Download Hypothalamus-pituitary-thyroid axis interactions with pineal gland in

Hypothalamus-pituitary-thyroid
axis
interactions
with pineal gland in the rat
G. L. BRAMMER,
J. E. MORLEY,
E. GELLER,
A. YUWILER,
AND J. M. HERSHMAN
BRAMMER, G. IL., J. E. MORLEY, E. GELLER, A. YUWILER,
AND J. M. HERSHMAN. Hypothalamus-pituitarythyroid axis
ilzteractions
with pineal gland in the rat. Am. J. Physiol.
236(4); E416-E420, 1979 or Am. J. Physiol,: Endocrinol.
Metab.
Gastrointest. Physiol. 5(4): E416-E420, 1979.-We
examined in
the rat several possible relationships
between the pineal gland
and the hypothalamus-pituitary-thyroid
axis. The pineal gland,
the retina, and the hypothalamus
exhibited a diurnal rhythm
in thyrotropin-releasing
hormone
(TRH) content with peak
values occurring around 1200 h. This rhythm in the hypothalamus was abolished by constant light but was not affected by
pinealectomy.
Nor did pinealectomy
affect hypothalamic
TRH
content, pituitary
content of thyroid-stimulating
hormone
(TSH) or prolactin;
serum levels of (TSH), triiodothyronine
(?I&), or thyroxine (T4), or serum free-thyroxine
index; or freetriiodothyronine
index. Melatonin
did not affect TSH or prolactin release from the anterior pituitary
or TRH release from
the hypothalamus
in vitro. Isoproterenol
did not affect the
TRH content of pineal glands in vitro; nor did TRH or T3 affect
basal or stimulated activities of serotonin N-acetyltransferase,
the presumed controlling
enzyme in melatonin production.
We
found no evidence for significant interactions
between the pineal gland and the hypothalamus-pituitary-thyroid
axis.
pineal gland of the rat follows a diurnal rhythm that may
be synchronized by environmental lighting. The rhythm
is suppressed in constant light, but is free running in
constant dark. Under diurnal lighting conditions, the
onset of dark is accompanied by sympathetic nerve activity resulting in an adrenergically mediated, increased
activity of serotonin N-acetyltransferase (SNAT), the
apparent controlling enzyme in the production of melatonin. The circadian periodicity of pituitary TSH in rat
(7) and in man (1) is well recognized. Constant dark
produces an increase in thyroid weight (19) and radioactive iodine uptake (30). The effects of constant dark
on TSH and PBI are controversial (20, 25). Vriend et al.
(30) have shown that blinding of hamsters leads to depression of the plasma free-thyroxine index and that this
effect can be reversed by pinealectomy or bilateral superior cervical ganglionectomy.
Because of the uncertainty in the literature concerning
the relationship of the pineal gland to the H-P-T axis, we
have undertaken a number of experiments in an attempt
to delineate this relationship more clearly. The availability of radioimmunoassays for TRH and TSH, as well as
thyrotropin-releasing
hormone; triiodothyronine
for the thyroid hormones, has enabled us to study the
entire H-P-T axis. Further, tissue culture techniques
have aIlowed us to look for an interaction between TRH
and factors in the pineal gland influencing melatonin
THE RELATION OF THE PINEAL GLAND to the hypothalamus-pituitary-thyroid
(H-P-T) axis is unclear. In 1972 production and to examine the effect of melatonin both
Relkin (20) postulated that the pineal gland had an on the release of pituitary TSH and on the release of
inhibitory influence on thyrotropin-l’eleasing hormone hypothalamic TRH. We have particularly examined the
(TRH) secretion. This postulate was based on his obser- following: 1) the effect of lighting conditions on the TRH
vations that 3 days after pinealectomy in the rat there content of the hypothalamus, the pituitary, the retina,
was an acute increase in serum thyroid-stimulating hor- and the pineal gland and on thyroid function; 2) the
mone (TSH) and protein-bound iodine (PBI), which was effect of pinealectomy on the TRH content of the hypono longer present by day 6. More recently, in support of thalamus and on thyroid function; 3) the effect of phis postulate, Relkin (22) has reported decreased plasma noradrenergic stimulation on the TRH content and the
TSH after intraventricular injection of melatonin in the effect of added TRH or T3 on basal and stimulated
rat, Ishibashi et al. (8) found that pinealectomy increased SNAT activity in cultured pineal glands; and 4) the effect
thyroxine secretion rate; Rowe et al, (25), however, ob- of melatonin on the release of TSH from acutely cultured
served no effect of pinealectomy on plasma TSH or PBI anterior pituitary glands and on the release of TRH from
acutely cultured hypothalami.
levels.
Environmental lighting influences both the pineal
gland and the thyroid, which also suggests a pineal- METHODS
thyroid relationship. The production of melatonin in the
Male Sprague-Dawley rats were housed in a temperaE416
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on September 19, 2016
Neurobiochemistry
Research Laboratory
T-85, Veterans Administration
Brentwood
Hospital,
Los Angeles 90073; Department
of Psychiatry and Biobehauioral Sciences, School of
Medicine, University
of California, Los Angeles 90024; and Endocrinology
Research Laboratory,
Veterans Administration
Wadsworth
Hospital, Los Angeles, California
90073
THYROID
AXIS
INTERACTIONS
WITH
PINEAL
E417
GLAND
a gyratory water bath shaker at 37°C under 5% CO2 in
02. The preincubation medium was then replaced by 1
ml preoxygenated medium containing either no additions, melatonin (lo-’ M), TRH (1.4 x 10B8M), or melatonin with TRH. There were five flasks in each group.
After 1 h of further incubation at 37”C, the mediums
were colIected and stored frozen until assayed for TSH
and prolactin at a 1:20 dilution. The wet weight of pituitaries in each flask was recorded.
Hypothalami (excluding the mammillary bodies) were
removed from male rats and halved, and the halves were
divided between control and treated groups. Pairs of
hypothalamus halves were incubated in heat-treated,
bacitracin-containing medium under the conditions described above for pairs of pineal glands. The preparation
of the hypothalamus halves took; approximately 15 min.
The tissue pieces were preincubated for 15 min in medium already equilibrated to the culture conditions. After
the preincubation period, tissue and support screenswere
removed, blotted, and placed in equilibrated medium
containing no additions or 10Y4M melatonin for a 30-min
test period. The medium was then frozen for TRH assay
and the wet; weight of the hypothalamus pairs recorded.
Assays. TSH was measured by radioimmunoassay
with the National Institute of Arthritis, Metabolic, and
Digestive Diseases (NIAMDD)
rat TSH RP-1 used as a
reference preparation. Thyroxine (Td) and triiodothyronine (Tz) were measured by radioimmunoassay as described previously (l), The unsaturated binding capacity
of serum proteins was assessedby a triiodothyronine
uptake test (TTJJ) (15). The free-thyroxine index (FTJ)
and free-triiodothyronine
index (FTJ) were obtained by
calculating the product of the Td or T3 and total TJJ
values. TRH was measured by the method of Bassiri and
Utiger (2) with antibody kindly supplied by Dr. Robert
Utiger of the University of Pennsylvania. Prolactin was
measured with a radioimmunoassay kit supplied by
NIAMDD, Pineal gland SNAT activity was measured by
the method of Deguchi and Axelrod (6) as modified by
Pa&t et al. (13).
Results reported in the text are means t SD (n).
Tabular values are means t SD. The significance of
differences between means was determined by Student’s
t test.
RESULTS
The immunoreactive TRH contents of the pineal
gland, retina, and hypothalamus were significantly higher
at 1200 h compared with 2400 h in rats housed under
diurnal lighting conditions (Fig. 1). Serum Ts and TSH
levels were also significantly higher at 1200 h, but no
difference in pituitary TRH or serum Td was observed
between the two time points.
Constant light abolished the difference between measures of hypothalamic TRH and serum TSH and Ts on
samples taken at 1200 h and 2400 h (Table I).
Pinealectomy did not abolish the diurnal difference in
hypothalamic TRH or serum T3 (Table 2), but did abolish the diurnal difference observed in serum TSH (Tables
2 and 3). A significant difference was observed in serum
T3 levels between pinealectomized and sham-pinealec-
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on September 19, 2016
ture- and light-controlled room (lights on 0500-1700 h)
with food and water ad libitum. Photic experiments made
use of rats from our colony weighing ZOO-250g. Pinealectomized and sham-pinealectomized rats were obtained
commercially (Zivic-Miller, Allison Park, PA). Completeness of pin .ealectomy was confirmed at post mortem.
Animals were decapitated and trunk blood was collected. Serum samples were stored frozen until assayed.
The anterior pituitary glands were weighed and disrupted
by sonication in 1 ml 50 mM phosphate-buffered saline
(pH 7.45). The hypothalamus, the retina, and the pineal
gland were dissected and extracted in methanol, as previously described (18).
Photic experiments. a) Animals were killed at 1200 h
and 2400 h. b) Animals were killed at 2400 h after 67 h
constant light and at 1200 h the next day.
Pinealectomy experiments. a) Pinealectomy was performed on animals weighing 150 g, Twelve days after
surgery, pinealectomized and sham-pinealectomized animals were killed at 1200 h and pinealectomized animals
only were killed at 2400 h. b) Pinealectomy and sham
procedures were performed on 60- to 80-g rats. At 13
days after surgery, animals were lightly etherized at 1300
h and blood samples were withdrawn from the external
jugular vein, At 60 days after surgery, the same animals
were killed at 1300 h. In addition to the other tissues,
thyroid glands were also dissected free and weighed.
Pineal glands for culture experiments were taken from
45- to 60-day-old rats of either sex from our colony at
0900-1000 h, Culture conditions were essentially as described by Pa&t et al. (14) except that the final concentration of glutamine in the culture medium was 2 mM. In
brief, pairs of pineal glands were supported on a nylon
mesh screen in a 16-mm-diameter well containing 0.15 ml
medium. Test compounds were added in volumes of 10
pl and incubations were carried out at 37OC in an atmosphere of 5% CO2 in 02. Because TRH is degraded by
protease activity in many mediums containing serum
fractions, in some experiments examining the release of
TRH or the effects of TRH on SNAT activity complete
medium was heated at 56°C for 30 min and bacitracin
was then added to a level of 50 U/ml before the medium
was used for culture.
Pineal culture experiments. a) After a 30-min preincubation period in complete medium, pineals were transferred to medium with or without 2 x IO-” M DL-isoproterenol. After 3 h pineal glands were removed and frozen
on solid CO:! in groups of 3 or 4 TRH assay. b) Pineal
glands were placed in culture with somatostatin or TRH
and with or without isoproterenol. After 6 h glands were
individually frozen until assayed for SNAT activity. c)
After a 50-min preincubation period in complete medium
with or without T3, pineal glands were transferred to Lnorepinephrine- containing mediu m wi.th or witho ut T3
for 6 h and then were individually frozen until assayed
for SNAT activity.
Anterior pituitaries were removed from male rats and
quartered, and the quarters were randomized. Three
quarters were placed ii lo-ml stoppered Erlenmeyer
flasks containing 1 ml modified Gey and Gey medium (4)
that had been equilibrated to the culture conditions. The
tissue portions were individually preincubated for 2 h in
E418
BRAMMER
I
p<.Ol
100
AL,
SNAT activity stimulated in cultured pineal glands by
either lo-’ M or lOm6M isoproterenol (Fig. 2). Triiodothyronine at a concentration of 7.7 x 10wl’ M (50 ng/dl)
had no effect on the dose-response curve of norepinephrine-stimulated SNAT activity in cultured pineal glands
(Fig. 3).
In anterior pituitary cultures, 10B5M melatonin, alone
or in conjunction with 1.4 x IO-’ M TRH, had no
A TRH (pg/mg)
200
ET
2
1
PINEAL
HYPOTHALAMUS
RETlNA
PITUITARY
3. Effect ofpinealectomy
and 60 days after surgery
-. --
13 days
TABLE
8 SERUM
13 days
Sham
60 days
PX”
PX*
Sham
429 * 93
403
t
73
TRKpg/w
FIG. 1. Diurnal
rhythms
in TRH content
and measures
of H-P-T
axis, A: TRH content of different
tissues. B: serum measures of H-P-T
axis function.
Open bars indicate
samples
taken at 1200 h; hatched
bars, samples
at 2408 h. Vertical
lines indicate
1 SD; n = 12 for all
groups.
Pituitary
TSH,
mu/w
Pituitary
wmg
Serum
Serum
6.8 AI 3.6
prolactin,
376
TSH, &J/ml
prolactin,
92 &41
88 -t 38
T3, ng/dl
T+ pg/dl
TT&J
FTJ
FTJ
wt, mg
wt, mg
59 -t 8
5.5 * 0.7
53 t 9
4.8 k 0.9
t
173
5.4 k 4.3
270
81 -t- 54
28 t 18
t, 223
90 t, 62
17k12
@ml
TABLE
1. Effect of constant light
,---_ - -~--.-.
.- - .--1200 h
Hypothalamic
TRH,
Serum TSH, @J/ml
Serum Ts, ng/dl
Serum Tq, pg/dl
Values
are means
pg/mg
275t
153k
55t
-..----
2400 h
28
94
6
299Ik
31
122 k 61
52 zk 14
6*0 * 1.0
5.9 k 1.9
2. Effect ofpinealectomy
after surgery
_ .
_--_
.-.--- ^
_----.~------px*
n=
mg
k6
k 1.0
t 0.04
IL 9.8
9.8 k 1.9
10.5
26.5
are means
A SE; n = 11 for all groups.
* 1.4
k 5.3
45t
5.0 t
1.78 t
80 k
8.9 t
10.5 *
25.5 k
11
1.2
0.04
18
2.0
1.4
4.2
* Pinealectomized.
IO
12 days
1200 h
Pituitary
Serum
Serum
Serum
Serum
Values
47
5.6
1.74
82
t SD; n = 12.
TABLE
Hypothalamic
.~
Serum
Serum
Serum
Serum
Serum
Pituitary
Thyroid
TRH,
pg/
TSH, mU/mg
TSH, $J/ml
prolactin,
rig/ml
Ts, ng/dl
T4, pg/dl
_ __-- --- -
Values are meins -t- SD,
pared with PX at 1200 h.
313-+
8,2 t
82-+
13.4 -+
50tn
5.4 t
2400 h
Sham
10
32
2.6
47
12
1.9
7.6
100
13,9
35
3.9
* Pinealectomized.
t15
t 1.7
t48
t 5.8
*12t
t 1.9
6
(8)
px*
n=9
322
W
0
n=9
161
7.6
74
7.6
39
4.4
AL sot
t 1.1
t
xk
k
k
TRH
@ TRH
+ ld8M
GUPROTERENOL
l
TRH
+ 16%
ISOPROTERENOL
0
SOMATOSTATIN
48
10
St
2.0
t P < 0.01 com-
tomized animals at 1200 h 12 days after surgery in one
experiment (Table Z), but not at 13 days or 60 days after
surgery in another experiment (Table 3). No significant
differences were observed in any other measures comparing pinealectomized animals at 1200 and 2400 h or
comparing pinealectomized and sham-pinealectomized
animals at 1200 h at 12 days or at 1300 h at 13 or 60 days
after surgery (Tables 2 and 3).
The several experiments in which pineal glands in
culture were used yielded no significant treatment differences. Pineal glands cultured for 3 h with either no
additions or lo-” M isoproterenol had TRH contents
(pghg) of 2.8 t 2.2 (8) and 4.3 t 3.8 (8), respectively.
Somatostatin or TRH, each tested uver a range of concentrations, had no effect on basal SNAT activity in
cultured pineal glands, nor did TRH have any effect on
NONE:
-13-12
HORMONE
-9 -8
-5 -4
(log M)
FIG. 2. Effect
of hormones
on serotonin
N-acetyltransferase
in cultured
pineal glands. Vertical
lines indicate
I SD,
samples in parentheses;
where not indicated,
n = 4.
activity
Number
of
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on September 19, 2016
Hypothalamic
THYROID
AXIS
INTERACTIONS
PINEAL
-5
E419
GLAND
-4
NOREPINEPHRINE
3. Effect of T3 on norepinephrine-stimulated
tyltransferase
activity
in cultured
pineal glands,
I SD; n. = 4 for all groups.
FIG.
(log M)
serotonin
N-aceVertical
lines indicate
4. Effect of melatonin on release of TSH
and prulactin
in anterior pituitary
cultures
~- .--------.. TABLE
TSR, pU/mg
Control
Melatonin
(10s5 M)
TRW (1.4 x lO+ M)
Melatonin
+ TRH
Values
are means
per ml
493 t 213
874 t 725
760 & 261
676 +- 411
Praiactin,
ng/mg per
ml
t
196 -4
207 t
176 k
156
33
32
64
25
* SD; 12 = 5 for all groups.
significant effect upon the
(Table 4). In hypothalamic
had no significant effect on
into the medium (control,
treated, 17.3 t 6.6 (4)).
release of TSH or prolactin
cultures, 10B4 M melatonin
the amount of TRH released
25.5 -t 8.5 (6) pg/mg per ml;
DISCUSSION
The hypothalamic
trophic hormones, TRH, luteinizing
hormone-releasing
hormone (LHRH),
and somatostatin,
have all been shown to be present in the pineal of a
variety of mammalian species (31). Our results show the
presence of a diurnal rhythm of immunoreactive
TRH in
the pineal of the rat. In addition, we confirm that there
is a diurnal rhythm of TRH in the retina (26) and the
hypothalamus
(5).
The diurnal rhythm
in hypothalamic
TRH content
and serum T3 level was abolished by constant light but
was not affected by pinealectomy.
Diurnal lighting, but
not diurnal melatonin production,
seems correlated with
the diurnal rhythm in both hypothalamic
TRH content
and serum T3 level. The diurnal rhythm in serum TSH
level was abolished by both constant light and pinealectomy. The TRH content of the hypothalamus,
peaking
during the light period, is phase reversed from pineal
melatonin production, peaking during the dark period. If
the reduced hypothalamic
TRH content is a reflection of
functional release of TRH, then the phase reversal of
TRH content and melatonin production seems inconsistent with the postulate
that melatonin inhibits TRH
release from the hypothalamus.
Compared with sham-operated
controls, pinealectomy
had no effect on hypothalamic
TRH content, TSH levels,
or measures of thyroid function. The difference in serum
T3 levels between sham-operated
and pinealectomized
animals observed in one experiment
(Table 2) was not
observed in another experiment (Table 3). These differing results may be due to chance, but we cannot exclude
an effect of pinealectomy
on T3 production.
Thus our
data confirm and extend the work of Rowe et al. (25),
which showed no effect of pinealectomy
on plasma TSH
or PBI levels. We conclude that it is unlikely that the
pineal gland plays a physiological
role in the regulation
of thyroid function.
TRH has been reported to be distributed
throughout
the brain (9), the retina (%), the gastrointestinal
tract
(12), and the placenta (27). It has been suggested that
TRH is a ubiquitous
neurotransmitter
(9, 12) that has
been co-opted by the pituitary to provide a stimulus to
maintain basal TSH levels. Our observation of a diurnal
rhythm in rat pineal gland TRH content that is phase
reversed
from the adrenergically
regulated
diurnal
rhythm in melatonin production
and reports of interactions between constituents
of the H-P-T axis and adrenergically induced responses suggested to us the possibility of an interaction between H-P-T constituents
and
the regulation
of melatonin production.
We found no
evidence for an interaction between pineal TRH content
or exogenous TRH or T3 and the adrenergic
SNAT
regulatory system in the rat pineal gland. The lack of an
inhibitory
effect of TRH on norepinephrine
stimulation
of pineal SNAT activity is contrary
to the report of
Tsang and Martin (28) indicating an inhibitory effect of
TRH on norepinephrine
stimulation of cyclic AMP levels
in the rat pineal gland because increased cyclic AMP
seems to mediate the increase of SNAT activity.
A number of studies have shown that pinealectomy
depresses basal prolactin levels in rats (21,24) and blocks
the prolactin rise that occurs in constant darkness (23).
Pineal extracts (3), melatonin (11), and arginine vasototin (29), a polypeptide recently identified in the pineal
gland of rats (17) and man (16), are all capable of elevating prolactin in vivo. Although
mean prolactin
levels
were lower in our pinealectomized
rats compared with
the sham-operated
controls, at no time were the levels
significantly
different. Similarly there was no significant
difference in pituitary prolactin content, Peak prolactin
levels have been shown to occur between 0400 and 0500
h (23) and it is possible that a significant difference could
have been observed at that time. In addition, the “basal”
prolactin levels reported
by Ronnekleiv
and McCann
(24) were very high (between 40 and 100 rig/ml) and this
could have represented
an effect of pinealectomy
on
decreasing the stress-related
prolactin
rise. Melatonin
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.3 on September 19, 2016
-6
WITH
E420
produced no effect on prolactin or TSH release from the
anterior pituitary in vitro. This result is in keeping with
earlier studies suggesting that the action of melatonin on
prolactin release is at the hypothalamic
rather than at
the pituitary
level (11). Our results suggest that the
pineal gland does not play a major physiological
role in
the control of prolactin.
BRAMMER
The skilled technical
assistance
of J. Briggs,
A. Reed is gratefully
acknowledged.
This study was supported
by the Veterans
worth
Medical
Research
Service,
the Veterans
wood Research
and Development
Service, and
Health Grant HD-71-81.
Received
15 August
1978; accepted
in final
form
E. Martin,
ET
J. Park,
AL.
and
Administration
WadsAdministration
BrentNational
Institutes
of
22 November
1978.
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1. AZUKIZAWA,
M., A. E. PEKARY,
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AND D. C.
PARKER. Plasma thyrotropin,
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