0022-3042/78/0601- I357S02.00/0
Journal of Neurochemislry Vol. 30, pp. 1357-1361
Pergamon Press Ltd. 1978. Printed in Great Britain
8 International Society for Neurochemistry Ltd.
ACCELERATION OF CHOLINE UPTAKE AFTER
DEPOLARIZATION-INDUCED ACETYLCHOLINE RELEASE
I N RAT CORTICAL SYNAPTOSOMES
ROBERTROSKOSKI,JR.
Department of Biochemistry, The University of Iowa, Iowa City, IA 52242, U.S.A.
(Received 10 November 1977. Accepted 20 December 1977)
Abstract-Activation of nerve elements in uivo and in vitro is associated with an increased rate of
choline uptake by a Na+-dependent high affinity transport system. Following the methodology of
BARKER(1976),rat cortical synaptosomes were depolarized (37"C, 10 min) by 25 mM-KCI in the presence
of CaClz (I mM) or other divalent cations. After reisolation by centrifugation, the rate of 3H-choline
uptake (1.25 p ~ was
) measured by Millipore filtration. KCl treatment alone failed to accelerate the
rate of uptake in the reisolated synaptosomes. CaCI2, BaC12 or SrCI2 (but not MgC12 or MnCI2)
were necessary ( l m ~ to
) observe the KCI induced acceleration. Moreover, RbCI, but not LiCl or
CsCI, also produced the calcium-dependent rate enhancement in the reisolated synaptosomes. The
conditions mediating the enhanced rate of choline uptake correlated strongly with those associated
with neurotransmitter release. To test this possibility, synaptosomal acetylcholine content was measured
in response to the various salt treatments. Treatment with KCI (25 mM) and CaCI2 (1 mM), but not
KCl alone, reduced the synaptosomal acetylcholine content from 154 to 113 pmol/mg protein. The
respective rates of choline uptake increased about 60%. The increased rate was reversed by incubation
with 50 pM-choline followed by synaptosome reisolation. This procedure also normalized the acetylcholine content. In summary, the rate of choline uptake by the high affinity choline uptake system
is inversely related to the synaptosomal acetylcholine content.
ACETYLCHOLINE biosynthesis is closely linked to
acetylcholine release. COLLIER& KATZ (1974), for
example, showed that acetylcholine synthesis keeps
pace with release in the rat superior cervical sympathetic ganglion. POTTER
(1970) has reached similar conclusions using the rat phrenic nerve-diaphragm preparation. In the rat CNS, SIMONet al. (1976) demonstrated that increases or decreases in neuronal activity
in specific regions were accompanied by corresponding changes in the Na+-and temperature-dependent
high affinity choline uptake system. Furthermore,
BARKER(1976) demonstrated that after K + depolarization of rat brain synaptosomes in uitro, choline
uptake was enhanced. He reported an increase in the
V,,, of transport, but no change in K,. JENDENet
al. (1976) reported that there was an inverse correlation of choline uptake and acetylcholine content
(altered by treatment in uivo) in mouse brain synaptosomes. They suggested that synaptosomal acetylcholine inhibits the high affinity choline uptake system.
Following the lead of BARKER(1976) and JENDEN
et a!. (1976), the mechanism of the K + mediated increase in the rate of choline uptake was examined.
In contrast to BARKER'S
results (1976), initial experiments indicated that increased choline uptake
occurred under conditions associated with previous
acetylcholine release. To confirm this notion, acetylThis work was supported by US. Public Health Service
Grant NS-11310.
choline content in synaptosomes in response to K +
depolarization in the presence or absence of CaZ+
and other agents was measured. Increases in the rate
of choline uptake occurred only when acetylcholine
depletion was found.
MATERIALS AND METHODS
Depolarization of synaptosomes. Using male SpragueDawley rats ( 1 5ck2OO g), cerebral cortical synaptosomes
were prepared by the procedure of COTMANet al. (1976).
They were suspended in Ringer solution (glucose, IOmM;
KCI, 1 mM; MgSO,, 1.2 mM; Na2HP04, 2 . 4 m ~ ;NaCI,
150 m ~ Tris-HCI,
;
10 mM; CaCIZ, 0 mM; all adjusted to
pH 7.4) and incubated 10min at 37°C prior to all subsequent manipulations. Unless otherwise noted, the following protocol was used for depolarization. Portions of
2 M-KCI and 0.2 M-CaCI, to give the final specified concentration were added to the synaptosomes (1 mg protein/ml)
suspended in Ringer solution at 0". No addition was made
to the reference synaptosomes which were carried through
all the incubations and centrifugations in parallel with the
treated synaptosomes. After the specified addition, the suspensions were incubated for 10min at 37°C. The tubes
were centrifuged (5000 #, 10 min, 0°C) and the pellets were
resuspended in the original vol of Ringer solution by vortexing and then recentrifuged. The pellets were resuspended in the initial vol of Ringer solution and transport
experiments were then performed (pilot studies showed
that the vortexing did not produce increased liberation of
lactate dehydrogenase into the supernatant).
Portions of synaptosomes (90 p1 of 1 mg protein/ml)
were pre-incubated at 37°C (5 min) in triplicate. Then 10 pl
1357
1358
ROBERTROSKOSKI,
JR.
of 12.5 p ~ - ~ H - c h o l i (69
n e C/mmol) in Ringer solution were TABLE 1. DIVALENTCATION SPECIFICITY I N MEDIATING
added (1.25p~ final) to initiate transport. (Pilot studies DEPOLARIZATION-ENHANCED CHOLINE UPTAKE INTO RAT
CORTICAL SYNAPTOSOMES
showed that uptake was nearly linear for 30min and plateaued by 60min. To stop the reaction at Smin,
Uptake
150 mM-NaCl(5 ml) were added to each test tube. The sus(pmol/min/mg
pensions were applied to glass fiber filters (GFA) (preTreatment
protein)
viously soaked in 150 mM-choline chloride) on Millipore
filtration assemblies (water aspirator pump, 0.5 ml flow 1. Ringer
4.63 f 0.41
5.17 0.42
rate/s of filtrate). The tube was rinsed once with 5 m l of 2. KCI (25mM)
7.38 f 0.81*
150mM-NaC1 solution. The filters were then washed 6 3. KCI (25 mM), CacI2 (1 mM)
7.61 f 0.74*
(1 mM)
,
times with 5 ml portions of the NaCl solution. The filters 4. KCI ( 2 5 m ~ )BaCI,
6.53 f 0.537
5. KCI (25 mM), SrCI2 (1 mM)
were numbered, dried 5 min at I I O T , and radioactivity
4.51 f 0.40
was measured following the addition of 5ml of Tritosol 6. KC1 (25 mM), MgCI, (1 mM)
4.74 f 0.51
7. KCI (25 mM), MnCI2 (I mM)
(FRICKE,
1975).
Using glass fiber filters briefly rinsed in 150 mM-chohne
Portions of synaptosomes (1 mg protein in 1 ml) were
decreased the background from a 900-1300 c.p.m. to a treated with the specified components (lOmin, 37°C). The
more constant 300 c.p.m. (248,000 c.p.m. input). Since cho- synaptosomes were isolated by centrifugation and then
line fails to exhibit homoexchange (SIMONet al., 1976), the resuspended. This was repeated. Choline transport was
unlabeled choline does not displace intrasynaptosomal then measured as described in Methods and Materials. The
choline. To assess Na+-dependence of uptake, pilot experi- values represent the means f S.E.M. of 6 determinations.
* P < 0.01.
ments showed that uptake of 3H choline from Ringer solut P < 0.05.
tion where LiCl replaced NaCl was less than 10% that
of the control with choline concentrations from 0 . 2 5 ~ ~
to 8 p ~ .
KCl-CaCl, treatment (not shown). The increased rate
To measure the acetylcholine content, the specified treat- of transport is reflected in the V,,, (16.3 vs. 9.5 pmol/
ments were performed and the synaptosomes were centri- min/mg protein). Assuming 20 mg of synaptosomal
fuged, resuspended in Ringer solution, centrifuged, resus- protein/g wet wt this is also in close agreement with
pended and preincubated for 5min (37°C) as described
the results of BARKER(1976). Since a Ca2+-depenabove. Then 0.8 ml portions were centrifuged (13,OOO g,
dence
was consistently observed during the present
1 min, ambient temperature, Eppendorf microfuge), and the
supernatants were aspirated. Then 1 5 0 ~ 1of acetone: experiments, and not observed previously (BARKER,
1 M-formic acid solution (85:15) were added to the pellet 1976), additional studies were performed with other
and the synaptosomes were dispersed by sonication. After divalent metal ions. BaCI, and SrCI, (1 mM each)
30min (on ice) the suspension was centrifuged (13,OOOg, were able to replace CaCl, during KCI treatment
1 min) and 130 PI were taken for acetylcholine determina- (Table 1). These divalent cations alone, however,
tion. Tetraphenylboron extraction was performed by the failed to enhance choline uptake. MgCI, and MnC1,
method of GOLDBERG
& MCCAMAN
(1973) and acetylcho- were unable to replace CaCI,. These results parallel
line was measured radiometrically (I4C acetylcholine) the divalent metal requirements for neurotransmitter
using choline acetyltransferase from human placenta (Rosrelease from synaptosomes (COTMAN
et al., 1976).
KOSKI et al., 1975) as outlined by APRISON et al. (1974).
The
specificity
of
the
monovalent
cations
was then
'The final yield of exogenous 3H acetylcholine (10,OOO
tested.
In
the
presence
of
CaCl,,
RbCl
also
mediates
c.p.m.) added to parallel synaptosome fractions was used
the
enhanced
choline
uptake
(Table
2).
RbCl
alone,
to calculate recovery.
Protein was measured by the procedure of LOWRYet however, does not bring about this rate acceleration.
Moreover, treatment with LiCl or CsCl also fails to
al. (1951).
3H-choline (69.5 Ci/mmol) and 'H acetylcholine (60 Ci/ speed up choline transport. The effect of the monomol) were purchased from New England Nuclear, Boston, valent cations in mediating the acceleration of choline
MA.
CATION SPECIFICITY MEDIATING
Student's t-test was used to assess possible differences TABLE2. MONOVALENT
ENHANCED CHOLINE UPTAKE
& COCHRAN,
1967).
(SNEDECOR
RESULTS
Depolarization mediated acceleration of choline transport into synaptosornes
Although there may be a small increase in transport after incubation with KCl ( 2 5 m ~ ) there
,
is a
substantial increase in choline uptake in synaptosomes isolated after treatment with CaCl, (1 mM) and
KCI (25 mM) as shown in Table 1. The rate of uptake
is usually @loo% greater than the control depending on the preparation. In agreement with BARKER
(1976), the apparent K,'s for choline transport are
1 . 5 1 . 7 p ~and are unaltered in response to the
Treatment
1.
2.
3.
4.
Ringer
KCI ( 2 5 m ~ )
KCI (25 mM), CaCl, (1 mM)
RbCl ( 2 5 m ~ )
5. RbCl (25mM), CaC1, (1 mM)
6. LiCl (25 mM), CaC1, (1 mM)
7. CSCI (25 mM), cac12 (1 mM)
Uptake
(pmol/min/mg
protein)
+
4.11 0.32
4.43 f 0.44
6.87 0.64*
4.38 f 0.41
6.74 0.62*
4.02 f 0.38
4.19 f 0.43
After the specified treatment, the synaptosomes were isolated and choline transport was measured as outlined in
Table 1. The values represent the mean f S.E.M. of 6 determinations.
* P < 0.01.
Synaptosomal choline uptake
1359
conditions. Choline uptake was increased about 54%
in the KC1-CaCl2 treated synaptosomes, but not after
the other treatments.
Uptake
In a second experiment, synaptosomes were treated
(pmol/min/mg
with KCI-CaC12, isolated, incubated with 50 pi-choTreatment
protein)
line (10 min, 37"C), isolated, and then acetylcholine
1. Ringer
4.32 f 0.34
content and choline uptake were measured. There is
7.19 f 0.80*
2. KCI (25 mM), CaCI, (1 mM)
normalization of acetylcholine content in the depolar3. KCI (25 mM), CaCl, (1 mM),
ized synaptosomes (Table 5). Furthermore, the rate
4.82 f 0.42
MgClz (15 mM)
of choline uptake decreased t o control values in as4. KCI (25 mM), CaCI, (1 mM),
MnCI, (15 mM)
4.52 f 0.51
sociation with the normalized acetylcholine content.
4.69 f 0.43
5. KCI (25 mM)
This shows that the depolarization enhanced choline
After the specified treatment, the synaptosomes were iso- uptake is reversible. An experiment was performed
lated and choline transport was measured as outlined in to show that the two washes following the incubation
Table 1. The values represent the means f S.E.M.of 6 deter- with 50 pM-choline effectively removed the choline
minations.
prior to the transport experiments. When the synapP < 0.05.
tosomes were incubated with 'H choline ( 5 0 ~ and
)
reisolated as described in Table 5, the washing prouptake in reisolated synaptosomes parallels their cedure removed greater than 99.2% of the label from
the medium. To further show that dilution of the
effectiveness in depolarizing nerves (SHANES,1958).
radioactivity by exogenous choline was not responInhibition of enhanced choline uptake by Mg2+ and sible for the decreased rate of uptake, choline was
Mn2+
added to a control tube after the 10min incubation
Based on electrophysiological studies in uiuo (DEL (0°C). After the usual reisolation, this addition did
CASTILLO
& KATZ, 1954; KATZ & MILEDI,1969) and not decrease uptake of 3H choline.
release studies in uitro (BLUASTEIN,1975; COTMANet
General characteristics of the depolarization induced inal., 1976), Mg2+ and Mnz+ block Ca2+-dependent
crease in choline transport
neurotransmitter release. These compounds also inThe depolarization induced acceleration of choline
hibit the Ca2+-dependent acceleration of choline
uptake
occurred within 4 min, was maximal at 10 min
uptake in reisolated synaptosomes (Table 3). All these
results seem consistent with the idea that acetylcho- (Table 6), and was not any greater at 15 or 20min
line release occurs prior to the observed acceleration (not shown). Furthermore, the effect was not present
if the treatment was performed at 0°C (Table 6). The
of choline uptake in the reisolated synaptosomes.
25 mM-KCl and 1 mM-CaC1, concentrations were also
Synaptosomal acetylcholine content and choline uptake optimal.
Two experiments were performed to test this hypothesis. First, synaptosomes were treated with KCl
(25 mM) alone or with CaCI, (1 mM), or with CaC12
DISCUSSION
(1 mM) and MgC1, (15 mM). The synaptosomes were
BARKER(1976) discovered that treatment of synapisolated and preincubated at 37°C (5 min). Transport
of choline and acetylcholine content were determined tosomes with KCl (35mM) for 10min in uitro
in parallel. There is an inverse relationship between enhanced the rate of choline uptake after reisolation.
choline uptake and acetylcholine content (Table 4). The rapid onset of this rate acceleration by treatment
The acetylcholine content was decreased about 30% in uitro appears amenable to neurochemical characafter treatment with KCl (25 mM), CaCl, (1 mM). Ace- terization. The rapid onset of this response renders
tylcholine was not significantly reduced in the other unlikely a role for protein synthesis. Another possible
TABLE
3. BLOCKADE
OF THE
ENHANCED CHOLINE UPTAKE BY
MAGNESIUM AND MANGANESE
TABLE4. COMPARISON
OF SYNAPTOSOMAL ACh
Treatment
1.
2.
3.
4.
Ringer
KCI (25mM)
KCI (25 mM), CaCI, (1 mM)
KCI (25 mM), CaCI, (1 mM)
MgC12 (15 mM)
CONTENT AND CHOLINE UPTAKE
ACn
(pmol/mg protein)
174 f
161 f
108 f
154 f
11
10
8*
12
Choline Uptake
(pmol/min/mg)
3.87
4.19
6.14
4.21
f 0.37
f 0.42
f 0.53'
f 0.39
The synaptosomes were treated as specified and choline transport was measured
as outlined in Table 1. Acetylcholine content after reisolation was measured by procedures given in Materials and Methods. The values represent the mean
S.E.M.
of 6 determinations.
*
*P
< 0.05.
1360
ROBERTROSKOSKI,
JR.
TABLE5. EFFECTOF
INCUBATION WITH CHOLINE ON THE DEPOLARIZATION ENHANCED TRANSPORT
First treatment
Second treatment
1. Ringer
None
50 pM-Choline
50 pM-Choline
(0")
None
50 pM-chohne
50 pM-Choline
(0")
2. KCI (25mM), CaCI, (1 mM)
Uptake
(nmol/min/mg protein)
ACn Content
(pmol/mg protein)
5.19 & 0.42
4.81 & 0.43
4.93 f 0.51
154 & 7
163 4
158 6
8.31 0.71*
5.46 0.49
8.1 1 & 0.83*
113 & 4
143 4
118 f 3
+
+
Synaptosomes (8 mg protein in 8.00 ml) were first incubated at 37°C (10 min) under control or depolarizing conditions
(KCI, CaC12).After centrifugation (10 min, SO00 8). the pellet was suspended in 8.00 ml of Ringer solution. The suspension
was recentrifuged and resuspended. Aliquots were incubated with 50 pM-choline (10 min, 37°C; or 0 min at 0°C) where
specified. The suspensions were centrifuged and resuspended. This was repeated and then transport and acetylcholine
content were measured as described in Materials and Methods. The values represent the mean & S.E.M.of 6 determinations.
* P < 0.05.
mechanism for altering the transport rate might involve covalent modification of proteins by phosphorylation. Experiments were performed to ascertain
whether cAMP dependent protein phosphorylation
might be involved. The rate enhancement, however,
could not be mimicked by treatment of the synaptosomes in vitro with CAMP,mono- or dibutyryl cAMP
or adenosine (each at 0.1 mM) for 10min at 37°C followed by reisolation (ROSKOSKI
unpublished). Synaptic membranes undergo a Ca2+dependent phosphorylation (DE
LORENZO,
1976) so that a phosphorylation mechanism independent of cAMP cannot be
excluded.
The present results largely confirm and extend
those reported by BARKER(1976). The acceleration
occurs within 5 min, is maximal at 10 min, and is not
enhanced after 20 min of KCl-CaCI, treatment. The
response is temperature-dependent and is associated
with an increase in the V,,, of transport without a
change in the apparent K,,, for choline. The major
difference in the two studies is the present demonstration of Ca2+-dependence.BARKER
(1976) did find,
however, that MgCI2 (10 mM) inhibited the enhanced
TABLE
6. TIMEAND TEMPERATURE
DEPENDENCE OF DEPOLARIZATION ENHANCED CHOLINE UPTAKE
1.
2.
3.
4.
5.
Time,
Temperature
Transport
% control
10
10
10
0
25
37
37
0
104
154
179
161
107
5
0
The synaptosomes were treated with Ringer (control) or
with KCI (25m~)-CaCI, (1 mM) for the specified time at
the indicated temperature. They were isolated as outlined
in Table 1 and transport was measured as described in
Materials and Methods. Each value represents the mean
of triplicate determinations. The control (100%) was
4.87 & 0.51 pmol/min/mg protein. The values represent the
mean of 6 determinations.
rate of choline uptake and suggested that Ca2+ may
be involved. The Ca2'-dependence was consistently
found in the present study. One possible explanation
for the difference might be related to the somewhat
more highly purified synaptosome preparation
employed in the present study. The extrasynaptosoma1 Ca2+ content, for example, may be diminished
during the additional steps involved in the purification procedure.
MURIN& KUHAR
(1976) and VACA& BEACH(1977)
also report that CaZ+is required for the depolarization (KCl or veratridine) induced increase in high
affinity choline uptake in hippocampal synaptosomes
and the avian ciliary nerve-iris preparation, respectively. In a subsequent study, however, MURINet al.
(1977) report that Mg2+ will substitute for Ca2+ during the depolarization induced acceleration of choline
transport. In the perfused cat superior cervical preparation, COLLIER
& ILSON(1977) report that during
nerve stimulation, Ca2+ must be present to observe
an increase in uptake of two choline analogues
(homocholine and triethylcholine). MgS04 (18 mM),
which decreased acetylcholine release by two-thirds,
did not block the nerve stimulation induced increase
in choline analogue uptake. Additional work to
clarify the Ca2+ and Mg2+ effects in these different
preparations seems warranted.
To further document a role for Ca" in the rate
acceleration, the effect of some of its congeners were
tested. Sr2+ and Ba2+, but not Mg2+, substitute for
CaZ+ in stimulating neurotransmitter release (COTMAN et al., 1976). Similar specificity occurs in accelerating the transport rate (MURINet al. (1977) and
Table 1). Furthermore, neurotransmitter release from
synaptosomes is inhibited by Mg2+ and M n Z + ;these
divalent cations also abolish the depolarization induced choline uptake.
The monovalent cation specificity in enhancing
choline transport into synaptosomes parallels their
effect on enhancing CaZ+ uptake and stimulating
acetylcholine release (BLAUSTEIN,
1975). Rb+ in addi-
Synaptosomal choline uptake
tion to K + treatment accelerate choline uptake, but
Li' and Cs' are without a measurable effect [MURIN
& KUHAR(1976) and Table 21.
There appeared to be a rather good correlation
between conditions associated with acetylcholine
release and acceleration of choline uptake in vitro.
To test this idea more directly, acetylcholine content
of synaptosomes treated with KCl in the presence of
divalent cations was measured. An inverse correlation
of acetylcholine content and the initial rate of choline
uptake was found. Furthermore, the effect was reversible. After KCI-CaCl, treatment, acetylcholine content was decreased about 35% and choline uptake
was accelerated. Upon incubation with choline
(50 p ~ ) ,the acetylcholine content returned toward
normal and the rate of choline uptake was correspondingly diminished.
In addition to its role as a precursor for the synthesis of the neurotransmitter acetylcholine, choline
is ubiquitously found as a component of the phospholipid phosphatidylcholine. In the present studies, and
those of JENDENet al. (1976) and POLAK
et al. (1977),
there is an inverse correlation between acetylcholine
content and the rate of choline uptake. In acetylcholine biosynthesis (Acetyl-CoA choline + acetylcholine + CoA), choline and acetylcholine are both
within cholinergic synaptosomes. Since choline is the
transported substance, intrasynaptosomal choline
may inhibit exogenous choline uptake. Acetylcholine,
an analogue, may also inhibit uptake. Other metabolic consequences occur in response to synaptosome
depolarization so that other mechanisms for the
acceleration of transport are not excluded (DE
BELLEROCHE8z BRADFORD,1972).
+
REFERENCES
APRISONM. H., SHEAP.A. & RICHTERJ. A. (1974) Choline
1361
and Acetylcholine. Handbook of Chemical Assay Methods
(HANINI., ed.) pp. 63-80. Raven Press, New York.
BARKERL. A. (1976) Life Sci. 18, 725-732.
BLAUSTEIN
M. P. (1975) J. Physiol. 247, 617-655.
COLLIERB. & KATZ H. S. (1974) J. Physiol. 238, 639655.
COLLIERB. & ILSON D. (1977) J . Physiol. 264, 489-509.
COTMANC. W., HAYCOCK1. W. & WHITEW. F. (1976)
J . Physiol. 254, 475-505.
J. S. & BRADFORD
H. F. (1972) J . NeuroDE BELLEROCHE
chem. 19, 585-602.
DE LORENZO
R. J. (1976) Biochem. biophys. Res. Commun.
71, 59C597.
DEL CASTILLO J. & KATZ L. (1954) J. physior. 124,
553-559.
FRICKE
U. (1975) Analyt. Biochem. 63, 555-558.
GOLDBERG
A. M. & MCCAMAN
R. E. (1973) J. Neurochem.
20, 1-8.
JENDEND. J., JOPE R. S. & WEILERM. H. (1976) Science
194, 635637.
KATZ B. & MILEDIR. (1969) Publ. Staz. zool. Napol. 37,
30S310.
LOWRY0. H. ROSEBROUGH
N. J., FARRA. L. & RANDALL
R. J. (1951) J. biol. Chem. 193, 265-275.
MURINL. C. & KUHARM. J. (1976) Molec. Pharmac. 12,
1082-1090.
MURINL. C., DE HAVENR. N. & KUHARM. J. (1977)
J . Neurochem. 29, 681-688.
ROSKOSKI
R. JR., LIMT.Y.& ROSKOSKIL. M. (1975) Biochemistry 14, 5105-51 10.
POLAK
R. L., MOLENAAR
P. C. & VAN GELDERM. (1977)
J. Neurochem. 29, 477486.
POTTERL. T. (1970) J. Physiol. 206, 145-166.
SHANESA. M. (1958) Pharmac. Rev. 10, 59-164.
SIMONJ. R., ATWEHS . & KUHARM. J. (1976) J. Neurochem. 26, 909-922.
SNEDECOR
G. W. & COCHRAN
W. G. (1967) Srarisrical
Methods, pp. 69-71, 549. The Iowa State University
Press, Ames, IA.
VACAK. & BEACHR. (1977) Trans. Am. SOC. Neurochem.
8, 168.