39
0.16 2.83 9.42 24.37 85.47 176.93 329.41 465 33 618.53 1401.52 1692.14 1568.81
12.06 18.83 29.86 33.22 71.15 93.5i 91.11
10 10 10 10 10 10 10
0.33 0.65 0.64 0.45 2.00 0.59 3.49
2.43 7.5’2 11.51 22.02 3’2.82 36.63 41.95
10 10 10 10 10 7 10
0.30 0.44 0.45 ‘2 75 1. 0.82 0.69 1.09
7.25 13.18 20.79 27.62 51.99 65.10 61. .53
42.96 60.30 73.75 120.41 184.27 167.81
10 10 8 10 10 10
1.65 2.77 2.20 4 ‘2“1 5.95 .3 56
16.61 29.09
9 10 10 10 8 10
0.96 1 .02 3.25 1.88 3.43 2.79
29.79 44.70 55.57 99.72 143.29 134.92
errors
of the mean
35.39 is.03 102.31 102.03
a hlenns (x), number of determinations (S), (SE) are shown separately for bakhes A and B. b Average of the means of batches A and B.
and st,andard
MAO
IN
DEVELOPING
&3l0j?tlS
371
listed separately in Table 1. After stage 25, batch B is always significantly lower than batch A. In spite of this, the shapes of the curves derived from each batch are essentially the same. This batch difference will be discussed more fully belo\v. Table 1 includes a section which was derived l)y averaging the means of headed “Average” batches A and R. The data in Fig. 1 and Table 2 were computed from these averages and the: drv nrcights. The probabilities in Tables 3 and 4 are shover separatel!~ for each batch. Dry n-eights are givren in Table 5.
FIG.
1.
Monoamine oxidase activity
acetic acid formed
per hour per milligram
expressed as moles of 5-hyclroxyindoledry weight per structure.
In Table 1 the data are presented as micromicromoles of 5-HIAA formed per hour per structure. There is an increasing enzyme activity in all structures from the time of earliest measurement until stage 47, after which there is a leveling off. Whole embryo activity at stage 12 is almost undetectable, but slowly increases to stage 35. The level rises sharply between stages 35 and 38, the period of hatching. It con-
272
PETER
C.
BAKER
tinues increasing to stage 46. After this there is a decline followed by leveling off after stage 47. Eyes and brain show much lower levels, and a rather even rise to stage 47, after which they too level off. Most of the changes seen in Table 1 are statistically significant as noted in Table 3. Figure 1 and Table 2 show changes as micromicromoles 5-HIAA produced per milligram dry weight per structure per hour. Brain shows clearly as a center of high MAO activity through all stages. Eyes too concentrate MAO activity but only from stage 40 through TABLE
2
MONOAMIXE OXIDASE ACTIVITY EXPRESSED AS MICROMICROMOLES OF 5-HIAA FORMED PER HOCR PER MILLIGRAM DRY WEIGHT PER STRUCTCRE~
stage
Whole
12 20 25 30 35
0.36 6 .31) 21.4L x5 89 106.94
3x 40 41 48 46 47 48
407 ,67 76!) 65 1118.5!) 1501 20 3.584.45 4464 75 4183.40
a These values are derived
Eyes
Brain
-
-
725 00 1318.00 2079 00 2iB’L. 00 3!)!X) .23 3255.00 c? 2RO’. 61
from the average v&es
2291 .15 3438.08 4’2i4.62 63%. 50 6513.18 5X66.09
shown on Table 1.
stage 42. The statistical significance of eye and brain concentrations as compared to whole embryo are to be found in Table 4. Thin layer chromatograms made at stages 20, 25, 30, 35, 38, 41, 46, and 48, establish the incubation product as 5-HIAA for all three structures, except whole embryo at stage 20, where the yield was so low it did not rise above background. The RI values determined for 91; 5-hydroxytryptophol, the three possible products were: 5-HIAA, 80; and N-acetylserotonin, 36. DISCUSSION
The developmental pattern of MAO activity is in many respects similar to the pattern of other enzymes acting in the same pathway. 5-Hydroxytryptophandecarboxylase ( 5-HTPDC ) , which effects the
I’ Hl:tllk qxtces represent no measurement, as in stage 3X for brain, or levels I)elow whole ernhryo, 3s in stages 46, 4’7, :x11(14S for eyes. P (prol~:tldily) derived from Stutlelrt-l:isher t. XS = not signifiant. V:dues have IWSL determined sep:rr:ttely for 1xttc11es h :mtl R.
final step in S-EIT synthesis methyltransferase (HIOMT), synthesis from SE-IT, have Xenopus. In whole embryos
from tryptophan, and hydroxyindole-Owhich effects the final step in melatonin already been measured in developing these two enzymes showed low relative
274
PETER
C. BAKER
levels before hatching, sharp rises during hatching, continued increase to stage 47, and leveling off thereafter. This is similar to the changes shown here for MAO. These two enzymes also showed a lowered level of activity at stage 25, around the time of first spontaneous movement. This lowering of activity is not seen here with MAO. 5-HTPDC and HIOMT activity in eyes and brain generally was high, and these two structures seem to be centers of activity for the two enzymes. For all three of these enzymes the extent of increased activity in whole embryo between posthatching, stage 38, and limb bud, stage 47, is seven- to tenfold. The absolute levels of activity are of differDRY
JVEIGHTS
I~XPRESRED AS,)
12 20 2.5 30
HRAIX
AS MILLIGRAFJS FROM STAGE
OF Q'IIOLE 12 TO k&GE
EMBRYO,
F:YE PAIR,
48
0.450 0 .4G 0.440
:v5 38 49
0 4x 0.434 0 ,434 0 4%
41 4’2 46
0.416 0.412 0, 301
0.010 0.010 0.01:3
0 OlB 0.013 0.013 0.016
47 48
0. :3x 0 .355
0.020 0.023
0.0%2 0.033
0 010
0 010
ing orders of magnitude, but the relative increases are not. With minor exceptions all three enzymes show much the same shape of curve. The high concentration of this system in embryonic brain is very much in keeping with high concentrations found in adult brain. In vertebrates the central nervous system is second only to the gastrointestinal tract as a center of 5HT metabolism (Douglas, 1965; Erspamer, 1961; Sharman, 1965). The lateral eyes have received far less consideration in this respect. Welsh (1964) has found 5-HT in the retina and pigment epithelium of four vertebrate classes, and HIOMT in the retina of a variety Quay ( 196513) h as demonstrated of lower vertebrates. The indole compounds being considered here are derived from tryptophan (Udenfriend et al., 1956). Investigations of amino acid
metabolism in developing amphibians have not included tryptophan (Dcuchar, 1856, 1963), although assay by M’allace (1963) indicates that tryptophan is a yolk constituent. In a recent study of yolk utilization in developing Xcnopw, Sclman and Pawsey (1965) reported that SCVNI out of twelve tissues initiated yolk lm~akdown around the time of hatching. Tlic oiisct of increased indolc metal~olism at tlw same time may bc related to tryptophan rcleaw 1)): yolk. In this same c’ontest. tlw obserwd hatch difhwncc in 1IAO activity might lw the result of \wiatioiis in volk constitution from batch to hatch. A sin&u lwtcll diffcrcwcc was.fountl in data for S-IITPDC and IIIO\IT measurcmcwts. although not so pronomwrd. Lccpcr et rrl. ( lHfj8 ) and l~ellman and Roth ( 19655) havr shon-n that \I,40 is not sul,strate spc~cific~for 5-IIT. Oswald and Strittnratter ( 1963) haw iudicatctl that thcw may 1~ distinct J1.40~ for each tissuri. \\‘ith thcwh ~onsidclatiolls in mind tlw method of \\.urtman ancl Axctlrod ( 1963 ) \vas modified to Lw $5-IIT. not trvptarnilw, as the substlatr~.
Quantitative comparison of the ill citro cnpacit)- for making 5IIT ( 5-HTPDC activity) and destroying 5I1T ( 11,40 activity) indicates that the drstructiw wpacit) is about tenfold greater in developing Xcn0pu.s. Sinw 3-HT has lwcn fom~d. the capacity for destruction must somehow 1~ isolated. Other investigations have shown that there is a structural separation of some parts of the pathway. Bodanski et al. (195’7) have fomld that 5-HTPDC is located in the supcrnatant fraction, and Rodriguez dc Lores Arnaiz and De Robertis ( 1962) have shown that 11AO is associated with the mitochondria. Two clear conclusions cali be drawn. There is an incrcasc in MAO activity in dewloping Xenopzls embryos, and a concentration of that activity in the brain. Previous measurements in other parts of the same pathway support these conclusions. SUhlhfARY
Monoamine oxidase ( MAO ) activity was measured in whole embryos, brains, and lateral eyes of developing Xennpzrs km&. ‘iC-Shydroxytryptamine precursor was introduced into an in citro system, and after incubation radioactive S-hydroxyindoleacetic acid product was extracted and measured in a liquid scintillation counter. The MAO activity of whole embryos, measured from gastrula to premetamorphic larva, row from almost undetectable to very high
276
PETER
C. BAKER
levels, which leveled off at limb bud stage, Eyes, measured from immediately after hatching, showed low activity which rose to Iimb bud stage and then leveled off. Brains, measured from somewhat later after hatching, had about twice the activity found in eyes but followed about the same pattern. Brains showed a higher concentration of MAO activity than whole embryos. These results were considered in relation to other enzymes measured in this same pathway and on the same animal. There appears to be an increase in indole enzyme activity during development and a concentration of this activity in the brain. The possible relation between available substrate released from stored yolk and increased enzyme activity was discussed. I would like to thank Dr. W. B. Quay for the time, offered during the cnwse of this investigation.
consideration,
ancl help
REFERENCES BAKER, P. C. (1965). Acta Embr~yol. Morphol. Exptl. in press. BAKER, P. C. (1966). Neuroendocrinology in press. BAKER, P. C., QUAY, W. B., and AXELHOD, J. ( 1965). Development of hydroxyindole-0methyltmnsfernse activity in eye and brain of the nmphibian, Xenopzls laevis. Life Sci. 4, 1981-1987. BENNETT, D. S., and GIAR~~AN, N. J. ( 1965). Schedule of appearance of 5hydroxytryptamine ( serotonin ) and associated enzymes in the developing rat brain. .I. Nezcrochem. 12, 911-918. BODANSKI, D. F., WEISSBACH, H., and WDENFHIEND, S. (1957). The distribution of serotonin, 5-hydroxytryptophnn decnrboxylase and mononmine oxidase in brain. J. Netwochem. 1, 272-278. BOURNS, B. B. ( 1965). Metabolism of amines in the brain of the chick during embryonic development. Life Sci. 4, 583-592. BUZNIKOV, G. A., CHUDAKOVA, I. V., and ZVEZUINA, N. D. (1964). The role of nemohumors in early embryogenesis. I. Serotonin content of developing embryos of SE;I urchin and loach. J. Emhryol. Exptl. Morphol. 12, 563-573. DEUCHAII, E. M. (1956). Amino ncids in developing tissues of Xrnopus laevis. J. Emlqol. Exptl. Morphol. 4, 327-346. DEUCHAH, E. hl. ( 1963). Amino acids and differentintion in animal embryos. Symp. Sot. Exptl. Bid. 17, 58-73. DOUGLAS, W. W. (1965). 5.IIydroxytryptamine and antagonists. In “The Phar(L. Goodman and A. Gilman, ed.), pp. macological Basis of Therapeutics” 644-664. Macmillan, New York. ERSPAMER, V. ( 1961). Recent research in the field of 5-hydroxytryptamine and related inclolealkylamines. Progr. Drug Res. 3, 151-367. inhibition of amine FELL~UAN, J. H., and ROTH, E. S. (1965). Th e competitive oxidase with special reference to the carcinoid syndrome. Cnn. J. Biochem. 43, 909-914.
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md
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the
mctnl)olism of norepinephrine, epincphrine and their O-methyl analogs hy partially purified enzyme lxcpxxtions. Arch. Biochcm. 77, 417-427. NIEUWKOOP, I’. D., and FABEK, J. (1956). “Normal Table of Xenoprrs Zocvi,s ( Dwdin) .” North-IIolland l’nldishing Co., Amsterdam. OSHIKA, H., and N.sKAI, K. ( 1965). Effect of \IAO inhibitor on drvelopentnl mechanism in the amphibian. Jnpn. J. Pharmncol. 15, 82-87. OSWALD, E. O., and STIUTTMATTEH, C. F. ( 1963). Comparative studies in the characterization of monoamine oxidnses. Proc. Sot. Exptl. Hid. Med. 114, 668-673. QUAY, 1:‘. R. ( 1969). Differential extractions for the sp-~trophotofluorometric measun’rmcnt of diverse Fj-hytlrouy and li-methoxy indo!es. And. Riochev1. 5, 51-59. Qu~\Y, 11’. 13. ( 1365a). Intlole derivatives of pined ant1 related neural and retinal tissues. ~klT~Jl~Jcd. Rec. 17, 321-345. QUAY, TV. B. ( 19651)). Retinal ant1 pineal lr\droxyindolc-0-methYltr~lnsf(~r~lse activitv in vertebrates. Life Sci. 4, 989-991. QUAY, G7. R., and BACNAHA, J, T. ( 1964). R c,I,‘1t’IVC potencies of indolic and relatctl co~nlx~unds in the body-lightening reaction of lar\al Xenoprs. Ad. Itrfrrn. Phtrr,,lcrcotl!/n. 150, 137-148. Roo~rrcu~z I)E LCHIES AI
R. A. ( 196:‘). Studies on ampl~il~ian yolk. IL’. AII anal!.~is of thr nt;lillhotly componnnt of Lolk platelets. Niocl~int. Ru)ph!/.~. Adu 74, 5OEi-518. ‘Il’er.s~r, 1. II. ( 1964 ) The quantitative distribution of .5-h~dro\!trvptamine in thca IICYVOLIS system, eyes and other organs of some vertebrate<. 11; “Comprative Neurochemistry” ( D. Richter, cd.), pp. 355-3366. l’crgamon Press, London. \%‘UKThIAN, R. J., ant1 AXEI.IW~, 1. (1983). A sensitiw and specific assay for the estimation of monoamine oxidase. Bioclrem. Phurnlmol. 12, 14398-1441, ~fl’ALLACE,