COLBURNt, ANDNGUYEN THOAt *MedicalService, Veterans Administration Hospital, Philadelphia, Pennsylvania 19104, and Department ofMedicine,MedicalCollegeofPennsylvania, Philadelphia, Penns
Trang 1Proc Nat Acad Sci USA
Vol 73, No 3, pp 941-944, March 1976
Medical Sciences
brain
(thyroid hormones/adrenergic nervoussystem/synaptosomes/neurotransmitters/catecholamines)
MARY B DRATMAN*, FLOYL CRUTCHFIELD*, JULIUSAXELRODt, ROBERT W COLBURNt, ANDNGUYEN THOAt
*MedicalService, Veterans Administration Hospital, Philadelphia, Pennsylvania 19104, and Department ofMedicine,MedicalCollegeofPennsylvania, Philadelphia, Pennsylvania 19129; t National Institute of Mental Health, Bethesda, Maryland 20014
Contributed byJuliusAxelrod, January 5,1976
ABSTRACT Radioactive triiodothyronine reaching the
rat brain after intravenous administration israpidly and
se-lectivelytakenup inthe nerve ending fraction.A
concentra-tion gradient ofradioactivityfrom brain cytosol to
synapto-somes is observed at 5 min,increases linearlyoverthe first
hour, and is maintained for at least 10 hr Radioactivity in
thesynaptosomes is due totriiodothyronine (90%)plusa
sin-gle unidentified metabolite (10%).Approximately85% of the
synaptosomalradioactivity isreleasedbyosmoticdisruption
of the particles.The process of selective uptake,
concentra-tion, and retentionoftriiodothyronine in nerveterminals of
theratbrainmaybe related to thesympathomimeticand
be-havior-altering effectsof thethyroid hormones
Thyroid hormonesexertmarked centralstimulatingand
pe-ripheral sympathomimetic effects, which are notexplained
byincreased catecholamine production orenhanced
adren-ergic receptorsensitivity Onthe contrary,circulatinglevels
ofcatecholamines (1,2),turnoverratesofnoradrenalinein a
number of tissues (3-5), and sensitivity of atleastsome
ad-renergic receptors (6-8) are reported tobe inversely
corre-lated with the thyroid state To explain their
sympathomi-meticactions, wehaveproposedthatiodothyronines,like
ty-rosineand other tyrosine analogues, may betransformed to
adrenergicneurotransmitters(9)
The metabolic pathway leading from precursor amino
acidtoadrenergicneurotransmitter occurswithin thenerve
terminal Therefore, if iodothyronines enter this pathway,
their uptakeand metabolism in normallyinnervated tissues
should be alteredbyadrenergicdenervation.To testthis
ex-perimentally, endogenous triiodothyronine (T3)
concentra-tionanduptakeof[125I]labeled T3werecomparedin
inner-vated and denerinner-vatedsalivaryglandafterunilateral superior
cervical ganglionectomy The results demonstrated that
ad-renergic denervation significantly reduced the uptake and
retentionof T3intheratsubmaxillary salivary gland (M B.
Dratman,F L Crutchfield,J Axelrod,R D Utiger,andH
Menduke, manuscriptsubmitted) Thisevidence, while
con-sistent with uptakeand retentionof T3 inadrenergicnerve
terminals, nevertheless provided no direct information
re-gardingthis process.Methodsforisolatingnerveterminal
el-ementsinsalivary glandandotherperipheraltissues are not
available However, since enriched nerve ending
prepara-tions (synaptosomes) canbe obtained bymeansof
subcellu-lar fractionation of brain (10), this method was used to
ex-amine ratbrains afterintravenousadministration of[125I]T3
The results demonstrate that T3 is selectivelytaken up and
retained within synaptosomes
METHODS
Experiments were performed in 250gadult male rats
pre-Abbreviations: T3, triiodothyronine; S1, supernatant phase of first
centrifugation (1000X g for 10min)of rat brainhomogenate
viously surgically thyroidectomized (Zivic-Miller Laborato-ries) Animals were provided with 4% calcium lactate in theirdrinking water To maintain the euthyroidstate, they
weregiventhyroxine,15gg/kgbody weight daily,up tobut
not including the day of termination of the experiment Some experiments were performed in intact rats; results
werenodifferent from thoseinthyroidectomizedeuthyroid
animals(unpublished observations)
Approximately 50jiCi of [125IIT3labeled in the phenolic
ring[obtained fromB.J.Green ofAbbottsLaboratories,
spe-cific activity approximately 500 jiCi/jig in 50% (vol/vol)
propylene glycol] or 50% propylene glycol was administered
as asingle doseintravenously and animals were decapitated
5, 20, 60, 180, and 600 min later Blood was collected from thedecapitation site and the serum wasseparated and
ana-lyzed forradioactivity and radioactive iodocompounds Sub-cellularfractions of whole brain minus cerebellum were pre-pared according to the method of Whittaker et al (10)
Briefly, following 1000 X gcentrifugation of the brain ho-mogenate for 10 min, nuclei and cellular debris were dis-carded, and the supernatantphase(Sl fraction) was layered
on adiscontinuous sucrosedensity gradientconsisting of1.2,
0.8, and 0.32 M sucrose, andcentrifuged in a swinging
buck-et rotorat50,000X gfor 1 hr Individual gradientfractions
including myelin, synaptosomes, and mitochondria were separated (see diagram, Table 1A) and diluted with 10-18 volumes of isotonic Krebs buffer (11), and pellets were sepa-ratedby centrifugation at 20,000 X g for 20 min To
deter-mine the extent of translocation of labeled T3 during the fractionation procedure, brains of animals which received intravenouspropylene glycol without isotope were homoge-nized at 40 in sucrose containing 0.05jgCi of [125I]T3. Ap-proximately 75% of added ['25I]T3 was recovered in the cy-tosol (plus microsomes); the remainder was distributed among thevarioussubcellular organelles as shown in Table
1B Allbrainfractions labeled in vivo were corrected for in
vitrouptakeat40
Radioactivity in individual subcellular fractions and in
serum was studied by means of paper chromatography in threesolvent systems:butanol:ethanol:0.5Mammonia, 5:1:2; butanol:acetic acid:water, 4:1:1; and tertiary amyl alcohol:2
M ammonia:hexane, 5:6:1 Added carrier compounds were identified by means of ultraviolet light at 259 nm After de-velopment, the radioactivity in each 1 cm segment of the
chromatogramwascounted foratleast 10 min
RESULTS
Followingintravenous administration of[125IIT3torats,
lev-els ofradioactivityintheserumdecreasedoverthe 10 hr
pe-riod, whereas, the concentration in the brain increased, reaching a plateau after the first hour (Fig 1) Discontin-941
Trang 2942 Medical Sciences: Dratman etal.
Table 1. Radioactivityfrom['25I]T3 in subeellular
Time after intravenous [ 25 I]T3 Number of animals
-
-Cytosol (containing
Subcellular particles
p/s
p/s -synaptosomes
p/s -post synaptosomes
p/s
-m -mtochondria
p/s (A) Distribution of subcellular components on discontinuous sucrose gradient; M refers to sucrose concentration (B) Animals received only intravenous propylene glycol;S1 was derived from brains after homogenization at40 in 0.32 M sucrose containing approximately 0.05 UCi of [125I]T3 (C) Animals received intravenous ['251]T3and were decapitated at intervals as indicated; all values were corrected for
con-uous sucrosedensity gradientseparationof theSi fraction of
brain showed that radioactivity from [125I]T3 wastaken up
intoall subcellularparticles (Table 1CandFig 2) However,
within5minandthroughout thefirst hour after [125I]Ts,
ra-dioactivityassociated with the nerveendingswas morethan
2-fold greater than that exhibited by any other particulate
subcellular component, and was stillmorethan 50% greater
at the end of the3 hr period (Fig 2) Concentration of
ra-dioactivity in synaptosomes was calculated (11) and
com-pared withconcentration in thecytosol (see legend, Fig 3);
a ratioofsynaptosomal tocytosol radioactivitygreaterthan
onewasobserved at 5 min; the ratio increasedlinearlyover
the firsthour,andwasmaintainedfor at least10hr(Fig 3)
No corrections were made for the presence of microsomal
particles in the cytosol However, such corrections would
4,000r
_ 3,000
E
a2,000
1,0001
cm
E
E
UL
70C
50C
30C
Serum
f
) - Brain
10
FIG 1 Radioactivity in serum and brain after a single
intrave-dose of [125IT3 Vertical bars indicate SEM.
have increased further the cytosol to synaptosome concen-trationgradient
To verify that the organelle identified as synaptosomes did, in fact, exhibit functional properties of nerve ending preparations, brain fractions from untreated animals were incubated in the presence of 0.4 ,uM [3H]norepinephrine The synaptosomal component behaved as a nerve ending preparation, exhibiting a highly temperature-iependent up-take ofnorepinephrine (40-fold increase in uptake at 370)
Todetermine the identity of the radioactive compounds
inindividual subcellular components, suspensions ofmyelin, mitochondria, microsomes, and synaptosomes were applied
to the origin of paper strips and the chromatograms were
Hours
FIG 2 Radioactivity in subcellular particles of Si fraction of
rat brain at various time intervals after intravenous [125IT3; frac-tions were separated according to diagram, Table 1; data are
ex-pressed as mean 4 SEM (vertical bar); synaptosomes: 0 0; my-elin: 0-0; post-myelin: A - A; post synaptosomes: A - A,
mi-tochondria: X - -X.
A
0.8 M
1 2 0- ,"
1.2 M
l
B
0 min 5
65.5 ± 1.20
9.5 ± 0.55 2
3.05 ± 0.32 1.5 6.85 ± 0.22 3 1.7 ± 0.06
1
1.9 ± 0.11 2
0 I Z 3
Hou rs
Proc Nat.Acad.Sci USA 73(1976)
Trang 3Proc Nat Acad Sci USA 73(1976) 943
particles ofSi fraction of rat brain homogenate
C
5 min
4
20 min
4
50.1 ± 0.86
2.9 ± 0.71
4
3.9 ± 0.20
4
8.6 ±0.27
11
1.9 ± 0.24
3
2.0 ± 0.42
5
45.3 ± 0.72
5.0 ± 1.23
5
5.2 ± 0.20
4
10.2 ± 0.19 12
2.2 ± 0.25
4 2.6 ± 0.49
5
60 min
4
42.5 ± 1.17
5.7 ± 1.11
6
4.8 ± 0.61
6
12.75 ± 0.46 13
1.9 ± 0.13
4
1.75 ± 0.38
4
180 min
5
39.2 ± 0.71
8.7 ± 0.29 4.5
4.4 ± 0.40 4 13.6 ± 0.95
10
2.0 ± 0.32 3 0.85 ± 0.08 3
600 min
4
36.6 ± 0.57
10.4 ± 0.85
5
4.8 ± 0.33 4 13.3 ± 1.13 8 2.15 ± 0.25 3 1.7 ± 0.07
3 centration of [125I]T3 in vitro at 4° as in (B) Data are expressed as mean % 4o SEM of total cpm/mg of brain applied to gradient; p/s = ratio
of particle-bound to nonparticle-bound radioactivity in individual fractions separated on gradient.
developed in three separate solvent systems At both 1 and 3
hrafter[125I]T3,labeledT3accounted forvirtuallyall of the
radioactivity associated with subcellularparticlesother than
synaptosomes A second small peakofradioactivitywas
ob-served in chromatograms of the synaptosomal fraction,
0
26
24
c,
-'D
0
C.)
|.
0
I4
l.2
i
Ii
I.
0
0
Minutes
.
0
Hours
FIG 3 Concentration of radioactivity in synaptosomes
rela-tive to brain cytosol after intravenous [1251]T3, calculated as
fol-lows:
cpm in synaptosomes/mg brain cpm in cytosol/mg brain
where brain density = 0.8 ml/g and synaptosomal density = 0.184
ml/g, derived from data in Fig 4 of ref 11 The concentration
gra-dient relationship to time is described by means of a least squares
straight line, the slope of which is + 0.0116 concentration gradient
units/min (r = 0.93) The relationship is not linear after the first
hour; differences between 1, 3, and 10 hr ratios are not significant.
* = ratio in individual animals; = mean value at each time iii
terval.
amounting to approximately 10%, as compared with
ap-proximately90%Ts
Synaptosomal membranes rupturewhensubjectedto
hy-potonic conditions, resulting inrelease of contained precur-sor aminoacids andneurotransmittervesicles(12) To
deter-mine whether osmotic disruption would release
synaptoso-mal radioactivity derived from T3, portions of the nerve
endingfraction werecollected onMillipore filters; radioac-tivity on the filters and in the filtrate was measured
fol-lowing washingwitheitherisoosmoticbufferorwithwater.
Synaptosomesexposedtohypoosmotic conditions lost85%of their radioactivity as compared with samples treated with buffer (Table 2). Thus, [1251]T3 appears to be contained
within, rather than attached to, the membranes of the
sy-naptosomalparticles.
DISCUSSION The results ofthe present experiments demonstratethat the hormonetriiodothyroninereaching the brain bywayofthe
systemic circulation is differentially distributed among all
Table 2 Effect of osmotic disruption on retention of
synaptosomalradioactivity
Conditions Experiment n Isoosmotic Hypoosmotic Difference
Rats were given intravenous [1251]T3 and decapitated 1 hr later Brains were homogenized and SI fractions were separated Por-tions (0.2 ml) of the synaptosomal fractions were applied to Milli-pore filters, pore size 0.8 Am, and each pellet was washed three times with 5 ml portions of either isoosmotic buffer or water Data
are expressed as mean cpm retained on Millipore filter + SEM.
MedicalSciences: Dratmanetal
Trang 4944 Medical Sciences: Dratman et al.
thesubcellularorganelles,butisselectivelytaken upintothe
nerveending (synaptosomal) fraction Particle-bound
radio-activity isvirtuallyall accounted forbyT3, except foran
ad-ditional unidentified metabolite in the synaptosomal
frac-tion A concentration gradient ofradioactivityfrom cytosol
to synaptosomes isevident at 5min, increases linearlyover
the first hour, and is maintained for at least 10 hr after
ad-ministration of labeled hormone Morethan 80% of the
ra-dioactivity in the synaptosomes is released by osmotic
dis-ruption of theparticles.These observationsprovideevidence
thatT3istaken up,concentrated, and retained withinnerve
endings Thepresenceofametabolite ofT3 detectedonlyin
the synaptosomes suggests that the hormone may be
trans-formed within nerveterminals Although evidence derived
from experiments with salivary glandsuggests arole for T3
in peripheral adrenergic nerves, there is no information
available from the present experimentsregardingthenature
of the nerve endings which concentrate the hormone in
brain
The aromatic amino acids, tyrosine and phenylalanine,
are actively taken up into nerveterminals (13) and form a
variety of adrenergic neurotransmitters including
norepi-nephrine, dopamine, epinephrine, and also octopamine,
phenylethylamine, and phenylethanolamine (14) Tyrosine
analogues (e.g., a-methyl-m-tyrosine) are also concentrated
innerveendings,wheretheyareconvertedtofalse
adrener-gic neurotransmitters (15). The possibility that
iodothyron-inesmayundergo analogous metabolictransformations,
sug-gested by their aromatic amino-acid structure, their potent
central and peripheral adrenergic effects, and the
suscepti-bility of these effects to modification by sympatholytic
drugs,is nowgivenadditional supportbyevidence that T3is
taken up,concentrated, retained, andprobablymetabolized
withinnerveterminals of theratbrain
We thank Dr Francis H Sterling for his many contributions to
this work and Mses Effie Erlichmanand Bonni Wisdow for their
expert technical assistance This work was supported by funds from
the Medical Research Service, Veterans Administration Hospital,
Philadelphia, Pa and National Institutes of Health Grant no.
AM16420.
1. Christensen, N J (1973) "Plasma noradrenaline and adrena-line in patients with thyrotoxicosis and myxoedema," CGn Sci Mol Med 45, 163-171.
2 Stoffer, S S., Jaiang, N., Gorman, C & Pikler, M (1973)
"Plasma catecholamines in hypothyroidism and hyperthyroid-ism," J Clin Endocrinol 36, 587-589.
3 Landsberg, L & Axelrod, J (1968) "Influence of pituitary,
thyroid and adrenal hormones on norepinephrine turnover and metabolism in the rat heart," Circ Res XXII, 559-571.
4 Klawans, H L., Jr & Shenker, D M (1972) "Observations on the dopaminergic nature of hyperthyroid chorea," J Neurol Transm 33,73-81.
5 Beley, A., Rochette, L & Bralet, J (1973) "Influence du traitement par la thyroxine et le propylthiourcile sur le taux
de renouvellement de la noradre'naline dans huit organes peri-pheriques du rat," Arch Int Physiol Biochim 81, 287-298.
6 Aoki, V S., Wilson, W R & Theilen, E 0 (1972) "Studies of the reputed augmentation of the cardiovascular effects of cat-echolamines in patients with spontaneous hyperthyroidism,"
J Pharmacol.Exp.Ther 181, 362-368.
7 El Shahawy, M., Stefadouros, M A., Carr, A A & Conti, R (1975) "Direct effect of thyroid hormone on intracardiac con-duction in acute and chronic hyperthyroid animals," Cardio-vasc Res 9,524-531.
8 Spaulding, S W & Noth, R H (1975) "Thyroid-catechol-amine interactions," Med Clin North Am 59, 1123-1131.
9 Dratman, M B (1974) "On the mechanism of action of thy-roxine, an amino acid analog of tyrosine," J Theor Biol 46, 255-270.
10 Whittaker, V P., Michaelson, I A & Kirkland, R J A (1964)
"The separation of synaptic vesicles from nerve-ending parti-cles ('Synaptosomes')," Biochem J 90, 293-303.
11 Colburn, R W., Goodwin, F K., Murphy, D L., Bunney, W E., Jr & Davis, J M.(1968) "Quantitative studies of norepi-nephrine uptake by synaptosomes," Biochem Pharmacol 17, 957-964.
12 Whittaker, V P (1965) "The application of subcellular frac-tionation techniques to the study of brain function," Prog Biophys 15, 39-96.
13 Guroff, G (1972) in Basic Neurochemistry, eds Albers, R W., Siegel, G J., Katzman, R & Agranoff, B W (Little Brown & Co., Boston, Mass.), pp 197-198.
14 Axelrod, J & Saavedera, J M (1974) "Aromatic amino acids
in thebrain,"in Ciba Found Symp 22 (new ser.) (American Elsevier, New York), pp 51-59.
15 Kopin, I J (1968) "False adrenergic transmitters," Annu Rev Pharmacol 8, 377-394.
Proc Nat.Acad.Sci USA73(1976)