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Our results and discussion
In our experiments on rats we assumed that an interrelationship exists
between the influence of thyroidal hormones and estrogens on the secretion
of dopamine. We estimated the effect of estrogens and thyroidal hormones
on AP concentrations of DA and its precursor DOPA, its metabolite DOPAC,
concentrations of NE in the AP, and on DA-2 dopamine receptors in membrane
fraction of AP cell after a chronic administration (21 days) of estradiol
benzoate (in an aqueous microcrystal suspension, Agofolin-Depot, Biotika,
Prague), triiodothyronine (MERCK), or both substances together. In agreement
with the findings of Schreiber et al. (Schreiber, Pribyl, Rohácová,
1970; Schreiber, 1979), we observed that the AP of EB-treated rats were
larger than those of T3, EB+T3, or control rats, whether or not related
to body weight (P>0.01) (Tab. 1). EB-treated rats had lower AP levels of
DA, DOPA, and NE, whereas treatment with T3 alone had no significant effects
on AP levels of these substances (Tab. 1). When T3 was administered in
combination with EB (EB+T3), however, EB-induced reductions of AP concentrations
of DA, DOPA, and NE were attenuated significantly (Tab. 1). Administration
of 3-hydroxybenzylhydrazine dihydrochloride (NSD-1015), an irreversible
inhibitor of L-aromatic amino acid decarboxylase, significantly increased
DOPA concentrations in the AP in all experimental groups (Tab. 1). In EB-treated
rats, NSD-1015-induced DOPA accumulation in the AP was significantly less
than in control animals (P>0.01). T3 treatment had no significant effect
on NSD-1015-induced DOPA accumulation in the AP, nor did it affect the
reduced DOPA accumulation in EB-treated rats (Pacák et al., unpublished
results). EB significantly increased serum levels of prolactin and reduced
serum concentrations of thyroxine. TSH content in the AP remained unchanged.
The content of prolactin and cAMP in the AP was increased after EB treatment.
T3 alone did not affect AP cAMP level but reduced AP TSH and plasma T4
levels. Serum prolactin levels were enhanced after T3 administration. EB+T3
attenuated the EB-induced increases in prolactin levels in the AP and plasma
(Tab. 2). A chronic treatment with EB increased the number of AP DA-2 receptors
and T3 attenuated these increases, without altering DA-2 receptor affinity
(Fig. 1), (Nedvídková et al., in press).
Since catecholamines in the AP are derived at least partly from the
hypophyseal portal blood, and the ME is a main site of release of catecholamines
into portal blood, the results suggest that estrogen treatment decreases
activity of dopaminergic and noradrenergic neurons terminating in the ME
and that T3 partially blocks these inhibitory effects. In general, these
results are consistent with the hypothesis that mechanisms of EB-induced
AP growth include release from dopaminergic restraint.
Administration of T3 alone did not affect AP content of DA, DOPA, DOPAC,
or NE, consistent with previous reports that hyperthyroid rats have normal
DA content in the arcuate nucleus (Fuxe and Hokfelt, 1969) and normal concentrations
and release of DA into hypophyseal portal blood and in the ME (Rondeel
et al, 1988., Yang and Pan, 1994)). Simulataneous administration of T3
with EB partially prevented EB-induced pituitary enlargement and attenuated
EB-induced decreases in AP concentrations of DA, DOPA, DOPAC, and NE levels.
These data suggest an inhibitory interaction between estrogen and thyroid
hormones in influencing synthesis and turnover in catecholaminergic neurons.
Since both DA and NE inhibit prolactin secretion (Ishibashi and Yamaji,
1984; Hall, Harvey, Chadwick, 1984), EB-induced decreases in DA and NE
in the AP could contribute to estrogen-induced prolactin hypersecretion
and pituitary enlargement, and thyroid hormone administration could block
these effects of estrogens by preventing EB-induced catecholaminergic inhibition.
On the other hand, Wang et al. (1994) observed that both in presence or
absence of estradiol, thyroxine replacement in oophorectomized and thyroidectomized
rats decreased portal plasma TRH and arterial plasma TSH to euthyroid levels.
Combination of the injection of thyroxine and EB in vivo caused significantly
decreased levels of portal plasma dopamine and increased arterial plasma
PRL. On the basis of these results the authors suggested that the negative
feedback control of thyroid hormones on the secretion of pituitary TSH
is due, at least in part, to inhibition of secretion of TRH into hypophyseal
portal blood, and that thyroxine potentiates the stimulatory effect of
estradiol on the secretion of pituitary prolactin by inhibition of secretion
of dopamine into hypophyseal portal blood. The authors, however, did not
follow the weights of the AP. Several possibilities could explain this
contradictory result. First, we conducted experiments on male rats and
in contrast to our thyrotoxic rats, their rats obtained T4 substitution
to an euthyroid status. It is possible that only pathophysiologically high
concentrations of thyroidal hormones elicit inhibiting interaction with
estrogens influencing synthesis and turnover incatecholaminergic neurons
and plasma levels of prolactin to return to normal levels. Estrogen-induced
effects on central dopaminergic systems depend on sex, dose, duration of
treatment, and endocrine status. Thyroidectomy diminishes the concentrations
of circulating prolactin and treatment by thyroxine restores plasma prolactin
to euthyroid levels (Tang, Wang S., Wang P., 1986). On the other hand,
it is also known that hypothyroid patients have high plasma levels of prolactin
and after treatment with thyroidal hormones return to normal levels.
The in vivo findings contrast with results of in vitro studies, where
thyroid hormones have been reported to enhance growth and tumor transformation
of AP cells exposed to estrogens (Zhou-Li et al, 1991; Koga, Nakao, Sato,
1992). The present study confirms the previous in vivo reports and supports
a catecholaminergic basis for this results. Results are supported by the
observation of Hordon and Wright (1994) that pituitary hyperplasia in 12-year-old
girl may be associated with primary hypothyroidism and therefore it is
vital to have endocrine investigation prior to consideration of surgical
removal of an apparent pituitary tumor.
Membrane-bound dopaminergic (DA-2) receptors determine the actions of
DA in the anterior pituitary (Caron et al, 1978; Sibley, De Lean, Creese,
1982). Alterations in cell membranes, mutations of pituitary transcription
factors, or changes in G-protein/cAMP pathways leading to defective DA-2
receptor number or function have been suggested to contribute to pituitary
tumorigenesis (Munemura, Aquie, Sibley, 1989; Woods, Johnston J., Johnston
D, 1991; Lin et al., 1992; Ruvkin, 1992). Our results demonstrate that
chronic treatment with EB increases the number of anterior pituitary DA-2
receptors and that T3 attenuates these increases, without altering DA-2
receptor affinity. Since T3 inhibited EB-induced anterior pituitary hyperplasia
and EB-induced increases in PRL levels in AP and plasma, these results,
coupled with previous neurochemical evidence of EB-induced depletion of
tissue DA in the AP (Di Paolo, Daigle, Barden, 1985), help to explain why
administration of DA-2 agonist can prevent pituitary tumor formation and
hyperprolactinemia in rats treated with estrogens.
The findings that EB treatment increased the number of DA-2 receptors,
T3 alone altered neither the number nor affinity of DA-2 receptors, and
that EB+T3 administration attenuated the EB-induced increases in DA-2 receptor
number (Nedvídková et al., in press) could have reflected
compensatory rather than primary changes in DA-2 receptors. (This could
be explained by our observations that estrogen inhibits the synthesis and
release of DA from TIDA systems and catecholamine contents in the AP and
T3 prevents these effects.) The calculated changes in DA-2 receptor number
after EB treatment, however, may also represent increased density of lactotrophs.
As reported previously, numbers of lactotrophs increase up to twofold after
chronic estrogen treatment (Dušková and Schreiber, 1989). Since
in the present experiment EB increased DA-2 receptor number 3.6-fold when
compared to control rats, we do not think that EB-induced increases in
DA-2 receptor numbers were due only to increases in the number of lactotroph
cells. Rather, EB may also have increased the number of receptors per cell
as reported previously (Bression et al, 1983). Hruška and Nowak (1988)
using binding studies observed not only an increase in dopaminergic DA-2
receptors 3-10 days after a single estrogen treatment but also that 6-hydroxydopamine
treatment was able to potentiate these hormone effects.
EB, T3, or EB+T3 did not affect the affinity of AP DA-2 receptors, confirming
previous reports (Di Paolo, Daigle, Barden, 1985; Ali and Peck, 1985; Levesque,
Di Paolo, 1991). EB also increased cAMP levels in the AP and T3 potentiated
this effect, suggesting that estrogens cause a functional uncoupling of
AP DA-2 receptors through modification of the receptor or the G proteins
(Munemura, Aquie, Sibley, 1989). Although estrogen can cause G-protein
mutations (Munemura, Aquie, Sibley, 1989; Friedman et al, 1994) and can
promote pituitary tumor formation, this does not imply that cAMP directly
regulates cell division in the AP since T3 attenuated EB-induced tumorigenesis
but augmented EB-induced cAMP generation. The results suggest that pituitary
tumor formation does not depend in any simple way on local cAMP production.
On the other hand we may suggest that cGMP play the role in EB-induced
tumorigenesis. We observed significantly enhanced cGMP content in the AP
after EB administration (Nedvídková and Schreiber, 1992).
It is known that the lactotrophic cells responds to a secretagogue stimulus
only in the presence of calcium ions (Zofková and Nedvídková,
1988), which operated through phospholipide metabolism with cGMP product.T3
administration also attenuates EB-induced hyperprolactinemia. Findings
of estrogen-induced increases in cAMP and prolactin levels in the AP and
estrogen-induced decreases in DA levels in the AP (Pacák and Nedvídková,
1994) could fit with cAMP-induced stimulation of the prolactin gene or
DA-induced inhibition of secretory activity of prolactin cells (Gibbs and
Neill, 1978; Murdoch, Rosenfeld, Evans, 1982; Demarest, Riegle, Moore,
1984; Sarkar, Gottschall, Meites, 1984; Casanueva et al. 1982). Previously
we observed by estradiol-induced changes in adenohypophyseal beta-adrenergic
receptors, positively coupled with adenylate-cyclase system (Pacák
et al. 1991). Thyroid hormones attenuate estrogen-induced hyperplasia,
activation of prolactin cells, and decreases in delivery of DA to AP cells
(Schreiber, Pribyl, Rohácová, 1970; Dušková and Schreiber,
1989; Pacák and Nedvídková, 1994) but do not affect
estrogen-induced increases in cAMP levels in the AP. Thus, the same mechanisms
that participate in T3 inhibition of estrogen-induced tumorigenesis may
play a role in EB-induced stimulation of prolactin - namely via dopaminergic
system but not via cAMP.
One possible explanation for the DA-2 receptor changes in response to
EB, T3, and EB+T3 is that EB inhibits DA receptor occupation in the AP
and that T3 prevents this inhibition. Thus, EB would lead to upregulation
of local DA-2 receptors, and T3 would prevent this upregulation. This explanation
leads to the prediction that administration of a DA-2 agonist should prevent
EB-induced tumorigenesis. The finding of bromocriptine-induced inhibition
of pituitary tumor formation in estrogen-treated animals is in line with
this prediction (Kalberman et al. 1980; Elias and Weiner, 1987; Bevan et
al. 1992).
In conclusion, estrogen-induced AP growth is associated with decreased
TIDA synthesis and metabolism of catecholamines, and thyroid hormone treatment
attenuates both the estrogen-induced growth and estrogen-induced catecholaminergic
inhibition. The results therefore support a mutually antagonistic interaction
between thyroid hormones and estrogens on catecholaminergic function in
influencing AP growth.
We also observed that alaptide, a drug chemically derived from prolyl-leucyl-glycin
amide (PLG) reduced adenohypophyseal weight a serum levels of prolactin
in rats. On the other hand, it significantly increased the binding of 3H-spiperone
to dopamine DA-2 receptors in AP membrane preparations and the administration
of alaptide together with EB resulted in an aditional increase of dopamine
DA-2 binding sites. We assume that alaptide has a weak dopaminergic activity
on the AP of male rats (Nedvídková et al. 1994), but we cannot
at this time explain the synergistic effect of alaptide with EB on the
number of DA-2 receptors and the opposite effect on serum levels of prolactin.
We observed very interesting results with oral administration of methylene
blue (MB), the inhibitor of quanylate cyclase activity and of the production
of cGMP. MB raised the blood thyroxine level in both intact and carbimazole-fed
animals. MB reduced serum and AP content of TSH administered alone or together
with carbimazol. Slightly, but significantly, the AP weight in intact animals
fell (Nedvídková et al. 1995). To which extent thyroidal
hormones are involved in this result remains an open question. MB inhibition
of serum TSH levels, possibly as a result of the negative feedback by increased
thyroxine, could be an important finding. However, although MB raised the
reduced serum T4 values observed after carbimazole, in our experiments
the T4 level did not reach 50% of control level, so that the marked inhibitory
effect of MB on TSH secretion, with resultant reduction of hypertrophy
of the thyroid is still an open question. We can suppose some direct effect
of MB at the hypothalamohypophyseal level.
Similar to thyroid hormone, MB also affected estradiol-benzoate decrease
of AP concentration of DA and serum level of prolactin (Nedvídková
et al., unpublished observations). We may suggest that the effect of MB
on the dopaminergic system, especially on the concentrations of dopamine
and the changes in the DA-2 receptors may be affected by the increase of
thyroid hormones. (Nedvídková et al. 1995). Decreased plasma
and the AP concentration of prolactin correlate well with changes in the
AP dopamine levels. The mechanism of this action should be a result of
MB interaction in thyroid hormone metabolism and their binding activity
on specific regulatory elements.
The use of a substance which does not belong to those naturally occurring
in the organism, as for example MB, but interacting with some natural regulators
might serve as an instrument for the study of some mechanisms involved
in the regulation of hormone secretion or growth activity of the anterior
pituitary.
Acknowledgement
This work was supported by grant IGA-1825 from Grant Agency of the Czech
Ministry of Health and GA CR of the Czech Republic, 311/93/0987.
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