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An alternative route for biosynthesis of norepinephrine
Life Sciences No . 10, pp . 523-526, 1962 . Pergamon Press, Inc . Printed in the United States .
AN ALTERNATIVE ROUTE FOR BIOSYNTHESTS OF NOREPINEPHRINE Cyrus R . Creveling, Morton Levitt and Sidney Udenfriend National Heart Institute, Betheada, Maryland (Received 20 September 1962) IT is now well established that tyrosine is the dietary precursor of norepinephrine and that 3,4-dihydroxyphenylalanine (dope) and 3,4-dihydroxyphenethylamine (dopamine) are intermediate products in the biosynthesis . In an earlier publication from this laboratory
it was found that tyramine-C14 was not incorporated into adrenal catecholamines .l At that time this finding was considered sufficient
to rule out tyramine as a possible precursor of the sympathetic hormone . However, the following observations which were made subsequent to those studies made it necessary to reconsider tyramine as an intermediate in norepinephrine biosynthesis : (1) the isolation of the phenol analog of norepinephrine, norsyaephr~Tq (natopamine) from octopus salivary gland by Erspamer Z .and demonstration tYiat it was converted to norepinephrine
by oxidation in air ; (2) demonstration that norsynephrine and its N-methyl analog occur normally in mammalian urine ;3+ 4 (3) demonstration that dopamine -oxidase converts tyramine to norsynephrine, as effectively as it does dopamine to norepinephrine ; (4) demonstration that mammalian aromatic L-amigo acid decarboxylase converts tyrosine to tyramine ;5 (5) demonstration that tyramine is a normal constituent of
mammalian urine and tissues . 6 These observations make it highly likely +.hat the following (Fig . 1) metabolic conversions must also occur, to some extent,
in animal tissueff . Studies with tyramine-2-C 14 and noraynephrine-3-H3 were carried out to investigate these possibilities . Male rata (150 g) were injocted with the monoamine oxidase inhibitor, Marsilid, 150 mg/kg i .p . at 48 hr intervals . Twenty-four hours after the first dose of Marsilid radioactive amine was administered . 523
52 4
BIOSYNTHESIS OF NOREPINEPHRINE
No . 10
HH HH OHH -C-C-0OOH -C-C-IQH 2 -C-C-NH 2 H~ H$ ~~nHH -~ oH~ -~. ~2 1/2 02 -c02 tyrosine
~
norsynephrine
tyramine
1/2 1/2 0 2 02
v
HOHo-
$H
-~-c-~2 HH
____~, 1/2 02
dopamine
OHH HO- ~ -c-c-1~2 HO- ~ H x norepinephrine
FIG. 1 tyramine-2-C 14 was given in divided doses of 0 .63 mg/day, for 5 days, for a total of 44 .8 x 106 c .p .m. Norsynephrine-7-H3 was given in doses of 0 .5 mg/day, for 5 days, for a total of 48 .3 x 106 c .p.m. Urines were collected and pooled for each compound and the animals were killed at the end of the fifth day ; the adrenal glands were removed and homogenized in 2 ml of 10 per cent trichlaroacetic acid . To each urine sample 10 moles of carrier nor-
epinephrine and normetanephrine were added and the urines treated with ~-glucuronidase . 7 Norepinephrine and epinephrine were isolated on an alumines column, further purified on a Dowex-50-H+ column according to Bertler et a1 . 8 , and then subjected to chromatography on Whatman #3 paper using butanol-acetic acidNorepinephrine fractions, obtained from urines experiment, had specific activities of 648 and at the last txo stages of purification . In one
water (120 :30x50) . of the tyramine 575 c .p .m .%mole
experiment with norsynephrine comparable stages gave values of 1095 and 1085 c .p .m./ftmole . Normetanephrine, in the effluent of the alumines columns, was chromatographed on a Dowex-50 column? and then purified by chromatography on Whatman ~l paper using the butanolacetic acid-water system described above . The normetanephrine area was eluted and rechromatographed on paper using isopropanol-1 N
ammonia (822) . A further purification by chromatography with butanol-acetic acid-water was carried out . Normetanephrine in the last three chromatographic steps of the tyramine experiment had specific activities of 8280, 8350 and 8250 c .p .m./mole . Radioactivity iras assayed with a scintillation spectrometer on aliquot portions dis-
No . 10
HIOSYNTHESIS OF NOREPINEPHRINE
525
solved in Brayts solution .9 Norepinephrine and epinephrine were assayed fluorometricallyl 0 and normetanephrine by the periodate procedure,ll
The data summarized in Table 1 are from one experiment . Comparable findings have been made in other studies . As can be seen
neither tyramine nor norsynephrine were incorporated to any measur able extent into adrenal epinephrine . The results r~rith tyramine are in agreement with earlier studies . l Tn this instance the use of a monoamine oxidase inhibitor did not improve incorporation. TABLE 1 Conversion of Tyramine and Norsynephrine to Norepinephrine and Normetanephrine _ T ____ Compound isolated
Total Radioactivity
Fraction of urinary radioactivity
c .p .m . (Tyramine-2-C 14 ; 44 .8 x 106 c .p .m .)* O Epinephrine (adrenal) 5780 Norepinephrine (urine)
Metanephrine (urine)
83,009
Total radioactivity (urine) 30 x 106 (Norsynephrine-7-H3 ; 48 .3 x 106 c .p .m .) ** Epinephrine (adrenal) O 10,950 Norepinephrine (urine) Normetanephrine (urine) Total radioactivity (urine)
367,000
per coat
0 .02 0 .28
0 .04 1 .5
25 x 106
*California Corporation for Biochemical Research . **Prepared by Y . Kanaoka, Laboratory of Chemistry, National Institute of Arthritis and Metabolic Diseases, Bethesda~ Md . Although adrenal catecholamines mere not labelled urinary norepinephrine and normetanephrine were highly labelled . Although the fractions of tyramine and noraynephrine which appeared in the norepinephrine and its O-methylated product sere small, the amounts were significant . Furthermore, it is known that the bulk of aorepinephrine is excreted in the urine as vanillyl mandelic acid and other acidic metabolites . From the studies of Goodall e,~t~a1 . 12 one would expect the acid metabolites to contain several times as
526
BIOSYNTHESIS OF' NOREPINEPHRINE
No . 10
much activity as is found in norepinephrine and normetanephrine combined . If so, then the present experiments suggest that several per cent of the tyramine in the tissues may be metabolized via norepinephrine and that an appreciable proportion of this may go through norsynephrine . Of course, it may also be that some tyramine is converted to dopamine, as shown in Fig . l . In any event tyramine and norsynephrine, both of which are normally found in tissues, can be converted to norepinephrine in vivo .Just how important this alternate route (or routes) may be is not readily apparent . Since tyramine is present in tissues, it
may prove to contribute significantly to norepinephrine formation under normal conditions . Alternatively it may merely represent a minor pathway with little physiological significance, as would appear from the results on adrenal catecholamines . It may, however, be highly significant in evaluating the pharmacological actions of t~"ramine . Although some aspects of the pharmacology of tyramine may be mediated through norepinephrine release from depots, l2 others mray actually be due to direct conversion to norepinephrine through the pathways discussed above . This would be true particularly at the higher doses of tyramine . Studies are currently under way to evaluate the significance of this pathway with respect to norepinephrine biosynthesis in intact animals and to compare it with the established dopa route .
2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
References S . UDENFRIEND and J .B . WYNCAARDEN, Biochim . Biophys . Acta ., s , 48, (1956) . Y, ERSPAMER, Nature, Lond . ~, 375 (1952) " J .J . PISANO, J .A . OATES, A . ARMEN, A . SJOERDSMA and S . U'ilENY , 898 (1961) . FRIEND, J . Biol . Chem . Y. KAKIMOTO and M.D . ARMS NC, J . Biol . Chem . ~2 , 208 (19G2) . W. LOVENBERC, H . WEISSBACH and S . UDENFRIEND, J . Biol . Cïiem. 8g (1962) . S. ECTOh, K . MELMAN, W . LOVENBERC and A . SJUERDSb:A, fharesaçolo Rt _4 _. ]48 (1962) . I .J . OPI , -J . AXELROD and E . CORDON, J . Biol . Chem . 2~z, , 2109 (1961) . A . BERTLER, A . CARLSSON and E . ROSENCREN, Acta Physiol . S .:and . 44, 273 (1958) . C.~RAY, Anal, Biochem . 1, 279 (1960) . J .R . CROUT, C .R . CREVELI~C and S . UDENFRIEND, J . Pharm . E:cp . Ther . ~, 269 (1961) . J .J . fISANO, Clin. Chim . Acta , 406 (1960) . bicC . COODALL, N. KIRSHNER and ~ . ROSEN, J . Clin . Invest . ~, 707 (1959) . J .H . BURN, Brit . Med . J . 1(123 (1961) .
AN ALTERNATIVE ROUTE FOR BIOSYNTHESTS OF NOREPINEPHRINE Cyrus R . Creveling, Morton Levitt and Sidney Udenfriend National Heart Institute, Betheada, Maryland (Received 20 September 1962) IT is now well established that tyrosine is the dietary precursor of norepinephrine and that 3,4-dihydroxyphenylalanine (dope) and 3,4-dihydroxyphenethylamine (dopamine) are intermediate products in the biosynthesis . In an earlier publication from this laboratory
it was found that tyramine-C14 was not incorporated into adrenal catecholamines .l At that time this finding was considered sufficient
to rule out tyramine as a possible precursor of the sympathetic hormone . However, the following observations which were made subsequent to those studies made it necessary to reconsider tyramine as an intermediate in norepinephrine biosynthesis : (1) the isolation of the phenol analog of norepinephrine, norsyaephr~Tq (natopamine) from octopus salivary gland by Erspamer Z .and demonstration tYiat it was converted to norepinephrine
by oxidation in air ; (2) demonstration that norsynephrine and its N-methyl analog occur normally in mammalian urine ;3+ 4 (3) demonstration that dopamine -oxidase converts tyramine to norsynephrine, as effectively as it does dopamine to norepinephrine ; (4) demonstration that mammalian aromatic L-amigo acid decarboxylase converts tyrosine to tyramine ;5 (5) demonstration that tyramine is a normal constituent of
mammalian urine and tissues . 6 These observations make it highly likely +.hat the following (Fig . 1) metabolic conversions must also occur, to some extent,
in animal tissueff . Studies with tyramine-2-C 14 and noraynephrine-3-H3 were carried out to investigate these possibilities . Male rata (150 g) were injocted with the monoamine oxidase inhibitor, Marsilid, 150 mg/kg i .p . at 48 hr intervals . Twenty-four hours after the first dose of Marsilid radioactive amine was administered . 523
52 4
BIOSYNTHESIS OF NOREPINEPHRINE
No . 10
HH HH OHH -C-C-0OOH -C-C-IQH 2 -C-C-NH 2 H~ H$ ~~nHH -~ oH~ -~. ~2 1/2 02 -c02 tyrosine
~
norsynephrine
tyramine
1/2 1/2 0 2 02
v
HOHo-
$H
-~-c-~2 HH
____~, 1/2 02
dopamine
OHH HO- ~ -c-c-1~2 HO- ~ H x norepinephrine
FIG. 1 tyramine-2-C 14 was given in divided doses of 0 .63 mg/day, for 5 days, for a total of 44 .8 x 106 c .p .m. Norsynephrine-7-H3 was given in doses of 0 .5 mg/day, for 5 days, for a total of 48 .3 x 106 c .p.m. Urines were collected and pooled for each compound and the animals were killed at the end of the fifth day ; the adrenal glands were removed and homogenized in 2 ml of 10 per cent trichlaroacetic acid . To each urine sample 10 moles of carrier nor-
epinephrine and normetanephrine were added and the urines treated with ~-glucuronidase . 7 Norepinephrine and epinephrine were isolated on an alumines column, further purified on a Dowex-50-H+ column according to Bertler et a1 . 8 , and then subjected to chromatography on Whatman #3 paper using butanol-acetic acidNorepinephrine fractions, obtained from urines experiment, had specific activities of 648 and at the last txo stages of purification . In one
water (120 :30x50) . of the tyramine 575 c .p .m .%mole
experiment with norsynephrine comparable stages gave values of 1095 and 1085 c .p .m./ftmole . Normetanephrine, in the effluent of the alumines columns, was chromatographed on a Dowex-50 column? and then purified by chromatography on Whatman ~l paper using the butanolacetic acid-water system described above . The normetanephrine area was eluted and rechromatographed on paper using isopropanol-1 N
ammonia (822) . A further purification by chromatography with butanol-acetic acid-water was carried out . Normetanephrine in the last three chromatographic steps of the tyramine experiment had specific activities of 8280, 8350 and 8250 c .p .m./mole . Radioactivity iras assayed with a scintillation spectrometer on aliquot portions dis-
No . 10
HIOSYNTHESIS OF NOREPINEPHRINE
525
solved in Brayts solution .9 Norepinephrine and epinephrine were assayed fluorometricallyl 0 and normetanephrine by the periodate procedure,ll
The data summarized in Table 1 are from one experiment . Comparable findings have been made in other studies . As can be seen
neither tyramine nor norsynephrine were incorporated to any measur able extent into adrenal epinephrine . The results r~rith tyramine are in agreement with earlier studies . l Tn this instance the use of a monoamine oxidase inhibitor did not improve incorporation. TABLE 1 Conversion of Tyramine and Norsynephrine to Norepinephrine and Normetanephrine _ T ____ Compound isolated
Total Radioactivity
Fraction of urinary radioactivity
c .p .m . (Tyramine-2-C 14 ; 44 .8 x 106 c .p .m .)* O Epinephrine (adrenal) 5780 Norepinephrine (urine)
Metanephrine (urine)
83,009
Total radioactivity (urine) 30 x 106 (Norsynephrine-7-H3 ; 48 .3 x 106 c .p .m .) ** Epinephrine (adrenal) O 10,950 Norepinephrine (urine) Normetanephrine (urine) Total radioactivity (urine)
367,000
per coat
0 .02 0 .28
0 .04 1 .5
25 x 106
*California Corporation for Biochemical Research . **Prepared by Y . Kanaoka, Laboratory of Chemistry, National Institute of Arthritis and Metabolic Diseases, Bethesda~ Md . Although adrenal catecholamines mere not labelled urinary norepinephrine and normetanephrine were highly labelled . Although the fractions of tyramine and noraynephrine which appeared in the norepinephrine and its O-methylated product sere small, the amounts were significant . Furthermore, it is known that the bulk of aorepinephrine is excreted in the urine as vanillyl mandelic acid and other acidic metabolites . From the studies of Goodall e,~t~a1 . 12 one would expect the acid metabolites to contain several times as
526
BIOSYNTHESIS OF' NOREPINEPHRINE
No . 10
much activity as is found in norepinephrine and normetanephrine combined . If so, then the present experiments suggest that several per cent of the tyramine in the tissues may be metabolized via norepinephrine and that an appreciable proportion of this may go through norsynephrine . Of course, it may also be that some tyramine is converted to dopamine, as shown in Fig . l . In any event tyramine and norsynephrine, both of which are normally found in tissues, can be converted to norepinephrine in vivo .Just how important this alternate route (or routes) may be is not readily apparent . Since tyramine is present in tissues, it
may prove to contribute significantly to norepinephrine formation under normal conditions . Alternatively it may merely represent a minor pathway with little physiological significance, as would appear from the results on adrenal catecholamines . It may, however, be highly significant in evaluating the pharmacological actions of t~"ramine . Although some aspects of the pharmacology of tyramine may be mediated through norepinephrine release from depots, l2 others mray actually be due to direct conversion to norepinephrine through the pathways discussed above . This would be true particularly at the higher doses of tyramine . Studies are currently under way to evaluate the significance of this pathway with respect to norepinephrine biosynthesis in intact animals and to compare it with the established dopa route .
2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
References S . UDENFRIEND and J .B . WYNCAARDEN, Biochim . Biophys . Acta ., s , 48, (1956) . Y, ERSPAMER, Nature, Lond . ~, 375 (1952) " J .J . PISANO, J .A . OATES, A . ARMEN, A . SJOERDSMA and S . U'ilENY , 898 (1961) . FRIEND, J . Biol . Chem . Y. KAKIMOTO and M.D . ARMS NC, J . Biol . Chem . ~2 , 208 (19G2) . W. LOVENBERC, H . WEISSBACH and S . UDENFRIEND, J . Biol . Cïiem. 8g (1962) . S. ECTOh, K . MELMAN, W . LOVENBERC and A . SJUERDSb:A, fharesaçolo Rt _4 _. ]48 (1962) . I .J . OPI , -J . AXELROD and E . CORDON, J . Biol . Chem . 2~z, , 2109 (1961) . A . BERTLER, A . CARLSSON and E . ROSENCREN, Acta Physiol . S .:and . 44, 273 (1958) . C.~RAY, Anal, Biochem . 1, 279 (1960) . J .R . CROUT, C .R . CREVELI~C and S . UDENFRIEND, J . Pharm . E:cp . Ther . ~, 269 (1961) . J .J . fISANO, Clin. Chim . Acta , 406 (1960) . bicC . COODALL, N. KIRSHNER and ~ . ROSEN, J . Clin . Invest . ~, 707 (1959) . J .H . BURN, Brit . Med . J . 1(123 (1961) .