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N-acetylhistidine in developing embryos of the killifish, Fundulus heteroclitus and the zebrafish, Brachydanio rerio
LIFE SCIENCES Vol . 6, pp . 609-614, 1967 . Printed in Great Britain.
Pergamon Press Ltd.
N-ACETYLHISTIDINE IN DEVELOPING EMBßYOS OF TAE &ILLIFISH, FUNDULUS RETEROCLITUS AND TAE ZEBBAFISR, BRACHYDANIO $E1tI0 Morris R . Baslove and George Ruggieri, S .J .
(Received 23 December 1966) School of Medicine, University of Aawa ü, Honolulu, Rewa ü and Osborn Laboratories of Marine Sciences, New York Aquarium, Brooklyn, lfew York Introduction N-acetylhistidine (NAcR)
ie a compound occurring in the brain of bony
fish, amphibians and reptiles only (1-5) .
It has also been found in extra-
cerebral tissues of poikilothermal vertebrates and in larval amphibians (2,4,6) .
NAcH is found in high concentration in the brain, with levels up
to 2 mg/g in fish .
However, phylogenetic progression up to the reptiles
shows a gradual decrease in the percentage of this compound ae a function of the total extractable imidazolea (1,2,4) .
NAcH has been found to be
synthesized by brain enzymes from L-hietidine and acetyl coenzyme A (7) and is degraded by a highly specific acylase (S) .
While the function of NAcH
is unknown, a role in acetylation processes (3,9) and in permeability phenomena in the nervous system have been suggested (10) .
It appeared of
interest therefore to try to correlate NAcR content with the embryonic level of development of the central nervous system in fish . Methods Embryological studies were carried out on the developing embryos of killifish,
Fundulua heteroclitus , a marine fish, and on the embryos of the
zebrafiah,
Brachydanio rerio , a freshwater fish .
ta med after natural fertilization (11) .
Zebrafiah eggs ware ob-
Killifish eggs were obtained by
stripping msture females and externally fertilized as described by
N-ACETYLHISTIDINE
61 0
Costello et al . (12) .
Vol . 6, No . 6
Developmental stages for ubrafieh are according to
Hísaokt ~ Battle (11) and for killifish as described by Armstrong and Child (13) .
For NAcH analysis, e~bryoe were smashed on lihatman #1 paper,
immediately heat dried, and then saturated with 95x ethano1,0 .1 N HC1 (1 :1) solution and redried prior to chromatography (14) .
For the ubra-
fieh, the wet weight per egg we found to be 1 .0 mg, for hatched fry, 0 .3 n8 .
In this species, 15-25 embryos were used to assay each stage .
Killifish eggs weighed 4 .5 nig each and hatched fry 2 .3 mg . 3-5 killifish embryos were used . in Kg HAcH per 1000 embryos .
For analysis,
Hesulta for both species are reported
To eliminate the possibility of an eaogenoue
source of NAcA, the experimental animals were not fed after hatching and the experiment was terminated just prior to cgmplete reeorption of the yolk . 1QAcH was measured after paper chromatography in 1-butanol, acetic acid, water (4 :1 :5) and development with Pauly reagent .
The red color
formed was analysed spectrophotometrically at 510 m~ after elution with 1Z butanol (7) . To determine the effect of an anti-cholinesterase on McH concentrations, ubrafieh and killifish embryos were treated with Melathion (5 mgs x), a potent inhibitor of fish brain cholinesterase (15) .
Dis
appearance of coordi~ted movements just after a period of convulsion served as the end point for these experimnnte . BReulte and Discussion The appearance and concentration of 1fAcH in developing embryos of the sebrafish and the killifish have been inveetígated and the results are presented in Figures 1 and 2 .
Vol .
6, No .
6
N-ACE1'YLHISTIDINE
611
FIG . 1 RAcH concentration vs, developmental etage in embryos of Brachydanio rerio .
Day
5faq,
b ~b
1
20
22
2
23
3
24
4 25
5
FIG . 2 NAcH concentration vs . developmental etage in embryos of Fundulus heteroclitus .
61 2
N-ACETYLHISTIDINE
Vol . 6, No . 6
Appearance of NAcH in these embryos has been observed at a time just prior to that stage in which the first twitches occur indicating a possible relationship between this compound and CNS control over somatic movements . No NAcH is measurable in embryos of Fundulus heteroclitus at stages 25-28 when only contractions of somatic musculature have been initiated .
After
the first appearance of NAcH, there ie a rapid increase in the concentration of this substance which continues to rise for several days after hatching . It is of interest that the appearance of acetylcholineaterase (AChE) activity in embryos of Fundulua heteroclitus occurs upon development of a CNS-somatic reflex arc also (16) .
In addition, Uesugi and Yamazoe (17)
have observed the appearance of acetylcholineaterase activity in embryos of the rainbow trout Salmo gairdnerü , and Gabella (18) has measured acetylcholineaterase activity and nervous system formation in developing embryos of the guppy, Lebietea reticulatus .
In these cases, AChE activity
parallels the development of nervous tissue and the neuromuscular junction . Since NAcH is found in high concentration in the CNS and appears at a time in embryonic fish development when the enzyme AChE is also first observed, the effect of inhibition of this enzyme on NAcH content was determined . In these experiments, using the AChE inhibitor Malathion, ít was found that the treated embryos had from 6-55X less NAcH than the controls as seen in Table 1 . Since NAcH appearance parallels the first appearance of acetylcholineaterase it is interesting to find that the concentration of the nmino acid falls when this enzyme is inhibited .
However, this may represent a non
specific response to Malathion induced disruption of CNS organization, with no direct relationship between the cholinergic system and NAcH .
While the
actual role of NAcH in the CNS of poikilothermic vertebrates remains obscure, it is observed that the appearance and increase in the concentration
Vol . 6, No . 6
N-ACETYI,HISTIDINE
613
of this substance closely parallels the functional, enzymatic and anatomical development of the CNS in I+tindulue heteroclitua and Brac~ anio rerio . TABLE 1 The Effect of Malathion on NAcH Concentration in Embryos of Brachydanio rerio and P~ndu lus heteroclitua Incubation with Brachydanio rerio l control Malathion 5 mga
x
control Ma lathion 5 mgr
NAcH as X of ata~ control
Stage 22
100
22
45
Poet-hatch 120 hr .
x
"
"
"
100
"
74
Poat-hatch 24 hr .
100
Fundulus heteroclitus 2 control Malathion 5 mga
x
"
"
" "
94
l Incubation time ; 60 minutes ; temperature 26 ° C ; analysis of 15-25 anímele (means of duplicates) . 2 Incubation time ; 20 minutes ; temperature 26°C ; analysis of 3 animale per assay . Summer The appearance of the amino acid oC-N-acetyl-L-hietidine in snbryps of the killífiah, lgundulus heteroclitua a salt voter fish and the sebrafieh Brachydanio rerio , a fresh~ter fish have been investigated .
In both species,
appearance of NAcH parallels the appearance of acetylcholineetersae activity which occurs just prior to the establishment of the C11S-somatic reflea arc .
In the kíllifieh, there is a sharp rise in 1fAcH concentration just
a few days before hatching which is not observed in the rebrafish .
Ia both
species, the NAcH content continues to rise for several days after hatching . A single treatment with Ma lathion, a potent AChB inhibitor has bean found to decrease the content of IiAcH in these eabryoa froo 6-SSx of control le~ela .
614
N-ACETYLHISTIDINE
Yol . 6, No . 6
Ac kaovledgemente This research vas supported by National Science Foundation Research Grant GB-3607 and by a grant from the John A . Hertford Foundation Inc ., to the Nev York Aquarium . References 1.
M . H . BASLOW, J . Fish, Res . Bd . Canada
~, 107 (1964) .
2.
M . H . BASLOW, Zoologica
3.
A, ANASTASI, P, OORiQ±ALE and V . ERSPAMER, J . Neurochem . _11, 63 (1964) .
4.
V . ERSPAMER, M, BOSEGHIIiI and A, ANASTASI, J . Neurochem .
5.
P . CORREALE, Holl . Soc . Ital . Biol . Sper . _40, 170 (1964) .
6.
Y . 101HODA and T . IRMA, Science
7.
M . H. BA SLOW, brain Res . (in press-1966) .
8.
M. H . BASLOW and J, F, LENNEY, Can . J . Biochem . (in press-1966) .
9.
A, HANSON, Acta Chem . Scand . _20, 159 (1966) .
5`, 63 (1965) .
2, 123 (1965) .
152 , 1241 (1966) .
10 .
M. H . BASLOW, Am . Zool . ~, 230 (1965) .
11 .
K. 1C, HISAORA and H, I . BATTLE, J . Morph . 1~, 311 (1958) .
12 .
D, P . COSTELLO, M. S, DAVIDSON, A, EGGERS, M, H . FOR and C . HENLEY, Methods for Obtaining and Handling Marine Egge end Embryos p . 247 Lancaster Press, Inc ., Lancaster, Pa . (1957) .
13 .
P, B . A1H3STRO1fG and J . S, CHILD, Biol . Bull . 1~, 143 (1965) .
14 .
G . D, IaiGCIERI, S .J M . H . BASIAW and R . F . NIGRELLI, Am . Zool . 5, 740 (1965) .
15 .
3 1, 580 (1959) . C . M. i~ISS, Sewage and Induct . Waetee ~
16 .
C, H . SAWYER, J . Cell, and Comp . Physiol . ~, 71 (1944) .
17 .
S . IIESIIGI and S, YÁMAZOE, Gunma J . Mad . Sci . 1~ 3, 91 (1964) .
18 .
G . GAHELLA, Atti Acc . llaz . Lincei ~, 86 (1964) .
Pergamon Press Ltd.
N-ACETYLHISTIDINE IN DEVELOPING EMBßYOS OF TAE &ILLIFISH, FUNDULUS RETEROCLITUS AND TAE ZEBBAFISR, BRACHYDANIO $E1tI0 Morris R . Baslove and George Ruggieri, S .J .
(Received 23 December 1966) School of Medicine, University of Aawa ü, Honolulu, Rewa ü and Osborn Laboratories of Marine Sciences, New York Aquarium, Brooklyn, lfew York Introduction N-acetylhistidine (NAcR)
ie a compound occurring in the brain of bony
fish, amphibians and reptiles only (1-5) .
It has also been found in extra-
cerebral tissues of poikilothermal vertebrates and in larval amphibians (2,4,6) .
NAcH is found in high concentration in the brain, with levels up
to 2 mg/g in fish .
However, phylogenetic progression up to the reptiles
shows a gradual decrease in the percentage of this compound ae a function of the total extractable imidazolea (1,2,4) .
NAcH has been found to be
synthesized by brain enzymes from L-hietidine and acetyl coenzyme A (7) and is degraded by a highly specific acylase (S) .
While the function of NAcH
is unknown, a role in acetylation processes (3,9) and in permeability phenomena in the nervous system have been suggested (10) .
It appeared of
interest therefore to try to correlate NAcR content with the embryonic level of development of the central nervous system in fish . Methods Embryological studies were carried out on the developing embryos of killifish,
Fundulua heteroclitus , a marine fish, and on the embryos of the
zebrafiah,
Brachydanio rerio , a freshwater fish .
ta med after natural fertilization (11) .
Zebrafiah eggs ware ob-
Killifish eggs were obtained by
stripping msture females and externally fertilized as described by
N-ACETYLHISTIDINE
61 0
Costello et al . (12) .
Vol . 6, No . 6
Developmental stages for ubrafieh are according to
Hísaokt ~ Battle (11) and for killifish as described by Armstrong and Child (13) .
For NAcH analysis, e~bryoe were smashed on lihatman #1 paper,
immediately heat dried, and then saturated with 95x ethano1,0 .1 N HC1 (1 :1) solution and redried prior to chromatography (14) .
For the ubra-
fieh, the wet weight per egg we found to be 1 .0 mg, for hatched fry, 0 .3 n8 .
In this species, 15-25 embryos were used to assay each stage .
Killifish eggs weighed 4 .5 nig each and hatched fry 2 .3 mg . 3-5 killifish embryos were used . in Kg HAcH per 1000 embryos .
For analysis,
Hesulta for both species are reported
To eliminate the possibility of an eaogenoue
source of NAcA, the experimental animals were not fed after hatching and the experiment was terminated just prior to cgmplete reeorption of the yolk . 1QAcH was measured after paper chromatography in 1-butanol, acetic acid, water (4 :1 :5) and development with Pauly reagent .
The red color
formed was analysed spectrophotometrically at 510 m~ after elution with 1Z butanol (7) . To determine the effect of an anti-cholinesterase on McH concentrations, ubrafieh and killifish embryos were treated with Melathion (5 mgs x), a potent inhibitor of fish brain cholinesterase (15) .
Dis
appearance of coordi~ted movements just after a period of convulsion served as the end point for these experimnnte . BReulte and Discussion The appearance and concentration of 1fAcH in developing embryos of the sebrafish and the killifish have been inveetígated and the results are presented in Figures 1 and 2 .
Vol .
6, No .
6
N-ACE1'YLHISTIDINE
611
FIG . 1 RAcH concentration vs, developmental etage in embryos of Brachydanio rerio .
Day
5faq,
b ~b
1
20
22
2
23
3
24
4 25
5
FIG . 2 NAcH concentration vs . developmental etage in embryos of Fundulus heteroclitus .
61 2
N-ACETYLHISTIDINE
Vol . 6, No . 6
Appearance of NAcH in these embryos has been observed at a time just prior to that stage in which the first twitches occur indicating a possible relationship between this compound and CNS control over somatic movements . No NAcH is measurable in embryos of Fundulus heteroclitus at stages 25-28 when only contractions of somatic musculature have been initiated .
After
the first appearance of NAcH, there ie a rapid increase in the concentration of this substance which continues to rise for several days after hatching . It is of interest that the appearance of acetylcholineaterase (AChE) activity in embryos of Fundulua heteroclitus occurs upon development of a CNS-somatic reflex arc also (16) .
In addition, Uesugi and Yamazoe (17)
have observed the appearance of acetylcholineaterase activity in embryos of the rainbow trout Salmo gairdnerü , and Gabella (18) has measured acetylcholineaterase activity and nervous system formation in developing embryos of the guppy, Lebietea reticulatus .
In these cases, AChE activity
parallels the development of nervous tissue and the neuromuscular junction . Since NAcH is found in high concentration in the CNS and appears at a time in embryonic fish development when the enzyme AChE is also first observed, the effect of inhibition of this enzyme on NAcH content was determined . In these experiments, using the AChE inhibitor Malathion, ít was found that the treated embryos had from 6-55X less NAcH than the controls as seen in Table 1 . Since NAcH appearance parallels the first appearance of acetylcholineaterase it is interesting to find that the concentration of the nmino acid falls when this enzyme is inhibited .
However, this may represent a non
specific response to Malathion induced disruption of CNS organization, with no direct relationship between the cholinergic system and NAcH .
While the
actual role of NAcH in the CNS of poikilothermic vertebrates remains obscure, it is observed that the appearance and increase in the concentration
Vol . 6, No . 6
N-ACETYI,HISTIDINE
613
of this substance closely parallels the functional, enzymatic and anatomical development of the CNS in I+tindulue heteroclitua and Brac~ anio rerio . TABLE 1 The Effect of Malathion on NAcH Concentration in Embryos of Brachydanio rerio and P~ndu lus heteroclitua Incubation with Brachydanio rerio l control Malathion 5 mga
x
control Ma lathion 5 mgr
NAcH as X of ata~ control
Stage 22
100
22
45
Poet-hatch 120 hr .
x
"
"
"
100
"
74
Poat-hatch 24 hr .
100
Fundulus heteroclitus 2 control Malathion 5 mga
x
"
"
" "
94
l Incubation time ; 60 minutes ; temperature 26 ° C ; analysis of 15-25 anímele (means of duplicates) . 2 Incubation time ; 20 minutes ; temperature 26°C ; analysis of 3 animale per assay . Summer The appearance of the amino acid oC-N-acetyl-L-hietidine in snbryps of the killífiah, lgundulus heteroclitua a salt voter fish and the sebrafieh Brachydanio rerio , a fresh~ter fish have been investigated .
In both species,
appearance of NAcH parallels the appearance of acetylcholineetersae activity which occurs just prior to the establishment of the C11S-somatic reflea arc .
In the kíllifieh, there is a sharp rise in 1fAcH concentration just
a few days before hatching which is not observed in the rebrafish .
Ia both
species, the NAcH content continues to rise for several days after hatching . A single treatment with Ma lathion, a potent AChB inhibitor has bean found to decrease the content of IiAcH in these eabryoa froo 6-SSx of control le~ela .
614
N-ACETYLHISTIDINE
Yol . 6, No . 6
Ac kaovledgemente This research vas supported by National Science Foundation Research Grant GB-3607 and by a grant from the John A . Hertford Foundation Inc ., to the Nev York Aquarium . References 1.
M . H . BASLOW, J . Fish, Res . Bd . Canada
~, 107 (1964) .
2.
M . H . BASLOW, Zoologica
3.
A, ANASTASI, P, OORiQ±ALE and V . ERSPAMER, J . Neurochem . _11, 63 (1964) .
4.
V . ERSPAMER, M, BOSEGHIIiI and A, ANASTASI, J . Neurochem .
5.
P . CORREALE, Holl . Soc . Ital . Biol . Sper . _40, 170 (1964) .
6.
Y . 101HODA and T . IRMA, Science
7.
M . H. BA SLOW, brain Res . (in press-1966) .
8.
M. H . BASLOW and J, F, LENNEY, Can . J . Biochem . (in press-1966) .
9.
A, HANSON, Acta Chem . Scand . _20, 159 (1966) .
5`, 63 (1965) .
2, 123 (1965) .
152 , 1241 (1966) .
10 .
M. H . BASLOW, Am . Zool . ~, 230 (1965) .
11 .
K. 1C, HISAORA and H, I . BATTLE, J . Morph . 1~, 311 (1958) .
12 .
D, P . COSTELLO, M. S, DAVIDSON, A, EGGERS, M, H . FOR and C . HENLEY, Methods for Obtaining and Handling Marine Egge end Embryos p . 247 Lancaster Press, Inc ., Lancaster, Pa . (1957) .
13 .
P, B . A1H3STRO1fG and J . S, CHILD, Biol . Bull . 1~, 143 (1965) .
14 .
G . D, IaiGCIERI, S .J M . H . BASIAW and R . F . NIGRELLI, Am . Zool . 5, 740 (1965) .
15 .
3 1, 580 (1959) . C . M. i~ISS, Sewage and Induct . Waetee ~
16 .
C, H . SAWYER, J . Cell, and Comp . Physiol . ~, 71 (1944) .
17 .
S . IIESIIGI and S, YÁMAZOE, Gunma J . Mad . Sci . 1~ 3, 91 (1964) .
18 .
G . GAHELLA, Atti Acc . llaz . Lincei ~, 86 (1964) .