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On the mechanism of norepinephrine depletion by aramine
Life Sciences Vol . 3, pp . 695-702, 1984. Pergamon Press, Inc. Printed in the United States.
ON THE MECHANISM OF NOREPINEPH1tIAiE DEPLETION BY ARaMrrrn Sidney Udenfriend and Perola 2altzman-Nirenberg Laboratory of Clinical Biochemistry, National Heart Institute, Bethesda, Md .
(Received 15 June 1964) It is now generally agreed that cX-methyl-3, 4-dihydroxy-L-phenylalanine (Aldomet) and
cx-methyl-3-hydroxy-L-phenylalanine (CZ-methyl-mete-tyrosine, g -
MMT) lower tissue norepinephrine levels only when they are decarboxylated to their corresponding amines and subsequently oxidized to the corresponding ß hydroxylated amines (1,2,3,4) .
The product derived from (X-MMT, aramine, has
been shown to be a potent norepinephrine releasing agent when administered by itself and because it does not interfere with norepinephrine assay it has been used to study the mechanism of the release .
In a previous paper from
this laboratory (4) it was pointed out that following administration of CY MMT or aramine to guinea pigs, aramine appeared in the heart in relatively large amounts but that most of it disappeared fairly rapidly .
Twenty-four
to 96 hrs subsequent to administration norepinephrine levels were markedly depleted yet little aramine could be detected .
By contrast Carlsson et al .
(2) reported that they could find large amounts of aramine in tissues for several days subsequent to administration of
C~MMr or aramine and suggested
that the presence of aramine in the norepinephrine depots prevented repletion with norepinephrine .
They also suggested a stoichiometric relationship
existed between norepinephrine depletion and the aramine level .
The present
studies were undertaken to re-investigate the stoichiometry of the norepinephrine depletion due to aramine .
When these studies were underway an
abstract by Shore and Alpers appeared (5) concerning comparable studies .
898
MECHANISM OF NOREPINEPHRINE DEPLETION
Vol . 3, No. 7
Methods and Materials Aremine (metsraminol) ~ad was aäministered to
was
loudly
s upp].ied by Merck Sharp and Dohme
~inP a pigs by the intraperitoneal route (3 mg/kg) .
Extraction of amines from tissues .
Three to four hearts were transferred
to a stainless steel homogenizer containing 15 ml of O.1E M perch].oric acid and homogenization was carried out for 2 min .
The homogenate wsa transferred
to s tube and centrifuged far 15 min . at 8000 r .p .m.
The supernatant fluid
was decanted into a beaker and the precipitate was returned to the homogenizer and re-extracted with 10 ml of perchloric acid.
After centrifugation the
second perchlaric acid extract was pooled with the first and the mixture was divided into two parts] three fourths for aramine assay and one-fourth for norepinephrine assay . Aramine assay .
The perclal.oric acid extract was neutralized with 10~
potassium carbonate end then centrifuged.
A~`ter centrülagation the super-
notant solution was appLted to a Dowexr50 Na+ column (200-1100 mesh 0.6 x iF .S
cm):.
The columns were washed with 10 ml of water and eluted with 6 ml each
oß 0 .$ N~ 1 E and 2 ft~ in that order .
The final 6 ml elvate~ containin9 the
aremiae, was evaporated to àryness under vacuum, redissolved in 1 ml of water* and 0 .9 ml was taken Par calorimetric assay using (~ibb~s reagent, as described previoue],y .
IL~.^+ P was recovered to the extent of 75~ and values were
corrected for this recovery . Norepin~~ine sassy .
The ~10~ extract was neutralized with S2C03
to PH ~ in the presence of 0 .04
bromphenol blue .
After centrifugation
the residue was washed with about 2 ml of water and the washes were combined with the extract .
These solutions were then edjusted to P$ 8 .1+ in the
presence of alumins and assayed by a modification of the tri2~ydro~indole method (6) . Care must be taken that the solution is neutral at this point .
Vol . 3, No. 7
MECHANISM OF NOREPIIIEPHRINE DEPLETION
697
Aramine was administered rather than Oc-I~A~ in order to eliminate the problem of metabolic co~ersion .
The guinea pig heart was chosen because
aramine penetrates readi.]~y and because control norep in ~ ne levels in this organ are high (1 .8 ~,g~g or 11 .1 II}lmOlee/g) .; making the balance studies more meailingßul .
Measurements oß aramine and norepinephrine were made on aliquots
oß the ssme pooled tissue extract, to Bomber reduce chauces oß error . Results and Discussion As can be seen in Fig . 1 the aramine taken up by guinea pig heart is released ßirst rapidly (halß-11ße^, 6 hrs) and then quite slow],y (halß-11ße
~+5
hrs) .
Montancri et a7. . (7) have reported ca~mpaxable release oß nor-
epiaephrine~3 .
However the halß-11ße ßor t11e long lived component oß nor-
epinephrine was much shorter (about aramine .
15
hrs) than the value we observed ßor
Shore and Alpers (5) have also reported a long lived component ßor
aremine in a number oß animal tissues .
It is obvious that several days aßter
administration oß aramine signißicant quantities oß the aramine remain in the tissues .
(75
Twenty-Boor to ~+8 hrs ßollowing administration oß G~-I~ß~II'
mg~kg) to guinea pigs 2-4 m .Tles oß aramine were also bound per g oß heart . Failure to detect these p+~rn~T+ tB Oß aramine resulting ßrom the amino acid precursor, in previous studies in this laboratory (~+)~ were due to the relatively high tissue blanks obtained in the trichloroacetic acid extracts which were used ßor aramine assgy in those studies* . The persistence oß aramine and the prolonged depletion oß norepinephrine Suggest that the two are interrelated .
In ßact Carlsson and Lindquist (2) .
have suggested that they are gveutitatively interrelated and Shore and Alpers (5) have presented evidence that during the prolonged depletion oß norepinephrine aremine can be Bound in the tissues in amounts exactly equivalent to the norepin ephrinP deßicit .
In our studies it has not been
The act that trichloroa~cetic acid extracts are not suitable ßor assay oß smell amounts oß aremine by the Gibbis reagent was suggested by A . Carlsson (personal camDnmication) .
698
MECHADTISM OF NOREPINEPHRII~iE DEPLETION
Vol. 3, No. 7
Ty~ ~ 6 Hou~s 7 6
a
w Q
5 4
0 0 U
W 2
2
i
Q Q
I
20
I
40
I
60 HOURS
I
ß0
I
f00
FIG . 1
The release of aramine following its uptake by guinea pig heart. The data for plotting the curve represents the averages of all the values shown in Table 1 . The half-life values estimated for the slopes .
(T
1/2) were
Yol . 3, No . 7
MECHANLSM OF NOREPINEPHRINE DEPLETION
899
possible to demonstrate a continued equivalence between the aramine level and the norepinepàrine deficit .
As shown in Table 1 the only approach to
equivalence was observed early in the experiment .
Howevers at all other
TABLE 1 Norepinephrine and Aramine in Guinea Fig Heart Following Aramine Administration Time nr
Number of Animal s
o 1"5
3
5
3 3 4
21+
21F 21+
1+
21F 21+ 31 31 31 31 31 31 31
1F
~ 1} >+
1+
4 >+
1+
>+ 3 3 1+ >+
88 88 88 s8 88
1+ 1+
>+
Norepinephrine
Norepinephrine Deßicit m~.moles g
Ll.l ± 0.9 6 .0
Aramin e
0 5.1
0 9 "7
~+ .0 .. 2 .8 1 .8 2.>+ 2"3 2..7 2.1+ 2.3 2.9 2.8 2.8 3 .0 2.5 2.3 6 .1 7.7 6"3
7.1 _ 8.3 9"3 8 .7 8 .8 8 .4 8 .7 8 .8 8 .2 8 .8 8 .8 8 .1 8 .6 8 .8 5 .0 3.1+ >+ .8
6 .5
-
-
-
-
31+ .0 1} .7 1+ .2 5"5 3.1 3 .0 2.9 3.2 3.0 2.3 2.1 1 .8 l .o 0 "7
Except for the zero time values each line represents a single experiment comprising a pooling of the number of animals indicated in column 2. In experiments where values for both aramine and nor epinephrine are shown they were carried out on aliquots oß the same extract oß pooled tissue . Twenty animals representing 5 experiments were carried out sianaltaaeous]~y with these experiments as zero time controls . The values obtained were in agreement with hundreds oß such controls run in this laboratory by the same investigators . The range (± S .D .~ shown in line 1+ represents the results obtained in this laboratory on hundreds oß animals over a period oß years . points the amounts of aramine did not account for the deficit in norep inephrine . At 31 and
48
hrs the aramine levels were on],y
35
to ~ of the norepinephrine
deficit and in ßact tale deficit was greater thaw the total oß norepinephrine
700
MECHANISM OF NOREPINEPHRINE DEPLETION
and examine combined .
Vol. S, No. 7
These data are not indicative oß a continuous
equivalence between the depleting agent and the norepinephrine dsßicit . It should be pointed out that a balance study oß this kind is meaningful only when mar}y factors are taken into consideration . important the amounts of examine injected
(3
First and most
mg~kg~ i .p .~ were the mini~i
856.
amounts required to produce norepinephrine depletion oß about 80 to With
1.5
mg~ kg depletion was less .
With amounts of aramine larger than
3 mg~kg there was little additional release .
Increasing the dose of
ß~~i nP introduced more examine into the tissue with little eßßect on norepinephrine . The second factor concerns the choice of animal and tissus .
We
deliberately used the guinea pig heart because it is richer in norepinephrine than most tissues containing releasable norep inephrine . kg~ is administered the norepin ~ n e deficit is about
When examine (3 mg~
9
msmnl es~g .
Even
with such a large deficit the remainder can still be measured with precision . The large amounts oß examine that would be required to achieve equivalence can also be measured with precision . Thirdly
the stoichiometry oß examine action can be studied most sim*,~
with examine rather than its precursor
a"~.
However, with aramine~ studies
must be limited to peripheral tissues since the amine does not penetrate into the central nervous system to any greater extent . The results of this study indicate that examine is taken up by sympathetic tissue .
Initially there is a sufßicient amount of examine
present to account ßor a male per mole displacement .
Whether the initial
release involves a mole for mole displacement cannot be verißied as yet . However
a large proportion of the examine disappears rapidly coinciding
with the most rapid phase oß norepinephrine release .
By 2~+ hrs the residual
o+ninP a no longer add up to the original 11 .1 mFtmales and ßor two days or longer the examine level is about equal to the residual norepinephrine ; the
Vol . 3, No. 7
MECHANISM OF NOREPINEPHRINE DEPLETION
70 1
sum of the two accounting for only 50~ of the control norepinephrine level . The question as to whether or not the long lasting aramine depletion is due to an exact mole for mole replacement is of more than academic interest .
If this were the case, then one must conclude that sympathetic
tissues contain a fixed number of storage sites for norepinephrine which cannot be exceeded .
However, norepinephrine levels can be increased after
norepinephrine infusion (8) and after administration of monoamine oxidase inhibitors,
Furthermore, one would have to conclude that after several
days the same norepinephrine sites become available as were initially filled by aramine and that in the interim there was no destruction and reaynthesis of binding sites . There is no question that administered aramine markedly delays norepinephrine repletion .
The experimentally determined half-life of nor-
epinephrine in normal guinea pig heart is about 12-14 hra (7) .
Yet after
aramine depletion it takes many days far norepinephrine to return to control values .
Since part of the aramine which reaches the heart disappears with a
half-life of 45 hra, it is reasonable to consider that this residual aramine in some way inhibits norepinephrine repletion .
Although it would be convenient
to find an exact equivalence between residual aramine and the norepinephrine deficit the data for guinea pig heart do not support this .
The observed
equivalence in some tissues of rats and rabbits reported by Shore and Alpera (5) suggest that differences may exist among animal species .
It ie even more
probable that the differences reflect differences in experimental design .
The
conclusions to be drawn from experiments of this type are of importance with respect to the function of the sympathetic nervous system and we would suggest that final judgement be reserved until more detailed experiments become available .
702
MECHArTIBM OF NOREPIIIEPI~II~IE DEPLETION
Vol . 8, No. 7
References 1 . W . LOVENBERG, H, WEISSBACH, and S . UDENFRLEND, Biochem . Biophys . Res . Camonun . 3, 225 (1960) . 2 . A . CARLSSON AND M. LII~QVIST, Acta Physiol . Scand . 54, 87 (1962) . 3 . G .L . CESSA, E, COSTA, R. KUNTZMAN and B .B . BRODLE, Life Sciences 1, 353 (1962) . 4 . S . UDENFRIEI~ and P . ZALTZMAN-NIRENBERG, J . Pharm . Exp . Therap . 138, 194 (1962), 5 . P .A . SHORE and H, ALPERS, Fed . Proc . i, 350 (1964) . 6 . J .R, CROUT, C .R . CREVELING and S . UDENFRIEND, J . Pharm . Exp . Therap . 1~, 269 (1961) . 7, R, j+~NTANART , E, 232 {1963) .
M,A . BEAVEN and B .B, BRODIB, Life Sciences _2, _COSTA,
8 . S . SPECTOR - to be published,
ON THE MECHANISM OF NOREPINEPH1tIAiE DEPLETION BY ARaMrrrn Sidney Udenfriend and Perola 2altzman-Nirenberg Laboratory of Clinical Biochemistry, National Heart Institute, Bethesda, Md .
(Received 15 June 1964) It is now generally agreed that cX-methyl-3, 4-dihydroxy-L-phenylalanine (Aldomet) and
cx-methyl-3-hydroxy-L-phenylalanine (CZ-methyl-mete-tyrosine, g -
MMT) lower tissue norepinephrine levels only when they are decarboxylated to their corresponding amines and subsequently oxidized to the corresponding ß hydroxylated amines (1,2,3,4) .
The product derived from (X-MMT, aramine, has
been shown to be a potent norepinephrine releasing agent when administered by itself and because it does not interfere with norepinephrine assay it has been used to study the mechanism of the release .
In a previous paper from
this laboratory (4) it was pointed out that following administration of CY MMT or aramine to guinea pigs, aramine appeared in the heart in relatively large amounts but that most of it disappeared fairly rapidly .
Twenty-four
to 96 hrs subsequent to administration norepinephrine levels were markedly depleted yet little aramine could be detected .
By contrast Carlsson et al .
(2) reported that they could find large amounts of aramine in tissues for several days subsequent to administration of
C~MMr or aramine and suggested
that the presence of aramine in the norepinephrine depots prevented repletion with norepinephrine .
They also suggested a stoichiometric relationship
existed between norepinephrine depletion and the aramine level .
The present
studies were undertaken to re-investigate the stoichiometry of the norepinephrine depletion due to aramine .
When these studies were underway an
abstract by Shore and Alpers appeared (5) concerning comparable studies .
898
MECHANISM OF NOREPINEPHRINE DEPLETION
Vol . 3, No. 7
Methods and Materials Aremine (metsraminol) ~ad was aäministered to
was
loudly
s upp].ied by Merck Sharp and Dohme
~inP a pigs by the intraperitoneal route (3 mg/kg) .
Extraction of amines from tissues .
Three to four hearts were transferred
to a stainless steel homogenizer containing 15 ml of O.1E M perch].oric acid and homogenization was carried out for 2 min .
The homogenate wsa transferred
to s tube and centrifuged far 15 min . at 8000 r .p .m.
The supernatant fluid
was decanted into a beaker and the precipitate was returned to the homogenizer and re-extracted with 10 ml of perchloric acid.
After centrifugation the
second perchlaric acid extract was pooled with the first and the mixture was divided into two parts] three fourths for aramine assay and one-fourth for norepinephrine assay . Aramine assay .
The perclal.oric acid extract was neutralized with 10~
potassium carbonate end then centrifuged.
A~`ter centrülagation the super-
notant solution was appLted to a Dowexr50 Na+ column (200-1100 mesh 0.6 x iF .S
cm):.
The columns were washed with 10 ml of water and eluted with 6 ml each
oß 0 .$ N~ 1 E and 2 ft~ in that order .
The final 6 ml elvate~ containin9 the
aremiae, was evaporated to àryness under vacuum, redissolved in 1 ml of water* and 0 .9 ml was taken Par calorimetric assay using (~ibb~s reagent, as described previoue],y .
IL~.^+ P was recovered to the extent of 75~ and values were
corrected for this recovery . Norepin~~ine sassy .
The ~10~ extract was neutralized with S2C03
to PH ~ in the presence of 0 .04
bromphenol blue .
After centrifugation
the residue was washed with about 2 ml of water and the washes were combined with the extract .
These solutions were then edjusted to P$ 8 .1+ in the
presence of alumins and assayed by a modification of the tri2~ydro~indole method (6) . Care must be taken that the solution is neutral at this point .
Vol . 3, No. 7
MECHANISM OF NOREPIIIEPHRINE DEPLETION
697
Aramine was administered rather than Oc-I~A~ in order to eliminate the problem of metabolic co~ersion .
The guinea pig heart was chosen because
aramine penetrates readi.]~y and because control norep in ~ ne levels in this organ are high (1 .8 ~,g~g or 11 .1 II}lmOlee/g) .; making the balance studies more meailingßul .
Measurements oß aramine and norepinephrine were made on aliquots
oß the ssme pooled tissue extract, to Bomber reduce chauces oß error . Results and Discussion As can be seen in Fig . 1 the aramine taken up by guinea pig heart is released ßirst rapidly (halß-11ße^, 6 hrs) and then quite slow],y (halß-11ße
~+5
hrs) .
Montancri et a7. . (7) have reported ca~mpaxable release oß nor-
epiaephrine~3 .
However the halß-11ße ßor t11e long lived component oß nor-
epinephrine was much shorter (about aramine .
15
hrs) than the value we observed ßor
Shore and Alpers (5) have also reported a long lived component ßor
aremine in a number oß animal tissues .
It is obvious that several days aßter
administration oß aramine signißicant quantities oß the aramine remain in the tissues .
(75
Twenty-Boor to ~+8 hrs ßollowing administration oß G~-I~ß~II'
mg~kg) to guinea pigs 2-4 m .Tles oß aramine were also bound per g oß heart . Failure to detect these p+~rn~T+ tB Oß aramine resulting ßrom the amino acid precursor, in previous studies in this laboratory (~+)~ were due to the relatively high tissue blanks obtained in the trichloroacetic acid extracts which were used ßor aramine assgy in those studies* . The persistence oß aramine and the prolonged depletion oß norepinephrine Suggest that the two are interrelated .
In ßact Carlsson and Lindquist (2) .
have suggested that they are gveutitatively interrelated and Shore and Alpers (5) have presented evidence that during the prolonged depletion oß norepinephrine aremine can be Bound in the tissues in amounts exactly equivalent to the norepin ephrinP deßicit .
In our studies it has not been
The act that trichloroa~cetic acid extracts are not suitable ßor assay oß smell amounts oß aremine by the Gibbis reagent was suggested by A . Carlsson (personal camDnmication) .
698
MECHADTISM OF NOREPINEPHRII~iE DEPLETION
Vol. 3, No. 7
Ty~ ~ 6 Hou~s 7 6
a
w Q
5 4
0 0 U
W 2
2
i
Q Q
I
20
I
40
I
60 HOURS
I
ß0
I
f00
FIG . 1
The release of aramine following its uptake by guinea pig heart. The data for plotting the curve represents the averages of all the values shown in Table 1 . The half-life values estimated for the slopes .
(T
1/2) were
Yol . 3, No . 7
MECHANLSM OF NOREPINEPHRINE DEPLETION
899
possible to demonstrate a continued equivalence between the aramine level and the norepinepàrine deficit .
As shown in Table 1 the only approach to
equivalence was observed early in the experiment .
Howevers at all other
TABLE 1 Norepinephrine and Aramine in Guinea Fig Heart Following Aramine Administration Time nr
Number of Animal s
o 1"5
3
5
3 3 4
21+
21F 21+
1+
21F 21+ 31 31 31 31 31 31 31
1F
~ 1} >+
1+
4 >+
1+
>+ 3 3 1+ >+
88 88 88 s8 88
1+ 1+
>+
Norepinephrine
Norepinephrine Deßicit m~.moles g
Ll.l ± 0.9 6 .0
Aramin e
0 5.1
0 9 "7
~+ .0 .. 2 .8 1 .8 2.>+ 2"3 2..7 2.1+ 2.3 2.9 2.8 2.8 3 .0 2.5 2.3 6 .1 7.7 6"3
7.1 _ 8.3 9"3 8 .7 8 .8 8 .4 8 .7 8 .8 8 .2 8 .8 8 .8 8 .1 8 .6 8 .8 5 .0 3.1+ >+ .8
6 .5
-
-
-
-
31+ .0 1} .7 1+ .2 5"5 3.1 3 .0 2.9 3.2 3.0 2.3 2.1 1 .8 l .o 0 "7
Except for the zero time values each line represents a single experiment comprising a pooling of the number of animals indicated in column 2. In experiments where values for both aramine and nor epinephrine are shown they were carried out on aliquots oß the same extract oß pooled tissue . Twenty animals representing 5 experiments were carried out sianaltaaeous]~y with these experiments as zero time controls . The values obtained were in agreement with hundreds oß such controls run in this laboratory by the same investigators . The range (± S .D .~ shown in line 1+ represents the results obtained in this laboratory on hundreds oß animals over a period oß years . points the amounts of aramine did not account for the deficit in norep inephrine . At 31 and
48
hrs the aramine levels were on],y
35
to ~ of the norepinephrine
deficit and in ßact tale deficit was greater thaw the total oß norepinephrine
700
MECHANISM OF NOREPINEPHRINE DEPLETION
and examine combined .
Vol. S, No. 7
These data are not indicative oß a continuous
equivalence between the depleting agent and the norepinephrine dsßicit . It should be pointed out that a balance study oß this kind is meaningful only when mar}y factors are taken into consideration . important the amounts of examine injected
(3
First and most
mg~kg~ i .p .~ were the mini~i
856.
amounts required to produce norepinephrine depletion oß about 80 to With
1.5
mg~ kg depletion was less .
With amounts of aramine larger than
3 mg~kg there was little additional release .
Increasing the dose of
ß~~i nP introduced more examine into the tissue with little eßßect on norepinephrine . The second factor concerns the choice of animal and tissus .
We
deliberately used the guinea pig heart because it is richer in norepinephrine than most tissues containing releasable norep inephrine . kg~ is administered the norepin ~ n e deficit is about
When examine (3 mg~
9
msmnl es~g .
Even
with such a large deficit the remainder can still be measured with precision . The large amounts oß examine that would be required to achieve equivalence can also be measured with precision . Thirdly
the stoichiometry oß examine action can be studied most sim*,~
with examine rather than its precursor
a"~.
However, with aramine~ studies
must be limited to peripheral tissues since the amine does not penetrate into the central nervous system to any greater extent . The results of this study indicate that examine is taken up by sympathetic tissue .
Initially there is a sufßicient amount of examine
present to account ßor a male per mole displacement .
Whether the initial
release involves a mole for mole displacement cannot be verißied as yet . However
a large proportion of the examine disappears rapidly coinciding
with the most rapid phase oß norepinephrine release .
By 2~+ hrs the residual
o+ninP a no longer add up to the original 11 .1 mFtmales and ßor two days or longer the examine level is about equal to the residual norepinephrine ; the
Vol . 3, No. 7
MECHANISM OF NOREPINEPHRINE DEPLETION
70 1
sum of the two accounting for only 50~ of the control norepinephrine level . The question as to whether or not the long lasting aramine depletion is due to an exact mole for mole replacement is of more than academic interest .
If this were the case, then one must conclude that sympathetic
tissues contain a fixed number of storage sites for norepinephrine which cannot be exceeded .
However, norepinephrine levels can be increased after
norepinephrine infusion (8) and after administration of monoamine oxidase inhibitors,
Furthermore, one would have to conclude that after several
days the same norepinephrine sites become available as were initially filled by aramine and that in the interim there was no destruction and reaynthesis of binding sites . There is no question that administered aramine markedly delays norepinephrine repletion .
The experimentally determined half-life of nor-
epinephrine in normal guinea pig heart is about 12-14 hra (7) .
Yet after
aramine depletion it takes many days far norepinephrine to return to control values .
Since part of the aramine which reaches the heart disappears with a
half-life of 45 hra, it is reasonable to consider that this residual aramine in some way inhibits norepinephrine repletion .
Although it would be convenient
to find an exact equivalence between residual aramine and the norepinephrine deficit the data for guinea pig heart do not support this .
The observed
equivalence in some tissues of rats and rabbits reported by Shore and Alpera (5) suggest that differences may exist among animal species .
It ie even more
probable that the differences reflect differences in experimental design .
The
conclusions to be drawn from experiments of this type are of importance with respect to the function of the sympathetic nervous system and we would suggest that final judgement be reserved until more detailed experiments become available .
702
MECHArTIBM OF NOREPIIIEPI~II~IE DEPLETION
Vol . 8, No. 7
References 1 . W . LOVENBERG, H, WEISSBACH, and S . UDENFRLEND, Biochem . Biophys . Res . Camonun . 3, 225 (1960) . 2 . A . CARLSSON AND M. LII~QVIST, Acta Physiol . Scand . 54, 87 (1962) . 3 . G .L . CESSA, E, COSTA, R. KUNTZMAN and B .B . BRODLE, Life Sciences 1, 353 (1962) . 4 . S . UDENFRIEI~ and P . ZALTZMAN-NIRENBERG, J . Pharm . Exp . Therap . 138, 194 (1962), 5 . P .A . SHORE and H, ALPERS, Fed . Proc . i, 350 (1964) . 6 . J .R, CROUT, C .R . CREVELING and S . UDENFRIEND, J . Pharm . Exp . Therap . 1~, 269 (1961) . 7, R, j+~NTANART , E, 232 {1963) .
M,A . BEAVEN and B .B, BRODIB, Life Sciences _2, _COSTA,
8 . S . SPECTOR - to be published,