Salma Aslam Kundi, Mary Forsling*, Halluck Kleimestur*, Saleem Wazir,
Taj Muhammad
Khan**,
Background: Estrogens modulate
the release of Arginine Vasopressin (AVP). There is
an increased level of AVP in plasma and pituitary under osterogen
hormones. Methods: Ten rats were experimental and ten were controls. The
ten experimental rats were injected tamoxifen daily
subcutaneously for three days in does of 1 mg/kg body weight in 0.1 ml
vegetable oil base. The ten control rats were given 0.1 ml of vegetable oil
vehicle only subcutaneously. On the morning of the fourth day all the twenty
rats were decapitated. Blood was centrifuged at 2500 rev/min for 15 min at 40C.
Plasma was stored at –20oC for assay of AVP. Pituitary gland was
removed and homogenized in a test tube containing 1 ml of 0.2 molar acetic acid
& stored at –20oC for analysis of pituitary AVP. Uteri of all
rats were removed by careful dissection & weighed. Results: Uterine
weight and plasma AVP levels were decreased but the pituitary AVP level was
unaffected. Conclusion:Under Tamoxifen (anti-estrogen) effect the plasma vasopressin
decreased but the level in the posterior was not affected. The effect of osterogen on Hypothalamo
Pituitary axis (HPA is measurable in the hypothalamus therefore probably anti-oestrogenic effect would operate more at hypothalamic level
rather than posterior pituitary. This needs further investigation at the
Hypothalamus level.
Key Words: Vasopressin, Tamoxifen,
Pituitary
INTRODUCTION
There
has been considerable interest expressed over the past two decades in the
relationship between steroid hormones and the secretion of vasopressin in both
animals and humans. Ovarian sex steroids play a major role in the mechanisms
underlying the changes in body fluid observed during the ovarian cycles of both
humans and animals.
Arginine Vasopressin (AVP) is a neuroendocrine hormone synthesized in the hypothalamus, and
is stored and secretedby the posterior pituitary
gland in response to stimuli such as plasma hypertonicity
and hypotension. It causes antidiuresis in man and
most other mammals. Secrection of the hormone is
affected by changes associated with the menstrual cycle in women and oestrous cycle in rats.
In primates the sexual cycle is menstrual, its most conspicuous feature
is the periodic vaginal bleeding that occurs with the shedding of the uterine
mucosa (menstruation).
In mammals the sexual activity of the male is more or less continuous,
but in most species the sexual activity and several different cells are
involved at different points in the menstrual cycle. Most of the time the
female avoids the male and repulses his sexual advances. Periodically however, there is an abrupt change in behaviour and the female seeks out the male, attempting to
mate. These short episodes of heat or oestrous are so
characteristic that the sexual cycles in mammalian species that do not
menstruate is named the estrous cycle. This change in female sexual behaviour is brought on by a rise in the circulating blood
estrogen level. In spontaneous ovulating species with estrous cycle, such as
the rat, there is no episodic vaginal bleeding but the underlying endocrine
events are the same as those in the menstrual cycle.
Two main steroid hormones produced are oestradiol
(oestrogen) and progesterone which has progestational activity. The naturally occurring oestrogens are steroids that are secreted by the theca interna and granulosa cells of
the ovarian follicles, by the corpus luteum, by the feto-placental unit, and in small amounts by the adrenal
cortex and the testis. The biosynthetic pathway involves their formation from
androgens. They are also formed by aromatization of androstenedione
in the circulation. Almost all the oestradiol comes
from ovary, oestrogens have many actions on the body,
one of it is some degree of salt and water retention as noticed just before
menstruation. It mediates these effects by direct action on the renal tubules and
probably also by the release of the hormone vasopressin which is involved in
the regulation of intravascular volume and composition 1,2 . It is
possible that increased AVP secretion contributes to the premenstrual fluid
retention.
Progesterone is a C21 steroid secreted by the corpus luteum
and the placenta. It is an important intermediate in steroid biosynthesis in
all tissues that secrete steroid hormones, and small amounts apparently enter
the circulation from the testis and adrenal cortex. Progesterone has an anti oestrogenic effect on myometrial
cells, decreasing their excitability, their sensitivity to oxytocin,
and their spontaneous electrical activity, while increasing their action
potential. It decreases the number of oestrogen
receptors in the endometrium and increases the rate
of conversion of 17β oestradiol to less active oestrogens. Large doses of progesterone inhibit LH
secretion and potentiates the inhibitory effects of
estrogens. Large doses of progesterone produce natriuresis,
probably by blocking the action of aldosterone on the
kidney.
Some investigators have failed to observe any significant effect of
these gonadal steroids on osmoregulation
or AVP secretion.3 In
contrast others have documented changes in plasama
AVP concentration. During the rat oestrous cycle or
alterations in rat pituitary content of AVP in response to exogenous oestradiol and progesterone4. Skowsky and colleagues5 demonstrated that large
doses of oestradiol increased basal serum AVP
concentration and decreased serum osmolity in rats,
suggesting that osmoregulation was influenced by gonadal steroid hormones.
Changes in the AVP concentration due to alterations in wather balance were also seen over the estrous cycle of
rat. Increased neurosecretory activity in supra-optic
and para ventricular nuclei has been observed during
the pro-oestrous and oestrous
in cyclic animals 6. Variations in AVP concentration during oestrous cycle reflects the ovarian steroid concentrations
in plasma, being highest when oestradiol concentration
are highest and lowest when progesterone concentrations are increased 1.
Little is known of the manner in which ovarian steroids may influence
AVP release but the study of Barron7 suggests that oestrogens alters the slope of the plot of plasma AVP
against plasma osmolality. Oestradiol
receptors are shown in the hypothalamic nuclei and neurohypophysis
8 and gonadal steroids have also been
shown to influence AVP pathways 2. A combination of audio radiogrpah and immunohistochemistry
technique has indicated that oestradiol can
concentrate in magnocellular neurons 8,9
and have a stimulatory effect on activity of neurons in paraventricular
nucleus 10.
Oestradiol recepters have also been shown to be located in cells of arcuate nucleus and the tuberoinfundibular
dopaminergic neurons originating from this nucleus
innervates the megnocellular nuclei11furthermore
estrogen have been shown to modify catecholamine content in paraventricular
nucleus4 and to concentrate in the nuclei of noradrenergic neurons
that project as paraventicular nuclei 12.
The influence of eastrodiol on vasopressin release is
not however established. While some5, described a stimulatory effect
others groups have found no effect. We set out to examine the effect of
chemical castration due to tamoxifen and its effect
on vasopressin release from the posterior pituitary.
Tamoxifen (Nolvodex, property of imperial chemical industries limited)
was introduced in early 1970. This nonsteroidal anti
estrogen, was used as a subcutaneous injection in our study.
MATERIAL AND METHODS
Twenty
Srague Dawley femal virgin rats weighing 240-280 grams were selected from
the Animal House of the department of physiology,
Ten rats were experimental and ten were controls. The ten experimental
rats were injected tamoxifen daily subcutaneously for
three days in does of 1 mg/kg body weight in 0.1 ml vegetable oil base. The ten
control rats were given 0.1 ml of vegetable oil vechicle
only subcutaneously. On the morning of the fourth day all the twenty rats were
decapitated.
The rats were rapidly decapitated by a guillotine, care being taken to
avoid squeezing the thorax during execution 13. The blood issuing
from the vessels of the trunk was collected in chilled heparinised
tubes. Small aliquots were drawn immediately into capillary tubes for microhematocrit determination.
The remaining blood was centrifuged at 2500 revolutions/minute for
fifteen minutes at 4° C, in refrigerated centrifuge (MSE, coldspin).
The plasma was separated and osmolality and,
electrolyte concentration determined. The remaining plasma was stored at - 20°
C for subsequent extraction and assay of plasma vasopressin.
The pituitary gland of each rat was also removed immediately after death
of the rat by careful dissection and homogenised in a
test tube containing I ml of 0.2 Molar acetic acid and stored at - 20° C. it
was stored for subsequent extraction and analysis of Pituitary vasopressin (Pt
AVP). The uteri of all the rats were also removed by careful dissection and
weighed.
The glass tubes containing the posterior lobes of the pituitary glands
initially had 1 ml of 0.21 M acetic acid. The gland was homogenised
in it with a glass rod. 1 ml acetic acid was added again and further homogenised. Finally 0.5 ml acetic acid was added and final
homogenization took place to make up the final amount of 2.5 ml and boiled for
five minutes. This mixture was centrifuged at 2100 revolutions/minute for 20
minutes. The supernatant was removed into another tube and stored in a deep
freezer at -20 C until assay.
The plasma was also stored for AVP assay in the deep freezer at -20 C
until assay. Assay on both pituitary AVP levels and plasma AVP levels was to be
done at a later date when all the samples were collected.
The radioimmunoassay for AVP were performed by Dr. Mary Forsling, using techniques the use of Sap Paks for AVP extraction, the use of a radio labeled 125
I-AVP and an antibody raised against AVP conjugated to thyroglobulin.
The techniques employed were basically the same for the plasma and pituitary
assays, except that a more sensitive antibody is needed for the former because
of the much lower AVP concentrations involved.
The procedure for the pituitary AVP sampling involved the dilution of 50
micro litre extract with a 5.95 ml of buffer (TRIS+human serum albumin) from which duplicate 100 micro litre neat and double diluted samples were prepared. To
each of these was added 100 micro litre of
standardized labeled AVP solution and 25 micro litre
of anti body solution, the sample tubes being vortexed
after each addition. After incubation at 4 C over night, the unbound AVP was
removed by addition of charcoal, the remaining solution then being removed for
radioactive counting by suction pipette following centrifugation.
The same
procedure was followed with a standard AVPsolution to
produce 12 samples of known concentrationeach being
half the strength of the other. The radioactive counts obtained with these
references samples (LKB Wallac Mine Gamma Counter
Model 1275) were then plotted as a semi log graph of percentage bound 125 I AVP
against the amount of AVP/sample.
This
standard curve was then used to construct parallel curves throught
the experiment points obtained with the pituitary AVP samples and to determine
the amount of AVP present (in units U defined by the First International Standard
for AVP, 77-501). The plasma concentration of AVP is presented in the text in
micro U / ml of plasma and the pituitary AVP content in UM/gland (milli units/gland).
The significance of the differences between the means of groups were
determined by means of students t test for paired or unpaired samples, as
appropriate. Most of the results were analyzed by computer (Commodore CBM Model
8096) and the probabilities read from tables. Probability values <0.05 are
taken to be significant and values between 0.05 and 0.10 possibly significant.
RESULTS
1.
Uterine
Weight:
The
uterine weight was significantly decreased due to tamoxifen
treatement. This reductionis
statistically highly significant P < 0.001 by student’s t-test.
2.
Plasma
AVP (PAVP)
The
PAVP concentration in the unchallenged and untreated control rat was 1.147 +
0.16 micro units/ml (S.E.M) where as in the tamoxifen
treated rat it was 0.38 + 0.1 micro units/ml (S.E.M). this reduction under tamoxifen influence is highly significant statistically (P
< 0.001) by a student’s t-test.
3.
PIT-AVP:
The tamoxifen treated group compared to the unchallenged,
untreated control rats show no significant statistical difference.
Discussion
The
Decrease in plasma vasopression (AVP) after ovariectomy did not appear to be due to the removal of
ovarian source of vasopressin since although ovarian vasopressin has been
reported to have rapid turnover 14 the content is only 0.004% of
that in the pituitary. There was no decreased in the pituitary vasopressin to
account for the reduced plasma vasopressin concentration. Pituitary AVP content
was also shown 15 to
be similar in sham operated and ovariectomized rats,
confirming that removal of gonadal steroids has no
significant effect on AVP storage in posterior pituitary. Neither did the
decrease in Plasma AVP concentration appear to be due to factors normally
assumed to suppress vasopressin release, namely a blood volume expansion or
decrease in plasma osmolality. Although one cannot
rule out subtle modulation.
A fall in the plasma AVP could result from removal of the effect of
ovarian steroids on the pathways controlling release.
Decarboxylase
(ODC) activity as a result of oestradiol stimulation
can cause an increase release of AVP from neurohypophysis
16.
Table-1: Comparison of the
control and experimental groups
|
|
Control |
Experimental |
Pooled
Variance Estimate |
||
|
T-Value |
Degree of Freedom |
2-Tail Probability |
|||
|
Uterine*
Weight |
206.9+10.18 |
142.2+10.34 |
4.45 |
15 |
<0.001 |
|
PLAVP |
1.15+0.16 |
0.38+0.1 |
4.065 |
12 |
<0.001 |
|
PITAVP |
583.4+52.97 |
568.7+24.8 |
0.25 |
12 |
0.85 |
*
Wt/100 grm body weight.
Uterine weight in
millgrams/100 grm body weight. PLAVP=micro units/milli liters. PITAVP=milli
units/gland
Area postrema the most caudal circum-ventricular organ located on dorsal
surface of medulla is also involved in the regulation of AVP. The AVP synthesis
& release are tonically stimulated by Area postrema in basal state and
regulated in the stimulated states.17
The action of sex steroids on the hypothalamus-pituitary-adrenal (HPA)
axis is measurable in the hypothalamus.18 Sheep treated with tamoxifen were compared to controls. Estrogen significantly
increased hypothalamic corticotropin releasing factor
and AVP concentration. The result demonstrate that the effect of estrogen on
HPA axis is measurable in the hypothalamus, and is therefore not primarily at
the pituitary.
Our studies support their results and we need to do further investigation
at the hypothalamic level as well. The set points for vasopressin release in
response to increasing plasma osmolality and hypovolaemia alter with reproductive status, in a study23
vasopressin release following ovariectomy and oestrogen replacement was done. It was found that
vasopressin release in response to both stimuli was reduced in ovariectomized rats and the response was restored by oestradiol replacement. We need to do more work involving
the Hypothalmo Pituitary axis (HPA) and the level of
AVP under anti-estrogen effects.
References
1.
Butcher RL, Collins WE, Fugo
NW. Plasma concentrations of LH, FSH, prolactin,
progesterone and oestradiol throughout the 4-day oestrous cycle of the rat. Endocrinology 1974;94:1704-7.
2.
De Vries GJ, Buijs RM, Sluiter AA. Gonadal hormone action on the morphology of the vaspressinergic innervation of
the adult rat brain. Brain Research 1984;298:141-5.
3.
Durr JA, Stamoutsos BA, Lindheimer MD.Journal of Clinical Investigation 1981;68: 337-46
4.
5.
Skowsky WR, Swan L, Smith
P. Effects of sex steroid hormones on arginine
vasopressin inintact and castrated male and female
rats. Endocrinology 1979;104:105-106.
6.
Swabb DF, Jongkind OF. The hypothalamic neurosecretory
activity during the estrous cycle, pregnancy, parturition and persistent
estrous after gonadectomy in the rat. Neuroendocrinology 1970;6:133-145.
7.
Barron WM, Schreiber J, Lindheimer
MD. Effect of ovarian sex steroids on osmoregulation
and vasopressin secretion in the rat. Endocrine Metab.
American Journal of Physiology 1986;250: E352-E361.
8.
9.
Sar M, Stumpf WE. Simultaneous localization of Hestradiol
and neurophysin I or orginine
vasopressin in hypothalamic neurons demonstrated by a combined technique
10. Negoro HS, Visessuwan S,
11. Grant
LD, Stumpf WE. Hormone uptake sites in relation to
central nervous sysem biogenic amine systems. In:
Anatomical Neuroendocrinology.
12.
13. Husain, M. K, Manger WM, Rock TW, Weiss RJ, Frantz AG.
“Vasopressin release due to manual restraint in the rat: role of body
compression and comparison with other stressful stimuli”. Endocrinology
1979;104:641-4.
14. Watches
DC, Swann RW,
15. Barnafi L, Croxatto H. Vasopressin
and oxytocic activities in the posterior lobe of the
pituitary gland of male and female rats following adrenalectomy
and gonadectomy. Acta Endorinologicu 1966;52, 3-6.
16. Khan
MA, Khan MA, Aslam M. Food intake and water retention over the oestrus cycle of the rat treated with difluormethylornithine
(DFMO). 1995
17. Arima H, Kondo K, Murase T, Yokoi
H, Iwasaki Y, Saito H, Oiso Y. Regulation of
vasopressin synthesis and release by area postrema in
rats. Endocrinology 1998;139(4):1481-6.
18. Wood
CE, Saoud CJ, Stoner TA, Keller-Wood M. Estrogen and
androgen influence hypothalamic AVP and CRF concentrations in fetal and adult
sheep. Regul Pept.
2001;98(1-2):63-8.
19.
______________________________________________________________________________
Address for Correspondence:
Salma Aslam Kundi, Department of Physiology,
Phone: +92-992-381907 Ext: 3074
Email: salmakundi@hotmail.com